Human Lysosomal Sulphate Transport Martin David LEWIS B.Sc. (Hons) Thesis Submitted For the Degree Of Doctor of PhilosoPhY rn The University of Adelaide (Faculty of Medicine) May 2001 Lysosomal Diseases Research Unit and Department of P aediatrics PathologY Faculty of Medicine Department of Chemical 'Women's Women's and Children's HosPital and Children's HosPital South Australia South Australia 11 Elliot, Sømuel and Millie Table of Gontents Abstract xl1 Declaration xiv Acknowledgments XV Abbreviations xvi List of Figures xxi List of Tables xxiii 1. INTRODUGTION. """""""" 1 1.1.1 1,1.2 Membrane proteins. """"""""2 1.1.3 Types oftransporters. """"""'4 5 t.t.4 Carrier transport mechanisms. 1.1.5 1.2 Sutphate metabolism. 8 1.2.1 Phosphoadenosinephosphosulphatesynthesis"" I 1 1.2.2 The roles of sulphate within the cell' ' "" " " 1.2.2.1 A structuralrole of sulphation. ..""""' """""""""""' 11 t2 1.2.2.2 Metabolic and regulatory roles of sulphate' """""" 1.2.3 Intracellular sulphate pools...'........ """"""12 12 1.2.3.1 The origins of intracellular sulphate pools"" 1.2.3.2 Metabolism of sulphate from cysteine """""""""""" 15 1.2.3.3 Regulation of sulphate pools 1.3 The definition and function of the lysosome' """""""""""'19 1.3.1 Structure of the lysosome. ....'......... """""'20 1.3.1.1 Lysosomal hydrolysis of glycosaminoglycans"""' """"""""""""'21 25 1.3.1.1.1 Sulphatases 1.3 .2 Lysosomal biogenesis. 1.3.2.1 Targeting of lysosomal luminal proterns' 27 1V t.3.2.2 Targeting of lysosomal membrane proteins. """""""28 28 1.3.2.3 Lysosomal membrane Proteins 30 1.3.3 Lysosomal transporters 32 1.3.3.1 Proton pump (H*-ATPase) 33 1.3.3.2 Lysosomal cystine üansport............. 34 1 .3 .3.3 Lysosomal sialic acid transport. '.... 1.3.3.4 Lysosomal cobalamin tansport. """""'35 1.3.3.5 Lysosomal sulphate transport..'.'.. """"'35 t.3.4 Consequences of lysosomal dysfunction. 40 1.4 Non-lysosomalanionexchangers. 40 1.4.1 Functions of the chloride / bicarbonate transporters"""""""""' 40 l.4.l.l The erytlrocyte anion transporter 1.4.1.2 Structure and stability of Band 3. 1.4.1.3 Transport properties of Band 3. ............""' """"""""42 1.4.1.4 Transport of sulphate by Band 3' ."""""""" """""""42 43 1.4.1.5 common properties of Band 3 and the lysosomal sulphate transporters 1.4.2 Mitochondrial anion transport' """""""""44 1.5 Mammalian sulphate specific transporters """""""""""""'45 45 1.5.1 Sodium-dependentsulphatetransporter' 48 I .5.2 The sodium independent sulphate anion transporter family 1.5.2.1 Distribution and gene location of SAT-1' """""""""49 50 1.5.2.1J SAT-1 and cr-L-iduronidase genes overlap 50 1.5.2.2 Diastrophic dysplasia sulphate transporter""" 51 t.5.2.3 A sulphate transporter 'Down Regulated in Adenoma' """""""""' 52 1.5.2.4 Pendrin, a thyroid-specific sulphate transporter s4 1.6 Othersulphate-specifictransporters....."""' ..54 1.7 A chronological perspective ofthis study"""""""""' V 2. MATERIALS AND GENERAL METHODS........... """"""' 55 2.1 Materials. 55 2.1.1 Biological materials (Tissues, cells and serum) 55 2.1.2 Tissue culture media and enzFe. 56 2.1.3 Radiochemicals................ """"""""""""" 56 2.r.4 Immunological materials, reagents and peptides' """""' 57 2.1.5 Electron microscopy and photographic materials 2.1.6 Chromatographic and other medta.' """""'57 ........................... 