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Metalloproteinase cleavage of versican at the fibroblast cell surface Maurice, Sean Bertram 2009

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 METALLOPROTEINASE CLEAVAGE OF VERSICAN AT THE FIBROBLAST CELL SURFACE  by  Sean Bertram Maurice B.Kin., University of Calgary, 1999  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies (Dental Science)  THE UNIVERSITY OF BRITISH COLUMBIA  October, 2009 © Sean Bertram Maurice, 2009  ii ABSTRACT  Versican is a large aggregating proteoglycan expressed in the pericellular matrix of fibroblast cells.  It is highly expressed during development and remodeling.  The regulated synthesis and degradation of versican are associated with physiological remodeling.  Versican is expressed in fibroproliferative lesions of human pulmonary fibrosis and atherosclerosis.  Stromal expression of versican is associated with many forms of cancer and may be predictive of poor prognosis.  Abnormal persistence of the versican-rich matrix may contribute to fibroproliferative and oncogenic processes.  The process of versican degradation is not understood, but as versican is a pericellular molecule, physiological degradation likely involves cell surface-associated proteolysis. As such, the overarching hypothesis for this work is that regulated versican turnover involves the cell surface-associated metalloproteinases ADAMTS-2, MMP-2 and MT1-MMP, that are expressed in versican-rich remodeling lesions.  ADAMTS-2 is a procollagen N-propeptidase involved in collagen fibrillogenesis.  As procollagen is synthesized in a versican-rich matrix, it was hypothesized that ADAMTS- 2 might bind and process versican.  MMP-2 and MT1-MMP in complex with TIMP-2, are activated at the cell surface during wound healing, pulmonary fibrosis and cancer.  Versican was purified from human fetal lung fibroblast cultures for in vitro proteolysis experiments.  The purified versican preparation was characterized by electrophoresis, chromatography, spectrophotometry and mass spectrometry.  ADAMTS-2 and versican localization in normal and fibrotic human lungs were investigated.  ADAMTS-2 was shown to co-purify with versican from human fetal lung fibroblasts.  Bovine ADAMTS-2 was purified from fetal calf skin and shown to cleave purified human versican.  The plant lectin concanavalin-A (ConA) induces a matrix degradative phenotype and is used here to investigate the process of versican degradation relative to apoptotic  iii events in human fetal lung fibroblast cultures.  Con-A induced increased expression of MMP-2 and MT1-MMP in human fetal lung fibroblasts and a concomitant loss of versican from the matrix.  Microarray analysis was used to investigate expression of possible versican-degrading enzymes and their inhibitors, expressed in response to ConA.  Recombinant MMP-2 and MT1-MMP were shown to process purified versican in vitro.  This work expands upon the body of knowledge of versican turnover and should help in the search for therapeutic avenues to treat fibroproliferative and oncogenic processes.  iv TABLE OF CONTENTS Abstract ....................................................................................................................ii Table of Contents ....................................................................................................iv List of Tables ...........................................................................................................ix List of Figures .......................................................................................................... x Abbreviations......................................................................................................... xiii Dedication..............................................................................................................xiv Acknowledgements ................................................................................................xv Co-authorship Statement.......................................................................................xvi  CHAPTER 1 – BACKGROUND 1.1.  Versican in normal physiology .................................................................... 1 1.1.1.  Development ............................................................................................ 2 1.1.2.  Reproduction............................................................................................ 4 1.2.  Versican in wound healing disorders.......................................................... 5 1.2.1.  Wound healing and fibrosis...................................................................... 5 1.2.2.  Pulmonary fibrosis.................................................................................. 10 1.2.3.  Atherosclerosis....................................................................................... 13 1.2.4.  Cancer stroma........................................................................................ 15 1.2.5.  Arthritis ................................................................................................... 19 1.2.6.  Tendinopathies....................................................................................... 19 1.3.  Versican structure ....................................................................................... 20 1.3.1.  Hyalectans ............................................................................................. 21 1.3.2.  Gene organization and regulation .......................................................... 21 1.3.3.  Splice variants........................................................................................ 24  v 1.3.4.  Glycosaminoglycans .............................................................................. 27 1.3.5.  N-terminus/G1........................................................................................ 30 1.3.6.  C-terminus/G3........................................................................................ 30 1.4.  Versican turnover in tissue remodeling .................................................... 31 1.4.1.  Matrix metalloproteinases ...................................................................... 32 1.4.2.  ADAMTS ................................................................................................ 37 1.4.3.  Known and unknown versican proteolytic events .................................. 40 1.4.4.  Versican proteolysis ............................................................................... 41 1.4.5.  Glial hyaluronate binding protein............................................................ 41 1.4.6.  Hyaluronectin ......................................................................................... 43 1.4.7.  Matrikines............................................................................................... 44 1.4.8.  Potential mechanisms of altered proteolysis in aberrant tissue remodeling ........................................................................................................ 45 1.5. Rationale ....................................................................................................... 45 1.5.1. Overarching hypothesis .......................................................................... 48 1.5.2. Specific aims ........................................................................................... 48 1.6.  References ................................................................................................... 50  CHAPTER 2 – PURIFICATION AND CHARACTERIZATION OF VERSICAN 2.1.  Summary ...................................................................................................... 78 2.2.  Introduction.................................................................................................. 78 2.3.  Experimental procedures ........................................................................... 80 2.3.1.  Cell culture ............................................................................................. 80 2.3.2.  Immunofluorescence staining and microscopy ...................................... 81 2.3.3.  Isolation of versican ............................................................................... 82  vi 2.3.4.  Electrophoretic techniques..................................................................... 82 2.3.5.  Western blotting ..................................................................................... 83 2.3.6.  Quantification of chondroitin sulfate concentration with the DMMB assay................................................................................................................. 84 2.3.7.  Gel filtration chromatography ................................................................. 84 2.3.8.  Characterization of proteolytic fragments............................................... 84 2.3.9.  Enzyme incubation................................................................................. 85 2.3.10.  Proteomic identification ........................................................................ 85 2.4.  Results.......................................................................................................... 86 2.4.1.  Versican purification from human fetal lung fibroblast cells ................... 86 2.4.2.  Optimization of versican purification ...................................................... 89 2.4.3.  Gel filtration analysis of versican............................................................ 91 2.4.4.  Characterization of versican degradation............................................... 91 2.4.5.  Mass spectrometric characterization of versican ................................... 93 2.4.6.  Versican glycosylation and characterization .......................................... 95 2.5.  Discussion ................................................................................................... 97 2.6.  References ................................................................................................. 100  CHAPTER 3 – VERSICAN-ADAMTS-2 INTERACTIONS IN HUMAN   PULMONARY FIBROSIS 3.1.  Summary .................................................................................................... 106 3.2.  Introduction................................................................................................ 107 3.3.  Experimental procedures ......................................................................... 109 3.3.1.  Patient samples.................................................................................... 109 3.3.2.  Histology .............................................................................................. 110  vii 3.3.3.  Immunohistochemistry ......................................................................... 110 3.3.4.  Release of ADAMTS-2 from normal lung tissues................................. 111 3.3.5.  Cell culture ........................................................................................... 111 3.3.6.  Isolation of versican ............................................................................. 112 3.3.7.  Co-purification of versican and ADAMTS-2 ......................................... 113 3.3.8.  Separate elution of ADAMTS-2 and versican ...................................... 113 3.3.9.  Electrophoretic techniques................................................................... 113 3.3.10.  Purification of fetal bovine skin ADAMTS-2 ....................................... 115 3.3.11.  Versican digestion and ADAMTS-2 incubations ................................ 115 3.4.  Results........................................................................................................ 116 3.4.1.  Versican and ADAMTS-2 co-localize in normal human lungs.............. 116 3.4.2.  Versican and ADAMTS-2 localization in BOOP ................................... 116 3.4.3.  Versican and ADAMTS-2 are localized to remodeling areas in UIP .... 118 3.4.4.  Release of ADAMTS-2 from normal human lung tissue ...................... 118 3.4.5.  ADAMTS-2 co-purifies with versican at physiological pH .................... 120 3.4.6.  ADAMTS-2 co-purification with versican is pH dependent................... 123 3.4.7.  Purification of bovine ADAMTS-2......................................................... 125 3.4.8.  ADAMTS-2 degrades versican............................................................. 127 3.4.9.  Versican inhibits auto-degradation of ADAMTS-2................................ 129 3.5.  Discussion ................................................................................................. 131 3.6.  References ................................................................................................. 135  CHAPTER 4 – VERSICAN DEGRADATION AT THE CELL SURFACE BY   MMP-2 AND MT1-MMP 4.1.  Summary .................................................................................................... 141  viii 4.2.  Introduction................................................................................................ 142 4.3.  Experimental procedures ......................................................................... 144 4.3.1.  Tissue culture....................................................................................... 144 4.3.2.  Immunofluorescence staining and microscopy .................................... 145 4.3.3.  SDS-PAGE........................................................................................... 146 4.3.4.  Gelatin zymography ............................................................................. 147 4.3.5.  RNA preparation .................................................................................. 147 4.3.6.  Microarrays .......................................................................................... 148 4.3.7.  Enzyme assays .................................................................................... 149 4.4.  Results........................................................................................................ 150 4.4.1.  ConA induces degradation of versican ................................................ 150 4.4.2.  ConA induces changes in MMP-2 and MT1-MMP localization ............ 150 4.4.3.  Microarrays .......................................................................................... 153 4.4.4.  Caspase and MMP inhibition alters the apoptotic response of fibroblast cells to ConA ................................................................................... 155 4.4.5.  MMP-2 and MT1-MMP cleave versican in vitro. .................................. 157 4.4.6.  MT1-MMP cleaves and disaggregates recombinant versican G3........ 159 4.5.  Discussion ................................................................................................. 159 4.6.  References ................................................................................................. 165  CHAPTER 5 – CONCLUDING REMARKS AND FUTURE DIRECTIONS ......... 172 5.1.  References ................................................................................................. 179 Appendix 1 – Complete data set of CLIP-CHIP proteinase, inhibitor and control spot fold changes and p-values.......................................................... 183  ix LIST OF TABLES Table 1.1  Versican expression in cancer.............................................................. 16  x LIST OF FIGURES Figure 1.1  Structures of interstitial proteoglycans revealed by electron microscopy .............................................................................................................. 3 Figure 1.2  Granulation tissue in a cutaneous wound at five days post injury......... 7 Figure 1.3  Myofibroblasts in normal and pathological wound healing.................... 9 Figure 1.4  Versican expression associated with proliferating fibroblasts in idiopathic pulmonary fibrosis ................................................................................. 12 Figure 1.5  Versican localization in atherosclerosis............................................... 14 Figure 1.6  Hyalectan family members in the central nervous system .................. 22 Figure 1.7  Versican splice variants showing domain composition and potential glycosylation sites.................................................................................................. 23 Figure 1.8  Versican V0 primary sequence and potential glycosylation attachment sites ....................................................................................................................... 25 Figure 1.9  Glycosaminoglycan monomer constituents......................................... 28 Figure 1.10  MMP family domain organization, active site consensus sequence and propeptide ‘cysteine switch’............................................................................ 33 Figure 1.11  Common and divergent pathways of regenerative versus pathological wound healing ....................................................................................................... 35 Figure 1.12  ADAMTS family domain organization and evolutionary relationships .......................................................................................................... 39 Figure 1.13  Detection of versican proteolytic fragments ...................................... 42 Figure 1.14  Normal and aberrant proteolysis of versican at the cell surface ....... 46 Figure 2.1  Versican at the cell surface of human fetal lung fibroblasts ................ 87 Figure 2.2  Purification of versican from human fetal lung fibroblast cells at pH 7.5 .................................................................................................................... 88  xi Figure 2.3  Chondroitin sulfate concentration measured by the DMMB assay ..... 90 Figure 2.4  Analysis of versican separated by gel filtration ................................... 92 Figure 2.5  N-terminal sequencing of versican degradation product ..................... 94 Figure 2.6  Tryptic versican peptides detected in MS/MS ..................................... 96 Figure 2.7  Versican cleavage sites and peptides detected in proteomics................ experiments compared with potential glycosylation sites ...................................... 98 Figure 3.1 Versican and ADAMTS-2 localization in normal lungs and in bronchiolitis obliterans organizing pneumonia (BOOP)....................................... 117 Figure 3.2 Versican and ADAMTS-2 localization in usual interstitial pneumonia (UIP) .................................................................................................................... 119 Figure 3.3  Release of ADAMTS-2 from normal human lung tissue.................... 121 Figure 3.4  ADAMTS-2 and versican co-purify .................................................... 122 Figure 3.5  ADAMTS-2 co-purification with versican is pH dependent................ 124 Figure 3.6  Purification of bovine ADAMTS-2 from fetal calf skin........................ 126 Figure 3.7  ADAMTS-2 degrades versican in vitro. ............................................. 128 Figure 3.8  Versican inhibits ADAMTS-2 auto-degradation................................. 130 Figure 4.1  Concanavalin A induces degradation of versican concomitant with fibroblast cell apoptosis ....................................................................................... 151 Figure 4.2  Concanavalin A induced changes in metalloproteinase expression and localization.................................................................................................... 152 Figure 4.3  Microarray analysis of differentially expressed fibroblast proteases and inhibitors in response to Concanavalin-A ..................................................... 154 Figure 4.4  Caspase and MMP inhibition alters the apoptotic response of fibroblast cells to Concanavalin-A ....................................................................... 156 Figure 4.5  MMP-2 and MT1-MMP cleave versican in vitro................................. 158  xii Figure 4.6  MT1-MMP cleaves and disaggregates recombinant versican G3..... 160  xiii ABBREVIATIONS ADAMTS-2 a disintegrin and metalloproteinase with thrombospondin motifs-2; MMP-2    matrix metalloproteinase-2; MT1-MMP  membrane-type matrix metalloproteinase-1; IPF    idiopathic pulmonary fibrosis; CSPG   chondroitin sulfate proteoglycan; BOOP   bronchiolitis obliterans organizing pneumonia; HUVEC  human umbilical vein endothelial cell; BSA    bovine serum albumin; HFL-1   human fetal lung fibroblast; ECM    extracellular matrix; PBS    phosphate-buffered saline; TBS    tris-buffered saline; UIP     usual interstitial pneumonia; DMEM   Dulbecco’s modified Eagle medium; CM    conditioned medium; DMMB   1,9-dimethylmethylene blue chloride. ConA   concanavalin A; Z-FA-FMK  carboxybenzyl-phenylalanine, alanine, fluoromethylketone; Z-VAD-FMK carboxybenzyl-valine, alanine, aspartate, O-methylated      fluoromethylketone; SAM    significance analysis of microarrays.  xiv DEDICATION I am deeply indebted to many people who have helped, encouraged and supported me over the past 6 years.  Above all, I would like to dedicate this work to my family:  To my wonderful, long-suffering wife Andrea, who fulfills my heart and soul, and has enabled me to live up to my fullest potential.  To my children, Zachary, Mary-Grace and Susan, for giving me a break from my work, for keeping my worries in perspective, for making me smile, laugh and play, when I might otherwise forget to.  To my parents who worked hard to provide a decent upbringing for me and my sisters, who challenged me to be my best and at the same time allowed me to learn from my own mistakes.  To the Lord God, without whom nothing would be possible and by who's grace I live each day.   xv  ACKNOWLEDGEMENTS  I am forever indebted to Dr. Clive Roberts for many years of dedicated supervision. Over the last six years Clive has always been generous with his time, offering extensive guidance and thought provoking suggestions.  This work would not have been possible without the kind supervision, trust and congeniality that was offered to me in the lab.  It has been a privilege having Clive as a mentor.  I am indebted to members of the Roberts lab, Saloumeh Pourmalek and Rendi Yan for friendship and technical assistance in the lab.  I also thank prior lab member Dennis Lee who performed significant groundwork on versican purification that benefited my studies enormously.  I am extremely grateful to Dr. Chris Overall for his assistance, advice and encouragement throughout my thesis work.  I am also grateful to Dr. Overall and Dr. Roberts for the collaborative atmosphere that I was able to train in.  I thank my committee members Dr. Ed Putnins and Dr. Doug Waterfield for taking time to meet one on one, for offering thorough and thoughtful feedback on my work and for their encouragement.  I would like to thank the many members of the Overall lab for providing advice, mentorship and friendship, and allowing me to feel like a member of their lab, more than just a collaborator.  I am also grateful to Dr. Ross MacGillivray and the Centre for Blood Research that is an outstanding interdisciplinary facility and a privilege to work in.  My future as a scientist is brighter due to the diverse scientists and techniques that I was exposed to within this centre.  xvi CO-AUTHORSHIP STATEMENT Chapter 2 - I was involved in all aspects of the experimental design, data analysis and writing of the manuscript, with assistance from Dr. Clive Roberts, Dr. Chris Overall and Dr. Alain Doucet.  I performed all the experiments described with the exception of the purification of versican at pH 6.0 that was performed by Dennis Lee and which was used for most of the experiments described.  I thank Dr. Richard Dean and Dr. Oded Kleifeld for intellectual discussion surrounding the proteomics work.  I thank Suzanne Perry for expert assistance with Edman Degradation chemical N-terminal sequencing.  Chapter 3 - I was involved in all aspects of the experimental design, data analysis and writing of the manuscript with assistance from Dr. Clive Roberts.  I performed all experiments described with the exception of the immunohistochemical staining and lung tissues extractions which were performed by Rendi Yan.  Chapter 4 - I was involved in all aspects of the experimental design, data analysis and writing of the manuscript with assistance from Dr. Clive Roberts, Dr. Chris Overall, Dr. Reinhild Kapplehoff and Dr. Alain Doucet.  I performed all experiments described with assistance from Reinhild Kapplehoff in performing and analyzing the microarray experiments; and assistance from Alain Doucet in performing and analyzing the proteomics experiments.  Yili Wang expressed and purified recombinant MMP-2 and soluble recombinant MT1-MMP.  Recombinant versican His-G3 and His-LC constructs were cloned, expressed, purified and refolded by Clive Roberts, Heidi Kai and Saloumeh Pourmalek.  Anti-His-LC antibody LC2 was generated by Clive Roberts and Saloumeh Pourmalek.   xvii   This work was supported by grants from the Canadian Institutes for Health Research (MT 15171) and the British Columbia Lung Association.  CHAPTER 1 – BACKGROUND  1.1.  Versican in normal physiology  Versican is expressed in many tissues during development and also in adult tissues. First named because it appeared to be a 'versatile proteoglycan' (Zimmermann & Ruoslahti, 1989), versican is indeed associated with many different tissues and functions. Versican is expressed in the brain, vasculature, intervertebral discs, liver, myometrium and prostate (Dours-Zimmermann & Zimmermann, 1994).  It is associated with elastic microfibrils in the skin and loose connective tissue of many organs (Bode- Lesniewska et al, 1996; Zimmermann et al, 1994).  Versican contributes to fibrous networks in the pancreas and biliary tracts (Fukata et al, 1989).  Versican performs several mechanical and biochemical functions.  Its glycosaminoglycan side chains are involved in creation of a hydrated space that allows the resistance to stretch and compression through the reversible redistribution of water, a property termed visco-elasticity (Kinsella et al, 2004).  The physical size of the molecule allows it to alter accessibility of the cell surface and thereby indirectly alter cell surface binding and signaling (Kinsella et al, 2004; Roberts, 2003).  Creation of an expanded and hydrated matrix is also associated with facilitation of cell migration and proliferation prior to collagen deposition and tissue remodeling.  Versican appears to aid in the ordered deposition of collagen during normal wound healing.  However versican is also associated with contributing to disorganization of collagen fibrils during cervical dilation (Uldbjerg & Malmstrom, 1991), suggesting that the concentration and localization of the molecule is critical to determining its effect on collagen organization.  Versican binds numerous ligands through its protein domains 1  and its glycosaminoglycan side chains.  Variable ligand binding may allow versican to alter matrix permissiveness and glycosaminoglycan binding of cytokines contributes to ECM cell signaling and chemotactic gradients.  Versican has been referred to as an anti-adhesive molecule and it is specifically excluded from sites of focal adhesion (Yamagata & Kimata, 1994; Yamagata et al, 1993).  Because of its association with cell migration and proliferation, versican is proposed to be a haptotactic factor, promoting cell movement through creation of a gradient of cell-extracellular matrix adhesions (Cattaruzza & Perris, 2005).  Versican’s multiple functions are accomplished through its composite structure that contains globular and linear protein domains with a substantial mass of glycosaminoglycans.  Compared to the related proteoglycan aggrecan, versican is less glycosylated, but it is still a very large hydrodynamic component of the ECM.  As visualized by electron microscopy, versican’s core protein is extended through the glycosaminoglycan attachment domains, the glycosaminoglycans are fairly linear and the globular terminal domains are small compared to the size of the whole protein (Fig. 1.1).  1.1.1.  Development  Versican was first identified because of its association with skeletal development in the embryo (Kimata et al, 1986).  A transgene insertional mutation into mouse chromosome 13 identified a locus critical to endocardial cushion formation in the heart (Yamamura et al, 1997).  The homozygous mice are embryonic lethal by day 10.5 and the gene responsible was identified as versican (Mjaatvedt et al, 1998).  Hyaluronan synthase-2 knockout mice develop a very similar phenotype of cardiac malformation suggesting a crucial interaction (Camenisch et al, 2000).  Versican proteolysis is also 2 Figure 1.1.  Structures of interstitial proteoglycans revealed by electron microscopy. Bovine interstitial proteoglycans analyzed by glycerol spraying/rotary shadowing electron microscopy.  A. Cartilage proteoglycan.  E. Aorta proteoglycan (versican).  Adapted from Morgelin, M. et. al.  Shared and distinct features of interstitial proteoglycans from different bovine tissues revealed by electron microscopy.  © J. Biol. Chem. 1989; 264: 12080-12090. Copied under licence from Access Copywright.  Further reproduction prohibited. 3  critical in heart development where cleavage products alter cell behaviour differently from the intact molecule (Kern et al, 2007; Kern et al, 2006).  Versican appears to guide neural crest cell (NCC) migration and axon outgrowth through inhibitory interactions (Landolt et al, 1995; Schmalfeldt et al, 2000). Overexpression the transcription factor Pax3 leads to overexpression of versican and defective NCC migration in mice (Henderson et al, 1997).  Purified versican inhibits NCC attachment and migration in vitro. (Perris et al, 1996; Schmalfeldt et al, 2000). However, versican coated membranes attract migrating NCC and co-polymerization of versican with collagen increases NCC migration (Perissinotto et al, 2000).  Versican may thus direct NCC migration through a combination of permissive and inhibitory interactions construed by different parts of the molecule.  Specific proteolytic events may be necessary to end versican’s role in facilitating NCC migration and proliferation (Dutt et al, 2006).  1.1.2.  Reproduction  In most normal adult tissues little is known about versican turnover.  However significant production and loss of versican is associated with numerous aspects of reproduction. Versican is associated with loose connective tissue and blood vessel walls of the normal ovary (Voutilainen et al, 2003).  In both the pre-ovulatory follicle and in the cytoplasm of granulosa cells after ovulation versican is a stromal component adjacent to the basement membrane (Irving-Rodgers et al, 2006a).  Stromal versican is widely dispersed in the bovine corpus luteum (Irving-Rodgers et al, 2006b).  Versican expression and cleavage are associated with maturation of the cumulus-oocyte complex in vivo (Dunning et al, 2007). 4   A versican-like proteoglycan assists human sperm cell migration in vitro (Eriksen et al, 1994), and versican has since been shown to be a significant component of human follicular fluid (Eriksen et al, 1999).  High levels of stromal versican are observed in development of the prostate, but expression is decreased greater than 10 fold during puberty (Sakko et al, 2007).  A large hyaluronan binding chondroitin sulfate proteoglycan (CSPG) similar to versican has been suggested to contribute to the disorganization of collagen fibrils during cervical dilation (Uldbjerg & Malmstrom, 1991).  Indeed versican expression increases in association with matrix remodeling and collagen degradation in the cervical connective tissue just before birth (Westergren-Thorsson et al, 1998).  1.2.  Versican in wound healing disorders 1.2.1.  Wound healing and fibrosis  Wound healing processes can result in a spectrum of end results under different circumstances.  In oral wound healing or dermal wound healing in the fetus, tissue regeneration is complete.  In normal dermal wound healing, limited scar formation occurs and in dermal scarring disorders such as hypertrophic scarring and keloids, scarring is sustained and proliferative.  These are just two of a large group of disorders that involve prolonged scarring and proliferation of connective tissue.  Fibroproliferative diseases include pulmonary fibrosis, systemic sclerosis, hepatic fibrosis, renal fibrosis, atherosclerosis and some aspects of cancer metastasis (Wynn, 2007).  In each, normal organ tissue is progressively replaced by functionally abnormal tissue.  Despite common mechanisms underlying many of these diseases, there are no currently approved therapies that directly target mechanisms of fibrosis (Wynn, 2007). 5   There are three overlapping phases of wound healing: inflammation, tissue formation and tissue remodeling (Singer & Clark, 1999).  In response to a wounding event, injured epithelial and endothelial cells become activated and trigger coagulation and inflammation.  Activated cells secrete growth factors, cytokines, chemokines and proteinases that participate in the chemical and physical recruitment of inflammatory cells.  This includes the regulated proteolysis of ECM proteins of the basement membrane and fibrin clot.  Fibroblast cells become activated to myofibroblasts that migrate and proliferate, expressing α-smooth muscle actin (α-SMA).  In the tissue formation phase, myofibroblasts secrete ECM components that form a temporary scaffold to assist inflammatory cell recruitment and neovascularization.  This temporary extracellular matrix is rich in fibrin, fibronectin, hyaluronan and versican.  It is designated the provisional matrix to indicate its normal expression is both essential and transient (Clark et al, 1982).  The new tissue rich in myofibroblast cells and the provisional matrix they synthesize is termed the granulation tissue (Fig. 1.2).  The granulation tissue allows neovascularization, wound closure and tissue remodeling by proteases including the MMP’s.    During tissue remodeling, the provisional matrix is gradually replaced by a collagenous matrix.  Collagen deposition is necessary for the structural arrangement of the remodeling tissue and for a return to normal tissue function, however excess deposition of collagen leads to fibrosis.  A balance between synthesis and catabolism of collagen is necessary for appropriate resolution of wounding.  Catabolism is controlled by different groups of metalloproteinases during the different phases of wound healing (Madlener et al, 1998). 6 Figure 1.2.  Granulation tissue in a cutaneous wound at fi ve days post injury. Activated myofi broblasts synthesize a provisional matrix and together form the gran- ulation tissue in which reepithelization, neovascularization and tissue remodeling occur. Adapted from Singer, A.D. & Clark, R.A.F.  Cutaneous Wound Healing.  © N. Engl. J. Med. 1999; 341: 738-746.  Copied under licence from Access Copywright. Further reproduction prohibited. 7   As α-SMA expressing myofibroblasts shorten collagen networks to effect wound closure, proteolytic removal of excess ECM molecules and cells is necessary (Tomasek et al, 2002).  While it is the activated myofibroblasts that are principally responsible for resolution of the granulation tissue provisional matrix, changes in extracellular matrix molecules can influence the ability of myofibroblasts to remodel the extracellular matrix (Clark et al, 1995; Xu & Clark, 1996).  Therefore ECM remodeling in the resolution of wound healing both regulates and is regulated by activated myofibroblasts.  Myofibroblasts eventually cease α-SMA expression and contractility, and disappear by apoptosis (Desmouliere et al, 1995).  