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The roles of ext1 and ext2 in heparan sulfate polymerization and hereditary multiple exostoses McCormick, Craig 2000

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THE ROLES OF EXT1 AND EXT2 IN HEPARAN SULFATE POLYMERIZATION AND HEREDITARY MULTIPLE EXOSTOSES By CRAIG McCORMICK B.Sc. (Hons), University of New Brunswick, 1995 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Microbiology and Immunology) We accept this thesis as conforming to the required standard University of British Columbia April 2000 © Craig McCormick, 2000 Wednesday, April 5, 2000 UBC Special Collections - Thesis Authorisation Form Page: 1 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of y \ • c r»Bi »l The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada http://www.library.ubc.ca/spcoll/thesauth.html ABSTRACT A human cDNA library was screened for the ability to restore susceptibility to herpes simplex virus (HSV) infection in sog9 cells, a normally HSV-resistant murine L cell line. After extensive screening, a single cDNA was isolated that could completely restore the HSV-susceptibility of this cell line. The cDNA was sequenced and found to be identical to EXT1, a previously identified putative tumour suppressor gene involved in hereditary multiple exostoses (HME). Analysis of the protein product of the EXT1 cDNA revealed that EXT1 is a type II transmembrane glycoprotein that is decorated with N -linked oligosaccharides, localized predominantly to the endoplasmic reticulum. Examination of E X T 1-expressing sog9 cells revealed that HSV-susceptibility is restored in these cells due to a restoration of the primary HSV-receptor, heparan sulfate (HS), to the cell surface. Taken together, these results suggested that EXT1 is likely involved in HS biosynthesis. H M E is an autosomal dominant disorder caused by mutations in either EXT1 or EXT2. EXT2, like EXT1, is localized predominantly to the endoplasmic reticulum (ER), but EXT2 is unable to restore heparan sulfate to the surface of sog9 cells, suggesting that EXT1 and EXT2 do not share redundant functions in the cell. When these two proteins were co-transfected into the same cell, there was a striking re-location of EXT1 and EXT2 to the Golgi apparatus, and stable E X T 1/EXT2 complexes could be isolated. This EXT1/EXT2 complex had a greater cumulative HS polymerizing activity than either protein alone, suggesting that the complex is the more biologically active form of the enzyme. To determine whether the HS polymerizing activity of EXT1/EXT2 was relevant to H M E , disease-causing E X T missense mutants were constructed and tested for complex formation, subcellular localization, and function. These missense mutants fall into two categories, those that form inactive hetero-oligomeric complexes that are retained in the ER, and those that can form Golgi-localized hetero-oligomeric complexes that are deficient in one of the two transferase activities necessary for the polymerization of HS. Moreover, none of the E X T mutants could restore HS to the surface of sog9 cells, as i i measured by the sensitive HSV-1 infection assay. Thus, defects in EXT1 or EXT2 inactivate the EXT1/EXT2 complex. These findings provide a rationale to explain why mutations in either of the two genes can cause H M E . During the course of this study, the parental mouse L cell line was stably transfected with EXT1 to observe the effects of overexpression of one member of the EXT1/EXT2 complex. These L-EXT1 cells displayed an altered, more stellate cell morphology, and anion exchange chromatography of glycosaminoglycans revealed that their cell surface HS was less negatively charged and more heterogeneous than L cell HS. However, the most remarkable phenotype of L-EXT1 cells was a dramatic increase in the ability of progeny HSV to spread from cell-to-cell. These L-EXT1 cells also displayed a decreased affinity for certain extracellular matrices. Taken together, these observations suggest that disrupting the balance of the members of the EXT1/EXT2 complex leads to changes in the architecture of the cell surface that allow for more efficient cell-to-cell spread of HSV, possibly through altered cell-cell contacts. iii Table of Contents ABSTRACT II TABLE OF CONTENTS IV LIST OF TABLES VIII LIST OF FIGURES IX LIST OF ABBREVIATIONS J XI PREFACE XV ACKNOWLEDGEMENTS XVI CHAPTER 1: INTRODUCTION 1 1.1 O V E R V I E W 1 1.2 G L Y C O S A M I N O G L Y C A N S & P R O T E O G L Y C A N S 1 1.3 G L Y C O S A M I N O G L Y C A N BIOSYNTHESIS 2 1.3.1 Heparan sulfate/Heparin 5 1.3.2 Chondroitin sulfate : 6 1.4 G L Y C O S A M I N O G L Y C A N - PROTEIN INTERACTIONS 6 1.5 HS AS A P O R T A L O F E N T R Y FOR H U M A N PATHOGENS 7 1.6 T H E HERPESVIRUSES 9 1.7 C L I N I C A L P A T H O L O G Y O F H S V - 1 A N D H S V - 2 9 1.8 H S V S T R U C T U R E 10 1.9 A B RIEF O V E R V I E W O F T H E H S V LIFE C Y C L E 10 1.9.1 HSV: the lytic cycle 10 1.10 H S V : E N T R Y 13 1.10.1 Binding 15 1.10.2 Stable attachment 18 1.10.2.1 HveA / HVEM / TNFRSF14 19 1.10.2.2 HveB / Prr2 / nectin 2 20 1.10.2.3 HveC/Prrl/nectin 1 and HveD/Pvr/CD155 20 1.10.2.4 Highly 3-O-sulfated heparan sulfate 21 1.10.3 Penetration 22 1.11 H S V EGRESS 23 1.12 C E L L T O C E L L SPREAD O F H S V 24 iv 1.13 C H A R A C T E R I Z A T I O N OF G L Y C O S A M I N O G L Y C A N - D E F I C I E N T / H S V RESISTANT C E L L LINES ..: 2 4 1.14 B O N E D E V E L O P M E N T 27 1.14.1 Hereditary multiple exostoses 2 9 1.14.2 HME-linked malignant transformation 29 1.14.3 S K E L E T A L D Y S P L A S I A A N D C E L L S U R F A C E ARCHITECTURE 3 0 1.15 H Y P O T H E S E S A N D OBJECTIVES 3 3 C H A P T E R 2: M A T E R I A L S A N D M E T H O D S 35 2.1 M A T E R I A L S 35 2.2 C E L L S A N D V I R U S E S 35 2.3 V I R A L STOCK PRODUCTION 35 2.4 D E T E R M I N A T I O N OF VIRUS TITER 3 6 2.5 ISOLATION OF R A D I O L A B E L E D VIRUS 3 6 2.6 V I R U S A T T A C H M E N T A S S A Y 3 7 2.7 C D N A SCREENING 37 2.8 CONSTRUCTION O F E X T FUSION PROTEINS . 4 0 2.9 ANTIBODIES 4 2 2 . 1 0 INDIRECT I M M U N O F L U O R E S C E N C E MICROSCOPY 4 2 2.11 I M M U N O B L O T T I N G 43 2 . 1 2 IMMUNOPRECIPITATION OF R A D I O L A B E L E D E X T PROTEINS 4 4 2 .13 P U L S E - C H A S E L A B E L I N G E X P E R I M E N T S 4 4 2 .14 A S S A Y OF C E L L U L A R G L Y C O S Y L T R A N S F E R A S E ACTIVITIES 45 2 .15 A N I O N E X C H A N G E C H R O M A T O G R A P H Y OF G L Y C O S A M I N O G L Y C A N S 45 C H A P T E R 3: I D E N T I F I C A T I O N O F EXT1 A S A T Y P E II T R A N S M E M B R A N E G L Y C O P R O T E I N I N V O L V E D I N H E P A R A N S U L F A T E B I O S Y N T H E S I S 47 3.1 INTRODUCTION 4 7 3.2 R E S U L T S 48 3.2.1 Screening of a cDNA library for HSV-1 entry-restoring cDNAs 48 3.2.2 Identification ofEXTl as a HSV-1 infection enhancing cDNA 57 3.2.3 Virus attachment to EXT1-transfected cell monolayers 5 2 3.2.4 Anion exchange chromatography of glycosaminoglycans from EXT 1-expressing cells 5 5 3.2.5 Construction of myc-epitope tagged and GFP tagged EXT1 fusion proteins 58 3.2.6 Immunofluorescence analysis ofEXTl fusion proteins : 58 3.2.7 Immunoblotting of EXT1 fusion proteins 61 3.3 DISCUSSION 63 C H A P T E R 4: A N A L Y S I S O F T H E R E L A T I O N S H I P B E T W E E N EXT1 A N D EXT2 66 V 4.1 I N T R O D U C T I O N 6 6 4 . 2 R E S U L T S 6 8 4.2.1 EXT1 and EXT2 are Functionally Distinct Proteins 68 4.2.2 EXT1 and EXT2 Accumulate in the Golgi Apparatus 70 4.2.3 EXT1 and EXT2 Form Homo- and Hetero-oligomeric Complexes 77 4.2.4 EXT1-EXT2 Complexes Possess Enhanced Glycosyltransferase Activity 79 4 . 3 D I S C U S S I O N ; 8 1 CHAPTER 5: ANALYSIS OF AETIOLOGIC MUTATIONS IN EXT1 AND EXT2 86 5.1 I N T R O D U C T I O N 8 6 5 . 2 R E S U L T S 8 6 5.2.1 Construction of aetiologic EXT1 and EXT2 mutants 86 5.2.2 Disease causing mutations destroy EXT1 activity in sog9 cells 89 5.2.3 Aetiologic EXT1 and EXT2 mutants have defects in intracellular trafficking 91 5.2.4 HME-linked Mutant Constructs Lack Glycosyltransferase Activities 95 5 . 3 D I S C U S S I O N 9 7 CHAPTER 6: A ROLE FOR HEPARAN SULFATE IN C E L L - C E L L TRANSMISSION OF HERPES SIMPLEX VIRUS 99 6.1 I N T R O D U C T I O N 9 9 6 . 2 R E S U L T S 9 9 6.2.1 Effect ofEXTl on cellular susceptibility to HSV-1 99 6.2.2 Analysis of cell-to-cell spread of virus 103 6.2.3 Anion exchange chromatography of GAGs from L-EXT1 cells 104 6.2.4 Analysis ofL-EXTl cell attachment to different matrices 108 6.2.5 Analysis of cell-cell spread of HSV on different matrices 109 6 .3 D I S C U S S I O N 1 1 2 CHAPTER 7: DISCUSSION 114 7.1 E L U C I D A T I O N O F E X T 1 F U N C T I O N 1 1 4 7 . 2 P R O T E O G L Y C A N S A N D B O N E D I S O R D E R S 1 1 5 7 . 3 I N D I A N H E D G E H O G , P A R A T H Y R O I D H O R M O N E R E L A T E D P R O T E I N A N D E X T I N B O N E D E V E L O P M E N T 1 1 6 7 . 4 O T H E R B O N E T U M O U R S 1 1 9 7 . 5 P R O S P E C T S F O R T H E F U T U R E 1 1 9 REFERENCES 121 APPENDIX 1 139 vi CHEMICAL REAGENTS AND LABORATORY SUPPLIES : 139 LIST OF SUPPLIERS 142 vii LIST OF TABLES T A B L E l . l O C C U R R E N C E A N D P R O P E R T I E S O F G L Y C O S A M I N O G L Y C A N S 4 T A B L E 1.2 M O D I F I C A T I O N S A N D F U N C T I O N S O F T H E H S V - 1 E N V E L O P E G L Y C O P R O T E I N S 12 T A B L E 1.3 R E L A T I V E I N F E C T I V I T I E S O F HSV O N C O N T R O L A N D M U T A N T C E L L L I N E S 26 T A B L E 4.1 G L C N A C - T A N D G L C A - T A C T I V I T I E S O F I M M U N O P R E C I P I T A T E D EXT1-EXT2 C O M P L E X E S 80 T A B L E 5.1 G L C N A C - T A N D G L C A - T A C T I V I T I E S O F I M M U N O P R E C I P I T A T E D E X T l -EXT2 M U T A N T C O M P L E X E S 96 T A B L E 6.1 R E L A T I V E I N F E C T I O N O F HSV-l(KOS) M U T A N T V I R U S E S O N G L Y C O S A M I N O G L Y C A N M U T A N T C E L L L I N E S 101 T A B L E 6.2 P L A Q U E S I Z E O F G E / G I M U T A N T V I R U S E S O N C E L L M O N O L A Y E R S 106 viii LIST OF FIGURES F I G . 1.1 T H E S U G A R U N I T S A N D T H E I R C O R R E S P O N D I N G G L Y C O S Y L T R A N S F E R A S E S I N V O L V E D I N H S A N D CS B I O S Y N T H E S I S 3 F I G . 1.2 A N E X A M P L E O F T H E R O L E T H A T H E P A R A N S U L F A T E P R O T E O G L Y C A N S C A N P L A Y I N F A C I L I T A T I N G R E C E P T O R - L I G A N D I N T E R A C T I O N S A T T H E C E L L S U R F A C E 8 F I G . 1.3 E L E C T R O N M I C R O G R A P H O F T H E HSV P A R T I C L E (x 100,000) 11 F I G . 1.4 S C H E M A T I C D I A G R A M O F T H E P R O C E S S O F H S V E N T R Y 14 F I G . 1.5 F U N C T I O N A L D O M A I N S O F T H E H S V - 1 G L Y C O P R O T E I N S G C - 1 , GB - 1 A N D G D - 1 17 F I G . 1.6 S C H E M A T I C M O D E L O F B O N E D E V E L O P M E N T 28 F I G . 1.7 T H E M U L T I P L E E X O S T O S E S P H E N O T Y P E : 32 R G . 2.1 S C H E M A T I C R E P R E S E N T A T I O N O F T H E C D N A S C R E E N I N G P R O T O C O L 39 F I G . 3.1 E F F E C T O F E N T R Y M E D I A T O R (EM) cDNA E X P R E S S I O N O N HSV-1 I N F E C T I O N O F SOG9 C E L L S . 50 R G . 3.2 PCR A M P L I F I C A T I O N A N D R E S T R I C T I O N E N Z Y M E A N A L Y S I S O F P U T A T I V E H S V - E N T R Y M E D I A T I N G (EM) CDNA 53 R G . 3.3 P R O T E I N S E Q U E N C E O F EXTJ, T H E P U T A T I V E H S V - E N T R Y M E D I A T I N G C D N A 54 F I G . 3.4 A N I O N E X C H A N G E C H R O M A T O G R A P H Y O F G L Y C O S A M I N O G L Y C A N S F R O M EXTl E X P R E S S I N G C E L L L I N E S 56 R G . 3.5 A N I O N E X C H A N G E C H R O M A T O G R A P H Y O F H E P A R I T I N A S E - D I G E S T E D G L Y C O S A M I N O G L Y C A N S F R O M E X T l - E X P R E S S I N G S O G 9 C E L L S 57 F I G . 3.6 S U B C E L L U L A R L O C A L I Z A T I O N O F G F P - T A G G E D F U S I O N P R O T E I N S 60 R G . 3.7 I M M U N O B L O T T I N G O F EXTl F U S I O N P R O T E I N S 62 R G . 3.8 C O M M O N T O P O L O G Y O F M A M M A L I A N G O L G I G L Y C O S Y L T R A N S F E R A S E S 65 F I G . 4.1 A M I N O A C I D A L I G N M E N T A N D P R E D I C T E D S E C O N D A R Y S T R U C T U R E F O R H U M A N EXTl (746 A . A . ) A N D H U M A N EXT2 (718 A .A . ) 67 F I G . 4.2 A N A L Y S I S A N D R E S C U E O F T H E EXTl D E F E C T I N SOG9 C E L L S 69 R G . 4.3 P U L S E - C H A S E A N D E N D O G L Y C O S I D A S E H D I G E S T I O N O F EXT2 72 F I G . 4.4 I N T R A C E L L U L A R L O C A L I Z A T I O N O F EXTl A N D EXT2 73 F I G . 4.5 I N T R A C E L L U L A R L O C A L I Z A T I O N S O F C O - T R A N S F E C T E D EXT F U S I O N P R O T E I N S . . 74 R G . 4.6 I N T R A C E L L U L A R L O C A L I Z A T I O N O F SOG9 C E L L D E R I V E D EXTl 75 F I G . 4.7 I N T R A C E L L U L A R L O C A L I Z A T I O N O F O T H E R T Y P E I I M E M B R A N E P R O T E I N S 76 R G . 4.8 R A D I O I M M U N O P R E C I P I T A T I O N A N A L Y S I S O F EXT1/EXT2 C O M P L E X E S 78 F I G . 4.9 T H E S U G A R U N I T S A N D T H E I R C O R R E S P O N D I N G G L Y C O S Y L T R A N S F E R A S E S I N V O L V E D I N H S A N D C S B I O S Y N T H E S I S 82 R G . 5.1 T O P O L O G Y O F EXTl A N D EXT2 I N T H E ER M E M B R A N E 88 F I G . 5.2 H S V - 1 I N F E C T I O N O F SOG9 C E L L S T R A N S F E C T E D W I T H A E T I O L O G I C EXTl M U T A N T cDNAs 90 R G . 5.3 I N T R A C E L L U L A R L O C A L I Z A T I O N O F A E T I O L O G I C EXTl M U T A N T S 92 R G . 5.4 I N T R A C E L L U L A R L O C A L I Z A T I O N O F A E T I O L O G I C EXT2 M U T A N T S 93 ix F I G . 5.5 R A D I O I M M U N O P R E C I P I T A T I O N A N A L Y S I S O F M U T A N T EXT2 O L I G O M E R I C C O M P L E X E S 94 F I G . 6.1 HSV-1 A T T A C H M E N T A S S A Y 102 F I G . 6.2 P L A Q U E F O R M I N G E F F I C I E N C Y O F HSV-l O N E X T l - T R A N S F E C T E D C E L L L I N E S 105 F I G . 6.3 P L A Q U E F O R M I N G E F F I C I E N C Y O F G E - A N D G I - D E F I C I E N T V I R U S E S O N E X T l - E X P R E S S I N G A N D C O N T R O L C E L L L I N E S 106 F I G . 6.4 A N I O N E X C H A N G E C H R O M A T O G R A P H Y O F G L Y C O S A M I N O G L Y C A N S F R O M E X T l - T R A N S F E C T E D L C E L L S 107 F I G . 6.5 L I G H T M I C R O S C O P Y O F L A N D L - E X T l C E L L S P L A T E D O N D I F F E R E N T M A T R I C E S 111 F I G . 7.1 A M O D E L F O R T H E R E G U L A T I O N O F C H O N D R O C Y T E D I F F E R E N T I A T I O N 117 X LIST OF ABBREVIATIONS 3-OST 3-O-sulfotransferase a.a. amino acid A A V adeno-associated virus bEXT bovine exostosin B H K baby hamster kidney B H V bovine herpes virus B M P bone morphogenetic protein B S A bovine serum albumin cDNA complementary deoxyribonucleic acid CHO Chinese hamster ovary cpm count per minute CS chondroitin sulfate D E A E diethylaminoethyl-D M E M Dulbecco's modified Eagle medium D N A deoxyribonucleic acid DS dextran sulfate dsDNA double-stranded deoxyribonucleic acid E B V Epstein-Barr virus E C L enhanced chemiluminesence E D T A ethylenediaminetetraacetic acid endoH endoglycosidase H E M entry mediator ER endoplasmic reticulum E X T exostosin E X T L exostosin-like FBS fetal bovine serum Fc fragment crystallizable (of immunoglobulins) FGF-2 fibroblast growth factor-2 FITC fluorescein isothiocyanate xi F M D V type O foot and mouth disease virus G A G glycosaminoglycan Gal galactose GalNAc N-acetylgalactosamine GFP green fluorescent protein GlcA glucuronic acid GlcA-T glucuronic acid transferase GlcNAc N-acetylglucosamine GlcNAc-T N-acetylglucosamine transferase GlcNS0 3 N-sulfoglucosamine GPI glycosylphosphatidylinositol h hour H A hyaluronic acid H B D heparin binding domain H C M V human cytomegalovirus HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid Hh hedgehog H H V human herpesvirus HIV human immunodeficiency virus H M E hereditary multiple exostoses HPLC high performance liquid chromatography HRP horseradish peroxidase HS heparan sulfate HS-Pol heparan sulfate polymerase HSV-1 herpes simplex virus type 1 HSV-2 herpes simplex virus type 2 Hve herpesvirus entry mediator IdoA iduronic acid IgG immunoglobulin G Ihh Indian hedgehog IP immunoprecipitation xii Kb kilobase pair kDa kiloDalton KS keratan sulfate K S H V Kaposi's sarcoma-associated herpesvirus L B Luria broth LGS Langer-Giedion syndrome L O H loss of heterozygosity m milli M molar M6P mannose-6-phosphate M6PR mannose-6-phosphate receptor M A b monoclonal antibody mEXT murine exostosin ug microgram uJ microlitre min minute MOI multiplicity of infection M W molecular weight NDST N-deacetylase-N-sulfotransferase NP-40 Nonidet P-40 OPG osteoprotegerin P A G E polyacrylamide gel electophoresis PAPS adenosine 3' phosphate 5' phosphosulfate PBS phosphate buffered saline PFU plaque forming units PG proteoglycan PMSF phenylmethylsulfonyl fluoride Prrl poliovirus receptor related protein-1 Prr2 poliovirus receptor related protein-2 PRV pseudorabies virus Ptc patched xiii PTHrP parathyroid hormone related protein PVR poliovirus receptor RIPA radioimmunoprecipitation assay R P M revolutions per minute SDS sodium dodecyl sulfate ST swine testes TGF-B transforming growth factor-B T G N trans Golgi network TNF tumour necrosis factor Ttv tout-velu U unit UDP uridine diphosphate UTR untranslated region V Z V varicella zoster virus w.t. wild type xiv P R E F A C E The work presented herein is the culmination of research efforts from 1995 to 2000. Below is the list of papers which have been published as a result of this work, and the contributions made by the candidate: • McCormick, C , Leduc, Y , Martindale, D , Mattison, K , Esford, L. E , Dyer, A. P , & Tufaro, F. The putative tumour suppressor E X T l alters the expression of cell-surface heparan sulfate. Nature Genet. 19,158-161 (1998). The candidate is responsible for the majority of the work in this study, with the exceptions of Figures Id, 2, and 3, which were a heparin inhibition assay, HPLC of glycosaminoglycans, and immunofluorescence experiments, respectively. • Lind, T , Tufaro, F , McCormick, C , Lindahl, U , & Lidholt, K. The putative tumor suppressors E X T l and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate. J. Biol. Chem. 273,26265-26268 (1998). The candidate helped the first author with interpretation/planning of experiments, providing reagents, and writing of the manuscript. • McCormick, C , Duncan, G , & Tufaro, F. New perspectives on the molecular basis of hereditary bone tumors. Moi. Med. Today 5, 481-486 (1999). The candidate is responsible for the writing of this review. The data presented in Figure 3 was generated by the candidate. • McCormick, C , Duncan, G , Goutsos, K. T , & Tufaro, F. The putative tumor suppressors E X T l and EXT2 form a stable complex that accumulates in the Golgi and catalyzes the synthesis of heparan sulfate. Proc. Nat. Acad. Sci., 97, 668-673 (2000). The candidate is responsible for the majority of the work presented in this study, with the exception of Fig. la, b, which describe the identification of the EXTl defect in sog9 cells. Dated Frank Tufaro, Ph.TXTProfessor and Graduate Supervisor xv ACKNOWLEDGEMENTS There are many individuals whom I would like to thank for their support throughout the past five years. First, I would like to thank my supervisor, Dr. Frank Tufaro, for providing a positive working environment, significant creative freedom, and important lessons in scientific writing. Furthermore, I would like to thank the members of my committee, Dr. Caroline Astell, Dr. Pauline Johnson and Dr. Gerry Weeks, for their guiding hands throughout this project and in the preparation of this thesis. Thanks go to Dr. Ulf Lindahl and the members of his group for cooperation on the E X T project. I would like to thank the members of the Tufaro lab for their friendship and support. I will always remember us all sitting around the table at Dim Sum, very much like a family. A special thanks goes to my family who provided loving support, and would have been proud regardless of the outcome of this research. Finally, I would like to thank Andrea for joining me on this adventure, and hopefully, all of the adventures that will follow. xvi CHAPTER 1: Introduction 1.1 Overview Viruses are opportunistic pathogens that are unable to replicate on their own, and therefore must enter a host cell and use the host cell energy supplies and biosynthetic machinery to replicate. Because of their dependence on the host cell, the study of viruses has consistently lead to a greater understanding of the host cell biology. Viruses often target critical regulatory events in the host cell; thus knowledge of the processes exploited by viruses has often highlighted cellular regulatory mechanisms. To gain entry into the host cell, viruses use host cell surface receptors that normally serve as receptors for other molecules. Herpes simplex virus (HSV) uses heparan sulfate (HS) glycosaminoglycans (GAGs) as receptors for initial attachment to the host cell surface. I have used the HS binding ability of HSV-1 to identify a cellular gene, EXTl, which is involved in HS polymerization. The cellular factors that viruses interact with are often key regulators of the cell cycle and E X T l is no different - human beings with inherited mutations in EXTl have developmental defects that lead to bone tumours (hereditary multiple exostoses - HME) and sometimes chondrosarcomas. The following introduction addresses the structure and properties of GAGs, with an emphasis on HS. I also review the process of HSV infection, highlighting viral entry and cell-to-cell spread, processes that depend upon HS GAGs. HS plays a role in a multitude of other biological processes, including the development of bone, which will be discussed. Finally, the genetics and pathology of H M E will be reviewed, setting the stage for a discussion of the potential links between HS biosynthesis and this important hereditary disease of bone. 1.2 Glycosaminoglycans & Proteoglycans Proteoglycans are complex macromolecules consisting of a protein core to which GAGs are covalently linked. Despite the diversity of their core proteins, proteoglycans share some common features due to their G A G side chains, which are highly negatively charged and occupy a relatively large amount of space (for a comprehesive review of 1 proteoglycan structure, see (Kjellen and Lindahl, 1991)). The localization of proteoglycans to the plasma membrane and extracellular matrix makes them important intermediates between cells and their environment. They have been implicated to play a role in cell-cell (Dietrich et al, 1977) and cell-matrix (LeBaron et al, 1989) interactions, organization of basement membranes (Kleinman et al, 1983), control of macromolecular diffusion (Kanwar et al, 1980), and also interactions with a variety of ligands such as growth factors, hormones, and neurotransmitters (Kjellen and Lindahl, 1991). 1.3 Glycosaminoglycan biosynthesis The biosynthesis of HS and chondroitin sulfate (CS) GAGs occurs by the concerted action of several membrane bound enzymes located in the endoplasmic reticulum and Golgi apparatus (Silbert and Sugumaran, 1995; Salmivirta et al, 1996). Most sugar nucleotides involved in these glycosylation steps are synthesized in the cytoplasm of the cell and require specialized transporters to translocate them to their site of synthesis within the lumen of these organelles (Hirschberg and Snider, 1987; Abeijon et al, 1997). With few exceptions, GAGs are O-linked to a serine residue in the core protein, via a common tetrasaccharide of xylose-galactose-galactose-glucuronic acid (Fransson etal, 1985; Bourdon etal, 1987; Kitagawa^aZ, 1998)(Fig. 1.1). Most GAGs differ in the composition of their repeating disaccharide subunits. Heparan sulfate, heparin, and hyaluronic acid (HA) have repeating N-acetyl glucosamine and uronic acid subunits, while CS and dermatan sulfate have N-acetyl galactosamine and uronic acid subunits, and keratan sulfate (KS) has N-acetyl glucosamine and galactose subunits (Table 1.1). It is generally accepted that the generation of the saccharide sequences is dictated by the specificity and availability of biosynthetic enzymes, rather than by use of a template. 2 Heparan sulfate GlcNAc a 1—4 GlcA pi GlcNAc a l — 4-3 GalNAc pi n GlcA Bl-f3 GalNAc pi-n linkage region Chondroitin sulfate core — protein Fig. 1.1 The sugar units and their corresponding glycosy transferases involved in HS and CS biosynthesis. The non-reducing ends are to the left. Abbreviations are as follows; Ser (serine), X y l (xylose), Gal (galactose), GlcA (glucuronic acid), GlcNAc (N-acetylglucosamine), GalNAc (N-acetylgalactosamine). 