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Investigation of the collagenolytic activity of cathepsin K complexes by site-directed mutagenesis Takimoto, Shinako 2013

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 Investigation of the Collagenolytic Activity of Cathepsin K Complexes by Site-Directed Mutagenesis   by Shinako Takimoto  B.S., The University of Georgia, 2000  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2013  ? Shinako Takimoto, 2013 ii  Abstract  Cathepsin K (catK) is a lysosomal cysteine protease predominantly expressed in osteoclasts.  It is the most potent collagenase and elastase in human and involved in a variety of physiological functions including bone degradation, wound healing, maturation of hormones, and a range of important proteolytic activities required for normal cellular function.  The main organic constituent of bone, type I collagen, has a highly organized and tightly packed structure and is resistant to proteolysis by most proteases.  CatK is the only human protease that efficiently cleaves triple helical collagen, leading to the complete degradation of bone organic matter.  This enzyme is the main protease expressed by osteoclasts and is responsible for bone degradation during bone resorption.  In recent years, it has been shown that catK forms a complex with bone associated glycosaminoglycans (GAGs) to gain this collagenase activity.  However, precise mechanism remains unclear.  Due to the major role in bone resorption, catK has been a pharmaceutical target for osteoporosis treatment.  Several catK inhibitors have been developed, yet adverse side effects remain a concern.  A major issue of the active site inhibitors is its interference to other functions of this enzyme.  Gaining the insight of mechanical details of collagenolytic activity of catK can lead to substrate-targeting specific inhibitors that can treat osteoporosis with minimum side effects.  There are two catK-GAG complex models based on x-ray crystallography developed in our laboratory; the dimer and the tetramer models. In this study, mutant proteases were made to assess the role of specific protein interaction sites in these proposed models.  Mutation at one residue in particular, N99, exhibited 40~50% reduction of degradation activity toward soluble and insoluble collagen without affecting regular proteolytic activity.  The atomic force microscopy analysis revealed that the complex formation, iii  observed in the wild type enzyme, was compromised in this mutant protease.  This indicates that N99 contributes to the protein interaction necessary for the collagenolytic catK complex formation.  Additionally, two mutant proteases showed facilitated collagenase activity against insoluble fibers.  These finding contribute to gain better understanding of catK?s collagenolytic activity and will lead development of exosite inhibitors to treat osteoporosis in the future. iv  Preface  This research was designed, carried out, and analyzed by myself under general supervision of Dr. Dieter Br?mme except for the atomic force microscopy experiment (AFM).  AFM experiment was carried by Dr. Preety Panwar.  The two structural models used in this project were developed by Dr. Adeleke Aguda and Xin Du of the laboratory. v  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Tables ................................................................................................................................ ix List of Figures .................................................................................................................................x List of Symbols ............................................................................................................................ xii List of Abbreviations ................................................................................................................. xiii Acknowledgements ......................................................................................................................xv Chapter 1: Introduction ................................................................................................................1 1.1 Human cathepsin K ......................................................................................................... 1 1.2 Bone remodeling and its regulation ................................................................................ 2 1.3 Collagen structure ........................................................................................................... 4 1.4 CatK and collagen degradation ....................................................................................... 5 1.5 Osteoporosis and catK inhibitors .................................................................................... 6 1.6 Project overview ............................................................................................................. 7 Chapter 2: Materials and Methods ............................................................................................12 2.1 Enzyme synthesis .......................................................................................................... 12 2.1.1 Subcloning/mutagenegsis ......................................................................................... 12 2.1.2 Yeast transformation and protein expression ............................................................ 12 2.1.3 Enzyme activation ..................................................................................................... 14 2.1.4 Protein purification ................................................................................................... 14 vi  2.1.5 Western blotting ........................................................................................................ 15 2.1.6 E-64 Titration?active enzyme concentration determination ................................... 16 2.2 Validation of enzyme .................................................................................................... 16 2.2.1 Michaelis-Menten kinetics ........................................................................................ 16 2.2.2 Stability ..................................................................................................................... 16 2.2.3 Thermal shift assay ................................................................................................... 17 2.3 Enzymatic activity assay ............................................................................................... 17 2.3.1 Z-FR-MCA hydrolysis assay .................................................................................... 17 2.3.2 Collagen degradation ................................................................................................ 17 2.3.2.1 Degradation of soluble collagen ....................................................................... 18 2.3.2.2 Degradation of insoluble collagen .................................................................... 18 2.3.2.3 SDS-PAGE analysis.......................................................................................... 18 2.3.2.4 SEM analysis .................................................................................................... 19 2.3.3 Elastin degradation.................................................................................................... 19 2.3.4 Gelatin degradation ................................................................................................... 19 2.4 Validation of complex formation .................................................................................. 20 Chapter 3: Results........................................................................................................................21 3.1 Mutation at P88, M97, N99, and T101 ......................................................................... 21 3.1.1 Kinetics and stability................................................................................................. 22 3.1.2 Collagenase and elastase activity .............................................................................. 23 3.1.3 Atomic Force Microscopy (AFM) ............................................................................ 27 3.2 Mutation at N175 and K176 ......................................................................................... 28 3.2.1 Kinetics and stability................................................................................................. 29 vii  3.2.2 Collagenase and elastase activity .............................................................................. 30 3.3 Mutation at K106 and R108 .......................................................................................... 32 3.3.1 Kinetics and stability................................................................................................. 32 3.3.2 Collagen and elastin degradation .............................................................................. 34 3.4 Mutation at S138, L139, T140, N199, and K200 ......................................................... 36 3.4.1 Mutation at S138, L139, and T140 ........................................................................... 37 3.4.2 Mutation at N199 and K200 ..................................................................................... 42 Chapter 4: Discussion ..................................................................................................................45 4.1 Effects of MUTN99A ...................................................................................................... 45 4.2 Effects of T101, K106 and R108 mutation ................................................................... 48 4.3 Potential significance of T 101, K106, and R108 ......................................................... 48 4.4 Destabilizing mutations ................................................................................................ 51 4.4.1 Mutation at S138, L139, and T140 ........................................................................... 51 4.4.2 Mutation at N199 and K200 ..................................................................................... 53 Chapter 5: Conclusion .................................................................................................................55 References .....................................................................................................................................57 Appendices ....................................................................................................................................65 Appendix A Cloning ................................................................................................................. 65 A.1 Primers ...................................................................................................................... 65 A.2 PCR site-directed mutagenesis ................................................................................. 66 A.3 pUC19N .................................................................................................................... 68 Appendix B Protein purification ............................................................................................... 69 B.1 Purification buffer ..................................................................................................... 69 viii  B.2 FPLC program .......................................................................................................... 69 B.3 SDS-PAGE gel.......................................................................................................... 70 Appendix C Culture medium recipes for Pichia pastoris ......................................................... 72  ix  List of Tables  Table 1.1 Mutation overview ..................................................................................................... 