Myofibroblast contractility is important for physiological tissue remodeling but excessive contractility can also lead to tissue deformations as seen in fibrosis (Hinz, 2007).  The timely loss of excess ECM molecules and myofibroblasts allows a return to normal architecture, whereas, persistence of the myofibroblasts and the provisional matrix may contribute to fibrosis (Fig. 1.3).  Compared to fetal wound healing, myofibroblasts involved in adult wound healing show an increased expression of α-SMA and increased contractile capacity (Estes et al, 1994; Moulin et al, 2001).  Exogenous TGF-β induces scar formation in healing fetal wounds suggesting a role for this growth factor in fibrotic processes (Lin et al, 1995; Sullivan et al, 1995).  Rapid re-epithelialization appears to be critical to scarless healing of fetal wounds. (Martin, 1997). In fetal wound healing, high expression of chondroitin sulfate is associated with scarless healing and may alter collagen fibril formation (Whitby & Ferguson, 1991).  However, high levels of versican and reduced fibroblast apoptosis are associated with hypertrophic scarring (Armour et al, 2007; Scott et al, 1996).  These observations suggests that versican expression and degradation are 8 Figure 1.3.  Myofi broblasts in normal and pathological wound healing. Adapted from Tomasek, J.T. et. al.  Myofi broblasts and Mechanoregulation of Connective Tissue Remodelling.  © Nat. Rev. Mol. Cell Biol. 2002; 5:349-363.  Copied under licence from Access Copywright.  Further reproduction prohibited. 9  essential components of normal wound healing and that persistence of versican may lead to aberrant healing.  Fibrosis is invariably preceded by inflammation, however fibrogenic processes are largely independent of inflammatory processes (Wynn, 2004).  Therefore improved therapies will need to be directed at fibrogenic pathways rather than at the primary causes of the fibrosis (Friedman, 2007; Wynn, 2007).  1.2.2.  Pulmonary fibrosis  Pulmonary fibrosis is a scarring disorder of the lungs involving progressive replacement of lung parenchyma by non-functional collagen-rich scar tissue.  Idiopathic pumonary fibrosis (IPF) is a specific form of pulmonary fibrosis differing from the other idiopathic interstitial pneumonias (Travis et al, 2002).  It is identified by the histopathological pattern of usual interstitial pneumonia and exhibits characteristic fibroblastic foci (King et al, 2000).  IPF is driven largely if not entirely by non- inflammatory mechanisms (Selman et al, 2001; Travis et al, 2002).  Median survival of patients with idiopathic pulmonary fibrosis is 3 to 5 years and there is no currently effective therapy (Khalil & O'Connor, 2004).  The current 'standard of care' therapy of prednisone and azathioprine is in fact potentially detrimental to patients as these cytotoxic agents have not been proven to be effective for IPF (Hunninghake, 2005).  In addition to chronic injury, genetic factors contribute to the susceptibility to pulmonary fibrosis (Chung et al, 2003).  One such genetic susceptibility factor in humans is mutations in telomerase genes (Armanios et al, 2007).  In pulmonary fibrosis, myofibroblasts are primarily responsible for the excess deposition of collagen (Zhang et al, 1994).  In fact myofibroblasts are the effector cells 10  that are primarily responsible for all synthesis of ECM proteins and immune mediators that perpetuate pulmonary fibrosis (Thannickal et al, 2004).  In the developing lung, versican is the predominant chondroitin sulfate bearing proteoglycan and its expression is associated with alveolar tissue volume changes (Faggian et al, 2007).  Versican is expressed in the most prevalent forms of human lung fibrosis, including those associated with non-granulomatous inflammation: organizing diffuse alveolar damage in patients with adult respiratory distress syndrome; usual interstitial pneumonia in patients with IPF and idiopathic BOOP (Bensadoun et al, 1996).  Versican is also expressed in association with myofibroblasts in granulomatous forms of lung fibrosis including the lesions of tuberculosis, sarcoidosis and extrinsic allergic alveolitis.  In all of these forms of human lung fibrosis, versican is found in association with migratory, proliferating alpha actin-positive myofibroblasts (Fig. 1.4) (Bensadoun et al, 1997).  Myofibroblasts synthesize type 1 procollagen within the versican-rich matrix (Bensadoun et al, 1996; Bensadoun et al, 1997).  Versican is a significant component of the expanded interstitial tissue in the interstitial lung disease lymphangioleiomyomatosis (Merrilees et al, 2004). Interestingly, it has been suggested that asthma bears certain similarities to fibrosis (Roberts, 1995) and increased versican expression contributes to an expanded bronchial ECM that is detrimental in asthma (Potter-Perigo et al, 2004; Roberts & Burke, 1998).  In patients with mild asthma, activated fibroblasts producing versican are present in bronchoalveolar lavage fluid and appear to contribute to peribronchial fibrosis (Larsen et al, 2004).  Pulmonary fibrosis seems to be perpetuated by a proteolytic imbalance.  The tissue inhibitor of metalloproteinases (TIMPs) are all highly expressed in IPF compared to normal and they may therefore inhibit normal proteolytic activity required for normal 11 Figure 1.4.  Versican expression associated with proliferating fi broblasts in idiopathic pulmonary fi brosis.  Versican stains strongly in association with proliferat- ing myofi broblasts in the thickened interstitium and in a subepithelial fi broblast rich focus which is in the process of colonizing an alveolus.  Scale bar = 100 µm.  Image courtesy of Dr. Clive R. Roberts. 12  remodeling (Ramos et al, 2001; Selman et al, 2000).  Yet there are also many MMP and ADAMTS proteases that are up-regulated in IPF (Pardo et al, 2008).  It remains to be clarified which enzymes and inhibitors are critical for normal resolution.  It is also unclear which enzymes and inhibitors function in a predominantly pro-fibrotic versus anti-fibrotic manner.  While the up-regulation of these proteases as a whole can be assumed to indicate their role in facilitating the progression of IPF, it is also possible that in response to the events of fibrosis the body is attempting to restore a proteolytic balance and encourage remodeling through increasing protease expression.  The reality is quite likely a complex interplay of these two opposing processes. While inflammation is often believed to precede and lead to pathways of fibrosis, it is possible that in IPF inflammation occurs subsequent to the formation of fibroblastic foci and that the pathology is driven by chronic epithelial injury leading to aberrant fibroblastic wound healing (Selman & Pardo, 2002).  Numerous new therapies to combat IPF by inhibition of fibrotic events independent of inflammation are now in development and clinical trials (Rogliani et al, 2008).  1.2.3.  Atherosclerosis  Versican is a major component of smooth muscle pericellular matrix in the normal aorta (Yao et al, 1994).  Yet versican expression is also abundant in atherosclerotic lesions (Evanko et al, 1998; Halpert et al, 1996), transplant arteriopathy (Lin et al, 1996a; Lin et al, 1996b) and in restenotic lesions after angioplasty (Wight et al, 1997). Dermatan sulfate glycosaminoglycans on versican bind platelets and may contribute to platelet accumulation at ruptured atherosclerotic plaques (Mazzucato et al, 2002). Under normal conditions, versican is part of a hydrated viscoelastic matrix that assists in normal vessel functioning, yet its pathological expression appears to be at the centre 13 Figure 1.5.  Versican localization in atherosclerosis. A. Movats staining of human artery with early intimal thickening shows proteoglycans in light blue.  B. Versican immunostaining shows reactivity in same thickened intima. C. Human coronary atherosclerotic plaque staining shows proteoglycans adjacent to a thrombus.  D. Versican staining is abundant in the same layer.  Adapted from Wight, T.N. and Merilees, M.J.  Proteoglycans in atherosclerosis and restenosis, key roles for versi- can.  © Circ. Res. 2004; 94:1158-1167.  Copied under licence from Access Copywright. Further reproduction prohibited. 14  of many of the events of atheroslerosis and restenosis  (Wight, 2008; Wight & Merrilees, 2004).  High concentrations of versican are transiently associated with myofibroblasts in the adventitia and neointima during coronary artery repair (Shi et al, 2000).  In atherosclerosis, versican staining is abundant in the thickened intima (Fig. 1.5) (Wight & Merrilees, 2004).  Vascular smooth muscle cells require a versican-rich matrix for migration and proliferation (Evanko et al, 1999).  Likewise, antisense inhibition of versican synthesis reduces cell proliferation in injured rat carotid arteries (Huang et al, 2006).  Versican binding to fibulin-2 appears to contribute to this promotion of growth and migration (Olin et al, 2001; Strom et al, 2006).  Versican proteolysis is associated with neointimal regression (Kenagy et al, 2005). Versican turnover in the neointima appears to involve multiple proteasese acting in sequential steps (Kenagy et al, 2005; Kenagy et al, 2006).  Fragments of versican are detected in normal and diseased blood vessels (Formato et al, 2004; Sandy et al, 2001; Theocharis et al, 2003a) indicating that versican turnover occurs in both normal and pathological vessel remodeling.  1.2.4.  Cancer stroma  Versican was first identified as a cancer-related gene in 1990.  It was observed to be hypomethylated in colorectal cancer, potentially allowing its excessive expression to contribute to malignancy (Adany & Iozzo, 1990).  Since then there have been many more reports of versican as a major component of the stroma surrounding tumors in most if not all the bodies organ systems.  A number of reports have found that versican expression is correlated with poor prognosis and reduced incidences of relapse-free survival (Table 1.1).  In fact expression of versican has been found to be predictive of 15   P re se nt  in  tu m ou r s tro m a A ss oc ia te d w ith  gr ow th  &  m et as ta si s A ss oc ia te d w tih  in va si ve  p ot en tia l A ss oc ia te d w ith  an gi og en es is  R el at ed  to  d is ea se  fre e su rv iv al  P ot en tia l i nd ic at or  O f p oo r p ro gn os is  Brain  √      (Paulus et al, 1996) Oral squamous cell carcinoma  √ √     √   √  (Pukkila et al, 2007) Oral malignant melanoma  √ √  √    (Banerjee et al, 2005; Docampo et al, 2007) Salivary gland  √      (Nara et al, 1991) Odontogenic  √ √      (Ito et al, 2002; Zhao et al, 1999) Laryngeal squamous cell  √ √   √     (Skandalis et al, 2006b; Skandalis et al, 2004; Stylianou et al, 2008; Vynios et al, 2008) Pharygeal squamous cell  √ √     (Pukkila et al, 2004) Esophageal  √ √     (Hao et al, 2006) Non small cell lung  √ √     (Pirinen et al, 2005) Gastric  √      (Theocharis et al, 2003) Pancreatic   √  √          (Fukata et al, 1989; Koninger et al, 2004; Mauri et al, 2005; Skandalis et al, 2006a) Liver  √ √  √    (Lin et al, 2007) Breast  √ √   √ √   √   √  (Castronovo et al, 2007) Prostate  √      √   √  (Ricciardelli et al, 1998; Ricciardelli et al, 2007; Sakko et al, 2003) Testicular  √ √  √ √   (Labropoulou et al, 2006) Cervical  √ √     √   √ (Kodama et al, 2007a) Endometrial  √ √      √   √ (Kodama et al, 2007b) Ovarian  √ √     √  (Voutilainen et al, 2003) Uterine  √      (Catherino et al, 2004; Malik & Catherino, 2007) Colorectal  √ √  √    (Adany & Iozzo, 1990; Lin et al, 2007; Mukaratirwa et al, 2004; Theocharis, 2002; Tsara et al, 2002) Basal cell carcinoma  √       (Karvinen et al, 2003) Cutaneous malignant melanoma  √ √ √     √ (Docampo et al, 2007; Touab et al, 2003; Touab et al, 2002) Leukemic monocytes  √     (Makatsori et al, 2003) Chondromyxoid fibroma √      (Romeo et al, 2007) Yolk sac tumour √      (Isogai et al, 1996; Nakashima et al, 1990; Sobue et al, 1989) Table 1.1.  Versican expression in cancer 16  poor prognosis in oral (Pukkila et al, 2007), breast (Ricciardelli et al, 2002; Suwiwat et al, 2004), prostate (Ricciardelli et al, 1998), cervical (Kodama et al, 2007a), endometrial (Kodama et al, 2007b) and cutaneous (Touab et al, 2003; Touab et al, 2002) cancers. Versican expression may contribute to anchorage independence that precedes neoplastic conversion (Oba-Shinjo et al, 2006).  Modifications of versican are associated with aggressiveness and metastatic potential in laryngeal, gastric, pancreatic, colon and rectal tumor stroma (Skandalis et al, 2006a; Skandalis et al, 2006b; Theocharis, 2002; Theocharis et al, 2003b; Tsara et al, 2002).  Modifications observed include a lower hydrodynamic size with a higher percentage of chodroitin sulfate chains, higher percentage of 6 sulfated dissacharides and reduced average molecular weight of both chondroitin and dermatan sulfate side chains.  It has long been known that growing tumors induce the formation of extracellular matrix surrounding them in a process known as the stromal reaction (Ioachim, 1976). Within an appropriately permissive ECM a tumor can grow, recruit vasculature, evade host immune response and eventually metastasize.  Tumor derived factors have been shown to induce versican expression in cultured benign prostatic and pancreatic stromal cells (Koninger et al, 2004; Sakko et al, 2001).  Creation of a versican-rich matrix may also facilitate neo-angiogenesis that is required for continued tumor growth (Brown et al, 1999; Koyama et al, 2007), suggesting that versican is permissive for tumor growth.  Host fibroblasts are likely responsible for most versican synthesis in response to tumor signaling.  However, there is evidence of cancer cells secreting versican into the stroma (Bouterfa et al, 1999; Dobra et al, 2000; Mauri et al, 2005; Touab et al, 2002). 17   Tumor stroma and the granulation tissue of healing wounds share a number of properties.  Through the release of vascular permeability factor, tumor cells induce fibrinogen extravasation and formation of a fibrin-fibronectin gel (Dvorak, 1986). Proliferation of fibroblasts, recruitment of inflammatory cells and angiogenesis gradually transform the fibrin-fibronectin gel into a vascular tumor stroma that is much like wound granulation tissue (Dvorak, 1986).  The forming granulation tissue exhibits strong expression of CSPGs (Yeo et al, 1991).  Since tumor growth is dependent on a suitable stroma, the altered tumor stromal itself can be considered carcinogenic and it is likely that successful cancer therapies of the future will need to address normalization of the tumor microenvironment (Bissell & Radisky, 2001).  Myofibroblasts play a central role in cancer invasiveness (De Wever et al, 2008). The induction of activated fibroblasts by dysregulation of proteases is proposed to be similar in fibrosis and tumor microenvironments (Radisky et al, 2007).  Type 1 collagen is a metastasis-associated gene that is suggested to be a potential anti-metastasis target (Fingleton, 2007; Ramaswamy et al, 2003).  As in fibrosis the excess deposition of type 1 collagen occurs in a versican-rich matrix.  While host induction of a permissive stroma seems to be a consistent requirement of cancer progression and this stroma is clearly versican-rich under most circumstances, it is not necessarily the case that versican expression promotes growth and metastasis in all cases.  In advanced laryngeal cancer, increased expression of versican is seen concomitant with a decrease in high molecular weigh versican extractable from the tissue and an increase in versican fragments (Stylianou et al, 2008).  Thus it is possible that versican expression  is part of a host encapsulation reaction that the tumor must circumvent through proteolytic means in order to continue growth and progression.  As the tumor stroma is complex and is stimulated by various host-tumor interactions that 18  are not fully understood, we need to be cautious in assigning functions to highly expressed molecules (Iozzo, 1995).  Thus, elucidating versican’s role and turnover in the tumor stroma is crucial to the design of future therapies that would target the aberrant tumor stroma.  1.2.5.  Arthritis  The prototypical cartilage proteoglycan is aggrecan, however versican is also involved in cartilage growth and turnover.  Versican was first identified due to its association with limb chondrogenesis (Kimata et al, 1986).  It is highly expressed in the perichondrium and presumptive joint interzone (Matsumoto et al, 2006; Shepard et al, 2007).  Versican expression is up-regulated by chondrogenic stimuli and knock-down results in compromised mesenchymal chondensations (Kamiya et al, 2006; Shepard et al, 2008).  Human mesenchymal stem cells undergoing osteoblast differentiation express versican (Foster et al, 2005).  Increased versican to aggrecan ratio is associated with osteoarthritis and dedifferentiation of chondrocytes (Martin et al, 2001).  Collagen-induced arthritis in rats results in increased versican production by peripheral blood mononuclear cells (Shou et al, 2006).  It may be that increased expression of versican occurs with proliferative elements of arthritic processes.  1.2.6.  Tendinopathies  Versican is a component of normal achilles tendon (Corps et al, 2006) and the scapholunate interosseous ligament (Milz et al, 2006).  Versican expression is associated with small arteries and in the neointima of severe carpal tunnel syndrome 19  (Tsujii M et al, 2006) and with proliferating fibroblasts and microvessels in patellar tendonosis (Scott et al, 2008).  Several catabolic products of versican have been detected and characterized in bovine tendon (Samiric et al, 2004) and many more have been detected in human patellar tendons of patients with tendinosis (Scott et al, 2008).  The role of versican degradation relative to tendinosis disease progression is not clear.  1.3.  Versican structure  The spatial orientation of versican's larger isoforms was demonstrated in a seminal paper two decades ago that analyzed large interstitial proteoglycans including aorta proteoglycans by electron microscopy (Fig. 1.1) (Morgelin et al, 1989).  The aorta proteoglycans appears to be versican V0 and V1 as the size and domain compositions are appropriate and we know that these isoforms are highly expressed in the aorta. These were later refered as 'versican type' proteoglycans (Morgelin et al, 1994) and based on our current knowlege, there is no other logical candidate proteoglycan.  This early work demonstrated that versican has more diversity in the length of its glycosaminoglycans and an average glycosaminoglycan length about twice that of aggrecan's (Morgelin et al, 1989).  Due to its fewer glycosaminoglycan side chains that create less electrostatic repulsion, versican's core protein was less extended than aggrecan's and contracted less upon glycosaminoglycan digestion.  Based on its composite structure, versican may span between two extracellular binding interactions as 'molecular bridge' (Miosge et al, 1998; Ruoslahti, 1996) with variable size and degrees of glycosylation determined by expression of different splice variants.  20  1.3.1.  Hyalectans  In addition to versican, there are several proteoglycans that share a homologous C- terminal domain with a selectin-like domain composition.  These include versican, aggrecan, neurocan and brevican; and they have been referred to as ‘lecticans’ to indicate proteoglycans with lectin-like domains (Ruoslahti, 1996).  The term ‘hyalectans’ has since been dubbed to designate proteoglycans with lectin-like domains and hyaluronan binding N-terminal domains (Iozzo, 1998).  This hyalectan family appears to offer a repertoire of proteoglycans that may bind identical substrates. Through the altered expression of versican splice variants and hyalectan family members, cells could regulated expansion or contraction of the extracellular matrix and alter its chemotactic properties (Fig. 1.6).  1.3.2. Gene organization and regulation  The human versican gene lies on chromosome 5 and is composed of 15 exons (Iozzo et al, 1992; Ito et al, 1995; Naso et al, 1994; Zako et al, 1995).  All the versican isoforms contain N and C terminal globular domains termed G1 (comprising exons 1-6) and G3 (comprising exons 9-15) respectively.  Exon 7 encodes the smaller of the GAG attachment domains termed GAG-α and exon 8 encodes the larger one termed GAG-β (Dours-Zimmermann & Zimmermann, 1994; Zimmermann & Ruoslahti, 1989).  The isoform containing both GAG-α and GAG-β is called V0, containing only GAG-β is V1, only GAG-α is V2, and V3 is the smallest (Figs. 1.6, 1.7)  Reduced activity of the tumor suppressor gene TP53 results in decreased versican expression (Yoon et al, 2002).  Dihydrotestosterone increases versican expression 21 Figure 1.6.  Hyalectan family members in the central nervous system. Versican splice variants compared with other hyalectan family members showing puta- tive glycosaminoglycans only.  Adapted from Bandtlow, C.E. and Zimmermann, D.R. Proteoglycans in the Developing Brain: New Conceptual Insights for Old Proteins.  © Physiol. Rev. 2000; 80:1267-1290.  Copied under licence from Access Copywright.  Fur- ther reproduction prohibited. 22 Figure 1.7.  Versican splice variants showing domain composition and potential glycosylation sites.  Domain composition: Ig = Ig-like V-type; Link = link domain; GAG-α = glycosaminoglycan attachment domain alpha; GAG-β = glycosaminoglycan attachment domain beta; E = Calcium-binding EGF-like domain; Lect = C-type lectin-like domain; CCP = complement control protein, short consensus repeat or sushi domain.  Arrows illustrate potential attachment sites for glycosaminoglycans (black arrows), O-linked glycosylations (red arrows) and N-linked glycosylations (blue arrows).  Based on Zimmermann & Ruoslahti, 1989; Dours-Zimmermann & Zimmermann, 1994. V0 V1 V2 V3 23  during puberty (Sakko et al, 2007).  Versican expression in smooth muscle cells is regulated by a beta-catenin-T-cell factor complex (Rahmani et al, 2005).  Versican is upregulated by TGF-β in a variety of cells (Arslan et al, 2007; Kahari et al, 1991; Robbins et al, 1997; Schonherr et al, 1991; Venkatesan et al, 2002; Wolf et al, 1994; Zhao & Russell, 2005).  Though TGF-β is sometimes considered a pro-fibrotic cytokine, it is also essential in tissue remodeling wherein excess or inadequate TGF-β are linked to numerous disease states (Blobe et al, 2000).  TGF-β null mice exhibit multiple defects of development and the inflammatory system (Kaartinen et al, 1995; Proetzel et al, 1995; Sanford et al, 1997; Shull et al, 1992).  It is possible that excess TGF-β contributes to fibrosis through stimulating excess versican synthesis, but the data is not yet conclusive.  1.3.3.  Splice variants  Alternative splicing of versican mRNA results in four different gene products with identical N- and C-terminal globular domains but different sized central glycosaminoglycan-attachment domains due to the inclusion of one or both or neither of the GAGα and GAGβ domains (Ito et al, 1995; Naso et al, 1994; Zako et al, 1995). The first cloning of the versican gene involved a partial V1 transcript (Krusius & Ruoslahti, 1986) that was later fully cloned (Zimmermann & Ruoslahti, 1989).  The alternatively spliced GAG-beta domain was later discovered and cloned, uncovering both the V0 and V2 isoforms (Dours-Zimmermann & Zimmermann, 1994).  There are as many as 41 potential glycosaminoglycan attachment sites in versican V0 though it is predicted that in vivo V0 has between 17-23 glycosaminoglycans attached, V1 has 12-15, V2 has 5-8 and V3 has none (Fig. 1.7 & 1.8) (Dours- 24 Figure 1.8.  Versican V0 primary sequence and potential glycosylation attachment sites. Versican V0 primary sequence with colours representing different protein domains.  The N-terminal globu- lar domain is composed of the signal peptide (black), Ig-like domain (blue), Link 1 (light green) and Link 2 (dark green).  GAG-α is yellow and GAG-β is light orange.  Note in vivo these two glycosaminoglycan attachment domains are considerably extended due to glycosaminoglycan hydrophilic interactions and repulsion.  The C-terminal globular domain is composed of two EGF domains (brown and orange), the C- type lectin domain (red) and the complement regulatory protein-like domain (purple).  Potential attachment sites are marked for glycosaminoglycans (hexagons) and N- and 0-linked glycosylations (circles).  Based on Zimmermann & Ruoslahti, 1989 and Dours-Zimmermann & Zimmermann, 1994. 25  Zimmermann & Zimmermann, 1994; Zimmermann & Ruoslahti, 1989).  Versican V1 and V3 may also occasionally retain and express intron 14 in the carboxy terminal, presumably altering its binding properties (Lemire et al, 1999).  While V3 may be completely void of glycosylations, it does contain potential glycosylation and glycosaminoglycan attachment sites (Fig. 1.7 & 1.8).  Whether V3 might be glycosylated in vivo has yet to be determined.  Alternative splicing is common in ECM proteins (Boyd et al, 1993), yet the majority of known alternative splicing events add or subtract protein-protein interaction domains resulting in modest changes in molecular size (Resch et al, 2004).  Alternative splicing of versican's large and heavily glycosylated central domains is thus a unique case of alternative splicing that has dramatic effects on the hydration and viscoelastic properties of the ECM with a 20 fold size difference between the largest and smallest isoform (Figs. 1.6 - 1.8).  Numerous studies have documented RNA levels of versican transcripts in different tissues and at different stages, though there is some conflict between reports. Generally, V0 and V1 seem to be associated with cell proliferation and migration where they are expressed at high levels, followed by a drastic reduction that is necessary for tissue maturation.  V0 and V1 are associated with guiding neural crest cell migration (Dutt et al, 2006).  V0 and V1 are expressed in melanoma cell lines and V0 predominates with the less differentiated cells (Touab et al, 2002).  The larger isoforms are still detectable at lower levels in mature blood vessels (Bode-Lesniewska et al, 1996; Yao et al, 1994) and skin (Zimmermann et al, 1994).  V2 is a major component of the brain ECM and the predominant versican isoform present in the brain (Paulus et al, 1996; Schmalfeldt et al, 2000; Schmalfeldt et al, 1998).  The V3 transcript can be expressed by cytokine stimulated endothelial cells (Cattaruzza et al, 2002).  V3 26  overexpressing cells grow more slowly, are less extended and adhere more firmly (Lemire et al, 2002; Serra et al, 2005).  Overexpression of V3 perturbs vascular elastic fibre assembly (Merrilees et al, 2002).  In the developing Cornea, V0 is the most expressed isoform during the early postnatal period, V1 and V2 are expressed moderately and V3 is expressed more abundantly in later periods (Koga et al, 2005).  Versican V3 may have an opposite effect on cell proliferation and migration from the larger isoforms.  Since V3 would shorten the putative ‘molecular bridge’ between the two terminal binding domains it would vastly alter the hydrodynamic properties of the pericellular matrix compared with a proliferative matrix rich in versican V0 and V1.  Wagner disease is a rare, autosomal dominant, vitreoretinopathy.  Recent work has identified mutations in versican's exons 7 and 8, and intron 7, in several families with this disease (Kloeckener-Gruissem et al, 2006; Miyamoto et al, 2005; Mukhopadhyay et al, 2006).  These mutations appear to result in a splice variant imbalance, potentially the inability to synthesize V0, that disrupts the ultrastructure of the vitreous gel and leads to the characteristic liquefaction.  1.3.4. Glycosaminoglycans  Glycosaminoglycans are specialized glycans that differ from N- and O-linked glycosylations by being consistently linear, repeating dimers usually containing one uronic acid (glucuronic acid or iduronic acid) and a hexosamine residue (Fig. 1.9). Proteoglycans like versican have glycosaminoglycans bound to the core protein through a four residue linker attached to a serine residue (Sugahara et al, 2003). Glycosaminoglycans are increasingly being recognized to bind protein substrates in the ECM.  Post-translational structural fine tuning through epimerization of uronic acids and variable sulfation patterning provides an enormous variability and alters substrate 27 Figure 1.9.  Glycosaminoglycan monomer constituents. Disaccharide monomers of the four classes of glycosaminoglycans showing their variable epimerization of glucuronic acid to iduronic acid and positions of variable sulfation.  Adapted from Bandtlow, C.E. and Zimmermann, D.R.  Proteoglycans in the Developing Brain: New Conceptual Insights for Old Proteins.  © Physiol. Rev. 2000; 80:1267-1290.  Copied under licence from Access Copywright.  Further reproduction prohibited. 28  binding ability (Raman et al, 2005).  As these side chains are also capable of being modified in a number of ways, they have been referred to as the most information- dense biological molecules (Turnbull et al, 2001) and it has been proposed that their modifications may form a code necessary for proper development (Bulow & Hobert, 2006).  Mechanical properties of glycosaminoglycans include binding water to create a hydrated matrix.  Just as versican deficiency abolishes the capacity to form endocardial cushions in heart morphogenesis (Mjaatvedt et al, 1998), chondroitin synthesis is necessary to create osmotic swelling pressure in caenorhabditis elegans tissue morphogenesis (Hwang et al, 2003).  This suggests that versican’s glycosaminoglycans are directly responsible for the its space filling and viscoelastic properties (Kinsella et al, 2004).  In addition to binding water, versican’s glycosaminoglycans bind numerous substrates and further affect the physical and chemical composition of the pericellular matrix.  Versican's glycosaminoglycans bind various chemokines and growth factors (Hirose et al, 2001).  Dermatan sulfate glycosaminoglycans on versican bind platelets and may contribute to platelet accumulation at ruptured atherosclerotic plaques (Mazzucato et al, 2002).  Versican may aid in lymphocyte homing through glycosaminoglycan binding to L- and P-selectin (Kawashima et al, 2000; Kawashima et al, 1999) with binding involving over-sulfated portions of the glycosaminoglycan chain (Kawashima et al, 2002).  The 70kDa heavy-chain component of inter-α-trypsin inhibitor (ITI) binds to follicular fluid versican (Eriksen et al, 1999).  Changes in size, sulfation and epimerization of glycosaminoglycans are all mechanisms that fine tune versican’s functions in the ECM.  In advanced laryngeal squamous cell carcinoma there is a reduction in 6-sulfated disaccharides and an 29  increase in 4-sulfated disaccharides (Skandalis et al, 2004).  Versican’s glycosaminoglycans have been measured at different masses and with different sulfation patterns (Skandalis et al, 2006a; Skandalis et al, 2006b; Theocharis et al, 2003b).  Versican's hydrodynamic size changes on stimulation with platelet-derived growth factor or TGF-β in a manner that does not alter the core protein size (Schonherr et al, 1991).  1.3.5.  N-terminus/G1  Versican's N-terminus contains an immunoglobulin-like domain followed by two link domains.  This is domain organization is consistent throughout the hyalectan family. This domain binds hyaluronan (HA) with high affinity through a ternary complex with link protein (LeBaron et al, 1992; Matsumoto et al, 2003; Shi et al, 2004).  We now know there to be four different link protein family members (Spicer et al, 2003). Interestingly these four link proteins are located on chromosomes next to the four hyalectan family members versican, brevican, aggrecan and neurocan respectively, suggesting several early gene duplications.  However, it does not appear that the link protein adjacent to a given hyalectan is the physiological link used.  Link protein-3 (HAPLN-3) colocalizes and is coordinately upregulated with versican in arterial smooth muscle cells (Ogawa et al, 2004), despite HAPLN-1 and versican being paralogous genes (Spicer et al, 2003).  1.3.6.  C-terminus/G3  Understanding the binding activities of the G3 domain of versican is of significance importance since it could form the second end of the putative ‘molecular bridge’ with 30  hyaluronan.  Additionally, the selectin-like domain composition of the G3 domain suggests a possible cell surface binding activity that has yet to be described. A number of different ECM binding partners have been documented and it seems plausible that binding to different ECM proteins or cell surface ligands is regulated and plays an important role in modulating the hydration and visco-elastic properties of the ECM.  Versican has been shown to bind fibrillin-1 (Aspberg et al, 1999; Isogai et al, 2002) and HA-versican-fibrillin-1 complexes seem to be critical to maintaining physiological properties of the ciliary and vitreous bodies in the eye (Ohno-Jinno et al, 2008).  Versican has also been shown to bind fibulin-2 (Olin et al, 2001), supporting previous observations that versican colocalizes with fibulin-1 and -2 during heart development and appears to make a switch in molecular associations as development proceeds (Miosge et al, 1998).  Versican forms a complex with HA and tenascin-R in the brain (Aspberg et al, 1997; Ruoslahti, 1996).  In addition to mediating binding with matrix or cell surface ligands, the G3 domain has been reported to self associate in a calcium dependent manner (Ney et al, 2006).  1.4.  Versican turnover in tissue remodeling  Versican is clearly a ubiquitious proteoglycan that is critically involved in development, remodeling and homeostasis of numerous tissues.  As versican is heavily expressed in remodeling tissues and present at more modest levels under homeostatic conditions, its regulated turnover is necessary for formation of normal tissue architecture.  Versican expression and turnover are involved in both normal and pathological remodeling.  Therefore elucidating the proteolytic pathways of normal versican turnover is essential to determining the dysregulated events that contribute to aberrant remodeling. 31  1.4.1.  Matrix metalloproteinases  Tissue remodeling involves not only the synthesis of new matrix but also the regulated proteolysis of matrix proteins and bioactive molecules (Page-McCaw et al, 2007).  Proteases perform irreversible, post-translational modifications to proteins and thereby govern many aspects of normal and aberrant cell physiology.  Proteolysis is necessary for infiltration of inflammatory cells, migration and proliferation of the granulation tissue, as well as resolution of the provisional matrix.  There are over 560 proteases and homologues in the human body including 194 metalloproteinases to date (Overall & Blobel, 2007; Puente et al, 2003).  The metalloproteinases or metzincins are characterized by a HExxHxxGxxH catalytic zinc binding motif and conserved methionine turn (Stocker et al, 1995).  This family includes the astacins, adamalysins, matrix metalloproteinases (MMPs) and the serralysins.  The first MMP was discovered in 1962, detected during tadpole morphogenesis for an ability to cleave fibrillar collagen, which is otherwise largely resistant to proteolysis (Gross & Lapiere, 1962).  MMPs are synthesized as inactive zymogens that contain a propeptide “cysteine switch” complexed to the active site zinc atom (Figure 1.10) (Wart & Birkedal-Hansen, 1990).  As the known spectrum of MMP substrates grew for several decades, our understanding of MMP functions was largely limited to their roles in degrading ECM structural proteins.  