3 Table 1.1 Occurrence and properties of glycosaminoglycans Polysaccharide M W (xlO-3) Repeating period monosaccharides Sulfate per disaccharide unit Examples of occurrence in mammalian tissues Hyaluronic Acid 4-8000 D-glucuronic acid D-glucosamine 0 various connective tissues, skin, vitreous humour, synovial fluid, umbilical cord, cartilage Chondroitin 4-and 6-sulfates 5-50 D-glucuronic acid D-galactosamine 0.1-1.3 cartilage, cornea, bone, skin, arterial wall Dermatan sulfate 15-40 L-iduronic acid D-galactosamine 1.0-3 skin, heart valve, tendon, arterial wall Heparan sulfate 30a D-glucuronic acid or L-iduronic acid D-glucosamine 0.4-2 lung, arterial wall, ubiquitous on cell surfaces Heparin 6-25 D-glucuronic acid or L-iduronic acid D-glucosamine 1.6-3 lung, liver, skin, intestinal mucosa (Mast cells) Keratan sulfate 4-19 D-galactose D-glucosamine 0.9-1.8 cartilage, cornea, invertebral disc Table reproduced from (Lindahl and Hook, 1978). "Average M W of HS chain (Nieduszynski, 1989) 4 1.3.1 Heparan sulfate/Heparin Once an N-acetyl glucosamine sugar is added to the tetrasaccharide linker of a growing G A G chain, it is committed to synthesis of a HS backbone(Fritz et al, 1994) (Fig. 1.1). Subsequent steps involve the alternating transfer of GlcNAc and D-glucuronic acid (GlcA) monosaccharide units, from the corresponding UDP-sugar nucleotides to the nonreducing termini of the growing chains (Helting and Lindahl, 1971; Helting and Lindahl, 1972; Lidholt and Lindahl, 1992) (Table 1.1, Fig. 1.1). The mechanism for termination of the chain is unknown. Following polymerization of the heparin/HS backbone, the polymer is modified through a series of consecutive reactions that is initiated by N-deacetylation and N-sulfation of GlcNAc, both of which are catalyzed by a single bi-functional enzyme in the Golgi (Hashimoto et al, 1992; Eriksson et al, 1994; Aikawa and Esko, 1999). This sulfotransferase reaction, and those reactions that follow, obtain sulfate from the sulfate transporter 5'-phosphosulfate 3'-phosphoadenosine (PAPS). Next, adjacent glucuronic acid subunits are converted to iduronic acid (IdoA) by an epimerase (Lindahl et al, 1972). The presence of IdoA residues in the G A G chain adds conformational flexibility to the polymer, and therefore, allows the HS polymer to adopt a greater range of structures in three dimensions, providing more potential binding sites for different proteins (Lindahl et al, 1998). The epimerization step is followed by extensive 2-O-sulfation of GlcA/IdoA (Bienkowski and Conrad, 1985; Kobayashi et al, 1996) and 6-O-sulfation of N-sulfoglucosamine (Habuchi etal, 1995; Habuchi etal, 1998). Some minor reactions include 3-O-sulfation of GlcNAc (Kusche et al, 1988; Razi and Lindahl, 1995; Shworak et al, 1997; Shworak et al, 1999). The substrate specificities of the enzymes involved are generally such that structural modifications introduced in one particular reaction will be prerequisite to substrate recognition at a subsequent step. On the other hand, most of the modification reactions are incomplete, and only a fraction of the potential target units are actually attacked by the corresponding enzymes. This selection may, in turn, restrict the availability of substrate in later reactions. These cooperative and incomplete enzymatic reactions lead to an extremely heterogeneous saccharide sequence with a broader range of biological properties (Lindahl etal, 1998). 5 1.3.2 Chondroitin sulfate Once an N-acetyl galactosamine sugar is added to the tetrasaccharide linker of a growing G A G chain by the GalNAc-Tl enzyme, it is committed to synthesis of a CS backbone. The polymerization step of CS biosynthesis occurs by a similar mechanism as for HS biosynthesis, with the stepwise addition of GlcA and GalNAc units from the corresponding UDP-sugar, to the non-reducing end of the growing polysaccharide (Silbert and Reppucci, 1976; Kitagawa et al, 1995; Kitagawa et al, 1997). 1.4 Glycosaminoglycan - protein interactions Proteoglycans are primarily localized in two major areas: associated with the plasma membrane or with basement membranes. Proteoglycans are associated with the plasma membrane in three different ways; intercalation of the core protein into the plasma membrane (Marynen etal, 1989; Saunders etal, 1989), intercalation through a glycosylphosphatidylinositol (GPI) anchor covalently attached to the core protein (Ishihara et al, 1987; Yanagishita and McQuillan, 1989; Carey and Stahl, 1990; David et al, 1990; Brunner etal, 1991), or interactions between the G A G side chains and other molecules on the cell surface. Typical concentrations of HS proteoglycans on the cell surface as measured in various cell culture systems are in the range of 105-106 molecules/cell (Hook et al, 1984). For many proteoglycans, proper functioning requires the interaction of the core protein with extracellular or intracellular ligands, along with interactions between G A G side chains and extracellular ligands. Binding of proteins to GAGs is generally electrostatic in nature, although other types of interactions may occur. The G A G binding regions in proteins are usually clusters of basic amino acids. These G A G binding regions often have a preference to interact with the more highly sulfated, and therefore highly negatively charged, regions on GAGs. The best-described examples of this specificity are the pentasaccharide sequences recognized by antithrombin and fibroblast growth factor-2 (FGF2) (Lindahl et al, 1994). Binding of proteins to HS chains may serve a variety of functional purposes, from simple immobilization or protection against proteolytic degradation to distinct modulation of biological activity (Fig. 1.2). Because of such interactions, HS 6 proteoglycans are critically involved in a variety of biological phenomena at various levels of complexity, including organogenesis in embryonic development, angiogenesis, regulation of blood coagulation and growth factor/cytokine action, cell adhesion and lipid metabolism (Lindahl etal., 1994; Salmivirta etal., 1996; Rosenberg etal., 1997). 1.5 HS as a portal of entry for human pathogens The structural complexity of GAGs, in addition to their abundance on the surface of almost all mammalian cells, has made them attractive targets for a number of important pathogens. These pathogens include bacteria, like Chlamydia trachomatis and Neisseria gonorrhoeae as well as protozoans like Leishmania (Rostand and Esko, 1997). For the related herpesviruses, HSV (WuDunn and Spear, 1989), human cytomegalovirus (HCMV) (Compton et al., 1993), pseudorabies virus (PrV) (Mettenleiter et al, 1990), and bovine herpes virus type 1 (BHV-1) (Okazaki et al, 1991), the initial interaction of virus with cells is binding of virus to cell-surface HS. Recent reports have also implicated HS as a receptor for human immunodeficiency virus (HIV) (Patel et al, 1993), type O foot and mouth disease virus (FMDV) (Jackson et al, 1996), respiratory syncytial virus (RSV) (Krusat and Streckert, 1997), Dengue virus (Chen etal, 1997), Sindbis virus (Klimstra et al, 1998), vaccinia virus (Chung et al, 1998), porcine reproductive and respiratory syndrome virus (Jusa et a/., -1997), equine arteritis virus (Asagoe et al, 1997), and adeno-associated virus (AAV) (Summerford and Samulski, 1998). Studies of the nature of the interactions between these microbes and the cell surface GAGs reveals that most of the microbe binding activity consists of reversible, low-affinity interactions. The microbe requires subsequent high-affinity interactions with specific cell surface receptors to complete the process of entry into the host cell. 7 h e P a r a n syndecan suliate chains inactive FGF2 A A A * \ A \ protease cleavage site FGF receptor o o glypican y GPI-linkage site signal transduction Fig. 1.2 A n example of the role that heparan sulfate proteoglycans can play in facilitating receptor-ligand interactions at the cell surface. In the figure, fibroblast growth factor-2 (FGF2, mauve triangles) is only recognized by FGF receptors after it has been activated (mauve circles) by interaction with heparan sulfate chains (Spivak-Kroizman et al., 1994). Ligand binding to FGF2 receptors results in a ternary complex, and these activated FGF receptors then form dimers initiating a signal transduction cascade via the cytoplasmic C-termini. Both syndecans and the glycosylphosphatidylinositol (GPI)-linked glypicans are actively shed from the cell surface which might facilitate the diffusion of growth factors and other molecules between cells (Edgren et al., 1997; Liu et ah, 1998). 8 1.6 The Herpesviruses The family Herpesviridae comprises a group of large, enveloped double-stranded D N A (dsDNA) viruses that infect a broad range of tissues from an equally broad range of species. The herpesvirus family consists of three subfamilies, the alpha-, beta-, and gamma- herpesvirinae, which share common features of size, genomic homology, and structure (Roizman and Sears, 1996). Alphaherpesvirinae, which include HSV-1, HSV-2, varicella zoster virus (VZV), pseudorabies virus (PRV), and bovine herpesvirus (BHV-1), are characteristized by a relatively short, 24-hour replicative cycle, and a preference for epithelial and neural tissues. Betaherpesvirinae consist of human cytomegalovirus (HCMV), human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7), and typically have longer replicative cycles. The gammaherpesvirinae are lymphotrophic, targeting B and T lymphocytes, and are represented by Epstein-Barr virus (EBV) and the recently identified Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV). 1.7 Clinical pathology of HSV-1 and HSV-2 The herpes simplex viruses were the first herpesviruses to be discovered and are the most extensively characterized members of their family. Viral infection in humans, the natural host, usually remains localized to cells of the epidermis and peripheral nervous system, except in newborn infants, who are more prone to disseminated infection. There are two serotypes of HSV, designated HSV-1 and HSV-2. HSV-1, which has an estimated prevalence of 70-90% worldwide (Roizman, 1993) is primarily associated with recurrent facial lesions in adults, but it also can cause encephalitis in certain individuals. HSV-2, with a prevalence of 20.8% worldwide (Aurelius, 1998) is primarily associated with severe recurrent genital lesions, but is also associated with neonatal infections. Infection with HSV-1 and to a lesser extent HSV-2 is the leading cause of infectious corneal blindness in North America, with over 550,000 cases annually. Worldwide, 70% of people over the age of forty have antibodies against HSV-1 (Roizman and Sears, 1996). 9 1.8 HSV structure The HSV virion consists of four elements (see Fig. 1.3); (i) an electron-opaque core, which contains the -150 Kb dsDNA genome (ii) an icosadeltahedral capsid surrounding the core, (iii) an amorphous tegument surrounding the capsid, consisting of a group of virus-encoded proteins that are critical for the establishment of infection and transcription of early genes, and (iv) an outer envelope exhibiting spikes on its surface, which consist of 10 glycoproteins, glycoprotein B (gB), gC, gD, gE, gH, gl, gj, gK, gL and gM, of which only gB, gD, gH, gK and gL are essential for productive infection in cultured cells (Table 1.2). These glycoproteins are involved in the processes of virus entry, egress, cell-to-cell spread and immune evasion (Roizman and Sears, 1996). 1.9 A brief overview of the HSV life cycle The viral replicative cycle consists of two distinct phases; the lytic phase in which progeny virus is produced and the host cell is destroyed, and the latent phase, in which the HSV genome is able to remain in a quiescent episomal form for years, until an as-yet-unknown stimulus reactivates the virus and causes a lytic infection. 1.9.1 HSV: the lytic cycle For HSV the process of infection begins with the productive, lytic infection of epithelial cells. To initiate infection, the virus must attach to cell surface receptors. Fusion of the envelope with the plasma membrane rapidly follows initial attachment (see section 1.10 for details). The de-enveloped capsid is then transported to the nuclear pores and the viral D N A is released into the nucleus, where transcription, replication of viral D N A , and assembly of new capsids takes place. Viral D N A is transcribed throughout the reproductive cycle by host RNApolII, with concomitant participation of viral transcription factors. Viral gene expression is coordinately regulated via tegument proteins that promote the transcription of a sequentially ordered cascade of 'immediate-early', 'early' and 'late' gene products. Meanwhile, viral D N A replication is carried out by a rolling circle mechanism, yielding concatemers that are cleaved into monomers and packaged into capsids (Roizman and Sears, 1996). 10 F i g . 1.3 E lec t ron micrograph o f the H S V particle (x 100,000). The H S V v i r ion consists o f a double stranded linear D N A genome contained wi th in an icosahedral caps id ( A ) . Surrounding the capsid is the amorphous tegument (B) . The v i r ion envelope (C) contains g lycoprote in spikes w h i c h are indicated by the arrows. Th i s image was obtained f rom the Unive r s i ty o f Cape T o w n website (http:/ /www.uct.ac.za/depts/mmi/stannard/emimages.html). and or ig ina l ly publ ished by (S tannardefa / . , 1987). 11 Table 1.2 Modifications and functions of the HSV-1 envelope glycoproteins HSV-1 glyco-protein Essential for replication in cell culture? Post-translational modifications Function gB Yes -N-linked oligosaccharides -homodimer Binding; penetration; cell-to-cell spread; neuroinvasive determinant gC No -N-linked oligosaccharides -O-linked oligosaccharides -homotetramer Binding; C3b receptor gD Yes -N-linked oligosaccharides -O-linked oligosaccharides -homodimer Stable attachment; penetration; cell-to-cell spread; neuroinvasive determinant gE No -heterodimer with gl Cell-to-cell spread; Fc receptor gG No Unknown gH Yes -heterodimer with gL Penetration; egress; cell-to-cell spread gl No -heterodimer with gE Cell-to-cell spread; Fc receptor gJ No Unknown gK Yes Egress gL Yes -heterodimer with gH -proteolytic cleavage of transmembrane region Penetration; cell-to-cell spread gM No Possibly cell-to-cell spread 12 Assembly occurs in two stages. After packaging of D N A into pre-assembled capsids, the virus matures and acquires infectivity by budding through the inner lamella of the nuclear membrane (Darlington and Moss, 1968). After leaving the inner nuclear membrane, the route of egress of infectious virus particles is uncertain, but clearly involves the host cell secretory apparatus (for more detail, see section 1.11). This entire reproductive cycle occurs in approximately 18 to 20 hours (Roizman and Sears, 1996). 1.10 H S V : Entry HSV infects a broad range of cells and can cause disease in a variety of tissues from diverse animal species. This relatively broad host range suggests that cellular receptors for HSV may be common to many different cell types and/or that there may be more than one pathway by which the virus can enter a cell. Furthermore, with eleven different viral glycoproteins, several of which project as distinct spikes from the membrane surface (Stannard et al, 1987) (Fig. 1.3), it seems likely that binding of the virus to a cell and the subsequent steps leading to virion-cell fusion may require sequential or simultaneous interactions between multiple virion proteins and several different sites or receptors on the cell surface. The process of H S V entry into the host cell can be divided into three separate events; (i) a reversible initial attachment to cell glycosaminoglycans; (ii) stable attachment to a protein receptor on the cell surface; (iii) penetration by fusion of viral and cellular membranes (Fig. 1.4). 13 initial stable penetration attachment attachment 1 Entry event Initial attachment Stable attachment Penetration Cell receptors HS,CS HveA, B , C, 3-0-sulfated HS ? Viral glycoproteins gC, gB gD g H - g L , gB Fig. 1.4 Schematic diagram of the process of HSV entry Adapted from (McClain and Fuller, 1994). 14 1.10.1 Binding The initial binding of HSV to the cell surface is mediated by interaction of viral envelope glycoproteins with HS and/or CS G A G chains of cell surface proteoglycans (Spear, 1993). Evidence for the interaction of HSV with GAGs stems from studies in which the cell surface concentration of HS is reduced by enzymatic digestion, resulting in a reduction in HSV infection (WuDunn and Spear, 1989). In addition, animal cell mutants with defects in G A G synthesis all show some degree of resistance to HSV infection (Shieh et al, 1992; Gruenheid et al, 1993; Banfield et al, 1995). Moreover, HSV infection is reduced by over 90% when soluble heparin or HS are present during inoculation (Gruenheid et al, 1993), and soluble dermatan sulfate (chondroitin sulfate B) has the ability to inhibit HSV-1 infection of both CS and HS/CS expressing mouse cell lines (Banfield et al, 1995). Although the presence of soluble GAGs reduces HSV infection, they do not appear to permanently inhibit the infectivity of the virion. Therefore, it appears that the soluble GAGs act as competitive inhibitors of virus binding to the cell surface. Experiments employing a biosensor system to observe the real-time binding of a protein to an immobilized ligand have shown that complexes between HSV-2 glycoprotein B and GAGs are reversible (Williams and Straus, 1997). This leads to speculation that interaction between viral glycoproteins and cell surface GAGs may be sufficiently strong to promote binding to the cell surface, but still weak enough to allow release of the virion if viral entry does not occur within a given time frame. Supporting evidence for this model of temporary virus adsorption to the cell surface is provided by study of G A G deficient cell lines. In these cells, exogenous dextran sulfate (a G A G analog) is able to form an alternative adsorption matrix for HSV-1 binding in the absence of other GAGs (Dyer etal, 1997). Several studies involving the alphaherpesviruses, HSV-1, bovine herpesvirus type 1 (BHV-1) and pseudorabies virus (PRV) were undertaken to identify the virion component responsible for initial binding to cell surface GAGs. Deletion of the gC coding genes of HSV-1, BHV-1 or PRV results in mutants that are significantly impaired in their ability to bind to cells (Schreurs et al, 1988; Zuckermann et al, 1989; Herold et 15 al, 1991; Liang et al, 1991). In addition, the gC homologs from each of these viruses bind to affinity columns coated with heparin under physiological salt concentration (Mettenleiter et al, 1990; Sawitzky et al, 1990; Herold et al, 1991; Okazaki et al, 1991). Moreover, neutralizing antibodies specific for the gC homolog of each of the three viruses can block binding of virus to cells (Zuckermann et al, 1989; Okazaki et al, 1991; Svennerholm et al, 1991), while most neutralizing antibodies specific for other glycoproteins only block viral penetration. These studies strongly suggest that the primary interaction responsible for virus binding to the cell surface is between gC and HS (Fig. 1.5). However, an inconsistency with the role of gC in virus binding is the fact that it is not essential for infection (Langeland et al, 1990; Herold et al, 1991; Sears et al, 1991). Experiments showing that infection by gC-deficient HSV-1 can be inhibited by soluble heparin suggest that other viral glycoproteins may participate in the initial G A G binding event (Herold et al, 1991). HSV-1 virions lacking both gC and gB are far more impaired in binding to cells than are virions that lack only gC. This suggests that gB, which also binds to soluble heparin, may also have a role in the initial binding of viruses to the cell surface (Herold etal, 1994) (Fig. 1.5). 16 gB-1 H B D 143-150 HBD 33-123 HBD 247 COOH 511 a.a. gC-1 HBD Oligomerization 68-76 626-653 N H ; — - s *— m COOH Penetration ^03 a.a. 241-441 HveA HveA/HveC g L M binding binding H 2 — 1 — g | _ _ | - COOH Penetration 369 a.a. 231-244 Fig. 1.5 Functional domains of the HSV-1 glycoproteins gC-1, gB-1 and gD-1. These proteins adopt a type I topology in cellular/viral membranes. Shaded boxes represent hydrophobic transmembrane regions. The location of the important regions on these proteins is indicated in amino acids (a.a.). HBD, heparin binding domain. 17 1.10.2 Stable attachment According to the current model of HSV entry, binding of the virus to GAGs is followed by a stable form of attachment to a second receptor. Early evidence for a secondary receptor came from the study of Chinese hamster ovary (CHO) cells and swine testes (ST) cells (Subramanian et al, 1994), which despite expressing normal amounts of HS, were highly resistant to HSV infection. While gB and gC are associated with initial binding to cell surface GAGs, it appears that HSV glycoprotein D (gD) facilitates stable attachment. Functional gD is essential for entry, since gD-negative virions and virions treated with anti-gD neutralizing antibodies fail to enter cells (Fuller and Spear, 1987). gD mediates infection at least in part by binding to a cell surface receptor as shown by the ability of UV-irradiated, gD-bearing virions or soluble gD to block infection by subsequently added virus in a saturable manner (Addison et al, 1984; Johnson and Ligas, 1988; Johnson and Spear, 1989). Moreover, cell surface gD expressed either during a productive infection or from a plasmid is able to block infection in a process called gD-mediated interference (Campadelli-Fiume etal, 1988; Johnson and Spear, 1989; Campadelli-Fiume et al, 1990). It was suggested that these excess amounts of cell-surface gD bind to most of the available endogenous stable attachment receptors on the cell surface, thus preventing infection (Campadelli-Fiume etal, 1988; Johnson and Spear, 1989; Campadelli-Fiume etal, 1990; Dean etal, 1994). One early candidate for a gD-binding stable attachment receptor was the mannose-6-phosphate receptor (M6PR). Glycoprotein D - l (gD-1) has mannose-6-phosphate (M6P) on a fraction of its oligosaccharides and interacts with the mannose-6-phosphate receptor (M6PR) component of most mammalian cell surfaces (Brunetti et al., 1994). However, when the M6PR is not present on the surface of susceptible mouse cells, infection is not inhibited (Brunetti et al, 1995). Furthermore, it was shown that antibodies directed against the M6PR and a soluble form of the receptor block infection only to a small degree. Thus, although the M6PR may yet play a role in the life cycle of HSV, it has been effectively disproved as a stable attachment receptor for HSV. 18 1.10.2.1 HveA / H V E M / TNFRSF14 The search for stable attachment receptors for HSV, as with the search for initial attachment receptors, was made possible by employing HSV-resistant cell lines. By screening a human cDNA expression library for genes that could mediate entry into HSV-resistant CHO-K1 cells, a single cDNA was isolated which rendered the cells 100-1000 times more susceptible to HSV-1 upon transfection (Montgomery etal., 1996). Sequencing of the cDNA revealed that it encoded a 283 amino acid protein, with three complete and one partial cysteine-rich repeats characteristic of members of the tumour necrosis factor/nerve growth factor receptor family. The protein was designated Herpesvirus entry mediator (HVEM), later renamed Herpesvirus entry mediator A (HveA), and officially named tumour necrosis factor receptor superfamily 14 (TNFRSF14) to indicate its place in an established family of proteins. HveA-transfected CHO-K1 cells also became susceptible to HSV-2, but not to the related PRV, indicating that HveA is not a general mediator of alphaherpesvirus entry. The failure of the M6PR study made it clear that to be considered a bona fide stable attachment receptor for HSV, certain criteria needed to be met. First, the absence of the receptor must significantly inhibit infection. Second, antibodies to the receptor must block infection. HveA meets these two criteria (Montgomery et al, 1996). Additional experiments have shown that truncated soluble forms of HveA could bind directly to HSV-1 gD (gD-1) in vitro, in a molar ratio of 2:1 (Whitbeck et al, 1997) (Fig. 1.5). The discovery of a novel member of the TNF/NGF receptor superfamily, and the demonstration of its ability to mediate HSV entry into activated T cells (Montgomery et al, 1996), was of considerable interest in the field of immunology. Although HSV primarily infects cells of epithelial and neural origin, several reports had described the replication of HSV in activated T cells (Pelton et al, 1977; Rinaldo et al, 1978; Teute et al, 1983) and the presence of infected lymphocytes in biopsies of cutaneous lesions (Boddingius et al, 1987). Taken together, these results raised the possibility that infection of T cells by HSV could serve as an anti-immune response mechanism. This hypothesis was strengthened by the discovery that the normal cellular ligands for HveA are secreted lymphotoxin a, and a new cytokine, called LIGHT (Mauri et al, 1998). The 19 remarkably descriptive acronym, LIGHT, refers to its homology to lymphotoxins, exhibition of inducible expression, and that it competes with HSV glycoprotein D for HveA, a receptor expressed on T lymphocytes. The fact that LIGHT is able to block HveA-dependent HSV-1 infection of T lymphocytes indicates that gD is a membrane anchored virokine (a viral cytokine), that mimics LIGHT. A number of viruses, including adenovirus (Shisler et al, 1997), hepatitis C (Zhu et ali 1998), as well as the herpesviruses E B V (Mosialos et al, 1995), and HHV-8 /KSHV (Bertin et al, 1997), target the TNFR for immunomodulation. Taken together, these interactions suggest an attractive model for HSV-1 infection of activated T lymphocytes, whereby the virus, through gD, modulates HveA signaling activities, perhaps blocking extracellular signals for apoptosis. 1.10.2.2 HveB/Prr2/nectin2 Certain mutant strains of HSV-1 are unable to use HveA as a stable attachment receptor. By repeating the screening of a human cDNA library for HSV-entry mediating cDNAs, using the mutant HSV-1 KOSridl as the selective agent, a single cDNA was isolated that could confer susceptibility to infection (Warner et al, 1998). This cDNA could mediate infection of several mutant strains of HSV-1, wild type HSV-2, and PRV, but not wild type HSV-1 or BHV-1 . Sequencing of this cDNA revealed that it was identical to a previously described member of the immunoglobulin superfamily, poliovirus receptor related protein 2 (Prr2) (Eberle et al, 1995). This protein was designated Herpesvirus entry mediator B (HveB), indicating its newfound function. HveB/Prr2 was expressed in some human neuronal cell lines, fibroblastic cells, keratinocytes, and, like HveA, primary activated T lymphocytes, although antibodies to HveB did not block infection of many of these cells. The observed differences in the ability of HSV-1 and HSV-2 strains to use HveB may account, in part, for differences in viral tissue tropisms. 