11 Table 3.1 Michaelis-Menten kinetics of MUTM97A/N99A/T101A, MUTP88A, MUTM97A, MUTN99A, MUTT101A ..................................................................................................................................... 22 Table 3.2 Melting temperature (Tm) of MUTM97A/N99A/T101A, MUTN99A, MUTT101A, MUTN175A/K176A, MUTK106A/R108T ................................................................................................. 23 Table 3.3 Michaelis-Menten kinetics of MUTN175A/K176A ......................................................... 30 Table 3.4 Michaelis-Menten kinetics of MUTK106A/R108T .......................................................... 33 Table 3.5 Structural stability prediction on single mutation .................................................. 40  x  List of Figures  Figure 1.1 Cathepsin K structure .................................................................................................... 2 Figure 1.2 Osteoclast regulation and bone resorption .................................................................... 4 Figure 1.3 Collagen structure .......................................................................................................... 5 Figure 1.4 Complex formation with CS-A ..................................................................................... 9 Figure 1.5 Roles of different cathepsin K complexes in collagen degradation ............................ 11 Figure 3.1 Molecular location of P88, M97, N99 and T101 ......................................................... 21 Figure 3.2 Stability assay of MUT97/99/101, MUTM97A, MUTN99A, MUTT101A ................................ 23 Figure 3.3 SDS-PAGE analysis of type I collagen degradation ................................................... 25 Figure 3.4 SEM images of type I collagen fiber treated with various mutants ............................ 26 Figure 3.5 Elastin and gelatin degradation by MUTM97A/N99A/T101A, MUTM97A, MUTN99A and MUTT101A ...................................................................................................................................... 27 Figure 3.6 AFM imaging of MUTN99A in the presence or absence of CS-A ................................ 28 Figure 3.7 M175 and K175 molecular location ............................................................................ 29 Figure 3.8 MUTN175A/K176A stability .............................................................................................. 30 Figure 3.9 MUTN175A/K176A activity ............................................................................................... 31 Figure 3.10 Molecular location of K106/C107/R108 interface .................................................... 32 Figure 3.11 MUTK106A/R108T stability ............................................................................................. 33 Figure 3.12 MUTK106A/R108T activity.............................................................................................. 35 Figure 3.13 Molecular location of residues S138/L139/T140 and N199/K200 ........................... 36 Figure 3.14 Western blotting for MUTS138G/L139H/T140E ................................................................. 38 Figure 3.15 Sequence alignment for S138, L139, and T140 ........................................................ 41 xi  Figure 3.16 MUTN199A/K200A activation by western blotting ......................................................... 42 Figure 3.17 Sequence alignment of N199 and K200 .................................................................... 44 Figure 4.1 T101 and K106/R108 role in collagen degradation .................................................... 49 Figure 4.2 Structural comparison between catK with CS-A and MMP-1 .................................... 50 Figure 4.3 Structural analysis of S138, L139, T140 region of catK ............................................. 52 Figure 4.4 Structural analysis of N199 and K200......................................................................... 54  xii  List of Symbols  All mutant enzymes generated in this study were symbolized as MUT, followed by original residue-residue number in active form-new residue in subscript.  For instance, when methionine at 97 was mutated into alanine, it was named MUTM97A. Mutant Names Residue Location in Active Enzyme Original Residue Residue Mutated To MUTP88A 88 Proline (P) Alanine (A) MUTM97A/N99A/T101A 97 99 101 Methionine (M) Asparagine (N) Theronine (T) Alanine (A) MUTM97A 97 Methionine (M) Alanine (A) MUTN99A 99 Asparagine (N) Alanine (A) MUTT101A 101 Threonine (T) Alanine (A) MUTN175A/K176A 175 176 Asparagine (N) Lysine (K) Alanine (A) MUTN199A/K200A 199 200 Asparagine (N) Lysine (K) Alanine (A) MUTN199A 199, Asparagine (N) Alanine (A) MUTK200A 200 Lysine (K) Alanine (A) MUTK106A/R108T 106 108 Lysine (K) Arginine (R) Alanine (A) and threonine (T) MUTS138G/L139H/T140E 138 139 140 Serine (S) Leucine (L) Threonine (T) Glycine (G) Histidine (H) Glutamic acid (E) MUTS138G/T140E 138 140 Serine (S) Threonine (L) Glycine (G) Glutamic acid (E) MUTL139H/T140E 139 140 Leucine (L) Threonine (T) Histidine (H) Glutamic acid (E)   xiii  List of Abbreviations  AFM: Atomic force microscopy AOX: alcohol oxidase BMGY: buffered glycerol complex medium BMMY: buffered methanol complex medium CatK: Cathepsin K CS-A: chondroitin sulfate A CTL: control DMSO: Dimethyl sulfoxide DTT: dithioerythreitol EDTA: ethylenediaminetetraacetic acid FPLC: fast protein liquid chromatography GAGs: glycosaminoglycans HRP: horseradish peroxidase IgG: immunoglobulin G kDa: kilodalton  MD: minimal defined MM: minimal medium MMP: matrix metalloprotease MW: molecular weight MWCO: molecular weight cut off PBS: phosphate buffered saline xiv  PCR: polymerase chain reaction rpm: revolutions per minute RT: room temperature SDS-PAGE: sodium dodecyl sulfate polyacrylamide electrophoresis SEM: scanning electron microscopy TBS: Tris buffered saline Tm: melting temperature WT: wild type ZFR-MCA: carbobenzoxy-phenylalanine-arginine-4-methylcoumarin-7-amide xv  Acknowledgements  I offer my gratitude to the department of biochemistry and molecular biology and particularly Dr. Dieter Br?mme, who took me into the laboratory and support me through my graduate program.  I am thankful to my advisory committee members, Dr. Filip Van Petegem and Dr. Francis Jean.  I owe special thanks to Dr. Preety Panwar, who offered me endless support and tremendous help during my study in Dr. Br?mme lab.  Also, I thank all laboratories that allowed me to invade their space and equipment, Van Petegem Lab, Eltis Lab, Murphy lab, Jan lab, Hongbin Li lab, and The Center for High-Throughput Phenogenomics. Also I appreciate the support from my friends, Dr. Sanja Arandjelovic, Dr. Alban Gaultier, and Dr. Minji Jo who inspired me to be a better researcher and person.  1  Chapter 1: Introduction  1.1 Human cathepsin K Cathepsins are papain-like proteases involved in multiple biological processes 1-3.  There are 11 human cathepsins known 1, 2.  The focus of my project, cathepsin K (catK), is a lysosomal cysteine protease that degrades extracellular matrix proteins.  It is known to possess strong gelatinase, collagenase, and elastase activities.  It has been also linked to specific diseases such as osteoporosis, atherosclerosis, neurological disease, cancer, and arthritis 1, 2, 4-7.  CatK is one of the most potent mammalian collagenases and can completely degrade type I and type II collagen, which are the main organic components of bones and cartilages 8, 9.  CatK is predominantly expressed in osteoclasts, one of the bone resident cells which degrade the bone matrix, and play vital roles in bone homeostasis 9-12.  Overactive osteoclasts and elevated activity of catK can lead to diseases related to low bone density such as osteoporosis.  On the other hand, deficiency of catK causes a rare autosomal recessive disease known as pycnodysostosis which is characterized by osteosclerosis 11. All the cysteine cathepsins share structural similarity.  Particularly cathepsin S (catS) and cathepsin L (catL) have similar amino acid sequences and structures, yet those cathepsins lack collagenolytic activity.  Cathepsins are expressed as preproproteins from which the propeptide is cleaved during activation, revealing the active site of the protein.  This propeptide can act independently as a catK inhibitor by binding to the active site 13. CatK has two distinctive domains, a right domain and a left domain with an active cleft in between (Figure 1.1).  The right domain has four beta sheets in barrel structure with one of the catalytic diad residues, H162, and the left domain has three alpha helices with the other catalytic 2  diad residue, C25.  One of the distinctive features of catK is that it is positively charged on the opposite side of the active cleft, unlike other cathepsins such as catL or catS.  There are three disulfide bonds, two in the left and one in the right domain.  Two of them (one on each side) are located in close proximity to the active site.     1.2 Bone remodeling and its regulation Bone remodeling is a highly regulated process consisting of two parts; resorption and formation14.  Resorption is performed by osteoclasts, and osteogenesis is carried out by osteoblasts.  Both osteoclasts and osteoblasts are differentiated from bone marrow monocytes/macrophage 15.  Osteoclastogenesis is promoted by binding of the receptor activator Figure 1.1 Cathepsin K structure R domain in light grey and L domain in dark grey.  Catalytic diad is in red (C25 and H162), and disulfide bonds are in yellow. C25 H162 3  of nuclear factor kB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) to their receptors expressed and present on osteoclast precursor cell surfaces 16 (Figure 1.2).  Both RANKL and M-CSF are expressed by bone marrow stromal cells and osteoblasts which are required for osteoclastogenesis 16.  Osteoprotegerin (OPG) can decelerate this process by binding to RANKL competitively to inhibit the RANK-RANKL interaction 17.  On the other hand, parathyroid hormone (PTH), tumor necrosis factor (TNF), and lipopolysaccharide (LPS) are known osteoclastogenic factors acting through upregulation of RANKL 18-20.  Interleukin 1 (IL-1) is also an osteoclastogenic factor acting through upregulation of M-CSF 21.  Estrogen interferes with this upregulation of M-CSF and inhibits osteoclastogenesis 21. Osteoclasts first attach themselves onto the bone surface via cell adhesion and create a microenvironment called the resorption lacuna 22, 23.  This cell adhesion creates a tight seal, visualized by the so-called actin ring 23, 24, which induces the reorganization of the cytoskeleton and initiates bone resorption (Figure 1.2) 25, 26.  More specifically, cells are polarized and create a ruffled membrane and acidify the resorption lacuna via an ATPase proton pump on the ruffled membrane 27, 28.  This acidification has two roles: i) demineralization of the bone matrix and ii) establishment of functional environment for lysosomal proteases.  CatK, along with other lysosomal cathepsins and MMPs, is released into the lacuna to degrade the organic matter of bone after the demineralization 22, 29.  CatK is known to be the most potent collagenase among those proteases and is responsible for collagen degradation in bones 14, 30.  4   1.3 Collagen structure Bone is a rigid structural organ consisting of three major components; 70% inorganic minerals, 22% organic matter and 8% water 31.  Organic components include adhesion proteins, but the major part of the organic matter is collagen, particularly type I collagen, which represents 90% of the organic matter 31.  Collagen is the most abundant protein in the human body, and type I collagen is the most abundant form of collagen.  It is a fibrous protein and has two ?1 chains and one ?2 chain to form a triple helix 32-35.  