In the last decade we have seen numerous examples of MMPs participating in cell signalling through the precise modification of bioactive molecules.  The first example of an MMP processing a bioactive molecule was MMP-2 processing of monocyte chemoattractant protein-3 that converted a pro- inflammatory chemokine into a chemokine receptor antagonist through the removal of four N-terminal amino acids (McQuibban et al, 2000).  This conversion had kinetic 32 Figure 1.10.  MMP family domain organization, active site consensus sequence and propeptide ‘cysteine switch.’  Adapted from Cauwe, B. et. al.  The biochemical, biological, and pathological kaleidoscope of cell surface substrates processed by matrix metalloprotei- nases.  © Crit. Rev. Biochem. Mol. Biol.  2007; 42: 113-185.  Copied under licence from Ac- cess Copywright.  Further reproduction prohibited. 33  parameters that were greater than for MMP-2 cleavage of gelatin, the substrate for which MMP-2 is commonly entitled (gelatinase-A).  Since then there have been numerous reports of various MMPs cleaving and altering bioactivity of substrates through cleavages of similarly small numbers of amino acids (Wolf et al, 2008).  A recent report demonstrates not only conversion of a chemokine from a receptor agonist into an antagonist, but also loss of glycosaminoglycan binding upon a second C- terminal cleavage (Cox et al, 2008).  Dysregulation of MMPs is correlated with many pathologies, especially cancer where MMPs contribute to all stages of cancer growth, including but not limited to invasion and metastasis (McCawley & Matrisian, 2000).  Based on the high levels of MMP expression and correlations with progression in many cancers, clincal inhibition of MMPs was a rational and promising approach to treating cancer (Stetler-Stevenson et al, 1996).  Numerous MMP inhibitor clinical trials were attempted for cancer treatment but all were unsuccessful for a variety of reasons, not the least of which is that MMPs are involved in a much greater diversity of processes than merely degrading ECM to facilitate metastasis (Coussens et al, 2002; Egeblad & Werb, 2002).  Likewise in rheumatoid arthritis and osteoarthritis, current MMP inhibitors have been unaffective due our lack of a precise and complete understanding of MMP activities in these diseases (Murphy & Nagase, 2008).  MMPs and inhibitors are necessary for normal wound healing but an imbalance of MMPs to TIMP inhibitors is a associated with pathological scarring (Fig. 1.11).  Despite the dissapointing results of many clinical trials for MMP inhibition in cancer, improving our understanding of the precise roles of individual proteases and their substrates may lead to the development of more specific inhibitors that will provide effective therapies.  As MMPs can have 'dual personalities' in inflammatory processes it 34 Figure 1.11.  Common and divergent pathways of regenerative versus pathological wound healing.  Adapted from Wynn, T.A.  Common and Unique Mechanisms Regulate Fibrosis in Various Fibroproliferative Diseases.  © J. Clin. Invest. 2007; 117:524-529.  Copied under licence from Access Copywright.  Further reproduction prohibited. 35  is clearly important to understand which MMPs are beneficial and which are detrimental in a particular disease state before improved therapies will be possible (Le et al, 2007; Lopez-Otin & Matrisian, 2007; Overall & Kleifeld, 2006).  For example, MT1-MMP is highly expressed and plays a major role in cancer (Sabeh et al, 2004; Sato et al, 2005). Based on its critical importance in tumor progression, it has been suggested that MT1- MMP is a promising target for future attempts at more specific inhibition in cancer (Lah et al, 2006).  Yet on the other hand, it would be wise to consider MT1-MMP a potential cancer anti-target until its role in adults is more thoroughly elucidated (Overall & Kleifeld, 2006), since it plays a critical role in development and cell signalling pathways (Holmbeck et al, 1999; Tam et al, 2004).  Thus much work is needed to understand the spectrum of roles performed by individual proteases before we can design more rational targeted therapies.  In addition to realizing that MMPs exert their effects on diverses classes of extracellular substrates, we are also in the process of elucidating how MMPs act more in precisely regulated cell surface-associated microenvironments than as soluble enzymes (Cauwe et al, 2007).  Thus understanding the constituents of cell surface associated microenvironments may be more relevant to physiological proteolysis than merely knowning if a given protease is capable of processing a particular substrate.  Regulation of MMP activation is extremely complex, involving regulation at multiple levels.  MMPs do not act in isolation but act in webs and cascades, necessitating an understanding of the interplay between proteases (Keller et al, 2007; Overall & Blobel, 2007; Overall & Kleifeld, 2006).  MT1-MMP activity and localization are altered through a variety of post-translational mechanisms.  MT1-MMP is internalized through both caveolae and dynamin dependent endocytosis, is recycled to the cell surface or degraded, is involved in activating other proteases, cleaving non-protease substrates 36  itself, autocatalytically cleaving itself to generate non-catalytic soluble forms and being cleaved by other proteases to relase catalytic soluble forms (Cauwe et al, 2007; Itoh & Seiki, 2004).  An additional level of complexity in metalloproteinase activation is conferred by the tissue inhibitors of metalloproteinases (TIMPs).  These physiological inhibitors of metalloproteinases are also sometimes necessary for MMP activation.  TIMP-2 for example forms a membrane activation complex with MT1-MMP in order to activate MMP-2 and either inadequate or excessive TIMP-2 impairs MMP-2 activation (Will et al, 1996).  TIMPs are multifunctional and in addition to direct interaction with proteases, can regulate signaling events through direct binding of cell surface receptors and initiating signaling cascades (Chirco et al, 2006; Stetler-Stevenson, 2008).  TIMP-3 is unique in its ability to bind to ECM and appears to be the primary TIMP involved in regulating ADAMTS activities (Kashiwagi et al, 2001).  TIMP-3 is the only TIMP to inhibit ADAMTS-2 and this inhibition is improved by glycosaminoglycan binding (Wang et al, 2006).  Increased expression of TIMP-3 may contribute to the accumulation of versican in prostate cancer stroma by inhibiting versican-degrading ADAMTS enzymes (Cross et al, 2005).  1.4.2.  ADAMTS  The 'a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs' (ADAMTS) family of enzymes are members of the adamalysin family and differ from the related membrane bound ADAM enzymes in their ancillary domains that contain glycosaminoglycan binding thrombospondin domains (Apte, 2004).  These enzymes have only fairly recently been described, with ADAMTS-1 being identified in 1997 (Kuno et al, 1997).  ADAMTS enzymes have been found to perform many of the 37  roles previously thought to be exclusively performed by MMPs.  The search for the putative aggrecan degrading MMP responsible for arthritis resulted in the eventual discovery of ADAMTS-5 as an essential enzyme for mouse arthritis (Glasson et al, 2005; Stanton et al, 2005).  Both MMP and ADAMTS enzymes seem to be involved in different aspects of normal and pathological aggrecan turnover though all the details are still not worked out (Fosang et al, 2008; Sandy, 2006).  As with the MMPs, regulation of ADAMTS activation appears to be complex, involving processing by other metalloproteinases or autolysis (Colige et al, 2005; Flannery et al, 2002; Gao et al, 2004; Tortorella et al, 2005).  ADAMTS-2 processing by by proprotein convertases and C-terminal processing results in 7 different forms of the enzyme (Colige et al, 2005).  In breast and lung cancer, ADAMTS expression is dysregulated (Porter et al, 2004; Rocks et al, 2006).  As with the MMPs, it is unclear whether these enzymes promote or inhibit cancer growth, but several ADAMTSs have been found to suppress tumour growth (Lopez-Otin & Matrisian, 2007).  ADAMTS-1, -4, & -9 have been shown to cleave versican (Jonsson-Rylander et al, 2005; Sandy et al, 2001; Somerville et al, 2003; Westling et al, 2004).  They are considered to form a proteoglycan processing super-clade that process hyalectans and are evolutionarily distinct from the other ADAMTS enzymes (Fig. 1.12) (Apte, 2004). The remaining 'super-clade' contains the procollagen processing enzymes ADAMTS-2, -3 and -14, along with 8 other enzymes: ADAMTS-6, 7, 10, 12, 16-19; with few or no known substrates.  Based on homologies to other proteins that are critical in development and cell signaling, it has been suggested that ADAMTS-2 likely plays important roles in development and cell signaling independent of its role in procollagen processing (Prockop et al, 1998).  Corresponding with this observation, ADAMTS-2 expression appears to be in excess of that required for its role in procollagen 38 Figure 1.12.  ADAMTS family domain organization and evolutionary relationships. A. ADAMTS domain organization in the minimal structure of ADAMTS-4 with only one thrombos- pondin motif.  All other ADAMTS family members contain additional thrombospondin motifs C-ter- minal to the spacer domain.  B. Domain structure in the procollagen N-propeptidases.  C. Evolu- tionary relationships of human ADAMTS enzymes - ADAMTS-1, 4, 5, 8, 9, 15 & 20 are thought to form a “super-clade” of proteoglycan processing enzymes.  ADAMTS 2, 3 & 14 are procollagen N-propeptidases with no other known substrates to date.  ADAMTS-13 is the von Willebrand fac- tor cleaving protease.  The other proteases (ADAMTS-6, 7, 10, 12, 16-19) have few if any known substrates.  Adapted from Apte S.S.  A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs: the ADAMTS family.  © Int. J. Biochem. Cell Biol. 2004; 36:981- 985.  Copied under licence from Access Copywright.  Further reproduction prohibited. A B C 39  processing in several tissues (Colige et al, 1997) and it can exist in multiple activation states, again suggesting the possibility of other roles than processing procollagen (Colige et al, 2005).  1.4.3.  Known and unknown versican proteolytic events  Several MMPs have a versican-degrading capacity but few have been well characterized.  MMP-1, -2, -3, -7 and -9 have all been shown to process versican preparations in vitro (Halpert et al, 1996; Passi et al, 1999; Perides et al, 1995). However, no characterization of proteolytic products has yet been done.  Just as aggrecan cleavage at specific sites is performed by numerous MMPs (Fosang et al, 1992), it is probable that numerous MMPs are capable of cleaving versican in vitro, but the physiological relevance remains to be worked out.  Several members of the ADAMTS family cleave versican.  There are four currently known and characterized versican cleavage sites produced by ADAMTS metalloproteinases (Jonsson-Rylander et al, 2005; Sandy et al, 2001; Westling et al, 2004).  These cleavage sites were discovered using synthetic peptides representing a portion of versican that was predicted to be cleaved and neo-epitope antibodies to the predicted cleavage sites.  These cleavage sites have been confirmed in vivo and in the case of DPEAAE there is much evidence for the cleaved product to exist in different remodeling processes.  Versican cleavage occurs at several stages of cardiac development and produces the DPEAAE neo-epitope in an asymmetric pattern, suggesting a possible function for the cleavage product (Kern et al, 2007; Kern et al, 2006).  Proteolysis may affect cell migration wherein an anti-DPEAAE antibody inhibits TGF-β2 induced cell migration (Arslan et al, 2007).  40  1.4.4. Versican proteolysis  In addition to the well known cleavage sites, it is likely that numerous as yet uncharacterized cleavages are relevant to versican’s normal and aberrant turnover (Kenagy et al, 2006).  We and others have observed multiple fragments of versican detected in cell culture media of when extracted from tissues (Fig. 1.13).  Multiple versican degradation proucts are detected in the stroma of pancreatic carcinoma (Skandalis et al, 2006a).  N- and C-terminal cleavage products of versican have recently been documented in the vitreous body of the eye (Ohno-Jinno et al, 2008).  An increase in versican expression and versican fragments corresponds with increasing stages of laryngeal cancer (Stylianou et al, 2008; Vynios et al, 2008).  Cytokine stimulation of human umbilical vein endothelial cells (HUVEC) results in different N- and C-terminal proteolytic fragments of versican depending on the cytokine used (Cattaruzza et al, 2002).  Numerous proteolytic fragments of versican can be immunoprecipitated from human brain astrocytoma with a C-terminal antibody (Zheng et al, 2004).  Degradation products of versican are detected in the vitreous body of the eye (Ohno-Jinno et al, 2008).  1.4.5.  Glial hyaluronic acid binding protein  Glial hyaluronic acid binding protein (GHAP) was first identified as a 60 kDa glycoprotein that could be reduced to 47kDa with enzymatic removal of N- and O- linked glycosylations (Perides et al, 1989).  Versican was found to colocalize with GHAP in the brain (Perides et al, 1992).  It was later suggested to potentially be a metalloproteinase induced versican fragment (Perides et al, 1995) and eventually identified as a versican V2 cleavage product with cleavage at the Glu405-Gln-406 bond (Westling et al, 2004).  Production of GHAP corresponds with a reduction in the 41 Figure 1.13.  Detection of versican proteolytic fragments. Versican purifi ed by ion exchange at physiological pH exhibits numerous fragments detected by electrophoresis and Western blotting.  Large arrows indicate aggregate and monomer versican, small arrows indicate proteolytic fragments.  A. Proteoglycan staining with Alcian Blue.  B. Versican immunostaining with 2B1 antibody.  C. Versican immunostaining with LC2 antibody.  D. Versican purifi ed by ion exchange from bovine and mouse aorta, digested with chondroitinase ABC and detected by Western blotting shows intact versican V0 and V1 along with numerous proteolytic fragments.  Part D adapted from Kenagy, R.D. et. al.  Versican degradation and vascular disease.  © Trends Cardiovasc. Med. 2006; 16:209-215.  Copied under licence from Access Copywright.  Further reproduction prohibited. A B C D 220 120 60 kDa 100 50 80 220 120 60 kDa 100 50 80 220 120 60 100 50 80 kDa 42  extracellular space, suggesting that versican proteolysis is required for normal brain development (Bignami et al, 1993).  1.4.6.  Hyaluronectin  Hyaluronectin is a hyaluronan binding glycoprotein that has been found in the central nervous system (Delpech et al, 1989) and is expressed by fibroblasts and smooth muscle cells (Ponting & Kumar, 1995).  It exists as a range of different protein sizes, with the major protein being approximately 60 kDa (Delpech et al, 1989; Ponting & Kumar, 1995).  Hyaluronectin is strongly expressed in the intima and surrounding deposits in human atherosclerotic lesions (Levesque et al, 1994).  It is produced and lost from the neointima to a greater extent in young rats than old in response to aortic injury (Chajara et al, 1998).  Hyaluronectin is associated with benign and malignant mesenchymal carcinomas (Girard et al, 1988); is in gliomal and menigiomal stroma (Delpech et al, 1993); and is elevated in invasive areas of breast carcinoma compared to non-invasive areas (Bertrand et al, 1992).  N-terminal sequencing of brain derived hyaluronectin revealed numerous sequences of versican’s N-terminal domain (Delpech et al, 1997).  Consistent with hyaluronectin being a fragment of versican, immunoprecipitation of fibroblast culture media with an anti-hyaluronectin polyclonal antibody detects a large protein product greater than 200 kDa (Ponting & Kumar, 1995).  Higher levels of hyaluronectin appear to be beneficial in cancer (Delpech et al, 1997; Delpech et al, 1993), suggesting that versican proteolysis might be a host response that limits tumor growth.  Whereas, mice injected with cells expressing high levels of hyaluronectin grew larger tumours and had more metastases than control mice (Paris et al, 2006).  Interestingly, cells expressing low levels of hyaluronectin had fewer metastases than control mice, but the significance of this finding is not 43  understood (Paris et al, 2006).  Together, these studies indicate that versican proteolysis is related to cancer progression and that multiple proteolytic fragments of versican are detectable in tumor stroma.  1.4.7.  Matrikines  The discoveries that GHAP and hyaluronectin are versican degradation products serves to underscore the extent and significance of versican proteolytic events that are yet to be described and understood.  Thus, elucidating the inventory of versican- degrading proteases and their cleavage events is crucial to future improvements in diagnosis and therapy of many fibroproliferative disorders.   Extracellular matrix proteins were historically considered as structural entities with few biologically interesting functions.  However, we now know that ECM proteins participate in signaling cascades through binding to cell surface receptors; can alter the location and availability of bioactive cytokines, chemokines and growth factors; and can in fact contain cryptic bioactive components that may be released upon proteolysis. The term matrikines was coined to denote bioactive ECM products produced by regulated proteolysis (Maquart et al, 2004).  Well known matrikines include the angiogenesis inhibitors angiostatin and endostatin that are proteolytically derived from plasminogen and type XVIII collagen respectively (O'Reilly et al, 1997; O'Reilly et al, 1994).  Endorepellin is an angiogenesis inhibitor derived from the heparan sulfate proteoglycan perlecan through BMP-1/Tolloid-like proteinase cleavage (Gonzalez et al, 2005; Mongiat et al, 2003).  Just as we know that MMPs themselves can have pleiotropic roles (Overall & Dean, 2006), single ECM proteins and proteoglycans can perform opposite functions under different stimuli (Bix & Iozzo, 2005). 44  1.4.8.  Potential mechanisms of altered proteolysis in aberrant tissue remodeling  Based on the detection of numerous proteolytic fragments of versican, it is predictable that versican produces cryptic matrikines that have yet to be described. Versican directs cell migration through haptotaxis (Cattaruzza & Perris, 2005) and such guidance could be specifically altered or even reversed through proteolytic events. Thus, there are multiple different potential pathways for versican turnover wherein its properly regulated turnover allows a return of normal tissue architecture and its aberrant turnover appears to contribute to the excess deposition of collagen and fibrosis (Figure 1.14).  Normal and pathological turnover may vary in the activity and activation state of the proteases involved.  Differences could be merely in the level of protease expression or the ratio of proteases to inhibitors present.  Differences could also involve different proteolytic pathways stimulated in the different remodeling conditions, just as aggrecan turnover in disease and normal remodeling appears to involve different groups of metalloproteinases (Sandy, 2006).  Clearly elucidating the relevant pathways and alterations is critical to understanding aberrant versican turnover in disease.  1.5.  Rationale  Versican is a large chondroitin sulfate proteoglycan associated with cell migration and proliferation in development.  Its expression allows creation of a hydrated and expanded extracellular space that is critical for endocardial cushion formation in the heart (Mjaatvedt et al, 1998).  Likewise, versican-rich barrier tissues are involved in guidance of neural crest cell migration (Dutt et al, 2006; Landolt et al, 1995). Proteolysis of versican appears to regulate these developmental processes both through the loss of the expanded extracellular space and through the creation of 45 Figure 1.14.  Normal and aberrant proteolysis of versican at the cell surface. Schematic representation of versican at the cell surface of fi broblast cells where it may bind ADAMTS-2 and contribute to collagen fi brillogenesis and deposition.  Excess and persistent versican expression may contribute to excessive collagen deposition as seen in fi brosis. Alternatively, appropriate versican proteolysis may be critical to physiological resolution of remodeling and a return to normal tissue architecture.  Metalloproteinases MT1-MMP and MMP-2 form an activation complex at the cell surface and ADAMTS-2 binds to sulfated glycosaminoglycans.  All three enzymes are well localized to contribute to versican degradation at the cell surface.  Proteolysis may release large or small portions of the C-terminal domain, releasing fi broblast cells from the matrix which versican is bound to and altering its hydrodynamic and chemotactic properties.  Proteolysis may also result in partial or complete loss of versican from the matrix and further degradation intracellularly. 46  cleavage products which may themselves be functional (Dutt et al, 2006; Kern et al, 2007; Kern et al, 2006).  Versican expression is associated with fibroproliferative remodeling in many forms of human pulmonary fibrosis (Bensadoun et al, 1996; Bensadoun et al, 1997; Roberts, 2003).  It is also abundant in atherosclerotic and restenotic lesions (Evanko et al, 1998; Halpert et al, 1996; Wight et al, 1997).  In numerous cancers, stromal versican expression is predictive of poor prognosis (Kodama et al, 2007a; Kodama et al, 2007b; Pukkila et al, 2007; Ricciardelli et al, 2002; Ricciardelli et al, 1998; Suwiwat et al, 2004; Touab et al, 2003; Touab et al, 2002).  Whereas versican turnover appears to be an essential part of physiological tissue remodeling, dysregulation of versican turnover may contribute to pathological remodeling.  Proteolytic pathways of versican turnover are to date poorly understood. Improving our understanding of proteolytic events involved in versican turnover is essential to understanding how versican turnover might be pathologically dysregulated. As versican is a pericellular molecule, regulated turnover of versican likely involves proteolysis in proximity to the cell surface.  In human pulmonary fibrosis, procollagen is synthesized within a versican-rich matrix (Bensadoun et al, 1996).  Thus it was hypothesized that the procollagen N- propeptidase ADAMTS-2 might bind and process versican.  MMP-2 and MT1-MMP are activated at the cell surface during wound healing (Okada et al, 1997; Overall et al, 2000), pulmonary fibrosis (Garcia-Alvarez et al, 2006) and many cancers (Sato et al, 2005).  These enzymes were likewise hypothesized to play a role in versican proteolysis.  Concanavalin-A (ConA) is a lectin that is known to bind fibroblast cell surface receptors and induce a matrix degrading phenotype that includes up-regulation of MMP-2 and MT1-MMP (Overall & Sodek, 1990; Yu et al, 1995). 47  1.5.1.  Overarching hypothesis  Regulated versican turnover involves the cell-surface associated metalloproteinases ADAMTS-2, MMP-2 and MT1-MMP, that are expressed in versican-rich remodeling lesions.  1.5.2.  Specific aims  1. To purify versican from human fetal lung fibroblast cultures  2. To investigate the potential of the procollagen N-propeptidase ADAMTS-2 to   cleave versican  3. To investigate mechanisms of versican degradation in the fibroblast degradative   phenotype induced by concanavalin-A  4. To assess the capacity of the cell-surface associated metalloproteinases MMP-2   and MT1-MMP to degrade versican in vitro  5. To characterize proteolytic fragments of versican   In order to investigate versican proteolysis in vitro, versican was purified from human fetal lung fibroblast cultures.  Versican was found to be labile and contain degradation products at physiological pH, allowing insight into contaminating proteases and leading to improved purification conditions.  Following previous work performed in the lab, conditions for optimized purification of versican were characterized and described. Attempts were made to use mass spectrometry to detect versican fragments.  To investigate ADAMTS-2 as a potential versican processing enzyme, the immuno- histochemical localization of both was determined in lung tissues of patients with idiopathic pulmonary fibrosis and in normal lung tissues.  The co-purification of versican 48  and ADAMTS-2 from human fetal lung fibroblast cultures was investigated and a pH dependence of co-purification was shown.  ADAMTS-2 was purified from fetal calf skin and used to investigate its capacity to process purified human versican in vitro.  ConA stimulation of human fetal lung fibroblasts was employed as a possible tissue culture model of versican degradation.  Loss of versican from the culture media was assessed concurrent with the up-regulation and activation of MMP-2 and MT1-MMP. Microarray analysis was used to investigate expression of possible versican-degrading enzymes and their inhibitors, expressed in response to ConA.  Using recombinant MMP-2 and MT1-MMP, versican proteolysis by these enzymes was investigated.  Proteolytic production of different degradation products was investigated with two different antibodies and with purified versican and a recombinant versican C-terminal construct.  Before findings of versican degradation can be translated from the laboratory into diagnostic or therapeutically valuable information, detailed characterization of proteolytic events including cleavage sites is required.  As such, traditional N-terminal sequencing was attempted and in light of the challenges of using this technique with a large proteoglycan, new mass spectrometry based techniques were investigated.  It is hoped that improving our knowledge of versican degrading proteases and characterizing degradation processes will lead to improved diagnosis and therapy for many fibroproliferative disorders. 49  1.6.  References Adany R, Iozzo RV (1990) Altered methylation of versican proteoglycan gene in human colon carcinoma. 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Mol Vis 11: 603-608  Zheng PS, Wen J, Ang LC, Sheng W, Viloria-Petit A, Wang Y, Wu Y, Kerbel RS, Yang BB (2004) Versican/PG-M G3 domain promotes tumor growth and angiogenesis. FASEB J 18(6): 754-756  Zimmermann DR, Dours-Zimmermann MT, Schubert M, Bruckner-Tuderman L (1994) Versican is expressed in the proliferating zone in the epidermis and in association with the elastic network of the dermis. J Cell Biol 124(5): 817-825  Zimmermann DR, Ruoslahti E (1989) Multiple domains of the large fibroblast proteoglycan, versican. EMBO J 8(10): 2975-2981   77 * A version of this chapter will be submitted for publication Sean B. Maurice, Clive R. Roberts, Alain Doucet and Christopher M. Overall, Purification and characterization of versican. 	
   CHAPTER 2 – PURIFICATION AND CHARACTERIZATION OF VERSICAN*  2.1.  Summary In development and normal wound healing, versican is highly expressed in association with migrating and proliferating mesenchymal cells and then rapidly cleared during the resolution of remodeling.  Versican is also a major component of the provisional matrix in a number of wound healing disorders. Alterations in its metabolism may contribute to the development of pulmonary fibrosis and atherosclerosis, and to cancer growth and metastasis.  It is a challenging molecule to characterize biochemically, yet understanding molecular events involving versican metabolism is key to assessing the utility of potential interventions that would target faulty proteolytic pathways.  Here I report that versican purified from human fetal lung fibroblast cells contains numerous proteolytic fragments, potentially as a result of a co-purifying proteolytic enzyme.  An optimized protocol for versican purification is presented.  I characterize a versican fragment with N-terminal sequencing and discuss challenges to characterization, including the use of mass spectrometry.  2.2.  Introduction  Versican expression is associated with migrating and proliferating, mesenchymal cells.  In development and normal tissue remodeling versican seems to be required for formation of a hydrated matrix that is conducive to cell migration.  Versican ablation is lethal due to defects in endocardial cushion formation in heart morphogenesis (Mjaatvedt et al, 1998).  During wound healing, activated fibroblast cells synthesize a 78  	
   versican-rich provisional matrix in which wound closure and tissue remodeling occur. The persistence of versican in the provisional matrix is associated with fibrotic remodeling.  Versican is a major component of the provisional matrix in all the major granulomatous and non-granulomatous forms of pulmonary fibrosis (Bensadoun et al, 1996; Bensadoun et al, 1997).  Versican is highly expressed in atherosclerotic and restenotic lesions (Evanko et al, 1998; Wight et al, 1997) and transplant arteriopathy (Lin et al, 1996).  Versican expression has been observed surrounding many types of human tumors. In fact versican expression is suggested to be predictive of poor prognosis in oral (Pukkila et al, 2007), breast (Ricciardelli et al, 2002; Suwiwat et al, 2004), prostate (Ricciardelli et al, 1998), cervical (Kodama et al, 2007a), endometrial (Kodama et al, 2007b) and cutaneous (Touab et al, 2003; Touab et al, 2002) cancers.  In the context of wound healing disorders, cancer displays several attributes of non-healing wounds (Bissell & Radisky, 2001; Dvorak, 1986).  It is possible that versican plays a role in perpetuating certain cancers which is similar to its role in sustaining fibrotic processes in pulmonary fibrosis and vascular remodeling.  As versican expression is essential in development yet correlated with a number of wound healing disorders, its role in aberrant remodeling may involve impaired degradation leading to its persistence.  Therefore characterization of molecular events involved in versican metabolism is of great physiological interest.  This is prerequisite for the creation of therapies that target the aberrant degradative events without interfering with the appropriate proteolytic events that are necessary for restorative remodeling.  We now know from clinical trials that relatively broad protease inhibition is not helpful in cancer and is sometimes even detrimental to patients (Coussens et al, 79  	
   2002).  This is presumably due to the variety of tasks performed by each proteinase and highlights the need for more accurately targeted therapies along with a more thorough understanding of the precise proteolytic events involved (Fingleton, 2008). Improved characterization of versican metabolism is also crucial to allow disease process monitoring with biomarkers.  Several versican cleaving proteases have been described and four cleavage sites have been characterized to date (Jonsson-Rylander et al, 2005; Sandy et al, 2001; Westling et al, 2004).  Yet there is evidence that more versican cleavage sites exist than have been described thus far.  In order to better understand differences between normal and aberrant versican turnover, there is a need to improve characterization of versican proteolysis.  In addition to maintaining versican in the pericellular matrix, fibroblasts secrete relatively large amounts of versican into culture media providing a suitable source for purification and subsequent studies (Zimmermann et al, 1994).  Here I describe purification of versican and versican fragments from human fetal lung fibroblast cells.  I characterize several proteolytic fragments of versican and discuss opportunities to improve characterization with tandem mass spectrometry.  2.3.  Experimental procedures 2.3.1.  Cell culture  Human fetal lung fibroblasts (HFL-1) were obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 20 mM HEPES and 10% (v/v) Cosmic Calf Serum (Hyclone, Logan, UT).  Cells at low passage number were grown to approximately 70% of confluence 80  
 before fixation and staining for microscopy or harvesting of serum-free conditioned medium (CM).  2.3.2.  Immunofluorescence staining and microscopy  Cells were fixed for 10-15 minutes with 4% para-formaldehyde in phosphate buffered saline (PBS), pH 7.5, then rinsed with PBS.  Cells were permeabilized in TBS-triton (20 mM Tris, pH 7.5, 0.9% NaCl, 0.2 % triton X-100) with 2% (w/v) bovine serum albumin (BSA).  Blocking was performed with 5% (v/v) normal goat serum and 2% (w/v) BSA in TBS-triton.  Washing was performed in 0.2% (w/v) BSA in TBS-triton and antibodies were diluted in the same buffer.  The primary antibody was mouse monoclonal anti- versican C-terminal domain, 2B1 (Isogai et al, 2002; Isogai et al, 1996) (Seikagaku, Tokyo, Japan), dilution 1:500.  Alexa Fluor 594 goat anti—mouse IgG, highly cross- adsorbed secondary antibody was used (Molecular Probes, Eugene, OR). Counterstaining for F-actin was with Alexa Fluor 488 phalloidin stain (Molecular Probes) and nuclear counterstaining was with Hoescht 33342 (Molecular Probes). Stained cells were mounted under coverslips with Prolong Gold antifade reagent (Molecular Probes) and stored at -20°C.  Microscopy was performed on a Leica DMRA2 automated microscope (Leica Microsystems GmbH, Wetzlar).  In the antibody labeled channel, three-dimensional images were acquired and image stacks were deconvolved using the Nearest Neighbour Deconvolution algorithm (Improvision, Coventry, UK).    81  
 2.3.3.  Isolation of versican  Cells at 70% of confluence were rinsed in serum-free media followed by incubation in serum-free media for 24 hours.  Cells were grown a minimum of 24 hours in serum- supplemented media before incubating in serum-free conditions again.  This cycle was performed a maximum of three consecutive times before discarding cultures.  Serum- free CM were collected and centrifuged at 1500 x g for 20 minutes to remove cellular debris.  Urea was added to 7 M before loading onto Q-Sepharose Fast Flow ion exchange resin (Amersham Biosciences, Piscataway, NJ) at approximately 1 litre CM per 5 mls resin.  The column was equilibrated with 0.1 M Tris, pH 7.5, 7M urea and eluted with 0.1 M Tris, pH 7.5, 7M urea, 1.5 M NaCl.  Later purifications optimized to reduce co-purification of proteases and proteolytic fragments were performed at pH 6.0 with additional salt in the start and equilibration buffer.  150 mM NaCl was added to bring the salt concentration to 400 mM before loading onto Q-Sepharose.  The column was equilibrated in 0.1 M sodium acetate, pH 6.0, 7 M urea, 0.4 M NaCl and eluted with 0.1 M sodium acetate, pH 6.0, 7 M urea, 1.5 M NaCl.  2.3.4.  Electrophoretic techniques Samples in non-reducing sample buffer (125 mM Tris-HCL, pH 6.8, 2.0% SDS, 2.0 M urea, 0.05% bromophenol blue) were separated on discontinuous SDS-PAGE gels with 4% (stacking) and 10% (separating) acrylamide.  Stacking and separating gels were kept during Western blotting to monitor high molecular weight versican aggregates within the stacking gel.  Relative molecular mass (measured in kDa) was estimated based on mobility of molecular weight markers: MagicMark XP (Invitrogen) 82  