1.10.2.3 HveC/Prrl/nectinl and HveD / Pvr / CD155 Considering the ability of HveB/Prr2 to act as a stable attachment receptor for HSV, the next logical step was to test the Prr2 homologs, Poliovirus receptor (Pvr) (Mendelsohn etal, 1989) and poliovirus-related receptor 1 (Prrl) (Lopez et al, 1995). 20 Pvr-expressing CH0-K1 cells were more susceptible to the non-primate herpesviruses PRV and BHV-1 , but remained resistant to HSV-1 and HSV-2 (Geraghty et al, 1998). Pvr was named Herpesvirus entry mediator D (HveD), to indicate its new role. By contrast, Prrl-expressing CHO-K1 cells mediated entry of HSV-1 and 2, PRV and B H V -1, the widest range of inter-strain specificity observed thus far. Prrl was renamed herpesvirus entry mediator C (HveC). Despite their abilities to act as herpesvirus entry mediators, HveA and HveB do not display the tissue distribution necessary to mediate infection of the epithelial and neural tissues that HSV normally infects. Remarkably, HveC is expressed in high levels in human cells of both epithelial and neural origin (Geraghty et al, 1998). Thus, HveC is the prime candidate for the secondary receptor that allows both HSV-1 and 2 to infect epithelial cells on mucosal surfaces and spread to cells of the nervous system. 1.10.2.4 Highly 3-O-sulfated heparan sulfate The previously identified gD-binding receptors for HSV, HveA, HveB, and HveC are all cell surface proteins, encoded by cDNAs isolated from a human cDNA library. Surprisingly, yet another round of screening cDNAs, this time from a mouse cDNA library, for the ability to mediate entry into CHO-K1 cells resulted in the isolation of a cDNA closely related to the human gene 3-O-sulfotransferase 3B (3-OST-3B)(Shukla et al, 1999; Shworak et al, 1999), which modifies HS late in biosynthesis (Lindahl et al, 1998). CHO-K1 cells normally express significant amounts of HS and CS, which HSV uses for initial attachment via gC and gB (Shieh et al, 1992), but entry of virus is blocked due to a lack of stable attachment receptors. However, expression of 3-OST-3B, one of a set of isoforms of the enzyme, in these cells results in the generation of highly sulfated disaccharide repeats in the HS polymer, namely, 2-O-sulfated iduronic acid and 3-0-sulfated/6-0-sulfated glucosamine, allowing HSV to bind more efficiently to the primary receptor (Liu et al, 1999; Shukla et al, 1999). Several lines of evidence demonstrated that HSV-1 entry mediated by 3-OST-3B is dependent upon gD and in the generation of gD-binding sites in HS. First, HSV mutants with single amino acid substitutions in gD were unable to infect cells expressing 3-OST-3B (Shukla et al, 1999). Second, both membrane-bound and soluble gD 21 competed with virus for the receptors generated by 3-OST-3B-modified HS, but not to the enzymes themselves. Finally, pretreatment of surfaces of 3-OST3B-expressing CHO-K l cells with HS-degrading enzymes (heparitinases) eliminated binding of soluble gD to the cell surface. These results raise the possibility that HSV-1 entry into cells could be mediated entirely by HS, provided the appropriate sites for virus binding and gD binding were present in the HS. Unlike the HveA, B and C stable attachment receptors, 3-OST-3B is widely expressed in human tissues, suggesting that in tissues that do not express any of the Hve-receptors, 3-OST-3B modifications of HS may compensate and permit virus entry. 1.10.3 Penetration Enveloped viruses enter cells by fusion with cellular membranes. Some viruses fuse with the plasma membrane while others are endocytosed and fuse with endosomal membranes. Agents that block endocytosis do not inhibit HSV entry, which suggests that HSV fuses with the plasma membrane (Fuller et al, 1989). Nonetheless, electron microscopic evidence suggests that HSV may be able to enter by endocytosis as well (Morgan et al, 1968). However, HSV entry by endocytosis results in degradation of the virus and non-productive infection (Campadelli-Fiume et al, 1988). Studies using mutant viruses and monoclonal antibodies show that HSV-1 gB, gD, gH, gK, and gL are essential for infection (Cai et al, 1988; Ligas and Johnson, 1988; Fuller et al, 1989; Forrester et al, 1992; Roop et al, 1993; Hutchinson et al, 1995). In the case of viruses missing any one of these glycoproteins, virions still attach to permissive cells, but do not enter the cells (Cai et al, 1987; Ligas and Johnson, 1988; Forrester et al, 1992; Roop et al, 1993). However, the attached virions can be delivered into the cells in the presence of the polyethylene glycol, a chemical fusogen, suggesting involvement of these glycoproteins in fusion activity (Fuller and Spear, 1987; Fuller et al, 1989). Thus far, the only confirmed fusogenic glycoprotein is gB. There is no clear evidence regarding the location of the fusogenic domain on gB, but recent work with BHV-1 gB truncation mutants suggests that this domain may be present in the second of the three membrane spanning regions (Li et al, 1997). 22 1.11 HSV egress The egress of herpesvirus particles from an infected cell is believed to initiate with the envelopment of nucleocapsids at the inner lamella of the nuclear membrane (Darlington and Moss, 1968), where virions can be observed budding through the inner nuclear membrane into the perinuclear space. However, subsequent routes taken by these particles as they travel out of the cell are less well understood. One view is that enveloped virions move via the secretory pathway by vesicular transport from the ER to the Golgi complex and trans-Golgi network, ultimately reaching the plasma membrane where they are released. This mechanism of egress implies that the virion acquires a full complement of transmembrane glycoproteins at the inner nuclear membrane and that these immature glycoproteins are processed in situ during egress through the Golgi compartment. Evidence in favor of this model was reported by Johnson and Spear (Johnson and Spear, 1982), who showed that ionophores which disrupt the budding of vesicles from the Golgi, such as monensin, inhibit the transport of HSV progeny virions to the cell surface and lead to an accumulation of enveloped particles in what are believed to be Golgi-derived vacuoles. The view that herpesvirus egress involves a single envelopment process has the intuitive virtue of economy, and this model finds favor in most standard texts. An alternative model for virus egress involves the fusion of enveloped virus particles in the perinuclear cisternae or the ER lumen with the outer nuclear membrane, thus releasing nucleocapsids into the cytoplasm. Capsids are often observed in herpesvirus-infected cells adjacent to membrane-bound vesicular structures, and it has been suggested that these represent virions in the process of re-envelopment by Golgi-derived vacuoles. Data supporting this pathway have been described for varicella-zoster virus (Gershon et ai, 1994; Zhu et ai, 1995), and for PRV (Whealy et ai, 1991). However, arguments against this pathway include analysis of a mutation in HSV-1 glycoprotein D which results in the accumulation of large numbers of unenveloped capsids in the cytoplasm. This mutation also causes reduced yields of extracellular virus, and it has therefore been proposed that naked cytoplasmic nucleocapsids represent a dead-end rather than a stage in the route of virus egress (Campadelli-Fiume etal., 1991). 23 1.12 Cell to cell spread of HSV HSV uses two separate routes to spread to new host cells: production of extracellular virions and direct cell-to-cell transmission. The production of extracellular HSV likely plays an important role in dissemination to other hosts. However, HSV antibodies do not contain HSV spread in epithelial tissues, suggesting that direct cell-to-cell spread is an important property of HSV pathogenesis. Spread of HSV by both the extracellular and cell-to-cell routes requires viral glycoproteins gB, gD, and gH-gL, the same set of glycoproteins that are essential for virus entry. This suggests that these glycoproteins play similar roles in cell-to-cell spread as they do for entry. Indeed, there is evidence that the lysine-rich heparin-binding domain of gB, but not gC, facilitates direct cell-to-cell spread of HSV-1 in cultured cells (Laquerre et al, 1998), while antibodies directed against gD, gH, and gL, prevented HSV-1 induced cell-cell fusion (Gompels and Minson, 1986; Highlander etal, 1987). Glycoproteins gE and gl form a hetero-oligomeric complex (Johnson and Feenstra, 1987; Johnson et al, 1988), that aids HSV in the process of immune evasion by binding to the Fc region of human immunoglobulin G (IgG) causing IgG aggregation (Johnson etal, 1988; Bell etal, 1990; Hanke etal, 1990). However, there have also been reports that deletion of gE or gl from HSV-1 severely inhibited the cell-to-cell spread of virus both in cell culture and in mice, the latter producing IgG which can not be recognized by the gE/gl Fc receptor (Balan et al, 1994; Dingwell et al, 1994). In epithelial cells gE/gl accumulated in cell junctions, co-localizing with the adherens junction protein B-catenin on lateral, but not basal or apical cell surfaces (Dingwell and Johnson, 1998). Together these results support a model in which gE-gl mediates transfer of HSV across cell junctions by interacting with cell junction components. 1.13 Characterization of glycosaminoglycan-deficient/HSV resistant cell lines As part of a broad study to identify host cell factors that facilitate HSV infection, a selection procedure to isolate HSV-resistant mouse cells (gro mutants) was developed (Tufaro et al, 1987). In this procedure, mouse L cells were infected with HSV-1 at an MOI of 1-3 PFU/cell, and after three days, any surviving cells were clonally expanded. It has been shown previously that HSV infection of a gro mutant, gro2C, which synthesizes 24 CS but not HS, is 90% resistant to infection relative to parental control cells (Gruenheid et al, 1993) (Table 1.3). The residual infection remained sensitive to inhibition by HS, however, suggesting that gro2C cells posess a GAG-dependent pathway of infection. Considering the levels of residual infection of gro2C cells, it was proposed that it should be possible to select for a variant of gro2C cells less susceptible to infection that was defective in additional components of the virus entry pathway. When gro2C cells were challenged by 'herpes selection', a cell line was isolated that was nearly uninfectible by HSV-1. This cell line, termed sog9, harbors additional defects in the G A G synthesis pathway such that no HS or CS GAGs are expressed on the cell surface (Table 1.3). HSV-1 infection of these cells is independent of GAGs, and can be restored to the level of gro2C cell infection by incubating the cells in a low concentration of dextran sulfate (Banfield et al, 1995; Dyer et al, 1997). This study provides compelling genetic evidence that HSV contains a 'second' pathway for infection, and that CS as well as HS moieties function as non-essential elements in the initial attachment of the virus to the host cell. 25 Table 1.3 Relative infectivities of HSV on control and mutant cell lines virus Relative infectivity(%)a L(control) gro2C sog9 HSV-l (KOS) 100 10 0.3 HSV-1(F) 100 20 0.8 HSV-2(G) 100 3.3 1 Titers of serial 10-fold dilutions of virus stocks grown in Vero cells were determined on cell monolayers. Results from at least three determinations were averaged and expressed relative to control L cell infection. This data first appeared in (Banfield et al, 1995). 26 1.14 Bone development Bone is a complex tissue composed of cells, collagenous matrix and inorganic elements. The growth, development and maintenance of bone are influenced by a wide variety of cytokines, growth factors and hormones. Long bones grow by extension at either end in growth zones situated between the marrow-filled diaphysis (which is bone) and two growth plates, which separate bone from the bulbous, cartilagenous ends (called epiphysis) (Fig. 1.6) (Poole, 1991). These bones grow via a strictly coordinated event, involving chondrocytes and osteocytes in the growth plate. Chondrocytes, cells that synthesize cartilage matrix in the epiphyses, proliferate, become larger and eventually apoptose. This leaves cavities that are subsequently invaded by osteocytes, present in the diaphyses, which replace cartilage with bone. The hypertrophy and apoptosis of the chondrocytes in the growth plate is thought to be caused, at least in part, by the presence of the perichondrial ring that surrounds the growth plate and prevents diffusion of nutrients and oxygen into the cartilage matrix. This ring contains bone produced by cells of the periosteum (a thin layer of connective tissue that lines the outside of bone); its outward advance precedes bone formation in the growth plate. As the bone-cartilage boundaries move outwards towards the bone ends, osteoclasts follow, reabsorbing bone and leaving a porous infrastructure in which marrow resides (Rodan, 1992). Considering this degree of complexity and strict coordination of events, it should not come as a surprise that disruptions in chondrocyte and osteocyte cell surface architecture often lead to developmental abnormalities. 27 a epiphysis growth plate metaphysis diaphysis metaphysis growth plate epiphysis Reserve zone (resting chondrocytes) Proliferative zone (chondrocytes start proliferating) Transition zone Upper hypertrophic zone Lower hypertrophic calcification of cartilage invading capillary osteoblast calcified trabecule A advancing A I growth plate | Fig. 1.6 Schematic model of bone development. Adapted from (Stickens and Evans, 1998). 28 1 1.14.1 Hereditary multiple exostoses Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by skeletal malformations, which manifest as bony, benign tumours (exostoses or osteochondromas) that develop from the growth plate of endochondral bone (Solomon, 1963) (Fig. 1.7). The development of exostoses during childhood and early puberty appears, in some cases, to take place at the expense of linear bone growth, thus accounting for short stature, which is a common clinical feature. Surgical intervention is sometimes required when these tumours interfere with joint movement or exert pressure on nerves. The first breakthrough in understanding the underlying genetic basis of H M E stemmed from observations that multiple exostoses are also seen in Langer-Giedion syndrome (LGS), a contiguous gene syndrome that maps to chromosome 8q24.1 (Buhler and Malik, 1984; Ludecke et al, 1991; Parrish et al, 1991). This suggested that the gene involved in H M E might also map to this region, and be responsible for the exostoses seen in LGS patients. Genetic linkage analyses of this region confirmed the presence of a locus for H M E , EXTl (Cook et al, 1993), and additional EXTloci on chromosomes 1 l p l 1-13 and 19p designated EXTl and EXT3 (LeMerrer et al, 1994; Wu et al, 1994). The human and mouse EXTl (Ahn et al, 1995; Lin and Wells, 1997) and EXTl (Stickens et al, 1996; Wuyts et al, 1996; Clines et al, 1997) genes have been cloned and sequenced, and their expression pattern indicates broad tissue distribution. At the time of their discovery, EXTl and EXTl shared significant homology with each other, but no homology with any other known genes. 1.14.2 HME-linked malignant transformation The most serious complication of H M E is malignant transformation of the exostoses to chondrosarcomas, which occurs in 0.5-2% of cases (Leone et al, 1987) (Hennekam, 1991) and, more rarely, to osteosarcomas (Schmale etal, 1994; Luckert-Wicklund et al, 1995). This disorder has an estimated prevalence of 1:50,000 to 1:100,000 in Western populations (Hennekam, 1991; Cook et al, 1993; Schmale et al, 1994) but might be as high as 1:1000 in Chamorros, the indigenous people of Guam and the Mariana Islands (Krooth et al, 1961). 29 It has been suggested that the EXT genes function as tumour suppressor genes, based on loss of heterozygosity (LOH) on chromosomes 8,11 and 19 in chondrosarcomas arising from sporadic or multiple exostoses (Hecht et al, 1995; Raskind et al, 1995). However, L O H analysis has also identified additional mutations in HME-derived chondrosarcomas on chromosome 3q and chromosome 10 (Hecht et al, 1997). These observations suggest a model in which inactivation of one EXT gene leads to formation of an exostosis, with subsequent inactivation of a second EXT gene or, possibly, another gene altogether, causing malignant transformation. Recently, several £XT-like genes (EXTL1, EXTL2 and EXTL3) have been identified that share significant sequence homology with the E X T genes (Wise et al, 1997; Wuyts et al, 1997; Van Hul et al, 1998). Although none of these genes has been implicated as a causative agent in H M E , they are localized to chromosomal regions associated with other forms of cancer. For example, EXTL1 and EXTL2 are localized to lp36 and l p l l -pl2, regions of the genome deleted frequently in a variety of tumour types (Wise et al, 1997; Wuyts et al, 1997), whereas EXTL3 is a candidate for the breast cancer locus at 8pl2-22 (Van Hul et al, 1998). Also of particular interest is the region 8q23-24, which contains the EXTl gene, as well as the genes encoding osteoprotegerin (OPG), a member of the tumour necrosis factor (TNF) receptor superfamily that acts as a soluble factor in the regulation of bone mass (Simonet et al, 1997), bone morphogenetic protein 1 (BMP-1), an enzyme involved in matrix deposition that might have a role in skeletal development (Kessler et al, 1996), and syndecan-2 (fibroglycan), an important HS proteoglycan present in developing bone (Marynen et al, 1989). The close proximity of these genes to each other raises the possibility that this region of chromosome 8 might harbour a gene cluster involved in the regulation of bone development. 1.14.3 Skeletal dysplasia and cell surface architecture Bone development is a highly regulated process sensitive to a wide variety of hormones, inflammatory cytokines and growth factors. Many skeletal dysplasias (the abnormal development of bone tissue) result from disruptions in cell surface architecture that perturb the cell's ability to interact with these developmental signaling molecules. Several HS proteoglycans, including glypican, betaglycan, perlecan and members of the 30 syndecan family, are components of the extracellular matrix in developing bone and participate in a wide variety of important biological processes, including critical involvement in cell signaling pathways (Bernfield et al, 1992; De Luca and Baron, 1999). There are numerous examples of skeletal dysplasias resulting from defects in cell surface GAGs. Achondroplasia, the most common form of chondrodysplasia in humans, characterized by short-limbed dwarfism and macrocephaly, is caused by mutations in the gene encoding the fibroblast growth factor (FGF) receptor 3, a cell-surface protein that binds FGF only in the presence of HS (Shiang et al, 1994). In mice, targeted disruption of the gene for biglycan, a small extracellular matrix CS proteoglycan enriched in bone, results in an osteoporosis-like phenotype (Xu et al, 1998). Moreover, diastrophic dysplasia is caused by mutations in a novel sulfate transporter gene, which probably results in undersulfated glycoproteins and GAGs (Wallis, 1995). Taken together, these studies suggest that bone development is a process exquisitely sensitive to changes in the composition of the cell surface, and in particular, proteoglycans. 31 F i g . 1.7 The mul t ip le exostoses phenotype. (a) A dissected right knee jo in t o f a 67-year-old man d isp lay ing exostoses projections on the femur, t ib ia and f ibula , (b) A section through the p rox ima l end on the right femur displays a hereditary mul t ip le exostoses ( H M E ) lesion, as w e l l as wid th and trabecular pattern changes. Reproduced, wi th permiss ion, f rom (Schmale et al, 1994). 32 1.15 Hypotheses and Objectives At the time this study was initiated, the mechanism of HSV-1 attachment to host cell surfaces was not well understood. The Tufaro lab has focused on the study of selected HSV-resistant cell lines which harbour defects in G A G biosynthesis (Gruenheid et al, 1993; Banfield et al, 1995), and the demonstration of the importance of these molecules in HSV entry into the host cell. In particular, sog9 cells were shown to have separate defects in CS and HS biosynthesis, resulting in a cell surface devoid of GAGs (Banfield et al, 1995). However, G A G biosynthesis is a complex multistep process, and it was not known which steps in HS and CS biosynthesis were defective in these cells. To address this problem, a human cDNA library was screened for genes that could restore susceptibility to HSV infection in sog9 cells. I reasoned that these cDNAs would either correct the defect in HS biosynthesis, or correct the defect in CS biosynthesis, or encode a HSV receptor that did not require the presence of cell surface GAGs to function. Multiple rounds of cDNA screening and subpooling resulted in the isolation of a single cDNA, which would restore HSV infection to near wild type levels by correcting the defect in HS biosynthesis. This cDNA was sequenced and identified as EXTl, a putative tumor suppressor involved in hereditary multiple exostoses, a disease characterized by the formation of benign bone tumours during bone development. Following the isolation of EXTl, the focus shifted to address the role of this protein in HS biosynthesis. Through a fruitful collaboration with Dr. Ulf Lindahl's lab, it was determined that E X T l , and another HME-linked protein, EXT2, formed a Golgi-localized complex which had enzymatic activities characteristic of a HS polymerase, which catalyzes the transfer of GlcNAc and GlcA sugars into a growing HS chain. I hypothesized that if proper HS biosynthesis is critical to the development of bone, then disease-causing mutations in E X T l and EXT2 should disrupt the glycosyltransferase activities of the complex. In fact, every disease causing mutation in E X T l or EXT2, including some relatively conservative missense mutations, abrogated HS-Pol activity in isolated complexes. Thus, defects in HS biosynthesis are almost certainly the underlying cause of H M E . 33 The results presented in this thesis provide strong evidence for the identity of the EXT genes and their relationship with each other, and should prove extremely useful for future studies of the role of HS in proper bone development and the defective process in H M E . 34 CHAPTER 2 : Materials and Methods 2.1 Materials A complete list of chemical reagents and suppliers appears in Appendix 1. 2.2 Cells and Viruses The parental L cell used was the clone ID line of Lmtk" mouse fibroblasts (Tufaro et al, 1987). The procedures for the isolation of the mutant gro2C and sog9 cell lines were described previously (Gruenheid et al, 1993; Banfield et al, 1995). Vero cells were a gift from S. McKnight. B H K cells were obtained from the A T C C . B H K , Vero, L , gro2C, sog9 cells were grown at 37° in D M E M supplemented with 10% FBS in a 5% C 0 2 atmosphere. The parental CHO-K1 and mutant CHOpgsA745, CHOpgsB761, CHOpgsD677, CHOpgsE605 and CHOpgsF606 cell lines (Esko et al, 1985) were a gift from J. Esko, and were grown in Ham's F12 media supplemented with 10% FBS. A l l stable EXTl-expressing or EXT2-expressing cell lines were generated by lipofectAMINE-mediated transfection with relevant plasmids, followed by selection in media supplemented with 700 u.g/ml Geneticin (G418). Clonal versions of these cell lines were derived from individual transfected colonies. R8102, a mutant HSV-1 F strain which has the B-galactosidase gene inserted between UL3 and UL4, under the ICP27 promoter, was a gift from B. Roizman. R8102 displays LD50 values similar to wild type HSV-1 strain F (B. Roizman, personal communication). HSV-1 KOS strain was a gift from D. Coen. The KOS-strain derived mutant viruses K C Z , KgBpK", KgBpKgC", KgBpK R gC" and KgBpKgC* (Laquerre et al, 1998) were a gift from J. Glorioso. F-gEBR, F-gEB (Dingwell et al, 1994) and F-US7kan (Johnson et al, 1988) were obtained from D. Johnson. A l l viruses were propagated and titered on Vero cells. 2.3 Viral stock production Subconfluent monolayers of Vero cells in 150 mm dishes were incubated with virus at a multiplicity of infection (MOI) of 0.05 plaque forming units per cell in D M E M . 35 After 1 hour, cells were washed once with PBS and overlaid with DMEM/10% FBS for 3 days. Cells and media were collected into a 50 ml conical vial, and centrifuged at 250 x g for 10 min. Supernatant was collected, except for 1 ml which was used to resuspend the cell pellet. The resuspended cell pellet was then subjected to 3 freeze-thaw cycles in a dry ice/ethanol bath. The cell lysates were centrifuged at 250 x g for 10 min, and the virus-containing supernatants were pooled. The combined supernatants were distributed into aliquots and stored at -80°C. 2.4 Determination of virus titer To determine the titer of virus stocks in various cell lines (i.e. Vero, L , sog9, etc.), 1 x 106 cells were plated in 6-well dishes and infected 18 hours later with serial 10-fold dilutions of virus stock in D M E M . After a 1 hour adsorption period, the inoculum was removed and cell monolayers were washed three times with PBS to remove unbound virus. Cell monolayers were then overlaid with D M E M containing 4% FBS and 0.1% pooled human IgG. As the infection progressed, the IgG neutralized any extracellular virus, while intracellular virus could still be transmitted by direct cell-to-cell contact, allowing a plaque to form. At 3-4 days post-infection, cell monolayers were washed with PBS and plaques were visualized by staining with 70% methanol/5% methylene blue for 5 minutes. 