"IN the NAME of ALLAH, MOST GRACIOUS, MOST MERCIFUL” Proquest Number: 10609317

"IN THE NAME OF ALLAH, MOST GRACIOUS, MOST MERCIFUL” ProQuest Number: 10609317

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ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 I dedicate this thesis to my family, especially my parents Faiz and Maryam and to my sister Lubna and my brother Imran, for all their love and support. BIOCHEMICAL AND IMMUNOLOGICAL STUDIES ON GAP JUNCTIONS

Salman Rahman

This thesis is submitted in partial fulfilment

of the requirements of the

University of London

for the degree of

Doctor of Philosophy

Laboratory of Protein Structure June 1991 National Institute for Medical Research Mill Hill, London NW7 1AA SUMMARY

A panel of polyclonal anti-peptide antibodies were generated towards selected sequences of the major rat liver gap junction polypeptide, connexin 32. The amino acid sequences were derived from different putative intra- and extracellular domains based on a low resolution, two- dimensional topographical model in which connexin 32 traverses the junctional membrane four times. Six anti peptide antibodies that recognized the parent polypeptide were generated from the use of eleven peptide immunogens. Within this panel of antibodies were reagents capable of recognizing both denatured and native forms of connexin 32.

In topographical studies of connexin 32 these antibodies directly demonstrated the cytoplasmic disposition of the amino and carboxyl termini of connexin 32, by immunolocalization to 'intact' and 'split' gap junctions. Similarly, the cytoplasmic disposition of a protease hypersensitive loop peptide, predicted to connect the second and third transmembrane domains, was also demonstrated. By characterizing the peptide products of connexin 32 generated by the controlled proteolysis of gap junctions, these site- directed reagents further supported the immunolocalization studies, and in addition, identified a disulphide bridge(s) between the two predicted extracellular domains of connexin 32. Taken together these studies endorse and extend previous topographical models of the arrangement of connexin 32 in the junctional membrane.

In separate studies, the subcellular distribution of connexin 32 in liver was analysed. These investigations implied that after synthesis on ribosomes, connexin 32 is transferred to the Golgi complex, where it accumulates without detectable modification by glycosylation, en route to the cell surface. In addition, biochemical and immunological approaches have identified several putative novel connexins in liver and brain tissues. ACKNOWLEDGEMENTS

I would firstly like to thank those people who have made a technical contribution to the work described in this thesis. For experiments performed at University College

London, I thank Dr. Colin Green and Dr. Robert Gourdie for their assistance in immunofluorescence experiments, and

Professor Anne Warner for performing the antibody perturbation assays. I am grateful to Ralph Foulkes (N.I.M.R.) for the synthesizing and assessing the quality of the synthetic peptides, and to Alan Harris (N.I.M.R.) for carrying out the microsequencing. Finally, I am very grateful to Elizabeth

Hirst (N.I.M.R.) for processing all samples for electron microscopy and to Rosa Alique (N.I.M.R.) for help with immunofluorescence experiments.

I wish to thank various members of the laboratory for their help and support, in particular Graeme Carlile, Ge

Kalapothakis, and Peter Tabona. I am also grateful to Dr.

Carlos Enrich (University of Barcelona) and Stamatis

Stamatoglou (N.I.M.R.) for kindly donating electron micrographs of liver tissue. Lastly, I'm very grateful to

Dr. Howard Evans (N.I.M.R.) for giving me the opportunity to work in his laboratory and for his conscientious supervision throughout my research. I thank the M.R.C. for the studentship and Dr. Bryan Winchester (Institute of Child

Health) for supervision. ABBREVIATIONS

o A Angstroms approx. approximately BSA bovine serum albumin degrees Celsius °c ^ . Ca2 + free calcium CNS central nervous system Cx Connexin cDNA complimentary deoxyribonucleic acid CAMP cyclic adenosine monophosphate Da Daltons DMSO dimethylsulphoxide E 280 extinction at 280nm E max " " maxima EDTA ethylenediaminetetraacetic acid FITC fluorescein isothiocyanate FMOC 9-fluorenylmethoxycarbonyl g acceleration due to gravity KHz Kilohertz KLH keyhole limpet haemocyanin MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester mRNA messenger ribonucleic acid MOPS (3 - [ N-morpholino ] propanesulfonic acid) PBS phosphate buffered saline 1OmM NaPhos (pH 7.4), 150 mM NaCl. PMSF phenylmethylsulphonylfluoride PTH phenylthiohydantoin r .p.m. revolutions per minute TFA trifluoroacetic acid TPA 12-0-tetradecanoyl-phorbol-13-acetate Tris [2-amino-2-(hydroxymethyl) propane-1,3-diol, (tris)] v/V volume by volume w / V weight by volume w/w weight by weight TABLE OF CONTENTS

Title Page

Summary 1 Acknowledgements ii Abbreviations iii

Chapter 1 : General Introduction

1 .1 The Plasma Membrane; a functional perspective 1 1 .2 Intercellular Junctions 8 1.2.1 The Desmosome 10 1.2.2 Adherens Junctions 14 1.2.3 Tight Junctions 16 1 .3 Gap Junctions And Direct Intercellular Communication 19 1.3.1 The Re-Evaluation Of Cell Theory 19 1.3.2 Morphology Of Gap Junctions 22 1.3.3 Biochemical Characterisation Of Gap Junctions 26 1.3.3a Components Of Liver (Epithelial) Gap Junctions 27 1 .3.3b Components Of Cardiac And Smooth Muscle Gap Junctions 29 1 .3.3c Components Of Lens Gap Junctions 30 1.3.4 The Connexin Family Of Gap Junction Channel-Forming Polypeptides 32 1 .3.4a Topography Of Connexin Polypeptides 34 1.3.5 Lipid Composition Of Gap Junctions 39 1.3.6 Functional Role Of Gap Junctions 42 1.3.7 Permeability Of Gap Junctions 45 1.3.8 Gating Of Gap Junctions 45 1 .4 Aims Of The Project 48

Chapter 2 : Materials and Methods

2.1 Generation Of Anti-Peptide Antisera 50 2.1.1 Synthesis Of Oligopeptides 50 2.1.2 Conjugation Of Peptides To Keyhole Limpet Heamocyanin 50 2.1.2a Conjugation Using Gluteraldehyde 52 2.1.2b Conjugation Using m-Maleimidobenzoyl N-Hydroxysuccinimide Ester 52 2.1.3 Immunization Of Rabbits 53 2.1.4 Affinity Purification Of Antisera 54 2.1.4a Preparation Of Immunosorbents; CNBr- Activated Sepharose CL-4B 54 3.1.1 The Development Of Anti-Peptide Antibody Technology 80 3.1.2 Selection Of Peptides For Immunization 82 3.2 Results 88 3.2.1 Production Of Anti-Peptide Antisera 88 3.2.2 Affinity Purification Of Anti-Peptide Antisera 89 3.2.3 Characterisation Of Antisera 89 3.2.4 Immunocytochemistry 96 3.2.5 Inhibition Of Intercellular Communication 99 3.3 Discussion 1 02

Chapter 4 : Topography Of Connexin 32 In The Gap Junction 109

4.1 Introduction 109 4.2 Results 113 4.2.1 Immunological Characterization Of Connexin 32 Proteolytic Products 113 4.2.1a Intact (Double Membrane) Gap Junctions 113 4.2.1b 'Split' (Single Membrane) Gap Junctions 1 20 4.2.2 Anti-Peptide Antibody Immunolocalisation To Intact And Split Gap Junctions 121 4.2.3 Evidence For A Disulphide Bridge(s) Connecting The Extracellular Loops Of Connexin 32 126 4.3 Discussion 129 4.3.1 Evidence For The Cytoplasmic Disposition Of The Amino Terminus 132 4.3.2 Evidence For The Cytoplasmic Disposition Of The Carboxyl Terminus Of Connexin 32 134 4.3.3 Evidence That The Peptide Connecting The M2 And M3 Domains Of Connexin 32 Is Intracellular 136 4.3.4 Evidence For Two Extracellular Loop Peptides Connecting The Four Transmembrane Domains Of Connexin 32 1 40 4.3.5 A Model For Connexin 32 And The Organisation Of The Connexon 145 4.3.6 Channel Construction In The Connexon And Its Gating 148 4.3.7 Special Considerations For The Modelling Of Gap Junction Channels 152

Chapter 5 : Liver Gap Junction Polypeptides and Homologous Antigens In Brain 156

5.1 Introduction 156 5.2 Results 5.2.1 Preparation Of Rat Liver And Guinea Pig Liver Gap Junctions 159 5.2.2 Immunological Comparison of Rat And Guinea Pig Gap Junction Components 164 5.2.3 Biochemical Properties Of Connexins 166 5.2.3a Dimerization 166 5.2.3b Anomalous Electrophoretic Mobility 170 5 . 2 . 3c Differential Rates Of Electrophoretic Transfer Of Connexin 32 and 26 172 5.2.4 Microsequence Analysis Of Guinea Pig Liver Connexins 172 5.2.5 Immunological Analysis Of Liver And Brain Homogenates 173 5.3 Discussion 177 5.3.1 Biochemical Properties Of Liver Connexins 179 5.3.2 Comparison Of Rat And Guinea Pig Liver Connexins 5.3.3 Identification Of Novel Connexins 182

Chapter 6 : The Subcellular Distribution Of Connexin 32 In Liver 185

6.1 Introduction 185 6.2 Results 187 6.3 Discussion 192

Chapter 7 : Concluding Remarks 199

7.1 Gap Junction Specific Anti-Peptide Antibodies 200 7.2 Topography Of Connexin 32 202

References 211 LIST OF FIGURES

Figure Page

1 .1 Protein To Lipid Ratios For Various Cellular Membranes 2 1 .2 Categories Of Receptor Mediated Signal Transduction 5 1 .3 Morphological Architecture Of Intercellular Junctions 12 1 .4 Cell-Cell Channel Concept 21 1 .5 Electron Micrograph And Models Of The Gap Junction 23 1 .6 Freeze Fracture Replica And Negative Stain Appearance Of The Gap Junction 25 1 .7 Topographical Model Of Connexin 32 Proposed By Zimmer et al (1987) 36 2.1 Peptide Synthesis 51 2.2 Peptide-KLH Conjugation Via MBS 51 2.3 Preparation Of Subcellular Fractions From Rat Liver 74 3.1 Hydropathicity And Flexibility Profiles For Connexin 32 85 3.2 Topographical Localization Of Synthetic Peptides 87 3.3 Affinity Purification Of DES 1 Antibodies 92 3.4 Interaction Of Anti-Peptide Antisera With SDS-Denatured Connexin 32 94 3.5 Interaction Of Anti-Peptide Antisera With Free Peptides 95 3.6 Indirect Immunofluorescent Histochemical Localization To Frozen Liver Sections 97 3.7 Indirect Immunoperoxidase Localization In Wax-Embedded, Paraformaldehyde Fixed Liver 98 3.8 Indirect Immunofluorescent Localization In Wax-Embedded, Paraformaldehyde Fixed Liver 100 3.9 Immunogold Localization In Ultrathin Liver Sections 101 4.1 Oligomeric Structure Of Ion Channels 11 1 4.2 Intact And Split Gap Junctions 114 4.3 Endo1 Lys C And Chymotrypsin Digestion Of Intact Gap Junctions 115 4.4 Proteinase K And Trypsin Disgestion Of Intact Gap Junctions 116 4.5 Further Tryptic Digest Of Gap Junctions And The Evidence For An Intramolecular Disulphide Bridge 117 4.6 Diagrammatic Peptide Maps For Connexin 32 119 4.7 Immunolocalization Of DES 1 Antibodies To Intact And Split Gap Junctions 121 4.8 Immunolocalization Of Gap 9 Antibodies To Intact And Split Gap Junctions 1 22 4.9 Immunolocalization Of Gap 10 Antibodies To Intact And Split Gap Junctions 125 4.10 Immunolocalization Of Gap 7-M Antibodies To Intact And Split Gap Junctions 127 4.11 Intact Gap Junctions Labelled With Pre- Immune Serum 128 4.12 Identification Of An Interloop Disulphide Bridge 130 4.13 Mapping Of Proteolytic Cleavage And Antibody Binding Sites In Connexin 32 1 37 4.14 Favoured Hypothetical Organisation Of Disulphide Bridges 1 42 4.15 Flanking Sequences Of Putative Half- Cystines 144 4.16 Structure Of The Gap Junction 1 47 4.17 Gating Mechanism For Ion Channels 150 5.1 Preparation Of Liver Gap Junctions 1 61 5.2 Morphology Of Isolated Rat Liver Gap Junctions 1 62 5.3 Morphological And Biochemical Analysis Of Guinea Pig Liver Gap Junction 163 5.4 Immunological Comparison Of Rat And Guinea Pig Liver Gap Junction Connexins 165 5.5 Dimerization Of Connexin 32 1 67 5.6 Microsequence Analysis Of Electroeluted 47KDa Protein 169 5.7 Anomalous Migration Of Connexin 32 During SDS-PAGE 171 5.8 Purification Of Guinea Pig Liver Connexin 32 Tryptic Peptides By HPLC 1 74 5.9 Western Blot Analysis Of Tissue Homogenetes 176 5.10 Western Blot Analysis Of Rat Embryonic Liver And Brain Homogenates 1 78 6.1 Morphology Of Hepatocyte Plasma Membrane 186 6.2 Western Blotting Of Liver Subcellular Fractions 188 6.3 Subcellular Distribution Of Connexin 32 In Liver 189 6.4 Treatment Of Subcellular Fractions With Endoglycosidase F 191 6.5 Connexin 32 Degradation Products In Lysosomes 193 6.6 Trafficking Routes Of Connexin 32 In Liver 196 7.1 Membrane Topographies Of Various Channel Forming Polypeptides 204 7.2 Functional Topography Of Connexin 32 208 LIST OF TABLES

Table Page

1 .1 Cell-Cell Adhesion Molecules 9 1 .2 Molecular Components Of Intercellular Junctions 1 5 1 .3 Gap Junction Genes And Size Of Products 33 1 .4 Distribution Of Connexins In Various Tissues 38 1 . 5 Cholesterol And Phospholipid Contents And Ratios Of Gap Junction And Plasma Membrane Preparations 41 3.1 Hydropathy And Flexibility Scales 84 3.2 Properties Of Anti-Peptide Antisera To Connexin 32 90 3.3 Affinity Purification Of Anti-Peptide Antisera 91 4.1 Amino Acid Sequences Of Putative Pore-Lining A-Helices 1 49 4.2 Amino Acid Sequences Of Putative Connexin Extracellular Domains 1 54 5.1 Recovery Of Gap Junctions From Rat And Guinea Pig Livers 1 60 CHAPTER 1

GENERAL INTRODUCTION

1.1 The plasma membrane; a functional perspective

Mammalian cells contain a vast network of membrane systems which regulate and control cellular activities. The surface, or plasma membrane, is a highly specialised, differentiated and dynamic organelle which acts as an interface between the cell and its environment. The cell interacts with its environment through the plasma membrane, which in metazoans, may involve specialized regions developed for cell-cell interactions.

In common with intracellular membrane systems, the most basic function of the plasma membrane is compartmentation. The lipid component of membranes provides the essential insulating property permitting the generation and maintenance of specific compartmentalized environments.

