3.-r1b BY WARREN GARY HILL M.Sc. Thesis Submitted For The Degree Of Doctor of PhilosoPhY tn The University of Adelaide (Faculty of Medicine) Lysosomal Diseases Research Unit Department of Chemical PathologY Women's and Children's HosPital South Australia and Department of Paediatrics FacultY of Medicine Women's and Children's HosPital South Australia July 1995 ERRATUM The following corrections to this thesis should be noted On page 6 line l4;'lumenal' should read 'luminal' \ On page 9 figure l. l; the abbreviation CFTR was used but not defined. It should have been defined as Cystic Fibrosis Transmembrane Conductance Regulator On page 2l line 20; 'cAMP and phosphorylation activatable' should read 'oAMP- and phosphorylation-activatable' On page 2l line 24;'was lymphocytes' should read 'was the lymphocyte' Onpage32 lines I 8 &. 2l ;' forskolin stimulated' should read'forskolin-stimulated' On page 34 line2l,page36line22 and page 38 lines 2 &3; glcNAc and galNAc should read GlcNAc and GalNAc Onpage 44 line I l; the fullstop should be at the end of the previous line On page 69 line 18; IMDM unà OlvfEIVf should have been listed in the abbreviations as Iscove's Modified Dulbeccos Medium and Dulbecco's Modified Eagles Medium On page 70 line l' G418 should have been listed in the abbreviations as Geneticin On page 154 line 22;'(F508's' should read '(F508 cell lines' On page 159 line 16; UNC should be listed in the abbreviations as Universþ of North Carolina and the reference Snouwaert et al., (L992) added after'mouse' On page 187 line l8; a fullstop is missing Onpage224 Conclusions B, number l; the sentence should read 'Differences in [35S].[3H] ratios appear to be due to differences in sulphate-utilisation by CFPAC rather than glucosamine-utilisation by PANC' Onpage228 line 8;the equation [SOa2-]i: [SO42-]c + i.248lll2 + [Cl-]o should be expressed as [SO42-]i : [SO42-]c + 2.248/(ll2 + [Cl-]o) On page 256 line 6; 'cysteine sulphate' should read 'cysteine sulphur' There was a man who sat each day tooking out through a narrow vertical opening where a single board had been removed from a tall wooden fence' the Each day a witd ass of the desert passed outside the fence and across narrow openinglirst the nose, then the head, the forelegs, the long brown back, the hindlegs and tastty the tait. One day, the man leaped to his feet with "lt the light of discovery in his eyes and he shouted for all who could hear him: is obvious! The nose causes the tail!" Frank Herbert 'Heretics of Dune' DEDICATION This thesis is dedicated to the memory of Nicky May 1 972-1 988 LIST OF ABBREVIATIONS v¡¡ THESIS ABSTRACT ix STATEMENT xi ACKNOWLEDGEMENTS xiii CHAPTER ON E . INTRO DUCTION 1.1 CYSTIC FIBROSIS 1 1.2 PATHOPHYSIOLOGY OF CYSTIC FIBROSIS. ......................2 1.2.1 MECONIUM ILEUS: 3 1 .2.2 PANCREATIC INSUFFICIENCY:'.....".' 1.2.3 INFERTILITY: 1 .2.4 RESPIRATORY INVOLVEMENT:...'..."' 1.3 ROLE OF CHLORIDE CHANNELS lN EPITHELIA .........."""' 6 1.3.1 CHLORIDE TRANSPORT lN AIRWAYS:.. .7 I.3.2 CHLORIDE TRANSPORT lN PANCREAS:"""""" """"""""""' 8 I.3.3 CHLORIDE TRANSPORT lN SWEAT DUCT:"""" """"""""""10 1.4 THE ION TRANSPORT DEFECT IN CYSTIC FIBROSIS. ......10 1.4.1 SELECTIVE ADVANTAGE FOR CF CARRIERS:. 