2.5 Isolation of radiolabeled virus Subconfluent monolayers of Vero cells in 150 mm dishes were incubated with virus at a multiplicity of infection (MOI) of 0.1 plaque forming units (PFU) per cell in D M E M . After 1 hour, cells were washed once with PBS and labeled for 48 hours with 25 | i C i of [35S]-methionine in methionine-free medium containing 5% dialyzed FBS and 10% complete D M E M . Cells and media were collected into a 50 ml conical vial, and centrifuged at 250 x g for 10 min. Supernatant was collected, except for 1 ml which was used to resuspend the cell pellet. The resuspended cell pellet was then subjected to 3 freeze-thaw cycles in a dry ice/ethanol bath. The cell lysates were centrifuged at 250 x g for 10 min, and the virus-containing supernatants were pooled. The combined supernatants were distributed into aliquots and stored at -80°C. The radiolabeled virus 36 stocks were titered on Vero cells. Radiolabel incorporation was measured by liquid scintillation counting, and expressed as CPM/PFU. 2.6 Virus attachment assay Confluent monolayers of mutant and control L cells (containing approximately 5 x 105 cells) growing in 24-well dishes were rinsed with phosphate-buffered saline (PBS) and incubated for 1 h at 37° in adsorption medium (DMEM-1% BSA-20 m M HEPES [pH 7.4]). Dishes were removed from the incubator and placed on ice. The medium was removed from the monolayers, and 0.1 ml of [35S]-methionine (>1000 Ci/mmol, ICN) radiolabeled HSV-1 strain KOS (0.5 cpm/PFU) diluted in adsorption medium was added to the wells and incubated for 2 h on ice. Following this adsorption period, the monolayers were rinsed four times with 0.5 ml of PBS prior to solubilization in cold lysis buffer (lOmM Tris-HCl [pH 7.4], 150 mM NaCl, 1 % Nonidet P-40, 1% Na-deoxycholate). Lysates were added to scintillation vials, and the radioactivity associated with the monolayers was determined by liquid scintillation spectroscopy. 2.7 cDNA screening The approach to screening a cDNA library for HSV-1 entry restoring cDNAs began with the establishment of pools of cDNA in E. coli. A cDNA expression library derived from human cervical carcinoma (HeLa) cells and using the pcDNA3.1(+) plasmid backbone was chosen (Invitrogen). To make pools of cDNA, the library in E. coli was plated on 100 L B agar plates containing 100 ug/ml of ampicillin, at a density of approximately 100,000 colonies per plate, so that the entire library, comprising 1 x 107 clones, would be represented. The next day, colonies were manually scraped off each plate into 1 ml of LB/50% glycerol and flash-frozen in a dry ice/ethanol bath. In addition, grouped pools of cDNA in E. coli were made by taking 0.1 ml from each of 10 pools, combining and flash freezing. This resulted in 100 pools of cDNA in E. coli, and grouped pools representing pools 1-10,11-20, 21-30, etc., as well as grouped pools representing all of the pools with numbers ending in a 1 (1, 11,21, etc.), a 2 (2, 12, 22, etc.), and so on. With these grouped pools, all of the pools of cDNA were tested with only 20 transfections instead of 100 transfections. To prepare cDNA pools for transfection, 10 ml L B broth cultures were inoculated with 0.01 ml of the appropriate 37 glycerol stock, grown overnight, and plasmids were purified using the modified alkaline lysis miniprep protocol followed by precipitation with polyethylene glycol (Sambrook et al, 1987). Sog9 cells were transiently transfected with grouped pools of cDNA, representing 1 x 106 cDNAs each, for 30 hours, allowing for cDNA expression. After 30 hours, cell monolayers were infected with R8102, a phenotypically wild type herpes simplex virus that expresses B-galactosidase from the promoter for ICP27, an early gene. The sog9 cell monolayers were infected at a mulitiplicity of infection (MOI) of approximately 0.1 plaque forming units per cell. Considering the relative resistance of sog9 cells to HSV-1 infection, infection at an MOI of 0.1 translated to approximately 500 infected cells out of a total of 1 x 106 cells. Ten hours later, transfected cell monolayers were fixed and stained with X-gal, so that infected cells would be stained blue. Several cycles of screening and subdividing of cDNA pools in this manner resulted in the isolation of a single cDNA which was able to enhance susceptibility to HSV-1 infection by approximately 200-fold. 38 Fig. 2.1 Schematic representation of the cDNA screening protocol. 39 2.8 Construction of EXT fusion proteins p E X T l was isolated from a HeLa cell cDNA library in pcDNA3.1 (#A550-26, Invitrogen). pEXTlmyc was constructed by PCR amplification of the E X T l coding region using primers complementary to the translation start site (5'-CGG G A T CCC G C A G G A C A C A T G C A G GCC A A A A A A C G C TAT TTC A T C C-3') and the region preceding the translation stop site (5'-TTT TCC TTT TGC G G C C G C TTT TTT CCT T A A GTC GCT C A A TGT CTC GGT A-3'), which contained BamHI and NotI restriction enzyme sites, respectively. pANTMEXTlmyc was constructed using a forward primer complementary to the region of E X T l following the putative transmembrane domain (5'-CGG GAT CCC G C A C A T G C A GTT T A G G G C A T C G A G G A G C C A CAG-3') , and the same reverse primer used for pEXTlmyc. A translation start codon, A T G , follows the BamHI restriction enzyme site in the forward primer. p l26AAEXTlmyc was constructed using a reverse primer (5'-TCC C C G C G G G G A G A T TTT CTC CCC TTT TTG CTG-3') containing a NotI site, and the same forward primer used for pEXTlmyc. Following digestion with BamHI and NotI, the EXTlmyc, ANTMEXT1 myc and 126AAEXTlmyc PCR products were ligated into pcDNA3.1/Myc-His B (#V800-20, Invitrogen), such that the myc epitope tag and 6XHis tag were in frame for subsequent translation. pG339DEXTlmyc and pR340CEXTlmyc were constructed by PCR amplification using the same forward primer used for pEXTlmyc, and mutagenic reverse primers which cause a G to A transition ( G339D: 5 ' -CAA A G C CTC C A G G A A TCT G A A G G A C C C A A G CCT G C G A T C A C G A G G A A C CAG-3') or a C to T transition ( R340C: 5 ' -CAA A G C CTC C A G G A A TCT G A A G G A CCC A A G CCT G C A A C C A C G A G G A A C CAG-3' ) . The PCR products and pEXTlmyc were digested with BamHI and PpuMI (a natural restriction site adjacent to the mutations), and the PCR products were ligated into BamHI/PpuMI digested pEXTlmyc. The mutated regions of E X T l were confirmed by D N A sequencing. pEXTlgfp was constructed by excision of E X T l from pEXTlmyc-his with BamHI and Sstll, followed by ligation into the BgUl and Sstll sites in the pEGFP-Nl expression vector (Clontech). The bovine EXT2 constructs were constructed as previously described (Lind et al, 1998). pbEXT2myc was constructed by PCR amplification of the EXT2 40 coding region using primers 5 '-CGG G A T C C C GGT TTC ATT A T G TGT G C G T C A GTC A A G TCC A A C A-3 ' and 5'-GCT C T A G A G CTC A C A G A T CCT CTT CTG A G A T G A GTT TTT GTT C T A A G C TGC C A A TGT TGG-3' . Following digestion with BamHI and Xbal, the bEXT2myc PCR product was then ligated into pcDNA3.1/myc-his B. A murine EXT2 cDNA was a gift from M . Lovett. pmEXT2gfp was constructed by PCR using primers 5'- C G G G A T C C C GGT TTC ATT A T G TGT G C G T C A GTC A A G TCC A A C A -3' and 5'-TCC C C G C G G G G A T A A GCT G C C A A T GTT G G G G A A - 3 ' . The mEXT2 PCR product was ligated into T-tailed pBluescript (Stratagene), followed by digestion with Hindlll and EcoRI and subsequent ligation into pEGFP-Nl . pmEXT2myc-his was constructed by digestion of pmEXT2gfp with Hindlll and Sstll and ligation into the pcDNA3.1/myc-his B vector. To isolate human EXTL2, EXTL3, and murine N-deacetylase/N-sulfotransferase-2 (NDST2), total cellular R N A was isolated from confluent HeLa cell and L cell cultures using TRIZOL Reagent, reverse transcribed and amplified by PCR using primers 5'- C C G CTC G A G C G G A A T T A A ACT T C A A C A C A A T G -3' and 5 ' -GGG G T A C C C C T A TTT TTC TTT TGT A G T TGG CAT-3 ' for EXTL2, 5'- C C G CTC G A G C G G C A G GCT G C A G A G G A C T C A T -3' and 5'-GGG GTA C C C C G A T G A A C T T G A A G C A C T TGG TCT-3' for EXTL3 and 5'- G A A G A T CTT C C C A C C A T G CTC C A G CTG T G G A A G GT -3' and 5 ' -CGG A A T TCC GCC C A C A C T G G A A T G TTG C A A T-3* for NDST2. The digested PCR products were ligated into the appropriate sites in pEGFP-Nl . G339DEXTlgfp, R340CEXTlgfp, and ANTMEXTlgfp were constructed by excision of the appopriate E X T l mutant insert from pcDNA3.1myc-his with BamHI and Sstll, followed by ligation into the BgUI and Sstll sites in the pEGFP-Nl expression vector (Clontech). C85REXT2gfp was generated by amplification of the entire mEXT2gfp plasmid with the mutagenic forward primer 5 ' -CGG G G G G A T CTC A G C CGT A G A A T G C A T AC-3 ' and the mutagenic reverse primer 5 ' -GTA TGC ATT C T A C G G CTG A G A TCC C C C CG-3 ' , and digestion of template D N A with Dpnl prior to transformation of competent E. coli. Successfully mutated C85REXT2gfp plasmids were identified by digestion with PvuII (loss of one site), and confirmed by D N A sequencing. D227NEXT2gfp was generated by amplification of the entire mEXT2gfp plasmid with the mutagenic forward primer 5'-CTT A C C GGC A G G GCT A C A A T G T C A G T A TTC 41 C-3' and the mutagenic reverse primer 5 ' -GGA A T A CTG A C A TTG T A G C C C TGC C G G T A A G-3', and digestion of template D N A with Dpnl prior to transformation of competent E. coli. Successfully mutated D227NEXT2gfp plasmids were identified by digestion with BsmI (gain of one site), and confirmed by D N A sequencing. A l l primers and restriction enzymes (except PpuMI, New England Biolabs) were obtained from Life Technologies. 2.9 Antibodies Primary monoclonal antibodies directed against the 6xHIS tag were obtained from Invitrogen, and anti-Myc monoclonal antibodies were obtained from Invitrogen and the A T C C (9E10 hybridoma). A polyclonal rabbit antibody directed against the myc-epitope tag was obtained from M B L . Polyclonal antibody directed against the GFP fusion protein was obtained from Clontech. Antibodies directed against various organelle marker proteins included the ER-specific mouse anti-calnexin (Transduction Laboratories) and rabbit anti-GRP78(BiP) (StressGen), the cis-Golgi-specific mouse anti-GS28 (StressGen), the medial-Golgi-specific mouse anti-membrin (StressGen), and the trans-Golgi-network-specific mouse anti-syntaxin 6 (StressGen). Mouse anti-paxillin was obtained from Transduction Labs. Secondary antibodies used for immunofluorescence detection included goat anti-mouse IgG conjugated to Texas Red (Cedarlane), goat anti-mouse IgG conjugated to Fluorescein (KPL), goat anti-rabbit IgG conjugated to Texas Red (Jackson Immunochemicals), and goat anti-rabbit IgG conjugated to Fluorescein (KPL). Secondary antibodies used for immunoblotting procedures included Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (KPL) and HRP-conjugated goat anti-rabbit IgG (KPL). 2.10 Indirect immunofluorescence microscopy. Monolayers of CHO-K1, sog9 and B H K cells were grown on glass coverslips to 70% confluence in either Ham's F12-10% FBS or DMEM-10% FBS. Transfection of these cells with E X T constructs was carried out following the LipofectAMINE (Gibco BRL) protocol. At 24-30 h post-transfection, the cells were rinsed with PBS and fixed in 4% paraformaldehyde for 15 min, followed by a 15 min incubation in the blocking 42 solution (PBS with 1% BSA). After blocking, cells were incubated with anti-HIS monoclonal antibody (Invitrogen), or anti-myc monoclonal antibody (Invitrogen) at 1:100, and anti-Golgi 58K monoclonal antibody (Sigma) or anti-calnexin monoclonal antibody (Transduction Laboratories) at 1:50 in PBS-1% B S A with 0.25% saponin (Sigma) for 1 h. Cells were washed three times with PBS, then incubated with goat anti-mouse IgG conjugated to Texas Red (Jackson Immunochemicals) diluted 1:200 in PBS-1%BSA for 30 min. The cells were then rinsed with PBS and mounted on glass coverslips. Immunofluorescence staining was observed using a Bio-Rad M R C 600 confocal epifluorescence microscope. Confocal images were rendered using NIH Image Version 1.60 and colorized with Adobe Photoshop Version 5.0 (Adobe Systems Inc.). Standard control experiments were performed, including incubation with the secondary antibody only, and with mock infected cells. A l l fixation and antibody incubations were performed at room temperature. 2.11 Immunoblotting Cell lines were transfected with myc tagged E X T l constructs using LipofectAMINE. After 30 h, cells were washed three times with PBS and lysed in 60mM n-octyl-b-glucopyranoside-TNE (lOmM Tris pH 7.5, 150mM NaCl, and 2mM EDTA) containing 1 flg/ml aprotinin (Boehringer Mannheim) and lu,g/ml leupeptin (Boehringer Mannheim) and incubated on ice for 10 min. Lysates were clarified by centrifugation at 8000Xg at 4°C for 10 min. Portions of the lysates were digested with l m U endoH (Boehringer Mannheim), or mock digested in endoH buffer (85mM sodium citrate-lug/ml aprotinin-lug/ml leupeptin). Lysates were resuspended in an equal volume of 2X sample buffer, boiled and separated on 7.5% polyacrylamide gels, and transferred to PVDF Immobilon-P membrane (Millipore). Membranes were incubated with a 1/5000 dilution of mouse anti-myc Mab (Invitrogen) or l|ig/ml of 9E10, a mouse anti-myc M A b (ATCC) in TBST (0.1% Tween-20, 20 mM Tris pH 7.5, 150 mM NaCl) and 1%BSA for 3 hs, washed, and incubated with a 1/10,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse M A b in TBST for 1 h. Membranes were washed thoroughly, developed using the chemiluminescence assay according to the manufacturer's instructions (KPL LumiGLO Chemiluminescent Substrate Kit), and exposed to B i o M A X 43 M L film (Kodak). Prestained molecular weight markers (Bio-Rad) were run on gels and transferred to the PVDF membrane. 2.12 Immunoprecipitation of radiolabeled EXT proteins Cell lines were transfected with GFP or myc-his tagged E X T constructs. After 18 h, cells were radiolabeled with 100 jxCi/ml 35S-methionine (ICN) in methionine and cysteine-free D M E M (ICN) for 2 h at 37°C. Cells were washed with PBS and lysed in Triton lysis buffer (2% Triton X-100, 20 m M Tris-HCl pH 7.4, 150 m M NaCl, 0.1 T.I.U./ml aprotinin, 10 Ug/ml leupeptin and 10 Ug/ml pepstatin) at 4°C for 15 min. The lysates were centrifuged at 12,000 x g for 15 min, and pre-cleared for 30 min with 25 fil of protein G-Sepharose (Pharmacia) at 4°C. The lysates were then incubated with 0.5 ug of mouse anti-myc monoclonal antibody (Invitrogen) or 0.5 ug of mouse anti-GFP monoclonal antibody (Clontech) for 2 h, followed by incubation with 25 ul of Protein G-Sepharose for 1 h. The lysates were centrifuged at 12,000 x g for 10 s, and washed two times with 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.2% Triton X-100, two times with 10 mM Tris-HCl pH 7.4, 500 mM NaCl, 2 mM EDTA, 0.2% Triton X -100, and two times with 10 mM Tris-HCl pH 7.4. The pellet was suspended in 30 ul of SDS-PAGE sample buffer and boiled for 15 min, prior to SDS-PAGE. Gels were dried, or proteins transferred to Immobilon-P membranes, and exposed to B i o M A X M R film (Kodak). 2.13 Pulse-chase Labeling Experiments B H K cells were transfected with myc or GFP-tagged mEXT2 constructs using the LipofectAMLNE PLUS protocol (Life Technologies). After 18 h, cells were incubated in methionine and cysteine-free D M E M (ICN) for 30 min, followed by radiolabeling with 100 uCi/ml 35S-methionine (ICN) for 30 min. Excess methionine was added and the monolayers lysed and immunoprecipitated (as described above) at 0,0.5, 1, 2, and 5 hours post-chase. Immunoprecipitates were divided in half and digested with l m U endoglycosidase H (Roche), or mock digested in endoH buffer (85 m M sodium citrate -0.1 mM PMSF). Lysates were resuspended in an equal volume of 2X sample buffer, 44 boiled and separated on 7.5% polyacrylamide gels. Gels were dried and exposed to B i o M A X M R film (Kodak). 2.14 Assay of Cellular Glycosyltransferase Activities. B H K or mutant sog9 cells were transfected with E X T constructs. At 30 h post transfection, cells were washed in PBS and lysed in Triton-glycerol lysis buffer (2% Triton X-100, 50% glycerol, 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1 T.I.U./ml aprotinin, 10 ug/ml leupeptin and 10 ug/ml pepstatin) with gentle agitation at 4°C for 20 min. The lysates were centrifuged at 12,000 x g for 15 min, and a portion of the supernatant representing 5 x 105 cell equivalents was subjected to immunoprecipitation as described above. Prior to the final wash, the beads were split into two equal fractions and centrifuged. Each pellet was suspended in 10 pi of either GlcNAc-T reaction mix (20 pg [GlcA-GlcNAc] n acceptor, 0.04 uCi UDP- 3 H-GlcNAc, 10 m M M n C l 2 , 0.04% Triton X -100, 70 mM HEPES pH 7.2) or GlcA-T reaction mix (40 pg GlcNAc-[GlcA-GlcNAc] n acceptor, 0.032 uCi UDP- 1 4 C-GlcA, 10 mM MgCl 2 , 5 m M CaCl 2 , 0.04% Triton X-100, 70 mM HEPES pH 7.2), and incubated for 30 min at 37°C as described previously (Lind et al, 1998). The reaction products were suspended in 1 ml dH 2 0 and centrifuged at 12,000 x g for 1 min prior to loading on a 50 cm Sepharose G-25 column. Labeled oligosaccharides were eluted in 50 mM Tris pH 7.4, 1 M NaCl, 1% Triton X-100, and quantified by liquid scintillation spectroscopy. 2.15 Anion exchange chromatography of glycosaminoglycans Biochemical labeling of GAGs was performed by a modification of procedures described previously (Bame and Esko, 1989). Briefly, GAGs were radiolabeled by incubation of cells for 24h with 50 (tCi of [35S]-sulfate (carrier free, -43 Ci/mg, ICN) per ml in DMEM-FBS modified to contain 10 (iM sulfate. The cells were washed three times with cold PBS and solubilized with 1.5 ml of 0.1 M NaOH at 25° for 15 min. Extracts were adjusted to pH 5.5 by the addition of concentrated acetic acid and treated with 2 mg of protease (Sigma) per ml in 0.32 M NaCl-40 mM sodium acetate, pH 5.5, containing shark cartilage chondroitin sulfate (2 mg/ml) as carrier, at 37° for 12 h. For some experiments, portions of the radioactive material were treated for 12 h at 37° with 10 mU of chondroitin A B C lyase (Sigma) or 0.5 U of heparitinase (Sigma). The radioactive 45 products were quantified by chromatography on DEAE-Sephacel (Pharmacia) by binding in 50 mM NaCl followed by elution with 1 M NaCl. For HPLC analysis, the glycosaminoglycan samples were desalted by precipitation with ethanol. Following centrifugation, the ethanol precipitates were suspended in 20 mM Tris, pH 7.4, and resolved by anion exchange HPLC with a TSK DEAE-3SW column (15 by 75 mm; Beckman Instruments). Proteoglycans were eluted from the column with a linear 50 to 700 mM NaCl gradient formed in 10 mM K H 2 P 0 4 (pH 6.0). A l l buffers contained 0.2% Zwittergent 3-12 (Calbiochem) to extend the life of the column. The glycosaminoglycans in the peaks were identified by digestion of the sample with the relevant enzymes prior to chromatography. 46 CHAPTER 3: Identification of EXTl as a type II transmembrane glycoprotein involved in heparan sulfate biosynthesis 3.1 Introduction Many of the advances in the understanding of HSV-1 entry into host cells have come from the study of HSV-1 resistant cell lines. The Tufaro lab's approach to studying HSV-1 entry has been to generate HSV-1 resistant murine cells and characterize their defects. By infecting susceptible murine L cell fibroblasts, and isolating and culturing any surviving cells, gro2C cells were isolated, which were 90% resistant to HSV-1 infection (Gruenheid et al, 1993). Analysis of the cell surface of gro2C cells revealed that these cells synthesized normal amounts of CS G A G , but no HS G A G . This evidence confirmed that HS was indeed a receptor for HSV-1. However, the residual infection of gro2C cells was still sensitive to inhibition by exogenously-added HS, suggesting that gro2C cells possessed a GAG-dependent pathway of infection. A second round of selection of HSV-1 resistant cells was performed, using gro2C cells as the parental cell line, resulting in the isolation of a cell line that was almost completely uninfectable by HSV-1. This cell line, termed sog9, harbours additional defects in the G A G synthesis pathway such that no GAGs are expressed on the cell surface. Taken together, these cell lines show that, in the absence of HS, CS could be used an alternative receptor for the initial binding of HSV-1 virions to the host cell surface. The isolation of HSV-1 resistant cell lines that had defects in G A G biosynthesis demonstrated the importance of these molecules in the infection process. Using a similar technique as the method used to screen for HSV-1 receptors in CHO-K1 cells (Montgomery et al, 1996)(Fig. 2.1), I attempted to screen a human cDNA library for genes which could restore susceptibility to HSV-1 infection in the highly resistant sog9 cell line. I reasoned that these cDNAs would either correct the defect in CS biosynthesis, or correct the defect in HS biosynthesis, or encode an HSV-1 receptor that did not require the presence of cell surface GAGs to function. 47 3.2 Results 3.2.1 Screening of a cDNA library for HSV-1 entry-restoring cDNAs The approach to screening a library for HSV-1 entry restoring cDNAs began with the establishment of pools of cDNA in E. coli. A cDNA expression library derived from human cervical carcinoma (HeLa) cells and using the pcDNA3.1(+) plasmid backbone was chosen because Montgomery et al. had successfully isolated HveA from HeLa cell cDNAs, and also because HeLa cells have multiple chromosomal duplications (see the A T C C 'Cells and Viruses' catalog) which may mean that some low-copy number genes may be more strongly represented in the cDNA library. Although the target cell line, sog9 cells, are mouse cells, I reasoned that proteins expressed from a human cDNA library should still be expressed and function in a similar manner in mouse cells as they would in a human cell line, as had been the case in the CHO-K1 cells (Montgomery et ai, 1996). Sog9 cells were transiently transfected with grouped pools of cDNA, representing 1 x 106 cDNAs each, for 30 hours, allowing for cDNA expression. After 30 hours, cell monolayers were infected with R8102, a phenotypically wild type HSV that expresses B-galactosidase. The sog9 cell monolayers were infected at a low mulitiplicity of infection (MOI) such that approximately 500 cells out of a total of 1 x 106 cells would be infected. Ten hours later, infected cells would be stained blue with X-gal substrate. Cell monolayers transfected with the grouped pool representing pools 1-10, as well as cells transfected with the grouped pool representing pools 5, 15, 25, etc. showed a modest (approximately two-fold) increase in susceptibility to HSV-1 infection. This indicated that pool 5 may contain a cDNA with the ability to increase the HSV-1 susceptibility of sog9 cells. Transfection of sog9 cells with cDNAs from pool 5 alone (representing approximately 1 x 105 cDNAs) resulted in a six-fold increase in susceptibility to HSV-1 infection, compared to mock transfected cells. Subsequent screening lead to the identification of subpools of pool 5 that showed ever-increasing levels of HSV-1 infection-stimulating activity. As further subpools were made, and the infection-stimulating cDNA accounted for a larger fraction of the total transfected cDNA, the observed stimulation of HSV-1 infection of sog9 cells became greater. Several cycles of 48 screening and subdividing in this manner resulted in the isolation of a single entry mediator (EM) cDNA which was able to enhance susceptibility to HSV-1 infection by approximately 200-fold (Fig. 3.1a, b). To better examine the effects of this E M cDNA on sog9 cells, stably-expressing cell lines were selected by addition of Geneticin (G418) to the culture medium of transfected cells. Infection of these stable E M cDNA-expressing sog9 cells revealed that the level of infection was nearly equal to that of the GAG-decorated parental mouse L cell fibroblasts (Fig 3.1c). This dramatic enhancement of susceptibility to HSV-1 infection suggested that the unidentified E M cDNA likely encoded a protein which was critical for HSV-1 entry into the host cell. 49 cell lines HSV-1 Added (CPM x 100) F i g . 3.1 Effect o f entry mediator ( E M ) c D N A expression on H S V - 1 infection o f sog9 cel ls . Subconfluent sog9 ce l l monolayers were transfected wi th a control p l a smid (a) or E X T l (b) and after 30 h infected wi th R 8 1 0 2 , a B-galactosidase expressing H S V - 1 strain at 0.1 plaque fo rming units per ce l l . A t 10 h post infection, cells were stained wi th X - g a l . (c) Sog9 cel ls stably expressing the putative entry mediator c D N A were infected wi th H S V -l ( K O S ) to determine their susceptibil i ty to H S V - i n f e c t i o n . Plaques were v i sua l ized after 4 days by methylene blue staining, (d) T o determine the effect o f the entry mediator c D N A on the attachment phase o f H S V - 1 infection, ce l l monolayers were incubated wi th radiolabeled H S V - l ( K O S ) on ice for 2 h. The radioactivi ty associated wi th the monolayer was determined by l i qu id scint i l la t ion spectroscopy. Va lues are averages o f four experiments. Open squares, L cel ls ; open circ les , sog9 cel ls ; c losed circ les , sog9-E M c D N A cel ls . 