Triple helices are then staggered with neighboring helices to form microfibrils which are arranged in a quasi hexagonal orientation 36.  These microfibrils are then staggered and stacked laterally to form a collagen fiber 37-39.  All triple Figure 1.2 Osteoclast regulation and bone resorption Osteoclasts are differentiated from macrophages by osteoblast stimulation.  Mature osteoclasts adhere and create an acidified resorption lacuna into which catK is released.  (Figure adopted from Yasuda H. 19984) 5  helices are cross-linked upon assembly and tightly packed into collagen fibers that are highly stable and insoluble in the physiological environment (Figure1.3).   1.4 CatK and collagen degradation CatK is one of the most potent human collagenases, which efficiently degrades triple helical type I and type II collagen, the main organic constituents of bones and cartilage 8, 9.  CatK Figure 1.3 Collagen structure Two ?1 chains and one ?2 chain create a triple helical collagen monomer and assemble into collagen microfibril, and fibril39.  Fibrils then bundle together to form collagen fiber.  Due to staggering, microfibrils show dark gap region and lighter overlap region (combined region is called D-period)37.  Tropocollagens are cross-linked to each other to form a microfibril, and fibril bundles are tied together with GAGs to make the fiber insoluble within the physiological environment. 6  can cleave a collagen molecule in its triple helical region multiple times and degrades insoluble collagen completely 9.  In contrast, the best characterized collagenolytic enzymes, metalloproteases, only cleave collagen once, leaving 1/4 and 3/4 fragments of collagen molecules 40-44.  The mechanism of how catK degrades collagen is still unknown.  However, in the last decade, it has been shown that glycosaminoglycans (GAGs) are essential for catK collagenolytic activity 45, 46.  GAGs bind to positively charged areas of catK located on the opposing side of the active cleft to form an activity specific complex.  This complex formation is unique to catK and cannot be observed in any other collagenolytic proteases, such as MMPs 47.  Interestingly, catL or catS, which have 60% similarity in their amino acid sequences with catK but lack any collagenolytic activity, do not form such complexes, suggesting that this process is very important in catK collagenase activity 45, 46, 48.  Moreover, this complex is not required for the non-collagenolytic activity of catK such as its gelatinase and elastase activities 49, 50.  This complex structure still needs to be studied in further details.  It has a great potential to be a therapeutic target for the treatment of osteoporosis, since the disruption of this complex can lead to the specific inhibition of the enzyme?s collagenase activity. 1.5 Osteoporosis and catK inhibitors Osteoporosis is a common disease affecting 1 in 3 women and 1 in 5 men during their life time.  Deterioration of bone and low bone mass density are the main symptoms and can lead to osteoporotic fractures (Osteoporosis Canada, www.osteoporosis.ca).  Therapeutic treatments include hormone replacement therapy (HRT), bisphosphonate treatment, and hormone modulator or PTH derivatives treatment 49, 51.  HRT is a common treatment for post-menopausal patients.  Estrogen is an inhibitor of osteoclastogenesis, and thus withdrawal of estrogen is a major cause of osteoporosis for post-menopausal women.  Estrogen is administered to such patients as a part 7  of the treatment for osteoporosis.  However, HRT is now shown to have serious adverse effects including increase chances of heart attacks, strokes, and blood clots 52.  Bisphosphonates, which are currently the main treatment option, are anti-resorptive drugs which induce apoptosis in osteoclasts and block the small GTPase 51, 53.  Although they have been shown to be potent osteoporosis drugs, they are now linked to jaw, bone and tissue necrosis 54.  Some catK active site inhibitors such as Balicatib (Novartis), ONO-5334 (Ono Pharmaceutical), and Odanacatib (Merck & Co., Inc) were developed and progressed to clinical trials.  However, some of them lack the specificity and inhibit other cathepsins leading to morphea like syndrome, or interfere with typical anti-inflammatory drugs prescribed to osteoporotic patients 55-57.  More catK inhibitors are in development with tolerable adverse effects.  The most promising catK inhibitor yet, Odanacatib, is a highly specific and potent active site inhibitor of catK and just passed phase III clinical trials 58-60.  This inhibitor shows inhibition of bone resorption without affecting bone formation by osteoblasts as a preventative medication for postmenopausal women 58.  Development of novel catK inhibitors is in high demand, and activity specific inhibitors of catK could eliminate issues associated with active site inhibitors such as the blocking of essential non-collagenolytic activities of catK.  1.6 Project overview We have two complex models to explain the collagenolytic activity of catK based on x-ray diffraction crystallography done previously in our laboratory: the dimer and tetramer models (Figure 1.4).  In the dimer model, one catK molecule binds to GAG, and one catK molecule interacts with the other one already bound to GAG in a head to tail orientation, exposing their active sites to opposite ends of this complex (Figure 1.4A).  In contrast, in the tetramer model, two catK molecules bind to one strand of GAG and two of its subunits come together in head to 8  head orientation.  In this model, GAG is binding to a positively charged region of catK (Figure 1.4B).  Our hypothesis is that monomers, dimers, and tetramers are working together to degrade type I collagen in bone and other organs (Figure 1.5).  First, monomeric catK cleaves off the telopeptide from collagen fibers and hydrolyzes proteoglycans, releasing soluble GAGs from collagen fibers.  Then dimer complexes cleave the collagen cross-links, releasing soluble tropocollagen molecules and the disintegrate collagen fibers.  Finally, catK molecules form tetrameric complexes with released GAGs and degrade solubilized collagen into smaller peptides.  9   Figure 1.4 Complex formation with CS-A A) Dimer model.  Two catKs are in head to tail orientation with two strands of CS-A interacting with one of them.  Protein interfaces of dimer model are P88/M97/N99/T101 on one side in orange and N175/K176/N199/K200 on the other side in green. B) Tetramer model.  One strand of CS-A is binding to two catKs.  Two of this subunit interact in a head to head orientation to form the tetramer.  Protein interface residues are P88/M97/N99/T101 (orange), K106/C107/R108 (wine red) and S138/L139/T140 (lime green).  They are involved in interaction between two molecules along the same GAG molecule.  P88/M97/N99/T101 interface is involved in both the dimer and tetramer models. CS-A is in blue. A B N175 K176 K200 N199 M97 P88 N99 T101 K106 R108 S138 L139 T140 10  My project is to test the potential protein-protein interaction sites involved in these complex models.  By introducing mutations into these sites and studying the effects on activities, we gain structural and functional insights into the collagenolytic activity of catK, which in turn may lead to the design of selective anti-collagenase inhibitors of this multitasking protease for the treatment of osteoporosis.  Mutation sites were selected based on available x-ray structures of the dimer and tetramer models.  For the dimer model, eight residues (P88, M97, N99, T101, N175, K176, N199, and K200) were selected.  Those sites were mutated to alanine, which has a functional methyl group without the bulkiness that might affect the structure of the protein (Figure 1.4A).  For the tetramer model, five residues (K106, R108, S138, L139, and T140) were identified and mutated into the corresponding catL residues (Figure 1.4B).  Three residues (P88, M97, N99, T101), identified in the dimer model, seem to be involved in the tetramer model as well (Figure 1.4).  The overview of these mutations is found in table 1.1.  All mutants were kinetically evaluated and assayed for specific activities including their collagenolytic and elastolytic activities.  Degradation of collagen was visualized on SDS-PAGE and quantified.  Degradation of insoluble collagen isolated from rat tails was examined by scanning electron microscopy (SEM).  Complex formation was examined by atomic force microscopy (AFM).  Together, these studies gain a structural insight of catK in the association of GAGs and its collagenolytic activity.  11   Table 1.1 Mutation overview Residues Mutation Affecting model N175, K176, N199, K200 Alanine scan Dimer P88, M97, N99, T101 Alanine scan Dimer and tetramer K106, R108 catL like (A, T respectively) Tetramer S138, L139, T140 catL like (G, H, E respectively) Tetramer  Figure 1.5 Roles of different cathepsin K complexes in collagen degradation Monomeric catK cleaves off proteoglycans and telopeptides (red) from collagen microfibrils.  Dimeric catK removes the cross-links between the microfibrils.  Tetrameric catK with the released GAGs degrades solubilized toporocollagens. telopeptides GAGs Soluble GAGs monomer collagen fragments toporocollagen tetramer dimer 12  Chapter 2: Materials and Methods  2.1 Enzyme synthesis CatK and its variants were expressed as proenzymes in Pichia pastoris and activated by pepsin before purifying by affinity chromatography.  Active site concentrations of the proteins were determined by inhibitor (E-64) titration.   2.1.1 Subcloning/mutagenegsis In order to perform site-directed mutagenesis, first the catK cDNA was subcloned into the cloning vector, pUC19N (Appendix A.3), using Not I and EcoR I sites.  To create the Not I site in pUC19 (Genebank accession # L09137), PCR site-directed mutagenesis was performed, and the newly generated vector was named pUC19N.  After the subcloning of catK into pUC19N was completed, this construct was used to generate all of the catK mutants described in the studies.  PCR site-directed mutagenesis was performed to generate mutants, and once the mutations were confirmed by DNA sequencing (Genewiz, South Plainfield, NJ), the catK cDNA was subcloned into the pPIC9 vector (Life Technologies, Carlsbad, CA) for expression of the recombinant protein.  All primers were synthesized at Integrated DNA Technology (Coralville, IA), and the sequences are listed in Appendix A.1.  The conditions for the PCR site-directed mutagenesis are listed in Appendix A.2.  All the endonucleases and the T4 DNA ligase used for subcloning were purchased from New England Biolabs (Ipswich, MA).  The dNTPs and pfu DNA polymerase were purchased from BioBasic Inc. (Markham, ON). 2.1.2 Yeast transformation and protein expression The pPIC9 vector containing the catK mutant gene was purified and linearized with Sac I.  The digested plasmid was purified by ethanol precipitation and resuspended in 10 ?l of H2O.  13  The plasmid was then electroporated into Pichia pastoris GS115 (Life Technologies, Carlsbad, CA) at 2,000 V for 5 milli-seconds using the Eppendorf electroporator (Hamburg, Germany).  The electroporated cells were resuspended in sterile 1 M sorbitol and plated on minimal dextrose (MD) agar plates and incubated at 30?C for 3 days.  On day 3, colonies were re-streaked on MD plates and minimal methanol (MM) plates and screened for catK protein expression.  Since the pPIC9 vector carries the promoter for alcohol oxidase, only colonies with pPIC9 integrated into their genome can grow on MM plates.  Those colonies were used to inoculate 5 mL of buffered minimal glycerol complex medium (BMGY) in 50 mL conical tubes and cultured for overnight.  Next day, the cells were span down and resuspended in 5 mL of minimal methanol complex medium (BMMY) to start the induction.  Between day 3-7, the activity of the enzyme was monitored to see which colonies yield more protein.  