 and Kaleidoscope Prestained (Bio-Rad).  Alcian blue staining was used for visualization of sulfated glycosaminoglycans (Krueger & Schwartz, 1987).  2.3.5.  Western blotting Western blotting was performed using the XCell II blot module (Invitrogen) to PVDF membrane (Millipore, Billerica, MA).  Blocking was performed with a solution of 2% (w/v) casein, 2% (w/v) bovine serum albumin, 0.5% (w/v) PVP, 20 mM Tris, pH 7.5, 5 mM EDTA, 0.9% NaCl, 1 x PSN antibiotic mixture (Gibco, Grand Island, NY) and 0.3% (v/v) Tween 20.  The following antibodies were used: mouse monoclonal anti-versican N-terminal antibody 12C5 (Asher et al, 1991)(obtained from the Developmental Studies Hybridoma Bank (NICHD), the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242), 1:500 dilution; anti-versican C-terminal domain 2B1, 1:1000 dilution; rabbit polyclonal anti-versican C-terminal domain recombinant construct LC2 (Pourmalek & Roberts, 2008), 1:10 000 dilution; and rabbit polyclonal anti-PG40 (Brennan et al, 1984; Krusius & Ruoslahti, 1986), 1:500 dilution.  Antibodies were diluted in a solution of Tris-BSA with 0.05% (v/v) Tween 20.  Highly cross-adsorbed goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugate (Bio-Rad) secondary antibodies were diluted 1:5000.  Visualization of the peroxidase was performed with Enhanced Chemiluminescence Plus Western blotting reagents (Amersham Biosciences, Piscataway, NJ) and exposed to X-ray film (Kodak, New Haven, CT) or captured using the ChemiGenius-2 bio-imaging system and Gene Snap software (Perkin Elmer, Woodbridge, ON)   83  
 2.3.6.  Quantification of chondroitin sulfate concentration with the DMMB assay  1,9-dimethylmethylene blue chloride (DMMB) was used to measure concentration of sulfated glycosaminoglycans, in this case chondroitin sulfate, at a wavelength of 525 nm according to the established protocol (Farndale et al, 1986).  Shark cartilage chondroitin sulfate A was used to prepare standard concentrations of chondroitin sulfate for generation of a standard absorbance curve (Seikagaku, Tokyo, Japan). Log10 standard curve of the chondroitin sulfate concentrations was plotted with Graph Pad Prism 5 (La Jolla, CA).  2.3.7.  Gel filtration chromatography  Purified versican was analyzed by gel filtration chromatography over a Superose-6 column (Amersham Biosciences) in TBS (20 mM Tris, pH 7.5, 0.9 % NaCl).  Column fractions were filter concentrated by a factor of 10 using Amicon centrifugal filter units (Millipore).  Concentrated column fractions were treated or not treated with 0.5 U/ml chondroitinase ABC (Sigma) at 37°C for 40 minutes before electrophoresis.  2.3.8.  Characterization of proteolytic fragments  Proteolytic fragments were separated by electrophoresis and electroblotted to immobilon-PSQ membrane (Millipore) with CAPS pH 11.  Membranes were stained with coomassie R-250 to identify fragments to be excised for sequencing.  N-terminal amino acid sequencing was performed directly off PVDF membrane by Edman degradation at the Nucleic Acid Protein Service Unit at the University of British Columbia.  84  
 2.3.9.  Enzyme incubation  Soluble human MT1-MMP lacking the transmembrane and cytoplasmic tail was expressed and purified as described (Tam et al, 2004).  His-tagged recombinant versican C-terminal ‘G3’ domain construct was expressed in Escherichia coli, purified, refolded and verified by fluorescence anisotropy spectroscopy, N-terminal sequencing and mass spectrometry (Pourmalek & Roberts, 2009).  Purified versican or versican G3 domain constructs were incubated alone in enzyme buffer or incubated with recombinant MT1-MMP for 0 or 24 hours at 37°C.  2.3.10.  Proteomic identification   After enzyme assay or control incubations, proteins were reduced with dithiothreitol and alkylated with iodoacetamide.  Differential reductive dimethyl labeling was performed with formaldehyde (37% w/v) or formaldehyde-d2 (20% w/v)(Sigma) with sodium cyanoborohydride (ALD Coupling Solution, Sterogene Bioseparations, Carlsbad, CA) as catalyst (Hsu et al, 2003).  After labeling, proteins were digested with mass spectrometry grade Trypsin Gold (Promega, Madison, WI).  Peptides were purified over Sep-Pak Light C18 cartridges (Waters, Milford, MA) before lyophilizing and reconstituting.   Peptides were separated with nanoscale reverse phase high performance liquid chromatography and sprayed into LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA).  Alternately, peptides were sprayed into a QSTAR-XL hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems Inc., Foster City, CA).  Peak lists were searched against the SwissProt human protein database (releases 50.0 through 54.0) using Mascot version 2.2 (Matrix Science, London, UK, 85  	
   www.matrixscience.com).  Search parameters included semi-tryptic cleavage, fixed carbamidomethyl cysteine, variable oxidation of methionine, and variable light or heavy (+4) dimethylation of N-terminus and lysine.  Peptide and fragment mass tolerances were 0.2 Da, confidence level was greater than 99.5% and ion score cut-off was 10. The scoring scheme used was ESI-TRAP for the LTQ-Orbitrap or ESI-QUAD-TOF for the QSTAR-XL.  Peptide chromatograms were manually verified to contain a minimum of three consecutive y-ions.  2.4.  Results 2.4.1.  Versican purification from human fetal lung fibroblast cells  Versican is synthesized by migrating and proliferating fibroblast cells and is involved in development and wound healing in the lungs.  Therefore human fetal lung fibroblast (HFL-1) cells were chosen as a candidate cell line from which to harvest versican.  I found these cells to stain for versican in their extracellular matrix with the most abundant staining pericellularly (Fig. 2.1).  I also found that HFL-1 cells secreted adequate quantities of versican to allow purification.  Following standard proteoglycan purification techniques, I purified versican over ion exchange resin.  In initial work I found that I was able to purify versican but that the preparation was labile in the presence of urea (Fig. 2.2).  Versican is present as an aggregate and monomer in the denaturing polyacrylamide gels (Fig. 2.2. large arrows) and numerous proteolytic fragments of versican are detectable (Fig. 2.2. small arrows).  The presence of lower molecular weight versican fragments detected by Western blotting could be attributed to degradation products in the medium that were co-purified.  This suggested that a protease could be co-purifying with versican. 86 Figure 2.1.  Versican at the cell surface of human fetal lung fi broblasts. Versican (red) stains strongly in the pericellular matrix associated with proliferating myofi broblasts.  Scale bar = 25 µm.  Results shown are representative of at least three experiments, each analyzed in triplicate. 87 220 120 60 kDa 100 50 80 St ar t El ut io n fra ct io ns W as h Fl ow  th ro ug h 220 120 60 kDa 100 50 80 St ar t El ut io n fra ct io ns W as h El ut io n fra ct io ns 220 120 60 kDa 100 50 80 LC22B1 40 Alcian Blue Figure 2.2.  Purifi cation of versican from human fetal lung fi broblast cells at pH 7.5. Initial purifi cation attempts at pH 7.5 and with no additional salt added to starting material or equilibration buffer resulted in a preparation containing versican or other proteoglycan fragments (Alcian Blue - small arrows) and degradation products of versican (2B1 and LC2 antibodies - small arrows).  Position of versican aggregate and monomer are marked with large black arrows.  Results shown are representative of at least three experiments, each analyzed in triplicate. Fl ow  th ro ug h 88  	
   2.4.2.  Optimization of versican purification  In order to reduce the possibility of a protease co-purifying with versican and cleaving versican in the preparation, I experimented with conditions to optimize versican stability and selection.  Proteinase inhibitors were avoided in favor of obtaining a versican preparation suitable for subsequent enzymatic assays.  A pH of 6.0 was chosen to reduce the possibility of a co-purifying enzyme having substantial catalytic activity and to reduce positive charges in weakly acidic proteins that might bind the column resin at neutral pH.  Salt was added to the starting material and the wash buffer to select for higher affinity binding to the ion exchange resin and reduce co-purification of a putative versican binding and degrading enzyme.  In order to obtain high concentrations of versican, a dual ion exchange purification method protocol was used.  Initially a high volume of culture media was batch bound to a small volume of ion exchange resin.  Reactive elution fractions were pooled from multiple runs and then re-purified over a smaller volume ion exchange column. Concentration of sulfated glycosaminoglycans in versican reactive fractions was measured using the DMMB assay (Farndale et al, 1986).  In initial purification attempts at pH 7.5, I was able to obtain a maximum concentration of 0.08 mg/ml chondroitin sulfate.  With optimized conditions and dual ion exchange runs, a chondroitin sulfate concentration of 0.75 mg/ml was obtained in versican eluting fractions (Fig. 2.3 asterisk).  The Roberts lab and others have observed that preparations from proliferating fibroblast cultures such as these contain predominantly versican V0 and V1 (Roberts, C.R., unpublished observations).  Since these large isoforms contain approximately twice as much glycosaminoglycan mass as core protein mass, the concentration of versican was estimated to be 1.1 mg/ml or about 1.1 µM versican. 89 Figure 2.3.  Chondroitin sulfate concentration measured by the DMMB assay. Purifi ed versican was analyzed by the DMMB (1,9-dimethylmethylene blue) assay (Farndale et al, 1986).  Chondroitin sulfate A was used to make a standard absorbance curve at 525 nm and absorbance of the versican preparation was plotted on this curve to determine the concentration of chondroitin sulfate in the versican preparation. Results shown are representative of three experiments, each analyzed in duplicate. * 0.75 mg/ml 90  	
   Under these optimized conditions, versican was purified with only very minor degradation products present.  2.4.3.  Gel filtration analysis of versican  I used gel filtration chromatography to further characterize our versican preparation. Versican was separated over Superose-6 high molecular weight gel filtration resin. Versican eluted in the predominant peak at the void volume of the column (Fig. 2.4). Alcian Blue staining and Western blotting showed a high molecular weight proteoglycan that stains for versican with both the N- (2B1) and C-terminal domain (12C5) antibodies, indicating it was intact versican.  Versican was present as high molecular weight aggregate and monomer in the alcian blue stain.  After chondroitinase digestion, versican was present predominantly as monomers with V0 and V1 isoforms detectable. Silver staining indicated a co-purifying protein in the third lane that had an apparent molecular weight of 120 kDa that is reduced to a doublet of 43 and 45 kDa upon chondroitinase ABC digestion.  Since this profile matches that of decorin, previously known as PG40, I performed a Western blot with a PG40 antibody and identified this protein as decorin (Brennan et al, 1984; Krusius & Ruoslahti, 1986).  2.4.4.  Characterization of versican degradation  Under our optimized versican purification scheme I occasionally observed two versican degradation products faintly in Western blots with the 2B1 antibody (Fig. 2.5). These fragments were visible with or without incubation, suggesting that they were co- purifying with versican from the culture media.  The products observed were a doublet with molecular weights of about 42 and 50 kDa.  In repeated experiments I attempted 91 C’ABC PG40 PG40 Vt Vt Vt Ag Mo V0 V1 Alcian Blue Silver Silver + C’ABC PG40 + C’ABC12C5 + C’ABC2B1 + C’ABC S S S Figure 2.4.  Analysis of versican separated by gel fi ltration. A.  Gel fi ltration chromatography of purifi ed veriscan detected at 280nm.  B.  Starting material (S) shows a high concentration of aggregating (Ag) and monomer (Mo) versican by Alcian Blue glycosaminoglycan staining and silver staining.  Western blotting with anti-versican C-terminus (2B1) and anti-versican N-terminus (12C5) antibodies after chondroitinase ABC treatment shows versican V0 and V1 isoforms.  Silver staining after chondroitinase ABC treatment of samples shows a potential chondroitin sulfate proteoglycan with a double band at 40 kDa.  Western blot- ting with anti-PG40 antibody confi rmed the identity of the chondroitin sulfate proteoglycan as decorin which is known to have a molecular weight of 120 kDa and to form a doublet of 43 and 45 kDa after chondroitinase digestion (Brennan et al, 1984; Krusius & Ruoslahti, 1986).  Results shown are representative of three experiments, each analyzed in duplicate. V0S V0S V0S A. B. 92  	
   to obtain N-terminal sequencing information to characterize these degradation products.  One sequence was obtained for the 50 kDa product with 9 residues detected corresponding to cleavage at alanine 2963 - aspartate 2964 of versican V0 or V1 (Fig. 2.5).  This cleavage would release the 432 amino acids of versican compromising the entire C-terminal globular domain.  2.4.5.  Mass spectrometric characterization of versican  As chemical N-terminal sequencing was only occasionally successful in producing a definitive sequence, other methods to characterize versican and versican fragments were sought.  Tandem mass spectrometry offers much promise as a technique to precisely identify peptide sequences.  Versican was analyzed by mass spectrometry before and after enzyme digestion experiments in hopes of characterizing cleavage sites.  By dimethyl labeling N-termini with isobaric formaldehyde prior to tryptic digestion (Hsu et al, 2003), it is possible to discriminate between tryptic peptides, proteolytic peptides in the control and proteolytic peptides produced by the experimental conditions.  In attempts to reduce sample complexity, removal of tryptic, un-labeled peptides prior to analysis was employed (Keller et al, 2007; Kleifeld et al, 2009).  Unfortunately, in repeat experiments only ambiguous sequence assignments were obtained.  However it was possible to obtain high confidence sequence data for three tryptic versican peptides (Fig. 2.6).  All three of these are proteotypic peptides that have been detected multiple times in the global proteome machine database (www.thegpm.org).  As the starting material in these experiments was a purified preparation of versican, the low number of peptides detected underscores the challenges in characterization of this molecule. 93 Figure 2.5.  N-terminal sequencing of versican degradation product. Purifi ed versican at 0 or 24 hours incubation shows a prominent doublet of products at 42 and 50 kDa.  Arrow marks the 50 kDa band which was sequenced.  N-terminal sequencing by Edman degradation reveals 9 residues corresponding to a previously uncharacterized C-terminal cleavage between Ala2963 and Asp2964.  Result shown was obtained once. 180 110 75 kDa 50 40 30 0    24 hours VITTA2963^DEIELEGAT 94  	
   2.4.6.  Versican glycosylation and characterization  As versican is substituted with large sulfated glycosaminoglycans and N- and O- linked glycosylations, it is likely that these abundant substitutions interfere with and limit peptide purification.  It is also possible that substitutions or fragments that remain after purification would interfere with the elution, ion sorting and detection in the mass spectrometer.  To date there are four published versican cleavage sites produced by ADAMTS metalloproteinases (Fig 2.7A) (Jonsson-Rylander et al, 2005; Sandy et al, 2001; Westling et al, 2004).  These four cleavage sites, plus the newly identified proteolytic site, all occur in the two glycosaminoglycan attachment domains (Fig. 2.7). It is likely that there are more versican-degrading enzymes and cleavage sites yet to be characterized, but the extent of versican’s substitutions continues to hinder efforts.  The global proteome machine database (www.thegpm.org) was queried for versican to analyze detection of different regions of the molecule.  All detected versican peptides from approximately 400 experiments were compiled and compared to versican’s core protein domains and potential glycosylation sites (Fig. 2.7B)(adapted from (Dours- Zimmermann & Zimmermann, 1994; Zimmermann & Ruoslahti, 1989)).  The number of observations of each peptide were complied from all versican detecting experiments in the database (Fig 2.7C).  The most frequently detected peptides were all found in the N- and C- terminal globular domains.  Throughout the molecule the location of detected peptides largely corresponded with regions lacking potential substitution sites, underscoring the need to remove or otherwise accommodate these glycosylations prior to analysis in order to obtain better characterization.   95 Figure 2.6.  Tryptic versican peptides detected in MS/MS. A. Analysis of versican tryptic peptides detected three signifi cant peptides.  Lys and Arg residues preceeding cleavage sites are underlined.  Chromatograms and error plots for ion fragments of tryptic peptide YEINSLIR (B), LLASDAGLYR (C) and LATVGELQAAWR (D).  Mass error plots indi- cate difference between observed and expected mass for ions shown in adjacent chromatograms. Database searching in MASCOT identifi ed peptides and ion fragments as listed.  Results shown are representative of four separate experiments. YEINSLIR YEINSLIR mass error A. Protein   Mass  Ion (score)   delta  score   Peptide  Versican   0.0801   30   R.YEINSLIR.Y (80)   0.0756   36  K.LLASDAGLYR.C    0.0742   61   R.LATVGELQAAWR.N   B. C. D. LLASDAGLYR LATVGELQAAWR LLASDAGLYR mass error LATVGELQAAWR mass error 96  	
   2.5.  Discussion  Based on the ubiquitous expression of versican in remodeling tissues and diseases, there is much interest in characterizing molecular events involved in its metabolism.  In addition to being a large, glycosylated, negatively charged, hydrophilic molecule, versican exhibits a pronounced tendency to aggregate.  Versican self-aggregation is through core protein interactions that may be calcium dependent interations of its C- type lectin domains (Morgelin et al, 1989; Ney et al, 2006).  Yet despite these biochemical challenges, versican has been purified successfully by several groups.  Standard versican purification schemes exploit versicans negatively charged glycosaminoglycans and large molecular weight to purify by anion exchange and gel filtration chromatography (Sakko et al, 2003; Schmalfeldt et al, 1998).  Some more extensive strategies have been reported to reduce trace contaminants (Mazzucato et al, 2002) or separate versican isoforms (Dutt et al, 2006).  Here I describe a relatively straightforward but effective strategy that allows purification of high molecular weight versican aggregates with only minor contaminants.  The preparation was shown to contain high molecular weight proteoglycans that label with a versican antibody. Analysis by mass spectrometry indicated that only versican is abundant in the preparation with three peptides being identified.  At neutral pH we observed a number of versican proteolytic fragments that co-purify over ion exchange resin.  Others have noted that uncharacterized versican fragments are present in the vasculature (Formato et al, 2004; Kenagy et al, 2006; Sandy et al, 2001; Theocharis et al, 2003), eyes (Ohno-Jinno et al, 2008), pancreatic and laryngeal cancer (Skandalis et al, 2006; Stylianou et al, 2008; Vynios et al, 2008).  We sequenced a C-terminal fragment of versican that appears to be proteolytically derived 97 Figure 2.7.  Versican cleavage sites and peptides detected in proteomics experiments compared with potential glycosylation sites. A. Documented and newly described versican cleavage sites are shown with large arrows.  Proteolytic fragment with N-terminus at site Ala2963 - Asp2964 characterized by N-terminal sequencing.  B. Schematic of versican domain composition labelled with potential glycosyla- tion sites: black arrows (glycosaminoglycans), red arrows (O-linked) and blue arrows (N-linked).  C. Bar graph illustrates number of observa- tions of each versican peptide reported in the Global Proteome Machine database (thegpm.org).  Detected peptide locations also marked on versican schematic with black arrowheads. (Westling et al, 2004) IVSFE405^QKATV TSEVE 950 ^GLAFV (Jonsson-Rylander et al, 2005) PEAAE1428^ARRGQ (Sandy et al, 2001) PSVQY1410^INGKH (Jonsson-Rylander et al, 2005) VITTA2963^DEIEL A. B. C. 98  	
   and bound to intact purified versican.  There seems to be a spectrum of proteolytic events and fragments that have yet to be characterized and which are likely physiologically significant.   There are several known versican cleavage sites (Jonsson-Rylander et al, 2005; Sandy et al, 2001; Westling et al, 2004). As with the related hyalectan aggrecan, most of the cleavage sites have been identified using a hypothesis driven approach with synthetic peptide substrates.  Though cleavage sites have been validated in vivo, there remains much evidence of further cleavage sites and relevant enzymes.  Gaining a more thorough understanding of versican cleavage events requires new and unbiased approaches.  Future detection by mass spectrometry will need to improve methods to deal with glycosaminoglycans and glycosylations to obtain higher confidence assignments. While mass spectrometry combined with database searching is an extremely powerful tool for identification of unknown peptides, standard procedures rely on the hydrophobicity of most peptides and the reliable masses of unmodified amino acid residues.  Wherein versican contains an abundance of both acidic residues and substituted amino acids, improving analysis by mass spectrometry will require methods to deal with these atypical requirements.   99  
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J Cell Biol 124(5): 817-825  Zimmermann DR, Ruoslahti E (1989) Multiple domains of the large fibroblast proteoglycan, versican. EMBO J 8(10): 2975-2981    105 * A version of this chapter will be submitted for publication, Sean B. Maurice and Clive R. Roberts, Versican-ADAMTS-2 interactions in human pulmonary fibrosis: the proteoglycan versican binds the procollagen N-propeptidase ADAMTS-2 and regulates its activity.  CHAPTER 3 – VERSICAN-ADAMTS-2 INTERACTIONS IN HUMAN PULMONARY FIBROSIS*  3.1.  Summary  The chondroitin sulfate proteoglycan versican is transiently expressed in developmental and remodeling processes where its expression coincides with cell migration, proliferation and the formation of new tissue.  In idiopathic pulmonary fibrosis (IPF), versican is associated with proliferating fibroblasts that synthesize collagen-rich fibrotic matrix.  Collagen fibrillogenesis takes place in a versican-rich provisional matrix which is apparently resorbed concomitant with collagen deposition, by a process that is yet to be identified.  As the two processes are concurrent, it was hypothesized that the procollagen N-propeptidase ADAMTS-2 (a disintegrin and metalloproteinase with thrombospondin motifs-2) might contribute to versican degradation and thus, resorption of the provisional matrix.  In this report I show that: (i) ADAMTS-2 is found in versican- rich areas of normal human lungs; (ii) ADAMTS-2 staining is associated with proliferating fibroblasts in the versican-rich proliferative lesions of IPF; (iii) ADAMTS-2 is released from normal human lung tissue by incubation with chondroitinase ABC; (iv) ADAMTS-2 and versican co-purify in human fetal lung fibroblast culture media; (v) ADAMTS-2 does not auto-degrade in the presence of versican and (vi) versican is a substrate for purified ADAMTS-2 in vitro.  This data demonstrates that versican binding may regulate ADAMTS-2 biological activity and thus collagen assembly. 106 3.2.  Introduction  Pulmonary fibrosis is a dysregulated repair process that is often associated with but not necessarily perpetuated by inflammation (Thannickal et al, 2004).  Idiopathic pulmonary fibrosis (IPF) is distinct from the related idiopathic interstitial pneumonias, exhibiting the histologic pattern usual interstitial pneumonia (UIP) with characteristic fibroblastic foci and is driven largely if not entirely by non-inflammatory mechanisms (Selman et al, 2001; Travis et al, 2002).  Median survival of patients with IPF is 3 to 5 years and there is no currently effective therapy (Khalil & O'Connor, 2004).  Previous work in our laboratory has demonstrated a consistent expression of versican within the active fibroproliferative lesions of organizing diffuse alveolar damage associated with adult respiratory distress syndrome, idiopathic bronchiolitis obliterans organizing pneumonia (BOOP), UIP (Bensadoun et al, 1996); and the lesions of sarcoidosis, extrinsic allergic alveolitis and tuberculosis (Bensadoun et al, 1997).  The spatial and temporal association of versican with fibroblasts in fibroproliferative lesions suggests that versican plays a specific role in the cell biology of pulmonary remodeling that leads to fibrosis (Roberts, 2003).  Since de novo procollagen synthesis occurs in fibroblasts surrounded by versican (Bensadoun et al, 1996), collagen fibril formation occurs in a versican-rich matrix.  However, these studies showed that versican is absent from areas of mature collagenous fibrosis.  Therefore, it was hypothesized that enzymes involved in collagen assembly might degrade versican in the evolution of fibroproliferative lesions.  Versican is a large aggregating ‘hyalectan’ proteoglycan with hyaluronan-binding and lectin-like domains (Iozzo, 1998).  Versican was first identified because of its association with skeletal development (Kimata et al, 1986).  Versican has a wide tissue distribution (Bode-Lesniewska et al, 1996; Dours-Zimmermann & Zimmermann, 1994) 107 and exhibits diverse functions associated with migrating and proliferating cells in development and disease (Wight, 2002).  Through the reversible binding of water, versican occupies a very large hydrodynamic space influencing chemokine gradients and cell adhesive properties (Kinsella et al, 2004).  In the developing lung, versican is the predominant chondroitin sulfate bearing proteoglycan and its expression is associated with alveolar tissue volume changes (Faggian et al, 2007).  Alternative splicing of versican m-RNA results in four different gene products with identical N- and C-terminal globular domains but different sized central glycosaminoglycan-attachment domains due to the inclusion of one or both or neither of the GAGα and GAGβ domains (Ito et al, 1995; Naso et al, 1994; Zako et al, 1995).  The versican deficient hdf mouse dies due to severe cardiac defects by embryonic day 10.5 (Mjaatvedt et al, 1998) and the hyaluronan deficient Has2 -/- (hyaluronan synthase-2) mouse dies at embryonic day 9.5-10 with similar severe cardiac abnormalities (Camenisch et al, 2000).  In both phenotypes, cardiac defects appear to result from the absence of the hyaluronan and versican-rich matrix required for cell migration and proliferation, as has been shown for vascular smooth muscle cells in vitro (Evanko et al, 1999).  The ADAMTS proteinases are members of the metzincin superfamily of metalloproteinases (Stocker et al, 1995).  They are large multi-domain enzymes which exhibit multiple proteolytically processed variants produced through autocatalytic activation as well as processing by other metalloproteinases (Colige et al, 2005; Flannery et al, 2002; Gao et al, 2004; Tortorella et al, 2005). ADAMTS-1 and -4 have been shown to bind sulfated glycosaminoglycans through thrombospondin-like, cysteine-rich and spacer domain-dependent interactions (Flannery et al, 2002; Kuno & Matsushima, 1998).  Substrate recognition and cleavage 108 of aggrecan by ADAMTS-4 does not occur when the glycosaminoglycan side chains are removed (Tortorella et al, 2000).  Similarly, the absence of thrombospondin and ancillary domains in ADAMTS-9 ablates its proteoglycan degrading activity (Somerville et al, 2003).  A preliminary immunohistochemical screen of human IPF tissue sections revealed ADAMTS-2 as a proteinase of interest.  In this study, I investigated the localization of versican and ADAMTS-2 in normal and remodeling human lung tissue.  The results strongly suggest that ADAMTS-2 is bound to versican in lung matrix and that binding to versican localizes and may regulate the biological activity of ADAMTS-2.  ADAMTS-2 - mediated degradation of versican may be an important element in resorption of the provisional matrix in the evolution of fibroproliferative lesions in lung wound healing and fibrosis.  3.3.  Experimental procedures 3.3.1.  Patient samples Lung tissues used in this study were obtained as part of a lung fibrosis tissue registry as previously described (Bensadoun et al, 1996; Bensadoun et al, 1997).  Lung biopsy tissues were obtained at diagnostic biopsy of patients with a clinical diagnosis of idiopathic pulmonary fibrosis, and were entered into the study following histologic diagnoses of bronchiolitis obliterans organizing pneumonia or usual interstitial pneumonia.  Age-matched control tissues were obtained from normal-appearing lung tissue, obtained from lung lobes that were resected from individuals with small localized tumors, as previously described (Bensadoun et al, 1996). Tissues from 6 UIP, 6 BOOP and 6 control patients were studied.  From some of the control patients it was possible 109 to obtain small samples of unfixed lung tissue for biochemical studies; these were flash-frozen in liquid nitrogen and stored at -80°C until analysis.  3.3.2.  Histology Lung tissues were fixed overnight in 10% neutral buffered formalin, dehydrated, embedded in paraffin and serially sectioned at a thickness of 5µm.  Sections were stained with hematoxylin and eosin to visualize overall architecture, alcian blue to localize glycosaminoglycans and picrosirius red to localize collagen as previously described (Bensadoun et al, 1996).  3.3.3.  Immunohistochemistry Sections were deparaffinized and hydrated in Tris-buffered saline (TBS) for 5 minutes before being immersed in freshly prepared 0.6% hydrogen peroxide in methanol for 40 minutes to block endogenous peroxidase activity.  Sections were blocked with 10% normal goat serum in 2% BSA for 4 hours.  The following primary antibodies were used: mouse monoclonal anti-versican C-terminal domain, 2B1 (Isogai et al, 2002; Isogai et al, 1996) (Seikagaku, Tokyo, Japan), dilution 1:400; rabbit polyclonal anti-versican (LeBaron et al, 1992) used as previously described (Bensadoun et al, 1996; Bensadoun et al, 1997) dilution 1:500; rabbit polyclonal anti- ADAMTS-2 pro-domain (Chemicon International, Temecula, CA) dilution 1:100; and rabbit polyclonal anti-ADAMTS-2 C-terminus (Chemicon International) dilution 1:400. Antibodies were diluted in 2% bovine serum albumin (BSA) in TBS and washed 4 times in TBS.  Antibody labeling was visualized with the Vectastain Universal Elite ABC kit (Vector Laboratories, Burlingham, CA) and DAB (3,3’-diaminobenzidine) as substrate (Vector Laboratories) according to the manufacturer's instructions.  Sections were 110 counterstained with Gill's Haematoxylin.  Negative controls were treated identically with the inclusion of non-immune IgG at the same concentration or with omission of primary antibody.  For each antibody and detection system, conditions were established that allowed use of purified antibody or serum at concentrations that generated no staining with non-immune IgG or serum at equivalent concentrations.  All sections that were to be compared were processed and stained concurrently.  After staining, sections were mounted in Histochoice mounting medium (Amresco, Solon, OH).  3.3.4.  Release of ADAMTS-2 from normal lung tissues Normal human lung tissue (50 mg tissue per ml TBS) was finely diced in TBS (20 mM Tris, pH 7.5, 0.9% NaCl) in the presence of complete mini, EDTA-free proteinase inhibitor cocktail tablets (one tablet per 25 ml)(Roche, Indianapolis, IN) and incubated with or without 0.1 U/ml chondroitinase ABC lyase (MP biomedicals) for one hour at 37°C.  Samples were centrifuged to remove tissue, and supernatant was isolated, diluted with different ratios of sample to reducing sample buffer (containing 65 mM dithiothreitol) and analyzed by electrophoresis and Western blotting for versican and ADAMTS-2.  Alternatively, normal human lung tissue was finely diced in TBS containing proteinase inhibitor cocktail tablets as above and heated to 95°C for 5 minutes before electrophoretic separation and analysis.  3.3.5. Cell culture Human fetal lung fibroblasts (HFL-1) were obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 20 mM HEPES and 10% (v/v) Cosmic Calf Serum (Hyclone, Logan, UT).  Cells at low passage number were grown to approximately 70% of confluence 111 before being rinsed in serum-free media followed by incubation in serum free media for 24 hours.  Cells were grown a minimum of 24 hours in serum-supplemented media before incubating in serum-free conditions again.  This cycle was performed a maximum of three consecutive times before discarding cultures.  Serum-free conditioned media were collected and pooled and used for isolation and purification of versican.  3.3.6.  Isolation of versican Serum-free conditioned medium from fibroblast cultures (CM) was collected and centrifuged at 1500 x g for 20 minutes to remove cellular debris.  Urea was added to 7 M and NaCl was added to bring the salt concentration to 400 mM before loading onto Q-Sepharose Fast Flow ion exchange resin (Amersham Biosciences, Piscataway, NJ) at approximately 1 litre CM per 5 mls resin.  The column was equilibrated in 0.1 M sodium acetate, pH 6.0, 7 M urea, 0.4 M NaCl and eluted with 0.1 M sodium acetate, pH 6.0, 7 M urea, 1.5 M NaCl.  Fractions were monitored for versican content by alcian blue (Sigma, St. Louis, MO) staining of SDS-PAGE gels (Krueger & Schwartz, 1987) and by Western blotting.  Versican-containing fractions were pooled and concentrated over a smaller volume Q-sepharose column using the same buffers.  Concentration of purified versican was estimated using the dimethylmethylene blue (DMMB)(Serva, Heidelberg) assay to quantify sulfated glycosaminoglycans (Farndale et al, 1986), using known concentrations of chondroitin sulfate C as standards (Seikagaku). Chromatography was performed on an AKTA purifier (Amersham Biosciences) and protein elution was monitored at 215, 229 and 280 nm simultaneously.  All procedures were performed at 4°C.  112 3.3.7.  Co-purification of versican and ADAMTS-2 Serum-free CM from fibroblast cultures was collected and centrifuged at 1500 x g for 20 minutes to remove cellular debris.  Urea was added to 7 M.  Approximately 1 litre of culture media was loaded onto 5 mls ANX-Sepharose Fast Flow ion exchange resin (Amersham Biosciences).  The column was equilibrated with 0.1 M Tris, pH 7.5, 7M urea and eluted with 0.1 M Tris, pH 7.5, 7M urea, 1.5 M NaCl.  All procedures were performed at 4°C.  3.3.8.  Separate elution of ADAMTS-2 and versican Serum-free CM from fibroblast cultures was collected and centrifuged at 1500 x g for 20 minutes to remove cellular debris.  Urea was added to 7 M and EDTA to 10 mM. Approximately 1 litre of culture medium was loaded onto 5 mls ANX-Sepharose and equilibrated in 0.1 M Tris, pH 7.5, 7M urea.  The pH elution was performed with 0.1 M Tris, 0.1 M sodium acetate, pH 5.0, 7 M urea.  This was followed by a salt elution in 0.1 M Tris, 0.1 M sodium acetate, pH 5.0, 7 M urea, 1.5 M NaCl.  Column fractions were concentrated by a factor of 10 using Amicon Ultra-15 centrifugal filter devices with 30,000 molecular weight cut-off (Millipore, Billerica, MA).  All procedures were performed at 4°C.  3.3.9.  Electrophoretic techniques Samples in non-reducing (except where stated) sample buffer (125 mM Tris-HCL, pH 6.8, 2.0% SDS, 2.0 M urea, 0.05% bromophenol blue) were separated on discontinuous SDS-PAGE gels with 4% (stacking) and 10 or 12% (separating) acrylamide.  Stacking and separating gels were kept during staining and Western blotting to monitor high molecular weight versican aggregates within the stacking gel. 113 For some experiments, 3-8% NuPage Tris-Acetate gradient gels were used with appropriate sample, running and transfer buffers according to the manufacturer's instructions (Invitrogen, Carlsbad, CA).  