Membranes are permeable to a restricted range of small, uncharged molecules including water (which is an essential component of all membranes), oxygen, carbon dioxide, glycerol and urea. The creation and maintenance of specific enclosed environments is attributable to the vast array of catalytic functions performed by membrane-associated proteins. Indeed the most catalytically reactive membranes, such as the mitochondrial inner membrane, contain a characteristically high protein to lipid ratio. (Fig. 1.1). The plasma membrane maintains a controlled intracellular environment by

-1- Protein to Lipid Ratios for Various Cellular Membranes

Myelin (P.M.)

Mouse liver P.M.

Hum. Erythrocyte P.M

Amoeba P.M.

HeLa Cell P.M. -

Bov. Ret. Rod P.M.

Mlt. Outer Mem.

Sarcoplas. Reticulum -

Mlt. Inner Mem. -

Liver Gap Junctions -

2 3 Arbitary Units

Fig. 1.1

-2- regulating the intracellular osmolarity, electric potential, pH, temperature and the concentration of important regulatory molecules such as cAMP and calcium. Membrane-bound enzymes actively transport specific inorganic ions out of the cell against concentration gradients. For example, the (Na+/K+ )-

ATPase uses the free energy of ATP hydrolysis to pump out Na+ + 24- in exchange for K . Similarly Ca -ATPases maintain a sub- 2+ micromolar intracellular Ca concentration, amidst a millimolar physiological environment.

A variety of communication mechanisms have developed in metazoans which underly a number of physiological phenomena. For example, the regulation of tissue functions, the control of embryonic development, and the operation of nervous and immune systems depends upon various pathways of cell-cell communication. One of these pathways is the direct intercellular exchange of small molecules through a plasma membrane specialization called the 'nexus' or gap junction.

Other mechanisms of communication also occur between cells that are somatically juxtaposed (but not in contact, such as occurs at the synaptic and neuromuscular junctions) and between cells that are far apart. 'Long range' communication involves the secretion of hormones or growth/differentiating factors which migrate to target cells via circulatory networks. The secreted ligand participates in a highly specific interaction with a receptor protein on the surface of the recipient cell, which triggers a cascade of intracellular events leading to a physiological response.

Receptor proteins are an ever growing class of cell surface-

-3- associated proteins which are often oligomeric and glycosylated. Among the many functions of receptors include the internalization in liver of the iron-transferrin complex via the transferrin receptor, and the transcellular migration of substances from blood to body cavities, illustrated by transfer of polymeric IgA bound to its receptor-secretory component to the intestinal lumen. Furthermore, many receptors modulate the permeability of the plasma membrane by opening and closing ion channels. The transduction of external signals into the cell may often require the involvement of intermediary G-proteins and the activation of membrane bound enzymes which modulate the intracellular levels of second messengers (Fig. 1.2).

Receptor-ligand interactions are often accompanied by an ensuing endocytotic event, representing one of the energy consuming processes responsible for the turnover of the plasma membrane. This selective plasma membrane internalization occurs at specialized regions termed 'coated pits'. The receptor ligand complexes are thought to concentrate, by lateral movements in the plane of the membrane, at the site destined to be endocytosed. The cytoplasmic surface of this region of the plasma membrane becomes laiden with an oligomeric protein called clathrin, which is constructed from a heavy (180 KDa) and two light

(35-40 KDa) polypeptide chains, and a number of assembly proteins. The characteristic morphology of the coated pit

(or 'clathrin basket') and their subsequent isolation have elucidated the 'filtering' properties of this plasma membrane

-4- Fig.1.2

Categories of receptor mediated signal transduction across the plasma membrane and the ensuing cascade of intracellular events. (Adapted from Evans and Graham, 1989). External cAMP modulating Ca2+-m obilizing Growth stim ulus ligands ligands factors -o- £ Receptor Receptor Receptor Receptor

Transduction GTP- G-protein GTP- G-protcin m echanism s ♦ Enzyme Adenylate Phospholipasei Protein m odulation cyclase C phosphorylation

ATP Diacyl Inositol Ca2+ M essage cAMP glycerol phosphates release generation * * Protein Protein Enzyme kinase A kinase C activation

Biological Protein Protein response phosphorylation phosphorylation

- 5 - specialization. For example, the low density lipoprotein receptors are concentrated approx. 100-fold at the coated pit compared to surrounding plasma membrane, whereas other membrane proteins such as adenylate cyclase are largely excluded (Evans and Graham, 1989). After internalization, the clathrin-associated vesicle becomes associated with the endocytic compartment, into which the receptor ligand complexes are transferred, following the disassembly of the clathrin structure. Since the surface area of the plasma membrane remains relatively constant, the internalized membranes must be continually recycled. Regions of the plasma membrane are particularly active in secretion,

involving fusion with intracellular vesicles. For example, the blood facing region of the basolateral plasma membrane domain of hepatocytes actively secretes serum proteins. It

is not surprising that cells in general are estimated to regenerate the equivalent of their entire surface area every

3-4 hours.

A further subclass of cell surface receptors are

those which promote cell-cell and cel1-substratum

interactions. In addition to targeting cell adhesion to

specific extracellular matrix components and ligands on

adjacent cells, these receptors influence many catholic

processes including cellular growth, differentiation,

junction formation and polarity. Several families of

adhesion receptors have now been identified and include: a)

the integrins; heterodimeric integral membrane glycoproteins

which promote both cell-substratum and cell-cell adhesion; b)

-6- the cell-cell adhesion molecules (CAMs); these belong to the immunoglobulin superfamily, and are particularly important during embryonic development, wound healing and the inflammatory response; c) the cadherins; these are developmentally regulated, calcium dependent homophilic cell cell adhesion proteins; d) the LEC-CAMs; these are cell adhesion pseudolectins that mediate white blood cell/endothelial cell adhesion; and e) homing receptors that target lymphocytes to specific lymphoid tissue. Cell CAMs behave both as receptors and ligands (i.e. demonstrating homophilic binding) and their programmed expression during the development of the embryo is thought to provide positional information for establishing cell-cell

interactions during histiogenesis (Edelman, 1988). The cell

CAMs have been subdivided into two categories based upon their sequence similarity and their functional requirement for extracellular calcium (Takeichi, 1988; Edelman, 1988 and

Rustishauser and Jessel, 1988). Calcium-independent CAMs

including N-CAM, Ng-CAM, LI (Moos et al., 1988) and

Fasciclin II (Harrelson and Goodman, 1988) are all members of the immunoglobulin superfamily and are found in neural tissues. CAMs with a functional requirement for extracellular calcium (the cadherins), constitute a family of

structurally similar molecules of 723-748 amino acids in

length (Takeichi, 1988). The overall level of sequence

similarity between cadherins from different tissues and

species is between 50 and 60%. Based on genetic and

immunological criteria, the cadherins have been subdivided

-7- into three subclasses (Takeichi, 1988): a) E-cadherin, found in adult epithelia and synonymous to uvomorulin, Cell-CAM

120/80, Arc-1 or L-CAM; b) N-cadherins, found in adult neural tissues and muscle, also termed A-CAM or N-Cal-CAM; and c)

P-cadherins, found primarily in placental and epithelial tissues, but are also expressed transiently in other tissues during embryonic development. A list of characterized cell adhesion molecules is given in table 1.1.

Although cell-cell adhesion is underlied by the cell adhesion molecules described above, the strength of adhesion between cells in tissues is largely dependent upon the presence of intercellular junctions. Indeed, a similar adhesive mechanism appears to underly cellular adhesion derived from cell adhesion molecules and that resulting from intercellular junctions, as shall be discussed below.

1.2 Intercellular Junctions

Since their categorisation by Farquar and Palade in

1963 the components of the epithelial junctional complex have been studied extensively, especially by morphologists. It is only in recent years however, that the identification and characterization of the molecular constituents of these intercellular junctions has started to emerge. Whereas all

junctions contribute to cell-cell adhesion, different

junctions perform specific biological functions. Robertson

(1963) first identified the gap junction, which was later resolved in the junctional membrane complex by Revel and

Karnovsky (1967). Its structure asserted its candidacy as

-8- Table 1.1 Cell-Cell Adhesion Molecules (adapted from Albelda & Buck, 1990)

Receptor Fam ily kOa Distribution Ligand Function

> L -P A M -1 In tc g rin 160/130 Lymphocytes Lymphocytc-EC adhesion a 4 in /0 p

L-PAM-2/VLA-4 Intcgrin 160/130 Leukocytes, V C A M - 1 Lymphocytc-EC adhesion, ai/01 m elanom a cells I N C A M - 1 10 Tumor-EC adhesion

C D 1 I/C D 1 8 In te g rin Hetcrodimcr Cell-cell adhesion ( L E U -C A M s ) LFA-l(CDlla) 177/95 All leukocytes IC A M -1 Leukocytc-leukocytc adhesion. Icukocyic-EC adhesion

Cell-cell adhesion, MAC-l(MO-l. t 165/95 Neutrophils, monocytes, iC3b, Fibrinogen CR3. CDllb) some lymphocytes Factor X. ICAM-1. phagocytosis, LPS complement binding

gp 150/95 aJPi 150/95 Granulocytes. iC3b Complement binding (CR4, CDllc) monocytes

I-C A M -1 IgG supcrfamily 76-1M M an y cells: induced LFA-1 Leukocyte adhesion by inflamation

CD2 (LFA-2, O KTU) IgG supcrfamily i0 A ll T Cells LFA -3 T Cell adhesion

L FA -3 IgG supcrfamily 50-77 Widespread C D 2 T Cell adhesion ? EndoCAM. PECAM-I IgC supcrfamily 130 EC, platelets, Initiation of EC-EC adhesion, platclci-monocyic-EC adhesion (C D 3 1 ) fam ily leukocytes

V C A M -1 IgG supcrfamily 90 Activated EC V L A -4 (aji3.) Adhesion of WDCs to EC activated by 11 1 or TN F

Ca1*-Independent IgG supcrfamily DcvclopmcntaJly regulated CAM* cell-cell adhesion N - C A M ( 0 2 , 180,140,120 Brain, muscle, heart, Homophilic binding B S P -2) kidney Ng-CAM (LJ. 200 N e u ral and glial cells Homophilic binding NILE) (ncural-ncuraj) Hetcrophilic binding (neural-glial)

Ca**-dcpcndcnt C a d h c rin Homophilic binding Developmental^ regulated C A M s ccll-ccll adhesion N -C a d h c rin 135 Brain, muscle, lens (A-CAM, N - C a l- C A M ) E -C a d h c rin 120-125 Ep ith eliu m (L-CAM , Arc-1, Uvomorulin, C e ll C A M 1 2 0 /8 0 ) P -C a d h c rin 130 Placenta, epithelium, mcsoihclium

H-CAM (CD44. C a rtilag e 90 Lymphocytes Endothelial cell Homing RTR- lymphocytes to HE Hermes antigen, lin k muscosal addrcssin ccll-substratum adhesion R T R E C M R I I I ) protein (MECA 367 Ag)

gp90Mfx-i« Lcc -C A M 90 Lymphocytes HEV carbohydrate? Homing RTR-lymphocytc to HEV (m urine) of peripheral LNs Leu 8 (hum an)

gpIM’^'-1' Lcc-CAM 100 Neutrophils Endothelial cell, Initial adhesion of neutrophil to carbohydrate? EC adjacent to inflammation

ELAM-J Lcc-CAM 115 Activated EC ■> Adhesion of WISCi to EC activated by LPS. IL 1. or TNF

C M P 1 4 0 L cc-C A M 140 Platelets, neutrophils, Adhesion of activated platelets to (PADCEM) monocytes, EC monocytes and neutrophils Adhesion of WBCs to activated EC

CD36 (CPIV) i 88 EC , platelets, Thrombospondin Plaiclcl-monocyic adhesion 'monocytes

-9 - the intercellular junction responsible for the observed

electrical and metabolic continuity in tissues. As the morphology of other junctions were elucidated, specific

biological functions were assigned from the properties

predicted by their structure. The structure properties and

biological functions of these junctions are now reviewed.

1.2.1 The Desmosome

Desmosomes (maculae adherentes) are specialized

intercellular adhesion junctions forming focal contacts

between the plasma membranes of adjoining epithelial cells,

analogous to rivets or spot welds. Although first reported

by Porter (1954), their biological effect was observed much

earlier. In the 1920's a series of elegant experiments by

Chambers, employing the techniques of microdissection to pull

cells apart, demonstrated that in epithelial tissue the cells

could not be separated without destruction. Consistent with

observations made at the resolution of the light microscope,

it was concluded that these cells in tissues were in fact a

syncytium.

Studies employing electron microscopy have shown that

epithelial cells are autonomous entities (even at

intercellular junctions), surrounded by intact and discrete

lipid bilayers of 75 to 90A width. In ultrathin sections,

desmosomes appear as macular, electron dense structures

between plasma membranes of adjacent cells, separated by

approx. 30nm wide extracellular space. Filamentous material

filling this extracellular space is bisected by a dense

-10- region termed the central stratum. On the cytoplasmic aspect of the junctional membrane are disc-shaped plaques to which

o are attached intermediate filaments (tonofilaments? 100A diameter) originating from within the cytoplasm. Thinner filaments arising from within the plaques traverse the plasma membrane and connect the tonofilaments to the central stratum, apparently providing direct mechanical coupling between the cytoskeletal networks of adjoining cells (Fig.

1.3). In conjunction with evidence from freeze fracture studies, this model provides a basis for a continuous structural network spreading throughout the entire epithelium.

The biochemical characterization of the desmosome proceeded the development of protocols allowing the preparation of desmosome fractions from mainly bovine tongue and snout epithelia. The isolation strategy exploits the detergent-resistant properties of the desmosomal membranes relative to non-junctional membranes (Skerrow and Matoltsy,

1974? Drochmans et al., 1978? and Gorbsky and Steinberg,

1981). A list of desmosome-associated protieins is given in table 1.2. Desmosomal proteins identified thus far have been categorized into two groups, glycosylated and non glycosylated. The former, have been subdivided into two classes, desmogleins (dgl) and desmocollins (dg2 and dg3) based on immunological criteria (Cohen et al., 1983) and limited sequence comparison (Kapprell et al., 1985). These glycoproteins have been localized to the desmosomal core and are thought to promote the adhesive properties characteristic

-11- Fig.1.3 Morphological architecture of the intercellular-

iunctions.

(TOP) Model of a tight junction. The schematic diagram highlights the gross structure of the junction illustrating the adhesion between adjacent membranes, promoted by the sealing strands. Each sealing strand is composed of two rows of closely spaced particles (one row contributed by each cell), which adhere tightly and arranged so as to form a network. Associated with the cytoplasmic surfaces of the junctional membrane are microfilaments that connect to the sealing strands and thereby structurally reinforce the junction.