50 3.2.2 Identification ofEXTl as a HSV-1 infection enhancing cDNA To identify the cDNA which was able to restore the HSV-1 susceptibility to sog9 cells, PCR and D N A sequencing experiments were performed using primers directed against the T7 forward and B G H reverse primer binding sequences provided in the pcDNA3.1(+) expression plasmid. PCR-amplification of the cDNA insert produced a 3.3 kilobase (Kb) product (Fig. 3.2a). Excision of the cDNA insert from the pcDNA3.1 plasmid with Hindlll and Xbal, resulted in 2.2 Kb and 1.1 Kb fragments, indicating that the cDNA insert had an additional internal Hindlll or Xbal site (Fig. 3.2b). Subsequent experiments revealed that this additional site was a Hindlll restriction enzyme site (data not shown). D N A sequencing from the 5' (T7 forward primer) and 3' (BGH reverse primer) ends of the cDNA, in conjunction with B L A S T (Basic Local Alignment Search Tool, http://www.ncbi.nlm.nih.gov/BLAST) searches, revealed that our cDNA sequences were greater than 90% identical with the putative tumour suppressor gene, EXTl (reference sequence accession #:NM000127), a candidate gene for a genetic disorder called hereditary multiple exostoses. The EXTl gene encodes a 3.3 Kb mRNA transcript, consisting of a 2.2 Kb protein-coding region flanked by a 750 base 5' untranslated region (UTR) and a 300 base 3' UTR. The isolated E M cDNA and E X T l only differed significantly in the extreme 3' end of the 3' UTR (some heterogeneity has been observed in this region among the human EXTl sequences submitted to Genbank—accession #s; S79639, U70539). Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilage-capped tumors (exostoses) that develop from the growth plate of endochondral bone (Solomon, 1963). This condition can lead to skeletal abnormalities, short stature, and in some instances, malignant transformation from exostoses to chondrosarcomas (Leone et al, 1987; Hennekam, 1991) or osteosarcomas (Schmale et al, 1994; Luckert-Wicklund et al, 1995). Genetic linkage analysis has identified three different loci for H M E , EXTl on 8q24.1, EXTl on 1 l p l 1-13, and EXT3 on 19p (Cook et al, 1993; LeMerrer et al, 1994; Wu et al, 1994). Sequence analysis of cDNA clones for the EXT genes indicate significant similarity to each other but not to other known genes (for a detailed sequence alignment of E X T l and EXT2, see 51 Fig. 4.1). EXTl is predicted to encode a 746 amino acid protein (Fig. 3.3) from a 3.4 kb mRNA that has been shown to be expressed in a large number of tissues, with the highest expression in the liver (Ahn et al, 1995). The promoter region of EXTl contains GC and C A A T boxes, but no T A T A box, suggesting a form of regulation characteristic of housekeeping genes. This is consistent with the ubiquitous expression of EXTl (Ludecke et al, 1997). Despite this extensive background knowledge of E X T l genetics, its cellular function, and hence its role in promoting HSV-1 infection of sog9 cells, was not clear. 3.2.3 Virus attachment to EXTl-transfected cell monolayers EXTl has been shown to be a member of a multigene family responsible for H M E , and yet nothing has been reported about the EXTl gene product and the mechanism by which it may function as a tumor suppressor. It was therefore useful to investigate the relationship between EXTl-mediated alterations in HSV infection and H M E disease pathogenesis. It is known that HSV infects cells by first attaching to cell surface GAGs (Banfield et al, 1995). To test whether the rescue of HSV-1 infection conferred by E X T l expression was the result of enhanced expression of cell surface GAGs, monolayers of control L cells, sog9 cells, and EXTl-expressing sog9 cells (sog9-EXT1) were incubated at 4° with radiolabeled HSV-1 and the adsorbed radioactive virus was quantified by liquid scintillation spectroscopy (Fig. 3.Id). These assays showed clearly that E X T l expression resulted in a partial restoration of HSV-1 attachment to sog9 cells. Competition assays using soluble heparin also indicated that the attachment observed was mediated by a HS-like molecule (data not shown). 52 A E M c D N A (ng) r 1 K b 1 10 100 B 1 K b - + •3.3 Kb I - 5 . 4 K b I- 2 . 2 K b I - 1.1Kb F i g . 3.2 P C R ampl i f ica t ion and restriction enzyme analysis o f putative H S V - e n t r y mediat ing ( E M ) c D N A . (a) A 3.3 K b product was P C R ampl i f ied from the p c D N A 3 . 1 expression p lasmid , containing the c D N A insert, (b) Diges t ion o f the p c D N A 3 . 1 - E M p lasmid wi th Hindlll and Xbal y i e lded a 5.4 K b p c D N A 3 . 1 vector band, and 2.2 K b and 1.1 K b fragments compr i s ing the E M c D N A insert. 53 A M Q A K K R Y F I L L S A G S C L A L L FYFGGLQFRA SRSHSRREEH SGRNGLHHPS so PDHFWPRFPE PLRPFVPWDQ L E N E D S S V H I SPRQKRDANS S I Y K G K K C R M 1 0 0 E S C F D F T L C K K N G F K V Y V Y P Q Q K G E K I A E S Y Q N I L A A I E G SRFYTSDPSQ iso A C L F V L S L D T LDRDQLSPQY VHNLRSKVQS LHLWNNGRNH L I F N L Y S G T W 2 0 0 PDYTEDVGFD IGQAMLAKAS I S T E N F R P N F D V S I P L F S K D HPRTGGERGF 2 5 0 L K F N T I P P L R KYMLVFKGKR Y L T G I G S D T R NALYHVHNGE D V V L L T T C K H 3 0 0 GKDWQKHKDS RCDRDNTEYE KYDYREMLHN A T F C L V P R G R R L G S F R F L E A 3 5 0 LQAACVPVML SNGWELPFSE VINWNQAAVI G D E R L L L Q I P S T I R S I H Q D K 4 0 0 ILALRQQTQF LWEAYFSSVE K I V L T T L E I I Q D R I F K H I S R NSLIWNKHPG 450 G L F V L P Q Y S S Y L G D F P Y Y Y A N L G L K P P S K F T A V I H A V T P L V S Q S Q P V L K L soo LVAAAKSQYC A Q I I V L W N C D KPLPAKHRWP A T A V P V V V I E GESKVMSSRF sso L P Y D N I I T D A V L S L D E D T V L S T T E V D F A F T VWQSFPERIV GYPARSHFWD eoo NSKERWGYTS KWTNDYSMVL T G A A I Y H K Y Y H Y L Y S H Y L P A SLKNMVDQLA eso N C E D I L M N F L V S A V T K L P P I KVTQKKQYKE TMMGQTSRAS RWADPDHFAQ 7 0 0 RQSCMNTFAS WFGYMPLIHS QMRLDPVLFK D Q V S I L R K K Y R D I E R L 746 B 100 200 300 400 500 600 700 11M1111111M1111111111111111111111111111111111111M11 j 1111111111111111111 100 200 300 400 500 600 700 Fig. 3.3 Protein sequence of EXTl, the putative HSV-entry mediating cDNA. (a) Sequence is expressed in standard single-letter code. The putative transmembrane domain is highlighted in green, putative N-linked oligosaccharide addition sites are highlighted in red. (b) Kyte-Doolittle hydophobicity plot (generated using D N A Strider version 1.1) of E X T l . Proposed hydrophobic regions of the protein are expressed as positive values, while hydrophilic domains are expressed as negative values. 54 3.2.4 Anion exchange chromatography of glycosaminoglycans from E X T 1 -expressing cells To test directly whether E X T l expression altered the synthesis of GAGs in sog9 cells, radiolabeled GAGs were isolated from total cell extracts and analyzed by anion exchange HPLC (Fig. 3.4). As shown previously (Banfield et al, 1995), control L cells synthesize two major sulfated peaks representing HS (fractions 46-59) and CS (fractions 60-68) (Fig. 3.4a). When stable cell lines expressing E X T l were examined, there was a dramatic shift in the HS peak such that the HS moieties synthesized were relatively undersulfated and therefore eluted as a broad peak at lower salt. The effects of E X T l expression appeared to be specific because CS synthesis was not significantly altered. To further test the specificity o f E X T l activity in the HS biosynthesis pathway, gro2C cells were analyzed in a similar assay. Gro2C cells were derived previously from L cells by selecting for resistance to HSV-1 infection, and are deficient in HS but not CS synthesis (Gruenheid et al, 1993). Gro2C cells expressing the E X T l cDNA (Fig. 3.4b) synthesized a novel peak compared with control gro2C cells that eluted at a similar position as the EXT-1 mediated HS peak synthesized by control L cells. Once again, CS synthesis was relatively unaffected by E X T l . We then analyzed sog9 cells which, as it turns out, have lost the ability to synthesize any of the major G A G species (Fig. 3.4c). Sog9 cells expressing the E X T l cDNA synthesized a single major peak of GAGs that eluted at a similar position as the EXTl-mediated peaks synthesized by control L and gro2C cells (Fig. 3.4c). This peak was sensitive to digestion with heparitinase confirming its identity as a HS G A G (Fig. 3.5). Taken together, the H P L C data indicated that E X T l expression exerts a dominant effect on G A G synthesis such that a novel peak of HS G A G is synthesized. 55 0 4o^ rxxxo3?oooocc^ ^ 10 20 30 40 50 Fraction Number Fig. 3.4 Anion exchange chromatography of glycosaminoglycans from E X T l expressing cell lines. Cell monolayers were grown for 24 h in the presence of [35S]-sulfate. GAGs were isolated and fractionated by HPLC. HS, elution position of heparan sulfate; CS, elution position of chondroitin sulfate, (a) Open squares, L cells; closed squares, L-EXT1 cells, (b) Open diamonds, gro2C cells; closed diamonds, gro2C-EXTl cells, (c) Open circles, sog9 cells; closed circles, sog9-EXTl cells. HPLC was performed by Yves Leduc. 56 Fig. 3.5 Anion exchange chromatography of heparitinase-digested glycosaminoglycans from EXTl-expressing sog9 cells. 10% of the purified [35S]-labeled glycosaminoglycans from sog9-EXTl cells (depicted in Fig. 3.4c), were digested with 0.5 U of heparitinase for 12 h at 37°C, re-purified, and fractionated by HPLC. The remaining heterogeneous GAGs from fractions 10-40 are typical digestion products for the heparitinase enzyme, which rarely digests GAGs to monosaccharides. HS, elution position of heparan sulfate. HPLC was performed by Yves Leduc. 57 3.2.5 Construction of myc-epitope tagged and GFP tagged E X T l fusion proteins Analysis of the E X T l amino acid sequence suggested that it could be a transmembrane glycoprotein that assumes a type II configuration with a seven amino acid cytoplasmic amino-terminus, a nineteen amino acid signal anchor sequence, and a long, lumenal carboxy-terminus (Fig. 3.3a). This assessment is based on hydropathy plots (Fig. 3.3b), which show a strong hydrophobic region near the N-terminus, and the presence of the sequence M Q A K K R at the N-terminus, which is similar to the N-termini of other type II ER-localized transmembrane proteins such as p63 and the H L A class II associated invariant chain (Teasdale, 1996). The essential elements of this ER-targeting signal, the di-arginine or X X R R motif, include two arginine residues, possibly substituted by lysines, close to the N-terminus, located at positions 2 and 3, 3, and 4, or 4 and 5, or split by a residue at positions 2 and 4, or 3 and 5 (Schutze, 1994). E X T l also contains two putative N-glycosylation sites. To test the intracellular location, post-translational modifications, and topology of the E X T l protein, plasmid constructs containing a truncated 5' non-coding region and a carboxy-terminal myc-epitope (EXTlmyc) or green fluorescent protein (GFP) tag (EXTlgfp) were generated. These constructs retained full activity in the HSV entry assay, which indicated that the myc and GFP tags did not impede the function of this protein, and that the upstream non-coding region was not required for expression. 3.2.6 Immunofluorescence analysis of E X T l fusion proteins To investigate the subcellular localization of E X T l , cell monolayers were transfected with EXTlgfp and after 30 hours, visualized by fluorescence microscopy. Confocal micrographs (Fig. 3.6a) revealed a staining pattern characteristic of the endoplasmic reticulum and Golgi apparatus. This was confirmed by co-staining for the ER-resident protein calnexin and the Golgi resident protein, Golgi 58K (data not shown). Parallel experiments were performed with E X T l myc, which, when visualized with anti-myc monoclonal antibody and a Texas Red conjugated secondary antibody, revealed a similar ER/Golgi localization (data not shown). This pattern was detected in all cell types investigated. 58 The identification o f E X T l as an ER/Golgi-resident protein suggested that it exerted its effects on the cell surface by altering proteins traversing the secretory organelles en route to the cell surface. Moreover, the putative type II configuration hypothesized for E X T l resembled several ER/Golgi-resident membrane proteins (Teasdale, 1996) including several glycosyltransferases involved in carbohydrate metabolism (Paulson and Colley, 1989). To investigate this further, a construct was generated in which the amino-terminal 26 amino acids were deleted from the EXTlmyc (ANTMEXT1 myc) and EXTlgfp (ANTMEXT1 gfp) constructs. Because these 26 amino acids contained the putative signal anchor sequence, I reasoned that A N T M E X T 1 myc and A N T M E X T 1 gfp should not be translocated to the ER upon translation. Following transfection of A N T M E X T 1 gfp, the N-terminally deleted form o f E X T l was found in a diffuse cytoplasmic location (Fig. 3.6b), and did not function in the HSV-1 infection assays. Identical results were obtained for A N T M E X T 1 myc (data not shown). These results indicated that the amino terminal hydrophobic domain was important for E X T l localization and function. To test whether the N-terminal portion o f E X T l was responsible for ER targeting, a construct was made in which the carboxy-terminal 620 amino acids o f E X T l were deleted from the EXTlmyc construct (126AAEXTlmyc). Although this E X T l construct had no activity in the functional HSV-1 assays, it did assume an ER localization (data not shown). Thus, the amino terminal 126 amino acids of E X T l were sufficient for ER targeting, which was consistent with the hypothesis that the K K R motif at the N-terminus o f E X T l functions as an ER-retrieval signal. 59 F i g . 3.6 Subcel lu lar loca l iza t ion o f GFP- t agged fusion proteins. E X T l g f p (a), and A N T M E X T l g f p (b), were transfected into B H K cel ls . Af ter 30 h, cells were washed in P B S , f ixed in 4 % paraformaldehyde, mounted on glass covers l ips , and observed us ing a B i o R a d M R C 600 confocal epifluorescence microscope. 60 3.2.7 Immunoblotting of E X T l fusion proteins Western blots of E X T l myc isolated from transfected Chinese hamster ovary (CHO) cells revealed two forms of EXTlmyc: 91 and 88 kD (Fig. 3.7). By contrast, the polypeptide expressed from the ANTMEXTlmyc construct accumulated as a single band of 88 kD. Because E X T l has two consensus addition sites for N-linked oligosaccharides, it was likely that the 91 kD and 88 kD forms of EXTlmyc represented alternatively processed forms of the ER/Golgi localized protein. To test whether E X T l was a glycoprotein as predicted from the sequence, proteins were extracted from EXTlmyc expressing cells and incubated with endoglycosidase H (endoH) prior to immunoblotting. Endo H cleaves high mannose N-linked oligosaccharide moieties from the protein backbone, and as such is an excellent reagent for determining the relative extent of N -linked processing in mammalian cells. In these experiments, endo H trimmed the high-mannose N-linked oligosaccharides from both the 91 kD and 88 kD species, generating 88 kD and 85 kD products (Fig. 3.7), which indicated that E X T l was a glycoprotein modified by high mannose sugars characteristic of the ER and proximal Golgi elements. Moreover, glycosylation at these two sites indicated that they were in the ER/Golgi lumen, which is consistent with a type II membrane topology. By contrast, ANTMEXTlmyc , which was not translocated into the ER due to loss of the predicted ER-localization motif, was endoH resistant (data not shown). 61 Fig. 3.7 Immunoblotting of E X T l fusion proteins. CHO cells were transfected with myc-tagged E X T l constructs for 30 h. CHO cell lysates were digested or mock-digested with 1 U endoH and subjected to polyacrylamide gel electrophoresis. Western blots were incubated with an anti-myc antibody and detected by chemiluminescence. The sizes of the prestained protein molecular weight markers are indicated in kilodaltons. 62 3.3 Discussion These studies show that E X T l functionally alters the expression of cell surface GAGs. This role for E X T l was demonstrated using several assays, including a novel functional assay based on HSV type 1. It is known that HSV preferentially adsorbs to HS and in some instances CS moieties (WuDunn and Spear, 1989; Herold et al, 1991; Herold et al, 1994; Banfield et al, 1995), and it appears that E X T l induces the synthesis of novel forms of these sulfated polysaccharides. Alterations in G A G expression induced by E X T l were also shown directly by anion exchange chromatography. The ability of E X T l to alter G A G expression is specific in that the E X T l cDNA was isolated by screening a library comprising approximately 10 million clones. These studies demonstrate that E X T l is localized to the ER/Golgi and likely adopts a type II transmembrane topology similar to other glycosyltransferases and heparin/heparan sulfate N-sulfotransferase/N-deacetylases (Homa etal, 1993; Smith and Lowe, 1994; Fukuta et al, 1995; White et al, 1995; Bennett et al, 1996) (Fig. 3.7). The amino-terminus of E X T l contains the amino acid motiflysine-lysine-arginine, which is similiar to the di-arginine motif that functions as an ER retrieval signal in other type II membrane proteins (Teasdale, 1996). The ER/Golgi location of E X T l suggests that E X T l alters the cell surface indirectly, perhaps by modifying proteins that traverse the secretory organelles en route to the cell surface. It is also possible that some portion of E X T l is cleaved and secreted from the cell. Although small amounts of extracellular E X T l can be detected, conditioned medium from sog9-EXTl cells does not rescue sog9 cells in the HSV-1 infection assay. What could be the role of HS in HME? It is known that HS GAGs function as co-factors in several signal-transduction systems that affect cellular growth, differentiation, motility and adhesion. HS GAGs may also play a role in the malignant transformation of cells, tumor growth, tumor cell adhesion, invasiveness and metastasis (Bernstein and Liotta, 1994). Many tumor types display alterations in HS (Esko et al, 1988; Iozzo and Cohen, 1994; Iida etal, 1996; Schamhart and Kurth, 1997; Tuszynski etal, 1997). Some poorly differentiated lung tumors have markedly altered patterns of HS proteoglycan expression, which may contribute to their invasive phenotype (Nackaerts et 63 al, 1997). Some proteins involved in cell adhesion and cell signaling such as A P C (adenomatous polyposis coli), DCC (deleted in colon carcinoma), and NF2 (neurofibromatosis type 2) are tumor suppressor genes (Fearon et al, 1990; Rubinfeld et al, 1993; Trofatter et al, 1993). Thus, in H M E , changes in the structure of cell surface proteoglycans may interfere with the signal transduction systems that govern cellular growth and differentiation, resulting in tumor formation. In summary, this study demonstrates that E X T l is an ER/Golgi-localized transmembrane glycoprotein that alters the synthesis and display of HS GAGs in mammalian cells. The involvement of E X T l in HS biosynthesis indicates that the loss or inactivation of this gene may contribute to altered cell growth control in H M E . 64 catalytic domain hydrophobic transmembrane domain lumen membrane cytoplasm short hydrophilic amino terminus Fig. 3.8 Common topology of mammalian Golgi glycosyltransferases. 65 TH domain 70 EXTl MQAKKRYFIL LSAGSCLALL FYFGGLQFRA SRSHSRREEH SGRNGLHHPS PDHFWPRFPE PLRPFVPWDQ EXT2 MCASVKGPAL IPRMKTKHRI YYITLFSIVL LGLIA TGMFQFW PBSIESSNDW NVEKRSIRDP 2° EEEE EE-HHHHEEE EHH EEEE E 140 EXTl LENEDSSVHI SPRQKRDANS SIYKGKKCRM ESCFDFTLCK KNGFKVYVYP QQKGEKIAES YQNILAAIEG EXT2 WRLPADSPI PERGD LSCRM HTCFDVYRCG FNPIKVYIYA LKVSNTISRE YNELLMAISD 2° E EE BEE EEEEE HHHHH BBBBBBBBB-210 EXTl SRFYTSDPSQ ACLFVLSLDT LDRDQLSPQY VHNLRSKVQS LHLWNNGRNH LIFNLYSGTW PDYTEDVGFD EXT2 SDYYTDDINR ACLFVPSIDV LNQNTLRIKE TAQAMAQLSR . . . WDRGTNH LLFNMLPGGP PDYNTALDVP 2° E EEEEEEEE B HHHHHHHHHH HHH E EEEEE 280 EXTl IGQAMLAKAS ISTENFRPNF DVSIPLFSKD HPRTGGERGF LKFNTIPPLR KYMLVFKGKR YLTGIGSDTR EXT2 RDRALLAGGG FSTNTYRQGY DVSIPVYS. . .PLS..AEVD LP. EKGPGPR QYFLLSS... . QVGLHPEYR 2° HHHHHHHHH E EEEE E EEEEEE E EE H 350 EXTl NALYHVHNGE DWLLTTCKH GKDWQKHKDS RCDRDNTEYE KYDYREMLHK ATPCLVPRGK RLGSFRFLEA EXT2 EEALQVXBGE SVLVLDKCTN LSEGVLSVRK RCHKH Q VFDYPQVLQE ATFCWLRGA RLGQAVLSDV 2° HHHHHH EEEEEE BHHBEH EEEEE BHBBBBBBBB 420 EXTl LQAACVPVML SNGWELPFSE VINWNQAAVI GDERLLLQIP STIRSIBQDK ILALRQQTQF LMEAYFSSVE EXT2 LQAGCVPWI ADSYILPFSE VLDWKRASW VPEEKMSDVY SILQSIPQRQ IEEMQRQARW FWEAYFQSIK 2° BBB EEEE E E BBBBBBBBB BBBBBBBBBB BBBBBBBBBB BBBBBBBBBB BBBBBBBBBB 490 EXTl KIVLTTLEII QDRIFKHISR NSLIWNKBPG GLFVLPQYSS YLGDFPYYYA NLGLKPPSKF TAVIBAVTPL EXT2 AIALATLQII NDRIYPYAAI SYEEWNDPP. .AVKHGSVSN PLFLP LIP..PQSQF TAIVLTYRRV 2° BBBBBBBBBB BBBBBB EEE EEEEE EE EEEEEEE 560 EXTl VSQSQPVLKL LVAAAKSQYC AQIIVLWNCD KPLPAKHRWP ATAVPVWIE GESKVMSSRF LPYDNIITDA EXT2 ES LFRV ITEVSKVPSL SKLLWWKNK KNPPEDSLWP KIRVPLKWR TAENKLSNRF FPYDEIETEA 2° BBBBBB BBBBBBBBBB -EEEEEEE EEEEEE BBB 630 EXTl VLSLDEDTVL STTEVDFAFT VWQSFPERIV GYPARSHFWD NSKERWGYTS KWTNDYSHVL TGAAIYBKYY EXT2 VLAIODDIIL TSDELQFGYE VWREFPDRLV GYPGRLHLWD HEMNKWKYES EWTNEVSMVL TGAAFYHKYF 2° HHH BBBBBBB BBBBBBBBBB E EE EE EEEEE BBBBBBBBBB 700 EXTl HYLYSBYLPA SLKNHVDQLA NCEDILMKFL VSAVTKLPPI KVTQKKQYKE TMMGQTSRAS RWADPDHFAQ EXT2 NYLYTYKMPG DIKNWVDABM NCEDIAMNFL VANVTGKAVI KVTPRK..KF KCPECTAIDG LSLDQTHMVE 2° EEEEE B BBBBBBBBB BBBBBBBBB BBBBB E 746 EXTl RQSCMNTFAS WFGYMPLIBS QMRLDPVLFK DQVSILRKKY RDIERL EXT2 RSECINKFAS VFGTMPLKW EHRADPVLYK DDFPEKLKSF PNIGSL 2° HHHHHHHHHH HH HHHHH— Fig. 4.1 Amino acid alignment and predicted secondary structure for human E X T l (746 a.a.) and human EXT2 (718 a.a.). These ER-localized type II transmembrane glycoproteins have an N-terminal cytoplasmic tail, a transmembrane (TM) domain (underlined), a stem region and a long C-terminal lumenal tail. Amino acid positions known to be mutated in H M E patients in either E X T l or EXT2 are highlighted in yellow. Conserved amino acids are shown in red, and putative N-linked glycosylation sites are double underlined. Alignments and secondary structure (2°) were predicted using Jpred and can be interpreted as follows: H corresponds to predicted alpha helices, and E to predicted 6-strands. 67 CHAPTER 4: Analysis of the relationship between E X T l and EXT2 4.1 Introduction Previous studies using epitope-tagged constructs have demonstrated that E X T l is an EPv/Golgi-localized glycoprotein whose expression enhances the synthesis of cell surface HS (see Chapter 3). HS chains are composed of alternating residues of D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc) joined by 1—»4 linkages, and a recent study has shown that both E X T l and EXT2 harbor GlcA transferase (GlcA-T) and GlcNAc transferase (GlcNAc-T) activities that catalyze the polymerization of HS (Lind etal, 1998). E X T l and EXT2 are structurally similar to previously identified glycosyltransferases in that they are type II transmembrane glycoproteins comprising an N-terminal cytoplasmic tail, a transmembrane domain, a stalk, and a large globular domain that is likely to harbor enzymatic activity (Fig. 4.1)(Colley, 1997). Moreover, a truncated, active form of EXT2 is secreted from cells and can be isolated from serum (Lind et al, 1998), which is a fate common to other ER and Golgi-localized glycosyltransferases including EXTL2 , which is an E X T homolog shown to encode an a l , 4-A/-acetylhexosaminyltransferase (Kitagawa etal, 1999). However, several important questions are raised by these observations. E X T l , when overexpressed in a cell, appears to be localized predominantly to the ER (Chapter 3), whereas the biosynthesis of HS chains is thought to occur exclusively in the Golgi cisternae (Nuwayhid etal, 1986; Hirschberg and Snider, 1987; Silbert and Sugumaran, 1995; Fernandez and Warren, 1998). Moreover, if the EXTl and EXT2 genes encode functionally redundant HS-Polymerases (HS-Pol), it is not clear why mutations in either gene cause H M E . To address these questions, we overexpressed functional, epitope-tagged and native forms o f E X T l and EXT2 in cells and examined their subcellular localization and enzymatic activity. 66 4.2 Results 4.2.1 E X T l and E X T 2 are Functionally Distinct Proteins To test the in vivo function of the E X T proteins, we used an assay based on the ability of herpes simplex virus type 1 (HSV-1) to infect cells by attaching to cell surface HS (see section 2.7). The target cells, sog9 cells, are 99.5% resistant to HSV-1 infection compared with control cells because of their defect in HS biosynthesis (Banfield et al, 1995). Experiments performed by Dr. Gillian Duncan have shown that sog9 cells harbour a defect in the E X T l gene which results in a mis-splicing event, joining exon 1 to exon 5 in a +1 reading frame (Fig. 4.2a, b). This predicts that sog9 cells synthesize a truncated E X T l protein of 335 amino acids (Fig. 4.2b) which, as expected, is non-functional in the HSV-1 infection assay (data not shown). Transfection of sog9 cells with native forms or tagged forms (Fig. 4.2C) of human E X T l resulted in a 200 fold enhancement of HSV-1 infection due to a restoration of HS biosynthesis and subsequent expression of HS on the cell surface. By contrast the related protein EXT2 displayed no activity in this assay (Fig. 4.2Q. Because sog9 cells lack a functional E X T l , these results indicate that EXT2 alone is not able to catalyze the polymerization of HS in the absence of full length E X T l . Additional experiments also established that the E X T homologs EXTL3 (Van Hul et al, 1998) and E X T L 2 (Kitagawa et al, 1999), and the HS-modifying enzyme N-deacetylase/N-sulfotransferase-2 (NDST2) (Eriksson et al, 1994) were inactive in this assay (Fig. 4.2Q, indicating that they could not compensate for a lack of E X T l activity. 