Gels were analyzed by silver staining and coomassie blue staining, and alcian blue and alcian blue-enhanced silver staining for visualization of glycosaminoglycans (Krueger & Schwartz, 1987).  Relative molecular mass (measured in kDa) was estimated based on mobility of molecular weight markers: HiMark Prestained (Invitrogen), MagicMark XP (Invitrogen) and Kaleidoscope Prestained (Bio-Rad, Hercules, CA).  Western blotting was performed using the XCell II blot module (Invitrogen) to PVDF membrane (Millipore).  Blocking was performed with a solution of 2% (w/v) casein, 2% (w/v) bovine serum albumin, 0.5% (w/v) PVP, 20 mM Tris, 5 mM EDTA, 0.9% NaCl, 1 x PSN antibiotic mixture (Gibco, Grand Island, NY) and 0.3% (v/v) Tween 20.  The following antibodies were used: anti-versican C-terminal domain 2B1, 1:1000 dilution; anti-ADAMTS-2 pro-domain, 1:500 dilution; anti- ADAMTS-2 C-terminus, 1:2000 dilution; rabbit polyclonal anti-DPEAAE neo-epitope antibody (Sandy et al, 2001) (Affinity Bioreagents, Golden, CO) 1:500 dilution; and mouse monoclonal anti-versican N-terminal antibody 12C5 (Asher et al, 1991) (obtained from the Developmental Studies Hybridoma Bank (NICHD), the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242), 1:1000 dilution. Antibodies were diluted in a solution of 2% (w/v) bovine serum albumin, 20 mM Tris, pH 7.5, 0.9 % NaCl and 0.05% (v/v) Tween 20.  Highly cross-adsorbed goat anti-mouse horseradish peroxidase-conjugate (Bio-Rad) and highly cross adsorbed goat anti-rabbit horseradish peroxidase-conjugate (Bio-Rad) secondary antibodies were diluted 1:5000. Visualization of the peroxidase was performed with Enhanced Chemiluminescence Plus Western blotting reagents (Amersham Biosciences) and exposed to X-ray film 114 (Kodak, New Haven, CT) or captured using the ChemiGenius-2 bio-imaging system and Gene Snap software (Perkin Elmer, Woodbridge, ON).  3.3.10.  Purification of fetal bovine skin ADAMTS-2 Bovine ADAMTS-2 was purified from fetal bovine skin by potassium chloride extraction, ammonium sulfate precipitation, Concanavalin-A Sepharose (Con-A Sepharose) chromatography and heparin Sepharose  chromatography as previously published (Colige et al, 1995).  In the previous work, the authors showed that the enzyme was active towards α-I-procollagen-N-propeptides.  Based on SDS-PAGE gel silver staining, I estimated the concentration of enzyme purified to be 100 ng/ml or 800 ρM for the approximately 125 kDa enzyme purified.  3.3.11.  Versican digestion and ADAMTS-2 incubations Purified versican concentration was estimated using the DMMB assay (Farndale et al, 1986) to quantify sulfated glycosaminoglycan.  Versican V0 and V1 are the primary mRNA splice variants expressed in proliferating HFL-1 cultures (Roberts, C.R. unpublished observations).  The concentration of versican was estimated based on an average of 1.5 mg total proteoglycan per 1 mg sulfated glycosaminoglycan detected with a resultant concentration of 1.12 mg/ml or approximately 1.12 µM versican. Versican and ADAMTS-2 were incubated together at a 1:1 volume ratio (1:1400 - E:S ratio) or individually diluted in the respective buffers from 0 to 48 hours at 37°C. Hyaluronan, heparan sulfate and chondroitin sulfate A, B and C (Seikagaku), were suspended in TBS.  ADAMTS-2 was incubated with TBS alone or at a 1:1 volume ratio 115 with each glycosaminoglycan at final concentrations of 5 nM to 5 µM.  Detection was by Western blotting with ADAMTS-2 C-terminus antibody.  3.4.  Results 3.4.1.  Versican and ADAMTS-2 co-localize in normal human lungs In normal lungs, alcian blue and picrosirius red staining showed collagen throughout the airway, alveolar and blood vessel walls, and very little histochemically-apparent glycosaminoglycan present (Fig. 3.1A).  Versican staining was associated with smooth muscle of the blood vessel walls and airways (Fig. 3.1B).  ADAMTS-2 staining was similarly associated with smooth muscle of the blood vessel and airway walls.  Versican was also associated with tips of the alveolar septae and as previously described, there was a strong association between versican staining and α–smooth muscle actin (Bensadoun et al, 1996).  3.4.2.  Versican and ADAMTS-2 localization in BOOP Patient samples exhibiting the pathological pattern bronchiolitis obliterans organizing pneumonia (BOOP) showed characteristic intraluminal buds composed of loose connective tissue adjacent to a thickened interstitium (Fig. 3.1A).  Alcian blue staining showed that the intraluminal buds contained a matrix rich in glycosaminoglycans which also stained heavily for versican (Fig. 3.1A & B).  This is consistent with our previous demonstration that the glycosaminoglycan is chondroitin sulfate, that versican is the predominant proteoglycan within intraluminal buds and that the histochemical glycosaminoglycan staining and versican staining are highly congruent (Bensadoun et al, 1996).  ADAMTS-2 staining of serial sections demonstrated abundant deposition of the enzyme in the collagen-rich thickened interstitium and trace staining within the 116 Figure 3.1.  Versican and ADAMTS-2 localization in normal lungs and in bronchiolitis obliter- ans organizing pneumonia (BOOP).  A.  Hematoxylin and eosin staining shows a thickened inter- stitium and characteristic intraluminal buds (asterisks) in distal airspaces in BOOP, in contrast with normal lung morphology.  Alcian blue and picrosirius red staining show glycosaminoglycan rich fi bro- proliferative regions in blue and collagen in red.  BOOP sections stain abundantly for glycosamino- glycans within intraluminal buds and weak collagen staining in these glycosaminoglycan-rich areas. Scale bars = 200 µm.  B. The normal lung shows versican and ADAMTS-2 staining (brown) in blood vessel walls (arrows) and in the interstitium of airways and alveoli.  The intraluminal buds of BOOP stain strongly for versican (asterisks).  ADAMTS-2 staining is minimally detectable within the matrix of the intraluminal buds in BOOP and is more abundant in the collagen rich thickened interstitium. Lower panels show a BOOP intraluminal bud at higher magnifi cation.  Versican staining is strong and faint ADAMTS-2 staining is associated with the myofi broblasts (small arrows).   Scale bars = 200 µm, Scale bar (high power) = 30 µm.  Tissues from 6 BOOP and 6 control patients were studied and representative semi-serial sections are shown. Hematoxylin & Eosin Normal BOOP Alcian Blue & Picrosirius Red Versican Control ADAMTS-2 Normal BOOP  BOOP High Power A. B. 117 matrix of the intraluminal buds in BOOP patient sections (Fig. 3.1B).  At higher magnification, ADAMTS-2 staining was weakly detectable in close association with the cells (arrows).  Similar data were obtained using an antibody to the C-terminus of ADAMTS-2 (data not shown).  3.4.3.  Versican and ADAMTS-2 are localized to remodeling areas in UIP Sub-epithelial fibroblast foci in usual interstitial pneumonia (UIP) stained heavily for versican.  These foci also stained for ADAMTS-2, with the most intense staining closer to the centre of the lesions.  Higher magnification of a sub-epithelial fibroblast focus showed ADAMTS-2 staining in association with the fibroblasts (Fig. 3.2 arrows).  It was previously documented that a similar staining pattern occurs for intracellular α-1-(I) procollagen in the versican-rich fibroproliferative intraluminal buds of BOOP (Bensadoun et al, 1996) consistent with the studies of Kuhn and McDonald (Kuhn & McDonald, 1991).  A consistent observation in the patient samples was that in the fibroproliferative lesions of UIP staining for versican and ADAMTS-2 was strong, and that the versican-rich fibroproliferative lesions of BOOP showed weak staining for ADAMTS-2.  The intensity of ADAMTS-2 staining in UIP was variable between different lesions and different locations within a given lesion. The intima and media of arteries in the lung stained intensely for both versican and ADAMTS-2, with ADAMTS-2 staining being stronger in the media, and versican staining stronger in the intima (Fig. 3.2 arrows).  3.4.4.  Release of ADAMTS-2 from normal human lung tissue The apparent co-localization of versican and ADAMTS-2 in normal lung tissue (Fig. 3.1) led us to hypothesize that binding to the chondroitin sulfate side chains of versican 118 Figure 3.2.  Versican and ADAMTS-2 localization in usual interstitial pneumonia (UIP). Versican staining in UIP is abundant within sub-epithelial fi broblast foci (asterisks) and within blood vessel walls (arrows).  ADAMTS-2 staining co-localizes to the blood vessel walls and co-localizes in the sub-epithelial fi broblast foci.  Lower panels show a UIP sub-epithelial fi broblast focus at higher magnifi cation.  ADAMTS-2 staining is evident associated with the myofi broblasts in the versican-rich sub-epithelial fi broblast focus (small arrows).   Scale bars = 200 µm, Scale bar (high power) = 30 µm.  Tissues from 6 UIP patients were studied and representative semi-serial sections are shown. Versican ADAMTS-2 UIP UIP UIP High Power 119 could localize ADAMTS-2 in the tissue.  ADAMTS-2 release from normal human lung tissues was therefore attempted on 2 patients samples, by treating tissue with chondroitinase ABC, using samples from the same patients as paired controls.  Normal human lung tissue diced in TBS with proteinase inhibitor tablets and incubated for 1 hour at 37°C released into the supernatant several faint bands detectable with the ADAMTS-2 pro-domain antibody (Fig. 3.3A lanes 8-10 and 11-13).  In contrast, incubation of the tissue samples in chondroitinase ABC for 1 hour at 37°C resulted in an increased release of ADAMTS-2, particularly a band of approximately 42 kDa (Fig. 3.3A lanes 1-3 and 4-6).  When tissue was heated to 95°C for 5 minutes in reducing sample buffer, ADAMTS-2 was released from the tissue as a series of species, 150- 200 kDa and also a 42 kDa species (Fig. 3.3B).  This is consistent with the range of species observed in extracting the enzyme from chick and bovine tissues (Colige et al, 1995; Hojima et al, 1989; Hojima et al, 1994).  These data suggest that binding to chondroitin sulfate may localize ADAMTS-2 within human lung tissue.  3.4.5.  ADAMTS-2 co-purifies with versican at physiological pH Versican was purified by ion exchange chromatography at pH 7.5 from freshly harvested conditioned medium of HFL-1 fibroblast cultures.  Chromatography and medium processing was done at 4°C.  Following purification, all column fractions were filter-concentrated by a factor of 10 and separated by SDS-PAGE.  Western blotting with versican and ADAMTS-2 antibodies indicated that ADAMTS-2 and versican co- purified on the anion exchange resin (Fig. 3.4A & B).  Later elution fractions to 1.5 M NaCl were negative for both versican and ADAMTS-2.  Considerable immunoreactivity for ADAMTS-2 was present in the 4% ‘stacking’ portion of the discontinuous gel, where high molecular weight versican (1-2 MDa) was present.  This is a much higher 120 100 80 60 30 M W  m ar ke r kDa Figure 3.3.  Release of ADAMTS-2 from normal human lung tissue.  Normal human lung tissue from patient 1 & patient 2 was fi nely diced in TBS with proteinase inhibitor tablets and incubated for 1 hour.  A, Chondroitinase ABC lyase (0.1 U/ml) was added to some tissue samples (lanes 1 – 6) but not to controls (lanes 8 – 13).  Tissues were incubated for 1 hour before removal of supernatant for ADAMTS-2 detection by Western blotting.  1, 2, or 5 µl sample from each patient (left to right for each sample condition) was added to 4, 3, or 0 µl TBS and 5 µl reducing sample buffer followed by SDS PAGE separation (4/10%) and West- ern blotting.  B, Reducing sample buffer was added directly to diced tissue samples at a 1:1 ratio before heating to 95°C for 5 minutes and then Western blotting.  C, Identical Western blot with primary antibody omitted and identical contrast adjustments to show absence of secondary antibody cross-reactivity with tissue sample.  Results shown are representative of three experiments, each performed in duplicate. A. B. 40 50 130 190 60 30 40 50 Pa tie nt  1 Pa tie nt  2 Pa tie nt  1  +  C ’A BC Pa tie nt  2 +  C ’A BC kDa1   2   3  4   5   6  7   8  9 10 11 12 13 Pa tie nt  1  Pa tie nt  2 Pa tie nt  1  Pa tie nt  2 C. 130 190 60 30 40 50 kDa 121 Figure 3.4.  ADAMTS-2 and versican co-purify.  Immunoblots of serum-free conditioned media (CM) in 7 M urea purifi ed over ANX-Sepharose anion exchange resin.  HFL-1 serum- free conditioned medium (lane 1), unbound sample (lane 2), 0 M NaCl column wash (lane 3) and column fractions eluted with a 0 – 1.5 M NaCl gradient (lanes 4 – 9).  Fractions were concentrated by equal amounts with centrifugal fi lter concentrators and separated by SDS PAGE (4/10%) followed by Western blotting.  A, Immunoblot with 2B1 antibody.  B, Immu- noblot with anti-ADAMTS-2 C-terminus antibody.  Results shown are representative of four experiments, each analyzed in duplicate. Versican ADAMTS-2 A. 200 120 St ar t El ut io n fra ct io ns W as h Fl ow  th ro ug h kDa B. 1    2     3     4     5    6     7     8    9 200 120 St ar t El ut io n fra ct io ns W as h Fl ow  th ro ug h kDa 1    2     3     4     5    6     7     8    9 122 molecular weight than full length ADAMTS-2 (177 kDa) indicating that this ADAMTS-2 was present in a protein complex that is stable to denaturing conditions (2.0 % SDS and 2M urea).  These observations suggested that ADAMTS-2 was bound to versican and eluted from the column as a complex with versican.  This is consistent with the observations of Leung et al. (Leung et al, 1979) who found that chick tendon procollagen N-propeptidase appeared to be bound to other non-collagenous molecules in a very high molecular weight complex.  3.4.6.  ADAMTS-2 co-purification with versican is pH dependent I investigated the pH-dependence of versican and ADAMTS-2 co-purification.  An ion exchange column was loaded in the same manner as before at pH 7.5.  Prior to the salt elution, I performed a gradient from pH 7.5 to 5.0 in the same buffer.  I observed minimal release of versican from the column and a substantial release of ADAMTS-2 (Fig. 3.5A & B, lanes 4-6).  Whereas ADAMTS-2 co-purified with versican at pH 7.5 was apparent in a high molecular weight protein complex after denaturing electrophoresis, this pH elution released predominantly lower molecular weight species including a strong band near the 177 kDa full size of ADAMTS-2 (Fig. 3.5B lane 6). When I followed the pH elution immediately with a salt elution from 0 to 1.5 M NaCl at pH 5.0, we saw an abundance of versican released from the column (Fig. 3.5A, lane 7) but no further ADAMTS-2 released (Fig. 3.5B, lane 7).  This is consistent with versican binding to the ion-exchange column directly and ADAMTS-2 binding to versican in a pH-dependent manner.  These properties allowed us to separate versican and ADAMTS-2, and purify ADAMTS-2-free versican as a substrate for enzyme digestion experiments.  123 240 120 70 St ar t Sa lt El ut io n W as h kDa Figure 3.5.  ADAMTS-2 co-purifi cation with versican is pH dependent.  Serum-free CM in 7 M urea was purifi ed over ANX-Sepharose anion exchange resin and eluted with a pH elution from 0.1 M Tris, pH 7.5 to 0.1 M Tris / 0.1 M Acetate, pH 5.0, before being eluted with 0 to 1.5 M NaCl at pH 5.0.  Immunoblots of starting sample (lane 1), 0 M NaCl, pH 7.5 column wash (lane 2), fi rst elution from pH 7.5 to pH 5.0 (lanes 3-6), second elution from 0 to 1.5 M NaCl at pH 5.0 (lanes 7 & 8).  Analysis was by SDS PAGE (4/10%) and Western blotting with 2B1 antibody (A) or anti-ADAMTS-2 C-terminus antibody (B).  Results shown are representative of two experiments performed and each analyzed in duplicate. Versican ADAMTS-2 A. p H El ut io n 1      2     3     4     5     6     7     8 50 240 120 70 50 St ar t Sa lt El ut io n W as h kDa B. p H El ut io n 1      2     3     4     5     6     7     8 124 3.4.7.  Purification of bovine ADAMTS-2 Following the previously published protocol (Colige et al, 1995) and in light of more recent work (Colige et al, 2005; Wang et al, 2003), ADAMTS-2 was purified from fetal bovine skin.  Potassium chloride extraction, ammonium sulfate precipitation and Con-A Sepharose chromatography were performed as described (Colige et al, 1995), then samples from each step of the purification were separated by SDS-PAGE and detected by Western blotting with ADAMTS-2 N- and C-terminal antibodies.  The N-terminal antibody detected several faint bands present in the starting material and through the first two steps of the purification, but no N-terminal immunoreactivity was present after elution from Con-A Sepharose (data not shown).  The C-terminal antibody detected a band of approximately 125 kDa in the initial extract as well as several smaller bands, but only the 125 kDa product remained after chromatography on Con-A Sepharose (Fig. 3.6A).  This is consistent with proteolytic processing of the enzyme to yield processed fragments with differing binding activities.  Fractions 11-14 were pooled and dialyzed, then loaded onto a heparin-Sepharose column and eluted with a gradient to 1 M KCl giving a greatly increased concentration of the 125 kDa product (Fig. 3.6B). Fractions 7-10 from heparin-Sepharose were pooled and dialyzed for further experiments.  Based on SDS-PAGE gel silver staining (Fig. 3.6C), I estimate the concentration of enzyme purified to be 100 ng/ml or 800 ρM.  The product purified is consistent with the molecular weight determined in the original publication which showed the enzyme was catalytically active towards α-I-(I) procollagen N-propeptides (Colige et al, 1995) and other recent work showing the activity of the 125 kDa enzyme towards α-I-(III) procollagen N-propeptides (Wang et al, 2003).   125 240 120 80 kDa Figure 3.6.  Purifi cation of bovine ADAMTS-2 from fetal calf skin.  Following the previously published protocol (Colige et al, 1995) bovine ADAMTS-2 was purifi ed from fetal calf skin.  Sam- ples were analyzed by SDS PAGE (3-8% gradient) and Western blotting with anti-ADAMTS-2 C-terminus antibody.  A, Extraction with potassium chloride, ammonium sulfate precipitation and Con A-Sepharose purifi cation.  Lane 1, initial homogenate in wash buffer after centrifugation.  Lane 2, second wash.  Lane 3, centrifuge pellet after two extraction cycles.  Lane 4, supernatant from ammonium sulfate precipitation of pooled extracts.  Lane 5, re-dissolved ammonium sulfate precip- itate.  Lane 6, centrifugation pellet.  Lane 7, centrifugation supernatant/column start material.  Lane 8, sample fl ow through not bound to Con A-Sepharose column.  Lane 9, column wash.  Lanes 10-14, column fractions eluted with column buffer containing 0 to 0.5 M α-methyl-D-mannoside and observed at 280 nm absorption.  B, Purifi cation of pooled ADAMTS-2 reactive fractions (lanes 11- 14 above) over heparin-Sepharose.  Lane 1, starting material, pooled lanes 11-14 from Con A-Sep- harose elution.  Lane 2, sample fl ow through not bound to column.  Lane 3, column wash.  Lanes 4-13, fractions of gradient elution from 0 to 1 M KCl.  Fractions 7 through 10 were pooled, dialyzed and stored for subsequent experiments.  C, Silver stain equivalent to B, above.  ADAMTS-2 in pooled fractions enclosed in white box.  Results shown are representative of one experiment analyzed in triplicate. A. 1  2  3  4   5  6  7  8  9  10 11 12 13 14 kDa B. 240 120 80 1   2   3   4  5   6   7   8  9  10 11 12 13 1   2   3   4  5   6   7   8  9  10 11 12 13 120 240 80 40 kDa C. 126 3.4.8.  ADAMTS-2 degrades versican I observed early in these studies that versican purified at pH 7.5 was labile and hypothesized that this could be attributable to its co-purification with ADAMTS-2.  A method to isolate versican that was free of ADAMTS-2 was established (see above). To investigate whether ADAMTS-2 might be catalytically active towards versican, the two purified proteins were incubated at 37°C at a range of ratios and times.  Optimal detection of proteolytic fragments was found to occur with bovine ADAMTS-2 and human versican at an enzyme to substrate ratio of 1:1400.  ADAMTS-2 was found to cleave versican, producing C- and N-terminal fragments (Figs. 3.7A & B respectively). C-terminal cleavage of versican produced a number of high molecular weight products, a prominent doublet close to 50 kDa and another band close to 40 kDa (Fig. 3.7A).  N- terminal cleavage resulted in high molecular weight products and two bands at approximately 80 and 70 kDa (Fig. 3.7B).  A 70 kDa versican fragment produced by the action of ADAMTS-1 and -4 has previously been described and characterized with an antibody to the DPEAAE neo-epitope (Sandy et al, 2001).  To determine whether ADAMTS-2 cleavage of versican generated this same neo-epitope, I analyzed the same samples by Western blotting with the DPEAAE antibody but observed no immunoreactivity in triplicate experiments (data not shown).  Therefore, I concluded that the N- and C-terminal cleavages described here have not been previously characterized.  For both N- and C-terminal versican antibodies, an increased versican immunoreactivity was detectable in the stacking gel after enzymatic cleavage.  Since versican is a high molecular weight (~1.5 MDa) proteoglycan which aggregates to form multimers with hyaluronan, SDS-PAGE does not adequately resolve the high molecular weight aggregates and immunoblotting to a membrane may also be only poorly effective to transfer the aggregate.  Therefore, an increase in versican immunoreactivity 127 Figure 3.7.  ADAMTS-2 degrades versican in vitro.  A, purifi ed human versican (0.56 µM) was incubated alone or with purifi ed bovine ADAMTS-2 (0.4 nM) for 0 to 48 hours at 37°C as indicated, then separated by SDS PAGE (4/12%) followed by Western blotting with 2B1 anti- body.  B, purifi ed human versican (0.56 µM) alone, purifi ed bovine ADAMTS-2 (0.4 nM) alone, or versican and ADAMTS-2 together, were incubated for 24 hours at 37°C, then separated by SDS PAGE (4/10%) followed by Western blotting with 12C5 antibody.  Results shown are repre- sentative of three experiments, each analyzed in triplicate. A. B. Versican C-Terminus Versican N-Terminus 220 80 40 kDa 30 70 120 55 kDa 460 1 2 3 4 5 6 Versican + + + + + + ADAMTS-2 - - + + + + Time 37o (h) 0 48 0.5 4 24 48 1 2 3 Versican + - + ADAMTS-2 - + + Time 37o (h) 24 24 24 128 in the stacking gel may be indicative of proteolytic processing of the core protein which results in disaggregation and altered protein behavior in SDS-PAGE separation and Western blotting.  Interestingly, purified versican alone does undergo some degradation and production of a 50 kDa doublet (Fig. 3.7A lane 2).  This proteolytic fragment is of a similar size to that produced by purified bovine ADAMTS-2 in vitro, though ADAMTS-2 is not detectable in this preparation by Western blotting (Fig. 3.8 lanes 1 & 2).  3.4.9.  Versican inhibits autodegradation of ADAMTS-2 I found that ADAMTS-2 when incubated alone underwent autocatalytic processing which degraded the C-terminal epitope within 48 hours (Fig. 3.8A lanes 3-6).  I also observed that incubation of ADAMTS-2 with versican resulted in preserved immunoreactivity of the 125 kDa band after 48 hours (Fig. 3.8A lanes 7-10).  This experiment was performed in triplicate and a representative experiment is shown.  To investigate the specificity of versican for inhibition of ADAMTS-2 autocatalytic degradation, I incubated ADAMTS-2 with 5 different glycosaminoglycans.  After 24 hours with no glycosaminoglycan added, ADAMTS-2 autodegradation was evident (Fig. 3.8B) compared with 0 hours incubation (Fig. 3.8A lanes 3 & 5).  Addition of hyaluronan had no effect and chondroitin sulfate B (dermatan sulfate) and C (chondroitin-6-sulfate) resulted in mild inhibition of ADAMTS-2 autodegradation.  Chondroitin sulfate A (chondroitin-4-sulfate) provided stronger inhibition and heparan sulfate provided the strongest inhibition of autodegradation.  Glycosaminoglycan concentrations up to 5 µM had no further effect (data not shown). As glycosaminoglycans were able to inhibit ADAMTS-2 auto-degradation, this result suggested that versican inhibition of ADAMTS-2 was through glycosaminoglycan binding rather than the availability of a preferred substrate such as versican.  Thus, 129 Figure 3.8.  Versican inhibits ADAMTS-2 autodegradation.  A, Purifi ed human versican (0.56 µM) alone, purifi ed bovine ADAMTS-2 (0.4 nM) alone, or versican and ADAMTS-2 together were incubated for 0 to 48 hours at 37°C as indicated, then separated by SDS PAGE (3-8% gradient).  B, Purifi ed bovine ADAMTS-2 (0.4 nM) was incubated for 24 hours at 37°C, alone in TBS or with the following glycosaminoglycans in TBS (5 or 50 nM fi nal concentrations): hyaluro- nan (HA), chondroitin sulfate A (chondroitin-4-sulfate)(CS-A), chondroitin sulfate B (dermatan sulfate)(CS-B), chondroitin sulfate C (chondroitin-6-sulfate)(CS-C) and heparan sulfate (HS). Detection was by Western blotting with anti-ADAMTS-2 C-terminus antibody.  Results shown are representative of three experiments, each analyzed in triplicate. 170 120 80 kDa 1 2 3 4 5 6 7 8 9 10 Versican + + - - - - + + + + ADAMTS-2 - - + + + + + + + + Time 37o (h) 0 48 0 48 0 48 0 .5 24 48 HA CS-A CS-B CS-C HS (n M) 0 10 10 0 0 10 10 0 0 10 10 0 0 10 10 0 0 10 10 0 A. B. 130 physiological binding of ADAMTS-2 to versican may inhibit autocatalytic degradation of ADAMTS-2 resulting in preserved enzymatic activity.  C-terminal processing of ADAMTS enzymes has been to shown to occur through autocatalytic processing as well as processing by other proteases, resulting in altered substrate specificity and catalytic efficiency of the enzymes (Colige et al, 2004; Colige et al, 2005; Flannery et al, 2002; Gao et al, 2004; Somerville et al, 2003; Tortorella et al, 2005).  Further work will be needed to clarify the nature of the autocatalytic degradation observed for ADAMTS-2 and its influence on enzyme activity.  3.5.  Discussion The chondroitin sulfate proteoglycan versican is a pericellular matrix molecule that is important in development of the skeleton and cardiovascular system.  Versican is believed to influence cell behavior through creation of a pericellular matrix that is permissive for migration and proliferation of distinct cell types.  Versican also binds a number of structural macromolecules and bio-active molecules (Wight, 2002).  It was previously shown that versican is expressed in remodeling processes in the most prevalent forms of human lung fibrosis, including those associated with non- granulomatous inflammation: Organizing diffuse alveolar damage in patients with adult respiratory distress syndrome; Usual interstitial pneumonia and idiopathic BOOP (Bensadoun et al, 1996).  Versican is also expressed in association with fibroblasts in granulomatous forms of lung fibrosis including the lesions of tuberculosis, sarcoidosis and extrinsic allergic alveolitis.  In all of these forms of human lung fibrosis, versican is found in association with migratory, proliferating alpha actin-positive fibroblasts (Bensadoun et al, 1997).  These are the cells that synthesize type I procollagen and thus are considered to be primarily responsible for the synthesis of new collagenous 131 matrix (Kuhn & McDonald, 1991).  As collagen fibrillogenesis takes place in a versican- rich provisional matrix and as this matrix is apparently resorbed concomitant with collagen deposition, it was hypothesized that a type I collagen-processing enzyme might contribute to versican degradation. Immunohistochemical staining showed that the procollagen N-propeptidase ADAMTS-2 co-localizes with versican in normal human lungs.  Both the proteoglycan and the enzyme are predominantly associated with smooth muscle in normal human lung tissue.  Consistent with a functional association between versican and ADAMTS-2, ADAMTS-2 was released from normal tissue by incubation of tissue with chondroitinase ABC.  This suggests that ADAMTS-2 is bound to chondroitin sulfate proteoglycans, including versican, in normal human lung tissue.  Two pathologic patterns of idiopathic human lung fibrosis were studied in the current study.  In remodeling lung tissue, ADAMTS-2 was found in association with the fibroblasts of the subepithelial fibroblast foci and in association with the fibroblasts of the intraluminal buds, in the pathologic patterns usual interstitial pneumonia and in bronchiolitis obliterans organizing pneumonia respectively.  Staining for ADAMTS-2 in the remodeling lesions of UIP was consistently stronger than those of BOOP.  It is of interest that BOOP is characterized by even-aged lesions that can resolve spontaneously, or following corticosteroid therapy.  In contrast, UIP is associated with a higher rate of morbidity and is less responsive to therapy. Versican is clearly a major component of the matrix of these remodeling lesions. Consistent with a functional interaction between ADAMTS-2 and versican, the two molecules were found to be bound together in human fetal lung fibroblast culture media, in a form that was stable to 7M Urea.  I observed that versican preparations were labile, and that spontaneous degradation could be inhibited by procedures that 132 separated versican from contaminating ADAMTS-2.  Purified versican was shown to be a substrate for purified bovine ADAMTS-2 in vitro and versican was susceptible to cleavage at a number of sites.  Though some degradation products appeared similar to those found naturally in versican preparations, none of the products of ADAMTS-2 activity appeared to correspond to the products of degradation previously shown to arise from ADAMTS-1 and -4 (Sandy et al, 2001).  These studies showed ADAMTS-2 to be susceptible to auto-degradation and that addition of versican appeared to protect ADAMTS-2 from auto-degradation more potently than addition of glycosaminoglycans. If this occurs in vivo, versican may prolong the biological half-life of this procollagen N- propeptidase. ADAMTS-2, -3 and -14 are procollagen N-propeptidases with a known substrate profile limited to the amino-propeptides of types I, II, III & V procollagens.  This work is apparently the first report of degradation of a non-collagenous substrate by a member of this clade of enzymes. Recent work indicates that multiple processing events may alter catalytic activity and substrate specificity of ADAMTS-2 (Colige et al, 2004; Colige et al, 2005).  ADAMTS-2 expression seems disproportionate to apparent rates of collagen biosynthesis in several tissues (Colige et al, 1997).  Based on homologies to other proteins that are critical in development and cell signaling, it has been suggested that ADAMTS-2 likely plays important roles in development and cell signaling independent of its role in procollagen processing (Prockop et al, 1998). In contrast to the procollagen N-propeptidases, ADAMTS-1, -4, & -9 have been shown to cleave versican (Jonsson-Rylander et al, 2005; Sandy et al, 2001; Somerville et al, 2003; Westling et al, 2004).  ADAMTS-1, -4, -9, and by homology -5, -8, -15 & - 20, are considered to form a group of ‘hyalectanases’ that process hyalectans 133 (hyaluronan-binding proteoglycans including aggrecan and versican) and are evolutionarily distinct from the other ADAMTS enzymes (Apte, 2004).  Defects in the ADAMTS-2 gene result in the autosomal recessive connective tissue disorder Ehlers-Danlos (EDS) syndrome type VIIC, characterized by extreme skin fragility and joint laxity (Colige et al, 1999).  Collagen fibrils in the skin of these patients retain their N-propeptides and are thin, branched and irregular, appearing ‘hieroglyphic’ in cross section (Nusgens et al, 1992).  ADAMTS-2 null mice develop similar skin fragility and irregular collagen fibril formation, but they also exhibit decreased spermatogenesis and are sterile (Li et al, 2001).  The ADAMTS-2 knockout mouse shows altered lung architecture with a decrease in lung surface area due to enlarged distal airspaces.  This appears to result from disordered lung development (Le Goff et al, 2006). Our data suggest that versican binding of ADAMTS-2 may regulate collagen assembly and that ADAMTS-2-mediated degradation of versican may be important in resorption of the provisional matrix following inflammation.  ADAMTS-2 binding by versican is potentially important in the fibroproliferative lesions of human pulmonary fibrosis; this may also explain our observation that collagen biosynthesis occurred in cells surrounded by versican, and that versican is essentially absent, as is type 1 collagen synthesis, from areas of mature collagenous fibrosis. 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Maurice, Reinhild Kappelhoff, Alain Doucet, Christopher M. Overall and Clive R. Roberts.  Versican degradation at the cell surface by MMP- 2 and MT1-MMP.  CHAPTER 4 – VERSICAN DEGRADATION AT THE CELL SURFACE BY MMP-2 AND MT1-MMP* 4.1.  Summary  Versican is a pericellular proteoglycan that is associated with proliferating mesenchymal cells in development and a number of disease processes that involve tissue remodeling.  In normal wound healing, versican is an abundant element of the provisional matrix in which fibroblast cell proliferation occurs.  The provisional matrix is apparently degraded, concomitant with fibroblast apoptosis and a return to normal tissue architecture.  The persistence of the versican-rich matrix is seen in pulmonary fibrosis and atherosclerosis; and in the pericellular matrix of many cancers.  The process of versican degradation is poorly understood, but as versican is a pericellular molecule, physiological degradation likely involves cell surface proteolysis.  Using concanavalin A (ConA) induction of fibroblasts as a model of matrix degradation I found that both pericellular versican and secreted versican were degraded concomitant with ConA induced fibroblast cell apoptosis.  MT1-MMP and MMP-2 were apparently mobilized to the cell surface and MMP-2 was activated on ConA treatment.  Microarray analysis was used to investigate expression of possible versican-degrading enzymes and their inhibitors, expressed in response to ConA.  Metalloproteinase inhibitors prinomastat (AG3340) and GM6001 (Ilomastat/Galardin) impeded versican degradation, filamentous actin degradation and MMP-2 activation to a greater degree than an inhibitor of apoptosis, Z-VAD-FMK.  Recombinant MMP-2 and MT1-MMP were capable of degrading versican in vitro and MT1-MMP exhibited a more subtle activity against versican within the C-terminal domain.  These data suggest that resolution of 141 the versican-rich provisional matrix in tissue remodeling occurs through MMP-2 and MT1-MMP dependent proteolysis.  4.2.  Introduction Versican is a large proteoglycan of the pericellular matrix surrounding fibroblast cells.  It is highly expressed in tissue remodeling events of development, wound healing and cancer.  In development, versican is crucial for formation of a hydrated extracellular space in which cell migration and differentiation can occur.  Deletion of the versican gene prevents successful formation of the endocardial cushion swellings necessary for proper segmentation of the mouse heart and is lethal in development (Mjaatvedt et al, 1998). Versican is a member of the ‘hyalectan’ family of hyaluronan- binding proteoglycans with lectin-like domains (Iozzo, 1998).  In normal wound healing, versican is expressed as part of a provisional matrix in which collagen synthesis and wound contraction occur, followed by degradation of the versican-rich matrix and a return to normal tissue architecture.  High levels of versican in the provisional matrix are associated with all the major forms of pulmonary fibrosis arising from granulomatous and non-granulomatous processes (Bensadoun et al, 1996; Bensadoun et al, 1997).  