(BOTTOM) Gross structure of a spot desmosome. Tonofilaments

o (100A in diameter), form a tensile network that extends into the cell interior from plaque structures at the membrane surface. These plaques are connected through transmembrane linkers (constructed presumably of core proteins) which extend across the extracellular space. The junction therefore serves to couple the tonofilaments networks of adjacent cells, allowing the dissipation of shearing stresses throughout the tissue. MICROVILLUS

TlGH T-JUNCTION SEALING ELEMENT

NETW ORK OF CYTOPLASMIC FILAMENTS SEALING STRAND

CORE MICROFILAMENTS

INTERCELLULAR SPACE

TONOFILAMENTS CENTRAL STRATUM

INTER CELLULAR SPACE

TRANS- MEMBRANE LINKER

CYTOPLASMIC PLAQUE

PLASMA MEMBRANE -12- of the desmosome (Steinberg et al., 1987). The non glycosylated components, localized to the desmosomal plaque, include two major components desmoplakins I and II which are highly related, but distinct polypeptides of Mr 240 and 210

KDa respectively (Jones and Goldman^1985; Miller et al.,

1987). Further plaque components include desmocalmin a 240

KDa polypeptide unrelated to the despmoplakins which displays calmodulin and keratin filament binding properties (Tsukita and Tsukita, 1985), and plakoglobin which is a component of other symmetrical adhesive junctions (Cowin et al., 1986). A

further plaque component of Mr 680 KDa termed desmoyokin has been localized to the periphery of the plaque structure

(Heida et al., 1989), in contrast to the desmoplakins which

appear to be located towards its centre.

A 125 KDa polypeptide related to the epithelial

cadherin (E-cadherin) has been shown to be a minor component

of desmosomes and is distinct from the desmocollins (dg2 and

dg3) (Jones, 1988). Amino-terminal sequence analysis of both

dgl and 2 has revealed partial sequence similarity with the

amino termini of members of the cadherin family of calcium-

dependent adhesive polypeptides (Holton et al., 1990).

Furthermore, the amino termini of the desmocollins are

disposed in the extracellular space (Holton et al., 1990).

Analysis of the cDNA deduced primary sequence corresponding

to desmoglein (dgl) coupled with microsequence analysis of

peptides derived from proteolytic treatment, has revealed

that this integral membrane glycoprotein traverses the lipid

bilayer once, with its amino terminus also located in the

-13- extracellular space. These results further show that dgl is related to the members of the cadherin family, especially within the extracellular portion of the molecule. However, the cytoplasmic domain of dgl is approx. 220 amino acid residues longer. (Koch et al., 1990). It appears therefore that the core components of the desmosome belong to a growing cadherin superfamily, with members that probably share a similar calcium-dependent adhesive mechanism.

1.2.2 Adherens Junctions

Intercellular adherens junctions (fasciae adherentes) are desmosome-like structures found in a variety of cell- types in addition to epithelial cells. They are structurally similar to the zonulae adherentes (belt desmosome) of the epithelial junctional complex, but are less continuous

(Farquhar and Palade, 1963? Hull and Staehelin, 1979;

Geiger et a l ., 1983). These junctions (see table 1.2) contain a desmosomal plaque-associated protein of molecular mass 83 KDa called plakoglobin, but differ from desmosomes by their association with actin microfilaments in place of keratin intermediate filaments. The major plaque protein of the adherens junctions is vinculin (Geiger et al., 1981) and the integral or 'core' components identified thus far also appear to be cadherin or cadherin-related proteins.

Adherens junctions are found within the cardiac intercalated disc and also in the fibres and anterior epithelium of the lens (Geiger et al.,1985; Lo, 1988). In ultrathin sections of vertebrate lens, they possess a

-14- Table 1.2 Molecular Components of Intercellular Junctions

Tight junctiona

Peripheral Components

ZO-1 Cingulin Actin*

Intercellular adhesion junctions3

Peripheral Components Integral Membrane Components

Actin* ACAM* Myosin E-cadhenn Tropomyosin alpha-actin Vinculin Plakoglobin Polypeptides of 400, 240,235,82 & 70 KDa

Desmosome^

Peripheral ('plague') Components Integral ('core') Components

DPI (240 KDa) DG1 (150 KDa) DPI I (210 KDa ) DG2 (110 KDa ) DPIV( 75 KDa ) DG3 (100 KDa) Desmocalmin (240 KDa) DG4 ( 22 KDa ) Plakoglobin (DPI II, 83 KDa) 140 KDa Desmoyokin (680 KDa) 125 KDa (E-cadherin)

GAP junction

Only integral components have been identified thus far which are the connexins and MP70 (see text).

See Stevenson and Paul (1989) See Schwartz et al (1990) Localization not exclusive

-15- classical morphology with an intercellular space of approx.

15-20nm containing electron dense material and visible cross bridges (Lo, 1988). Found between lens fiber cells, lens epithelial cells and between fiber and epithelial cells, adherens junctions are thought to maintain the spherical shape of the lens and stabilize the structural integrity of lens cells during shape change.

1.2.3 Tight Junctions

Tight junctions are intercellular occlusion zones which selectively modulate the passage of molecules across the epithelial paracellular pathway. They appear in ultrathin sections as intercellular plasma membrane fusions which flank the apical surface of the cell. In reality, the plasma membranes of adjoining cells are so closely associated

(but are distinct) that the extracellular space disappears generating a trilaminate appearance indicative of membrane anastomosis. Tight junctions are prominent wherever cells act as barrier between lumens and serosae/connective tissue.

A well known example is the blood-brain barrier, which consists of the cells lining the cerebral blood vessels.

Unlike the cells of vascular endothelium elsewhere in the body, these endothelial cells possess tight junctions. The selective impedence of these tight junctions to molecules is manifested by the inability to control CNS infections with antibiotics although molecules such as ethanol, diacetylmorphine and lysergic acid permeate easily. This subtle selective property of tight junctions was demonstrated

-16- in LLC-PK1 cells, a renal carcinoma cell line that can differentiate between the molecular shapes of mannitol (molecular radii: 5.4 x 5.4 x 11A) and methyl glucoside (molecular radii: 5.6 x 8.0 x 9.0A) in the paracellular traffic (Mullin et al., 1986). The molecular basis underlying the mechanism this physiological filtration

is not understood.

The two-dimensional organization of tight junctional membranes was deduced from freeze fracture studies

(Stahaelin, 1968). In freeze fracture replicas, tight

junctional membranes are characterized by a network of ridges

(sealing strands) which appear to traverse the plasma membrane and associate in a mirror fashion with complimentary

ridges from the adjoining cell membrane (Fig. 1.3). These

ridges, thought to be composed of integral membrane proteins,

effectively 'seal7 the apposing membranes and possibly

provide the permeability barrier. Claude and Goodenough

(1973, 1978) suggested that the transepithelial resistance

of a tissue could be correlated with the density of sealing

strands in its tight junctions. An alternative hypothesis

suggests that the cytoskeleton, specifically actin

microfilaments, plays a role in the control of junction

permeability. For example, exposure of human epithelial

monolayers to Clostridium difficile toxin A (Hecht et al.,

1988), cyctochalasin D (Madara et al., 1988), or gamma-

interferon (Madara and Stafford, 1989), caused a specific

increase in tight junctional permeability and the disruption

of the submembranous actin localization.

-17- Although well characterized morphologically, the biochemical description of the tight junction has only begun recently. Thus far, two protein components of the junction have been isolated and partially characterized. ZO-1, a phosphoprotein of 225 KDa approx., is peripherally associated with the tight junction (Stevenson et al., 1986). The second and more abundant component, cingulin, is a variable mixture of 140 and 108 KDa forms depending on the tissue source

(Sabanay et al., 1988). It too is a peripheral protein with a similar but not identical localization to ZO-1. The role of these components in tight junction architecture and physiology is not well understood. A comparison of two strains of MDCK cells which differ over 30-fold in transepithelial resistance (and thus tight junction permeability), revealed no difference in ZO-1 content or distribution, or in the overall ultrastructure of the junction. Contrary to the hypothesis of Claude and

Goodenough (1973, 1978) mentioned above, the phosphate content of ZO-1 however, was two-fold higher in the low resistance strain, suggesting that the molecular characteristics of the junctional components are important factors in junction permeability (Stevenson et al., 1989).

In addition to providing a barrier to paracellular routes in epithelia, tight junctions are thought to contribute to the generation and maintenance of epithelial cell surface polarity by preventing the lateral diffusion of components between apical and basolateral membrane domains.

However, this concept remains controversial as recent studies

-18- have demonstrated that certain cell surface molecules, such as the (Na+/K+)-ATPase (Nelson and Hammerton, 1989) and a 135

KDa apical membrane associated glycoprotein (Ojakian and

Schwimmer, 1988) in MDCK cells, have restricted distribution due to interactions with submembraneous cytoskeletal elements. Furthermore, inhibition of ZO-1 synthesis and distribution during embryogenesis did not alter the development of cell polarity, as assayed by cell flattening and concanavalin A binding (Fleming et al., 1989).

1.3 Gap Junctions and Direct Intercellular Communication;

1.3.1 The Re-evaluation of Cell Theory

A major advance in understanding the organization of higher living systems came from the observations of the botanist Schleiden who in 1838 concluded that all higher organisms were aggregates of circumscribed, autonomous units.

This formed the basis of modern cell theory and was essentially verified morphologically by electron microscopy.

Whereas cells are genetically independent, research during the last 40 years has shown that cells in tissues can

interact and function as a unit rather than as discrete bodies. During the late 407s and early 507s chemical transmission in the CNS was demonstrated, opening the doors

of receptor biology and provoking the re-evaluation of the degree of autonomy exhibited by cells. In 1952 Weidmann

demonstrated that Purkinje cells of the heart were

electrically coupled, and later Furshpan and Potter (1959)

demonstrated rectified electronic coupling between the

lateral axon of the crayfish nerve cord and its giant motor

-19- axon. However, the biological implications of these observations were not realized as excitable cells were conceived as specialized for the transmission of electrical signals and so electrical coupling between these cells was not considered as a challenge to cell theory.

Kanno and Loewenstein (1964) serendipitously discovered that non-excitable epithelial salivary gland cells were electrically coupled. This low resistance pathway between epithelial cells was not restricted to inorganic

ions, but allowed free passage of fluorescein (430 Da;

Kanno and Loewenstein, 1964), but not macromolecules such as

F—2 phage RNA (MW ~ 10 Da). Furthermore, electrical measurements confirmed visual impressions that the pathway between adjoining cells was well insulated from the cell exterior (Loewenstein and Kanno, 1964). Loewenstein (1966), showing considerable foresight, proposed that the

intercellular pathway consisted of a transcellular channel which lay in the junctions between cells (Fig. 1.4). By

1967, the gap junction was resolved by electron microscopy

(Revel & Karnovsky, 1967) and the membrane particles

clustered on both sides of the structure became likely

candidates for transcellular channel halves or, as they were

later called, 'connexons7. Thus a novel form of

intercellular communication was established and a putative

specialized region of the cell surface, conferring this

capacity to communicate, identified. The occurrence of

direct intercellular communication was subsequently

demonstrated for a variety of cells including hepatocytes

-20- Fig.1.4 Cell to cell channel concept.

(A) Electronic model proposed in 1966. S and 0 represents the high resistance values across the channel wall in the

'gap7 and the plasma membrane respectively. C identifies the low resistance pathway through the channel proper.

(B) Digrammatic model of the gap junction transcellular channel proposed by Loewenstien (1974). B

7 ~ - pW m —d ►— : C : : : | C • • > • • ^ rrh K /.- /.- K : : S :: V V H -21- 4 - ‘A A - ’: ! 0 > (Penn, 1966). Furthermore, Subak-Sharp et al (1969) demonstrated that communicating cells were capable of exchanging metabolites as well as inorganic ions. When wild- type (HGPRT+) cells were co-cultured with mutant (HGPRT ) cells in the presence of tritiated hypoxanthine, incorporation of the radiolabelled metabolite was detected, by autoradiography, in wild-type cells and the few mutant cells in contact with wild-type cells, but not in isolated mutant cells. This phenomenon illustrated how healthy cells could possibly sustain the growth of abnormal cells by metabolic co-operation, presumably through gap junctions.

1.3.2 Morphology of Gap Junctions

In ultrathin sections, gap junctions appear as two closely apposed regions of the plasma membrane separated by a

2-3nm wide gap (Robertson 1963; Revel and Karnovsky 1967).

Depending on the resolution and guality of staining, they may display a penta- or heptalaminar appearance corresponding to whether the extracellular space, or 'gap', is clearly definable (Fig. 1.5). Revel and Karnovsky (1967) showed that, in such preparations, gap junctions do not impede the flow of heavy metal tracers (lanthanum hydroxide) through the extracellular space (in contrast to tight junctions) but in sections cut tangentially to the junction, the permeance of tracers was impeded by a hexagonal array of cylindrical structures in the gap junction. In freeze fracture replicas, gap junctions appear characteristically as a plague-shaped region of the cell surface containing a highly dense

-22- Fig.1.5 (A) Electron micrograph of a liver ultrathin

section showing the plasma membrane and a gap

junction.

The two closely apposed plasma membranes of adjoining cells are separated by a 2-3nm 'gap' into which stain has penetrated. In this section, prepared by low temperature

fixation procedure, the transcellular channels extending across the gap are arguably visible. The micrograph was kindly donated by Dr. C. Enrich, Urtiversity of Barcelona.

(B & C) Model of the vertebrate gap junction

proposed by Peracchia and Bernadini (1984^.

Based on morphological and ultrastructural data, the 'open' and 'closed' states of the channels are indicated to describe the mechanism of physiological uncoupling. -23- arrangement of intra-membranous particles of 7-10nm in diameter (Kreutzinger, 1968; Goodenough and Revel, 1970; Fig.

1.6 A) on the P-fracture face, and a complementary set of depressions on the E-fracture face. With the exception of the arthropod junction, in which the intramembranous particles appear approx. 20% larger, the freeze fracture morphology of gap junctions is relatively constant irrespective of the origin of the tissue and species (Berdan

& Gilula, 1989). Its appearance in ultrathin sections can vary depending on the tissue examined. For example, gap junctions of cardiac tissue, which reside in the intercalated discs, appear thicker than those of liver, often displaying

'fuzzy' cytoplasmic faces in isolated fractions (Manjunath et al., 1984). Isolated fractions from liver and lens epithelium display a near crystalline structure, when negatively stained and viewed en face. In such samples clusters of hexameric assemblies are visualized with a centre to centre spacing of approx. 8nm, and in some instances a 2nm wide electron dense core structure is visible that is thought to constitute the channel pore into which stain has penetrated (Fig. 1.6 B; Goodenough and

Stoeckenius, 1972; Schuetze and Goodenough, 1982; Miller and

Goodenough, 1985/86). However, negatively stained fractions from heart and lens fibres display a disordered configuration, but proteolytic removal of a 17 KDa peptide from heart junctions and a 38 KDa peptide from lens fibre preparations, can induce the transition from a non-

-24- Fig.1.6 (A) Freeze fracture replica showing P-fracture

face of a gap junction.

The plaque-like structure is composed of a tightly packed cluster of intramembranous particles of 7-10nm diameter.

Mag. x 150,000.

(B) An isolated rat liver gap junction viewed en

face bv electron microscopy, and stained with

sodium silicotunastate.

A lattice array of hexameric particles (connexons) of 8nm centre to centre spacing form the membrane plaque. The connexons are seen more clearly after optical filteration

(inset) which identifies the putative channel pore (1.5-2.0nm diameter) into which stain has penetrated. %

• T C y *

CT'CVv

: •.'■■ £ b > ' ■■

■\r*y-v/yS, < • < • I • I » I

V.'_ ;\- V w - <5 < 4 ,-.v %.“* ■K.