12 1.5 THE CYSTIC FIBROSIS GENE ..."....13 1.6 THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) 1.6.1 STRUCTURAL SIMlLARITY WITH ABC TRANSPORTERS: """""" 1.6.2 ION CHANNEL OR TRANSPORTER? 1.6.3 TRANSPORTER FUNCTIONS:'......... 1.6.4 REGULATION OF CFTR: 1.6.5 LOCALISATION OF CFTR: 1.6.6 MOLECULAR BASIS FOR CFTR DYSFUNCTION:"""""' 1 .6.7 G ENOTYPE/PHENOTYPE CORRELATION :'........" 1.7 OTHER FUNCTIONS OF CFTR 1 .7.1 STIMULUS/RESPONSE COUPLING:......... 1.8.1 MUCUS AND MUCIN: ...... 34 1 .8.2 GLYCOSAMINOGLYCANS (GAGS): 35 1.8.2.1 Structure and function of GAGs: ........ 35 1 .8.2.2 Biosynthesis of GAGs q 1.9 SULPHATE BIOCHEMISTRY...... 41 1.10 GLYCOCONJUGATE SYNTHETIC DEFECTS IN CF...... 46 .I.10.1 ALTERED GLYCOSYLATION IN CF: 46 1.10.2 ALTERED SULPHATION IN CF: ß 1.10.3 POTENTIAL ROLE OF SULPHATE lN CF: ............ 49 1.1 1 POTENTIAL PATHOPHYSIOLOGICAL CONSEQUENCES OF GLYCOCONJUGATE DEFECTS.. .52 1.11.1 PULMONARY INFECTION IN CYSTIC FIBROSIS: .52 1.11.2 WHY THE CF LUNG? ........ .53 1.12 RATIONALE FOR THIS STUDY....... 57 1.13 ArMS.. 59 CHAPTER TWO . MATERIALS AND METHODS 2.1 MATERIALS/REAGENTS......... 63 2.1.1 CELL LINES USED IN THESE STUDIES: 63 2.1 .2 RADIOCHEMICALS: ................. 64 2.1 .3 ENZYMES/ANTIBODIES:.......... ......64 2.1.4 CHROMATOGRAPHIC MEDIA: ......65 2.1.5 CELL CULTURE MATERIALS:.. ......65 2.1 .6 MISCELLANEOUS MATERIALS ......66 2.1.7 CHEMICALS:.,.. ......67 ......69 2.2.1 CELL CULTURE:. 69 2.2.2 CRYOPRESERVATION OF CELL LINES: 70 2.2.3 REVIVAL OF CRYOPRESERVED CELLS: 70 iii 2.2.4IMMUNOFLUORESCENT STAINING OF CELLS: ..'....'........'....71 2.2.5 MEASUREMENT OF CHLORIDE EFFLUX: ."'.'...'.' 71 2.2.6 SUBCELLULAR FRACTIONATION: 72 2.2.7 ø-HEXOSAMI N IDASE ASSAY:....'.. 73 2.2.8 ACIDIFICATION ASSAY: ..................74 2.2.9 SCINTILI-ATION COUNTING: ........ 7A 2.2.10 METABOLIC RADIOLABELLING OF ADHERENT CELLS 74 2.2.1 1 M ETABOLIC RADI OLABELLI NG OF LYM PHOBI-ASTS:.. ................75 2.2.12 ALKALI EXTRACTION OF GAGS: ...'..'....'.... ................76 2.2.13 GAG ASSAY: ................77 2.2.1 4 SEPHADEX GsO CHROMATOG RAPHY: ......77 2.2.1 5 ION-EXCHANG E CHROMATOGRAPHY: "..... ......78 2.2.16 HYALURONIDASE DIG ESTION ......79 2.2.17 NITROUS ACID CLEAVAGE OF HEPARAN SULPHATE:....' ......79 2.2.18 CHONDROITINASE ABC DIGESTION:....', 80 2.2.19 DESALTING OF CS/DS DISACCHARIDES 80 2.2.20 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) OF CS/DS DISACCHARIDES: .81 2.2.21GENE TRANSFECTION OF CELL LINES: ".'....'. .82 (CPC) ......84 2.2.22 CETYLPYR I DI N IUM CHLORI DE PRECI PITATION OF GAGS: 2.2.23 PROTEIN ASSAY: u 2.2.24 STATI STI CAL ANALYSES : ... "'....... ".... " 85 CHA R3.SULP HATION BY YSTIC FI BROSIS AND OL PANCREATIC CELLS 3.1 TNTRODUCTION.... .89 .90 3.2. 1 GENERAL CHARACTERISATION: .... 90 90 3.2. 1 .1 Anti-Q¡tokeratin Staining: ..... 3.2.1.2 Acidification of Lysosomes: .. ..91 3.2.1.3 Chloride TransPort: ..92 3.2.2 ANALYSIS OF GLYCOCONJUGATES ..98 IV 3.2.2.1 Metabolic Radiolabellin g, An ion-Exchange Chromatog raphy and Peak Assignment:......... ..........98 3.2.2.2 Analysis of Peak I on Sepharose CL4B ...106 3.2.2.3 ['uS]:['H] Ratios in Glycoconjugates Synthesised by CFPAC and PANC: ................106 3.2.2.4 ["S]:['H] Ratios in Chondroitin/Dermatan Sulphate Synthesised by CFPAC and P 109 3.2.2.5 Demonstration of Heparan Sulphate in Peak lll: ..... 