Similarly, versican deposition is associated with lesion severity in atherosclerosis and restenosis (Evanko et al, 1998; Wight et al, 1997). Versican is highly expressed in numerous cancers and is associated with poor prognosis in oral, breast, prostate, cervical, endometrial and cutaneous cancers (Kodama et al, 2007a; Kodama et al, 2007b; Pukkila et al, 2007; Ricciardelli et al, 2002; Ricciardelli et al, 1998; Suwiwat et al, 2004; Touab et al, 2003; Touab et al, 2002).  Versican is a multifunctional regulator of the pericellular matrix that alters the physical and chemical properties surrounding fibroblast cells.  By virtue of its size and 142 glycosylation, high levels of versican may create substantial steric resistance to certain binding interactions and thus regulate cell signaling and survival (Roberts, 2003).  Normal wound healing requires the regulated deposition of collagen to restore tissue architecture.  Excess deposition of collagen is associated with fibrosis.  In the resolution of normal wound healing, myofibroblasts disappear by apoptosis (Desmouliere et al, 1995).  The persistence of the myofibroblasts appears to be critical to sustaining fibrogenesis and may be related to the persistence of the versican-rich pericellular matrix.  Characterization of normal and dysregulated versican degradation is therefore critical to understanding aberrant events that contribute to fibroproliferative remodeling in pulmonary fibrosis, atherosclerosis, and cancer stroma.  As regulated ECM remodeling often occurs through cell surface focalized proteolysis (Basbaum & Werb, 1996), versican turnover may involve cell surface-associated proteolysis.  MMP-2, TIMP-2 and MT1-MMP form a tightly regulated cell surface proteolytic complex (Overall et al, 2000; Strongin et al, 1995; Will et al, 1996).  Under normal conditions MMP-2 and MT1-MMP are synthesized by fibroblasts but remain predominantly in their inactive zymogen form.  During remodeling, MMP-2 and MT1- MMP are activated and participate in the regulated proteolysis of the ECM.  MMP-2 and MT1-MMP are expressed at high levels in resolving granulation tissue (Madlener et al, 1998) and in idiopathic pulmonary fibrosis (Garcia-Alvarez et al, 2006; Selman et al, 2000).  MMP-2 is activated in tumor cells and appears to be involved in tumor invasion and metastasis (Brown et al, 1993a; Brown et al, 1993b).  MT1-MMP is expressed on the surface of invasive tumor cells  (Sato et al, 1994), is correlated with invasiveness (Gilles et al, 1996; Nakamura et al, 1999; Ueno et al, 1997; Yamamoto et al, 1996) and is critical in cancer cell migration (Sabeh et al, 2004). 143  Concanavalin-A (ConA) is a lectin from the jack bean plant known to induce fibroblast cell apoptosis, inducing synthesis of metalloproteinases and a matrix degradative phenotype (Overall & Sodek, 1990).  ConA binds cell surface α-D-glucose and α-D-mannose residues (Smith & Goldstein, 1967) causing receptor clustering and cell agglutination (Kulkarni & McCulloch, 1995).  ConA induces three modes of protease up-regulation including the rapid activation of  MT1-MMP from intracellular compartments (Jiang et al, 2001) and corresponding MMP-2 activation (Zucker et al, 2002).  Upregulation of MT1-MMP occurs post-translationally (Yu et al, 1995) and MMP-2 and MT1-MMP are also transcriptionally up-regulated (Overall & Sodek, 1990; Zucker et al, 2002).  As the provisional matrix in normal wound healing is degraded concomitant with fibroblast cell apoptosis, ConA stimulation of fibroblast cells offers a tissue culture model of matrix degradation that may mimic certain physiological events relevant to the proper resolution of wound healing.  Here I show that ConA treatment of fibroblasts induces versican degradation, concomitant with up-regulation of MMP-2 and MT1-MMP.  I show that versican degradation is reduced by metalloproteinase inhibitors with a corresponding inhibition of morphological events of apoptosis.  MMP-2 and MT1-MMP are shown to cleave versican in vitro.  I suggest that MMP-2 and MT1-MMP may be critical proteases involved in resolution of the versican-rich matrix in tissue remodeling.  4.3.  Experimental procedures 4.3.1.  Tissue culture Human fetal lung fibroblasts (HFL-1) were obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 20 mM HEPES and 10% (v/v) Cosmic Calf Serum (Hyclone, Logan, 144 UT).  Cells were seeded at 2 x 104 cells per ml and grown to approximately 70% confluence in Lab-Tek II chamber slides (Nunc, Rochester, NY), incubated one hour in serum free medium before addition of ConA diluted in serum free medium (Sigma, St. Louis, MO).  Inhibitors were diluted in serum free medium and added for one hour prior to ConA treatment.  The following inhibitors were used: Z-FA-FMK (Carboxybenzyl- phenylalanine, alanine, fluoromethylketone)(Calbiochem, San Diego, CA), Z-VAD-FMK (Carboxybenzyl-valine, alanine, aspartate, O-methylated fluoromethylketone) (Calbiochem), GM6001 (Ilomastat/Galardin) (Calbiochem), prinomastat (AG3340) (Agouron Pharmaceuticals, Inc., a Pfizer Company, La Jolla, CA).  Versican released into conditioned medium was concentrated by a factor of 10 using Amicon centrifugal filter units (Millipore, Billerica, MA) and retentate was treated with 0.5 U/ml chondroitinase ABC (Sigma) at 37°C for 40 minutes before electrophoresis.  4.3.2.  Immunofluorescence staining and microscopy Treated cells were fixed for 10-15 minutes with 4 % para-formaldehyde in phosphate buffered saline (PBS), pH 7.5, then rinsed with PBS.  Cells were permeabilized in TBS- triton (20 mM Tris, pH 7.5, 0.9 % NaCl, 0.2 % triton X-100) with 2% (w/v) bovine serum albumin (BSA).  Blocking was performed with 5 % (v/v) normal goat serum and 2% (w/v) BSA in TBS-triton.  Washing was performed in 0.2% (w/v) BSA in TBS-triton and antibodies were diluted in the same buffer.  The following primary antibodies were used: mouse monoclonal anti-versican C-terminal domain, 2B1 (Isogai et al, 2002; Isogai et al, 1996) (Seikagaku, Tokyo, Japan), dilution 1:500; rabbit polyclonal anti- MMP-2 C-domain, α72Ex12 (Overall et al, 1999), dilution 1:400; and mouse monoclonal anti-MT1-MMP catalytic domain, clone 5H2 (R&D Systems, Minneapolis, MN), dilution 1:400.  Alexa Fluor 594 goat anti—rabbit IgG and goat anti—mouse IgG, 145 highly cross-adsorbed secondary antibodies were used (Molecular Probes, Eugene, OR).  Counterstaining for F-actin was with Alexa Fluor 488 phalloidin stain (Molecular Probes) and nuclear counterstaining was with Hoescht 33342 (Molecular Probes). Stained cells were mounted under coverslips with Prolong Gold antifade reagent (Molecular Probes) and stored at -20°C.  Microscopy was performed on a Leica DMRA2 automated microscope (Leica Microsystems GmbH, Wetzlar).  In the antibody labeled channel, three-dimensional images were acquired and image stacks were deconvolved using the Nearest Neighbour Deconvolution algorithm (Improvision, Coventry, UK).  Exposure and deconvolution settings were identical for each fluorescent channel in each experiment, throughout the different concentrations of ConA.  4.3.3.  SDS-PAGE Samples in non-reducing sample buffer (125 mM Tris-HCL, pH 6.8, 2.0% SDS, 2.0 M urea, 0.05% bromophenol blue) were separated on discontinuous SDS-PAGE gels with 4% (stacking) and 10% (separating) acrylamide.  Stacking and separating gels were kept during Western blotting to monitor high molecular weight versican aggregates within the stacking gel.  For some experiments, 4-15% Tris-HCl precast gradient gels were used (Bio-Rad, Hercules, CA).  Relative molecular mass (measured in kDa) was estimated based on mobility of molecular weight markers: HiMark Prestained (Invitrogen), MagicMark XP (Invitrogen) and Kaleidoscope Prestained (Bio- Rad).  Western blotting was performed as previously described (Maurice & Roberts, 2008).   146 4.3.4.  Gelatin zymography SDS-PAGE gels were cast as above with the addition of 0.1% gelatin to the 10% acrylamide, separating portion of the gel.  Gels were incubated in exchange buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM CaCl2, 2.5 % Triton X-100) for one hour then washed three times in incubation buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM CaCl2) and incubated 16 hours in the same buffer at 37°C.  Staining and destaining was performed with Coomassie Blue R-250 following standard protocol. The following antibodies were used: mouse monoclonal anti-versican N-terminal antibody 12C5 (Asher et al, 1991)(obtained from the Developmental Studies Hybridoma Bank (NICHD), the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242), 1:500 dilution; anti-versican C-terminal domain 2B1, 1:1000 dilution; rabbit polyclonal anti-versican C-terminal domain recombinant construct LC2 (Pourmalek & Roberts, 2009), 1:10 000 dilution; and rabbit polyclonal anti-PG40 (Brennan et al, 1984; Krusius & Ruoslahti, 1986), 1:500 dilution.  4.3.5.  RNA preparation  Following incubations with or without ConA, RNA was prepared and labeled as described (Kappelhoff & Overall, 2007).  Cells were washed three times with phosphate-buffered saline (PBS) to remove all serum components.  RNA was extracted, purified and homogenized with RNeasy Mini Kits and QIAshredder spin columns (Qiagen, Hilden, Germany) according to the manufacturer's instructions.  RNA integrity was determined by running samples on a 1.2% native agarose gel and inspecting for distinct 18S and 28S ribosomal RNA bands at an approximate 2:1 ratio. RNA quantity was determined by A260 measurement.  Samples were maintained at 4°C 147 and used immediately or frozen at –80°C until use.  RNA was reverse transcribed and purified using MessageAmp II aRNA amplification kit (Ambion, Austin, TX) according to the manufacturer's instructions.  Amplified RNA (aRNA) was fluorescently labelled with the universal linkage system (Kreatech Diagnostics, Amsterdam, Netherlands) according to the manufacturer's instructions.  Control and test samples were separately labeled with Cy3- or Cy5-ULS and then purified.  Labeling efficiency was monitored at A260 and A550 (Cy3) or A260 and A650 (Cy5) using the NanoDrop spectrophotometer microarray measurement tool to ensure successful labeling (Thermo Fisher Scientific, Waltham, MA).  All procedures were performed in an RNase-free environment.  4.3.6.  Microarrays  The CLIP-CHIP aminosilane microarray slides contain 70-mer oligonucleotides to all human protease, inhibitor and homologue genes (http://www.clip.ubc.ca)(Kappelhoff & Overall, 2007).  Microarray slides were prehybridized and oligonucleotides denatured as described (Kappelhoff & Overall, 2007) before hybridizing with Cy3- and Cy5- labeled aRNA.  After hybridization, slides were washed under stringent conditions before scanning with an Affymetrix 428 microarray laser scanner (Affymetrix, Santa Clara, CA) using 532 nm for Cy3 and 635 nm for Cy5.  Three biological replicates were performed and analyzed on separate slides containing duplicate arrays for a total of six arrays.  Image files were acquired in Imagene 6.1 (Biodiscovery, El Segundo, CA). Data were normalized in CARMAweb using 'normexp' for background correction, print tip loess for within array normalization and the quantile algorithm for normalization between arrays (https://carmaweb.genome.tugraz.at/carma/).  Two dimensional SAM (significance analysis of microarrays) analysis with a 1.5 fold cutoff and paired T-tests 148 were performed in TM4 MultiExperiment Viewer (Saeed et al, 2003) (http://www.tm4.org/mev.html).  4.3.7.  Enzyme assays  Versican was purified from HFL-1 cells at approximately 70% of confluence. Cultures were rinsed in serum-free media followed by incubation in serum-free media for 24 hours.  Serum-free CM were collected and centrifuged at 1500 x g for 20 minutes to remove cellular debris.  Urea was added to 7 M, pH 6.0 and 150 mM NaCl was added to bring the salt concentration to 400 mM before loading onto Q-Sepharose Fast Flow ion exchange resin (Amersham Biosciences, Piscataway, NJ) at approximately 1 litre CM per 5 mls resin.  The column was equilibrated in 0.1 M sodium acetate, pH 6.0, 7 M urea, 0.4 M NaCl and eluted with 0.1 M sodium acetate, pH 6.0, 7 M urea, 1.5 M NaCl.  Versican reactive fractions were pooled and purified a second time on a smaller volume column with the same protocol.  Concentration of purified versican was estimated using the dimethylmethylene blue (DMMB)(Serva, Heidelberg) assay to quantify sulfated glycosaminoglycans (Farndale et al, 1986), using known concentrations of chondroitin sulfate A as standards (Seikagaku).  Recombinant human MMP-2 was expressed in mammalian cell cultures as previously described (Bigg et al, 2001).  Soluble human MT1-MMP lacking the transmembrane and cytoplasmic tail was expressed and purified as described (Tam et al, 2004).  His-tagged recombinant versican C-terminal ‘G3’ domain construct was expressed in Escherichia coli, purified, refolded and verified by fluorescence anisotropy spectroscopy, N-terminal sequencing and mass spectrometry (Pourmalek & Roberts, 2009).  Purified versican or versican G3 domain constructs were incubated alone in 149 enzyme buffer or incubated with recombinant MMP-2 or MT1-MMP for 0 or 24 hours at 37°C.  4.4.  Results 4.4.1.  ConA induces degradation of versican. As ConA is known to induce a matrix degradative phenotype accompanying an apoptotic response in fibroblast cells (Overall & Sodek, 1990), I investigated whether versican was degraded in response to ConA.  Under normal conditions, versican stains strongly in the pericellular matrix of human fetal lung (HFL-1) fibroblasts (Fig. 4.1A).  F- actin staining is strong and organized into elongated stress fibers.  Nuclei are mostly oval shaped with a regular granular pattern of staining.  In the presence of 40 µg/ml ConA for 16 hours, versican staining is dramatically reduced, f-actin staining is reduced, actin stress fibers are lost and nuclei are irregular, condensed and fragmented.  In addition to maintaining cell-associated versican, HFL-1 fibroblasts also secrete versican into the media (Fig. 4.1B).  In response to increasing concentrations of ConA, reactivity to versican 12C5 antibody (N-terminal domains) is lost from the media. These data indicate that both pericellular and secreted versican are degraded concomitant with apoptosis in the matrix degradative phenotype induced by ConA in HFL-1 fibroblasts.  4.4.2.  ConA induces changes in MMP-2 and MT1-MMP localization. MMP-2 and MT1-MMP immunofluorescent staining increased dramatically in response to ConA (Fig. 4.2).  MT1-MMP consistently stained very strongly at 4 hours (Fig. 4.2D) followed by a decrease at 16 hours (Fig. 4.2F).  This was correlated with moderate staining for MMP-2 at 4 hours (Fig. 4.2C) which increased at 16 hours (Fig. 150 kDa Figure 4.1.  Concanavalin-A induces degradation of versican concomitant with fi broblast cell apoptosis.  A. Human fetal lung fi broblasts (HFL-1) at sub-confl uence were incubated with or without 40 μg/ml ConA in serum free media for 16 hours.  DNA, F-actin and versican (using the antibody 2B1) were visualized by fl uorescence microscopy.  Nuclear condensation and fragmenta- tion, loss of organized actin stress fi bers and substantial loss of versican staining are apparent in response to ConA.  Exposure and deconvolution settings were identical in the antibody channel at the different concentrations of ConA. Scale bars = 25 μm.  B. HFL-1 conditioned media after 16 hours treatment with 0, 10, 20 or 40 μg/ml ConA was fi lter concentrated and treated with chondroi- tinase ABC before separation by SDS-PAGE and Western blotting for versican with 12C5 antibody. Results shown are representative of three experiments, each analyzed in duplicate. A. B.   0   10   20  40 Versican F-actin DNA Merge 0 40 μg/ml ConA 470 270 120 μg/ml ConA 12C5 151 Figure 4.2.   Concanavalin-A induced changes in metalloproteinase expression and local- ization.  Immunofl uorescent staining of HFL-1 cells for MMP-2 (A, C, E) and MT1-MMP (B, D, F). Cells were treated for 0 (A & B), 4 (C & D) or 16 hours (E & F) with 40 μg/ml ConA before fi xing and staining. Exposure and deconvolution settings were identical for each antibody at the different time points.  Scale bars = 25 μm.  G. Gelatin zymogram showing MMP-2 zymogen (pro-), interme- diate and active forms in HFL-1 conditioned media over time in the presence of 40 μg/ml ConA. Results shown are representative of three experiments, each analyzed in duplicate. Pro Intermediate Active   0   1    2    4    8  12  16  20  24   G. hours + ConA A B C D FE MMP-2 MT1-MMP 152 4.2E).  Both transcriptional and post-translational up-regulation of MMP-2 and MT1- MMP likely contributed to the observed staining (Overall & Sodek, 1990; Yu et al, 1995; Zucker et al, 2002).  Concomitant with this activation and the ensuing fibroblast cell apoptosis was a dramatic reduction in pericellular versican (Fig. 4.1A), suggesting that these enzymes could play a role in degrading versican in ConA stimulated HFL-1 cells. Consistent with the immunofluorescent staining observed, secreted MMP-2 was observed in its active form in the culture media, detected by gelatin zymography (Fig. 4.2G).  4.4.3.  Microarrays  Based on the observations of rapid cell surface presentation of MT1-MMP and activation of MMP-2, I next investigated corresponding gene expression changes for all MMPs using the CLIP-CHIP complete human protease, inhibitor and homologue oligonucleotide microarray.  It is well known that a net proteolytic phenotype is induced by ConA (Overall & Sodek, 1990).  Stringent hybridization and wash conditions have been previously established to provide optimum specificity of hybridization and minimal background noise (Kappelhoff & Overall, 2007).  Microarray spots intensities were corrected against the background, normalized within each array and normalized between arrays before analysis.  Three biological replicates were employed with two arrays per chip, giving 6 total arrays per condition. Two dimensional SAM (significance analysis of microarrays) analysis was performed according to Tusher et al (Tusher et al, 2001) using a 1.5 fold change cutoff and p- values were obtained by paired students T-test.  MMP-11 was found to exhibit the highest expression change in response to ConA.  This was followed by increased expression for MMP-1 and MT1-MMP respectively (Fig. 4.3).  In addition to MMP-11, 153 Figure 4.3.  Microarray analysis of differentially expressed fi broblast proteases and inhibitors in response to Concanavalin-A.  HFL-1 cells were treated with 40 μg/ml ConA (Cy5) or control serum free media (Cy3) for 4 or 16 hours.  Normalized data were ana- lyzed by two dimensional SAM analysis and genes with signifi cant expression changes are shown.  Fold change of expression between 4 and 16 hour timepoints is shown.  P-values were determined with a paired T-test.  All 8 tubulin positive control spots showed signifi cantly altered expression.  Results shown represent three biological replicates each performed on duplicate arrays. RefSeq Abbreviation Description Fold change p-value NM_000301 PLG plasminogen 1.809249 0.041620 NM_012413 QPCT glutaminyl cyclase 2.029997 0.020802 XM_166659 HSHIN4 Hin-4 1.621677 0.006825 NM_000094 COL7A1 collagen, type VII, alpha 1 1.582294 0.005767 NM_030808 NUDEL nuclear distribution element-like-oligo. 1.585110 0.012137 XM_036729 USP41 USP41 1.706400 0.023639 NM_004995 MMP14 MT1-MMP 2.163458 0.018383 AY040094 HTRA3 HTRA3 1.813415 0.012818 X16323 HGF hepatocyte growth factor 1.862170 0.015145 NM_001648 KLK3 kallikrein hK3 1.598505 5.69E-04 NM_000014 A2M a-2-macroglobulin 2.729719 0.016373 NM_005025 SERPINI1 neuroserpin/PI12 1.898028 0.003110 NM_001912 CTSL cathepsin L 1.634475 0.002243 NM_000789 ACE angiotensin-converting enzyme 1 1.772955 0.002470 NM_002421 MMP1 collagenase 1 1.649192 3.23E-04 NM_005940 MMP11 stromelysin 3 3.436513 3.26E-07 NM_000930 PLAT t-plasminogen activator 2.945938 4.12E-10 NM_006350 FST follistatin 0.309850 9.03E-08 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.370580 2.90E-09 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.363009 1.39E-08 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.364405 2.44E-08 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.357775 7.04E-08 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.369174 1.96E-07 NM_007173 SPUVE umbelical vein proteinase 0.434380 2.72E-06 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.410225 2.44E-04 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.454935 8.06E-05 NM_002402 MEST mesoderm specific transcript hom. 0.386115 1.61E-04 NM_003813 ADAM21 ADAM21 0.577993 6.96E-07 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.516300 7.00E-05 NM_007262 DJ-1 DJ-1 0.645218 1.85E-06 NM_003619 PRSS12 neurotrypsin 0.516020 0.001004 XM_052597 USP53 USP53 0.566877 0.002066 NM_021114 SPINK2 serine PI Kazal type 2 0.619857 0.003004 4 hours 16 hours 154 several genes not previously associated with ConA that may have contributed to the matrix degradative phenotype included cathepsin L, plasminogen and tissue-type plasminogen activator.  MMP-2 and MMP-7 up-regulation did not meet the significance threshold employed, but they were up-regulated with p=0.014608 and 0.000648 respectively (Appendix 1).  The complete list of proteases, inhibitors, homologues and control spots printed on the chip is in Appendix 1 with significant genes highlighted and remaining genes that did not meet the significance threshold (either in fold change in expression or probability) in shades of grey. All 8 tubulin control spots were significantly down-regulated.  4.4.4.  Caspase and MMP inhibition alters the apoptotic response of fibroblast cells to ConA. To investigate the relationship between degradation of the versican-rich matrix and the intracellular events induced by ConA, I inhibited caspase and metalloproteinase activities.  With the pan-caspase inhibitor Z-VAD-FMK (200 µM), nuclei were irregular but not condensed or fragmented; some filamentous actin staining was present and there was minimal versican staining compared to the negative control caspase inhibitor Z-FA-FMK (Fig. 4.4A). Inhibition with GM6001 (20 µM) provided protection to filamentous actin and cell morphology; and increased protection of versican staining compared to Z-VAD-FMK.  Nuclei were partially irregular and condensed or fragmented.  Whereas GM6001 is a broad spectrum (hydroxamate-based) MMP inhibitor, prinomastat is a structure based MMP inhibitor designed to potently and selectively inhibit MMP-2, -3, -9, -13 and MT1-MMP with Ki = 0.05, 0.3, 0.26, 0.03 and 0.33 nM respectively, having weaker affinities for MMP-1 and -7 of 8.3 and 54 nM respectively (Shalinsky et al, 1999).  Prinomastat (20 µM) gave the strongest staining 155 Versican F-actin DNA Z-FA-FMK Z-VAD-FMK GM6001 Prinomastat Merge A. Figure 4.4.  Caspase and MMP inhibition alters the apoptotic response of fi broblast cells to Concanavalin-A.  A. HFL-1 cells were treated with Z-VAD-FMK poly-caspase inhibitor and Z-FA-FMK as a negative control, GM6001 broad spectrum matrix metalloproteinase inhibitor or Prinomastat MMP-2, -3, -9, -13 and MT1-MMP inhibitor, prior to treatment with 40 μg/ml ConA. Exposure and deconvolution settings were identical in the antibody channel at the different concentrations of ConA.  Scale bars = 25 μm.  B. MMP-2 activity in the presence of Z-FA-FMK, Z-VAD-FMK, GM6001 or Prinomastat.  Results shown are representative of three experiments each analyzed in duplictae. B. Pro Intermediate Active Z- FA -F M K Z- VA D -F M K G M 60 01 Pr in om as ta t 156 for versican and filamentous actin; and reduced the number of nuclei that were condensed or fragmented. Gelatin zymograms show MMP-2 in the zymogen (pro-) form, intermediate and active form with Z-FA-FMK as well as Z-VAD-FMK; whereas with GM6001 and prinomastat, MMP-2 is exclusively in the zymogen form, indicating inhibition of activation (Fig. 4.4B).  4.4.5.  MMP-2 and MT1-MMP cleave versican in vitro.  As prinomastat inhibits versican degradation subsequent to ConA stimulation most effectively and is a potent inhibitor of MMP-2 and MT1-MMP, I investigated their proteolytic capacity toward versican in vitro.  Versican incubated alone was found to produce minor auto-degradation products of 42 and 50 kDa as detected by 2B1 (Fig. 4.5A & B).  In the presence of MMP-2, versican degradation was apparent with a loss of high molecular weight protein and numerous proteolytic fragments detected, especially in the 30 – 60 kDa range as detected by 2B1, indicating cleavage of the C- terminal globular domain (Fig. 4.5A).  Versican incubated with MT1-MMP did not show any cleavage products or any loss of high molecular weight protein as detected by 2B1 (Fig. 4.5B).  However, Western blotting with the LC2 polyclonal antibody did detect a more specific cleavage, a single fragment at approximately 100 kDa.  Both 2B1 and LC2 detect epitopes in versican’s C-terminal G3 domain, therefore a difference in epitope accessibility must have been responsible for the difference in immunoreactivity. It is also evident that intact high molecular weight versican was not detected well by LC2, which was raised against a recombinant G3 domain construct containing the lectin-like and complement regulatory-like domains (Pourmalek & Roberts, 2009).  157 180 110 75 kDa Figure 4.5.  MMP-2 and MT1-MMP cleave versican in vitro.  A. Purifi ed versican (0.45 mg/ml) from HFL-1 conditioned medium was not incubated (lane 1), incubated alone (lane 2), incubated with recombinant human MMP-2 (10 μg/ml) or incubated with recombinant human MMP-2 (20 μg/ ml) at 37º C for 24 hours before SDS-PAGE separation and Western blotting with 2B1 antibody. B. The same incubation was performed without or with recombinant human MT1-MMP (7 μg/ml) and (14 μg/ml) before SDS-PAGE separation and Western blotting with anti-versican antibodies 2B1 and LC2.  Results shown are representative of three experiments, each analyzed in triplicate. A. B. 2B1 2B1 LC2 50 40 30 180 110 75 kDa 50 40 30 0    0   10   20 0    0     7   14          0    0     7   14   μg/ml MMP-2 μg/ml MT1-MMP 158 4.4.6.  MT1-MMP cleaves and disaggregates recombinant versican G3.  To further investigate the nature of versican proteolysis, a recombinant versican C- terminal G3 domain construct was incubated with MMP-2 and MT1-MMP.  The His- tagged G3 construct was determined to have the expected 37.5 kDa molecular weight by MALDI-TOF MS (data not shown).  Under denaturing but non-reducing conditions in SDS-PAGE, the constructs were detected as an aggregate at the top of the gel (Fig. 4.6 & B) consistent with the self-association that has been described for this domain (Ney et al, 2006).  When incubated with MMP-2, there was no change in the appearance of the aggregated protein and no production of proteolytic fragments detected (Fig. 4.6A).  With MT1-MMP there was an increase in immunoreactivity at the top of the gel, numerous proteolytic products detected from 60 – 180 kDa, as well as a prominent band near the 37.5 kDa monomer molecular weight (Fig. 4.6B).  These data indicate that through a cleavage event that has a limited effect on the molecular weight of the G3 domain construct, MT1-MMP is able to disaggregate the protein aggregates into monomers and several smaller multimers.  4.5.  Discussion  ConA is known to induce a matrix degrading phenotype in human fibroblasts with an increase in metalloproteinase expression and a concomitant decrease in tissue inhibitor of metalloproteinase expression (Overall & Sodek, 1990).  Since versican is degraded from the provisional matrix accompanying fibroblast apoptosis in normal tissue remodeling, it was hypothesized that ConA induced apoptosis of HFL-1 fibroblasts would provide a useful tissue culture model of versican degradation.  Indeed both pericellular and secreted versican are degraded from the matrix in this model which also stimulates expression and activation of MMP-2 and MT1-MMP.  By microarray 159 Figure 4.6.  MT1-MMP cleaves and disaggregates recombinant versican G3. A. Recombinant human versican His-G3 constructs were incubated with 0, 4, 8, 12, 24, 50, 100 µg/ml recombinant human MMP-2 for 24 hours, followed by SDS- PAGE separation and detection by Western blotting with LC2 antibody.  B. His-G3 constructs were incubated with 0, 4, 8, 12, 24, 50, 100 µg/ml recombinant human MT1-MMP for 24 hours, followed by SDS-PAGE separation and detection by West- ern blotting with LC2 antibody.  Results shown are representative of three experi- ments, each analyzed in triplicate. A.   0     4     8   12   24  50  100 180 110 75 kDa 50 40 30 μg/ml MMP-2 μg/ml MT1-MMP 180 110 75 50 40 30   0     4     8   12   24  50  100 B. kDa LC2 LC2 160 analysis I was able to confirm expression changes in MMP-1 and MT1-MMP that were consistent with previous reports (Overall & Sodek, 1990; Yu et al, 1995; Zucker et al, 2002).  Though MMP-2 up-regulation did not meet the significance threshold in SAM analysis in these experiments, immunofluorescent staining confirmed the significant mobilization of both MMP-2 and MT1-MMP at the cell surface.  This may indicate a greater effect of ConA on MMP-2 localization and activation rather than expression changes.  I observed substantial ConA induced expression of MMP-11 (stromelysin 3) that has not been previously reported.  MMP-11 has a broad normal distribution, is expressed in normal wound healing and is associated with breast, cutaneous and colorectal carcinomas (Basset et al, 1990; Porte et al, 1995; Wolf et al, 1992).  MMP-11 contributes to cancer progression and poor patient prognosis through rapid temporal expression of the active enzyme that contributes to inhibition of cancer cell apoptosis (Rio, 2005).  It would be interesting to discern the role of MMP-11 in matrix resorption in response to ConA.  Moderate expression of Cathepsin L, plasminogen and t-plasminogen activator were also observed.  Cathepsin L, along with MT1-MMP, is suggested to be a promising target for cancer treatment (Lah et al, 2006).  Cathepsin L has recently been shown to cleave extracellular endorepellin and is associated with activated caspase-3 in apoptotic endothelial cells (Cailhier et al, 2008).  Plasmin(ogen) is implicated in activating numerous MMPs (Ra & Parks, 2007) and thus along with expression of its activator (tissue-type plasminogen activator), likely contributes directly to the proteolytic phenotype.  As ConA induced apoptosis requires the sustained presence of ConA (Wang et al, 1983) I wondered whether the early and late events were separable through protease 161 inhibition.  Caspases are intracellular proteases that are required for apoptosis, regardless of apoptotic stimuli (Reed, 2000; Salvesen & Dixit, 1997).  It was predicted that caspase inhibition would alter only later stage events in ConA induced apoptosis. As expected, caspase inhibition substantially protected nuclei from condensation and fragmentation, however minimal protection from versican degradation and cytoskeletal changes was observed.  The metalloproteinase inhibitors protected versican from degradation more robustly and also limited changes to the cytoskeleton.  I have shown that inhibition of matrix degrading enzymes subsequent to ConA stimulation significantly preserves the actin cytoskeleton and cell morphology.  This appears to partially protect cells from apoptotic nuclear changes, suggesting a link between matrix degradation and apoptosis.  The concept of ECM changes affecting gene expression is termed "dynamic reciprocity" and involves ECM communication with the nucleus through membrane receptors and cytoskeletal changes, which invoke gene expression that further regulates cell morphology (Bissell et al, 1982; Nelson & Bissell, 2006).  It may be that ConA induction of matrix degradation stimulates cytoskeletal changes that in turn effect gene expression and the induction of apoptosis through mechanisms of dynamic reciprocity.  As versican is a major component of the pericellular matrix of HFL-1 cells, its degradation could contribute to cell signaling pathways leading to apoptosis.  The inhibition of versican degradation provided by prinomastat suggested a more specific inhibition of versicanase enzymes.  While MMP-1 and MMP-7 are transcriptionally up-regulated in response to ConA (Overall & Sodek, 1990), prinomastat inhibits MMP-2, -3, -9, -13 and MT1-MMP much more potently than MMP-1 or -7.  Thus, the inhibition of versican degradation and apoptotic changes observed in 162 the presence of prinomastat is likely due to the more potent and selective inhibition of MMP-2 and MT1-MMP.  MMP-2 has previously been shown to process a versican preparation (Passi et al, 1999).  With purified versican and recombinant MMPs, I was able to better characterize cleavage by MMP-2 and show that MT1-MMP also cleaves versican, apparently more specifically.  MT1-MMP was also shown to play a role in disaggregation of versican's G3 domains, the selectin-like domain that may bind a number of ECM ligands and/or an as yet undescribed cell surface receptor.  Proteolytic release of versican's G3 binding interactions could have dramatic effects on the mechanical and biochemical properties of the pericellular matrix.  Through releasing C-terminal binding interactions and maintaining the bulk of the protein intact, MT1-MMP could signal subsequent events in versican degradation and could alter the ways in which versican contributes to the steric hindrance and cytokine binding capacity of the pericellular matrix (Roberts, 2003).  Knockdown of MT1-MMP produces a more substantial phenotype than any other MMP knockdown to date.  MT1-MMP deficient mice develop numerous connective tissue defects including soft tisue fibrosis, delayed endochondral ossification and defects in angiogenesis (Holmbeck et al, 1999; Zhou et al, 2000).  These defects have been attributed to decreased collagen turnover (Holmbeck et al, 1999) and certainly altered collagen turnover contributes as MT1-MMP is critical for collagen phagocytosis (Lee et al, 2006).  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Lab Invest 82(12): 1673-1684    171 CHAPTER 5 – CONCLUDING REMARKS AND FUTURE DIRECTIONS   This thesis has focused on elucidating novel mechanisms of versican proteolysis that may be relevant to normal and pathological versican turnover.  My studies shed light on versican synthesis and processing by human fetal lung cells in culture.  I have successfully demonstrated in vitro cleavage by three cell surface-associated metalloproteinases that may be involved in versican processing in vivo.  These findings advance our understanding of versican proteolytic processing and move the field further toward a complete understanding of physiological turnover of versican in normal and pathological remodeling.  ADAMTS-2 has historically been considered highly specific based on its sensitivity to substrate conformation.  Heat denaturation of type I or II collagen ablates N-propeptide cleavage (Tuderman et al, 1978).  This finding has since been taken to imply that ADAMTS-2 processes only procollagens.  Yet an alternative explanation for the specificity of ADAMTS-2 is that the enzyme performs a quality control function, only allowing properly folded procollagens to be assembled into fibrils (Prockop et al, 1998). In addition to types I and II collagen, ADAMTS-2 is now known to also process collagen types III and V (Colige et al, 2005; Wang et al, 2003).  Based on domain composition ADAMTS-2 appears to have be designed for functions in addition to procollagen processing (Colige et al, 1997; Prockop et al, 1998).  Expression levels also suggest the enzyme may have functions other than collagen processing (Colige et al, 1997). Surely a better understanding of the activation states of the enzyme will help immeasurably in further analysis of its capacity to cleave non-collagen substrates (Colige et al, 2005). 