-2 5 — crystalline to crystalline structure (Manjunath et al., 1987;

Kistler and Bullivant, 1988). These observations indicated that proteolytically accessible regions of the protein components of gap junctions, isolated from heart and lens fibres, interfered with the lateral association of the transmembrane channels, and suggested that the biochemical composition of gap junctions may vary between tissues.

1.3.3 Biochemical Characterisation of Gap Junctions

The biochemical characterization of gap junctions soon followed the development of protocols allowing the preparation of highly enriched subcellular fractions from various tissues including liver (Benedetti and Emmelot, 1968?

Goodenough and Stoeckenius, 1972? Evans and Gurd, 1972;

Culvenor and Evans, 1977? Hertzberg and Gilula, 1979?

Henderson et et al., 1979; Finbow et al., 1980? Nicholson et a l ., 1983? Sikerwar and Malhotra, 1983? Hertzberg, 1984), heart (Kensler and Goodenough, 1980? Manjunath et al., 1982,

1984? Harfst et al., 1990), uterus (Zervos et al., 1985) and lens (Kistler et al., 1985). Isolation procedures usually involve the preparation of a plasma membrane fraction, and its subseguent treatment with non-ionic detergents or mild alkali to differentially solubilize non-junctional membranes.

Elucidating the identity of the principal protein components of gap junctions from within these preparations has proved to be an arduous and sometimes confusing task, which periodically identifies novel components. These obfuscations have arisen predominately from the proclivity of gap junction

-26- components to proteolysis during isolation, their tendency to aggregate and migrate anomonously during denaturing gel electrophoresis, and the periodical refinement of preparative methods. In recent years however, it has become apparent that the biochemical composition of isolated gap junctions is comparatively simple, consisting predominantly of a single type of channel forming polypeptide. These polypeptides constitute a family of heterologous-related channel formers now called connexins.

1.3.3a Components of Liver (Epithelial) Gap Junctions

Liver gap junctions are composed of two channel forming polypeptides of molecular mass 32000 Da (Paul, 1986?

Kumar and Gilula, 1986) and 26,453 Da (Zhang and Nicholson,

1989) termed connexin 32 and 26 respectively. The relative abundance of each connexin within the junction varies strikingly depending on the species from which the liver was obtained. For example, in rat and mouse liver preparations, connexin 32 is predominantly expressed, comprising 90% and

67% of the junctional protein respectively (Nicholson et al.,

1987? Traub et al., 1989). In guinea pig livers however, the relative expression of these two connexins is reversed, with connexin 26 comprising approx. 75% of the junctional protein

(Takeda et al., 1988? Chapter 5 in this study). The physiological significance underlying this differential co expression of these connexins is not known.

Connexin 32 is the most extensively studied gap junction polypeptide and a catalogue of evidence has accrued

-27- to secure its candidacy as an integral component of the epithelial gap junction. Polyclonal and monoclonal antibodies generated against connexin 32 immunolocalized to isolated junctions (Zimmer et al., 1987), and to gap junctions in situ. as demonstrated by indirect immunofluorescent labelling of frozen liver sections

(Dermietzel et al., 1987) and by indirect immunogold labelling of ultra-thin liver sections (Stevenson et al.,

1986). Affinity purified polyclonal antibodies to connexin

32 were shown to disrupt intercellular communication, as assessed by dye transfer and measurements of intercellular conductance, when injected into Xenopus and murine embryos

(Warner et al., 1984; Lee et al., 1987), or hepatocytes

(Hertzberg et al., 1985). Furthermore, when these antibodies were bulk loaded into Hydra, specific patterning processes were disrupted (Frazer et al., 1987). These experiments illustrate that antibodies to connexin 32 'block' or interfere with the operation of gap junctions, thereby preventing the exchange between cells of diffusible substances and abrogating the establishment of morphogenetic gradients. Reconstitution of electrophoretically purified connexin 32 into lipid bilayers gave rise to channel properties approximating to those displayed by gap junctions in tissues. These include a 2+ reduced conductance on exposure to low pH, high Ca concentration or connexin 32 antibodies (Young et al., 1987).

Finally, when Xenopus oocyte pairs were injected with connexin 32 specific messenger RNA, a concomitant increase in intercellular communication followed the delay accountable to

-28- the synthesis and assembly of gap junction channels. These

channels displayed similar properties to those associated with gap junctions in situ (Dahl et al., 1987? Swenson et

al., 1989).

The criteria supporting connexin 26 as an integral

channel forming component of gap junctions is far less

extensive, and is based upon the molecular similarity between

the connexins as well as a number of immunohistochemical

studies. Double immunofluorescence and double immuno-

cytochemical staining of tissue sections have demonstrated

that connexins 32 and 26 co-exist within the same junctional

plaque (Nicholson et al., 1987; Traub et al., 1989).

However, Risek et al (1990) have pointed out that polyclonal

antibodies to connexins 32 and 26 can cross-react with either

polypeptide in immunohistochemical studies, despite behaving

discriminately under the conditions of Western blotting. It

is not clear therefore whether these connexins co-exist

within the same channel, or form discrete channels which co

exist within the same junctional plaque, or indeed form

entirely discrete gap junctions within the same cell.

1.3.3b Components of Cardiac and Smooth Muscle Gap Junctions

In mammals, gap junctions are essential for the

function of muscular tissues (with the exception of skeletal

muscle) by providing a pathway that can allow electrical

continuity between cells. Gap junctions from both the

myocardium and the myometrium have been extensively studied

using a variety of biochemical approaches. The principal

-29- component of these junctions is a polypeptide of molecular mass 43,036 Da, related to the epithelial connexins and termed connexin 43. Polyclonal antibodies with specificity towards connexin 43 stain gap junctions in the intercalated disc of the atrium and the ventricle (Yancey et al., 1989).

In the myometrium of the uterus, antibodies to connexin 43 also stain gap junction plaques (Risek et al., 1990).

Furthermore, these antibodies immunolocalize to isolated cardiac gap junctions and block the transfer of dye between neonatal cardiocytes (Yancey et al., 1989). Injection of connexin 4 3 specific mRNA into oocyte pairs has also induced the formation of channels with properties resembling those associated with gap junctions (Swenson et al., 1989).

Recently a novel, putative gap junction polypeptide of relative molecular mass 70,000 has been identified in heart

(Harfst et al., 1990). Immunologically related to connexin

43, this larger putative connexin is the major gap junction associated protein component in ventricular myocytes.

1.3.3c Components of Lens Gap Junctions

The components of gap junctions in mammalian lens

tissues have recently been clarified after a period of

confusion attributable to the presence of a variety of

specialized surface membrane appositions between adjacent

lens cells. These specialized membranes are similar in

appearance to gap junctions in thin sections, and are also

co-enriched during the production of gap junctions fractions.

Isolated bovine and ovine lens fiber plasma membranes contain

-30- a predominant polypeptide of 26 KDa, referred to a MIP 26, as well as numerous other polypeptides including a polypeptide of 70 KDa termed MP70 (Kistler et al., 1985). Amino terminal

sequence analysis has demonstrated that MIP 26 is unrelated to the connexin family of channel formers, despite evidence which ascribes a channel forming capacity to this polypeptide

(Nicholson et al., 1983? Zamphigi et al., 1985). MP70

however, possesses N-terminal sequence similarity with other

connexins and a clone isolated from a lens cDNA library, which predicts a polypeptide of molecular mass 46,000

approx., shares amino acid identity with the N-terminus of

MP70 (Beyer et al., 1988). The relationship between MP70 and

the putative connexin 46 remains unclear.

As inferred from the sequence data,

immunohistochemical approaches have demonstrated that MP70

specific antibodies immunolocalize symmetrically (i.e. to

both cytoplasmic faces) to 18-20nm wide junctions whose

channels are coaxially aligned i.e. gap junctions. In

contrast however, MIP 26 specific antibodies immunolocalize

asymmetrically to junctions of ll-13nm width, with putative

channels which are not aligned coaxially (Zamphigi et al.,

1989). It appears that these cell surface specializations,

containing MIP 26, are not intercellular communication

pathways but perhaps promote intercellular adhesion by

acting as volume-regulating channels that collapse the lens

extracellular space. These junctions are observed to flank

gap junctions and in some instances reside within a gap

junctional plaque. MIP 26 may also be involved in gap

-31- junction assembly by promoting plasma membrane apposition via a 'ball and socket' mechanism (Gruijters, 1989).

1.3.4 The Connexin Family of Gap Junction Channel-Forming

Polypeptides

The development of antibodies to the components of gap junctions and the availability of microsequencing data has facilitated the production of molecular probes allowing the isolation and characterization of cDNA's encoding several different gap junction polypeptides. Sequence comparison of their deduced primary structures has revealed that these polypeptides constitute a family of heterologous channel- forming proteins. Table 1.3 lists the isolated gap junction cDNA clones reported thus far. Two nomenclature systems are currently in operation for this family of polypeptides.

Firstly, the gap junction polypeptide, termed 'connexin', is identified by its deduced molecular mass in KDa i.e. a polypeptide of 42,964 Da is referred to as connexin 43 (Beyer et al., 1987). The alternative nomenclature divides all connexins, whose entire amino acid sequence are known, into one of two classes, namely alpha- and beta-connexins. These are distinguished by the presence of the amino acid motif

K-X-X-X-E for the alpha-class or R-X-X-X-E for the beta- class, located in the region of the polypeptide corresponding to the third transmembrane segment (Risek et al., 1990).

This nomenclature therefore ascribes a phylogenetic relationship between the connexins and further categorizes connexins into subclasses based on their order of reported discovery (Kumar, 1991). The differences between alpha- and

-32- Table 1-3 Gap Junction Genes and Size of Products H O0) Q ) 0 'O os 'O -P T3 O b Q 0)Q u (l) u u 0 ) 0 0) d Ci 0) 0 P< W ) 0 > 0) 0« H * SJ U TJ 0) A 0) oj •H H • ■H A m W P d ■P Q 0 W H H +> « 55 K CU H 0 *H M X M ' 0 -P 0 0) ) X ) 0 -H U N B 0 CU

X CO UD cn CO CM CM CO MCM CM »—< ” ' CO CO MCM CM rH CU OC MC CO CO CM CO o CO o o - r CD CM - E o i—H OS < 0Q rd P X X X I 1 --- V K » ( -H »—i i—I o X5 rH X cd P p d Dr co rd cD • rd P cr> p oE oo E co E ■ r—1 rH ^ rH MCO CM CM ID CM l - i r—1 c o u3 CO QP PQ PQ PQ O 2 D S>C OS X cd • P cn P rH i • m r cn •rH CT> 2 4 CM CM CM N CD CM n i r—t CM CD •—i X 0 0 ID

Sequence unpublished Protein not identified beta-connexins is also apparent in the sequences corresponding to extracellular portions of the polypeptide

(Chapter 4, table 4.2). In this thesis, connexins shall be referred to on the basis of their molecular weight.

1.3.4a Topography of Connexin Polypeptides

A number of algorithms have been devised to assess the hydropathy profiles of polypeptides of known amino acid sequence. They are based on either the physical characteristics of amino acid side chains, such as solubility in ethanol and vapour pressure, or on a statistical study of the distribution of each residue between the interior and the surface of globular proteins of known structure. Synthesis of several such scales coupled with adjustments to optimize prediction of potential transmembrane segments was proposed by Kyte and Doolittle (1982), which has received widespread application (see table 1, Chapter 3).

Analysis of connexin primary sequences using this algorithm predicts a common structural design with four hydrophobic domains of approx. 20-25 amino acids in length, corresponding to putative transmembrane segments (M1-M4).

Two hydrophilic domains, which diversify considerably in both size and sequence between different connexins, are located between the second and third hydrophobic segments and between the end of the fourth hydrophobic segment and the carboxyl terminus. Immunocytochemical and protein chemistry

approaches were combined to provide evidence supporting the predicted topography of rat liver connexin 32 (Zimmer et al.,

-34- 1987). These experiments led to the generation of a low resolution two-dimensional model of the arrangement of connexin 32 in the lipid bilayer (Fig. 1.7). Complying with hydropathy predictions, the polypeptide was proposed to traverse the lipid bilayer four times with its hydrophilic domains located in the cytoplasm. On the basis of their similar hydropathy profiles, all connexins are assumed to adopt similar topographies within the junctional membrane although further biochemical evidence has also been provided in relation to connexin 43 in heart gap junctional membranes

(Yancey et al., 1989).

Apart from their similar hydropathy profiles, the connexins share a number of other similar characteristics.

Whereas the amino terminal 20 amino acids display partial sequence similarity, the level of sequence similarity rises considerably within the ensuing transmembrane (M1-M4) segments, particularly within Ml, and the extracellular domains.

In general, comparison at both the nucleic acid and protein levels illustrates a high level of homology between gap junction component genes with a suggestion that this growing family of polypeptides may have evolved from one or two ancestral genes. With the recent discovery and cloning of two synaptic vesicle proteins, synaptophysin (Sudhof et al.,

1987) and synaptoporin (Knaus et a l ., 1990), which possess similar hydropathy profiles to the connexins and are thought to form hexamers within the vesicular membrane, a wider group or superfamily of polypeptides appears to be emerging,

-35- Fig.1.7 A two-dimensional topographical model of connexin

32 in the junctional membrane (Zimmer et al..

1987).

This particular diagram shows the amino terminus embedded in the membrane to account for its observed resistance to proteolytic attack. EXTRACELLULAR

CYTOPLASMIC

-36- related by their overall structural design.

1.3.4b Distribution of Connexins

The initial attempts to analyse the distribution of gap junction polypeptides were based on immunochemical approaches using antisera raised against the principal component of rat liver, namely connexin 32. By a combination of immunohistochemistry and Western blotting, a widespread distribution of an homologous antigen was observed across tissues and species (Dermietzel et al., 1984? Hertzberg and

Skibbens, 1984? Warner et al., 1984). Due to the cross- connexin class immunospecificity of these antibodies and the post-mortem proteolysis of gap junction components, a distorted consensus developed which perceived gap junctions as comprising of a catholic component of Mr 28-30 KDa. An amino terminal specific antibody to connexin 32 merely reinforced this consensus because it too displayed cross- connexin class immunospecificity (Zervos et al., 1985).

Nevertheless, these early investigations demonstrated that gap junction antigens were present in a wide variety of tissues including stomach, kidney, liver, heart and uterus, as well as in Xenopus. The isolation of cDNA clones extended the sphere of investigation to the mRNA level, and refined the immunological approach by providing the information necessary for the generation of anti-peptide antibodies with connexin-specific immunospecificity. Table 1.4 summarises the distribution of connexin mRNA and antigen in various tissues.

Although the detection of connexin mRNA is not

-37- j-ab1e 1-4 Distribution of Connexins in Various Tissues

(adapted from Kumar, “1991)

Tissue Immunochemical RNA

ai 0i 0 i 0 2

Hearc + _ 4 - _ _ Liver - + 4* — + 4-

. Myometrium + — 4 - — — Endometrium _ 4 - - 4* 4-

Ovary + — - 4- -- TesCes N.D. N.D. 4- 4-

Kidney + + + + 4 - 4- Pancreas (Exocrine) — + 4 - N.D. Pancreas (Endocrine) N.D. + ? N.D. Intestine 4- + + (-) 4* N.D. 4- Spleen N.D. - 4* ( ) 4- S toraach 4- + (-) N.D. (-) 4- Lung N.D. N.D. 4- 4-

Cornea + , N.D. N.D. Lens Epithelium + — N.D. N.D. Lens Fibre - - N.D. N.D.