Since protein is secreted, 100?l of activated condition medium (CM) were taken to monitor the activity using Z-FR-MCA, small synthetic peptide (methods were described in 2.1.3 and 2.3.1).  Since the alcohol oxidase (AOX1) promoter is driving the expression of the protein, culture was induced by daily addition of methanol to 0.5%.  The colonies with higher expression level were selected for larger scale expression.  For large volume expression, first, 5 mL BMGY was inoculated from fresh colony and cultured at 30?C with 200 rpm shake for 2 days.  Then 500 mL BMGY was inoculated with 5mL culture and grown for 2 days.  After 2 day, cells were collected and resuspended in 1 L BMMY to start protein production.  Protein expression was induced with daily addition of methanol to a final concentration of 0.5% for 5~7 days.  During induction period, potential contamination was monitored by light microscope examination, and expression levels were measured by Z-FR-MCA hydrolysis daily (2.3.1). 14  Culture was collected and spun down at 1,000 xg for 10 minutes using Beckman Coulter Avanti centrifuge (Pasadina, CA).  The supernatant was concentrated down to ~30 mL using Prep/Scale cartridge and Amicon concentrator with 3KMWCO membrane (EMD Millipore, Billerica, MA). The recipes of all media used for protein expression are listed in Appendix C. 2.1.3 Enzyme activation Prior to chromatographic purification, concentrated culture media was activated with 0.6 mg/ml pepsin (Sigma-Aldrich, St. Louis, MO) at pH 4.3 at 37?C.  Activation can be quite rapid once it starts as activated catK can activate proenzymes as well.  Activation was monitored using Z-FR-MCA every 5 minutes (method is described in 2.3.)  Once the activation slowed down, the mixture was moved on to the ice.  Pepsin activation was terminated by adjusting pH to 5.5 with potassium hydroxide. For screening, ~500 ?l of condition medium (CM) was taken out from the culture, and pH was adjusted to 4.0.  Then CM was activated with pepsin for 10 minutes, and activity was measured using 100 ?l of activated CM. 2.1.4 Protein purification Purification was done in 2-step chromatography utilizing FPLC (GE Healthcare Life Science, Cleveland, OH).  Then enzymes were first purified using an N-butyl Sepharose column followed by SP-Sepharose chromatography (GE Healthcare Life Science, Cleveland, OH).  Ammonium sulfate was added to final concentration of 2 M to activated catK solution to final volume of 50 mL.  The mixture was then centrifuged to remove any insoluble impurities at 6000 rpm for 10 minutes at 4?C and loaded onto a N-butyl Sepharose column at 1 mL/min.  The protein was eluted with a 2 to 0 M ammonium sulfate gradient in buffer B.    After N-butyl Sepharose purification, the active fractions were pooled, concentrated, and dialyzed against 15  Buffer B (Appendix B.1) for 2 hours at 4?C before loaded onto a SP-Sepharose column.  Here the protein was eluted using 0 to 2 M sodium chloride gradient in buffer B.  After SP-Sepharose purification, the protein was concentrated, and stored in 100 mM sodium acetate (pH 5.5) with 2.5 mM DTT and EDTA at -80?C in aliquots.  The elution of the proteins from the columns was monitored at 280 nm, and the presence of active catK was measured by Z-FR-MCA hydrolysis.  The FPLC program for the purification is listed in Appendix B.2., and coomassie stained gels of different steps of purification and all purified mutant enzymes are shown in Appendix B.3. 2.1.5 Western blotting Western blotting analysis was performed to confirm expressions and purifications of some of the mutant proteases.  A pre-activation sample, an activated sample, a N-butyl Sepharose purified sample, and a SP-Sepharose purified were run on a SDS-PAGE gel.  The proteins were transferred to PVDF membrane in wet-transfer system at 300 mA for 1.5 hours at 4?C.  The membranes were then blocked with 5% non-fat dry milk in TBST (0.05% Tween-20 in TBS buffer) for 1 hour at room temperature and incubated with anti-human catK rabbit polyclonal antibody (generated in Br?mme laboratory) for overnight at 4?C in 5% non-fat dry milk in TBST.  The membrane was then washed with TBST for 5 minutes three times.   Then membrane was blotted with goat secondary antibody conjugated with horseradish peroxidase (HRP) against mouse IgG (Southern Biotech, Birmingham, AL) in 1% non-fat dry milk in TBST.  The secondary antibody was visualized by Luminata classic western HRP substrate (EMD Millipore, Billerica, MA) with ChemiGenius2 Bioimaging system (Syngene, Frederick, MD). 16  2.1.6 E-64 Titration?active enzyme concentration determination The active site concentration of the purified enzymes was determined by E-64 titration (E-64 biding to enzyme at 1:1).  Briefly, 5 ?l of series of E-64 solutions were mixed with 5 ?l enzyme in appropriate dilution and incubated for 30 minute on ice in 90 ?l of 100mM sodium acetate (pH5.5) with 2.5 mM DTT and EDTA.  The residual activity was measured using 5-10 ?l of the mixture by Z-FR-MCA hydrolysis (2.3.1).  The active site concentration was calculated by plotting activity levels against E-64 concentration, and this active site concentration was used to calculate the protease molar concentration for all assays and kinetic studies. 2.2 Validation of enzyme This section describes the methods used to validate the enzymatic integrity of the enzymes. 2.2.1 Michaelis-Menten kinetics 2.5 nM of enzyme was used for kinetic study.  The assay contained 995 ?l 100 mM sodium acetate (pH5.5) with 2.5 mM DTT and EDTA in a plastic cuvette.  5 ?l of Z-FR-AMC (final concentration of 1, 2, 4, 8, 12, 15, 20, 25, 30, (50) ?M, extinction coefficient=508.155M-1cm) was added right before the activity measurement.  Vmax, Km, kcat, and kcat/Km were calculated using the Lineweaver-Burke plot. 2.2.2  Stability  The stability of the recombinant enzyme was tested at RT (~21?C), 28?C, and 37?C.  1 ?M of enzyme was incubated at those temperatures and the remaining activity was tested at different time points (0.5, 1, 2, 4, 8, 15, 24 hour) by Z-FR-MCA hydrolysis (2.3.1). 17  2.2.3 Thermal shift assay Thermal Shift assay was performed to evaluate the thermal stability of mutant proteins which showed significant difference in their activities when compared to WT.  The assays were done in 25 mM sodium acetate with 10% glycerol at pH5.5.  5 ?g protein was mixed with 5 ?l of 10x SyproOrange solution (Invitrogen, Carlsbad, CA) in up to 50 ?l sodium acetate buffer.  Samples were run for melting curve setting on real time PCR (MiniOpticon Real Time PCR Detection System, BIO-RAD, Hercules, CA) using SYBR green filter.  The temperature range was set from 20-95?C with 15 seconds hold every 0.5?C followed by a fluorescence reading.  The melting point was determined from the first derivative curve. 2.3 Enzymatic activity assay This section describes the various activity tested for the enzyme to see the effects of the mutations followed by the validation of the enzyme integrity. 2.3.1 Z-FR-MCA hydrolysis assay For standard condition, 5 ?l of 1mM ZFR-MCA (Bachem, Torrance, CA) were used as substrate in a total volume of 1 mL at RT (final concentration of 5 ?M Z-FR-MCA).  The activity was measured by release of amino-methylcoumarin (AMC) from the synthetic substrate (excitation 380nm, emission 450nm) for 30 seconds using a fluorospectrophotometer (Perkin Elmer Luminescence Spectrophotometer LS50B) at RT. 2.3.2 Collagen degradation Degradation of collagen by the protease was studies by using two different type of substrates, soluble collagen and insoluble collagen.  Soluble collagen was purchased from Sigma (St Louis, MO), and the insoluble collagen fiber was prepared from rat tail which is describe in 18  detail in 2.3.2.2.  Degradation was examined by SDS-PAGE (2.3.2.3), and insoluble collagen fiber was later examined by scanning electron microscopy (2.3.2.4). 2.3.2.1 Degradation of soluble collagen  200 nM of enzyme and 100 nM of CS-A (Sigma, St. Louis, MO) were mixed with 0.5 mg/ml of soluble bovine type I collagen (Affimetrix, Santa Clara, CA) in total volume of 50 ?l in 100 mM sodium acetate with 2.5 mM DTT and EDTA.  Digestion was carried out at 28?C under mild shaking for 1~4 hours depending on enzyme stability.  Degradation was terminated by addition of 5-fold excess of E-64 before the SDS-PAGE analysis.  2.3.2.2 Degradation of insoluble collagen  Tails from Sprague Dawley rats were cleaned with ethanol, and then the fiber bundles were pulled out from distal end one by one using a tweezer.  Isolated fibers were washed and cleaned in PBS and 70% ethanol.  The fibers were air dried and stored at -20?C until use. 50 ?l of 1 ?M enzyme was mixed with 1 mg of fibril cut into 5 mm length (7~10 pieces per reaction).  Digestion was carried at 28?C with mild shake for 2-4 hours depending on the enzyme stability.  Digestion was terminated by addition of 5-fold excess of E-64 for overnight at 4?C.  Fibers were removed gently from the tube at the end of the digestion and processed for SEM imaging.  The digestion mixture was then centrifuged at 13,000 rpm for 30 minutes, and the supernatant was used for SDS-PAGE analysis. 2.3.2.3 SDS-PAGE analysis Both soluble and insoluble collagen degradation mixtures were run on 10 % polyacrylamide gels to visualize the release or degradation of ?1 and ?2 chains by coomassie staining.  The bands were quantified using ImageJ (US National Institutes of Health, Bethesda, MD) densitometer program.  The ?1 and ?2 peptides are present on a gel without digestion in 19  case of soluble collagen.  All band densities were standardized against the no enzyme control (CTL) to monitor the overall degree of degradation.  However, for insoluble collagen, the ?1 and ?2 peptides are not present on a gel unless it is digested.  Thus, all the band densities were standardize against WT to see the degree of degradation when compared to the wild type enzyme mediated degradation. 2.3.2.4 SEM analysis The digested collagen fibers were washed with PBS and fixed with 2.5 % glutaraldehyde in PBS for 15-30 minutes at RT.  Then fibers were washed three times 5 minutes in water and subsequently dehydrated in series of ethanol (25, 50, 70 100%) for 10 minutes at each step.  The samples were dried and placed on stubs with carbon tape (TED Pella, Inc, Redding, CA) for overnight at 37?C oven.  The samples were then coated with 5 nm gold palladium using Leica EM MED020 coating system (Wetzlar Germany).  The images were taken using FEI Helio NanoLab 650 (FEI, Hillsboro, OR) operated at 1kV. 2.3.3 Elastin degradation 1 ?M of enzyme was mixed with 1mg of CongoRed Elastin (Sigma, St. Louis, MO) in 50 ?l total volume and incubated at 37?C with vigorous shake for overnight.  Release of CongoRed was measured at 490 nm using Vmax kinetic microplate reader (Molecular Device, Sunnyvale, CA). 2.3.4 Gelatin degradation Soluble collagen was heated at 95?C for 15 minutes before setting up the assay.  This pre-treated collagen was used as substrate, and the degradation was performed as described in the assay for the degradation of soluble collagen (2.3.2.1).  The mixture was then analyzed in SDS-PAGE. 20  2.4 Validation of complex formation Atomic force microscopy (AFM) was used to examine the complex formation and the molecular organization in the condition used in the activity assays.  All buffers and water used to prepare the samples were filtered through 0.22 ?m membrane.  All pipets and tubes coming in contact with samples were rinsed extensively with filtered water before handling samples.  CatK and CS-A were filtered separately before they were mixed together.  Mixture (10 pM CatK) was then placed on freshly cleaved mica (TED Pella Inc., Redding, CA) for 10 minutes at RT.  The mica was then extensively rinsed with filtered distilled water and air dried.  