58 2.1.7 Chemicals. 61 2.1.8 Equipment............' 61 2.t.9 Miscellaneous materials.... """"""""""""' 62 2.2.1 Preparation and collection of placentae' .'.'.....'......'....""'62 2.2.2 Preparation of cytoplasmic organelles from human placentae........ 2.2.3 Protein quantification and enzyme assays' " 63 2.2.3.1 Protein quantification. .... '.. '.... '.,.. " " "" activity 2.2.3 .2 Determination of B-N-acetylhexosaminidase (EC 3.2.1.52) 2.2.3.3 Assaying acetyl-coenzyme A cr-glucosaminide N-acefyltransferase (EC 2.3.1.78) activity""'64 64 2.2.3.4 Determination of cytochrome-c oxidase (EC 1.9.3.1) activity.' 2,2.3.5Determinationofmonoamineoxidase(EC1.4.3.4)activity..'... 65 65 2.2.4 Preparation of proteins from organelles"""""""" 65 2.2.4.1 Isolation of organelle membranes and associated proteins. 65 2.2.4.2 Solubilisation of membrane proteins for SDS-PAGE 66 2.2.4.3 Extraction of phospholipid from membrane proteins 66 2.2.5 Chromatography used to fractionate proteins' 66 2.2.5.1 Preparation of protein samples for chromatographic fractionation. 67 2.2.5 .2 Preparation and use of con A-Sepharose to fractionate proterns 2.2.5.3 Preparation and use of Red Dye to fractionate proteins 67 2.2.6 Polyacrylamide gel electrophoresis. 68 V1 69 2.2.6.2 Two-dimensional gel electrophoresis' 70 2.2.7 Visualisation ofproteins resolvedby electrophoresis"""""""' 2.2.7.l Brilliantblue G-colloidal stain.'.... 70 2.2.7.2 Silver stain. """""""""""70 7l 2.2.8 Dryingpolyacrylamidegels.....".' 7l 2.2.9 Electro-blotting of samples frompolyacrylamide gels to membrane filters' 2.2.10 Immunological methods. """""""""""""'72 1'.' 2.2.10.1 Polyclonalantibodyproduction' 2.2.10.1.1 Immunisationregime 72 73 2.2.10.1.2 Preparation of polyclonal antibody from serum' IJ 2.2.10.1.3 Enzyme linked immuno-sorbant assay (ELISA) 74 2.2.10.2 Detection of protein on Western blots 2.2.10.2.1 General Western blot method. """'74 74 2.2.10.2.2 Modified Cetus@ Western blot method' 2.2.10.3 Visualisation of a secondary antibody"""' """"""75 75 2.2.11 Western blot analysis visualised by Enhanced Chemiluminescence 3. CHARACTERISATION AND PARTIAL PURIFIGATION OF THE LYSOSOMAL SULPHATE TRANSPORTER. ."...............76 76 3.1 Introduction. 3.2 Experimental Aims. 80 3.3 Methods..... 80 3.3.1 Lysosomal proton pump assay.. """"""""' 80 3.3.1 .1 Washing and buffering of intact cytoplasmic organelles' 80 3.3.1.2 Proton pump ( H*-Atlase) measurements 81 3 .3.2 Electron microscoPY. 3.3.2.1 Negative staining of liposomes. 81 3.3.2.2 Fixation and section preparation of placental tissues' """"""""""' 81 v11 82 3.3.2.3 Processingtissucforimmunohistochemistry 82 3.3.2.4 Immunogold labelling. """"""""""""" 83 3.3.3 Sulphate tansport. """""""" 84 3.3.3.1 Sulphate influx into cytoplasmic organelles by a sulphate concentration gradient. 3.3.3.2 Sulphate influx into and efflux from cytoplasmic organelles, by counter transport 84 and truns-stimulation with sulphate. by co-transport or c¡s-stimulation with a 3 .3 .3.3 Sulphate influx into cytoplasmic organelles pH gradient. 85 85 3.3.3.4 Sulphate influx into proteoliposomes with a pH gradient" 85 3.3.3.5 Stopping suþhate transport'..... """"""' 3.3.3.5.1 Amicon vacuum manifold. ....."""" """"""""""" 85 3.3.3.5.2 Sizeexclusion........"....'