68 AUG UGA exon 1 2|3|4|5|6l7|a|9|l0| 11 exon 1 |5 T n w.t.mEXTl sog9 mEXTl UAA B w.t. mEXTl TAT|GAG AA|A TAT GjAT TAT CGG GAA ATG CTG CAC AAT GCC ACT TTC TGT C T G . . . . TGA donor 1 acceptor 1 splice junction (746 a.a.) TYR GLU LYS TYR ASP TYR ARG GLU MET LEU HIS ASN ALA THR PHE CYS LEU.. . STOP sog9 mEXTl TAT JGAG AAfc TTA TJTC AGG ACA GAA TAT TCA AGC ACA TAT CAC GTA ACA GTT TAA donor 1 acceptor 4 splice Junction (335 a.a.) TYR GLU LYS LEU PHE ARG THR GLU TYR SER SER THR TYR HIS VAL THR VAL STOP EXTl'. mock EXT2 EXTL2 EXTL3 NDST2 Fig. 4.2 Analysis and rescue of the E X T l defect in sog9 cells. Sog9 cells harbour a defect in the murine E X T l gene that results in a mis-splicing event, joining exon 1 with exon 5 (a), resulting in a truncated 335 amino acid protein (b). (c) Sog9 cells were transfected with indicated constructs and infected with HSV-1 to detect the presence of newly synthesized cell surface heparan sulfate. Infected cells stain blue in the presence of X-gal. 69 4.2.2 E X T l and EXT2 Accumulate in the Golgi Apparatus It has been shown previously that epitope-tagged human and murine E X T l proteins are localized predominantly to the ER when overexpressed in cells (Chapter 3 and (Lin et al, 1998)). To determine the intracellular localization of EXT2, a GFP-tagged murine EXT2 was expressed in B H K and sog9 cells and analyzed by confocal microscopy (Fig 4.4b). EXT2, like E X T l , was localized predominantly to the ER (Fig. 4.4a, b). Moreover, pulse-chase experiments showed that the majority of newly-synthesized EXT2 remained sensitive to endoglycosidase H (endoH) for at least 5 h post synthesis (Fig. 4.3). Thus, EXT2 appeared to be modified by high mannose N-linked oligosaccharide moieties characteristic of proteins retained in the ER or cis Golgi elements. The presence of N-linked glycans is also consistent with the type II membrane topology predicted for EXT2. To investigate the pattern of EXT2 expression in the presence o f E X T l , GFP-tagged versions of each protein, which retained full activity, were overexpressed in the same cell. Remarkably, the EXT proteins redistributed to the Golgi apparatus from the ER (Fig. 4.4c, g, h, i). The Golgi accumulation of the EXT1/EXT2 complex was confirmed by colocalization with a Golgi marker protein (Fig. 4.4d, e, f). Moreover, all combinations of epitope tagged and untagged full-length E X T polypeptides behaved in this manner (Fig. 4.5). However, a severely truncated form o f E X T l which lacks the carboxy-terminal 620 amino acids is unable to induce the migration of EXT2 to the Golgi (Fig. 4.5). Thus, it appears that accumulation o f E X T l and EXT2 in the Golgi cisternae resulted from the concomitant expression of both full-length proteins. There have been a number of reports indicating that the biosynthesis of HS chains occurs predominantly in the Golgi cisternae (Nuwayhid et al, 1986; Hirschberg and Snider, 1987; Silbert and Sugumaran, 1995; Fernandez and Warren, 1998). In this light, the data so far suggested that the relocation of E X T l and EXT2 to the Golgi may be required for a role in HS biosynthesis. I hypothesized that the truncated 335 amino acid protein produced by sog9 cells may therefore be non-functional due to an inability to redistribute to the Golgi apparatus. To test this hypothesis, 335AAEXTlgfp was transfected singly (Fig 4.6a), or co-transfected with myc-his tagged EXT2 (Fig. 4.6b, c, 70 d), and observed by immunofluorescence. These experiments show that sog9-cell derived E X T l is unable to re-distribute to the Golgi in the presence of EXT2, which may explain its lack of function in HS biosynthesis. Furthermore, this experiment reveals that the C-terminal 411 amino acids of E X T l are critical for migration to the Golgi in the presence of EXT2. As a control for these studies, it was determined whether several other closely-linked enzymes in the HS biosynthesis pathway, E X T L 2 and NDST2, as well as the EXT-like protein EXTL3 , behaved in this manner. When overexpressed individually in cells, GFP-tagged EXTL2, and EXTL3 were localized predominantly to the ER, whereas NDST2 localized to the Golgi (Fig. 4.7). The intracellular distribution of E X T L 2 , EXTL3 or NDST2 was not altered by concomitant overexpression of E X T l and/or EXT2 (data not shown). Thus, these proteins were transported to their respective intracellular locations without interaction with E X T l or EXT2. 71 chase 0 0.5 1 2 5 endoH - + - + - + - + - + *t g p M M M | ^B^!?gMi; WS£mm E X T 2 m y c ^ M I mm im mm m mm ^ * mm " " * v ' * **"* ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ E X T 2 g f p ^ ^ * ( M ^ i ^ M i M a i ,, , F i g . 4.3 Pulse-chase and endoglycosidase H digestion o f E X T 2 . M o n o l a y e r s o f B H K cel ls were transfected wi th m y c - or GFP- t agged murine E X T 2 constructs and, after 18 h, radiolabeled wi th 100 u C i / m l [ , 5 S]methionine for 30 min . Excess methionine was added and the monolayers harvested at 0, 0.5, 1, 2, and 5h post-chase. Port ions o f the lysates were treated wi th e n d o H (+) or m o c k digested (-) and subjected to S D S - P A G E . 72 A B C - A D E F tufa aim G / H I F i g . 4 . 4 Intracellular loca l iza t ion of E X T l and E X T 2 . Mono laye r s o f B H K cel ls were transfected with E X T l g f p (a), m E X T 2 g f p (b), or both (c). W h e n transfected into the same ce l l , E X T l g f p and E X T 2 g f p relocated to the G o l g i (c, d), whi le the G o l g i apparatus was immunolabe led wi th an an t i -Golg i 5 8 K monoclona l antibody and a Texas R e d conjugated secondary antibody (e). W h e n over la id , they show excellent co- loca l iza t ion (f). In a separate experiment, E X T l g f p (g) and E X T 2 m y c - h i s (h) were co-transfected into B H K cel ls , and the myc-his tagged E X T 2 was immunolabe led wi th mouse ant i -HIS and goat anti-mouse Texas R e d . W h e n images (g) and (h) are over la id , they show that E X T l and E X T 2 each local ize to the same subcellular compartment (i). 73 I \ I 1 ufp-l>I X 12 EXTlgfp-bEXT2myc E X r i s l p - F L A G b i ; x r 2 KXTl-niKX12g«p. K\rigtp-niEXI2myc 126 \ A K X 11 myc-mEX 12qfp Fig. 4.5 Intracellular localizations of co-transfected E X T fusion proteins. A variety of epitope tagged and native forms of human, bovine and murine E X T proteins were co-transfected in pairs into B H K cells, and GFP-localization analyzed by confocal microscopy at 488 nm. A l l combinations of full length constructs showed a distinct Golgi localization. The prefixes m and b indicate mouse and bovine, respectively. 74 Fig. 4.6 Intracellular localization of sog9 cell derived E X T l . Monolayers of B H K cells were transfected with 335AAEXTlgfp (a), or 335AAEXTlgfp and mEXT2myc-his (b, c, d). After 30 hours of expression myc-his tagged EXT2 proteins were detected using an anti-His monoclonal antibody and a Texas Red conjugated secondary antibody (c). When overlaid, 335AAEXTlgfp (b) and mEXT2myc-his (c) show excellent co-localization (d). 75 c 1 Fig. 4.7 Intracellular localization of other type II membrane proteins. Monolayers of B H K cells were transfected with GFP fusion constructs of the EXT homologs EXTL2 (a), EXTL3 (b), or the murine N-deacetylase/N-sulfotransferase-2 (NDST2), a key enzyme in HS biosynthesis (c). After 30 h, cell monolayers were fixed with 4% paraformaldehyde and coverslips mounted on glass slides. GFP fluorescence was observed by confocal microscopy at 488 nm. 76 4.2.3 E X T l and EXT2 Form Homo- and Hetero-oligomeric Complexes The data so far suggested that E X T l and EXT2 may form a complex that accumulates in the Golgi. To determine directly whether E X T l and EXT2 form homo-and/or hetero-oligomeric complexes in vivo, different combinations of myc or GFP tagged E X T constructs were transfected into cells, radiolabeled, and immunoprecipitated. SDS-PAGE analysis revealed that anti-GFP antibody brought down two forms of E X T l gfp, 113 kD and 115 kD, and a single 112 kD polypeptide representing EXT2gfp, while anti-myc antibody brought down two forms of EXTlmyc-his, 88 kD and 91 kD, and a single 85 kD polypeptide representing myc-his-tagged EXT2 (Fig. 4.8). When EXTlgfp and EXTlmyc-his were co-expressed and EXTlmyc-his was immunoprecipitated with anti-myc, EXTlgfp coprecipitated, indicating that E X T l was capable of forming homo-oligomers in vivo (Fig. 4.8a). The corresponding experiment using EXT2myc-his and EXT2gfp revealed that EXT2 is also able to form homo-oligomers in vivo, which can be immunoprecipitated using either the anti-myc or anti-GFP antibody (Fig. 4.8a). With regard to hetero-oligomer formation, SDS-PAGE analysis revealed that when EXTlgfp and mEXT2myc-his, or EXTlmyc-his and EXT2gfp were co-expressed and immunoprecipitated with antibody against one of the tags, the oppositely-tagged protein co-precipitated (Fig. 4.8b). These results indicate that E X T l and EXT2 form a complex in vivo. To ensure that the observed association between E X T l and EXT2 was not an artifact of our experiments, lysates from singly-transfected cells were mixed and immunoprecipitated (Fig. 4.8c). In this case no complex between E X T l and EXT2 was detected, indicating that the two proteins probably cannot associate ex vivo. Taken together, these data show that both E X T l and EXT2 are capable of forming both homo- and hetero-oligomeric complexes in vivo. 11 A construct EXTlgfp EXTlgfp EXTlmyc EXTlmyc antibody myc gfp gfp myc myc gfp EXTl gfp = EXTlmyc = -„ M B construct EXTlgfp EXTlgfp EXT2myc EXT2myc antibody myc gfp gfp myc myc gfp EXTlgfp = EXTlmyc -Hi c EXTlgfp construct EXTlgfp EXT2myc EXT2myc antibody myc gfp gfp myc myc gfp EXTl gfp= EXT2gfp EXT2gfp EXT2myc EXT2myc myc gfp gfp myc myc gfp mm — -EXT2gfp EXT2gfp EXTlmyc EXTlmyc myc gfp gfp myc myc gfp M i EXT2gfp = EXTl myc - EXT2myc F i g . 4.8 Radio immunoprec ip i ta t ion analysis o f E X T 1 / E X T 2 complexes . B H K cel ls were transfected wi th various combinat ions o f myc-h is or G F P tagged E X T l and E X T 2 constructs, radiolabeled wi th [ 3 5 S]methionine and immunoprecipi ta ted with the indicated ant i -myc or a n t i - G F P antibodies. (A) E X T l and E X T 2 homo-o l igomer analysis; (B) E X T 1 / E X T 2 hetero-oligomer analysis. These images are representative o f results obtained f rom several experiments. (C) T o determine whether E X T 1 - E X T 2 complex formation was able to occur outside o f the ce l l , lysates containing E X T l g f p or E X T 2 m y c - h i s were m i x e d ex vivo and immunoprecipi ta ted wi th either a n t i - G F P or anti-m y c antibody. 78 4.2.4 EXT1-EXT2 Complexes Possess Enhanced Glycosyltransferase Activity Recent evidence indicates that E X T l and EXT2 possess GlcA-T and GlcNAc-T activities in vitro (Lind et ah, 1998). To explore the possibility that the Golgi-resident EXT1-EXT2 complex represented the active form of HS-Polymerase (HS-Pol), GFP tagged E X T forms were expressed in B H K or sog9 cells, purified by immunoprecipitation, and assayed for GlcNAc-T and GlcA-T activities (Table 4.1). Both cell lines were analyzed because B H K cells express HS and therefore have some endogenous HS-Pol activity, whereas sog9 cells, which are deficient in HS biosynthesis, harbor a specific defect in E X T l (Fig. 4.2a, b). In all cases when E X T l was overexpressed in cells, a high level of HS-Pol activity was observed. By contrast, when EXT2 was overexpressed in B H K cells it exhibited low levels of GlcA-T activity and essentially no GlcNAc-T activity. Moreover, there was no detectable HS-Pol activity when EXT2 was overexpressed in sog9 cells, indicating that under these experimental conditions EXT2 overexpressed on its own does not exhibit HS-Pol activity. The highest enzymatic activity was isolated from cells overexpressing both E X T l and EXT2 (Table 4.1, GlcA-T activity), which suggests that the Golgi-resident EXT1-EXT2 complex represents the active form of HS-Pol in the cell. 79 Table 4.1 GlcNAc-T and GlcA-T activities of immunoprecipitated EXT1-EXT2 complexes Cell E X T l EXT2 3 H-GlcNAc-T 1 4 C-GlcA-T line construct construct activity(cpm)3 activity (cpm)a B H K - - 9 4 EXTlgfp - 349 770 - mEXT2gfp 19 63 sog9 - - 8 29 EXTlgfp - 691 592 - mEXT2gfp 20 20 EXTlgfp mEXT2myc 219 2538 EXTlgfp mEXT2gfp 303 3018 EXTlgfp bEXT2myc 573 1862 E X T l mEXT2gfp 750 3381 "EXT proteins were immunoprecipitated with the rabbit anti-GFP antibody. The GFP tags on E X T proteins are labeled in bold for clarity. The prefixes m and b indicate mouse and bovine, respectively. GlcA-T and GlcNAc-T activities were calculated as cpm of incorporated [ 3H]-GlcNAc and [ 1 4C]-GlcA per immunoprecipitate. The sog9 cell derived values represent the averages of at least two experiments, while the B H K cell derived values are derived from a single experiment. Further replicate experiments were not performed due to limited quantities of acceptor substrates. 80 4.3 Discussion The putative tumor suppressors E X T l and EXT2 were first identified because of their role in hereditary multiple exostoses (HME), which is an autosomal dominant disorder characterized by the formation of multiple, cartilage-capped tumors (exostoses) that develop from the growth plate of endochondral bone (Solomon, 1963). This condition can lead to skeletal abnormalities, short stature, and in some instances, malignant transformation from exostoses to chondrosarcomas (Leone et al, 1987; Hennekam, 1991) or osteosarcomas (Schmale et al, 1994; Luckert-Wicklund et al, 1995). Our demonstration that E X T l and EXT2 form a Golgi-resident complex, "the Golgi oligomer", comprising the GlcA-T and GlcNAc-T activities required for the polymerization of HS (Table 4.1) provides compelling evidence that H M E is caused by a deficiency in HS-Pol. Moreover, by using sog9 cells, which lack functional E X T l , we show that E X T l and EXT2 cannot substitute for each other in vivo. These results support a model in which E X T l works in concert with EXT2 to provide the HS-Pol activity in the cell (Fig 4.9). It is also possible that other polypeptides involved in HS biosynthesis may associate with the E X T proteins in the Golgi. An analysis of enzymatic activities associated with different E X T constructs (Table 4.1) suggests that a complex o f E X T l and EXT2 is required to elicit the maximal GlcA transferase activity observed. By contrast, E X T l , when overexpressed in our cell lines, possesses both GlcNAc and some GlcA transferase activity. Because a cell line deficient in EXT2 is not available, it is not yet possible to examine E X T l expression in the absence of EXT2. Taken together, the accumulated data suggests a model in which E X T l and EXT2 cooperate to generate the active enzyme responsible for the polymerization of HS. Although HS polymerization is not directly demonstrated by the in vitro assays, the herpes simplex virus adsorption assay (Fig. 4.2) detects the expression of cell surface HS moieties, and this has been shown to be facilitated by the restoration of E X T l activity in sog9 cells. Additional studies on purified polypeptides will be required to sort out the roles of individual components of the enzyme complex. 81 Heparan sulfate EXT1-EXT2 complex G^cNA^TO) (HSG1CXTII] E X T L 2 GlcNAc-TI GlcNAc a l ^ GlcA pi-U GlcNAc a l — / " X f N / "N / " " N * (GlcA-Tl) fGal-Tin (Gal-TI J f Xyl-T ) 4 GlcA pi—3 Gal pi—3 Gal pi—4 Xyl pi—Ser -+3 GalNAc Bl—4 GlcA 01- -3 GalNAc pl-ii linkage region Chondroitin sulfate core — protein Fig. 4.9 The sugar units and their corresponding glycosyltransferases involved in HS and CS biosynthesis. The non-reducing ends are to the left. Abbreviations are as follows; Ser (serine), X y l (xylose), Gal (galactose), GlcA (glucuronic acid), GlcNAc (N-acetylglucosamine), GalNAc (N-acetylgalactosamine). 82 These conclusions were facilitated by the analysis of HS deficient sog9 cells, which harbor a specific defect in the EXTl gene that results in the absence of HS-Pol activity. As such, sog9 cells represent the only stable cell line for which an EXTl defect has been characterized, and they provide a useful tool with which to determine the activity of EXT2 expressed on its own. Also critical to this study was the use of functional assays to assess E X T l activity. This allowed for the development of epitope-and GFP-tagged constructs that remained fully functional in vivo. By expressing combinations of functional tagged and untagged constructs in the sog9 cells, localization data could be correlated with activity. In this manner, we determined that EXT2 expression in sog9 cells does not rescue HS biosynthesis, as measured in the highly-sensitive HSV infection assay or in HS-Pol enzyme assays. In fact, the highest amount of HS-Pol activity was observed when E X T l and EXT2 were co-expressed. Because we have observed that the EXT1/EXT2 hetero-oligomeric complex is localized predominantly to the Golgi cisternae, our data suggest that the Golgi is the site of cellular HS-Pol activity. The relatively small amount of HS-Pol activity that results from the expression o f E X T l alone in sog9 cells is likely the result of complex formation between transfected E X T l and endogenous EXT2. It has been shown that EXT2 in sog9 cells accumulates in the ER due to the inability of the truncated E X T l to facilitate its redistribution to the Golgi (Fig. 4.6). Although we have observed some Golgi-localized E X T l in sog9 cells, most of the mass appears to be either retained in or recycled to the ER. It is interesting that E X T l contains a motif, K K R , in its short cytoplasmic tail that is similar to the consensus di-arginine ER retrieval signal described for several type II membrane proteins (Teasdale, 1996). A similar motif, K X R , is present in EXT2. Although preliminary data suggest that these motifs are important for ER localization, additional experiments will be required to elucidate the steps in the formation of functional E X T complexes. Glycosyltransferases are commonly secreted into the extracellular medium in truncated form, and a number of these enzymes have been cloned based on sequence information derived from soluble forms isolated from serum or milk (Paulson and Colley, 1989; Joziasse, 1992). In the case of EXT, it has been shown previously that a 70 kD truncated form of EXT2 isolated from bovine serum harbors the two transferase activities 83 required for the biosynthesis of HS (Lind et al, 1993; Lind et al, 1998). In light of our demonstration that E X T l and EXT2 form a functional hetero-oligomer, it may be that the soluble ectodomains of E X T l and EXT2 are sufficient to maintain the oligomeric complex through a number of purification steps. Alternatively, it is also possible that EXT2 harbors some enzymatic activity that is not detectable in any of our cell-based assays, or that EXT2 is somehow activated following its exposure to E X T l in the cell. In either case, the data support a model in which E X T l and EXT2 possess distinct activities that are not functionally redundant in mammalian cells. Moreover, the E X T enzyme complex must traverse the secretory organelles while exiting the cell, which is consistent with our demonstration of Golgi localization for the complex. Additional experiments to identify and characterize the putative extracellular EXT1-EXT2 complex should help to resolve the activities o f E X T l and EXT2 in this complex. The Golgi localization for the EXT1-EXT2 complex is consistent with what is known for other glycosyltransferases that modify proteins traversing the secretory organelles. Hetero-oligomer formation has been observed for other glycosyltransferases, and may represent a common mechanism by which protein complexes are retained in a particular cellular compartment required for their activity. The kin recognition hypothesis of Warren and colleagues suggests that enzymes residing in the same Golgi cisternae could form hetero-oligomers (Nilsson et al, 1993; Nilsson et al, 1994). In the case of the E X T proteins, overexpression of either one does not lead to substantial Golgi localization, which suggests that E X T homo-oligomers, which may form in the ER, are not sufficient to signal movement to or retention in Golgi cisternae. It may be the case that uncomplexed E X T l and EXT2 cycle between the ER and the Golgi, and that hetero-oligomer formation in the cis Golgi cisternae, or an earlier compartment, leads to Golgi retention. This model does not preclude an important role for E X T homo-oligomers or of other members of the putative enzyme complex, nor does it eliminate the possibility that the complexes form in the ER and move to the Golgi. It is likely, however, that the highly-active hetero-oligomer retains its function following cleavage and transport into the extracellular space. This would indicate that the complex is stable, and that it could retain at least partial activity in several different cellular compartments. In this manner, cells could control the expression of HS-Pol by downregulating a single component of the 84 complex. It would also resolve the issue of why mutations in either E X T l or EXT2 alone cause H M E , as both E X T l and EXT2 appear to be necessary to produce the fully active enzyme complex. The evidence that E X T l and EXT2 are glycosyltransferases that catalyze HS biosynthesis supports the hypothesis that alterations in the display of HS moieties contribute to altered cell growth control in H M E . It is known that glycosaminoglycans function as co-factors in several signal-transduction systems that affect cellular growth, differentiation, motility and adhesion, and may play a role in the malignant transformation of cells, tumor growth, tumor cell adhesion, invasiveness and metastasis (Bernstein and Liotta, 1994). Many tumor types display alterations in glycosaminoglycans (Esko etal., 1988; Iozzo and Cohen, 1994; Iida etal., 1996; Schamhart and Kurth, 1997; Tuszynski et al., 1997). Recently, several novel genes have been identified that share significant sequence homology with the EXT genes (Wise et ai, 1997; Wuyts et al, 1997; Van Hul et al, 1998). Although none of these genes have been implicated as causative agents in H M E , they are localized to chromosomal regions associated with other forms of cancer. For example, the a l , 4-N-acetylhexosaminyltransferase E X T L 2 (Kitagawa et al, 1999) is assigned to the lp21 region, where osteopetrosis, a dominant hereditary disease of bone, has been mapped by genetic linkage analysis (Saito et al, 1998). The E X T family may therefore represent a class of tumor suppressors that exert their effects indirectly by modifying proteoglycans destined for display at the cell surface. The recent discovery that Tout-velu, the Drosophila homolog o f E X T l , is critical for the diffusion of Hedgehog proteins, which are important developmental signaling proteins, suggests that HS biosynthesis may play a crucial role in many stages of development (Bellaiche et al, 1998; Ingham, 1998) (see Chapter 7, section 7.3 for more details). It will be useful to investigate whether modifications to cell surface HS occur in chondrocytes isolated from patients with H M E , and whether other E X T genes represent additional constituents of glycosaminoglycan biosynthesis pathways. Taken together, these results should prove extremely useful for elucidating the role of E X T and perhaps other families of glycosyltransferase genes in the development of human tumors. 85 CHAPTER 5: Analysis of aetiologic mutations in EXTl and EXT2 5.1 Introduction Hereditary multiple exostoses is a genetically heterogeneous disease, with three chromosomally distinct loci implicated thus far. These loci include E X T l on 8q24.1, EXT2 on 1 l p l 1-13 and EXT3 on 19p, with the majority of cases attributed to E X T l and EXT2 (Blanton et al, 1996; Raskind et al, 1998). Combined results from a number of studies that include all three E X T chromosomes indicate that the proportion of E X T families carrying mutations in E X T l , EXT2, and EXT3 is 0.57, 0.30, and 0.13, respectively (Blanton etal, 1996; Legeai-Mallet etal, 1997; Raskind etal, 1998). Most of the cases described thus far have involved frameshift mutations which predict premature chain termination in the E X T l or EXT2 proteins. However, there have also been reports of a number of aetiologic (disease-causing) single amino acid changes (Raskind, 1996; Hecht et al, 1997; Philippe et al, 1997), and a report of a single amino acid deletion (Raskind et al, 1998). Clearly, the regions where these mutations arise are critical for normal function of the E X T proteins. It has been shown that E X T l and EXT2 are type II transmembrane glycoproteins which form a hetero-oligomeric complex with the in vitro enzyme activities necessary for the polymerization of heparan sulfate (Chapters 3,4). I hypothesized that if HS polymerization is related to the disease pathogenesis of H M E , then E X T l and EXT2 constructs bearing disease-causing mutations should be defective in enzyme activity. Furthermore, I reasoned that these specific mutations may be useful for pinpointing the different functional domains of the E X T proteins, which may lead to a better understanding of the roles of these mutations in the pathogenesis of H M E . 