Images were taken using AFM microscope (Cypher Scanning probe microscope, Asylum Research, Santa Barbara, CA) in air using tapping mode, and images (512_512 pixel scans) were acquired at a scanning rate of 3 Hz. Silicon tips (Model- AC160TS, Asylum Research) with a radius of 7 nm were used to record the images at resonance frequency of 300 kHz and spring constant of 42 N/m.  21  Chapter 3: Results  3.1 Mutation at P88, M97, N99, and T101 A triple alanine mutant (MUTM97A/N99A/T101A) was first generated and evaluated.  These residues are located in the L domain of catK facing towards the backside of the protease and are depicted in Figure 3.1.  These residues are a part of the protein interface of the dimer and tetramer configurations of catK-GAG complexes (Figure 1.4). When the inhibitory effect on the collagenase activity by this mutant was observed, single mutants (MUTM97A, MUTN99A, and MUTT101A) were generated and examined for the same parameters to identify more specific residue(s) responsible for that effect.  Those sites were all individually mutated to alanine to study their involvement in both the dimer and the tetramer complexes.  MUTP88A was extremely unstable and did not allow any experiments to evaluate its activity, suggesting that this residue critically affects protein stability.   Figure 3.1 Molecular location of P88, M97, N99 and T101 P88/M97/N99/T101 (orange) are found on a loop in L domain and involved in both the dimer and tetramer complexes.  Yellow shows the disulfide bonds, and red shows the catalytic diad. H162 C25 P88 M97 N99 T101 22  3.1.1 Kinetics and stability The triple mutant (MUTM97A/N99A/T101A) and single mutant proteins (MUTM97A, MUTN99A, and MUTT101A) were evaluated for their enzyme kinetic parameters and stability to assess the protease integrity in comparison to wild type catK.  MUTN99A and MUTM97A were determined to have either slightly lower or higher enzyme efficiency, respectively (Table 3.1).  Table 3.1 Michaelis-Menten kinetics of MUTM97A/N99A/T101A, MUTP88A, MUTM97A, MUTN99A, MUTT101A kcat/Km values are comparable for all the mutants to the wild type enzyme.  [E]=2.5nM   Km (?M) kcat (s-1) kcat/Km (?M-1s-1) WT 4.1?1.1 15.3?5.9 3.8?1.0 MUTM97A/N99A/T101A 5.9?1.3 19.1?9.9 3.3?1.4 MUTM97A 5.9?2.7 28.0?10 4.8?2.4 MUTN99A 3.6?1.8 8.7?7.2 2.2?0.7 MUTT101A 3.2?0.7 7.3?5.5 3.0?1.8  The stability of the proteases at three different temperatures (RT, 28?C and 37?C) was tested over time in the absence of substrates or inhibitors.  The enzymes were incubated at each temperature, and the residual activities were subsequently tested for Z-FR-MCA hydrolysis at 0.5, 1, 2, 4, 8, and 15 hour time points.  The wild type enzyme retained its activity up to 8 hours at RT, 4 hours at 28?C, and 30 minutes at 37?C (Figure 3.2).  At RT and 28?C, MUTM97A/N99A/T101A and MUTN99A were less stable than wild type catK, whereas MUTM97A shows very similar activity level as the wild type protease.  Unexpectedly, MUTT101A was slightly more stable than wild type catK (Figure 3.2).  Thermal shift assays were performed to 23  determine differences in thermal stability of MUTM97A/N99A/T101A, MUTN99A, and MUTT101A, and the melting points (Tm) were evaluated further.  There was a slight shift in the melting point observed for MUTN99A, but it is still comparable to Tm of wild type enzyme (Table 3.2).  Because of the stability differences, 4 hour time points were selected for all of the activity assays.   Table 3.2 Melting temperature (Tm) of MUTM97A/N99A/T101A, MUTN99A, MUTT101A, MUTN175A/K176A, MUTK106A/R108T All the mutants have very similar Tm comparing to WT enzyme   WT MUTM97A/N99A/T101A MUTN99A MUTT101A MUTN175A/K176A MUTK106A/R108T Tm (?C) 55.0?0.5 54.7?0.3 52.7?0.3 54.7?0.3 55.1?0.3 56.2?0.3  3.1.2 Collagenase and elastase activity To test the collagenase activity of mutant enzymes, both soluble collagen and insoluble collagen were treated with enzymes and analyzed by SDS-PAGE.   As mentioned in 3.1.1, a 4 hour time point was set as standard for all collagenase and elastase assays with catK mutants.  To Figure 3.2 Stability assay of MUT97/99/101, MUTM97A, MUTN99A, MUTT101A Residual activities were measured at different temperatures as indicated above.  MUTM97A is very comparable to wild type catK whereas MUT97/99/101 and MUTN99A exhibit a slightly less stability.  MUTT101A is slightly more stable than the wild type enzyme at RT and 28?C. 24  efficiently degrade soluble collagen, catK needs supplementation by the chondroitin sulfate A (CS-A), whereas collagen fibers isolated from rat tails have sufficient GAGs associated to degrade it.  Normally, ?1 and ?2 chains (~140 and ~130 kDa) of type I collagen were degraded by wild type catK in the presence of GAGs as shown in Figure 3.3A.  To assess the effect of catK mutations on collagen degradation, the density of the ?1 and ?2 chains on SDS-PAGE gel were normalized against control collagen and compared.  MUTM97A and MUTT101A retained the same level of collagenolytic activity against soluble collagen (Figure 3.3A).  MUTM97A/N99A/T101A and MUTN99A, on the other hand, show much less degradation compared to the wild type enzyme (p<0.05) (Figure 3.3A).  Interestingly, MUTT101A shows slightly more degradation than the wild type enzyme in the absence of CS-A (Figure 3.3A).  This could be due to its higher stability.       25   Next, the collagenolytic activity of the mutants was examined using insoluble collagen.  Insoluble collagen is not visible on SDS-PAGE as only soluble constituents of the degradation assay were transferred to the polyacrylamide gels.  Once the insoluble collagen is digested by wild type catK, ?1 and ?2 chains appear on the gels as in Figure 3.3B.  Degradation of those ?1 and ?2 chains can be confirmed by the presence of smaller fragments on the gel as well.  The bands right below 75 kDa in all lanes are keratin contamination of insoluble collagen preparations from the rat tail.  In order to compare the collagenolytic activity against insoluble collagen among those mutants, the densities of ?1 and ?2 chains were normalized against the wild type catK mediated degradation and compared.  Control lanes contain collagen without added catK or its variants, assuring that there is no contamination of any type of collagenase in Figure 3.3 SDS-PAGE analysis of type I collagen degradation Densitometric data and representative gels of collagen degradation.  A) Degradation of soluble collagen with 200 nM enzyme.  B) Degradation of insoluble collagen fiber with 1uM enzyme.  Degradation was performed at 28?C for 4 hrs.  MUTM97A/N99A/T101A and MUTN99A show inhibition on both soluble and insoluble collagen degradation.  * indicate statistically significant difference when compared to wild type catK activity (p<0.05) 130- 95- 70- 62- A  B 26  the insoluble collagen preparations used in the experiment.  Similar to the degradation of soluble collagen, MUTM97A/N99A/T101A and MUTN99A show ~50% inhibition in degradation of insoluble collagen while MUTM97A retained full collagenolytic activity (Figure 3.3B).  Unexpectedly, MUTT101A has an almost 2-fold increase in degradation (p<0.05, Figure 3.3B).  This could be due to the extended stability of MUTT101A compared to the wild type catK. Degradation of insoluble collagen fibers was then further examined by scanning electron microscopy (SEM).  Rat tail collagen fibers were treated with catK variants for 4 hours before glutaraldehyde fixation and prepared for SEM imaging.  Collagen fibers in lower magnification did not show differences between fibers treated with different mutants.  However, observation at higher magnification revealed that the surface of the fibril bundles was disrupted in the presence of the wild type protease, MUTM97A, and MUTT101A (Figure 3.4).  In contrast, the fibers treated with MUTM97A/N99A/T101A and MUTN99A present more orderly fibril bundle structures comparable to those not treated with catK (Figure 3.4).   This observation is correlating well with the SDS-PAGE results.  Figure 3.4 SEM images of type I collagen fiber treated with various mutants MUTM97A/N99A/T101A and MUTN99A show more packed fibrils whereas wild type catK, whereas WT, MUTM97A and MUTT101A show a more distressed surface of fibrils.  Scale bar is 5?m. 27  In the analysis of elastin hydrolysis, MUTM97A/N99A/T101A and MUTN99A significantly inhibited the degradation of elastin (p<0.05, Figure 3.5A).  MUTT101A showed an increased elastase activity, yet this difference is not statistically significant.  Again, the increased activity of this variant is likely due to its increased stability.  Gelatin degradation assays showed that all mutants degrade gelatin completely, proving the overall integrity of the active site (Figure 3.5B).    3.1.3 Atomic Force Microscopy (AFM) CatK-GAG complexes were examined by Atomic Force Microscopy (AFM).  Monomeric catK was observed in the samples without CS-A for both wild type catK and MUTN99A (Figure 3.6).  In the presence of CS-A, a ring like conformation was confirmed for the wild type enzyme indicating the tetramer complex organization (Figure 3.6).  However, MUTN99A enzyme in the presence of CS-A exhibited deformed structures missing the central Figure 3.5 Elastin and gelatin degradation by MUTM97A/N99A/T101A, MUTM97A, MUTN99A and MUTT101A A) MUTM97A/N99A/T101A and MUTN99A exhibit a significant decrease in elastin degradation.  B) Gelatin degradation show that all mutant enzymes retained functional active cleft. * indicates statistically significant differences when compared to wild type catK activity (p<0.05). A B 28  pore (Figure 3.6), suggesting that Asn99 is in fact involved in protein-protein interaction in catK-GAG tetramer complex.  3.2 Mutation at N175 and K176 N175 and K176 are located at the N-terminal end of a ?-sheet in the R domain (Figure 3.7).  The double mutant at N175 and K176 was tested for the potential involvement in the dimer formation (Chapter 1.6, Figure 1.4).  Both residues were mutated into alanine.    Figure 3.6 AFM imaging of MUTN99A in the presence or absence of CS-A Wild type catK and MUTN99A are present as monomers without CS-A.  In the presence of CS-A, the wild type enzyme forms ring like formations, whereas deformed complexes can be observed in MUTN99A. WT MUTN99A Without CS-A With CS-A 29         3.2.1 Kinetics and stability The MUTN175A/K176A mutant was tested for its enzyme kinetic and stability.  Double mutation at N175 and K176 showed similar kcat/Km values indicating no structural interference with the integrity of the active site of this mutant (Table 3.3). Stability of MUTN175A/K176A was tested at RT, 28?C, and 37?C over time.  The enzyme was incubated at each temperature, and the activity was measured at 0.5, 1, 2, 4,8,15 hour time points by Z-FR-MCA hydrolysis.  At 37?C, both wild type and mutant enzymes lost activity by 30 minutes (Figure 3.8).  At RT and 28?C, MUTN175A/K176A sustained slightly more activity over time (Figure 3.8).  To examine the stabilities and the melting points of the enzyme, thermal shift assay was performed.  There was no difference observed in Tm value between the wild type and the MUTN175A/K176A mutant (Table 3.2).   Figure 3.7 M175 and K175 molecular location M175 and K175 (Green) are located on one of the ?-sheets of the R domain.  Disulfide bonds are in yellow, and the catalytic residues (C25 and H162) are in red. H162 C25 K176 N175 30  Table 3.3 Michaelis-Menten kinetics of MUTN175A/K176A  The mutant protein exhibited very similar efficiency of enzyme.  Km (?M) kcat (s-1) kcat/Km (?M-1s-1) WT 3.9?1.2 12.2?2.4 3.3?1.1 MUTN175A/K176A 1.8?1.5 3.2?1.9 2.0?0.8        3.2.2 Collagenase and elastase activity  The collagenase activities against both soluble and insoluble collagens were comparable to that seen for the wild type enzyme (Figure 3.9).  In case of insoluble collagen, MUTN175A/K176A had slightly increased activity, but the difference was statistically not significant (Figure 3.9B).  The slight increase in collagenolytic activity is again in line with the slightly increased stability of this variant (Figure 3.9B).  