172  While the specificity of ADAMTS-2 toward exclusively types I and II collagen was dogma in the field for over a decade, we now know that type III collagen is in fact processed by the same enzyme (Greenspan & Wang, 2005).  The procollagen C- propeptidases (bone morphogenetic protein-1 and the tolloid-like metalloproteinases) are well known to be involved in signaling events that occur in morphogenic and homeostatic events in the ECM (Ge & Greenspan, 2006).  Likewise, it was predicted that ADAMTS-2 not only processes versican but may have other actions that regulate tissue remodeling.  I have shown that versican is processed by ADAMTS-2 in vitro and provided strong evidence for a binding interaction between the two.  This work lays the ground work for future studies necessary to further characterize the pattern of co- localization and cleavage events described here.  Further work will need to confirm cleavage with a recombinant enzyme and investigate the effects of the 7 different enzyme activation states on its activity toward versican (Colige et al, 2005).  The use of ADAMTS-2 null mice could potentially be used to validate the in vivo significance of our claims but interpreting results would be a challenge.  ADAMTS-2 null mice have lungs that do not develop properly and resemble an emphysematous phenotype (Le Goff et al, 2006).  Additionally, there is not a good model for inducing chronic pulmonary fibrosis.  The most common model of pulmonary fibrosis is bleomycin induced fibrosis which denudes epithelium and induces a rapid, acute inflammatory response that is very different from human pulmonary fibrosis.  The current 'standard of care' therapy for idiopathic pulmonary fibrosis is of limited therapeutic benefit and the same is true for most forms of fibrosis.  Historically the deposition of collagen rich scar tissue was considered irreversible.  In the last decade numerous reports have shown a degree of reversibility in animal models.  However, the increased speed of disease progression in animal models may not accurately mimic the 173 human pathologies which can advance over a much more prolonged period (Friedman, 2007).  One problematic aspect of the more prolonged human disease may be the crosslinking of matrix molecules, particularly collagen, that occurs in more mature scars.  MMP-2 has previously been shown to cleave versican (Passi et al, 1999).  Thus as MT1-MMP activates MMP-2 in a cell surface activation complex (Will et al, 1996), I queried both MMP-2 and MT1-MMP cleavage of versican.  I found several detectable products of versican produced by MMP-2 and an apparently more specific cleavage by MT1-MMP.  In combination there are several newly identified products of versican that need to be further studied to determine if these products are produce in vivo and if they have physiological significance.  The singular cleavage event induced by MT1-MMP seems to affect versican's G3 domain aggregation.  If this is an in vivo role of MT1- MMP, it could have dramatic effects on the properties of the ECM.  MT1-MMP has a unique substrate binding preference from the other MMPs (Kridel et al, 2002).  It appears to play a dramatic role in cancer metastasis (Sato et al, 2005) and has developmental roles independent of MMP-2 activation (Oblander et al, 2005). MT1-MMP null mice have a more dramatic phenotype than any of the knockout mice to date and exhibit numerous skeletal abnormalities including soft connective tissue fibrosis (Holmbeck et al, 1999).  This connective tissue fibrosis is blamed on defective collagenolysis which is likely at least part of the phenotype.  But if MT1-MMP cleavage of versican is required to signal an end to the fibroproliferative phase, then MT1-MMP deficiency could lead to persistent versican and persistent collagen deposition that causes fibrosis. 174  In order to validate the in vivo significance of the in vitro proteolytic events described here, future work will need to characterize the N-terminal proteolytic sequences described here and determine the presence of these putative processing sites in vivo. Cleavage site neo-epitopes are difficult to determine with proteoglycan substrates, presumably because of interference from the glycosylations and glycosaminoglycans.  I have made numerous attempts to characterize cleavage products by Edman Degradation chemical sequencing but these attempts have been mostly frustrated by the concentration of protein transferred to sequencing membrane, the charged and glycosylated nature of versican’s side chains or possible binding interactions that are not solubilized by urea and SDS.  In order to circumvent the challenges inherent in chemical sequencing of proteoglycans, I have employed mass spectrometry based techniques to characterize versican degradation.  The results have shown some promise, but unfortunately are still confronted with difficulties that are presumably again related to the difficult nature of the protein and its substitutions.  However, with appropriate improvements in sample preparation that can accommodate this challenging substrate, it is possible that mass spectrometry will soon be able to offer great advances in proteoglycan characterization.  Mass spectrometry based proteomics is becoming a very powerful tool for biological and medical research.  As instruments and protocols improve and become more affordable, it is likely that this technology will in the future be used for clinical diagnosis. Thus, improved detection of versican is not only relevant to medical research and therapeutic avenues, but also to diagnosis and treatment.  Once pathways and consequences of versican processing are more fully elucidated, it may be possible to identify biomarkers of disease prognosis and progression, as is suggested for ECM metabolites in general (Moseley et al, 2004). 175  I have investigated degradation of versican in a model of apoptosis associated with extracellular matrix proteolysis.  While the expression of several metalloproteinases is well characterized in this model, including the putative versican-degrading enzymes MMP-2 and MT1-MMP, it is unclear what relative contribution these known enzymes make to the total matrix degradation observed.  I have attempted to investigate the complete repertoire of proteases induced by ConA in Human Fetal Lung fibroblasts. Additionally this work has identified several new candidate proteases that may be important in degrading versican in this model of apoptosis.  To date, versican cleavage has been documented by MMP-1, -2, -3, -7 and -9; and ADAMTS-1, -4, & -9 (Halpert et al, 1996; Jonsson-Rylander et al, 2005; Passi et al, 1999; Perides et al, 1995; Sandy et al, 2001; Somerville et al, 2003; Westling et al, 2004).  I have been able to expand this repertoire by two enzymes and provide some characterization of their cleavage products.  This is beginning to look like a long list of potential in vivo versican processing enzymes, yet the list is likely not yet complete, nor do we fully understand all the processing events of each of these enzymes. Recent work has predicted that many more enzymes and cleavages were likely to occur than the current state of knowledge describes (Kenagy et al, 2006).  Indeed much work has been done to identify a key aggrecan degrading enzyme and though no such enzyme has yet been identified, much insight into the complexity of turnover of this large proteoglycan has been gained (Fosang et al, 2008; Sandy, 2006).  Thus a large challenge lies ahead to inventory all proteases and cleavages before we can fully understand aberrant events in proteolytic pathways.  In addition to the complexity inherent in multiple proteases performing multiple cleavages, protease regulation is complex.  Though the search for a single protease to target for therapeutic intervention will remain tempting, it is increasingly clear that all 176 proteases are part of webs and cascades of interconnected pathways, often leading to pleiotropic roles for a given protease (McCawley & Matrisian, 2001; Overall & Dean, 2006; Overall & Kleifeld, 2006).  This does not mean that identification of a single critical enzyme that is dysregulated and therefore targetable in a certain disease is not possible.  But, in order to find this elusive target, we need to first understand the whole web that it is a part of.  It is now abundantly clear from the many clinical trials of MMP inhibition in cancer and arthritis that broad spectrum MMP inhibition isn't an therapeutic option (Coussens et al, 2002; Egeblad & Werb, 2002; Murphy & Nagase, 2008).  In addition to needing more specific inhibitors and a more detailed understanding of the proteases that are targets or anti-targets in cancer and other diseases, an understanding of protease balance in normal remodeling is also required.  Whereas all proteases presumably have a normal function and many are implicated in normal wound healing, it is perhaps the imbalance of proteases and inhibitors that is detrimental in pathological wound healing (Wynn, 2007).  In addition to the need to characterize the complete repertoire of proteases acting in complex physiological processes, it is also critical to understand the different paths of physiological remodeling that can lead to complete regeneration, partial scarring, or perpetuative scarring disorders.  These diverse but related processes may involve subtle alterations in expression and activity of the same proteases.  Interestingly, turnover of the cartilage proteoglycan aggrecan appears to involve different subsets of metalloproteinases that contribute to distinct remodeling processes (Sandy, 2006). Likewise, versican turnover under different conditions could involve different proteolytic pathways. 177  Though the field is complex, progress is being made and I feel confident that the work contained in this thesis will make a valuable contribution to our understanding of versican turnover and lead to more answers in the future.  As progress is made with new techniques like mass spectrometry, I expect we will be able to gather much more detailed information about precise cleavage events and in turn gain a more thorough understanding of molecular events of normal and aberrant remodeling.  This work represents one more step toward better diagnosis and the eventual rational design of therapies for dysregulated events of versican turnover.   178 5.1.  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Complete data set of CLIP-CHIP proteinase, inhibitor and control spot  fold changes and p-values. Signifi cantly regulated genes from Figure 4.3 are highlighted in orange or green for up- or down- regulation respectively.  Non-signifi cant genes are shaded grey with fold changes greater than one light and fold changes less than one dark.  Normalized data were analyzed by two dimensional SAM (signifi cance analysis of microarrays) analysis with fold change cutoff of 1.5 and p-values were determined with paired T-tests.  A, Proteinases, B, Inhibitors, C, Control spots. RefSeq Abbreviation Description Fold change p-value NM_022060 ABHD4 abhydrolase dom. containing 4 1.454261 0.000254 NM_001097 ACR acrosin 1.043162 0.473111 NM_001640 APEH acylaminoacyl-peptidase 0.884535 0.009870 NM_021794 ADAM30 ADAM 30 0.919071 0.071365 NM_145004 ADAM32 ADAM 32 1.403959 0.230837 AL117415 ADAM33 ADAM 33 1.134082 0.052602 NM_001110 ADAM10 ADAM10 1.134054 0.084147 NM_002390 ADAM11 ADAM11 1.278375 0.167937 NM_003474 ADAM12 ADAM12 1.029362 0.497379 NM_003815 ADAM15 ADAM15 0.879478 0.003346 NM_003183 ADAM17 ADAM17 1.042539 0.330290 NM_014237 ADAM18 ADAM18 0.942399 0.367394 NM_033274 ADAM19 ADAM19 1.062181 0.363387 NM_001464 ADAM2 ADAM2/Fertilin-b 1.048863 0.316374 NM_003814 ADAM20 ADAM20 1.002430 0.985725 NM_003813 ADAM21 ADAM21 0.577993 6.96E-07 NM_021723 ADAM22 ADAM22 1.048896 0.205405 NM_003812 ADAM23 ADAM23 1.075683 0.211801 NM_014265 ADAM28 ADAM28 1.133435 0.116771 NM_014269 ADAM29 ADAM29 0.844513 0.285773 AF215824 ADAM7 ADAM7 1.306727 0.175068 NM_001109 ADAM8 ADAM8 0.988350 0.799768 NM_003816 ADAM9 ADAM9 0.833627 0.011397 NM_006988 ADAMTS1 ADAMTS1 0.824290 0.000376 AF163762 ADAMTS10 ADAMTS10 1.143656 0.027877 NM_030955 ADAMTS12 ADAMTS12 0.715973 0.000259 AB069698 ADAMTS13 ADAMTS13 1.019927 0.611718 NM_080722 ADAMTS14 ADAMTS14 0.890824 0.037172 NM_139055 ADAMTS15 ADAMTS15 1.359314 0.001548 NM_139056 ADAMTS16 ADAMTS16 1.101893 0.088858 NM_139057 ADAMTS17 ADAMTS17 1.118995 0.015415 NM_139054 ADAMTS18 ADAMTS18 0.961064 0.463806 NM_133638 ADAMTS19 ADAMTS19 1.415382 0.195874 NM_014244 ADAMTS2 ADAMTS2 1.098380 0.075114 NM_025003 ADAMTS20 ADAMTS20 0.547807 0.873311 AB002364 ADAMTS3 ADAMTS3 1.398798 0.030722 NM_005099 ADAMTS4 ADAMTS4 1.155083 0.036036 NM_007038 ADAMTS5 ADAMTS5/11 1.152815 0.024532 NM_014273 ADAMTS6 ADAMTS6 0.931969 0.060252 NM_014272 ADAMTS7 ADAMTS7 1.180135 0.004580 NM_007037 ADAMTS8 ADAMTS8 1.268601 0.001373 AB037733 ADAMTS9 ADAMTS9 1.144522 0.327950 NM_001129 AEBP1 adipocyte-enhancer binding prot. 1 1.056484 0.301068 NM_006796 AFG3L2 Afg3-like protein 2 1.109291 0.095558 NM_004262 HAT airway-trypsin-like protease 1.003150 0.960339 NM_000666 ACY1 aminoacylase 1.086611 0.062162 A.  Proteinases 183 NM_001977 ENPEP aminopeptidase A 0.996591 0.896260 NM_020216 RNPEP aminopeptidase B 0.997609 0.844104 NM_018226 RNPEPL1 aminopeptidase B-like 1 1.011218 0.791509 NM_022350 AMPEP aminopeptidase MAMS 0.974787 0.662580 NM_001150 ANPEP aminopeptidase N 1.000585 0.930189 NM_032823 AOPEP aminopeptidase O 1.007141 0.946547 NM_022098 PEPP aminopeptidase P homologue 0.871418 0.000921 BC007579 XPNPEP1 aminopeptidase P1 1.010884 0.962761 AB011097 ARTS1 aminopeptidase PILS 0.924429 0.522424 AK075131 AQPEP aminopeptidase Q 0.920071 0.750169 NM_024663 NPEPL1 aminopeptidase-like 1 1.093503 0.201465 NM_006463 AMSH AMSH 0.701548 0.001395 AB037794 AMSH2 AMSH 2 1.589992 0.338583 NM_000789 ACE angiotensin-converting enzyme 1 1.772955 0.002470 NM_021804 ACE2 angiotensin-converting enzyme 2 0.976711 0.421599 NM_002770 PRSS2 anionic trypsin (II) 1.120854 0.066294 NM_005577 LPA apolipoprotein 0.979589 0.961044 NM_133463 AMZ1 Archeometzincin 1 1.062921 0.550218 NM_016627 AMZ2 Archeometzincin 2 0.969020 0.643988 NM_000049 ASPA/ACY-2 aspartoacylase-2 1.076493 0.260786 BC008689 ACY-3 aspartoacylase-3 1.230296 0.005368 NM_012100 DNPEP aspartyl aminopeptidase 1.341927 0.000581 NM_004993 MJD1 ataxin-3 1.029945 0.665557 XM_045705 ATX3L ataxin-3 like 1.075647 0.762251 NM_013325 AUTL1 autophagin-1 1.013331 0.889911 NM_052936 AUTL2 autophagin-2 1.002871 0.913473 NM_032852 AUTL3 autophagin-3 1.238541 0.177532 NM_032885 AUTL4 autophagin-4 1.076813 0.279442 NM_001700 AZU1 azurocidin 1.137931 0.083914 NM_032859 BEM46L1 BEM46-like 1 0.632998 0.167255 BC014049 BEM46L2 BEM46-like 2 1.026164 0.452129 BG74273 BEM46L3 BEM46-like 3 1.221964 0.221574 NM_032857 LACTB beta lactamase 0.864424 0.107739 NM_012104 BACE beta-secretase 1 1.042162 0.466283 AF178532 BACE2 beta-secretase 2 0.972128 0.952413 NM_000386 BLMH bleomycin hydrolase 1.068627 0.323763 NM_022119 PRSS22 brain serine proteinase 2 0.931212 0.150799 NM_024332 C6.1A C6.1A 0.779539 0.031573 NM_005186 CAPN1 calpain 1 0.818196 0.156850 NM_023087 CAPN10 calpain 10 1.408405 0.054289 NM_007058 CAPN11 calpain 11 1.130280 0.127778 XM_290840 CAPN12 calpain 12 0.736004 0.028707 AK027176 CAPN13 calpain 13 0.937641 0.705539 AK092257 CAPN14 calpain 14 1.107906 0.134747 NM_005632 CAPN15 calpain 15/Sol protein 1.102571 0.197625 NM_001748 CAPN2 calpain 2 0.819171 0.011195 NM_000070 CAPN3 calpain 3 1.268147 0.309727 NM_004055 CAPN5 calpain 5 0.944898 0.130228 NM_014289 CAPN6 calpain 6 1.030792 0.626466 NM_014296 CAPN7 calpain 7 1.067843 0.367554 AA043093 CAPN8 calpain 8 1.857922 0.180951 NM_006615 CAPN9 calpain 9 1.000595 0.951196 NM_001868 CPA1 carboxypeptidase A1 0.990094 0.715733 NM_001869 CPA2 carboxypeptidase A2 1.021823 0.655109 NM_001870 CPA3 carboxypeptidase A3 0.898208 0.038564 NM_016352 CPA4 carboxypeptidase A4 0.857495 0.089095 AF384667 CPA5 carboxypeptidase A5 1.233693 0.004121 NM_020361 CPA6 carboxypeptidase A6 0.973282 0.587763 NM_001871 CPB1 carboxypeptidase B 0.927342 0.452585 184 NM_001304 CPD carboxypeptidase D 1.166176 0.008111 NM_001873 CPE carboxypeptidase E 1.163725 0.042171 AF368463 CPM carboxypeptidase M 0.998476 0.977612 NM_001308 CPN carboxypeptidase N 1.052441 0.461802 NM_173077 CPO carboxypeptidase O 1.493100 0.031803 NM_001872 CPB2 carboxypeptidase U 1.277515 0.120754 NM_019609 CPX1 carboxypeptidase X1 1.021360 0.558988 XM_058409 CPX2 carboxypeptidase X2 0.974808 0.534689 NM_003652 CPZ carboxypeptidase Z 1.117924 0.000525 NM_033292 CASP1 caspase-1 0.901156 0.098538 NM_032977 CASP10 caspase-10 1.786867 0.157123 NR_000035 CASP12 caspase-12 1.528438 0.163008 NM_012114 CASP14 caspase-14 1.093792 0.686632 AF098666 CASP14L caspase-14-like 0.885609 0.025528 NM_032982 CASP2 caspase-2 0.825651 0.007925 NM_004346 CASP3 caspase-3 1.036027 0.589779 NM_033306 CASP4 caspase-4/11 1.128637 0.924746 NM_004347 CASP5 caspase-5 1.842041 0.086213 NM_001226 CASP6 caspase-6 0.903534 0.100255 NM_033339 CASP7 caspase-7 0.697202 0.177442 NM_033357 CASP8 caspase-8 1.095961 0.186674 NM_001229 CASP9 caspase-9 1.102391 0.243970 Y14039 CFLAR casper 1.451308 0.110366 NM_001908 CTSB cathepsin B 1.238410 0.002583 NM_001814 CTSC cathepsin C 0.968233 0.460786 NM_001909 CTSD cathepsin D 1.257334 0.001232 NM_001910 CTSE cathepsin E 0.989282 0.806077 NM_003793 CTSF cathepsin F 1.122462 0.154566 NM_001911 CTSG cathepsin G 0.971384 0.556059 NM_004390 CTSH cathepsin H 0.844747 0.005814 NM_000396 CTSK cathepsin K 1.101728 0.051209 NM_001912 CTSL cathepsin L 1.634475 0.002243 NM_001333 CTSL2 cathepsin L2 0.976291 0.772144 NM_001334 CTSO cathepsin O 1.134489 0.118764 AK024855 CTSS cathepsin S 1.186685 0.095733 NM_001335 CTSW cathepsin W 1.347526 0.198029 NM_001336 CTSZ cathepsin Z 0.875334 0.171538 NM_002769 PRSS1 cationic trypsin 0.870892 0.127983 NM_020205 CEZANNE Cezanne 0.902312 0.192495 AJ430383 LOC161725 Cezanne-2 1.117649 0.350853 NM_016006 CGI-58 CGI-58 0.871740 0.040012 NM_016014 CGI-67 CGI-67 protein 0.845476 0.395027 NM_031213 CGI-67L1 CGI-67-like protease-1 1.218480 0.002672 AL390079 CGI-67L2 CGI-67-like protease-2 1.069017 0.220878 AL137441 CGI77 CGI-77 0.913404 0.322111 NM_001836 CMA1 chymase 1.014554 0.721889 AJ272212 CTRL chymopasin 1.086110 0.880373 NM_001906 CTRB1 chymotrypsin B 0.949463 0.403485 NM_007272 CTRC chymotrypsin C 1.194942 0.265224 NM_000133 F9 coagulation factor IXa 1.013312 0.666829 NM_000131 F7 coagulation factor VIIa 0.964173 0.555779 NM_000504 F10 coagulation factor Xa 1.152671 0.003286 NM_000128 F11 coagulation factor XIa 1.029300 0.477540 NM_000505 F12 coagulation factor XIIa 1.013373 0.807388 NM_002421 MMP1 collagenase 1 1.649192 0.000323 NM_002424 MMP8 collagenase 2 0.971386 0.560481 NM_002427 MMP13 collagenase 3 1.197408 0.009373 NM_016546 C1RL complement C1r-homolog 1.136744 0.260458 NM_000063 C2 complement component 2 1.237043 0.102853 185 AK024951 C1R complement component C1ra 0.871277 0.026977 NM_001734 C1S complement component C1sa 1.200853 0.250167 NM_001710 BF complement factor B 0.832811 0.001992 NM_001928 DF complement factor D 0.955309 0.467658 XM_115647 DF2 complement factor D-like 1.298070 0.032828 NM_000204 IF complement factor I 1.085039 0.295383 NM_006833 COPS6 COPS6 0.746265 2.59E-06 NM_006587 PRSC corin 1.051322 0.263489 NM_015247 CYLD1 cylindromatosis protein 0.920750 0.434999 NM_006310 NPEPPS cytosol alanyl aminopeptidase 0.963657 0.555967 AK055994 DDI-RP DDI-related protease 1.007998 0.797404 NM_014479 ADAMDEC1 DECYSIN 1.024712 0.551656 NM_014058 DESC1 DESC1 protease 1.469035 0.223924 NM_021044 DHH desert hedgehog protein 1.114139 0.097488 NM_004341 CAD dihydroorotase 0.762871 0.002296 NM_001385 DPYS dihydropyrimidinase 1.061531 0.132688 NM_001313 CRMP1 dihydropyrimidinase-related prot. 1 1.110940 0.140675 U97105 DPYSL2 dihydropyrimidinase-related prot. 2 1.045365 0.415335 NM_001387 DPYSL3 dihydropyrimidinase-related prot. 3 0.741723 0.076881 NM_006426 DPYSL4 dihydropyrimidinase-related prot. 4 1.429597 0.010371 AK022795 DPYSL5 dihydropyrimidinase-related prot. 5 2.159467 0.224888 NM_004826 ECEL1 DINE peptidase 1.045482 0.471216 AB040925 DPP10 dipeptidyl-peptidase 10 0.897500 0.852863 NM_001935 DPP4 dipeptidyl-peptidase 4 1.293671 1.15E-06 NM_001936 DPP6 dipeptidyl-peptidase 6 1.030660 0.691847 NM_017743 DPP8 dipeptidyl-peptidase 8 0.949749 0.514363 BC000970 DPP9 dipeptidyl-peptidase 9 0.844169 0.008445 NM_013379 DPP7 dipeptidyl-peptidase II 1.029468 0.406123 NM_005700 DPP3 dipeptidyl-peptidase III 1.079052 0.099389 NM_007262 DJ-1 DJ-1 0.645218 1.85E-06 AK093336 DDI1 DNA-damage inducible protein 0.790787 0.894259 BN000122 DDI2 DNA-damage inducible protein 2 1.236150 0.282234 NM_022450 EGFR-RS EGF Receptor Related Sequence 1.438846 0.020862 NM_004771 MMP20 enamelysin 0.481213 0.317351 NM_006012 CLPP endopeptidase Clp 1.000976 0.988273 NM_001397 ECE1 endothelin-converting enzyme 1 1.136857 0.081053 NM_014693 ECE2 endothelin-converting enzyme 2 1.042765 0.355675 NM_002772 PRSS7 enteropeptidase 1.083461 0.442237 BQ638967 PRSS7L enteropeptidase-like 0.984907 0.840640 BN000134 ESSPL epidermis-specific SP-like 0.809350 0.080004 NM_032950 MMP28 epilysin 1.126129 0.076767 NM_005656 TMPRSS2 epitheliasin 0.903374 0.646202 NM_000120 EPHX1 epoxide hydrolase 1.000713 0.989957 NM_173567 EPHXRP epoxyde hydrolase related protein 1.412447 4.76E-05 NM_003756 EIF3S3 eukar. translation initiation F3S3 1.064378 0.401685 NM_003754 EIF3S5 eukar. translation initiation F3S5 0.863917 0.435274 XM_062387 EIF3S5B eukar. translation initiation F3S5B 0.963789 0.541981 NM_005857 FACE1 FACE-1/ZMPSTE24 0.929754 0.034499 NM_005133 FACE2 FACE-2/RCE1 0.878942 0.493130 NM_002569 PCSK3 furin 1.356860 0.001526 NM_003878 GGH gamma-glutamyl hydrolase 0.925619 0.047417 NM_013421 GGT1 gamma-glutamyltransferase 1 0.969636 0.664871 M30474 GGT2 gamma-glutamyltransferase 2 1.093889 0.004378 NM_004121 GGTLA1 gamma-glutamyltransferase 5 1.105785 0.208916 NM_153338 GGT6 gamma-glutamyltransferase 6 1.449491 0.085111 NM_080839 GGTL4 gamma-glutamyltransferase m-3 0.964341 0.282200 NM_052830 GGTL3 gamma-glutamyltransferase-like 3 1.018820 0.651485 NM_002652 PIP GCDFP15 7.145798 0.160822 NM_004530 MMP2 gelatinase A 1.244619 0.014608 186 NM_004994 MMP9 gelatinase B 1.013671 0.715787 NM_004994 MMP9 gelatinase B 0.935106 0.603452 NM_002056 GFPT1 Gln-fructose-6-P transamidase 1 0.781841 0.291443 NM_005110 GFPT2 Gln-fructose-6-P transamidase 2 1.051759 0.178847 XM_115871 GFPT3 Gln-fructose-6-P transamidase 3 1.130827 0.264770 U00238 PPAT Gln-PRPP amidotransferase 0.533084 0.145170 AK024471 CPGL glu-carboxypeptidase-like 1 0.867341 0.001391 NM_032649 CPGL2 glu-carboxypeptidase-like 2 1.049153 0.408566 NM_004476 FOLH1 glutamate carboxypeptidase II 1.004394 0.883932 NM_012413 QPCT glutaminyl cyclase 2.029997 0.020802 NM_017659 QPCT2 glutaminyl cyclase 2 0.967856 0.548463 NM_000027 AGA glycosylasparaginase 0.927215 0.677721 NM_025080 ASRGL1 glycosylasparaginase-2 0.961729 0.565542 NM_017714 AGA3 glycosylasparaginase-3 1.146179 0.179608 NM_006144 GZMA granzyme A 1.153341 0.342180 NM_004131 GZMB granzyme B 0.952086 0.525466 NM_033423 GZMH granzyme H 0.920577 0.200595 NM_002104 GZMK granzyme K 1.214102 0.247507 NM_005317 GZMM granzyme M 1.073179 0.614885 AK055872 HP haptoglobin-1 1.001206 0.985733 NM_020995 HPR haptoglobin-related protein 0.961388 0.335165 BN000133 HATL1 HAT-like 1 1.060462 0.233544 NM_207407 HATL4 HAT-like 4 1.140300 0.320269 XM_068002 HATL5 HAT-like 5 0.956217 0.585807 NM_182559 HATRP HAT-related protease 0.976751 0.996477 AK056446 HSPCA heat shock 90kDa protein 1, alpha 0.820919 0.020186 NM_007355 HSPCB heat shock 90kDa protein 1, beta 0.995373 0.872926 NM_016292 TRAP1 heat shock protein 75 0.865612 0.001331 X16323 HGF hepatocyte growth factor 1.862170 0.015145 NM_001528 HGFAC hepatocyte growth factor activator 0.957073 0.124110 NM_002151 HPN hepsin 0.937720 0.549485 NM_015281 HETFL HetF-like 1.026152 0.684515 AF022913 PIGK hGPI8 0.828745 0.199609 NM_017493 HSHIN1 Hin-1 0.853778 0.071094 NM_024810 HSHIN2 Hin-2 0.931537 0.109526 XM_170950 HSHIN3 Hin-3 0.951775 0.422968 XM_166659 HSHIN4 Hin-4 1.621677 0.006825 XM_054098 HSHIN5 Hin-5 1.026979 0.471303 XM_066765 HSHIN6 Hin-6 1.101814 0.046186 BI829009 HSHIN7 Hin-7 0.834376 0.586693 XM_072402 HMRALP HmrA-like protease 0.966737 0.728342 XM_495898 ICEYH homologue ICEY 1.256028 0.253802 AB067478 USP38 HP43.8KD 0.905724 0.653267 NM_013247 HTRA2 HTRA2 1.204027 0.001415 AY040094 HTRA3 HTRA3 1.813415 0.012818 NM_153692 HTRA4 HTRA4 0.897795 0.032727 NM_004132 HABP2 hyaluronan-binding ser-protease 0.917983 0.327131 NM_014263 YME1L1 i-AAA protease 0.980993 0.709621 NM_031943 IFP38 IFP38 1.114173 0.980057 XM_290345 IFP38L IFP38-like 0.837753 0.219770 L38517 IHH indian hedgehog protein 0.931088 0.019190 NM_004969 IDE insulysin 1.210310 0.364742 NM_006837 COPS5 JAB1 0.851506 0.231860 AB067502 JAMML1 jammin-like protease 1 0.979801 0.741203 NM_032868 JAMML2 jammin-like protease 2 1.025238 0.560262 NM_014876 JSPH1 josephin-1 1.124937 0.348931 NM_138334 SBBI54 josephin-2 0.924007 0.051575 NM_002257 KLK1 kallikrein hK1 1.014014 0.755107 NM_002776 KLK10 kallikrein hK10 1.150840 0.072977 187 NM_006853 KLK11 kallikrein hK11 1.469602 0.479766 NM_019598 KLK12 kallikrein hK12 0.880613 0.008479 NM_015596 KLK13 kallikrein hK13 1.041709 0.664638 NM_022046 KLK14 kallikrein hK14 0.866821 0.021344 NM_017509 KLK15 kallikrein hK15 1.047758 0.333588 AF188747 KLK2 kallikrein hK2 1.040085 0.934142 NM_001648 KLK3 kallikrein hK3 1.598505 0.000569 NM_004917 KLK4 kallikrein hK4 1.130866 0.131797 NM_012427 KLK5 kallikrein hK5 1.015698 0.708888 NM_002774 KLK6 kallikrein hK6 1.252756 0.184855 NM_005046 KLK7 kallikrein hK7 0.394917 0.174768 NM_007196 KLK8 kallikrein hK8 0.970183 0.596052 NM_012315 KLK9 kallikrein hK9 1.098943 0.090614 NM_000420 KEL Kell blood-group protein 1.053523 0.189189 NM_002343 LTF lactotransferrin 1.236181 0.037937 BC008004 LGMN legumain 0.949136 0.209814 NM_033029 LMLN leishmanolysin-2 1.195515 0.543459 NM_015907 LAP3 leucyl aminopeptidase 0.914305 0.006925 NM_005575 LNPEP leucyl-cystinyl aminopeptidase 1.403500 0.067507 NM_000895 LTA4H leukotriene A4 hydrolase 1.022613 0.569565 NM_000308 PPGB lysosomal carboxypeptidase A 1.125502 0.012386 NM_005040 PRCP lysosomal Pro-X carboxypeptidase 1.020909 0.701323 NM_002426 MMP12 macrophage elastase 1.025370 0.583689 NM_020998 MSP macrophage-stimulating protein 1.514111 0.019433 AF282732 TLL1 mammalian tolloid-like 1 protein 0.939397 0.298330 AB023149 TLL2 mammalian tolloid-like 2 protein 1.559307 0.022867 AK055576 MPN marapsin 0.971473 0.290059 BN000131 MPN2 marapsin 2 1.200812 0.925842 D17525 MASP1/3 MASP1/3 1.092833 0.080067 NM_006610 MASP2 MASP2 0.999048 0.950861 XM_063846 MASTIN mastin 1.223391 0.468394 NM_002423 MMP7 matrilysin 1.439942 0.000648 NM_021801 MMP26 matrilysin-2 1.091954 0.185303 NM_021978 MTSP1 matriptase 1.170041 0.087936 AL022314 TMPRSS6 matriptase-2 0.937635 0.361323 BN000125 TMPRSS7 matriptase-3 1.238426 0.916249 NM_004413 DPEP1 membrane dipeptidase 1.124531 0.046123 NM_022355 DPEP2 membrane dipeptidase 2 1.149583 0.142254 NM_022357 DPEP3 membrane dipeptidase 3 1.179464 0.001996 NM_032046 MSPL membrane-type mosaic Ser-prot. 1.215649 0.083991 NM_005588 MEP1A meprin alpha subunit 1.232047 0.498529 NM_005925 MEP1B meprin beta subunit 0.934135 0.139786 NM_002402 MEST mesoderm specific transcript hom. 0.386115 0.000161 NM_002771 PRSS3 mesotrypsin 0.734651 0.048182 NM_145243 MPRP-1 metalloprotease related protein 1 0.918582 0.020607 D42084 METAP1 methionyl aminopeptidase I 1.064125 0.249254 NM_006838 METAP2 methionyl aminopeptidase II 1.033054 0.250483 XM_171022 METAPL1 methionyl aminopeptidase-like 1 1.061225 0.413293 NM_032549 IMMP2L mitoc. inner membrane protease 2 0.851981 0.034280 NM_005932 MIPEP mitochondrial intermediate peptidase 0.971591 0.668752 NM_004279 PMPCB mitochondrial processing peptidase beta-subunit 0.959081 0.226366 D50913 INPP5E mitochondrial processing protease 1.048021 0.270769 AK057788 IMMP1 mitochondrial signal peptidase 0.664368 0.345235 NM_022790 MMP19 MMP19 1.128133 0.889510 NM_147191 MMP21 MMP21 1.005056 0.941916 NM_004659 MMP23A/B MMP23A/B 0.998273 0.928795 NM_022122 MMP27 MMP27 0.861858 0.853438 NM_004995 MMP14 MT1-MMP 2.163458 0.018383 NM_002428 MMP15 MT2-MMP 0.983133 0.902077 188 NM_005941 MMP16 MT3-MMP 0.812033 0.001099 NM_016155 MMP17 MT4-MMP 1.190632 0.255853 NM_006690 MMP24 MT5-MMP 1.111633 0.443901 NM_006690 MMP24 MT5-MMP 0.911841 0.029017 NM_022468 MMP25 MT6-MMP 0.945224 0.945125 NM_022468 MMP25 MT6-MMP 0.432952 0.244066 NM_005467 NAALAD2 NAALADASE II 1.037426 0.621333 XM_173084 NAALAD3 NAALADASE III 1.222454 0.023891 NM_005468 NAALADL NAALADASE L peptidase 0.908969 0.049063 NM_004851 NAP1 napsin A 1.082102 0.237901 NM_002525 NRD1 nardilysin 0.966886 0.597463 NM_007289 MME neprilysin 0.500905 0.072480 NM_033467 MMEL2 neprilysin-2 1.118694 0.086336 AB033052 NLN neurolysin 1.020092 0.641340 NM_003619 PRSS12 neurotrypsin 0.516020 0.001004 NM_001972 ELA2 neutrophil elastase 0.964628 0.583718 NM_030808 NUDEL nuclear distribution element-like-oligopeptidase 1.585110 0.012137 NM_017668 NDE1 nuclear distribution-oligopeptidase 1.103897 0.060491 NM_031474 NRIP2 Nuclear recept. interacting prot. 2 1.093463 0.508430 NM_020645 NRIP3 Nuclear recept. interacting prot. 3 0.805127 0.034787 NM_005387 NUP98 Nucleoporin 98kD 1.350546 0.164170 NM_032582 USP32 NY-REN-60 1.119869 0.321096 NM_017807 OSGEP O-sialoglycoprotein endopeptidase 0.959080 0.311587 AK027836 OSGEP2 O-sialoglycoprotein endopeptidase 2 1.019548 0.544832 AB058718 OJP Ojeda peptidase 1.043205 0.465846 NM_002775 HTRA1 osteoblast serine protease 1.484490 0.000301 NM_017670 OTUB1 Otubain-1 1.017346 0.602179 NM_023112 OTUB2 Otubain-2 1.301934 0.104560 BI061462 OVCN Ovastacin 0.904816 0.036898 BN000120 OVTN oviductin-like 1.212961 0.311264 BN000128 OVCH ovochymase-like 1.429653 0.000457 NM_002570 PCSK6 PACE4 proprotein convertase 1.017082 0.767492 NM_005805 POH1 Pad1-homolog 0.963119 0.812916 NM_033440 ELA2A pancreatic elastase II (IIA) 1.037992 0.472482 NM_015849 ELA2B pancreatic elastase II form B 1.124211 0.440893 NM_005747 ELA3A pancreatic endopeptidase E (A) 1.138915 0.019980 NM_007352 ELA3B pancreatic endopeptidase E (B) 1.064688 0.562000 U28727 PAPPA pappalysin-1 1.009101 0.850082 AF342989 PLAC3 pappalysin-2 1.038529 0.603443 NM_006785 MALT1 paracaspase 1.392879 0.057095 NM_003119 SPG7 paraplegin 1.345894 0.026691 AJ223812 PGA3/4/5 pepsin A 1.034250 0.890539 NM_002630 PGC pepsin C 1.109538 0.077240 NM_000444 PHEX PHEX endopeptidase 1.096208 0.624305 NM_004793 PRSS15 PIM1 endopeptidase 0.796414 0.030515 NM_006875 PIM2 PIM2 endopeptidase 1.014907 0.720152 NM_014889 PITRM1 pitrilysin metalloproteinase 1 0.753610 0.358606 NM_006102 PGCP plasma Glu-carboxypeptidase 0.998208 0.947075 NM_000892 KLKB1 plasma kallikrein 1.514657 0.084405 XP_116753 KLKBL1 plasma-kallikrein-like 1 1.170515 0.044606 XM_114622 KLKBL2 plasma-kallikrein-like 2 1.132022 0.623932 AL136785 KLKBL4 plasma-kallikrein-like 4 0.978601 0.498375 NM_000301 PLG plasminogen 1.809249 0.041620 AJ488946 TMPRSS9 Polyserase I 1.065656 0.743825 NM_007318 PSEN1 presenilin 1 1.033506 0.403327 NM_000447 PSEN2 presenilin 2 1.161450 0.116783 AL110147 PSH1 presenilin homolog 1/SPPL3 0.988963 0.717937 XM_091623 PSH2 presenilin homolog 2 1.131264 0.181913 BC008959 PSH3 presenilin homolog 3/SPP 1.032244 0.413360 189 AB040965 PSH4 presenilin homolog 4/SPPL2B 1.082414 0.478373 NM_032802 PSH5 presenilin homolog 5 1.389466 0.197330 NM_018622 PARL Presenilins associated rhomboid like 0.808326 0.009104 NM_002768 PCOLN3 procol. III N-endopeptidase 0.943641 0.522468 NM_006129 BMP1 procollagen C-proteinase 1.009797 0.844182 NM_006191 PA2G4 proliferation-association protein 1 0.736682 0.000327 NM_002726 PREP prolyl oligopeptidase 0.964686 0.229692 AB007896 PREP2 prolyl-oligopeptidase 2 0.636632 0.035987 NM_000439 PCSK1 proprotein convertase 1 1.015411 0.833113 NM_002594 PCSK2 proprotein convertase 2 1.059467 0.569288 AK057235 PCSK4 proprotein convertase 4 1.041521 0.461231 NM_006200 PCSK5 proprotein convertase 5 0.828836 0.140023 NM_004716 PCSK7 proprotein convertase 7 0.976692 0.971112 NM_174936 PCSK9 proprotein convertase 9 0.887406 0.016149 NM_002773 PRSS8 prostasin 0.976496 0.290852 NM_173502 PSTL1 prostasin-like 1 0.989622 0.767593 NM_024006 PSTL2 prostasin-like 2 1.010635 0.811424 NM_002786 PSMA1 proteasome alpha 1 subunit 0.802332 0.000258 NM_002787 PSMA2 proteasome alpha 2 subunit 1.049705 0.253316 NM_002788 PSMA3 proteasome alpha 3 subunit 0.797633 0.000971 AK055714 PSMA4 proteasome alpha 4 subunit 0.845925 0.005051 NM_002790 PSMA5 proteasome alpha 5 subunit 1.111348 0.046027 X59417 PSMA6 proteasome alpha 6 subunit 0.785520 0.000568 NM_002792 PSMA7 proteasome alpha 7 subunit 0.894487 0.207568 NM_144662 PSMA8 proteasome alpha 8 subunit 0.865805 0.353937 AK023290 PSMB1 proteasome beta 1 subunit 0.860735 0.000279 NM_002794 PSMB2 proteasome beta 2 subunit 0.978726 0.726067 NM_002795 PSMB3 proteasome beta 3 subunit 1.091757 0.147756 NM_002796 PSMB4 proteasome beta 4 subunit 0.936015 0.154752 XM_063287 LMP7L proteasome beta subunit LMP7-like 0.924460 0.164743 BF698890 PSMB6 proteasome catalytic subunit 1 0.916387 0.064043 NM_002800 PSMB9 proteasome catalytic subunit 1i 0.852746 0.022004 NM_002799 PSMB7 proteasome catalytic subunit 2 1.195697 0.139885 NM_002801 PSMB10 proteasome catalytic subunit 2i 0.974389 0.512615 NM_002797 PSMB5 proteasome catalytic subunit 3 1.026158 0.453165 NM_004159 PSMB8 proteasome catalytic subunit 3i 1.107383 0.086029 NM_000312 PROC protein C 0.979883 0.816247 AK027841 PROCL protein C-like 1.055244 0.458177 NM_003891 PROZ protein Z 1.055624 0.834986 NM_002777 PRTN3 proteinase 3 0.958978 0.416603 NM_006445 PRPF8 PRPF8 0.967341 0.966338 NM_002811 PSMD7 PSMD7 0.818327 0.061244 NM_017712 PGPEP1 pyroglutamyl peptidase I 1.162120 0.008243 XM_085243 PGPEP2 pyroglutamyl-peptidase II 1.064520 0.982954 NM_013381 TRHDE pyroglutamyl-peptidase II 0.801618 0.288159 NM_005045 RELN Reelin 1.044203 0.944445 NM_000537 REN renin 1.533507 0.366429 NM_003961 RHBDL rhomboid-like protein 1 1.222101 0.001525 NM_017821 RHBDL2 rhomboid-like protein 2 0.846682 0.249048 NM_138328 RHBDL4 rhomboid-like protein 4 1.774088 0.161399 NM_032276 RHBDL5 rhomboid-like protein 5 0.922198 0.137353 NM_024599 RHBDL6 rhomboid-like protein 6 1.131907 0.090738 NM_020684 RHBDL7 rhomboid-like protein 7 1.270785 0.011977 NM_015884 MBTPS2 S2P protease 1.191144 0.102409 NM_006590 USP39 SAD1 1.239193 0.001094 NM_014554 SENP1 sentrin/SUMO protease 1 1.108648 0.517573 AB037752 SENP2 sentrin/SUMO protease 2 0.948542 0.572702 NM_015670 SENP3 sentrin/SUMO protease 3 1.031751 0.526853 BC008589 SENP5 sentrin/SUMO protease 5 2.752479 0.054450 190 NM_015571 SENP6 sentrin/SUMO protease 6 0.938460 0.504407 AL136599 SENP7 sentrin/SUMO protease 7 0.847125 0.179761 AY008293 SENP8 sentrin/SUMO protease 8 1.096534 0.237534 NM_012291 ESPL1 separase 1.017833 0.908275 NM_004460 FAP Seprase 1.054997 0.202252 NM_021626 RISC serine carboxypeptidase 1 1.076623 0.236526 NM_014300 SPC18 signalase 18 kDa component 0.823789 0.019558 NM_033280 SPC21 signalase 21 kDa component 1.058889 0.062658 NM_144966 SPCL1 signalase-like 1 1.049542 0.771571 BC016840 SASP similar to Arabidopsis Ser-prot. 1.066196 0.124099 NM_153362 SPUVE2 similar to SPUVE 1.214683 0.184805 NM_003791 MBTPS1 site-1 protease 0.962031 0.434660 NM_000193 SHH sonic hedgehog protein 1.192162 0.328709 NM_030770 TMPRSS5 spinesin 0.937140 0.587066 NM_002422 MMP3 stromelysin 1 1.156270 0.027624 NM_002425 MMP10 stromelysin 2 0.259647 0.843173 NM_005940 MMP11 stromelysin 3 3.436513 3.26E-07 NM_007192 SUPT16H suppressor of Ty 16 homolog 0.922239 0.384692 NM_000930 PLAT t-plasminogen activator 2.945938 4.12E-10 NM_003184 TAF2 TBP-associated factor 2 0.850099 0.058997 AJ544583 TESSP2 testis serine protease 2 0.981679 0.846803 BN000137 TESSP5 testis serine protease 5 1.010511 0.905798 NM_013270 TSP50 testis-specific protein tsp50 1.502202 0.078141 NM_006799 PRSS21 testisin 0.929528 0.094963 NM_003249 THOP1 thimet oligopeptidase 1.023426 0.695655 NM_000506 F2 thrombin 3.580076 0.236128 NM_005865 PRSS16 thymus-specific serine peptidase 1.139589 0.304958 NM_022164 LCN7 TINAG related protein 1.104531 0.372483 NM_006290 TNFAIP3 TNFa-induced protein 3/A20 1.117219 0.025918 AJ252060 TRABID TRAF-binding protein domain 0.962997 0.439208 NM_003227 TFR2 transferrin receptor 2 protein (transferrin receptor 2) 1.324253 0.068687 NM_003234 TFRC transferrin receptor protein (transferrin receptor) 0.933878 0.589554 NM_032401 TMPRSS3 transmembrane Ser-protease 3 1.270711 0.369986 NM_019894 TMPRSS4 transmembrane Ser-protease 4 1.056896 0.054228 NM_000391 CLN2 tripeptidyl-peptidase I 1.213880 0.002512 NM_003291 TPP2 tripeptidyl-peptidase II 1.130411 0.008832 XM_059830 TRYX2 trypsin X2 0.708824 0.835672 NM_003294 TPS/TPSB1 tryptase alpha/beta 1 0.975988 0.734864 NM_024164 TPSB2 tryptase beta 2 0.850545 0.260166 NM_012217 TPSD1 tryptase delta 1 1.111942 0.115072 NM_012467 TPSG1 tryptase gamma 1 1.326345 0.008683 NM_152891 EOS tryptase homolog 2 1.015734 0.953286 BN000124 TESSP1 tryptase homolog 3 1.066833 0.522966 NM_014464 TINAG tubulointerstitial nephritis antigen 0.839493 0.899713 NM_003299 TRA1 tumor rejection antigen (gp96) 0.576270 0.046994 NM_002658 PLAU u-plasminogen activator 0.949939 0.625004 NM_004656 BAP1 ubiquitin C-term. hydrolase BAP1 1.170622 0.179090 NM_004181 UCHL1 ubiquitin C-terminal hydrolase 1 1.061961 0.325957 NM_006002 UCHL3 ubiquitin C-terminal hydrolase 3 0.887849 0.047070 NM_015984 UCHL5 ubiquitin C-terminal hydrolase 5 0.887117 0.030883 NM_003365 UQCRC1 UCR1 1.116582 0.007519 NM_003366 UQCRC2 UCR2 1.026141 0.447849 NM_007173 SPUVE umbelical vein proteinase 0.434380 2.72E-06 NM_003368 USP1 USP1 0.672616 0.007092 AL162049 USP10 USP10 0.973712 0.787306 NM_004651 USP11 USP11 0.926187 0.176778 AF022789 USP12 USP12 1.003746 0.954433 NM_003940 USP13 USP13 1.157484 0.267612 NM_005151 USP14 USP14 0.769938 0.015344 191 NM_006313 USP15 USP15 0.865988 0.407568 NM_006447 USP16 USP16 1.123125 0.181230 XM_172439 USP17 USP17 1.340944 0.192792 BN000116 USP17L USP17-like 0.915872 0.567893 NM_017414 USP18 USP18 1.164220 0.017610 AB020698 USP19 USP19 1.180446 0.279487 AK057225 USP2 USP2 1.547674 0.199746 NM_006676 USP20 USP20 0.846658 0.007529 NM_016572 USP21 USP21 1.168339 0.010950 AB028986 USP22 USP22 1.044551 0.433389 AB028980 USP24 USP24 0.909215 0.175331 AF170562 USP25 USP25 0.938393 0.512111 NM_031907 USP26 USP26 1.028510 0.504775 AW851065 USP27 USP27 0.939221 0.202904 NM_020886 USP28 USP28 1.037381 0.543870 NM_020903 USP29 USP29 1.473793 0.089794 NM_006537 USP3 USP3 1.191413 0.011935 NM_032663 USP30 USP30 1.031572 0.760051 AB033029 USP31 USP31 0.659025 0.028016 AK023845 USP34 USP34 0.869625 0.528234 AB037793 USP35 USP35 1.104436 0.144595 AB040886 USP36 USP36 0.990405 0.867564 AB046814 USP37 USP37 0.817328 0.245052 NM_003363 USP4 USP4 1.066400 0.888168 NM_018218 USP40 USP40 0.841031 0.136884 XM_036729 USP41 USP41 1.706400 0.023639 XM_166526 USP42 USP42 1.281985 0.219238 AK055188 USP43 USP43 1.035240 0.394008 NM_032147 USP44 USP44 0.745347 0.007580 NM_032929 USP45 USP45 0.920439 0.061584 NM_022832 USP46 USP46 1.241866 0.123345 AK027362 USP47 USP47 0.898842 0.038903 NM_018391 USP48 USP48 1.223463 0.002015 NM_018561 USP49 USP49 1.203964 0.306250 NM_003481 USP5 USP5 1.104538 0.719374 AI990110 USP50 USP50 0.832580 0.495060 BF741256 USP51 USP51 1.459642 0.055035 NM_014871 USP52 USP52 1.137918 0.257128 XM_052597 USP53 USP53 0.566877 0.002066 NM_152586 USP54 USP54 1.175494 0.007097 NM_004505 USP6 USP6 0.764128 0.284748 NM_003470 USP7 USP7 1.126001 0.175068 NM_005154 USP8 USP8 0.917845 0.745830 NM_004652 USP9X USP9X 0.849442 0.020622 NM_004654 USP9Y USP9Y 0.934081 0.635807 NM_025054 VCIP135 VCP(p97)/p47-interacting protein 1.315525 0.421193 AB029020 USP33 VDU1 1.006095 0.885476 NM_031311 CPVL vitellogenic carboxypeptidase-L. 1.041653 0.664587 NM_000285 PEPD X-Pro dipeptidase 1.070165 0.134920 NM_003399 XPNPEP2 X-prolyl aminopeptidase 2 1.052772 0.348058 192 B.  