Brain + 4 - 4 - (-) 4- 4- Astrocytes + - - N.D.

Oligodendrocytes - 4* - N.D. PinealocyCes - - + N.D.

Leptomeninges + - 4 - N.D. Ependyma 4- - 4* N.D. Brain Blood + N.D. N.D. N.D. Vessels

Data compiled from immunofluorescence and imraunobl'ot analysis (cable heading- immunochemical) and from Northern analysis (cable heading RNA) is shovm for primarily rac tissues.

References co original daca: Paul, 1986; Heynkes ec al., 1986; Beyer ec al.. 1987; 1988; 1989; Duponc ec al., 1988; Traub ec al., 1989; Zhang and Nicholson. 1989; Dermieczel ec al., 1989; Yamamoco ec al., 1990; Risek ec al., 1990.

Presence of produce is indicaced by (+); absence of produce is indicaced by (■*-). Samples for which no daca is available are labelled N.D. Symbols in brackets refer co confliccing daca.

-38- necessarily indicative of the presence of functional gap junction structures (due to post-transcriptional controls), there is, in general, good agreement between the detection of mRNA and the expression of its corresponding connexin. An exception however, is evident in the analysis of rat stomach.

Whereas immunological methods detect connexin 43 only (Dupont et al., 1988), Northern blot analysis failed to identify connexin 43 mRNA transcripts, although connexin 32 and connexin 26 transcripts were detected (Beyer et al., 1987;

Zang and Nicholson, 1989). It is possible that all three connexins are expressed, but in stomach, the relative abundance of the mRNA's and polypeptides exceed the limits of the detection methods. In addition, connexin 46 mRNA has been detected in rat heart, lens fibre cells and kidney but not in uterus, brain and liver (Beyer et al., 1988). It appears therefore that the distribution of connexins is inconsistent with the view that a specific connexin class is expressed in tissues derived from one particular germ layer.

There is, however, a preponderance of beta-connexins in endodermally derived tissues (epithelial) and the alpha- connexins in mesodermally derived tissues. The factors determining the cell specificity are not known, but are likely to consist of 'processing' events during embryonic development coupled to tissue-specific gene expression regulatory mechanisms present in the adult.

1.3.5 Lipid Composition of Gap Junctions

Compared with non-junctional membranes, the lipid to protein ratio of gap junctional membranes, like those of

-39- other intercellular junctions, is low. Lipids however, are an integral component of gap junctions but compositional analysis has suffered from drawbacks related to the use of detergents and alkali solutions to isolate gap junctional membranes from parent plasma membrane fractions. The use of such conditions for the preparation of gap junction fractions is not favourable for preserving junctional lipids in their native state, and probably 'strips' molecules peripherally associated with the junctional membrane.

Phospholipids are one of the predominant lipid constituents of gap junctional membranes, as might be expected. Mouse and rat liver gap junctions comprise of phosphatidylcholine as the major class of phospholipid, with smaller quantities of phosphatidyl-ethanolamine, phosphatidylserine and phosphatidylinositol (Evans and Gurd,

1972; Goodenough and Stoeckenius, 1972; Hertzberg and Gilula,

1979). Comparative studies have clearly shown that the recovery of phospholipid depends heavily upon the method adopted for the enrichment of gap junctions (table 1.5;

Henderson et al., 1979), with levels varying by as much as 4- fold. Rat liver gap junctions do not contain sphingomyelin

(Goodenough and Stoeckenius, 1972), in contrast to mouse liver preparations (Evans and Gurd, 1972). Sphingomyelin however, does appear to be the major phospholipid component of lens fiber gap junctions (Alcala et al., 1982/1983). A major role for sphingomyelin in the assembly of lens gap junctions has been implicated by the observation that major changes in the sphingomyelin composition of lens epithelial

-40- Table 1.5 (adapted from Malewicz et al 1990) CU O x Pa *-£3 .2 O i S ' ^ 3 CU ^ O O O X O “ X £ * TJ a e f X u v rv X °3 ”2 ~ X O X — o — ) e CO o cd v a ^ 2 ed a u>o a cu d a 2 H CU fl 2 u> d ‘-32 o O 3 0) ° *"5 2 ^ 2 fl ,2 o _ CO cd a 4> cd cd CU

f t C «♦-* X d n O X ja .2 X • a P PU Q - s X o PU X s X PU • rH 3 -»_a CD CD <1> cd o u, G o O cd co o O o U C f co PU o © ■ CO >-l cd o G CD

K ^ K r-C D C O H *> O H O D C iO lO oo o c— o oo O CN CO CN O CO H Tf H CO CD CO XT O CD CO D C D C H h O O O i O II (N H +1 CO +1 1 + 00 CO CO CO 00 cr> O C N C V Ol Tf lO lO +1 +1 4-1 C — CO C r—t —Cr D CD CD CO CO D CD CD CD O O O O O 6 G Q O O Q G G G O CO (D CO ) 0 CD CD

1 + n c £ o i 6 -41 -41 - 1 + l f f o o S o cd CD &> CO

H H N CO lO CO CO CD CO lO N H H 2 2 G G CD o CO D CD CD CD CD

LO - t O - Cs3 PQ O C X X X ^ a s r* S^ -» uo r D D > > > > G G G G

a a a a co oo oo -- U-t >+-< t - U <-+-« 1 ^ 5 D D CD CD CD o o CD G cr* r o £ g * o U UU o o S 6 S 2 I I II

O CO CO O CD 2 i-4 U

G 2

U O o PU CD CD >~~3

~3 *~ cd PQ cd CD plasma membranes occurs during cell elongation, an event accompanied by the massive formation of gap junctions

(Broekhuyse et al., 1978).

Little is known about the glycolipid content of gap junctional membranes, although Evans and Gurd (1972) showed that two types of glycolipid were present in vesicles produced as a by-product of gap junction enrichment. Among the neutral lipids found in gap junctions are small levels of free fatty acid, triglycerides and cholesterol esters.

However cholesterol, the major lipid component of gap junctions (table 1.5), is considerably enriched within the junctional membrane as compared with parent plasma membranes.

Furthermore, there is good agreement between junction cholesterol to protein ratios reported for most cell types, and this appears to be independent of the isolation procedure

(Henderson et al., 1979). This implies that cholesterol is more closely associated with the protein components of gap junctions than phospholipids, which may have important implications for reconstitution studies. If indeed high cholesterol levels are characteristic of gap junctions, a special requirement for cholesterol in ordering and anchoring connexons within junctional plaques is implicated (Malewicz et al . , 1990).

1.3.6 Functional Role of Gap Junctions

The ubiquity of gap junctions is a testament to their biological importance in metazoans. In electrically excitable tissues such as cardiac and smooth muscle, it is

-42- widely accepted that gap junctions underly the synchronous contraction of myocytes. Apart from their fundamental roles in the control of cellular growth (Mehta et al., 1986) and embryonic development (Green, 1987; Warner, 1988), gap junctions have been proposed to be required for endothelial migration into wound areas and for angiogenesis (Larsen and

Haudenschild, 1988; Pepper et a l ., 1989).

A role for gap junctions in cell growth control has been suggested by the observation that many cancer cells have decreased intercellular communication pathways (Loewenstein,

1979). Moreover, Enomoto and Yamasaki (1984) observed that the cells of a morphologically transformed focus, while capable of communicating with each other, were unable to establish communication with surrounding normal cells.

Interestingly, the growth of cancerous cells appears to be arrested by the formation of gap junctions between tumour and normal cells (Mehta et al., 1986). Indeed many studies examining junctional communication between cells infected with tumour promoting viruses, including SV40 (Azarnia and

Loewenstein, 1984) and Rous Sarcoma (Atkinson et al., 1981;

1986), are consistent with a role for gap junctions in regulating cell growth.

Morphological studies, supplemented by immunocytochemistry, have correlated liver regeneration, after partial hepatectomy, with a cycle of disappearance and reappearance of gap junctions (Yee and Revel, 1978; Yancey et al., 1979; Traub et al., 1983; 1989). Immunological studies have shown that the expression of both connexin 32 and

-43- connexin 26 were regulated similarly during regeneration, and

that a relationship between junction loss and cell division

exists (Traub et al., 1989; Dermieztel et al., 1987). Since

the expression of connexin 3 2 and the appearance of gap

junctions increased when cultured hepatocytes were

supplemented with components of the extracellular matrix,

including heparin and proteoglycans, gap junction modulation

may reflect the re-establishment of a normal matrix (Fujita

et al., 1987; Spray et a l ., 1987).

Direct intercellular pathways of molecular exchange

have often been implicated to underly certain developmental

phenomena (Wolpert, 1978). Direct evidence for the

involvement of gap junctions during embryogenesis has emerged

from a number of antibody perturbation experiments. Such

experiments have demonstrated specific developmental defects

including misplaced and even absent occular structure,

arising from microinjection of precursor cells with

polyclonal antibodies to connexin 32 (Warner et al., 1984),

the decompaction of communication-incompetent cells within

the murine embryo (Lee et al., 1987), and the disruption of head patterning processes in Hydra (Frazer et al., 1987).

Furthermore, this approach has demonstrated that gap

junctions may form communication pathways between the

polarizing region of the chick limb bud and the adjacent

anterior mesenchyme (Allen et al., 1990). Thus in the developing embryo, and also in differentiated adult tissues, gap junctions are responsible for the formation of

communication compartments which engenders tissue homeostasis and synchronized cellular activities.

-44- 1.3.7 Permeability of Gap Junctions

The permeability of gap junctions is based upon the

characteristics of its constituent channels. Junctional

permeability has been assessed either by electrical

conductance measurements under current or voltage clamp

conditions, or by the optical observation Of fluorescent dye

dispersal. The selectivity of the gap junction depends upon

the dimensions and charge distribution of its channels.

Microinjection of fluorescent molecules of known dimensions

into ventricular myocyte cell-pairs and the asessment of

their diffusion, has estimated a channel pore of 1.6nm diameter (Imanaga et al., 1987), with a molecular weight cut

off of 1200 (Simpson et al., 1976). This is consistent with ultrastructural estimates of a pore with a 1.5nm diameter

that widens to 2.5-3nm at its cytoplasmic mouth (Caspar et

al., 1977; Makowski et al., 1977; Unwin and Zamphigi, 1980;

Unwin and Ennis, 1984). Consistent with these estimates of pore size are single channel conductance values which,

depending on the cell type and experimental system, vary from between 50-165pS.

1.3.8 Gating of Gap Junctions

The cell can regulate the function of gap junctions

by controlling their synthesis and turnover, and by altering

the permeability of its constituent channels. The former

processes are highlighted by the rapid half-lives of

connexins, implying that gap junctions are highly dynamic

structures which are rapidly assembled and broken down. For

-45- example, the half-life of connexin 3 2 and connexin 26 was estimated to be approx. 1.3-2 hours in cultured murine hepatocytes (Traub et al., 1989), but in liver tissue, the half-life of connexin 32 was approx. 5.5 hours (Fallon &

Goodnough, 1981). Additionally, the half-life of connexin 43 has been estimated as approx. 2 hours and 1-2 hours for non- phosphorylated and phosphorylated forms respectively, in neonatal cardiac myocytes (Laird et al.,1991). It is noteworthy that these estimates are much lower than the 20-

40 hour half-lives of membrane glycoproteins.

The 'opening' and 'closing' of gap junction channels is influenced by a variety of physiological and external 24- agents. Increased intracellular levels of Ca , and decreases in cytoplasmic pH, both reduce junctional permeability i.e. close channels (Loewenstein, 1966; Turin and Warner, 1977). The pH and calciunt range influencing gap

junction permeability varies from system to system which might reflect different biochemical components. Although 24 both pH and Ca can reduce permeability independently, it is not known whether H+ directly affects channel activity or 24 indirectly, by altering the affinity of the channel for Ca

(Peracchia, 1990). The effects of calcium may be mediated by calmodulin (Hertzberg and Gilula, 1981) or intermediates of the phosphoinositide pathway (Yada et al., 1985). It is possible however, that calcium may act directly on the gap

junction channel for diffraction studies have demonstrated an alteration in the relative arrangement of channel subunits in 24 24 the junctional membrane between Ca -free and Ca -

-46- containing buffers, indicating that a gating mechanism is operational (Unwin and Ennis, 1984).

2+ + In contrast to Ca and H , cyclic AMP has been observed to elevate junctional permeability in most systems; the exception appears to be horizontal cells from fish retina

(Teranishi etal., 1983). This second messenger may exert its stimulatory effect by increasing the phosphorylation of junctional proteins (Loewenstein, 1985), thereby either extending the duration of the 'open7 channel state or increasing the probability of opening (De-Mello, 1986? Saez et a l ., 1986).

A variety of external agents reduce gap junctional permeability leading to the uncoupling of communication- competent cells. These include long chain alcohols and anaesthetics (Deleze and Herve, 1983; Ramon et al., 1985), neurotransmitters (Neyton and Trautmann, 1986) and regulators of protein kinases. Tumour promoting phorbol esters (TPA) and analogues of diacylgycerol activate protein kinase C which either directly or via a mediator(s) is responsible for reducing channel permeability (Yotti et al., 1979? Murray and

Fitzgerald, 1979). Since okadaic acid had no effect on intercellular communication, it appears unlikely that the inhibitory effect of TPA involves phosphatases type 2A or type 1 (Rivedal et al., 1990). It has been suggested that

TPA alters the rate of gap junction formation and that its effects are only significant on cells in which the rate of junction formation is already low (Pitts and Burk, 1987).

-47- Thus far, the only modulation of gap junction permeability by a gap junction specific agent has come from the employment of connexin-specific antibodies. Polyclonal antibodies to connexin 32 have been shown to disrupt junctional communication in a number of experimental systems including Xenopus and murine embryos (Warner et al., 1984;

Lee et al., 1987), Hydra (Frazer et al., 1987) and cultured hepatocytes (Hertzberg et al., 1985). Furthermore, antibodies to connexin 26 have also blocked dye coupling between cultured hepatocytes (Traub et al., 1989). Since co microinjection of dye and antibody rapidly inhibits the dye transfer, it is thought that antibodies exert their effect by binding directly to the channel, either sterically blocking access to the channel pore or inducing its closure. However, it is conceivable that antibodies can act at various levels that include interference with transport mechanisms and gap junction assembly or even the stimulation of gap junction turnover.

1.4 Aims of the Project

In this study, various aspects of hepatic gap junction biochemistry are addressed. In particular, this project has focused upon investigating the membrane topography of connexin 32. The success of this central aim was dependent upon the generation of a panel of effective site-directed, anti-peptide polyclonal antisera with specificity towards different intracellular and extracellular domains of connexin 32. Development of these antisera was allocated considerable investment considering the broad

-48- potential for such antibodies to inhibit gap junction channel activity and assembly. The following chapters describe the generation and characterization of a panel of polyclonal anti-peptide antisera directed towards connexin 32, their use in investigating the topography of connexin 3 2 in the junctional membrane, their detection of homologous antigens in different tissues and in liver across species, and their application in analysing the subcellular compartmentation of connexin 32 in liver.