Moreover, SEM analysis of MUTN175A/K176A treated rat tail collagen fibers indicates very similar degradation pattern when compared to the fibers treated with the wild type enzyme (Figure 3.9C).  MUTN175A/K176A showed a 4-fold increase in elastin degradation (p<0.05, Figure 3.9D), suggesting inhibitory activity of these residues towards elastase activity of the enzyme.  The effective gelatin degradation suggested that the enzymatic integrity of active sites of this mutant was intact (Figure 3.9E). Figure 3.8 MUTN175A/K176A stability Stability at RT, 28?C, and 37?C.  Mutant showed slightly increased stability. 31   ??2 ??1 Figure 3.9 MUTN175A/K176A activity A) Degradation of soluble collagen B) Degradation of insoluble collagen C) SEM image of a collagen fiber after 3 hour digestion.  Scale bar is 5?m D) elastin degradation.  Mutant enzyme showed comparable collagenase activity, and significantly increased elastase activity when compared to the WT enzyme (* p<0.05).  E) Effective gelatin degradation indicates the active site of the mutant is not altered and functional. * E C A D B 32  3.3 Mutation at K106 and R108 Interface residues K106/C107/R108 are found at the N-terminal end of a ?-sheet located in the L domain of catK (Figure 3.10).  Double mutation at K106 and R108 was examined in order to determine their involvement in the tetramer complex formation (Chapter 1.6 and Figure 1.4).  These residues were mutated into alanine and threonine (MUTK106A/R108T) mimicking residues present in non-collagenolytic Cathepsin L.  Cysteine at 107 was not mutated since it was conserved in the CatL amino acid sequence.   3.3.1 Kinetics and stability The double mutant, MUTK106A/R108T, was evaluated for enzyme kinetics and stability.  This mutant exhibited similar enzyme efficiency and, interestingly, a significantly increased stability at RT, 28?C and 37?C when compared to the wild type enzyme (Table 3.4 and Figure 3.11).  However, the melting curve indicated that the thermal stability of MUTK106A/R108T is Figure 3.10 Molecular location of K106/C107/R108 interface  Residues K106/C107/R108 (wine red) are located in the L domain facing downward.  Active site residues (C25 and H162) are in red, and disulfide bonds are in yellow. H162 C25 K106 R108 33  comparable to the wild type enzyme, and the melting point of this mutant was very similar to the Tm of wild type catK as well (Table 3.2).  This discrepancy may be from the fact that the thermal shift assay is determining thermal stability of the protein whereas the residual activity is taking auto proteolysis into account.  Table 3.4 Michaelis-Menten kinetics of MUTK106A/R108T The mutant protein exhibited very similar efficiency of enzyme.  Km (?M) kcat (s-1) kcat/Km (?M-1s-1) WT 4.1?1.1 12.2?2.4 3.8?1.0 MUTK106A/R108T 1.9?1.1 5.6?4.1 2.8?0.9  Figure 3.11 MUTK106A/R108T stability Protease stability was tested at RT, 28?C, and 37?C.  The mutant exhibited extended stability at all three temperatures over wild type catK.     34  3.3.2 Collagen and elastin degradation The degradation of collagen, elastin, and gelatin by MUTK106A/R108T were studied.  MUTK106A/R108T exhibited comparable collagen degradation potency against soluble collagen, and a 5-fold increased activity against insoluble collagen (Figure 3.12A and B).  Closer examination of MUTK106A/R108T treated rat tail insoluble collagen fibers by SEM did not confirm the difference in the degradation degree but rather showed similar degradation for both wild type catK and MUTK106A/R108T treated fibers (Figure 3.12C).  Interestingly, collagen degradation of soluble collagen without GAGs showed increased activity of MUTK106A/R108T as it was observed in the case of MUTT101A (Figure 3.12A).  This could be explained by the increased stability of this mutant.  However, further studies are required to conclude the effects on those mutations.  Furthermore, this mutant improved the elastin degradation by 4-fold (Figure 3.12D).   35   A Figure 3.12 MUTK106A/R108T activity A) Degradation of soluble collagen. The mutant variant has comparable activity as wild type catK. B) Degradation of insoluble collagen.  A 6-fold increase was detected in the variant?s activity when compared to the wild type protease.  C) SEM images of rat tail collagen after 3 hour digestion indicates quite similar degradation pattern for wild type catK and MUTK106A/R108T.  Scale bar is 5?m D) Elastin degradation.  The mutant exhibited a 4-fold increased activity when compared to wild type catK.  E) Gelatin degradation.  The mutant retained its gelatinase activity. * indicates statistical significance (p<0.05) D * E C 75- 135- 100- MW (kDa) B ??2 ??1 * CTL WT MUT106/106 * A 245- 150- 135- 100- 75- 63- MW (kDa) ??1 ??2 -           +          -          +           -         + CTL WT MUT106/106 CS-A 36  3.4 Mutation at S138, L139, T140, N199, and K200 Two interface sites in the catK complex were proposed to be studied by the mutagenesis approach.  The interface sites, S138/L139/T140 and N199/K200, were found in a loop region on the top part of R domain (Figure 3.13).  S138/L139/T140 is involved in the tetramer model across from residues K106/R108 (Figure 1.4B).  The other interface was N199/K200, which is involved in the dimer model across from T101 (Figure 1.4A).  Mutation at S138, L139, and T140 destabilized the enzyme, and mutation at N199 and K200 possibly damaged the active site cleft or caused mis-folding of protein leading to non-functional protease.  Structural observations, sequence alignments, and structural prediction analysis upon mutation were utilized to access the issues that arose with these mutant proteases.   N199 K200 C25 H162 S138 L139 T140 Figure 3.13 Molecular location of residues S138/L139/T140 and N199/K200 Residues S138/L139/T140 (lime) and N199/K200 (green) are located in a loop region of the R domain alongside of the N-terminus loop leading to the active site H162 residue.  Active sites are in red, and disulfide bonds are in yellow. 37  3.4.1 Mutation at S138, L139, and T140  S138, L139, and T140 residues were identified as protein-protein interface between two molecules of catK along one CS-A in the tetramer complex model (Figure 1.4).  A triple mutant, MUTS138G/L139H/T140E, was first generated.  These residues were mutated into the non-collagenolytic catL sequences (GHE).  Since catK and catL share a similar amino acid sequence and structure, replacement with catL analogues amino acid residues was not expected to cause major structural damage to the protein (Figure 3.14A).  Typically, purified mature catK run at ~27 kDa in SDS-PAGE as seen in Figure 3.14B.  However, in case of MUTS138G/L139H/T140E, no protein was detected at 27 kDa.  Western blot analysis was employed to examine the issue further more, and it revealed that pepsin-mediated processing yielded no protein at 27 kDa eventhough proproteins in higher molecular weight disappeared upon activation (Figure 3.14C).             38   Figure 3.14 Western blotting for MUTS138G/L139H/T140E A) Overlay of catK (grey) and catL (orange).  CatL amino acid sequence and the structure is very similar to catK even though catL does not possess collagenolytic function.  MUTS138G/L139H/T140E is in green, active sites (C25 and H162) are in red, and disulfide bonds are in yellow.  B) A coomassie stained gel of different steps of a typical purification.  Activated enzyme (~27 kDa) accumulate and carry on throughout the purification.  C) Western blot of different steps of MUTS138G/L139H/T140E purification.  Procathepsin K with higher molecular weight can be observed.  Once the activation occurred those higher molecular weight bands disappeared without accumulation of active enzyme around ~27 kDa. C B MW (kDa) 245- 180- 135- 100- 75- 63- 48- 35- 25- A H162 C25 S138/L139/T140 interface catK: dark and light greys catL: orange 39  Two online structure prediction programs, PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) and CUPSAT (http://cupsat.tu-bs.de/) were used to analyze the effect of the mutations.  PolyPhen2 (Polypmorphism Phenotypeing v2) algorithms can predict the stability of mutated proteins via the analysis of multiple sequence alignments and protein 3D-structures comprising the sequence, phylogenetic, and structural information.  The program utilizes the UniProt/Swiss-Prot database, UniRef100 database, protein database (PDB) and the dictionary of secondary structure in proteins (DSSP) to assess the effect of mutations on the important features such as hydrophobic core and electrostatic interactions, on secondary structure elements, solvent accessible surface area, phi-psi dihedral angles, and atomic contacts61.  CUPSAT (Cologne University Protein Stability Analysis Tool) uses protein environment specific mean force potentials (amino acid-atom potentials, torsion angle potentials) and energy distribution derived from torsion angles to predict protein stability.  4024 non redundant protein structures are extracted from recent PDB repository and used to calculate mean force potentials and torsion angle potentials62. L139H was predicted to be damaging mutation (Table3.5).  When the human catK amino acid sequence was aligned with human catL and catS sequences all three residues, S138/L139/T140, were not conserved (Figure 3.15A).  When human catK amino acid sequence was compared to catK sequences of other species, all three residues were highly conserved among mammals with little more variance in T140 (Figure 3.15B).  S138 has more variance in fish and amphibians, whereas L139 is the most conserved across the species (Figure 3.15B).  Considering those results, MUTL139H/T140E and MUTS138G//T140E were generated to see if any functional mutants could be obtained.  However, the activities for multiple colonies of any of the three mutants were barely measurable by Z-FR-MCA hydrolysis assay. 40  Table 3.5 Structural stability prediction on single mutation PolyPhen2 and CUPSAT 61, 62  Score 0-1, 1 being the most damaging in PolyPhen2   PolyPhen2 CUPSAT S138G 0.008  L139H 1 Probably damaging Destabilizing T140E 0.013  N199A 0.397 Stabilizing Unfavorable torsion K200A 0.589 Possibly damaging Destabilizing Unfavorable torsion 41   Figure 3.15 Sequence alignment for S138, L139, and T140  A) Those residues are not conserved among human cathepsins. B) Those residues are highly conserved among mammalians, and L139 is highly conserved over all among different species. MEROPES http://merops.sanger.ac.uk/ A B 42  3.4.2 Mutation at N199 and K200 MUTN199A/K200A is an interface mutant that was generated based on the dimer model.  This particular mutant did not have any activities by the Z-FR-MCA hydrolysis assay.  Western blot analyses of activated samples show relatively high levels of activated enzyme compared to the wild type enzyme culture at day 5 of induction (Figure 3.16).  Several attempts of selecting other colonies for protease expression by using the Z-FR-MCA assay resulted in no active enzymes.   When the human catK amino acid sequence was aligned with the sequences of human catS, both residues are conserved.  However, this region does not exist in human catL (Figure 3.17A).  The comparison of human catK amino acid sequence with catK sequences of different species showed that N199 and K200 residues are highly conserved (Figure 3.17B).  K200 has a little bit more of variance and is substituted with arginine, another basic residue, in fish (Figure 3.17B).  When those mutations were analyzed by PolyPhen2 and CUPSAT, N199A showed some minor problems (Table 3.5).  Although the mutation is not destabilizing, alanines may not have angle torsion potential to fit into the 199 and 200 position to substitute asparagine and Figure 3.16 MUTN199A/K200A activation by western blotting The western blot showed the same level of activated enzyme as wild type catK culture media. 43  lysine.  K200A on the other hand seems to be a bit more damaging according to those predictions.  The mutation was predicted to be damaging and causing destabilization (Table 3.5).  