Inhibitors RefSeq Abbreviation Description Fold change p-value NM_000014 A2M a-2-macroglobulin 2.729719 0.016373 NM_003381 VIP a-2-macroglobulin-family VIP 1.113748 0.200719 AK057908 A2ML a-2-macroglobulin-like 0.979668 0.863725 NM_001085 SERPINA3 a1-antichymotrypsin 1.115762 0.119054 NM_175739 SERPINA11 a1-antitrypsin member 11 0.967817 0.685694 NM_173850 SERPINA12 a1-antitrypsin member 12 0.949620 0.347089 NM_006220 SERPINA2 a1-antitrypsin member 2 0.963738 0.501943 NM_175739 SERPINA9 a1-antitrypsin member 9 1.180897 0.010430 AF113676 SERPINA1 a1-antitrypsin/a1-PI 2.069461 0.540468 NM_001633 AMBP a1-microglobulin/bikunin 0.963393 0.647342 NM_000934 SERPINF2 a2-antiplasmin 1.058816 0.539507 NM_001622 AHSG a2-HS-glycoprotein/fetuinA 1.131796 0.298448 BC032003 BUSI2/SPINK6 acrosin inhibitor 1.234841 0.282044 NM_016519 AMBN ameloblastin 1.156183 0.318906 NM_000484 APP amyloid-b precursor protein 1.221408 0.014118 NM_005166 APLP1 amyloid-b precursor-like prot. 1 1.217549 0.017650 NM_001642 APLP2 amyloid-b precursor-like prot. 2 1.373346 5.78E-05 NM_000029 SERPINA8 angiotensinogen/AGT 1.363918 1.30E-06 NM_003064 SLPI antileukoproteinase 1.241500 0.085895 NM_000488 SERPINC1 antithrombin III 0.820918 0.411764 NM_016252 BIRC6 apollon 1.069461 0.279690 NM_005024 SERPINB10 bomapin 1.422769 0.880646 NM_000062 SERPING1 C1 inhibitor 0.990278 0.811730 NM_001750 CAST calpastatin 1.150194 0.039511 NM_001166 BIRC2 cIAP1 0.840867 0.184977 AF070674 BIRC3 cIAP2 0.691155 0.303716 NM_004369 COL6A3 collagen, type VI, alpha 3 1.058041 0.537182 NM_000094 COL7A1 collagen, type VII, alpha 1 1.582294 0.005767 NM_001235 SERPINH1 colligin/CBP1 1.392081 0.217024 NM_000064 C3 complement component 3 0.916612 0.420624 NM_000715 C4 complement component 4 0.994905 0.840848 NM_001735 C5 complement component 5 1.274596 0.019929 BG568135 CPLP complement prec. like protein 0.989554 0.705651 NM_001756 SERPINA6 corticosteroid-binding glob. 1.557266 0.043683 AL096677 CST11 CRES2/cystatin 11 0.777972 0.692066 XM_166592 CVL2 (BMPER) crossveinless 2 1.051923 0.214969 AF494536 CST9 cystatin 9/CLM 1.104737 0.148970 NM_005213 CSTA cystatin A 1.110940 0.031630 NM_000100 CSTB cystatin B 0.952614 0.403644 NM_000099 CST3 cystatin C 1.229179 0.003679 NM_001900 CST5 cystatin D 0.972687 0.607470 NM_001323 CST6 cystatin E/M 1.003441 0.930285 NM_003650 CST7 cystatin F/leukocystatin 0.966097 0.673878 NM_005492 CST8 cystatin G 1.188810 0.366838 AK056477 CSTL1 cystatin L1 1.010096 0.843235 NM_001322 CST2 cystatin SA 1.083803 0.115995 NM_001898 CST1 cystatin SN 1.139818 0.093672 NM_002638 PI3 elafin precursor 1.030232 0.746807 AF419955 SERPINB11 epipin 0.980262 0.777802 NM_020398 SPINLW1 eppin 0.975148 0.924577 NM_032566 ECG2 esophagus cancer-rel. Prot. 2 1.296855 0.000104 NM_014375 FETUB fetuin B 1.043486 0.765349 NM_006350 FST follistatin 0.309850 9.03E-08 NM_007085 FSTL1 follistatin-like 1 0.667555 1.64E-06 NM_001553 IGFBP7 follistatin-like 2/IGFBP7 0.774831 0.000173 NM_005860 FSTL3 follistatin-like 3 1.002908 0.980355 NM_133493 CD109 Gov platelet alloantigens 1.173560 0.655916 AJ001696 SERPINB13 headpin/hurpin 1.496718 0.156616 193 NM_000185 SERPIND1 heparin cofactor II 1.080161 0.334504 NM_004684 SPARCL1 hevin 1.096283 0.057894 NM_003710 SPINT1 HGF activator inhibitor 1 1.109062 0.491511 NM_002159 HTN1 histatin 1 1.155191 0.089019 NM_000200 HTN3 histatin 3 1.578399 0.070932 NM_000412 HRG histidine-rich glycoprotein 1.029859 0.640591 NM_002864 PZP human pregnanzy-zone prot. 1.394909 0.133821 NM_000596 IGFBP1 IGF binding protein 1 1.258229 0.108997 NM_000597 IGFBP2 IGF binding protein 2 1.187733 0.004274 M35878 IGFBP3 IGF binding protein 3 1.011298 0.771358 NM_033341 BIRC8 ILP2 0.967029 0.719820 NM_000599 IGFBP5 insulin-like growth factor binding protein 5 1.073942 0.170618 NM_002178 IGFBP6 insulin-like growth factor binding protein 6 † 0.738959 0.000108 NM_006215 SERPINA4 kallistatin 0.998705 0.885007 NM_020116 FSTL5 kazal, EF-hand and Ig protein 1.013316 0.914660 NM_000893 KNG kininogen 1.191082 0.140830 NM_020169 LXN latexin/tissue caboxpep. Inhibitor 0.711593 0.182445 NM_002639 SERPINB5 maspin 0.889059 0.064913 NM_003784 SERPINB7 megsin 0.879306 0.152962 NM_004355 CD74 MHC II invariant gamma chain 1.008040 0.816759 BC007758 KAZALD1 MIG30-like protease inhibitor 0.960607 0.591541 NM_022161 BIRC7 ML-IAP 0.900314 0.061127 AJ001403 MUC5AC mucin type 5A/C 0.948732 0.068525 U06711 MUC5B mucin type 5B 0.971923 0.530027 NM_004536 BIRC1 NAIP 0.905093 0.061284 NM_005025 SERPINI1 neuroserpin/PI12 1.898028 0.003110 NM_002508 NID1 nidogen 1.049992 0.288353 NM_007361 NID2 nidogen 2 1.016664 0.706755 NM_003118 SPARC osteonectin 0.892947 0.057646 XM_495909 OVOS ovostatin 0.755928 0.567147 XM_495917 OVOS2 ovostatin-2 0.810840 0.507534 NM_006217 SERPINI2 pancpin 0.516027 0.133038 BC042057 PAPLN papilin 1.058672 0.340063 NM_002615 SERPINF1 PEDF 1.043587 0.546737 NM_021102 SPINT2 placental bikunin 1.253444 0.000317 NM_002575 SERPINB2 Plg activator inhibitor-2 0.965416 0.915980 NM_000602 SERPINE1 plg-activator inhibitor-1 0.911202 0.673847 NM_002728 PRG2 Pro-eosinophil major basic protein 1.000214 0.848812 NM_013271 PCSK1N proSAAS 1.037607 0.548793 NM_002567 PBP prostatic binding protein 1.032255 0.540805 NM_030666 SERPINB1 protease inhibitor 2 0.897819 0.147236 AK057138 SERPINB6 protease inhibitor 6/CAP 0.920037 0.077848 NM_002640 SERPINB8 protease inhibitor 8/CAP2 1.025011 0.680374 BC002538 SERPINB9 protease inhibitor 9/CAP3 1.106084 0.239791 BC008915 SERPINA5 protein C inhibitor 0.992072 0.815031 NM_016186 SERPINA10 protein Z-dependent PI 1.203672 0.134204 NM_005878 SERPINE2 proteinase nexin 1/GDN 1.347883 0.534628 NM_021111 RECK RECK 0.971845 0.703321 NM_002888 RARRES1 retinoic acid receptor responder 0.963711 0.899784 NM_006919 SERPINB3 SCC antigen 1 1.661538 0.152534 NM_002974 SERPINB4 SCC antigen 2 0.859302 0.151886 NM_003020 SGNE1 secretogranin V/7B2 1.020087 0.751839 X77166 SPINT3 ser. protease inh., Kunitz 3 1.216085 0.429040 NM_003122 SPINK1 serine PI Kazal type 1 0.913402 0.802950 NM_021114 SPINK2 serine PI Kazal type 2 0.619857 0.003004 NM_014471 SPINK4 serine PI Kazal type 4 1.273783 0.031096 NM_006846 SPINK5 serine PI Kazal type 5 1.356567 0.103273 BC032033 SPINK5L1 serine PI Kazal type 5-like 1 1.111233 0.368456 NM_001001325 SPINK5L2 serine PI Kazal type 5-like 2 1.132326 0.024687 194 XM_376433 SPINK5L3 serine PI Kazal type 5-like 3 1.212333 0.008169 AJ249900 SMOC SPARC related mod. Ca binding 1 0.952066 0.246799 AJ420521 SMOC2 SPARC related mod. Ca binding 2 0.957090 0.457938 NM_004598 SPOCK sparc/osteonectin, testican 1.463729 0.106900 NM_014767 SPOCK2 sparc/osteonectin, testican-2 1.090887 0.046304 NM_016950 SPOCK3 sparc/osteonectin, testican-3 0.639993 0.656148 NM_003154 STATH statherin 1.031822 0.711237 NM_001168 BIRC5 survivin 0.977001 0.493230 NM_005422 TECTA tectorin a 1.069791 0.251197 NM_080610 CST9L testatin 0.982269 0.795178 NM_003235 TG thyroglobulin 1.314124 0.218070 NM_000354 SERPINA7 thyroxine-binding globulin 1.195770 0.496546 NM_006287 TFPI tissue factor path. inhibitor 1.072906 0.099817 NM_006528 TFPI2 tissue factor path. inhibitor-2 0.900887 0.341419 NM_003254 TIMP1 tissue inhibitor of metalloprotease-1 1.146030 0.055343 NM_003255 TIMP2 tissue inhibitor of metalloprotease-2 1.226753 0.001573 NM_000362 TIMP3 tissue inhibitor of metalloprotease-3 0.829834 0.003184 NM_003256 TIMP4 tissue inhibitor of metalloprotease-4 1.065226 0.154352 NM_003256 TIMP4 tissue inhibitor of metalloprotease-4 0.994201 0.960888 NM_003692 TMEFF1 TMEFF1 1.165063 0.019164 NM_016192 TMEFF2 TMEFF2 1.212011 0.178170 BN000366 VTLGL1 vitellogenin-like 1 1.306186 0.126148 NM_000552 VWF von Willebrand factor 1.072439 0.228383 NM_080753 WFDC10A WAP four-disulfide core 10A 1.103681 0.415616 NM_172006 WFDC10B WAP four-disulfide core 10B 0.344524 0.224507 NM_147197 WFDC11 WAP four-disulfide core 11 1.008892 0.854191 NM_080869 WFDC12 WAP four-disulfide core 12 1.054683 0.082567 NM_172005 WFDC13 WAP four-disulfide core 13 0.831007 0.905993 NM_006103 WFDC2 WAP four-disulfide core 2 0.864907 0.006063 AL591713 WFDC3 WAP four-disulfide core 3 0.888224 0.416020 NM_145652 WFDC5 (WAP1) WAP four-disulfide core 5 1.298514 0.009845 AL031663 WFDC6 WAP four-disulfide core 6 0.927344 0.389578 NM_080827 WFDC8 WAP four-disulfide core 8 0.712735 0.315655 NM_147198 WFDC9 WAP four-disulfide core 9 1.066751 0.279610 XM_086637 WFDCL1 WAP four-disulfide core-like 1 1.016788 0.715165 NM_053284 WFIKKN WAP,FS,Ig,KU,NTR-containing prot. 1.306077 0.238757 NM_175575 WFIKKNRP WFIKKNRP-related protein 0.945046 0.147954 NM_001167 BIRC4 XIAP 1.076452 0.004785 AF411191 SERPINB12 yukopin 0.881319 0.299544 195 C.  Controls RefSeq Abbreviation Description Fold change p-value 3xSSC-Buffer-Control 9.101044 0.491734 3xSSC-Buffer-Control 2.153620 0.053697 3xSSC-Buffer-Control 2.117839 0.212673 3xSSC-Buffer-Control 1.683767 0.062998 3xSSC-Buffer-Control 1.682604 0.609299 3xSSC-Buffer-Control 1.541443 0.039585 3xSSC-Buffer-Control 1.522581 0.166693 3xSSC-Buffer-Control 1.504644 0.376851 3xSSC-Buffer-Control 1.487038 0.815125 3xSSC-Buffer-Control 1.455059 0.251017 3xSSC-Buffer-Control 1.443690 0.564168 3xSSC-Buffer-Control 1.401066 0.137379 3xSSC-Buffer-Control 1.394853 0.217646 3xSSC-Buffer-Control 1.350338 0.335351 3xSSC-Buffer-Control 1.333502 0.323246 3xSSC-Buffer-Control 1.321818 0.124892 3xSSC-Buffer-Control 1.318924 0.401541 3xSSC-Buffer-Control 1.293731 0.275640 3xSSC-Buffer-Control 1.269646 0.459571 3xSSC-Buffer-Control 1.265324 0.337944 3xSSC-Buffer-Control 1.255772 0.443341 3xSSC-Buffer-Control 1.253593 0.403036 3xSSC-Buffer-Control 1.213508 0.317620 3xSSC-Buffer-Control 1.201503 0.754413 3xSSC-Buffer-Control 1.200071 0.324825 3xSSC-Buffer-Control 1.199639 0.528563 3xSSC-Buffer-Control 1.198666 0.357011 3xSSC-Buffer-Control 1.184350 0.318411 3xSSC-Buffer-Control 1.176411 0.290846 3xSSC-Buffer-Control 1.175003 0.287960 3xSSC-Buffer-Control 1.165480 0.985294 3xSSC-Buffer-Control 1.155560 0.354897 3xSSC-Buffer-Control 1.150290 0.574551 3xSSC-Buffer-Control 1.147813 0.437894 3xSSC-Buffer-Control 1.132709 0.413412 3xSSC-Buffer-Control 1.131751 0.639678 3xSSC-Buffer-Control 1.120790 0.875669 3xSSC-Buffer-Control 1.119219 0.407661 3xSSC-Buffer-Control 1.111363 0.492707 3xSSC-Buffer-Control 1.109306 0.352026 3xSSC-Buffer-Control 1.108798 0.749334 3xSSC-Buffer-Control 1.108515 0.399319 3xSSC-Buffer-Control 1.104813 0.415468 3xSSC-Buffer-Control 1.093869 0.708624 3xSSC-Buffer-Control 1.086596 0.754336 3xSSC-Buffer-Control 1.085606 0.496637 3xSSC-Buffer-Control 1.081740 0.725755 3xSSC-Buffer-Control 1.074953 0.713016 3xSSC-Buffer-Control 1.074158 0.525540 3xSSC-Buffer-Control 1.070059 0.649994 3xSSC-Buffer-Control 1.069545 0.502262 3xSSC-Buffer-Control 1.069191 0.852549 3xSSC-Buffer-Control 1.068030 0.654231 3xSSC-Buffer-Control 1.065620 0.640266 3xSSC-Buffer-Control 1.060773 0.667300 3xSSC-Buffer-Control 1.054381 0.801154 3xSSC-Buffer-Control 1.049149 0.738078 3xSSC-Buffer-Control 1.048573 0.764230 196 3xSSC-Buffer-Control 1.048339 0.726058 3xSSC-Buffer-Control 1.045175 0.582461 3xSSC-Buffer-Control 1.045149 0.815620 3xSSC-Buffer-Control 1.037008 0.504428 3xSSC-Buffer-Control 1.036065 0.868471 3xSSC-Buffer-Control 1.030245 0.875102 3xSSC-Buffer-Control 1.024650 0.788535 3xSSC-Buffer-Control 1.024094 0.521891 3xSSC-Buffer-Control 1.023334 0.888356 3xSSC-Buffer-Control 1.018369 0.852364 3xSSC-Buffer-Control 1.016294 0.732374 3xSSC-Buffer-Control 1.012211 0.678951 3xSSC-Buffer-Control 1.011647 0.755222 3xSSC-Buffer-Control 1.009960 0.717156 3xSSC-Buffer-Control 1.002063 0.889794 3xSSC-Buffer-Control 1.001977 0.795394 3xSSC-Buffer-Control 0.998393 0.757535 3xSSC-Buffer-Control 0.992013 0.998461 3xSSC-Buffer-Control 0.981882 0.627969 3xSSC-Buffer-Control 0.979601 0.652281 3xSSC-Buffer-Control 0.979103 0.685563 3xSSC-Buffer-Control 0.973698 0.715474 3xSSC-Buffer-Control 0.969745 0.584018 3xSSC-Buffer-Control 0.964005 0.822468 3xSSC-Buffer-Control 0.958943 0.809208 3xSSC-Buffer-Control 0.958912 0.801799 3xSSC-Buffer-Control 0.958092 0.706562 3xSSC-Buffer-Control 0.944549 0.858589 3xSSC-Buffer-Control 0.938911 0.562575 3xSSC-Buffer-Control 0.905584 0.920803 3xSSC-Buffer-Control 0.902727 0.418808 3xSSC-Buffer-Control 0.902458 0.922249 3xSSC-Buffer-Control 0.899661 0.362540 3xSSC-Buffer-Control 0.898053 0.634489 3xSSC-Buffer-Control 0.895706 0.611872 3xSSC-Buffer-Control 0.890558 0.788501 3xSSC-Buffer-Control 0.881565 0.482852 3xSSC-Buffer-Control 0.876999 0.827741 3xSSC-Buffer-Control 0.844306 0.213852 3xSSC-Buffer-Control 0.828721 0.227371 3xSSC-Buffer-Control 0.825767 0.490169 3xSSC-Buffer-Control 0.821133 0.388954 3xSSC-Buffer-Control 0.817605 0.651503 3xSSC-Buffer-Control 0.816332 0.559286 3xSSC-Buffer-Control 0.814497 0.286290 3xSSC-Buffer-Control 0.813750 0.437126 3xSSC-Buffer-Control 0.811158 0.562574 3xSSC-Buffer-Control 0.800972 0.494220 3xSSC-Buffer-Control 0.798177 0.219879 3xSSC-Buffer-Control 0.790439 0.648159 3xSSC-Buffer-Control 0.781922 0.192736 3xSSC-Buffer-Control 0.781826 0.700627 3xSSC-Buffer-Control 0.781793 0.281725 3xSSC-Buffer-Control 0.777017 0.302385 3xSSC-Buffer-Control 0.767380 0.817716 3xSSC-Buffer-Control 0.761909 0.261408 3xSSC-Buffer-Control 0.760829 0.088047 3xSSC-Buffer-Control 0.746231 0.121717 3xSSC-Buffer-Control 0.732757 0.128131 197 3xSSC-Buffer-Control 0.720524 0.044995 3xSSC-Buffer-Control 0.691510 0.232009 3xSSC-Buffer-Control 0.687026 0.214044 3xSSC-Buffer-Control 0.656844 0.146509 3xSSC-Buffer-Control 0.654457 0.057955 3xSSC-Buffer-Control 0.648200 0.071157 3xSSC-Buffer-Control 0.589694 0.023626 3xSSC-Buffer-Control 0.576692 0.167650 3xSSC-Buffer-Control 0.562008 0.165418 3xSSC-Buffer-Control 0.554861 0.521925 3xSSC-Buffer-Control 0.441577 0.701282 NM_001101 ACTB Actin, cytoplasmic 1 (beta-actin) 1.320158 0.076795 NM_001101 ACTB Actin, cytoplasmic 1 (beta-actin) 1.260563 0.126537 NM_001101 ACTB Actin, cytoplasmic 1 (beta-actin) 1.214199 0.173790 NM_001101 ACTB Actin, cytoplasmic 1 (beta-actin) 1.163747 0.093199 NM_001101 ACTB Actin, cytoplasmic 1 (beta-actin) 1.143629 0.327883 NM_001101 ACTB Actin, cytoplasmic 1 (beta-actin) 1.108609 0.439689 NM_001101 ACTB Actin, cytoplasmic 1 (beta-actin) 1.048575 0.566668 NM_001101 ACTB Actin, cytoplasmic 1 (beta-actin) 0.990517 0.954436 NM_001658 ARF1 ADP-ribosylation factor 1 1.301575 1.97E-05 NM_001658 ARF1 ADP-ribosylation factor 1 1.179632 0.003460 NM_001658 ARF1 ADP-ribosylation factor 1 1.158393 0.000788 NM_001658 ARF1 ADP-ribosylation factor 1 1.120414 0.015584 NM_001658 ARF1 ADP-ribosylation factor 1 1.109894 0.054697 NM_001658 ARF1 ADP-ribosylation factor 1 1.107241 0.145408 NM_001658 ARF1 ADP-ribosylation factor 1 1.079506 0.140435 NM_001658 ARF1 ADP-ribosylation factor 1 1.061861 0.178591 NM_001916 CYC1 Cytochrome c-1 0.960322 0.591693 NM_001916 CYC1 Cytochrome c-1 0.888141 0.015703 NM_001916 CYC1 Cytochrome c-1 0.882977 0.006216 NM_001916 CYC1 Cytochrome c-1 0.858743 0.024970 NM_001916 CYC1 Cytochrome c-1 0.794529 0.000582 NM_001916 CYC1 Cytochrome c-1 0.787743 0.000360 NM_001916 CYC1 Cytochrome c-1 0.758082 0.000610 NM_001916 CYC1 Cytochrome c-1 0.688509 0.016184 Empty 1.342889 0.273144 Empty 1.316479 0.274844 Empty 1.272894 0.283970 Empty 1.269845 0.201692 Empty 1.261416 0.670280 Empty 1.251900 0.934110 Empty 1.202748 0.255891 Empty 1.193655 0.331574 Empty 1.113334 0.511300 Empty 1.111840 0.535238 Empty 1.053322 0.570632 Empty 1.032046 0.928316 Empty 1.015161 0.855747 Empty 1.006018 0.794495 Empty 0.989327 0.958162 Empty 0.975999 0.396827 Empty 0.966479 0.978768 Empty 0.930263 0.490321 Empty 0.914253 0.674592 Empty 0.906395 0.479707 Empty 0.890632 0.318396 Empty 0.882011 0.760864 Empty 0.880530 0.746988 Empty 0.879583 0.512697 198 Empty 0.871275 0.230929 Empty 0.870609 0.250286 Empty 0.869799 0.388612 Empty 0.869308 0.503993 Empty 0.859441 0.220139 Empty 0.852364 0.736347 Empty 0.847178 0.389354 Empty 0.839135 0.274108 Empty 0.838974 0.972312 Empty 0.834134 0.972503 Empty 0.830354 0.202315 Empty 0.824056 0.258456 Empty 0.821514 0.356099 Empty 0.816096 0.284541 Empty 0.813921 0.427722 Empty 0.809213 0.493912 Empty 0.800970 0.562223 Empty 0.798016 0.244291 Empty 0.794539 0.187829 Empty 0.793571 0.709491 Empty 0.792932 0.795721 Empty 0.785398 0.145544 Empty 0.782756 0.289911 Empty 0.779207 0.148956 Empty 0.767169 0.114927 Empty 0.762504 0.261767 Empty 0.754913 0.423930 Empty 0.747020 0.369506 Empty 0.743294 0.684023 Empty 0.741315 0.351065 Empty 0.738735 0.272198 Empty 0.719705 0.097783 Empty 0.719635 0.045603 Empty 0.718907 0.425930 Empty 0.704479 0.037835 Empty 0.702649 0.688181 Empty 0.700777 0.218482 Empty 0.683730 0.183219 Empty 0.676628 0.436179 Empty 0.665640 0.097736 Empty 0.663862 0.029969 Empty 0.652267 0.148114 Empty 0.649508 0.067682 Empty 0.640181 0.482898 Empty 0.628502 0.181282 Empty 0.626553 0.066332 Empty 0.624301 0.158246 Empty 0.619147 0.151247 Empty 0.613382 0.064076 Empty 0.611611 0.029449 Empty 0.604340 0.070956 Empty 0.596538 0.213151 Empty 0.585420 0.062434 Empty 0.582423 0.013252 Empty 0.581263 0.142787 Empty 0.579439 0.098209 Empty 0.579194 0.017104 Empty 0.578505 0.068985 Empty 0.572952 0.126854 199 Empty 0.570977 0.133955 Empty 0.566039 0.005847 Empty 0.551470 0.019429 Empty 0.538511 0.034281 Empty 0.531330 0.250666 Empty 0.518457 0.006085 Empty 0.507123 0.145761 Empty 0.496518 0.003269 Empty 0.479337 0.010037 Empty 0.453828 0.102864 Empty 0.413143 0.005362 Empty 0.410812 0.002063 Empty 0.055740 0.143100 GFP-Control 1.373794 0.041312 GFP-Control 1.362032 0.380011 GFP-Control 1.227926 0.595134 GFP-Control 1.221479 0.020548 GFP-Control 1.214157 0.027285 GFP-Control 1.211399 0.164803 GFP-Control 1.199223 0.002935 GFP-Control 1.193315 0.056545 GFP-Control 1.189702 0.126065 GFP-Control 1.177525 0.231654 GFP-Control 1.169067 0.263240 GFP-Control 1.164519 0.030689 GFP-Control 1.154977 0.047974 GFP-Control 1.144335 0.339825 GFP-Control 1.139460 0.375250 GFP-Control 1.136747 0.552272 GFP-Control 1.136351 0.110585 GFP-Control 1.133607 0.066904 GFP-Control 1.126970 0.307614 GFP-Control 1.126735 0.353548 GFP-Control 1.126402 0.384632 GFP-Control 1.124167 0.778590 GFP-Control 1.115585 0.165799 GFP-Control 1.103227 0.602188 GFP-Control 1.099573 0.310374 GFP-Control 1.098660 0.805795 GFP-Control 1.094874 0.277425 GFP-Control 1.085744 0.511572 GFP-Control 1.084966 0.361320 GFP-Control 1.081926 0.196239 GFP-Control 1.069167 0.481475 GFP-Control 1.065749 0.536470 GFP-Control 1.064340 0.290710 GFP-Control 1.062529 0.383850 GFP-Control 1.059041 0.558894 GFP-Control 1.056287 0.382762 GFP-Control 1.050203 0.477599 GFP-Control 1.048767 0.559015 GFP-Control 1.045609 0.555991 GFP-Control 1.044393 0.413498 GFP-Control 1.043899 0.701141 GFP-Control 1.042501 0.727120 GFP-Control 1.041797 0.596280 GFP-Control 1.030078 0.544586 GFP-Control 1.018080 0.746441 GFP-Control 1.014789 0.701207 200 GFP-Control 1.010149 0.958673 GFP-Control 1.006125 0.875940 GFP-Control 1.005197 0.899216 GFP-Control 1.004508 0.891268 GFP-Control 0.996329 0.890316 GFP-Control 0.990736 0.813793 GFP-Control 0.990362 0.994875 GFP-Control 0.989534 0.968705 GFP-Control 0.987680 0.981875 GFP-Control 0.984609 0.905031 GFP-Control 0.980034 0.836319 GFP-Control 0.975764 0.747441 GFP-Control 0.973944 0.864026 GFP-Control 0.971159 0.897367 GFP-Control 0.968959 0.888858 GFP-Control 0.962044 0.478087 GFP-Control 0.961799 0.934211 GFP-Control 0.959449 0.951717 GFP-Control 0.951498 0.480183 GFP-Control 0.949018 0.744483 GFP-Control 0.947818 0.612266 GFP-Control 0.940700 0.444380 GFP-Control 0.938334 0.178634 GFP-Control 0.936164 0.748720 GFP-Control 0.935277 0.518793 GFP-Control 0.933481 0.389017 GFP-Control 0.932744 0.908251 GFP-Control 0.930030 0.414937 GFP-Control 0.925838 0.669011 GFP-Control 0.919144 0.770480 GFP-Control 0.913763 0.353311 GFP-Control 0.912675 0.508359 GFP-Control 0.911817 0.875077 GFP-Control 0.907427 0.343631 GFP-Control 0.903184 0.114940 GFP-Control 0.899543 0.537866 GFP-Control 0.894978 0.399149 GFP-Control 0.869033 0.076159 GFP-Control 0.867638 0.419373 GFP-Control 0.850792 0.052546 GFP-Control 0.846430 0.002573 GFP-Control 0.814539 0.227671 GFP-Control 0.812311 0.020823 GFP-Control 0.804644 0.047511 GFP-Control 0.686506 0.187485 GFP-Control 0.538687 0.612090 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 1.006744 0.996547 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 1.002014 0.988162 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 0.975115 0.752235 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 0.974302 0.695841 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 0.951271 0.550169 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 0.857829 0.171651 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 0.855477 0.103813 NM_002046 GAPDH Glyceraldehyde-3-phosphate dehydrogenase 0.824674 0.044534 AF218029 H3F3B H3 histone, family 3B (H3.3B) 1.038948 0.495769 AF218029 H3F3B H3 histone, family 3B (H3.3B) 1.003586 0.961562 AF218029 H3F3B H3 histone, family 3B (H3.3B) 0.968885 0.711352 AF218029 H3F3B H3 histone, family 3B (H3.3B) 0.923480 0.124930 AF218029 H3F3B H3 histone, family 3B (H3.3B) 0.896015 0.084222 201 AF218029 H3F3B H3 histone, family 3B (H3.3B) 0.854087 0.010235 AF218029 H3F3B H3 histone, family 3B (H3.3B) 0.824989 0.010652 AF218029 H3F3B H3 histone, family 3B (H3.3B) 0.775796 0.005712 M15035 gpt MPA - Xanthine guanine phosphoribosyltransferase 1.213503 0.207676 M15035 gpt MPA - Xanthine guanine phosphoribosyltransferase 1.164360 0.600562 M15035 gpt MPA - Xanthine guanine phosphoribosyltransferase 1.059769 0.548813 M15035 gpt MPA - Xanthine guanine phosphoribosyltransferase 1.020576 0.539109 M15035 gpt MPA - Xanthine guanine phosphoribosyltransferase 0.962912 0.946339 M15035 gpt MPA - Xanthine guanine phosphoribosyltransferase 0.873381 0.797657 M15035 gpt MPA - Xanthine guanine phosphoribosyltransferase 0.866626 0.375594 M15035 gpt MPA - Xanthine guanine phosphoribosyltransferase 0.686178 0.409494 NDK1 NDK1 NDK1 1.842349 0.191870 NDK1 NDK1 NDK1 1.713619 0.102483 NDK1 NDK1 NDK1 1.521871 0.489858 NDK1 NDK1 NDK1 1.313271 0.331844 NDK1 NDK1 NDK1 1.276237 0.596352 NDK1 NDK1 NDK1 1.222647 0.803863 NDK1 NDK1 NDK1 1.214176 0.832524 NDK1 NDK1 NDK1 1.032623 0.957793 NDK1 NDK1 NDK1 1.017270 0.805858 NDK1 NDK1 NDK1 1.010801 0.956049 NDK1 NDK1 NDK1 0.920441 0.717328 NDK1 NDK1 NDK1 0.872578 0.289445 NDK1 NDK1 NDK1 0.794864 0.754110 NDK1 NDK1 NDK1 0.665884 0.271131 NDK1 NDK1 NDK1 0.619484 0.084678 NDK1 NDK1 NDK1 0.521094 0.932313 NC_N1 NC_N1 Negative Control_N1 1.564936 0.705817 NC_N1 NC_N1 Negative Control_N1 1.025611 0.688093 NC_N1 NC_N1 Negative Control_N1 1.005630 0.848389 NC_N1 NC_N1 Negative Control_N1 0.966209 0.391972 NC_N1 NC_N1 Negative Control_N1 0.964266 0.875656 NC_N1 NC_N1 Negative Control_N1 0.940094 0.163284 NC_N1 NC_N1 Negative Control_N1 0.920840 0.091896 NC_N1 NC_N1 Negative Control_N1 0.914413 0.385506 NC_N1 NC_N1 Negative Control_N1 0.911967 0.293816 NC_N1 NC_N1 Negative Control_N1 0.898627 0.006943 NC_N1 NC_N1 Negative Control_N1 0.885450 0.033359 NC_N1 NC_N1 Negative Control_N1 0.883065 0.036959 NC_N1 NC_N1 Negative Control_N1 0.876633 0.007467 NC_N1 NC_N1 Negative Control_N1 0.864734 0.228948 NC_N1 NC_N1 Negative Control_N1 0.862219 0.001460 NC_N1 NC_N1 Negative Control_N1 0.842646 0.020060 NC_N2 NC_N2 Negative Control_N2 1.041449 0.731445 NC_N2 NC_N2 Negative Control_N2 1.041330 0.534733 NC_N2 NC_N2 Negative Control_N2 1.014790 0.724225 NC_N2 NC_N2 Negative Control_N2 1.014354 0.769509 NC_N2 NC_N2 Negative Control_N2 0.968201 0.687824 NC_N2 NC_N2 Negative Control_N2 0.890440 0.202514 NC_N2 NC_N2 Negative Control_N2 0.885558 0.265160 NC_N2 NC_N2 Negative Control_N2 0.869849 0.708348 NC_N2 NC_N2 Negative Control_N2 0.833630 0.170708 NC_N2 NC_N2 Negative Control_N2 0.821774 0.307062 NC_N2 NC_N2 Negative Control_N2 0.790588 0.202369 NC_N2 NC_N2 Negative Control_N2 0.775770 0.137188 NC_N2 NC_N2 Negative Control_N2 0.768202 0.372975 NC_N2 NC_N2 Negative Control_N2 0.747658 0.009641 NC_N2 NC_N2 Negative Control_N2 0.734347 0.080367 NC_N2 NC_N2 Negative Control_N2 0.621109 0.038855 202 NC_N3 NC_N3 Negative Control_N3 1.221637 0.205127 NC_N3 NC_N3 Negative Control_N3 1.161713 0.608263 NC_N3 NC_N3 Negative Control_N3 1.143881 0.377324 NC_N3 NC_N3 Negative Control_N3 0.964219 0.568178 NC_N3 NC_N3 Negative Control_N3 0.950491 0.868757 NC_N3 NC_N3 Negative Control_N3 0.927004 0.888828 NC_N3 NC_N3 Negative Control_N3 0.923822 0.905672 NC_N3 NC_N3 Negative Control_N3 0.914573 0.894014 NC_N3 NC_N3 Negative Control_N3 0.908235 0.962994 NC_N3 NC_N3 Negative Control_N3 0.864487 0.226095 NC_N3 NC_N3 Negative Control_N3 0.823245 0.093444 NC_N3 NC_N3 Negative Control_N3 0.809570 0.356488 NC_N3 NC_N3 Negative Control_N3 0.803589 0.120472 NC_N3 NC_N3 Negative Control_N3 0.787581 0.140850 NC_N3 NC_N3 Negative Control_N3 0.773114 0.133649 NC_N3 NC_N3 Negative Control_N3 0.603433 0.421886 NC_N5 NC_N5 Negative Control_N5 1.443052 0.715605 NC_N5 NC_N5 Negative Control_N5 1.352722 0.135082 NC_N5 NC_N5 Negative Control_N5 1.270996 0.455543 NC_N5 NC_N5 Negative Control_N5 1.229399 0.253185 NC_N5 NC_N5 Negative Control_N5 1.166800 0.377805 NC_N5 NC_N5 Negative Control_N5 1.130391 0.346664 NC_N5 NC_N5 Negative Control_N5 1.011218 0.770438 NC_N5 NC_N5 Negative Control_N5 0.960458 0.694140 NC_N5 NC_N5 Negative Control_N5 0.952509 0.813063 NC_N5 NC_N5 Negative Control_N5 0.928456 0.812088 NC_N5 NC_N5 Negative Control_N5 0.915459 0.993516 NC_N5 NC_N5 Negative Control_N5 0.910951 0.872961 NC_N5 NC_N5 Negative Control_N5 0.817865 0.240898 NC_N5 NC_N5 Negative Control_N5 0.654316 0.274503 NC_N5 NC_N5 Negative Control_N5 0.525622 0.138899 NC_N5 NC_N5 Negative Control_N5 0.514356 0.346240 AF264723 neo NEOMYCIN - Aminoglycoside-3'-phosphotransferase 1.426174 0.252390 AF264723 neo NEOMYCIN - Aminoglycoside-3'-phosphotransferase 1.386859 0.293967 AF264723 neo NEOMYCIN - Aminoglycoside-3'-phosphotransferase 1.227263 0.718116 AF264723 neo NEOMYCIN - Aminoglycoside-3'-phosphotransferase 1.102149 0.880148 AF264723 neo NEOMYCIN - Aminoglycoside-3'-phosphotransferase 1.008823 0.358213 AF264723 neo NEOMYCIN - Aminoglycoside-3'-phosphotransferase 0.884944 0.890223 AF264723 neo NEOMYCIN - Aminoglycoside-3'-phosphotransferase 0.790242 0.928064 AF264723 neo NEOMYCIN - Aminoglycoside-3'-phosphotransferase 0.654853 0.246601 NM_016553 NUP62 Nucleoporin 62kD 1.643939 0.185881 NM_016553 NUP62 Nucleoporin 62kD 1.100782 0.134747 NM_016553 NUP62 Nucleoporin 62kD 1.097558 0.179589 NM_016553 NUP62 Nucleoporin 62kD 1.092638 0.087445 NM_016553 NUP62 Nucleoporin 62kD 1.006540 0.810547 NM_016553 NUP62 Nucleoporin 62kD 0.971338 0.604648 NM_016553 NUP62 Nucleoporin 62kD 0.969719 0.677006 NM_016553 NUP62 Nucleoporin 62kD 0.933934 0.366862 M25346 pac PURIMYCIN N-Acetyltransferase (S. alboniger) 3.657761 0.512893 M25346 pac PURIMYCIN N-Acetyltransferase (S. alboniger) 1.028002 0.815360 M25346 pac PURIMYCIN N-Acetyltransferase (S. alboniger) 1.026649 0.901336 M25346 pac PURIMYCIN N-Acetyltransferase (S. alboniger) 0.893954 0.253321 M25346 pac PURIMYCIN N-Acetyltransferase (S. alboniger) 0.808827 0.006837 M25346 pac PURIMYCIN N-Acetyltransferase (S. alboniger) 0.800525 0.023850 M25346 pac PURIMYCIN N-Acetyltransferase (S. alboniger) 0.765104 0.024010 M25346 pac PURIMYCIN N-Acetyltransferase (S. alboniger) 0.728418 0.006156 AK023419 RPL37a Ribosomal protein L37A 0.958284 0.527895 AK023419 RPL37a Ribosomal protein L37A 0.932828 0.180346 AK023419 RPL37a Ribosomal protein L37A 0.904768 0.109153 203 AK023419 RPL37a Ribosomal protein L37A 0.877932 0.054668 AK023419 RPL37a Ribosomal protein L37A 0.863956 0.029053 AK023419 RPL37a Ribosomal protein L37A 0.861970 0.001551 AK023419 RPL37a Ribosomal protein L37A 0.849491 0.016032 AK023419 RPL37a Ribosomal protein L37A 0.768271 0.002747 NM_021104 RPL41 Ribosomal protein L41 (60S, HG12) 1.036171 0.629180 NM_021104 RPL41 Ribosomal protein L41 (60S, HG12) 0.929887 0.120827 NM_021104 RPL41 Ribosomal protein L41 (60S, HG12) 0.919265 0.206726 NM_021104 RPL41 Ribosomal protein L41 (60S, HG12) 0.917678 0.038296 NM_021104 RPL41 Ribosomal protein L41 (60S, HG12) 0.899487 0.113507 NM_021104 RPL41 Ribosomal protein L41 (60S, HG12) 0.897576 0.057351 NM_021104 RPL41 Ribosomal protein L41 (60S, HG12) 0.793321 0.061383 NM_021104 RPL41 Ribosomal protein L41 (60S, HG12) 0.792411 0.005852 NM_001021 RPS17 Ribosomal protein S17 0.909104 0.230369 NM_001021 RPS17 Ribosomal protein S17 0.899736 0.105351 NM_001021 RPS17 Ribosomal protein S17 0.894875 0.097540 NM_001021 RPS17 Ribosomal protein S17 0.883096 0.655064 NM_001021 RPS17 Ribosomal protein S17 0.863176 0.028946 NM_001021 RPS17 Ribosomal protein S17 0.834702 0.002643 NM_001021 RPS17 Ribosomal protein S17 0.758972 0.000310 NM_001021 RPS17 Ribosomal protein S17 0.730181 0.000270 AL034379 RPS27 Ribosomal protein S27 1.128807 0.132651 AL034379 RPS27 Ribosomal protein S27 1.066045 0.196501 AL034379 RPS27 Ribosomal protein S27 1.047386 0.378683 AL034379 RPS27 Ribosomal protein S27 1.042813 0.563260 AL034379 RPS27 Ribosomal protein S27 1.029726 0.711324 AL034379 RPS27 Ribosomal protein S27 0.976316 0.510350 AL034379 RPS27 Ribosomal protein S27 0.962438 0.476288 AL034379 RPS27 Ribosomal protein S27 0.838590 0.010893 NM_001013 RPS9 Ribosomal protein S9 0.985912 0.869755 NM_001013 RPS9 Ribosomal protein S9 0.951073 0.394542 NM_001013 RPS9 Ribosomal protein S9 0.869374 0.040281 NM_001013 RPS9 Ribosomal protein S9 0.847976 0.000633 NM_001013 RPS9 Ribosomal protein S9 0.828876 0.002986 NM_001013 RPS9 Ribosomal protein S9 0.816042 0.001444 NM_001013 RPS9 Ribosomal protein S9 0.799754 0.000186 NM_001013 RPS9 Ribosomal protein S9 0.796959 0.003871 AK001313 RPLP0 Ribosomal protein, large, P0 1.138434 0.441591 AK001313 RPLP0 Ribosomal protein, large, P0 1.059897 0.229369 AK001313 RPLP0 Ribosomal protein, large, P0 1.042162 0.279066 AK001313 RPLP0 Ribosomal protein, large, P0 1.026556 0.435711 AK001313 RPLP0 Ribosomal protein, large, P0 1.012724 0.711921 AK001313 RPLP0 Ribosomal protein, large, P0 0.998578 0.997623 AK001313 RPLP0 Ribosomal protein, large, P0 0.976122 0.534154 AK001313 RPLP0 Ribosomal protein, large, P0 0.957830 0.216206 BI198347 RPLP1 Ribosomal protein, large, P1 1.351753 0.372986 BI198347 RPLP1 Ribosomal protein, large, P1 1.320017 0.166125 BI198347 RPLP1 Ribosomal protein, large, P1 1.223939 0.127324 BI198347 RPLP1 Ribosomal protein, large, P1 1.207411 0.414771 BI198347 RPLP1 Ribosomal protein, large, P1 1.115501 0.401144 BI198347 RPLP1 Ribosomal protein, large, P1 1.072582 0.707395 BI198347 RPLP1 Ribosomal protein, large, P1 1.041357 0.944258 BI198347 RPLP1 Ribosomal protein, large, P1 0.891959 0.935829 AK055976 TMSB4X Thymosin, beta 4, X chromosome 0.982417 0.731631 AK055976 TMSB4X Thymosin, beta 4, X chromosome 0.959842 0.470862 AK055976 TMSB4X Thymosin, beta 4, X chromosome 0.952760 0.397004 AK055976 TMSB4X Thymosin, beta 4, X chromosome 0.930307 0.317250 AK055976 TMSB4X Thymosin, beta 4, X chromosome 0.909007 0.406190 AK055976 TMSB4X Thymosin, beta 4, X chromosome 0.891040 0.632133 204 AK055976 TMSB4X Thymosin, beta 4, X chromosome 0.825296 0.072870 AK055976 TMSB4X Thymosin, beta 4, X chromosome 0.819117 0.042763 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.516300 7.00E-05 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.454935 8.06E-05 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.410225 0.000244 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.370580 2.90E-09 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.369174 1.96E-07 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.364405 2.44E-08 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.363009 1.39E-08 NM_006082 K-ALPHA-1 Tubulin, alpha, ubiquitous 0.357775 7.04E-08 205

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