-49- CHAPTER 2

MATERIALS AND METHODS

2.1 Generation Of Anti-Peptide Antisera

2.1.1 Synthesis Of Oligopeptides

Synthetic peptides corresponding to selected sequences of rat liver connexin 32 were prepared by R. Foulkes (Division of Peptide Chemistry N.I.M.R.) by FMOC-Polyamide Solid-Phase

Chemistry, using an LKB Biolynx Peptide Synthesizer. Sequence selection was based on the 2-D model describing the membrane topography of connexin 32 (Paul, 1986 and Zimmer et al., 1987).

After assembly, peptides were cleaved from the resin and deprotected by treatment with 95% aqueous TFA. Purity and quality of the preparations were assessed by HPLC and amino acid composition analysis. Free peptides were solubilized in either water or PBS and stored at -20°C. A diagrammatic description of peptide synthesis is shown in Fig. 2.1.

2.1.2 Conjugation Of Peptides To Keyhole Limpet

Heamocyanin (KLH)

Peptide-KLH complexes were produced using two crosslinking agents differing in their chemical group cross-linking specificities. Conjugation via free amino groups was performed with glutaraldehyde as a cross-linker and with greater specificity, using the bifunctional reagent m-maleimidobenzoyl-

N-hydroxysuccimide ester (MBS) Fig. 2.2.

-50- Fig.2.i Peptide Synthesis

Peptides were synthesized by FMOC polyamide solid-phase chemistry on Biolynx automated peptide synthesizers; the mechanism of synthesis is shown opposite. Completed peptides were released and deprotected by treatment with 95% aqueous

TFA.

Fig.2.2 Peptide-KLH Conjugation via MBS

Conjugation of peptides to KLH was achieved by a two step reaction in which KLH was activated with MBS by covalent modification of free amino groups via the carbonyl group of the benzoic acid moiety. The KLH-MBS complex was then reacted with free peptides via the sulphydrylL groups of cysteine residues, generating a stable crosslink as shown. •.Side chain car boxy and hydroxy Rv>ge o f reversible Polar solid support .'groups protected as;t-butyl linkage agents allowing use o f derivatives polar reaction media (eg D M F )

O C* H 2- C 0 2B u ‘ R ( O tt c h -c h 2- o -c -n h *c h -c o 2h + h 2n -c h -c *o -c h 2V y -0 -C H 2*C*Nlc-PolydifncihyIa crylamidc resin

’ Base-labile Fmoc Protected anuno acids activated Internal reference ■ amino.protecting as symmetrical anhydrides or am ino add group* activated esters

FMOC-Polyamide Solid-Phase Peptide Synthesis

Fig. 2.1

s - ch - c h - n h -)PEPTIDE c o h !

PROTEIN-n h

MBS CYS

Fig.2.2

- 5 1 - 2.1.2a Conjugation Using Glutaraldehyde

Peptide-KLH conjugates were generated using a modification of the procedure of Reichlin et al (1980). Peptide

(0.25umol) and KLH (0.005umol) (Sigma) were dissolved in PBS and mixed in a final volume of 1.5ml. While gently stirring, 1.3ml of 25% (w/v) glutaraldehyde was added dropwise and the reaction allowed to proceed overnight at room temperature. With most samples, the solutions became cloudy. Samples containing conjugate suspensions were dialysed extensively against PBS and then stored at -20°C.

In some samples the efficiency of conjugation was determined by incorporating a trace amount of iodinated peptide and determining the activity before and after dialysis.

A conjugation efficiency of 40% was routinely observed.

2.1.2b Conjugation Using m-Maleimidobenzoyl-N-

hydroxysuccinimide Ester (MBS)

Peptides were conjugated to KLH essentially as described by Green et al (1982). A KLH—MBS complex was first prepared by the addition of 20ul (0.7mg) of a 35mg/ml solution of MBS in dimethylformamide, to 1ml of a 5mg/ml solution (0.005umol) of

KLH dissolved in PBS. The MBS solution was added very slowly, with gentle stirring, to prevent the local concentration of dimethylformamide exceeding 30% (v/v), when KLH becomes

insoluble. The reaction proceeded with gentle stirring for 30 minutes at room temperature. Free MBS was separated from the

KLH-MBS complex by gel filtration through a Sephadex G-25 column

-52- (3cm x 0.5cm) equilibrated in 50mM sodium phosphate pH 6.0. The elution of the KLH-MBS complex was monitored at Eoon and the ^ o U initial peak fractions pooled giving an estimated recovery of

67%.

Conjugation (Fig. 2.2) was performed by the addition of

1.0ml of the peptide solution, containing 0.25umol of peptide dissolved in PBS, to the KLH-MBS complex giving a final reaction volume of 2.0ml (pH~7.0). The reaction proceeded for three hours at room temperature with gentle stirring and the pH monitored periodically. When necessary the pH was adjusted to approx. 7.0 with sodium hydroxide solution. Conjugation . . 125 efficiency was determined by the addition of I-labelled peptides in some samples, and after extensive dialysis was estimated to be approximately 40%.

2.1.3 Immunization Of Rabbits

A minimum of two Sandylop rabbits were immunized per conjugate and in some cases (GAP 8-KLH and DES 2-KLH) as many as four rabbits were used. Rabbits were given a total of three injections during their initial immunization cycle. The first two injections were administered intramuscularly at 10 day intervals in each thigh. The administered emulsions comprised of 150ug of peptide (per injection), in conjugated fdrm, containing equal amounts of the MBS and gluteraldehyde preparations, mixed with an equal volume of Freunds Complete

Adjuvant. A minimum period of 6 weeks elapsed before the final injection, administered sub-cutaneously at multiple sites along

-53- the rabbit's back. This injection was composed of a similar conjugate mixture emulsified with an equal volume of Freunds

Incomplete Adjuvant. Rabbits were bled from the ear vein 10 days later, and then at 4 day intervals.

Blood was collected directly into 30ml glass universal bottles and coagulation allowed to proceed at room temperature

for 1-2 hours and then overnight at 4°C. The coagulated material was discarded and the serum centrifuged at 8000 x g for

10 minutes to pellet any remaining insoluble material (mainly erythocytes). After centrifugation, serum was aliquoted and stored at -20°C or -70°C.

2.1.4 Affinity Purification

Antisera were affinity purified by immunoabsorption on columns containing their respective peptides covalently attached to either CNBr-activated Sepharose CL-4B (Pharmacia) or Separon

Hemacart 1000E (Anachem).

2.1.4a Preparation Of Immunosorbents; CNBr-Activated Sepharose

CL-4B

Approximately l.Og Of CNBr-activated Sepharose CL-4B was

hydrated by suspension in 30ml of ImM HC1 for 15 minutes at 4°C.

The swollen gel was then washed with 200ml of ImM HC1 by

suspension and filtration through a scintered glass filter (G3)

using a H20 vacuum pump. Conjugation was performed by

suspending the gel in 5ml of coupling buffer (0.1M NaHCO^, 0.5M

NaCl pH 8.3) containing 5-10mg of native peptide. The reaction

-54- proceeded for 2 hours at room temperature, with gentle agitation using a rolling platform. Excess unbound peptide was removed by washing the gel with 200ml of coupling buffer and the remaining active sites blocked by incubation in 5ml of 0.1M Tris-HCl (pH

8.0) for 2 hours at room temperature. A further three cycles of washes with 0.1M sodium acetate (pH 4.0), 0.5M NaCl followed by

0.1M Tris-HCl (pH 8.0), 0.5M NaCl were given before packing the gel into the glass column.

2.1.4b Separon Hemacart 1000E (Epoxy-activated)

Conjugation of peptides to Separon Hemacart 1000E

(Anachem) was performed according to the manufacturers specifications. Pre-packed columns were injected slowly with

5ml of H20 and then left for 2 hours at room temperature. The swollen gel was rewashed with 5ml of PBS and then injected with lml of the appropriate peptide (5mg/ml) dissolved in PBS.

Conjugation proceeded for 24 hours at 4°C. Excess unbound peptide was removed by washing the column with 10-20ml of H20.

Excess reactive groups were then blocked by injecting lml of

0.1M ethanolamine in 0.1M sodium borate (pH 9.0) and incubating overnight at 4°C. Columns were ready for use after extensive washes with PBS.

2.1.4c Preparation Of Affinity Purified Reagents

Antisera were applied to columns as two-fold concentrated gamma-globulin fractions, prepared by ammonium sulphate fractionation of crude sera. Samples were recycled through appropriate columns for 2-3 hours at a flow rate of

-55- l.Oml/min. Unbound protein was eluted with PBS, followed by a wash with 2M KC1. Anti-peptide antibodies were eluted using conditions of extremes of pH or mild denaturing agents

(see results-Chapter 3). Samples were then extensively dialysed against 7.5 mM K C 1 , 0.5mM Tris-HCl (pH 7.6) and concentrated ten-fold by rotary evaporation. Affinity reagents of between l-3mg/ml were routinely obtained and tested for immunoreactivity by Western blotting.

2.2 Antisera Characterisation

2.2.1 Dot Blots

Peptides were dissolved in PBS at appropriate concentrations and were absorbed to nitrocellulose strips under a gentle vaccum using a Schleicher and Schuell Minifold

II manifold. Sample loadings of lOOng and lug were made for each peptide. After absorption of the peptides, nitrocellose strips were dried completely before processing as described for Western blotting (Section 2.2.4), using antibody (serum) incubation at dilutions of 1/100 for 2 hours at 37°C.

2.2.2 Enzyme-Linked Immunosorbent Assay (ELISA)

Peptides were dissolved in PBS at appropriate concentrations and were loaded onto microtitre plates in a volume of lOOul. Serial loadings from lng to lug were made

for each peptide and then left overnight at 4°C to adhere to the plate. After removal of the PBS, samples were blocked with 1% BSA, 0.05% Tween 20 dissolved in PBS for 30 minutes at room temperature. After a cycle of 3x10 minute washes in

-56- 0.05% Tween 20 dissolved in PBS (PBS-T), the peptides were incubated with lOOul of their respective antisera diluted in

PBS for 2 hours at room temperature. Serial dilutions from

1/10 to 1/10,000 were used for each antiserum. Samples were then given a further cycle of washes in PBS-T, followed by an incubation with lOOul of a goat anti-rabbit horse radish peroxidase conjugate (Biorad Laboratories) diluted 1/3000 in

PBS-T, for 1 hour at 3 7°C. After a further cycle of washes with PBS-T, samples were developed by incubation in lOOul of developing solution: 0.4mg/ml o-phenylenediamine dihydrochloride, 24mM citric acid, 0.012% H 202 dissolved in

50mM sodium dihydrogen phosphate pH 5.0, for 15 minutes at room temperature in the dark. Colour development was stopped by the addition of 25ul of 2.5M H2S04 to each sample.

Samples were immediately analysed at 495nm using a Titerek

Multiskan MC automated plate reader.

2.2.3 Electrophoresis

Electrophoresis was performed in a discontinuous buffer system with 4.5% stacking gels and either 10%, 12.5% or 17.5% separating gels (Laemmli 1970). All samples analysed by denaturing electrophoresis were solubilized by a

30 minutes incubation in Laemmli buffer (7 5,'mM Tris-HCl pH 6.8, 2% SDS, 0.55M beta-mercaptoethanol, 0.003% bromophenol blue and 10% glycerol) at room temperature. For samples which had been pre-treated with alkylating agents

(2.3.1a), solubilization was performed using Laemmli buffer without m^rcaptoethanol. Electrophoresis was performed at a

-57- constant current of 1.5mA/cm and gels stained with either silver (Amersham hyperstain) or Coomassie brilliant blue G, or processed for Western blotting.

2.2.4 Western Blotting

Western blotting was performed essentially as described by Burnette (1981). Proteins were transferred to nitrocellulose membranes electrophoretically, (Schleicher &

Schuell O.lum) at an appropriate voltage and duration. After transfer, the nitrocellulose membranes were briefly washed in

PBS and then blocked in 5% skimmed milk dissolved in PBS for

1 hour at room temperature. Nitrocellulose membranes were then incubated in fifst antibody, diluted appropriately with

1% skimmed milk in PBS, for 2 hours at 37°C. Following two

10 minute washes in PBS, the nitrocellulose membranes were incubated in 0.2ug/ml iodinated protein A (6uCi/mg), diluted in 3% skimmed milk in PBS, for 1 hour at room temperature.

Finally, the nitrocellulose membranes were extensively washed in PBS containing 0.1% Triton X-100, dried, and exposed for autoradiography.

2.2.5 Immunofluorescence Microscopy

2.2.5a Frozen Sections

Rat livers were excised, sliced and rapidly frozen in powdered dry ice. Aproximately 6-10um thick sections were cut from frozen liver slices (these were kindly supplied by

R. Aligue), and mounted on gelatin-coated slides. Sections were given two 15 minute washes in PBS at room temperature to

-58- remove excess mounting material. Sections were not fixed,

for in previous experiments incorporating a variety of fixing

agents, antibody localization was not observed. Sections were blocked by an incubation in normal goat serum diluted

1/10 with PBS for 1 hour at room temperature. Sections were

then incubated in first antibody, appropriately diluted with

1% (v/v) normal goat serum in PBS, for 90 minutes at room

temperature. After three 10 minute washes with PBS, sections were incubated in goat anti-rabbit-FITC conjugated second

antibody (Sigma) diluted 1/50 with 1% (v/v) normal goat serum

in PBS. Second antibody incubations were performed for 1

hour at room temperature in the dark. After a further cycle

of washes in PBS, sections were mounted with a drop of lmg/ml

p-phenylenediamine dihydrochloride (Sigma) solution prepared

in 10% glycerol. Sections were viewed with an excitation

wavelength of 490nm, using a Zeiss (W. Germany) fluorescent

microscope unit.

2.2.5b Wax Embedded Sections

Wax embedded liver sections were kindly donated by

Dr C.R. Green of University College London. Rat liver tissue

was fixed in Zamboni's fixative (0.2% picric acid, 2%

paraformaldehyde, 0.1M phosphate buffer pH 7.4) and wax

embedded as described by Toshimori et al (1987).

Approximately 10-15um sections, mounted on slides, were

dewaxed by two 5 minute incubations in xylene. Sections were

then rehydrated by incubations in a decreasing ethanol series

- 90%, 70% and 50%, 5 minutes in each - and then washed with

-59- tap water for 5 minutes. Prior to first antibody exposure, sections were treated with 0.1% trypsin, 0.1% CaCl2 dissolved in 20mM Tris-HCl (pH 7.4) for 5-10 minutes at room temperature. After a brief wash with tap water and a 20 minute equilibration in PBS, sections were blocked with 0.1M lysine, 0.1% Triton X-100 dissolved in PBS for 45 minutes at room temperature. After a further 20 minute wash in PBS, sections were exposed to first antibody diluted 1/10 with PBS for 1 hour at 37°C. Following a 20 minute wash in PBS, sections were exposed to FITC-conjugated goat anti-rabbit secondary antibody (Sigma), diluted 1/20 in PBS, for 1 hour at room temperature in the dark. After a further two 20 minute washes with PBS, sections were mounted for fluorescent microscopy as described in section 2.2.5a. Sections were examined by confocal microscopy kindly performed by Dr R.G.

Gourdie of University College London.