Considering those results from amino acid alignment and structure prediction analysis, the single mutants, MUTN199A and MUTK200A, were generated to test if single amino acid replacement can tolerate the destabilizing effect of the mutations on the active site.  However, both single mutants did not produce enzymes with any activities detectable by Z-FR-MCA hydrolysis.  44   Figure 3.17 Sequence alignment of N199 and K200 A) Alignment among human cathepsins show those residues are not conserved.  B) Both residues are conserved among mammalian catKs.  MEROPS http://merops.sanger.ac.uk/ A B 45  Chapter 4: Discussion  This study was designed to test the potential protein?protein interaction sites in two independent oligomeric models of collagenolytic cathepsin K, based on x-ray diffraction crystallography data.  The residues potentially involved in the dimer complex model are P88, M97, N99, T101, N175, K176, N199 and K200.  The residues involved in the tetramer model are K106, R108, S138, L139 and T140, as well as P88, M97, N99 and T101, which are found in the dimer interaction site as well.  A total of six mutants were generated and examined; MUTM97A/N99A/T101A, MUTM97A, MUTN99A, MUTT101A, MUTN175A/K176A and MUTK106A/R108T.  Over all, most of mutations cause some stability shifts on the mutant proteases.  Notably, MUTP88A and MUTS138GL139H/T140E were destabilized, and no protein could be obtained.  Mutations at N199 and K200 to alanine may have affected the active site region, and no functional protein was purified to continue the study.  The reasons for this are discussed later in this chapter (4.4) 4.1 Effects of MUTN99A When mutant proteases were evaluated for degradation of soluble and insoluble collagen, MUTM97A/N99A/T101A showed significant inhibition in its activity against both soluble and insoluble collagens.  Subsequent experiments with single mutants at each residues (M97, N99, and T101) revealed that the mutation at N99 was responsible for this inhibition.  Mutation at N99 alone showed 40~50% inhibition of degradation of both soluble and insoluble collagens.  On the other hand, collagen degradation rates by MUTM97A and MUTT101A were very comparable to the wild type enzyme.  AFM analysis in the presence of GAGs showed that the formation of ring-like structures typically seen for the wild type enzyme is compromised with MUTN99A.  Ring 46  formation is indicative of the tetramer complex.  However, it should be noted that the resolution of AFM is low and that a detailed oligomeric structure of the complex cannot be identified.  Deformed complexes are observed within MUTN99A, and minor changes in the structures of the ring-like structures may have a significant effect on the collagenolytic activity.  The dimer structure, on the other hand, is not attainable by AFM analysis and might be present only on collagen fibers.  Thus the effect of MUTN99A on the dimer complex formation is unclear.  In our hypothesis, the dimer solubilize collagen fibers into tropocollagens, and the tetramers are responsible for the further degradation of soluble tropocollagen fragments.  Based on our hypothesis, it can be presumed that dimers were as well disrupted since solubilization of collagen fiber is also inhibited.  Further studies are required for the dimer complex formation. It is interesting to note that the inhibitory effect could be due to the protein stability issues.  The melting temperature of the mutant protein is determined to be 2?C lower than that of the wild type enzyme indicating that the mutation did not cause major stability issues to the protease.  However, when this mutant was tested for residual activities using Z-FR-MCA as substrate, it was ~40% less stable than the wild type enzyme at different temperatures measured (Figure 1.1).  To minimize the stability difference for the wild type and mutant proteases affecting the degradation assay, the collagen degradation assays were carried for four hours rather than twelve hours that was the standard protocol for collagen degradation with wild type catK in the laboratory.  Degradation at 28?C for four hours is the minimum to obtain the significant tropocollagen degradation visible on SDS-PAGE analysis.  Gelatin degradation assay carried out under the same conditions did not show any effect on the mutant hydrolysis activity suggesting that changes in stability are less of importance, and that the enzyme function is not 47  compromised.  In addition, the efficiency of the enzyme is marginally lower for MUTN99A than the wild type enzyme, yet the gelatinase activity shows that they were quite comparable. MUTN175A/K176A was generated and tested for the activity as these residues were thought to be involved in the dimer model on the other side of N99 in protein-protein interaction.  This mutant exhibited the same level of stability, and gelatin, elastin, and collagen degradations as the wild type enzyme, and no significant effect was observed. It is important to understand the structure of the catK-GAG complex as proposed by the models discussed earlier. However, this is hard to study due to a high tendency of the protein to form aggregates with sulfated GAGs.  The interaction between catK and sulfated GAGs to form functional complex is mainly due to electrostatic forces.  In a physiological environment, sulfated GAGs, such as chondroitin sulfate and dermatin sulfate, are negatively charged, whereas catK has positively charged residues clustered on the other side of the active cleft 46.  In the experimental mixture of GAGs and catK, it is hard to avoid this aggregation without disrupting the interaction necessary to create the functional complex.  In order to gain more insight into the complex formation and its activity, the complex can be examined by gel filtration, dynamic light scattering (DLS), cryo-electron microscopy, and x-ray crystallography. However, strict optimization of the conditions, such as pH, salt concentration and glycerol concentration, of complex formation is required to proceed to any of those experiments without aggregation occurring.  To avoid self-degradation and effect of inhibitor on the structure, an active site C25A mutant can be generated and used for any structural study in the future.  Cross-linking of the complex can be also considered for structural studies. 48  4.2 Effects of T101, K106 and R108 mutation Interestingly MUTT101A and MUT106/108 of catK displayed increased activity against insoluble collagen. (Figure 3.2 and figure 3.9).  No mutation at these sites has been reported related to any human diseases.  Protease stability might be a contributing factor for this effect.  Furthermore, surface study of digested collagen shows no differences between the wild type catK treated and these mutants treated collagen surface (Figure 3.3 and Figure 3.9).  Another unexpected result was that MUTT101A and MUT106/108 exhibit significant increases in the degradation of soluble collagen without external CS-A (Figure 3.2 and figure 3.9).  This was very intriguing since catK has been shown to require GAGs for efficient collagenase activity 47.  Due to a decrease in the charge of these residues, it is unlikely that the mutation (K106A and R108T) will increase the affinity of catK to GAGs.  It is possible that the increased activity observed is due to increases in substrate affinity of CatK. However, this result could have been due to potential GAG contamination.  It can be tested by using glycosidase treated collagen or synthetic collagen peptides as substrates in degradation assays to see if native GAGs are contributing factors in this increase in degradation. 4.3 Potential significance of T 101, K106, and R108 MUTT101A and MUT106/108 revealed an increased overall collagenolytic activity (figure 4.1A) with MUTT101A showing an about two fold increase and MUT106/108 showing an about six fold increase in collagen degradation when compared to the activity of the wild type enzyme.  This could be due to the increased protein stability of the mutant variants.  The mutations sites are located on one side of the enzyme involved in protein interaction of the dimer complex (Figure 4.1B) suggesting they might play a significant role in collagen degradation. 49   Mutations for all three residues caused change in surface charges causing disruption of electrostatic interactions and gaining the flexibility of the complex, which could be beneficial for collagen degradation.  One potential interaction the mutation could have disrupt is between residues K106/R108 and T140/S154 (Figure 4.2).  One theory is that two molecules of catK along the CS-A act as a dimer.  If that is the case, CS-A is the connecter between those two molecules, similarly to the linker region found in MMP-1 (Figure 4.2).  There is a CS-A binding site close to the active site based on our dimer model, and that site might be the docking site for the GAG/collagen.  In MMP-1, the two domains, hemopexin-like domain and the catalytic domain, bind to collagen independently and unwind collagen helix by conformational change when it was studied using a synthetic triple helical collagen peptide 63.  CatK complexes could be acting similarly to the hemopexin and catalytic domains of MMP-1.  Mutation of residues Figure 4.1 T101 and K106/R108 role in collagen degradation A) MUTT101A showed 2 fold increase whereas MUTK106AR108T exhibited 6 fold increase in degradation of collagen fiber when it compared to the wild type enzyme.  * indicates statistical significance (p<0.05).  B) All three resides are found to be on one side of the enzyme in L domain. * * A B T101 K106 R108 H162 C25 50  K106/R108 to alanines could have disrupted the interface between K106/R108 and T140/S154 and may have changed flexibility as a dimer and increased the binding and unwinding of the collagen.  To test this hypothesis, it would be interesting to carry out a full enzymological study determining the affinity to GAGs as well as collagen, and x-ray crystallography could be undertaken using the active site mutant (C25A) and synthetic peptide and GAGs 63.  Figure 4.2 Structural comparison between catK with CS-A and MMP-1 GAGs represented in blue in catK complex model can be equivalent of linker domain in MMP-1.  K106/C107/R108 region in maroon may lock the flexibility of two catK molecules by interact with T140 and S154.  Collagen peptide is in pink in MMP-1 crystal structure. catK with CS-A MMP-1 (PDB: 4AUO) Hemopexin- like domain Linker region Catalytic domain GAG binding site K106/R108 T140/S154 51  4.4 Destabilizing mutations The characterization of two other protein-protein interface sites failed due to the instability of the mutant proteins.  It is assumed that the mutations destabilized the overall enzyme structure or corrupted the active site cleft.  One interface site was S138/L139/T140, which is across from residues K106/C107/R108 in the tetramer model. 4.4.1 Mutation at S138, L139, and T140 A total of three mutant catK, MUTS138G/L139H/T140E, MUTS138G/T140E and MUTL139H/T140E, were generated to investigate the protein-protein interface involved in the tetramer model.  The catK amino acid sequence, SLT, was converted into the cathepsin L sequence, GHE.  Since this region is potentially interacting with K106/C107/R108, the activity of this mutant enzyme was expected to be similar to MUTK106A/R108T.  When the triple mutant, MUTS138G/L139H/T140E, was expressed, it had minimal peptidolytic activity.  To investigate this low activity, western blot analysis was performed.  The high expression level of proenzyme was confirmed, but relatively small amounts of active enzyme were detected as the propeptide was cleaved off (Figure 3.14C).  During the normal purification process, active enzyme accumulates (Figure 3.14B).  The propeptide is cleaved off, and as the purification continues, the concentration of purified active enzyme increases in intensity observed as an increase in the western blot signal (Figure 3.14B).  