5.2 Results 5.2.1 Construction of aetiologic E X T l and EXT2 mutants It has been reported recently that in some cases of H M E , the disease-causing mutations in E X T l are single base transitions which result in missense mutations 86 (Raskind, 1996; Hecht et ai, 1997; Philippe et ai, 1997). For E X T l , these mutations include an arginine to glycine/serine at amino acid 280, a glycine to aspartate change at amino acid 339, or an arginine to serine/cysteine/leucine/histidine change at amino acid 340 (Fig. 5.1). For EXT2, these mutations include a cysteine to arginine change at amino acid 85, and an aspartate to asparagine change at amino acid 227. I reasoned that if HS polymerization is related to the disease pathogenesis of H M E , then E X T l and EXT2 constructs bearing disease-causing mutations should be defective in polymerization of HS. To test this, constructs containing selected E X T l missense mutations at amino acid 339 (G339DEXTlmyc and G339DEXTlgfp) or 340 (R340CEXTlmyc and R340CEXTlgfp), or missense mutations at amino acid 85 (C85REXT2gfp) or 227 (D227NEXT2gfp) were generated by PCR using mutagenic primers (see section 2.8 for details). The mutations in these constructs were verified by restriction enzyme digestion analysis and D N A sequencing. 87 ER membrane E X T l N H , MQA^CKR EXT2 X-4—% * COOH R280 G339 R340 H627 N H , 0 = 0 f M C A S V K M K T K H R ii K H R COOH C85 D227 cytoplasm lumen Fig. 5.1 Topology of E X T l and EXT2 in the ER membrane. Disease-causing missense mutations (in green) are indicated by amino acid position on each protein. Locations of putative K X R ER-retrieval sequences are indicated on the N -termini of both proteins. Locations of proposed N-glycosylation sites are indicated with a Y . 88 5.2.2 Disease causing mutations destroy E X T l activity in sog9 cells The link between the EXTl gene and its role as an enzyme implicated in HS biosynthesis was first revealed by an in vivo functional assay (Chapter 3), using HS-deficient/EXTl-mutant sog9 cells (Banfield et al, 1995), and HSV-1 as a ligand for cell surface HS. Transfection of EXTl into sog9 cells resulted in the restoration of HS to the cell surface, which was then used by the virus to attach to the cell surface and initiate infection. To correlate the heparan sulfate polymerizing enzyme activity of E X T l to H M E , aetiologic mutant E X T l constructs were also tested in this functional assay. While the wild type E X T l and epitope tagged derivatives are able to restore HS to the cell surface and enhance HSV infection by approximately 200-fold, both the G339D and R340C mutant E X T l constructs were totally inactive in these assays (Fig. 5.2). These results provide strong evidence for a link between the heparan sulfate polymerizing activity of E X T l and the disease phenotype of H M E . As it has previously been shown that wild type EXT2 does not complement the E X T l defect in sog9 cells (Chapter 4), the HSV-1 based functional assay can not be used to determine the effect of the C85R and D227N missense mutations on EXT2 function. 89 Fig. 5.2 HSV-1 infection of sog9 cells transfected with aetiologic E X T l mutant cDNAs. Sog9 cells were transfected with indicated constructs and infected with HSV-1 to detect the presence of newly synthesized cell surface heparan sulfate. Infected cells stain blue in the presence of X-gal. 90 5.2.3 Aetiologic E X T l and E X T 2 mutants have defects in intracellular trafficking It has been shown that E X T l and EXT2 are predominantly localized to the ER when expressed alone in the cell, and when co-expressed, both proteins redistribute to the Golgi apparatus (Chapter 4). Interestingly, immunofluorescence analysis of E X T l constructs containing HME-linked missense mutations showed that G339D and R340C (Fig. 5.3) were localized predominantly to the Golgi apparatus when transfected alone. Moreover, EXT2 was translocated to the Golgi when co-expressed with these mutant forms of E X T l (Fig. 5.3). By contrast, the EXT2 missense mutations C85R and D227N were predominantly ER-localized when expressed alone in the cell, and were unable to redistribute to the Golgi apparatus when co-transfected with E X T l (Fig. 5.4). I reasoned that the observed defects in intracellular trafficking of mutant forms of EXT2 may result from an inability to form homo-oligomeric complexes or hetero-oligomeric complexes with E X T l . To test this, immunoprecipitations were performed on radiolabeled lysates from cells co-expressing selected mutant and wild type E X T proteins. The C85R EXT2 mutant was not impaired in its ability to form homo-oligomeric complexes with wild type EXT2 (Fig. 5.5a), nor was it impaired in its ability to form hetero-oligomeric complexes with wild type E X T l (Fig. 5.5b). The D227N EXT2 mutant was also not impaired in hetero-oligomer formation with E X T l (Fig. 5.5c). Taken together, these data suggest that the underlying basis for disease in patients harboring these specific EXT2 mutations is not due to a failure of EXT1/EXT2 complexes to form. However, in the case o f E X T l mutant proteins, their altered trafficking when expressed on their own may be indicative of a perturbation in folding or processing following synthesis in the ER, and a defective E X T l may result in a defective EXT1/EXT2 complex, thus explaining the loss of enzyme activity. In the case of the EXT2 mutant proteins, it appears that they form a complex with E X T l which is unable to exit the ER, possibly impairing enzyme activity due to incomplete processing of the complex, or because the complex is not exposed to the proper substrates normally located in the Golgi. 91 A B i c D E > F G H V Fig. 5.3 Intracellular localization of aetiologic E X T l mutants. Monolayers of B H K cells were transfected with G339DEXTlgfp (a), or R340CEXTlgfp (e), or co-transfected with G339DEXTlgfp and mEXT2myc-his (b-d), or R340CEXTlgfp and mEXT2myc-his (f-h). Myc-his tagged proteins were visualized by immunolabeling with mouse anti-His antibody and a goat anti-mouse conjugated to Texas Red. Images of GFP fluorescence (b, f) and Texas Red fluorescence (c, g) were overlaid to visualize co-localization of signal (yellow) (c, h). 92 Fig. 5.4 Intracellular localization of aetiologic EXT2 mutants. Monolayers of B H K cells were transfected with EXT2gfp (a), C85REXT2gfp (e), or D227NEXT2gfp (i), or co-transfected with EXT2gfp and EXTlmyc-his (b-d), or C85REXT2gfp and EXTlmyc-his (f-h), or D227NEXT2gfp and EXTlmyc-his Q-\). Myc-his tagged proteins were visualized by immunolabeling with mouse anti-Myc antibody and a goat anti-mouse conjugated to Texas Red. Images of GFP fluorescence (b, f, j) and Texas Red fluorescence (c, g, k) were overlaid to visualize co-localization of signal (yellow) (c, h, 1). 93 C85Rgfp C85Rgfp EXT2myc EXT2myc myc gfp gfp myc myc gfp B C85R _ EXT2gfp EXTlmyc = mmm *~~m ^^^^ mgL||ii ^ ^^^^ IP^P •• " C85Rgfp C85Rgfp EXTlmyc EXTlmyc myc gfp gfp myc myc gfp .C85R EXT2gfp •EXT2myc 1 "D227Ngfp D227Ngfp EXTlmyc EXTlmyc myc gfp gfp myc myc gfp mmmm 2T 5 • * jMWML'.'.g: Miiiii'iiuiiiiiiS C , i tg .D227N EXT2gfp : EXTl myc F i g . 5.5 Radio immunoprec ip i t a t ion analysis o f mutant E X T 2 o l igomer ic complexes . B H K cel ls were transfected wi th the indicated combinat ions o f mutant E X T 2 constructs and epitope tagged E X T l or E X T 2 constructs, radiolabeled wi th [ 3 5 S]-methionine and immunoprecipi ta ted wi th the indicated ant i-myc or a n t i - G F P antibodies. Immunoprecipi tated proteins were separated by S D S - P A G E , transferred to Immob i lon -P membranes, and exposed to B i o M A X M R f i l m , (a) C 8 5 R E X T 2 homo-o l igomer analysis; (b) C 8 5 R E X T 2 - E X T 1 hetero-oligomer analysis; (c) D 2 2 7 N E X T 2 - E X T 1 hetero-oligomer analysis. 94 5.2.4 HME-linked Mutant Constructs Lack Glycosyltransferase Activities The data so far indicated that E X T 1 and EXT2 form a hetero-oligomeric complex in the Golgi that, following isolation, possesses significantly more GlcA transferase activity, and therefore HS-Pol activity, than either polypeptide alone. These data are consistent with the aetiology of the disease in which patients having a hereditary mutation in either E X T l or EXT2 present with multiple exostoses. Interestingly, the G339D and R340C missense mutations in E X T l completely eliminated GlcA-T activity from the EXT1/EXT2 complex (Table 5.1), while GlcNAc-T activity remained relatively unchanged. When the C85R EXT2 mutant protein was isolated from EXTl-defective sog9 cells, it was completely inactive in both activities necessary for HS-polymerization (Table 5.1). Taken together, these data indicate that disease-causing mutant E X T proteins have defects in either one (EXTl ) or both (EXT2) HS-polymerizing activities, which further confirms that the pathogenesis of H M E stems from defects in HS synthesis. 95 Table 5.1 GlcNAc-T and GlcA-T activities of immunoprecipitated EXT1-EXT2 mutant complexes E X T l EXT2 3 H-GlcNAc-T 1 4 C-GlcA-T construct construct activity(cpm)3 activity(cpm)a EXTlgfp - 782 625 - mEXT2gfp N.D. 21 E X T l mEXT2gfp 495 2169 G339DEXTlgfp - 1220 14 R340CEXTlgfp - 1310 32 G339DEXTlgfp mEXT2myc 846 14 R340CEXTlgfp mEXT2myc 543 22 - C85REXT2gfp 16 44 E X T l C85REXT2gfp 3 3 "EXTl-defective sog9 cells were transfected with E X T constructs for 30 hours, lysed, and E X T proteins were immunoprecipitated with the rabbit anti-GFP antibody (GFP-fusion proteins are indicated in bold for clarity). The prefix'm' indicates 'murine'. GlcA-T and GlcNAc-T activities were calculated as cpm of incorporated [ 3H]-GlcNAc and [ 1 4C]-GlcA per immunoprecipitate. Data indicate results from a single experiment. Replicate experiments were not performed due to limited quantities of acceptor substrates. 96 5.3 Discussion In light of our findings that the aetiologic missense mutations in E X T l , G339D and R340C, do not prevent EXT1-EXT2 complex formation or redistribution of EXT2 to the Golgi apparatus, it is likely that these mutations alter the conformation of the HS-Pol heterocomplex, thereby inactivating the enzyme. This hypothesis is supported by the finding that these E X T l mutants are unable to compensate for the heparan sulfate synthesis defect in sog9 cells. Furthermore, in vitro enzyme assays show that E X T 1 -EXT2 complexes containing the G339D and R340C mutations are unable to catalyze the addition of GlcA to a growing HS chain, while retaining full GlcNac-transferase activity. Taken together, these results suggest that the G339D and R340C mutant forms o f E X T l are incorporated into defective EXT1-EXT2 complexes in vivo, which despite being Golgi localized, are inactive due to a loss of one of the two activities required for HS-polymerization. These results also suggest that the 339/340 region o f E X T l may be the domain involved in GlcA-transferase activity in the wild type EXT1-EXT2 complex. The known aetiologic missense mutations of EXT2, C85R and D227N, present a different set of problems. These mutations, like those described in E X T l , do not abrogate EXT1-EXT2 complex formation. However, these complexes do not re-locate to the Golgi apparatus, and are instead retained in the ER. In vitro enzyme activity of complexes containing the C85R EXT2 mutation are defective in both GlcNac-transferase and GlcA-transferase activities. These findings suggest that the formation of the E X T 1 -EXT2 hetero-oligomeric complex normally occurs in the ER, but if this complex is unable to re-locate to the Golgi apparatus, it is not active. It is formally possible that the abnormal ER-retention of these mutant complexes results from a mis-folding event which would prevent a molecular chaperone, like ERp72 (BiP), to release the complexes from the ER. Furthermore, it is possible that for the EXT1-EXT2 complex to be active, it may have to undergo post-translational modification in the Golgi, or perhaps undergo a conformational change in the more acidic Golgi microenvironment. Additional experiments to investigate the potential post-translational modifications and interactions with chaperones of the wild type and mutant E X T proteins will be necessary to answer these important questions. 97 H M E is inherited in an autosomal dominant fashion, such that affected individuals should have one mutant and one wild type copy at one E X T locus, and two wild type copies at a second E X T locus. Therefore, individuals with aetiologic mutations in one copy of E X T l or EXT2 would likely have two pools of EXT1-EXT2 complexes, one active and one inactive. The presence of the inactive E X T complexes in the ER/Golgi may be sufficient to cause deficiencies in the processing of proteoglycans destined for the cell surface or extracellular matrix. In the future, it will be important to investigate other HME-associated mutations to determine whether any of them are defective in complex formation, Golgi localization and/or glycosyltransferase activity. Such investigations may generate additional data on the functional domains and architecture of the EXT1-EXT2 complex. 98 CHAPTER 6: A role for heparan sulfate in cell-cell transmission of herpes simplex virus 6.1 Introduction HSV infection is initiated by the interaction of viral glycoproteins with cell surface HS and CS GAGs as well as cell surface proteins (Shieh et al, 1992; Gruenheid et al, 1993). Sog9 cells, which are defective in G A G biosynthesis, are greater than 99.5% resistant to HSV infection (Banfield et al, 1995). It has been shown that expression of a putative tumor suppressor, E X T l , directs the synthesis of a novel form of HS on the surface of sog9 cells, rendering them fully susceptible to HSV infection (Chapter 3). In fact these sog9-EXTl cells are the only tissue culture cell line known that express only HS and no CS. Using the E X T l cDNA, I was able to generate a complete panel of G A G mutant cell lines that express no GAGs (sog9), HS alone (sog9-EXTl), CS alone (gro2C), or HS and CS (gro2C-EXTl, L , and L-EXT1). The initial approach was to use this panel of cell lines to study HSV infection at a number of steps in order to ascertain the relative contributions of HS and CS to the various stages of the viral life cycle. 6.2 Results 6.2.1 Effect of EXTl on cellular susceptibility to HSV-1. To quantify the effect o f E X T l on HSV-1 infection, cell lines were selected which stably expressed E X T l . EXTl-expressing sog9 cells (sog9-EXTl) were approximately 150 times more susceptible to HSV-1 strain F infection than control sog9 cells, with an infection rate that was approximately 90% of that for the parental L cell (data not shown). Similar results were observed when sog9-EXTl cells were infected with HSV-1 strain KOS (Fig. 3.1c) or HSV-2 strain G (data not shown) indicating that this effect was not limited to a particular strain or type of HSV. Interestingly, E X T l expression in gro2C cells, which express CS, but not HS (Gruenheid et al, 1993), resulted in only mild increases in HSV susceptibility. HPLC analysis of the L , L -EXT1 , 99 and gro2C-EXTl cell lines indicated that all three synthesize HS and CS (Fig. 3.4). This suggests that the quantity and/or fine structure of the HS and CS GAGs on these cell surfaces can affect the efficiency of HSV attachment. It is well established that HSV-1 glycoprotein C (gC) and gB are capable of interaction with HS (Herold et al, 1991; Herold et al, 1994; Tal-Singer et al, 1995). To determine whether gC and/or gB are required for HSV-1 attachment to E X T l induced cell surface HS, a set of HSV glycoprotein mutants was obtained from J. Glorioso (University of Pittsburgh) (Laquerre etal, 1998). Infection of sog9 and sog9-EXTl cell monolayers with an HSV mutant which lacked the gB HS binding domain (KgBpK-) revealed relative infectivities very similar to those derived from the parental KOS strain, indicating that the lysine-rich gB HS binding domain is not required for attachment to EXTl-induced forms of cell surface HS (Table 6.1). By contrast, infection of these cell monolayers with an HSV mutant which was deleted for gC (KCZ) or both gC and the gB HS binding domain (KgCgBpK) , revealed that gC is essential for HSV-1 binding to E X T l induced forms of HS (Table 6.1). One curious aspect of the infection of this panel of cell lines was the mild, 2-fold increase in the infection rate of L-EXT1 cells compared to the parental L cell. L-EXT1 cells have been shown to synthesize a less negatively charged form of HS compared to L cells (Fig. 3.4) which should therefore be less able to bind HSV by electrostatic G A G -glycoprotein interactions. Furthermore, HSV-1 attachment assays suggest that the L -EXT1 cell surface is saturated more easily by excess virus as compared to the L cell (Fig. 6.1). Thus, while it is not clear why L-EXT1 cells are more susceptible to HSV infection, it may be that L-EXT1 cells express a certain HS G A G moiety which promotes infection by an unknown mechanism, perhaps by allowing greater access to stable attachment receptors. There are numerous situations in which different G A G modifications can either suppress or promote the process of receptor clustering on the cell surface. For example, clustering and activity of the lymphocyte homing receptor CD44, depends upon whether it is modified with HS or CS GAGs (Esford et al, 1998). Therefore, it is also possible that the altered HS that decorates the L-EXT1 cell surface may facilitate clustering of HS proteoglycans and/or secondary HSV receptors. 100 Table 6.1 Relative infection of HSV-1 (KOS) mutant viruses on glycosaminoglycan mutant cell lines relative infectivity of virus stock3 cell line phenotype KOS KgBpK" K C Z KgCgBpK-sog9 HS-CS- 1 1 1 1 sog9-EXTl HS+CS- 76 90 1 0.9 gro2C HS-CS+ 19 11 0.3 0.8 gro2C-EXTl HS+CS+ 48 41 0.6 1 L HS+CS+ 170 170 5 2 L-EXT1 HS+CS+ 360 300 10 2 "Titers of serial 10-fold dilutions of virus stocks grown in Vero cells were determined on cell monolayers using a plaque assay. Results from at least three determinations were averaged and expressed relative to control sog9 cell infection. HS, heparan sulfate; CS, chondroitin sulfate 101 0 100 200 300 400 500 HSV-1 Added (CPM x 1000) Fig. 6.1 HSV-1 attachment assay. Cell monolayers were incubated with radiolabeled HSV-l(KOS) on ice for 2 h. The radioactivity associated with the monolayer was determined by liquid scintillation spectroscopy. Values are averages of four experiments. Open squares, mouse L cells; closed squares, L-EXT1 cells; open circles, sog9 cells; closed circles, sog9-EXTl cells. 102 6.2.2 Analysis of cell-to-cell spread of virus In the course of this study, it was observed that E X T l expression in the parental L cell line caused only marginal increases in infectivity (Table 6.1). However, plaque assays revealed that expression of E X T l in L cells led to a dramatic increase in the cell-to-cell spread of the virus, as indicated by a significant increase in plaque size (Fig. 6.2). This enhancement in virus spread was not observed when E X T l was transfected into the mutant sog9 or gro2C cell lines, which suggests that this phenomenon is dependent upon the ability of the cells to synthesize the normal complement of HS and CS GAGs prior to transfection. To assess whether the effect of E X T l on cell-to-cell spread was applicable to primate cells, HS + CS + Vero cells were transfected with E X T l , and tested for enhanced susceptibility and spread. Vero-EXTl cells also showed a significant increase in cell-to-cell spread of the virus (Fig. 6.2). Several studies have implicated the HSV-1 gE/gl heterodimer as being involved in the process of cell-to-cell spread of virus (Dingwell et al, 1994; Dingwell et al, 1995). To test whether E X T l mediated increases in cell-to-cell spread of virus is dependent upon the presentation of gE and gl on the cell surface, cell monolayers were infected with gl deficient virus (F-US7kan), gE deficient virus (F-gEB), or a wild type revertant of the gE deficient virus (F-gEBR) (Fig. 6.3). Infection of any cell monolayer with gE or gl deficient viruses results in smaller plaques compared to the plaques resulting from wild type virus infection, confirming the role of gE/gl in normal cell-to-cell spread of virus. However, the plaques produced by gE and gl deficient viruses on L-EXT1 cells were still consistently larger than the plaques produced by these mutant viruses on wild type L cells. The fact that the spread of gE deficient or gl deficient viruses was not entirely abrogated suggests that HSV may use other interactions to mediate cell-to-cell spread in these cell lines. Consistent with previous observations of plaque assays, infection of sog9-EXTl cells with wild type virus results in plaques that are not significantly larger than the plaques produced on sog9 cells. Likewise, the plaques produced by gE and gl deficient viruses on sog9 and sog9-EXTl cells were not significantly different. Taken together, these data suggest that E X T l mediated stimulation of cell-to-cell spread of virus is independent of gE/gl heterodimer mediated interactions at the cell surface. It has been 103 shown previously that certain cellular G A G moieties play a role in several events in the HSV life cycle, including cell-to-cell spread (Shieh and Spear, 1994). These data suggest that gE/gl effects on cell-to-cell spread are not mediated through interactions with GAGs or modifications by GAGs. 6.2.3 Anion exchange chromatography of GAGs from L-EXT1 cells It has been shown previously that a clonally isolated EXTl-expressing L cell line synthesizes a heterogeneous, less negatively charged form of HS compared to the parental L cell (Fig. 3.4). I hypothesized that if E X T l enzymatic activity in transfected L cells resulted in the observed alterations in HS and HSV plaquing, then L cells transfected with non-functional E X T l mutants should be identical to the parental L cell with regard to these phenotypes. To test this hypothesis, L cells were transfected with the aetiologic mutants G339DEXT1 or R340CEXT1, radiolabeled with [35S]-sulfate, and GAGs were isolated and fractionated by anion exchange chromatography. Surprisingly, L cells transfected with G339DEXT1 or R340CEXT1 synthesizes a heterogeneous, less charged form of HS identical to L-EXT1 cell HS (Fig. 6.4). With regard to cell morphology, infectivity and plaque formation L-G339D cells and L-R340C cells are indistinguishable from L-EXT1 cells. Taken together, these data show that these mutations, which disrupt the HS-polymerization activity o f E X T l in in vivo and ex vivo assays (Fig. 5.2, Table 5.1), do not prevent the other observable phenotypes in these stably transfected L cells. 104 Fig. 6.2 Plaque forming efficiency of HSV-1 on EXTl-transfected cell lines. Cell monolayers were inoculated with approximately 100 PFU of HSV-l(KOS) for 1 h, washed 3 times with PBS and overlaid with medium containing 0.1% IgG. At four days post-infection, monolayers were rinsed and fixed with 5% methylene blue in 70% methanol, a, mouse L cells; b, L-EXT1 cells; c, Vero cells; d, Vero-EXTl cells. 105 F i g . 6.3 Plaque forming eff iciency o f g E - and gl-deficient viruses on E X T l - e x p r e s s i n g and control ce l l l ines. C e l l monolayers were infected wi th F - g E B R ( w i l d type revertant), F - g E B (gE-) or F -U S 7 k a n (gl-) for 1 h , washed 3 times wi th P B S and over la id wi th med ium containing 0 .1% I g G . A t four days post infect ion, the cel ls were rinsed and f ixed wi th 5% methylene blue in 7 0 % methanol. Statistical measurements o f plaque sizes can be found in Table 6.2. Table 6.2 Plaque size o f g E / g l mutant viruses on ce l l monolayers ce l l l ine phenotype size (in m m 2 ) o f plaques produced by H S V - 1 stock" F F (gE- ) F (g l - ) sog9 H S - C S - 0 . 0 6 7 ± 0 . 0 2 8 0 . 0 3 4 ± 0 . 0 1 2 0 . 0 3 4 ± 0 . 0 2 1 s o g 9 - E X T l H S + C S - 0 . 0 7 6 ± 0 . 0 3 4 0 . 0 4 0 ± 0 . 0 1 1 0.033+0.013 L H S + C S + 0 . 2 1 7 ± 0 . 0 7 9 0 . 0 9 7 ± 0 . 0 3 3 0.118+0.043 L - E X T 1 H S + C S + 1.311+0.239 0.322+0.094 0.536+0.183 "Results are averages o f 25 plaques selected randomly and captured wi th a Javel in chromachip camera mounted on a L e i t z Labover t microscope. Images were imported into N I H Image 1.60, where plaque margins were traced to obtain measurements o f surface area. 106 35000 30000 A 25000 A 1 20000 u cu Ck O U 15000 IOOOOA 5000 J 30 40 Fraction Number F i g . 6.4 A n i o n exchange chromatography o f g lycosaminoglycans from E X T l - t r a n s f e c t e d L cel ls . C e l l l ines were generated by l iposome mediated transfection o f E X T l constructs, fo l lowed by selection wi th 700 j i g / m l G 4 1 8 . C e l l monolayers were g rown for 24 h in the presence o f [ 3 5S]-sulfate. G A G s were isolated and fractionated by H P L C . H S , elution posi t ion o f heparan sulfate; C S , elut ion posit ion o f chondroi t in sulfate. 