2.2.6 Immunoelectron Microscopy

2.2.6a Immunocytochemistry on Isolate^ Gap Junctions

Gap junctions fractions were prepared as described in section 2.4.2. Labelling was performed adopting the pre embedding method described by Zimmer et al (1987), with minor modifications. Isolated junctions were washed in 75mM KC1,

5mM Tris-HCl pH 7.6 (Tris-saline) before incubation with first antibody. Gap junction derived single membranes or

'split junctions' were prepared by incubating gap junctions in 8M urea, lOmM Tris (pH 10) for 30 minutes at 37°C

(Manjunath et al., 1984). In certain experiments DTT was

-60- added to a final concentration of 20mM. Samples, equilibrated in Tris-saline, were exposed to primary antibody for 2 hours at room temperature or overnight at 4°C. Primary antibody incubations were performed using affinity purified reagents at concentrations of lmg/ml in Tris-saline suspensions of 100-200ul. In some experiments crude serum was used, diluted 1/10 with Tris-saline. Following primary antibody incubations, samples were pelleted in a microfuge for 10 minutes and subsequently given two further washes in

Tris-saline by resuspension and recentrifugation. Washed pellets were then resuspended in a lOOul of colloidal gold- labelled goat anti-rabbit secondary antibody (Janssen

Auroprobe EM GAR 5), diluted four-fold with Tris-saline, for

1 hour at room temperature. After a further three washes by suspension and recentrifugation in Tris-saline, samples were fixed as a pellet in 2% gluteraldehyde, 0.1M sodium cacodylate buffer (pH 7.2). Samples were then processed for electron microscopy as described in section 2.2.7.

2,2.6b Immunocytochemistry on Tissue-Sections

Ultrathin liver sections were labelled with affinity purified GAP 9 antibodies as described by Ali et al

(1990). Livers were perfused with 2% paraformaldehyde/0.1% gluteraldehyde in 0.1M sodium cacodylate buffer (pH 7.2) and

3% polyvinylpyrrolidone. The tissue was left overnight in fixative at 4°C and then washed sequentially for 60 minutes in PBS and 0.5M NH4C1/PBS, dehydrated, and embedded in

Lowicryl K4M. Thin sections were mounted on Formvar/carbon-

-61- coated nickel grids, and stained with 0.Olirig/ml GAP 9 antibodies diluted in 1% skimmed milk/PBS.

Immunohistochemical localization was visualized with lOnm colloidal gold-labelled Protein A (Janssen Pharmaceuticals,

Bearse, Belgium) diluted four-fold with PBS. Thin sections were then extensively washed with PBS and contrast stained with uranyl acetate for 5 minutes and then for 5 minutes with

lead citrate. Sections were viewed in a Philips EM300 electron microscope at 60KV.

2.2.7 Electron Microscopy of Gap Junction Fractions

Untreated and antibody labelled gap junction

fractions were prepared for electron microscopy by E. Hurst of N.I.M.R. as follows. Samples were pelleted in a microfuge and fixed overnight in 2% gluteraldehyde in 0.1M sodium cacodylate buffer (pH 7.2) at 4°C. Samples were post-fixed

in 1% Os04 in cacodylate buffer and then in 1% aqueous uranyl

acetate, for 1 hour each. Following dehydration in a graded

ethanol series, samples were embedded in Araldite and

sectioned. Sections were mounted on copper grids, stained

with uranyl acetate in 75% (v/v) ethanol for 20 minutes and

then in Reynold's lead citrate for 10 minutes. Sections were

viewed in a Philips EM300 electron microscope at 60KV.

2.3 Protein Chemistry

2.3.1 Chemical Modification of Liver Gap Junction Fractions

2.3.1a Alkylation of Liver Gap Junction Fractions

Gap junction pellets were resuspended in

-62- lOOul of reducing buffer; 30mM Tris-HCl pH 8.9, 8M urea, lmM

EDTA and lOOmM DTT, and incubated for 1 hour at 37°C.

Samples were then diluted to 1.5ml with distilled water and pelleted in a microfuge using a fixed angle (90°) rotor

(12000 x g) for 10 minutes. Samples were then washed by resuspension in 1ml lmM NaHC03, lmM DTT and recentrifugation.

Reduced gap junction pellets were then alkylated by resuspension with lOOul of lOmM N-ethylmaleimide (NEM) and incubated for 30 minutes at room temperature. Alkylated samples were then washed with lmM NaHCC>3, by dilution and centrifugation, solubilized in Laemmli buffer (non-reducing) and analysed by SDS-gel eletrophoresis (2.2.3).

2.3.lb Carboxymethylation and Succinylation of Guinea Pig

Liver Gap Junctions and the Purification of Tryptic

Peptides

Gap junctions (200mg approx), prepared from 14 guinea pig livers (approx 280g of tissue) were reduced with lOOmM

DTT as described in 2.3.1a. The reduced sample was then carboxymethylated with 5-iodoacetofluorescein(IAF; Miles

Scientific) by resuspension in 500ul of 20mM MOPS (pH 6.8),

80mM KC1, lmM CaCl2, 5mM MgCl2 containing an excess of IAF

(35mg of IAF; an approx IAF:cysteine ratio of 200:1). The sample was incubated for 30 minutes at room temperature with periodic agitation, and then subjected to three cycles of washes, by resuspension in 1.5ml of NH4HC03 (pH 7.8) and recentrifugation in a microfuge. The yellow/orange sample was succinylated by resuspending the pellet in 1ml of NH4HC03

-63- (pH 7.8) containing 400ug of succinic anhydride (2:1 w/w) and incubated for 20 minutes at room temperature, maintaining the pH between 7.5-8.5 by additions of 0.5M NaOH. The gap junctions were then pelleted and washed three times with 20mM

MOPS (pH 6.8), before recarboxymethylation as described above. The sample was given a further cycle of washes in

50mM Tris-HCl (pH 6.7), and then solubilized in non-reducing

Laemmli buffer for 30 minutes at room temperature. Analysis by SDS-PAGE was performed using a preparative 12.5% gel, followed by staining (as described in 2.3.4) and the region of the gel containing the 22 and 28 KDa polypeptides was excised.

Gel slices were diced using a razor blade and suspended in 600ul of 50mM NH4HCC>3 (pH 8.5), lOOmM CaCl2 , lmM

DTT containing 5.0ug of trypsin. After 2 hours incubation at

3 7°C a further aliquot of trypsin (5.0ug) was added to the reaction mixture, which was subsequently left overnight at

37°C. The reaction was stopped by the addition of a cocktail of proteolytic inhibitors (stock solution of pepstatin

0.05mg/ml, chymostatin 0.5mg/ml, antipain 0.5mg/ml, leupeptin 0.5mg/ml, diluted 1/100; lmM PMSF) and the gel pieces compressed by centrifugation at 20,000 x g. The supernatant was collected, and the gel slices reincubated in

400ul of buffer for a further 4 hours at 37°C to recover residual peptides. After further centrifugation, the supernatants were combined and stored at -20°C. Tryptic peptides were separated by reverse-phase HPLC using a Beckman

-64- 421 HPLC instrument equipped with a Beckman 125 variable wavelength detector. Peptides were resolved using a Zorbax

C8 (4.6mm x 15cm) column (DuPont) and eluted with a 1-90%

acetonitrile gradient in 12mM NH4HCC>3 (pH 6.0) (Buffer A

=14raM NH4HC03 (pH 6.0); Buffer B = lOmM NH4HCC>3 , 90%

acetonitrile, pH 6.0) at a flow rate of lml/min. Elution was monitored at 215nm and 480nm (the latter was the estimated

E for IAF in buffer A) and 1ml fractions were collected max 1 manually in microfuge tubes. Appropriate samples were

concentrated 20-fold, diluted to 1ml with 50% aqueous TFA and

reconcentrated 20-fold by rotary evaporation. Samples were

then characterized by microsequence analysis.

2.3.2 Proteolytic Digestion of Intact and Urea-Split Rat

Liver Gap Junctions

Gap junction samples were subjected to extensive

proteolytic treatment with four different enzymes; trypsin

(Sigma), chymotrypsin, endoproteinase Lys-C (Boehringer

Mannheim) and proteinase K (Sigma). For analysis by SDS-PAGE

and Western blotting, 2.5-5.0ug of a gap junction fraction

(amount per lane) was treated with either trypsin,

chymotrypsin or proteinase K (0.2ug) or with endoproteinase

Lys-C (0.4U), in a reaction volume of 25ul (giving an approx.

enzyme to susbstrate molar ratio of 1:25). Enzymes were

added in two aliquots; the second added approx. 1-2 hours

after enzyme exposure and the reaction allowed to proceed

overnight at 37°C. Reactions were carried out in buffers of

pH optimal for enzyme activity; trypsin and chymotrypsin in

-65- PBS (pH 7.4), proteinase K in 50mM sodium phosphate (pH 8.0)

and endo' Lys-C in lOOmM NH4HCC>3 (pH 8.5). Reactions were

stopped by the addition of a cocktail of proteolytic

inhibitors (see section 2.3.1b), except for endo' Lys-C digests which were stopped by the addition of an appropriate

amount of aprotinin in lOOmM NH4HCC>3 (pH 8.5). Samples were

solubilized by the addition of an equal volume of Laemmli buffer and incubated at room temperature for 15 minutes.

Peptides (both membrane bound and free) were analysed by SDS-

PAGE in 17.5% gels. For proteolytic treatment of 'split'

junctions, intact gap junctions were treated with $M Urea dissolved in lOmM Tris (pH 10) for 2 hours at 37°C. In some

experiments split junctions were alkylated by including lOOmM

DTT during incubation with urea, followed by exposure to NEM

(as described in 2.3.1a) prior to proteolytic treatment.

2.3.3 Treatment of Membranes with Endoglycosidase F

Lateral, sinusoidal and Golgi fractions, prepared

from rat liver homogenates, were treated with endoglycosidase

F (Boehringer Mannheim), which cleaves high mannOse side chains of N-linked glycoproteins and glycolipids.

Approximately 50ug of each membrane fraction was suspended in

50ul of 0.25M sodium acetate (pH 6.0), 20mM EDTA and lOmM 2- mercaptoethanol. A further 50ul of buffer containing 0.15U

of endoglycosidase F was added to each sample. Samples were

incubated overnight at 37°C and subsequently pelleted by

centrifugation for 10 minutes in a microfuge. The

supernatants were removed and the pellets solubilized in

-66- Laemmli buffer for 3 0 minutes at room temperature. Samples were then analysed by SDS-PAGE in 12.5% gels and detection of

connexin 32 (Mr 28 KDa) carried out by Western blotting using

GAP 9 antibodies.

2.3.4 Electroelution of SDS-PAGE Separated Polypeptides

Electroelution of electrophoretically separated

connexins was carried out essentially as described by

Hunkapiller et al (1983), for the purpose of obtaining microsequence data. To avoid artifactual chemical blockage

at the N-terminus, denaturing polyacrylamide gels were

prepared one day prior to use, and pre-electrophoresed for 1-

2 hours at 1.5mA/cm. Gap junctions (150ug) prepared from

guinea pig or rat livers, were solubilized in Laemmli buffer

at room temperature for 30 minutes and electrophoresis

performed in preparative 12.5% gels with O.lmM mercaptoacetic

acid in the cathode reservior buffer. After electrophoresis,

gels were stained in aqueous 1.0% (w/v) Coomassie Brilliant

Blue R250 for 5-10 minutes and rapidly destained in water

until bands corresponding to connexins and their oligomers

were visible. Appropriate bands were excised with a razor

blade and diced in a petri dish containing elution buffer

(50mM Tris-acetate pH 7.8, 0.1% SDS). After incubation for 5

minutes in elution buffer, the gel pieces were transferred

using a spatula to the large chamber of the elution cell, and

overlayed with soaking buffer (0.2M Tris-acetate pH 7.8, 2%

SDS) until just submerged. After a further 3 hour incubation

at room temperature, elution buffer was carefully overlayed

-67- until both chambers, and their connecting crossbridge, were filled. The elution cell was placed in the elution chamber and electroelution was carried out at 100V d.c. for 16-20 hours at 4°C, and then at 150V for a further 24 hours. The elution buffer was circulated between cathode and anode chambers and changed every 4 hours. After elution of the polypeptides was complete, samples were tested for purity by

SDS-PAGE and subsequently concentrated for microsequence analysis.

2.3.5 Microsequencing

Amino terminal sequence analysis of peptides (and electroeluted proteins) was kindly carried out by

Mr. A. Harris of N.I.M.R. Sequence analysis was performed with Applied Biosystems 470A gas-phase and 477A pulsed liquid-phase peptide sequenators. PTH-amino acids were analysed on-line with Applied Biosystems 120A analysers.

Data collection and analysis were done with an Applied

Biosystems 900A module calibrated with 25pmol PTH-amino acid standards.

2.3.6 Iodination of Synthetic Peptides and Protein A

2.3.6a Iodination of Synthetic Peptides

Peptides containing intrinsic or added tyrosine 125 - residues (GAP 7-M), were lodinated by the oxidation of I catalysed by Iodo-Gen (Pierce Chemicals). Approximately

150ug of peptide, dissolved in lOOul PBS was dispersed into a microfuge tube coated with 15ug of Iodo-Gen. The reaction

-68- 125 was initiated by the addition of 150uCi of Nal and allowed to proceed at room temperature for 5 minutes. The reaction was terminated by the removal of the reaction mixture from the tube, and labelled peptide seperated from free iodine by gel filtration through a Biogel P2 column (0.5cm x 3.0cm) equilibrated in PBS. Peptide elution was monitored at E2q o * 125 An approximately 50% incorporation of I was estimated.

2.3.6b Iodination of Protein A

Approximately 150ug of protein A, dissolved in 150ul 125 of PBS was mixed with lmCi of Nal (New England Nuclear) in a glass ampoule. The reaction was initiated by the addition of 5ul of a 5mg/ml solution of Chloramine T, and terminated by the addition of 50ul of sodium metabisulphite after 20 seconds. The iodinated protein A was separated from free iodine by gel filtration using a Sephadex G25 column (1cm x

10cm) equilibrated in PBS. The elution of protein A was monitored by gamma-ray detection, and comparison of labelled protein and free iodine peaks routinely indicated a greater 125 than 90% incorporation of I. Iodinated protein A was stored at -20°C until use.

2.4 Subcellular Fractionation Procedures

2.4.1 Preparation of Rat Liver Plasma Membrane Fractions

Liver plasma membranes were prepared from male

Sprague-Dawley rats (200g) as described by Evans et al

(1980). Ten rats were sacrificed by cervical dislocation and their livers rapidly removed and washed with lmM NaHCO^.

-69- Livers were then manually perfused with 0.9% NaCl and sliced, before homogenising using a Dounce homogenizer (volume 35ml, clearance 0.12mm; Blaessig, Rochester, N.Y.). Homogenisation was performed in lmM NaHCO^ containing lmM PMSF, with 8-10 strokes of a loose fitting pestle. A final homogenate volume of 1.0 to 1.2 litres was routinely obtained from approximately 140g of starting material. The homogenate was then filtered through 2 layers of muslin, followed by centrifugation at 1000 x g (3000 r.p.m.) in a Beckham JA14 rotor. The post-nuclear supernatant was carefully collected by aspiration (for the production of sinusoidal membranes

2.4.1b) and the pellet washed twice further, by resuspension into approx. 600ml of lmM NaHCO^/PMSF and recentrifugation at

2000 x g (6000 r.p.m.). The pellets were then resuspended into 80-85% (w/v) sucrose, to give a volume of 150-160ml and a final sucrose concentration of approx. 60% (w/v). The sample was carefully loaded onto 12 discontinuous sucrose

(w/v) gradients comprising of the sample (13ml), 54% sucrose

(7ml), 48% sucrose (6ml), 43% sucrose (6ml) and 8% sucrose

(5ml), prepared in Beckman 37.5ml polyallomer tubes. The sample was fractionated by centrifugation at 100,000 x g in a

Beckman SW28 rotor for 2 */2 hours and the parent plasma membrane fractions, equilibrated at the 48%-43% and the 43%-

8% interfaces, collected. These fractions were washed separately by dilution to 250ml with lmM NaHC03/PMSF and pelleted at 10,000 x g for 30 minutes in a Beckman JA14 rotor.