This suggests that MUTS138G/L139H/T140E active enzyme is not stable or folded correctly, and that it gets degraded rapidly by auto-proteolysis.  To examine the significance of these residues, the amino acid sequence of the human catK was compared to catK sequences of different species. All three residues were found to be highly conserved in mammals (Figure 3.15B).  T140 was the least conserved residue among those three.  When the mutation was tested by structure predication programs (PolyPhen261 and CUPSAT62), the mutation of L139 to histidine is 52  predicted to be damaging, whereas mutations at other two residues were predicted to be harmless for the structure of the protease (Table 3.5).  Based on those results, two double mutants, MUTS138G/T140E and MUTL139H/T140E, were generated.  However, the activities detected for those mutants were still minimal, based on hydrolysis of Z-FR-MCA.  Further investigation of the crystal structure indicates that this interface is only twelve amino acids away from the active site histidine residue (H152) and in close proximity to two conserved ?-sheets and a disulfide bond (Figure 4.3).  These results and observations strongly suggest that this region is essential to keep the structural integrity of the active site of catK.    Figure 4.3 Structural analysis of S138, L139, T140 region of catK S138/L138/T140 residues were less than 20 residues away from the active site, and other stabilizing features. H162 C25 S138 L139 T140 S-S bond to C204 ? helix ? sheet ? sheet 53  4.4.2 Mutation at N199 and K200 The interface comprised of residues N199 and K200 of catK was studied to ascertain its involvement in the dimer complex.  A total of three mutants, a double mutant (MUTN199A/K200A) and single mutants (MUTN199A and MUTK200A), were generated.  There was no activity detected by hydrolysis assay using Z-FR-MCA as a substrate.  Western blot analysis of this mutant revealed that this protease is in fact activated, and the level of activated enzyme for this mutant protein is very similar to that of the wild type enzyme from the same volume of expression cultures (Figure 3.16).  This result indicates that the mutations caused damage to the active cleft, and the protease is no longer functional.  To test the significance of those residues, first, the human catK amino acid sequence was compared to catK sequences in different species (Figure 3.17B).  Those two residues were highly conserved across species indicating that they play an important role in the function of catK.  Next the crystal structure was examined.  This region is almost fifty amino acids down stream of one of the active site, H162 (Figure 4.4).  The loop these residues are located on is between ? barrel and a disulfide bond, both of which are stabilizing the active cleft.  Then the mutation was tested by structure prediction analyses (PolyPhen2 61and CUPSAT 62), and the mutation at N199 or K200 were predicted to cause unfavorable torsion (Table 3.5).  Interestingly, there were three mutations (L195P, A197P, and R198G) within the region of interest reported in pycnodysostosis, which is characterized by the lack of functional catK 11, 64-66.  The sequence and structural analyses results together with the close location of the pycnodysostosis mutation sites to these residues indicate that this region is involved in keeping the physical integrity of the ? barrel (Figure 4.4). The torsion restraint to the loop caused by the mutations at N199 and K200 may be causing disruption to the active cleft, leading to a non-functional protease. 54   Figure 4.4 Structural analysis of N199 and K200 This site is less than 50 residues downstream of the active site H152 and just upstream from residues mutated in pycnodysostosis patients.  The pycnodysostosis mutation sites are highlighted in pink.  Yellows represent disulfide bond, and reds represent the active site. ?-sheet ?-sheet ?-sheet S-S bond  to C155 K200 N199 H162 C25 55  Chapter 5: Conclusion  Cathepsin K is a multifunctional protease which is involved in a wide range of important biological processes in our body.  In recent years more and more specific roles of catK have been discovered and studied extensively.  The best studied of all is its role in bone resorption.  Due to its pivotal role in bone homeostasis, it has been a major pharmaceutical target for the treatment of osteoporosis.  Development of catK inhibitors however has its difficulties.  Since all cathepsins share structural and in part substrate specificity similarities, selectivity of inhibitors has been an issue.  Also, inhibiting the active site can cause side effects due to its involvement in different types of physiological functions, from lysosomal protease function to hormone processing.  Development of activity specific exosite inhibitors of catK can eliminate some of these issues.  The mechanism of collagenolytic catK activity is still poorly understood, and gaining insights of how catK functions as collagenase can lead to exosite inhibitors that can treat osteoporosis and other bone resorption diseases more specifically. In this study, the structure and function relationship of collagenolytic catK has been studied by mutagenesis approach.  Based on the dimer and tetramer models developed previously in the lab, this study was focused on how mutation at protein interaction sites affects the collagenolytic activity.  Nine mutants were generated and evaluated.  Three mutants, MUTP88A, MUTN199A/K200A, and MUTS138G/L139H/T140E, could not be purified as functional proteins due to protein instability issues.  Four mutants, MUTN199A, MUTK200A, MUTS138G/T140E MUTL139H/T140E, could not be purified successfully.  Structural analysis led to the conclusion that these mutations are either destabilizing or deactivating the enzyme.  Two mutants, MUTM97A and MUTN175A/K176A, did not show any differences in their activities when they were compared to the 56  wild type enzyme.  One mutant, MUTM97A/N99A/T101A, showed significant inhibition of collagenolytic activity without an influence on its normal catalytic ability.  Single mutation experiments revealed that the mutation at N99 was the residue causing this inhibition.  This finding supports our models and identifies one of the residues essential to the collagenase activity of catK.  Unexpectedly, two of the mutants, MUTT101A and MUTK106A/R108T, facilitated the collagenolytic activity of catK.  This result can lead to the mechanical insight of how collagen degradation is carried out by catK in association with GAGs.  Even though further studies and analysis into the structural details of the complex are required, these results contribute to our understanding of the relationship between oligomeric status and the specific activity of catK.  Moreover, this can lead to a better elucidation of the mechanism by which catK unwinds tightly packed collagen and how it may initiate its degradation.  Based on these results and future studies, activity specific inhibitors of catK can be developed for the treatment of osteoporosis and other bone resorption diseases. 57  References  1. Turk, V., Turk, B., and Turk, D. 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Nishi, Y., Atley, L., Eyre, D., Edelson, J., Superti-Furga, A., Yasuda, T., Desnick, R., and Gelb, B. (1999) Determination of bone markers in pycnodysostosis: effects of cathepsin 64  K deficiency on bone matrix degradation, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 14, 1902-1908. 66. Hou, W., Br?mme, D., Zhao, Y., Mehler, E., Dushey, C., Weinstein, H., Miranda, C., Fraga, C., Greig, F., Carey, J., Rimoin, D., Desnick, R., and Gelb, B. (1999) Characterization of novel cathepsin K mutations in the pro and mature polypeptide regions causing pycnodysostosis, The Journal of clinical investigation 103, 731-738.  65  Appendices Appendix A  Cloning A.1 Primers Mutation Primer Design Not I introduction to pUC19 5?-GGG GAT CCT CTA GAG Gcg Gcc GCC AGG CAT GCA AGC TTG GCG-3? 5?-CGC CAA GCT TGC ATG CCT GGC ccC cgC CTC TAG AGG ATC CCC-3? P88?A 5?-CTC TGA AGc TGC CTA CGC ATA TGT GG-3? 5?-CCA CAT ATG CGT AGG CAc CTT CAG AG-3? M97/N99/T101 ?A/A/A 5'-GAA GAG AGT Tgc GTA Cgc CCC AgC AGG CAA GG-3' 5'-CTT GCC TGc TGG Ggc GTA Cgc AAC TCT CTT C-3' M97?A 5'- GGG ACA GGA AGA GAG TTG Tgc GTA CAA CCC AAC AGG C -3' 5'- GCC TGT TGG GTT GTA Cgc ACA ACT CTC TTC CTG TCC C -3' N99?A 5'- GGA AGA GAG TTG TAT GTA Cgc CCC AAC AGG CAA GGC -3' 5'- GCC TTG CCT GTT GGG gcG TAC ATA CAA CTC TCT TCC -3 T101?A 5'- GTT GTA TGT ACA ACC CAg CAG GCA AGG CAG C -3' 5'- GCT GCC TTG CCT GcT GGG TTG TAC ATA CAA C -3' N175K176?AA 5?-GGG ATA TGG AAT CCA GAA GGG Agc Cgc GCA CTG GA AAT TAA AAA CAG CTG GGG-3 5?-CCC CAG CTG TTT TTA ATT ATC CAG TGC gcG gcT CCC TTC TGG ATT CCA TAT CCC-3? N199K200?AA 5'-CCT CAT GGC TCG Agc Tgc GAA CAA CGC CTG TGG C-3' 5'-GCC ACA GGC GTT GTT Cgc Agc TCG AGC CAT GAG G-3' N199?A 5'- CCT CAT GGC TCG Agc TAA GAA CAA CGC CTG TGG -3' 5'- CCA CAG GCG TTG TTC TTA gcT CGA GCC ATG AGG -3' K200?A 5'- CCT CAT GGC TCG AAA Tgc GAA CAA CGC CTG TGG -3' 5'- CCA CAG GCG TTG TTC gcA TTT CGA GCC ATG AGG -3' K106CR ?ACT 5?-GGC AAG GCA GCT gcA TGC AcA GGG TAC AGA GAG ATC CCC G-3? 5?-CGG GGA TCT CTC TGT ACC CTG TGC ATG CAG CTG CCT TGC C-3? 66  Mutation Primer Design S138LT ?GHE  5?-CCT GTC TCT GTG GCC ATT GAT GCA gGC Cac gag TCC TTC CAG TTT TAC AGC AAA GG-3? 5?-CCT TTG CTG TAA AAC TGG AAG GAc tcg tGG CcT CA TCA ATG GCC ACA GAG ACA GG-3? L139T ?HE 5'- GGC CAT TGA TGC AAG CCa cga gTC CTT CCA GTT TTA CAG C -3' 5'- GCT GTA AAA CTG GAA GGA ctc gtG GCT TGC ATC AAT GGC C -3' S138LT ?GLE 5'- GGC CAT TGA TGC AgG CCT Gga gTC CTT CCA GTT TTA CAG C -3' 5'- GCT GTA AAA CTG GAA GGA ctc CAG GCc TGC ATC AAT GGC C -3'  A.2 PCR site-directed mutagenesis 10 ng of template plasmid 125 ng of each primers 250 ?M dNTP 500 ?M MgCl2 2.5% DMSO 1 U pfu DNA polymerase 2ul of 10x Buffer H2O to 20 ?l total volume        67  PCR program 1. Initial denaturing 95?C 3 minutes 2. Denaturing  95?C 30 seconds 3. Annealing  55?C 30 seconds 4. Extension  72?C 30 seconds 5. Repeat step 2~4 20 times 6. Final extension 72?C 10 minutes 68   A.3 pUC19N   69   Appendix B  Protein purification B.1 Purification buffer Buffer A Buffer B Buffer C 50 mM sodium Acetate 0.5 mM EDTA 2 M Ammonium sulfate  pH=5.5 50 mM Sodium Acetate 0.5 mM EDTA pH=5.5  50 mM Sodium Acetate 0.5 mM EDTA 2 M Sodium chloride  pH=5.5 0.5mM DTT was added right before the use for all purification buffers.  B.2 FPLC program N-Butyl sepharose 50 mL Buffer A 60 mL gradient from 100% Buffer A to 100% Buffer B 30 mL Buffer B SP-Sepharose 50 mL Buffer B 60 mL gradient from 100% Buffer B to 100% Buffer C 30 mL Buffer C *Speed of the flow was 1mL/min *3 mL fractions were collected throughout the process. *fractions were monitored for A280 and activity   70  B.3 SDS-PAGE gel  Typical purification gel  1. activated condition medium 2. N-butyl flow-through 3. N-butyl wash (2 M ammonium sulfate) 4. N-butyl wash (1 M ammonium sulfate) 5. N-butyl purified 6. SP-sepharose flow-through 7. SP-sepharose wash (0 M sodium chloride) 8. purified protein       catK proprotein (~45kDa) pepsin (~40kDa) 1    2 3   4 5 6  7 8 MW (kDa) Mature catK (~27kDa) 71  SDS-PAGE of purified mutant enzymes  1. wild type enzyme 2. MUTM97A/N99A/T101A 3. MUTM97A 4. MUTN99A 5. MUTT101A 6. MUTN175A/K176A 7. MUTK106A/R108T 100- 245- 135- 75- 63- 48- 35- 25- 20- 1     2        3         4  5     6         7 MW (kDa) 72   Appendix C  Culture medium recipes for Pichia pastoris MD:  1.34% Yeast Nitrogen Base with ammonium sulfate (YNB)   4x 10-5 % biotin 2% dextrose MM:  1.34% YNB   4x 10-5 % biotin 0.5% methanol BMGY: 1% yeast extract   2% peptone   100 mM potassium phosphate, pH6.0 1.34% YNB 4x 10-5 % biotin  1% glycerol BMMY: 1% yeast extract   2% peptone   100 mM potassium phosphate, pH6.0 1.34% YNB 4x 10-5 % biotin  0.5% methanol   

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