118 3.3 DrSCUSSrON.......... 125 CHAPTER 4. SULPHATION OF GLYCOSAMINOGLYCANS BY CYSTIC FIBROSIS LYMPHOBLASTS 4.1 INTRODUCTION 135 4.2 EXPERIMENTAL AIMS ..............1 38 4.2.1 CELL LINES: ..............1 38 4.2.2 SPECIFIC METHODS ..............1 39 4.3 RESULTS............. .........140 4.3.1 CHARACTERISATION OF GLYCOCONJUGATE SYNTHESIS BY LYMPHOBLASTS: ............. 140 4.3.2 ['-S]:['H] RATIOS lN GAGS PRODUCED BY CF AND CONTROL LYMPHOBLASTS: ......... 141 4.3.3 INFLUENCE OF CELL CYCLE SYNCHRONISATION ON LYMPHOBLASTS: .......... 146 4.4 DrSCUSSrON......... CHAPTER 5 . SULPHATION OF GLYCOSAMINOGLYCANS BY THE CFTRGÊ) MOUSE 5.1 tNTRODUCT|ON... 159 5..I.1 EXPERIMENTAL DESIGN 162 5.2 RESULTS f63 5.2.1 T'S]SULPHATE INCORPORATION INTO MURINE GAGS: 163 5.2.2 T'S]SULPHATE INCORPORATION INTO MURINE HEPARAN SULPHATE: ................. 166 5.2.3 ALTERATIONS IN CS/DS:HS RATIOS IN ORGANS FROM NORMAL AND CFTR(-/-) MICE: 172 5.2.4 ANALYSIS OF CS/DS DISACCHARIDES BY HPLC:" """"""'172 5.2.5 SPECIFIC ACTIVITY OF T'S]SULPHATE IN THE 45 DISACCHARIDE OF CS/DS: """"""176 5.3 DrSCUSSlON......... 181 5.3.1 CONCLUDING REMARKS: ....... 189 R PA CELLS AND THE EF FECT OF EXPRESSI ON ON SULPHATION 6.1 TNTRODUCTION.... 6.2 RESULTS: 4........ 6.2.1 GENERATION OF THE RECOMBINANT CFTR-EXPRESSING CELL LINE 'TR20': """"""'198 6.2.2 [..S]:[.H] RATIOS OF GAGS lN LONG TERM LABELLING STUDIES:......198 6.3 DISCUSSION: A............... """""""205 6.3.1 CONCLUSIONS: 4..... .211 6.4 RESULTS: 8........ 6.4.1 SHORT TERM LABELLING: INFLUENCE OF CLo AND p-D- XYLOSIDE:212 6.4.1.1 Experimental Design: ..............212 6.4.1.2 Results:..... ..............214 6.5.1 CONCLUSIONS: 8..' 224 6.6 RESULTS: C........ """"'226 6.6.1 SULPHATE UPTAKE AND POOL SIZE IN PANCREATIC CELLS:"..''""..226 6.6.1.1 Experimental Design: """"""226 6.6.1'2 Results:'.... """""227 6.6.2 CYSTEINE AS A SOURCE OF SULPHATE IN PANCREATIC CELLS: "..'2U 6.6.2.1 Experimental Design ...234 6.6.2.2 Results: ...236 ...238 6.7.1 CONCLUSIONS: C 249 6.8 RESULTS: D ................250 6.8.1 TNFLUENCE OF ALTERED SULPHATE POOLS AND p-D-XYLOSIDE ON ["S]:['H] RATIOS lN CS/DS:.. 250 6.8.1.1 Experimental Design: 250 6.8.1.2 Results: .250 258 6.9.1 CONCLUSIONS: D.... 259 6.10 OVERVIEW: A MODEL FOR THE EFFECT OF CFTR ON SULPHATE UTILISATION 260 CHAPTER SEVEN . REFLECTION 7.1 SULPHATION IN CYSTIC FIBROSIS .271 7.2 PHILOSOPHICAL CONSIDERATIONS .277 REFERENCES 283 VI ABBREVIATIONS ABC ATP binding cassette APS adenosine-5'-phosPhosulPhate ATP adenosine-5'{riPhosPhate bp base pair cAMP cyclic adenosine-5'-monophosphate CBAVD congenital bilateral absence of the vas deferens CF cystic fibrosis CFTR cystic f ibrosis transmem brane cond uctance reg ulator CSi DS chondroitin sulphate/dermatan sulphate CV coetficient of variation EBV epstein bar virus ECM extracellular matrix ER endoplasmic reticulum FBS loetal bovine serum GAG glycosaminoglycan galNAc N acetylgalactosamine glcNAc N acetylglucosamine HPLC high performance liquid chromatography HS heparan sulphate mRNA messenger