107 6.2.4 Analysis of L-EXT1 cell attachment to different matrices It has been shown previously that the cell-to-cell spread of HSV is accelerated in L-EXT1 cells compared to the wild type L cell line (Fig. 6.2). However, this accelerated virus spreading phenotype was retained even for HSV-1 glycoprotein mutants that have defects in GAG-binding (Table 6.1) or cell-cell spread of virus (Fig. 6.3, Table 6.2). Thus, the altered form of HS found on the surface of L-EXT1 cells is unlikely to cause enhanced virus spread through direct virus-GAG interactions. Therefore, it seemed possible that the observed enhancement in cell-to-cell spread of HSV was an indicator of a downstream effect of changing cell surface HS. A common feature of the L-EXT1 and Vero-EXTl cell lines is a more extended, stellate (star-shaped) cell morphology. It has long been known that cell morphology is largely controlled by changes in the actin cytoskeleton. The common links between the actin cytoskeleton and cell surface HS are focal adhesions (Woods etal., 1984; Burridge et ai, 1988), also known as adhesion plaques (Burridge et ai, 1987). Focal adhesions consist of clusters of plasma-membrane associated proteins of the integrin family, which probe the extracellular environment with their amino-termini, while signaling changes in the actin cytoskeleton via a variety of signaling molecules, including the tyrosine kinases ppl25FAK 1 (Schaller etal., 1992), pp60Src (Rohrschneider, 1980), and C-terminal Src kinase (Bergman et al, 1995), the serine/threonine kinases, protein kinase C (Woods and Couchman, 1992) and mitogen-activated protein kinase, the small G proteins Ras, Rho, and Rac, protein tyrosine phosphatase ID, phospholipase C, PI-3K, and adapter proteins such as paxillin and Grb2 (Turner et al, 1990; Miyamoto et al, 1995; Miyamoto et al, 1995). Interestingly, a number of studies have shown that HSV infection of cells causes radical changes in the actin cytoskeleton and cell-matrix interactions in the host cells (Heeg et al, 1981; Dienes et al, 1994). Thus, I hypothesized that the enhanced virus spread phenotype may have resulted from E X T l induced changes in cell-matrix interactions via the actin cytoskeleton and focal adhesions. As an initial test of the ability of L-EXT1 cells to interact with the extracellular matrix, L and L-EXT1 cells were seeded onto tissue culture dishes coated with collagen IV, fibronectin, laminin, poly-D-lysine, or the standard matrix used for previous 108 experiments, collagen I. After 3 hours, there appeared to be no difference between the ability of L cells and L-EXT1 cells to bind to the collagen I or fibronectin matrices, but L-EXT1 cells appeared to be marginally less able to adhere to collagen IV (data not shown). While L cells showed some adherence to laminin, L-EXT1 cells were much less able to bind to this matrix (Fig. 6.5 a, b). After 36 hours, the disparity between the cells' laminin binding abilities was increased (Fig. 6.5 c, d). Remarkably, 3 hours after L -EXT1 cells were seeded onto poly-D-lysine, they underwent a radical change in cell morphology, becoming amorphous, with a "fried-egg" appearance (Fig. 6.5 f, h). By comparison, L cells retained their polygonal shape on poly-D-lysine (Fig. 6.5 e, g). Taken together, these results suggested that E X T l expression in murine L cells caused radical changes in the cell's ability to interact with laminin, and the artificial ligand, poly-D-lysine. Because poly-lysine is commonly known to disrupt the formation of focal adhesions (Lo et al, 1998), and because two of the major cellular receptors for laminin are HS proteoglycans and the oc6Bl class of integrins (Bauer et al, 1992; Bazzoni et al, 1999), I hypothesized that focal adhesions may be altered in L-EXT1 cells. 6.2.5 Analysis of cell-cell spread of HSV on different matrices The data so far had suggested that interactions between the host cell and the extracellular matrix had been altered in L-EXT1 cells. To determine whether these changes in attachment to different matrices were relevant to the cell-to-cell spread of HSV, cells were plated on different matrices and infected with R8102, a B-galactosidase expressing HSV-1. Laminin was excluded from these experiments because L-EXT1 cells were unable to form a monolayer on this matrix. As expected, when cells were seeded on collagen I, HSV-1 was able to form significantly larger plaques on L-EXT1 cells than on L cells. Moreover, on the collagen IV and fibronectin matrices this effect was also evident, although the overall plaque size for both L and L-EXT1 cells on these matrices was reduced compared to that on collagen I. Plaque formation on the poly-lysine matrix, which disrupts focal adhesions (Lo et al, 1998), was severely inhibited for both L and L -EXT1 cell lines. Taken together, these results indicate that the extracellular matrix and focal adhesions play a major role in HSV-1 plaque formation. Despite these interesting 109 findings, it is not yet known if EXTl-induced changes in the adhesive properties of L cells is directly responsible for the accelerated HSV-plaquing phenotype in these cells. 110 F i g . 6.5 L i g h t microscopy o f L and L - E X T 1 cel ls plated on different matrices. Equa l numbers o f L and L - E X T 1 ce l l monolayers were t rypsinized, seeded onto l amin in (a, b, c, d) or p o l y - D - l y s i n e (e, f, g, h) coated plates, and observed at the indicated times and magnif icat ions. Images were captured wi th a Javel in chromachip camera mounted on a L e i t z Labover t microscope, imported into N I H Image 1.60, and processed wi th A b o b e Photoshop 5.0. Ill 6.3 Discussion E X T l was identified as a cellular factor which could make the HSV resistant cell line, sog9, susceptible to HSV infection. Assays based on the attachment of radiolabeled HSV-1 to cell monolayers at 4° revealed that the initial binding phase of HSV entry was restored in EXTl-expressing sog9 cells. E X T l expression caused sog9 cells to synthesize HS GAGs which corresponded to the elution position of HS. Although the levels of HSV attachment and infection of sog9-EXTl cells was comparable to that of the parental mouse L cell (Table 6.1, Fig. 6.1), sog9-EXTl cells make relatively small amounts of HS (Fig. 3.4). These data suggest that the specific G A G moieties induced by E X T l may have a relatively high affinity for HSV-1. It is known that the structural features of HS important for initial HSV-1 attachment include N-sulfation and 6-0-sulfation of the N-acetyl glucosamine subunits of HS (Herold et al, 1995). Furthermore, the generation of highly sulfated disaccharide repeats in the heparan sulfate polymer, namely, 2-O-sulfated iduronic acid and 3-0-sulfated/6-0-sulfated glucosamine appears to mediate the stable attachment phase of HSV-1 entry via gD (Liu et al, 1999; Shukla et al, 1999). Perhaps sog9-EXTl cells make a form of G A G which is enriched in these HSV binding moieties. An alternative interpretation is that a relatively higher proportion of the G A G synthesized in sog9 cells appears on the cell surface. Although E X T l expression in sog9 cells resulted in remarkable increases in cellular susceptibility to HSV infection, the plaques formed on sog9-EXTl cell monolayers were similar in size and appearance to the plaques formed on sog9 cells. Stable expression of EXTl in the parental L cell resulted in the opposite effect, with mild increases in cellular susceptibility to H S V entry, but significant increases in cell-to-cell spread of virus in the presence of IgG. These data suggest that E X T l must be changing the cell surface architecture at important junctions between L cells in order to facilitate cell to cell spread. Several previous studies have implicated the HSV-1 gE/gl heterodimer as being involved in the process of cell-to-cell spread of virus (Dingwell et al, 1994; Dingwell et al, 1995). I hypothesized that E X T l mediated increases in cell-to-cell spread of virus may be dependent upon the presentation of gE and gl on the cell surface. The plaques 112 produced by gE and gl deficient viruses on sog9 and sog9-EXTl cells are not significantly different (Fig. 6.3). However, the plaques produced by gE and gl deficient viruses on L-EXT1 cells are consistently larger than the plaques produced by these mutant viruses on L cells. Taken together, these data suggest that E X T l mediated stimulation of cell-to-cell spread of virus is independent of gE/gl heterodimer mediated interactions at the cell surface. In summary, E X T l was transfected into the parental mouse L cell line to observe the effects of overexpression of one member of the EXT1/EXT2 complex. These L -EXT1 cells displayed an altered, more stellate cell morphology, and anion exchange chromatography of GAGs revealed that their cell surface HS was less negatively charged and more heterogeneous than L cell HS. However, the most remarkable phenotype of L -EXT1 cells was a dramatic increase in the ability of progeny HSV to spread from cell-to-cell. These L-EXT1 cells also displayed a decreased affinity for certain extracellular matrices. Taken together, these observations suggest that disrupting the balance of the members of the EXT1/EXT2 complex leads to changes in the architecture of the cell surface that allow for more efficient cell-to-cell spread of HSV, possibly through altered cell-cell contacts. In H M E , defects in a single copy of E X T l or EXT2 cause disease, leaving at least one viable copy of each gene. Therefore, this disease may result from an imbalance between active and inactive EXT1/EXT2 complexes in the cell, resulting in changes to cell surface HS similar to the alterations observed in L-EXT1 cells. Thus, the secondary effects o f E X T l overexpression in L cells may be relevant to the disease of H M E . However, that is not to say that cultured L-EXT1 cells would be a good model for H M E . The relevant mutant cells in H M E are cartilage-synthesizing chondrocytes, while L -EXT1 cells are fibroblasts - the differences between them are considerable. The work presented in this thesis on EXTl-expressing sog9 and L cells provides the framework for future investigations into the E X T mutant chondrocytes - where the questions will be; (i) what types of GAGs do they produce? (ii) Are they susceptible to HSV infection? (iii) Are there cell morphology differences? And (iv) are there changes in the adherence of these cells to the extracellular matrix? 113 CHAPTER 7: Discussion 7.1 Elucidation of E X T l function The herpes simplex viruses are able to infect a broad range of human tissues, causing facial or genital lesions, and sometimes encephalitis or corneal blindness. Research efforts in the Tufaro lab have focused on the initial interactions between these viruses and their host cells. The specific approach to this research problem involved the isolation of HSV resistant tissue culture cell lines (gro2C and sog9) which harboured defects in cellular components necessary for viral infection (Gruenheid et ai, 1993; Banfield et al., 1995). These components were the ubiquitous HS and CS GAGs which promote initial HSV attachment to the cell surface. However, G A G biosynthesis is a complex multistep process, and it was not known which steps in HS and CS biosynthesis were defective in these cells. To address this problem, a human cDNA library was screened for genes that could restore susceptibility to HSV infection in sog9 cells. I reasoned that these cDNAs would either correct the defect in HS biosynthesis, or correct the defect in CS biosynthesis, or encode a HSV receptor that did not require the presence of cell surface GAGs to function. Multiple rounds of cDNA screening and subpooling resulted in the isolation of a single cDNA, which restored HSV infection to near wild type levels by correcting the defect in HS biosynthesis. This cDNA was sequenced and identified as EXTl, a putative tumor suppressor involved in hereditary multiple exostoses, a disease characterized by the formation of benign bone tumours during bone development (Chapter 3). Following the isolation of E X T l , the focus shifted to address the role of this protein in HS biosynthesis. Through a collaboration with Dr. Ulf Lindahl's lab, it was determined that E X T l , and another HME-linked protein, EXT2, formed a Golgi-localized complex which had enzymatic activities characteristic of a HS-polymerase, which catalyzes the transfer of GlcNAc and GlcA sugars into a growing HS chain (Chapter 4 and (Lind et ai, 1998; McCormick et al., 2000)). I hypothesized that if proper HS 114 biosynthesis is critical to the development of bone, then disease-causing mutations in E X T l and EXT2 should disrupt the glycosyltransferase activities of the complex. In fact, every disease causing mutation in E X T l or EXT2, including some relatively conservative missense mutations, abrogated HS-Pol activity in isolated complexes (Chapter 5). Thus, defects in HS biosynthesis are almost certainly the underlying cause of H M E . 7.2 Proteoglycans and bone disorders Perhaps it is not so surprising that H M E is caused by alterations in a gene involved in the biosynthesis of HS, a key molecule in maintaining cell surface architecture and biological activity. Heparan sulfate, along with several other GAGs, such as chondroitin sulfate, dermatan sulfate and keratan sulfate, are components of proteoglycans, which are present on the surface of most cells in multicellular organisms (see Table 1.1 for detailed tissue distributions). Proteoglycans are components, along with collagen, of bone and cartilage, where they have a major influence on tissue hydration, elasticity and cation composition. Several HS proteoglycans, including glypican, betaglycan, perlecan and members of the syndecan family, are components of the extracellular matrix in developing bone and participate in a wide variety of important biological processes, including critical involvement in cell signaling pathways (Bernfield et al, 1992; De Luca and Baron, 1999). For example, syndecan-3 participates in limb outgrowth and proliferation in response to the apical ectodermal ridge, and mediates cell-matrix and/or cell-cell interactions involved in regulating the onset of chondrogenesis. It also plays a role in regulating the proliferation of epiphyseal chondrocytes during endochondral ossification, and might be involved in regulating the onset of osteogenesis and joint formation (Kosher, 1998). Syndecan-1 (CD 138), a HS proteoglycan, enhances differentiation of bone-forming osteoblasts and reduces differentiation of bone-resorbing osteoclasts, a process that requires only the syndecan-1 HS chains (Dhodapkar et al, 1998). It seems likely that deficiencies in HS biosynthetic activity in H M E patients could lead to the display of altered forms of proteoglycans that impede proper bone development. It has been shown previously that a number of skeletal dysplasias are caused by changes in molecular interactions that rely on HS or other GAGs. Achondroplasia, the 115 most common form of chondrodysplasia in humans, characterized by short-limbed dwarfism and macrocephaly, is caused by mutations in the gene encoding the fibroblast growth factor (FGF) receptor 3, a cell-surface protein that binds FGF only in the presence of HS (Shiang et al, 1994). In mice, targeted disruption of the gene for biglycan, a small extracellular matrix CS proteoglycan enriched in bone, results in an osteoporosis-like phenotype (Xu etal, 1998). Moreover, diastrophic dysplasia is caused by mutations in a novel sulfate transporter gene, which probably results in undersulfated glycoproteins and GAGs (Wallis, 1995). Taken together, these studies suggest that bone development is a process exquisitely sensitive to changes in the composition of the cell surface, and in particular, proteoglycans. 7.3 Indian hedgehog, Parathyroid hormone related protein and EXT in bone development Advances in the understanding of the molecular biology of endochondral ossification have also proved valuable to the understanding of H M E . It is well established that proper bone development depends upon controlled chondrocyte proliferation, differentiation and apoptosis in the growth plate. Recent work has shown that two signaling molecules, Indian hedgehog (Ihh), synthesized by chondrocytes in the growth plate, and parathyroid hormone-related protein (PTHrP), acting via receptors expressed on chondrocytes, regulate chondrocyte maturation and hypertrophy in a coordinated way (Lanske et al, 1996; Vortkamp et al, 1996). In the current signaling model, the intermediate prehypertrophic chondrocytes within the growth-plate produce Ihh, which stimulates chondrocyte proliferation by up-regulating the expression of the second signaling molecule, PTHrP, by cells of the periarticular perichondral region (Fig. 7.1). Subsequently, PTHrP binds to the PTH/PTHrP receptor on a subpopulation of proliferating and prehypertrophic chondrocytes, postponing cell death by up-regulating Bcl-2, a well known anti-apoptotic protein. This allows for continued longitudinal cartilage growth, until a shift in the expression of Ihh or PTHrP disrupts the equilibrium. In support of this model, knock-out mice missing either the PTHrP gene or its receptor are small, with excessive and unmodulated bone formation leading to prematurely ossified growth plates (Lanske etal, 1996). 116 Perichondria! Region Fig. 7.1 A model for the regulation of chondrocyte differentiation. Indian hedgehog (Ihh) is produced by chondrocytes within the growth plate that are committed to hypertrophy. Either directly or indirectly, Ihh stimulates the production of parathyroid hormone-related protein (PTHrP) in the periarticular perichondrium, which diffuses to the prehypertrophic chondrocytes and induces production of Bcl-2, a well-known anti-apoptotic protein. Proper diffusion of Ihh depends on the cell surface transmembrane receptor patched (Ptc), and perhaps E X T l and EXT2 via their heparan sulfate proteoglycan products. Abbreviations: Pro, proliferating chondrocytes; Pre, prehypertrophic chondrocytes; Hyp, hypertrophic chondrocytes. Adapted from (Ingham, 1998). 117 Insights into the function of the EXT genes in the molecular signaling pathways governing bone development have come from an unexpected source, the common fruit fly, Drosophila melanogaster. Recently, a new member of the EXT gene family was identified called tout-velu (Ttv), meaning 'all-hairy' in reference to the phenotype observed in Drosophila Ttv mutants (Bellaiche et al, 1998). Like human E X T l , Ttv appears to be a glycosylated type II integral membrane protein. In Drosophila, Ttv is required for diffusion of an important segment polarity protein called hedgehog (Hh), which is a homolog of mammalian Ihh (Bellaiche et al, 1998; Ingham, 1998). Ttv is thought to act either by facilitating endocytosis of the secreted Hh protein, or by permitting Hh diffusion between responding cells (Bellaiche et al, 1998; Ingham, 1998). Based on its homology to E X T l , it was proposed that Ttv could be responsible for the synthesis of HS GAGs that specifically enhance Hh diffusion (Bellaiche et al, 1998). Although the mechanism of Ttv action in Hh diffusion is still unknown, a recent report demonstrates that total GAGs isolated from homozygous Ttv mutant Drosophila larvae show marked reductions in HS, but not CS (Toyoda et al, 2000). Evidence of an important role for HS GAGs in Hh diffusion and Drosophila development may have additional significance with regard to bone development in humans and H M E . If the human E X T proteins are indeed involved in the diffusion and/or efficient signaling of Ihh in the growth plate of developing bone, it may explain the aetiology of H M E (Bellaiche et al, 1998). According to this model, a defect in E X T l or EXT2 would block or misdirect Ihh diffusion across responding cells (as was seen with Ttv and Hh), disrupting the negative feedback loop that regulates signaling to the chondrocytes (Fig. 7.1). In support of this model, it has been suggested previously that integral membrane proteoglycans that bind growth factors could serve to deliver ligands to neighboring cells (Selleck, 1998). It has also been established that glycosylphosphatidylinositol (GPI)-linked molecules are capable of being transferred intact between the plasma membranes of cells that come into contact with one another (Kooyman et al, 1995). GPI-linked proteoglycans, like the glypicans (Fig. 1.2), could therefore provide a molecular mechanism for 'contact-mediated diffusion' of growth factors and signaling molecules 118 such as Hh. Considering the evidence at hand, disruption of this type of proteoglycan-mediated signaling might result in the localized aberrant bone growth observed in H M E . 7.4 Other bone tumours Although H M E is the hereditary bone tumour disorder best understood at the molecular level, other bone tumour loci are in the process of being characterized. For example, hereditary hyperparathyroidism-jaw tumour syndrome is an autosomal dominant disease recently mapped to chromosomal region Iq25-q31 (Hobbs et al, 1999). In Paget disease (oseitis deformans), a common metabolic disease resulting in rapid bone remodeling, approximately 1 % of affected individuals develop osteosarcoma, and recent work has suggested that the association between Paget disease and osteosarcoma is the result of a single gene or two tightly linked genes on chromosome 18 (Nellissery et al, 1998). Ewing's sarcoma, a common malignant primary bone cancer that occurs frequently in adolescents (approximately 20% of malignant bone tumours in Western populations), is caused by a number of different chromosomal translocations. The t(l I;22)(q24;ql2) translocation is present in up to 95% of cases of Ewing's sarcoma and results in the formation of an EWS-FLI1 fusion gene which encodes a chimeric transcription factor thought to activate as yet unknown target genes (Lin et al, 1999). Considering the importance of HS proteoglycans in H M E and other non-cancerous skeletal dysplasias, it would not be surprising if candidate genes linked to these bone tumours (or their target genes) were also found to be involved in formation of the cell surface architecture. 7.5 Prospects for the future The results presented in this thesis provide strong evidence for the identity of the E X T genes and their relationship with each other, and should prove extremely useful for future studies. These studies will undoubtedly involve fine mapping of the glycosyltransferase domains and intermolecular contact points of E X T l and EXT2, as well as the elucidation of the functions of the EXT-like genes E X T L 1 , L2 and L3. The realization that heparan sulfate, the most complex polysaccharide on the surface of mammalian cells, lies at a crucial intersection of the signaling pathway in bone development provides a new focus for research into hereditary bone tumours. The 119 greatest priority for future studies should be the analysis of the glycosaminoglycans produced by EXT-mutant chondrocytes derived from patient tissue. If the HS from these chondrocytes is altered in some measurable way, then we will be able to model the extracellular environment in the metaphyses of H M E patients, and perhaps, understand the downstream processes that result in tumour formation. 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Virol. 63: 3323-3329. 138 APPENDIX 1 Chemical reagents and laboratory supplies Acetic acid Acrylamide Agarose Ammonium persulfate(APS) Ammonium acetate Ampicillin Aprotinin Bis-acrylamide Bovine serum albumin (BSA) Bromophenol Blue Chloroform Chondroitin A B C lyase Chondroitin sulfate C Coomassie Brilliant Blue DEAE-Sephacel Dextrose Dimethyl sulfoxide (DMSO) Dulbecco's Modified Eagle Media (DMEM) D M E M without glutamine, methionine, cystine Dithiothreitol (DTT) EDTA Endoglycosidase F Endoglycosidase Ff Ethidium Bromide Fetal Bovine Serum (FBS) Geneticin (G418) Glycerol Ham's F-12 media Fisher Canadian Life Technologies Canadian Life Technologies Canadian Life Technologies Fisher Sigma Roche BioRad Roche V W R Fisher Sigma Sigma Bio-Rad Pharmacia Fisher Fisher Canadian Life Technologies ICN Canadian Life Technologies Fisher Roche Roche Sigma Canadian Life Technologies Canadian Life Technologies Fisher Canadian Life Technologies 139 Heparan sulfate Heparin HEPES Hydrochloric acid (HQ) Isoamyl alcohol Kodak BioMax M L film Kodak BioMax M R film Leupeptin L-glutamine LipofectAMPWE LipofectAMINE PLUS Lithium Chloride LumiGLO chemiluminescent substrate kit Luria broth base 2-mercaptoethanol Methanol Methylene Blue N , N'-methylene-bis-acrylamide N-octyl-b-glucopyranoside Nonidet P-40 (NP-40) Paraformaldehyde Paraformaldehyde (16% EM-grade) Pepstatin Phenol, buffer-saturated Phenylmethylsulfonyl fluoride (PMSF) Polyethylene glycol 8000 (PEG) Potassium Chloride Potassium Ferricyanide Potassium Ferrocyanide Potassium Phosphate (monobasic) Potassium Phosphate (dibasic) Sigma Sigma Fisher Fisher Fisher Interscience Interscience Roche Canadian Life Technologies Canadian Life Technologies Canadian Life Technologies ICN K P L Canadian Life Technologies Sigma Fisher Sigma Sigma Sigma Fisher Fisher Canemco Roche Canadian Life Technologies Sigma B D H Fisher Sigma Sigma Fisher Fisher 140 Pre-stained Protein Molecular Weight Markers Canadian Life Technologies Pronase Sigma 2-Propanol Fisher Protease Sigma Protein G-Sepharose-4 Fast Flow Pharmacia Saponin Sigma Scintillation cocktails (Ready Safe) Beckman Select Agar Canadian Life Technologies Sepharose CL4B Pharmacia 35S-methionine N E N Sodium Acetate Fisher, Mallinckrodt Sodium Chloride (NaCl) Fisher Sodium Deoxycholate Sigma Sodium Dodecyl Sulfate (SDS) Bio-Rad Sodium Hydroxide Fisher Sodium Phosphate (monobasic) Mallinckrodt Sodium Phosphate (dibasic) Fisher 35S-sulfate ICN Sucrose Fisher T E M E D Fisher Tissue culture flasks & dishes Canadian Life Technologies Trichloroacetic acid Fisher Tris Canadian Life Technologies Triton X-100 Sigma Tween-20 JT Baker Ultracentrifuge rotors & tubes Beckman 3mm Whatman filter paper V W R X-gal Canadian Life Technologies Zwittergent 3-12 Calbiochem 141 List of Suppliers Supplier Location Amersham Oakville, Ontario B D H Toronto, Ontario Beckman Palo Alto, California Becton Dickinson Mississauga, Ontario Bio-Rad Mississauga, Ontario Calbiochem San Diego, California Canadian Life Technologies Burlington, Ontario Canemco Lachine, Quebec Dupont-NEN Mississauga, Ontario Fisher Edmonton, Alberta ICN St. Laurant, Quebec Interscience Markham, Ontario JT Baker Hayward, California Kirkegaard & Perry Laboratories (KPL) Gaithersburg, Maryland Mallinkrodt Paris, Kentucky Millipore Mississauga, Ontario New England Biolabs Mississauga, Ontario Pierce Rockford, Illinois Pharmacia Baie d'Urfe, Quebec Sigma Mississauga, Ontario StressGen Victoria, British Columbia V W R Burnaby, British Columbia 142 

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