..' """"""""86 3.3.3.5.3 Anion exchange. """"' 86 86 3.3.4 Proteoliposome formation """"""""""""" 88 3.4.1 Isolation of lysosomes from human placenta""""" """" gradient. .."""""95 3.4.1.1 Density of placental cytoplasmic organelles separated in a Percoll@ densþ 91 3.4.2 Determination of lysosomal integrity by H*-ATPase activity 101 3.4.2.1 Assay conditions that effect proton purnping""" ""' 3.4.2.2 Determination of acidification rates for different cytoplasmic organelle fractions 105 separated in a Percoll@ gradient. 108 3.4.3 Morphology of placenta... """""""""""" 111 3.4.4 Sulphate transport in lysosomes and mitochondria""""""" """"""" 111 3.4.4.1 Development of sulphate transport assays' """"""" 3.4.4.2 Sulphate uptake into lysosomes and mitochondria' """""""""""112 tt6 3.4.4.3 Sulphate egress from lysosomes and mitochondria' 120 3.4.4.4 Co-transport and counter-transport in lysosomes and mitochondria' ....... '. '.............. " " " """ " 3.4.4.5 Trr,ns-stllr|ulation of sulphate transport with substrate analogues. .....'.""""""'124 3.4.4.6 Inhibition of sulphate transport with anion exchange inhibitors. 126 3.4.4.7 Differences and similarities between lysosomal and mitochondrial sulphate transporters. .... 128 vl11 t28 3.4.5 Reconstitution of lysosomal membrane proteins into phospholipid vesicles 3.4.5.1 Electron micrograph of proteohposomes. """"""""I29 t32 3.4.5.2 Development of reconstitution methodolo gy.'............ "' 133 3.4,6 Sulphate transport in proteoliposomes 138 3.4.7 Suþate transports differentþ in proteoliposomes than lysosomes' """"' 138 3.4,1.1 Sulphate interaction with phospholipid..'.......... """' 138 3.4.7.2 Thin layer chromatography ofphosphatidylcholine """"""""""" 140 3.5 General Discussion.. """""""""' 3.5.1 Sulphatetransport...... 4. IDENTIFICATION OF PROTE¡NS INVOLVED IN LYSOSOMAL SULPHATE TRANSPORT. ............. 143 143 4.1 Introduction......... 4.2 Experimental Aims........ 144 t44 4.3.1 Fluorography protein' 144 4.3.2 preparation of a polyclonal antibody against the human erythrocyte anion exchanger " 145 4.4 Results. 145 4.4,1 Characterisation of lysosomal membrane proteins by SDS-PAGE' """"""""' """ gradient' 4.4.1.1 Distribution and abundance of membrane proteins in a Percoll@ density ......".'."...145 149 4.4.1,2 Identification of a major protein isolated from lysosomal preparations 4.4.1,3 westem blot analysis of cholesterol desmolase in Percoll@ fractions....... ..,.".'.149 4.4.1.4 Affinity labelling of anion transporters.. """""""""152 155 4.4.2 Immunological search for the lysosomal anion exchanger 155 4.4.2.1 production of a polyclonal antibody against the anion exchanger Band 3 156 4.4.2.2 Inhibition of transport by antibody interaction. """' 156 4.4.2.3 Fractionation and preparation of lysosomal membrane proteins' """"""""""" 'Western 159 4.4.3 blot analysis of membrane fractions with an anti-Band 3 polyclonal antibody..'......'." 1)( 4.4.3.1 Identifrcation of Band 3 cross-reactive membrane proteins by SDS-PAGE and Western blot analysis. """"""""""""' 159 t67 4.4.3.2 Western blot anaþsis of lysosomal membrane proteins' 4.4.3.2.1 western blot analysis of lysosomal con A-sepharose eluate. r67 4.4.3.2.2 Investigation of western blot analysis to increase sensitivity t70 4.4.3.2.3 2-D Western blot analysis of Red Dye maûix flow through proteins.