-70- 2.4.1a Subfractionation of Plasma Membranes into Lateral and

Canalicular-Rich Membrane Fractions

Subfractionation of plasma membranes into lateral

(LatA and Latg) and canalicular-rich membrane fractions was performed as described by Ali et al (1990). Plasma membranes were resuspended in approx. 20-30ml of 8% (w/v) sucrose and further homogenized using a Dounce homogenizer with 20 strokes of a tight-fitting pestle (clearance 0.07mm). The homogenized sample was loaded onto discontiuous sucrose (w/v) gradients comprising of 8% sucrose (the sample), 37% sucrose,

43% sucrose and 49% sucrose. After centrifugation for 2 hours at 100,000 x g using a Beckman SW28 rotor, canalicular, lateral A and lateral B plasma membrane fractions were collected at the 8/37%, 37/43% and 43/49% sucrose interphases respectively, as described by Wisher and Evans (1975).

Further purification of canalicular membranes was achieved as follows (Ali et al., 1990). Crude canalicular membranes collected from the 8/37% interphase were pelleted (100,000 x g for 30 minutes) and resuspended in approx. 30ml of 8% (w/v) sucrose. The sample was then sonicated (6 x 15s bursts interspersed with 30s cooling periods in ice, 2um amplitude,

M.S.E. Soniprep with micro-tip probe) and loaded onto continuous gradients formed by mixing 12ml each of 10% (w/v) and 3 0% (w/v) Nycodenz (Nycomed U.K. Sheldon, Birmingham,

U.K.) dissolved in 0.25M sucrose. A 4ml 60% (w/v) sucrose cushion supported the gradients. After centrifugation at

100,000 x g for 2 hours (Beckman SW28 rotor), highly purified

-71- canalicular membranes, canalicular-depleted lateral A and canalicular-depleted lateral B membranes were visible as discrete bands in the gradient. These were collected manually, pelleted and stored in 8% (w/v) sucrose at -20°C.

2.4.1b Preparation of Liver Sinusoidal Membranes

Sinusoidal plasma membrane fractions were prepared from the post-nuclear supernatant (approx. 600-700ml) described in section 2.4.1, according to Wisher and Evans

(1975). Approx. 360ml of the supernatant was mixed with 40ml of 80% (w/v) sucrose solution generating a suspension with a final concentration of 8% (w/v). This suspension was centrifuged at 35,000 r.p.m. for 1 hour in a Beckman Type 45 rotor. The pellets were resuspended in 8% sucrose/5mM Tris-

HCl (pH 8.0) (sucrose/tris), with 5 strokes of an intermediate fitting Dounce homogenizer to give a final volume of approx. 200ml, and subsequently centrifuged at

19,500 r.p.m., for 15 minutes in a Beckman Type 30 rotor.

The top half of the supernatant was carefully discarded and the remaining portion carefully collected and stored on ice.

The pellets (containing mitochondria and lysosomes) were resuspended in 100ml of sucrose/tris and recentrifuged as before. The corresponding portion of the supernatant was collected and pooled with the primary post- mitochondrial/lysosomal supernatant, giving a final volume of approx. 150ml. The supernatant was then centrifuged at

30,000 r.p.m. for 1 hour in a Beckman Type 45 rotor and the pellets obtained were resuspended in twice the volume of 72%

-72- (w/v) sucrose giving a 40-60ml suspension in 47-48% (w/v) sucrose. Finally, the suspension was loaded as part of 38ml discontinuous sucrose (w/v) gradients consisting of 10ml of the sample (47%), 19ml of 39% sucrose and 8ml of 8% sucrose solution. The gradients were centrifuged at 20,000 r.p.m. overnight in a Beckman SW28 rotor. Sinunoidal membranes (M-

L) were collected at the 39/8% interface and residual microsomal membranes (M-Res) were collected at the 47/39% interface. Membranes were pelleted at 100,000 x g for 1 hour, resuspended in 8% (w/v) sucrose, and stored at -20°C.

2.4.2 Preparation of Hepatic Gap Junctions

Gap junction enriched fractions were prepared from the livers of male Sprague-Dawley rats (200g) and male

Hartley guinea pigs (250g), using the alkali-extraction procedure of Hertzberg (1984). The parent plasma membrane fraction was prepared as described in section 2.4.1 with the exception that during /■ the preparation of guinea pig membranes, fewer strokes (7-8) with the loose fitting pestle were required during the homogenization. Gap junctions were prepared from the membranes equilibrating 48/43% interface, or the combined 48-43% and 43-8% interfaces, if further subfractionation was not scheduled (see Fig* 2.3).

The plasma membranes recovered from approximately

140g of liver tissue were divided equally into two Beckman

JA20 thick-walled polycarbonate tubes, as a 5-7ml suspension in lmM NaHCO^/PMSF. An equal volume of 40mM NaOH was added

-73- Fig.2.3 Preparation Of Subcellular Fractions From Rat Liver

The flow diagram illustrates the fractionation and further sub-fractionation of liver homogenates giving rise to the preparation of enriched plasma membrane and intracellular membrane fractions. TJ "D UJ UJ

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These suspensions were then sonicated using a MSE Soniprep

150 (small probe) at 16 KHz for 10-20 seconds during which,

the appearance of the suspensions changed from turbid to

clear. A further volume of 20mM NaOH was added to each tube,

(final volume of 25ml). Crude gap junctions were then

pelleted by centrifugation at 17,600 x g for 15 minutes in a

Beckman JA20 rotor and given a further alkali wash by

resuspension in 20mM NaOH and recentrifugation. The pellets

were resuspended in 2ml of lmM NaHC03/PMSF and a further 10ml

of 54% (w/v) sucrose solution was then added. The resulting

suspension (12ral of an approximately 46% (w/v) sucrose

solution) was loaded as part of two discontinuous sucrose

(w/v) gradients, prepared in 17ml Beckman ultra-clear

polyallomer tubes. These gradients comprised of a 54%

sucrose cushion (4ml), the sample (6ml), 30% sucrose (4ml)

and 3ml of lmM NaHCC>3/PMSF. Centrifugation was carried out

at 100,000 x g for 90 minutes using a Beckman SW28 rotor.

Gap junctions were collected as a whitish band that

equilibrated at the 46%-30% (w/v) sucrose interface, and

analysed for protein using the method of Bradford (1965).

The quality of preparations were assessed by SDS-PAGE as

described in section 2.2.3.

2.4.3 Preparation of Endosome and Endosome-Depleted Golgi

Membranes

Endocytic membranes (Evans and Flint 198 5) and

endosome-depleted Golgi membranes (Evans 1985), were prepared

-75- from post-nuclear supernatants. Two rat livers (~15-20g net weight) were Dounce homogenized in 8% (w/v) sucrose using 10 strokes of a loose fitting pestle. Approx. 3 volumes of sucrose were used per gram of tissue. The homogenate was then filtered through nylon gauze (50-100 mesh) and re homogenized by 6 strokes with a tight-fitting pestle

(clearance 0.07mm). The re-homogenized filtrate was centrifuged at 1000 x g for 10 minutes in a Beckman JA20 rotor and the collected supernatant stored on ice. The nuclear pellets were washed twice further by resuspension (in

1.5 vol/g tissue of 8% (w/v) sucrose) and recentrifuged, and the post-nuclear supernatants collected and pooled (vol.-

100ml). The combined supernatants were centrifuged at 33,000 x g for 8 minutes at half-maximal acceleration (Beckman L5-

50) in a Beckman Type 30 rotor. After centrifugation, the supernatants were carefully collected, taking care not to disturb the loose material resting on the multilaminate pellet. The loose material was collected separately and stored on ice, and was subsequently used to prepare Golgi membranes (endome-depleted). The supernatants were layered onto a continuous sucrose (w/v) gradient comprising of a 1ml

70% sucrose cushion, 5ml of 43% sucrose and a continuous layer made by mixing 7.5ml each of 40% and 10% sucrose.

After centrifugation at 100,000 x g for 4 hours (Beckman

SW28), the gradients were unloaded as 1ml fractions and those . . 3 corresponding to densities between 1.095-1.117g/cm and 3 1.117-1.135g/cm collected and pooled as two endosomal fractions (D and E respectively). Fractions D and E were

-76- further subfractionated on continuous Nycodenz gradients contructed from a mixture of 6ml each of 27.6% and 13.8%

(w/v) Nycodenz, dissolved in 8% (w/v) sucrose and water respectively, and centrifuged at 100,000 x g (Beckman SW28 rotor) for 2 hours. Fractions corresponding to 'early',

'late', and receptor enriched endosomes were collected from fractions falling within the density ranges of 1.364-1.370, 3 1.358-1.364 and 1.373-1.390 g/cm respectively. Endosomal membranes were pelleted at 100,000 x g and stored at -20°C in

8% (w/v) sucrose.

Golgi fractions were prepared by resuspending the loose material, collected from the top of the multilaminate pellet, in approx. 50ml of heavy sucrose (80% w/v), generating a final sucrose concentration of 39% (w/v). The sample was then loaded as part of a discontinuous sucrose

(w/v) gradient comprising of equal volumes of 39% sucrose

(the sample), 29.5% sucrose, 20.5% sucrose and 8% sucrose.

After centrifugation at 100,000 x g (Beckman SW28 rotor) for

3 hours, three Golgi fractions were collected from the three sucrose interphases, corresponding to 'light', 'intermediate' and 'heavy' membranes. Membranes were pelleted and stored at

—20°C in 8% (w/v) sucrose.

2.4.4 Preparation of Whole Tissue Homogenates

Various organs from mice, chick, guinea pig and rat

embryos (18 day gestation) were excised, rapidly frozen in

powdered dry ice and stored at -70°C or homogenized

-77- immediately. Homogenization was performed in ice-cold lmM Na

HC03 containing a cocktail of proteolytic inhibitors (stock

solution of pepstatin 0.05mg/ml; chymostatin 0.5mg/ml;

antipain 0.50 I mg/ml; leupeptin 0.5mg/ml; diluted 1/100),

using an Ultra-Turrax homogenizer with 3 x 5 second bursts.

In some cases homogenates were alkali-extracted by the

addition of an equal volume of 40mM NaOH, followed by mild

sonication to shear released DNA. Alkali-extracted material was pelleted at 17,600 x g in a Beckman JA20 rotor and washed

in lmM NaHC03/PMSF by resuspension and recentrifugation.

Both non-alkali and alkali-extracted material was assayed for

protein using the method of Bradford (1965), before analysis

by gel electrophoresis and Western blotting.

2.4.5 Preparation of Rat Liver LysOsomes

Rat liver lysosomes were prepared essentially as

described by Wattiaux et al (1978). Four rat livers

(obtained from rats fasted overnight) were removed and

perfused with cold PBS and homogenised with 8 strokes of a

loose fitting Dounce homogeniser. An approx. homogenate volume of 200ml was obtained from 40-50g of liver tissue.

The homogenate was then filtered through 2 layers of nylon

gauze and re-homogenized with 5 strokes of an intermediate

Dounce homogeniser. The filtrate was centrifuged at 3500

r.p.m. (1000 x g) for 10 minutes using a Beckman JA20 rotor.

The supernatants were collected and stored on ice and the

pellets washed, by resuspension in 100ml of 8% (w/v) sucrose

and recentrifugation. The two supernatants were combined,

-78- giving a total volume of approx. 200ml, and then centrifuged at 6200 r.p.m. (3000 x g) for 10 minutes in a Beckman JA20 rotor. The supernatants was collected and recentrifuged at

11,500 r.p.m. (10,000 x g) for 20 minutes in a Beckman JA20 rotor, and the pellets washed by resuspension in 80ml of 8%

(w/v) sucrose and recentrifugation. The washed microsomal pellets were resuspended in 45% (w/v) Nycondenz, dissolved in

8% (w/v) sucrose, to give a final volume of 15ml approx. and density of not less than 1.18g/ml. Discontinuous Nycodenz

(w/v) gradients were prepared with 5ml of sample (45%),

6ml of 30% Nycodenz, 6ml of 26% Nycodenz, 8ml of 24%

Nycodenz, 8ml of 19% Nycodenz and 4ml of distilled water.

The gradients were centrifuged at 28,000 r.p.m. (100,000 x g) for 2 V hours in a Beckman SW28 rotor. Lysosome-enriched fractions were collected from the distilled water/19%

Nycodenz, 19/24% and 24/26% interfaces. The heaviest fraction (24/26% interface) contained the greatest enrichment of acid phosphatase activity compared to the homogenate.

Pellets were resuspended in 8% (w/v) sucrose and stored at -

2 0 ° C .

All centrifugation steps were carried out at 4°C and the protein concentrations for all fractions determined by the method of Bradford (1965).

-79- CHAPTER 3

PRODUCTION AND CHARACTERISATION OF ANTI-PEPTIDE ANTISERA DIRECTED TOWARDS SELECTED SEQUENCES OF CONNEXIN 32*

3.1 Introduction

3.1.1 The Development Of Anti-Peptide Antibody Technology

The first demonstration that an antiserum raised towards an artificial antigen possessed immunological reactivity towards a natural antigen was made by Goebel in 1938. He showed that diazotized p-aminobenzyl cellubiuronic acid conjugated to protein could elicit an immunological response producing a serum capable of immunoprecipitating type III polysaccharide and agglutinating typ^ III pneumococci. This approach was later extended to proteins by the demonstration that an antiserum raised egainst the C-terminal hexapeptide of the Tobacco Mosaic

Virus coat protein conjugated to BSA, was capable of immunoprecipitating infectious Tobacco Mosaic Virus (Anderer,

1963, Anderer and Schlumberger, 1965).

Anti-peptide antibody technology, despite its early

successes, failed to command general application due to a widely

held preconception that the production of anti-peptide antisera,

with reactivity towards the native parent protein, was possible

only if two criteria were fulfilled. Firstly, it was assumed

that prior knowledge of the regions of the polypeptide

corresponding to antigenic determinants was necessary. This

view developed mainly from the investigations with myoglobin and

-80- lysozyme (Atassi, 1975; Atassi and Lee, 1978) which showed that proteins contain a limited number of antigenic determinants and that only peptides corresponding to those regions could serve as suitable immunogens. These investigations also suggested that antigenic determinants on proteins were either 'sequential', composed of a linear stretch of amino acids, or 'assembled', comprising of amino acids located at discrete points on the polypeptide chain, brought into spatial juxtaposition as a consequence of protein folding. Secondly, it was believed that the antigenic determinant on the native protein and the corresponding peptide used for immunization must assume similar conformations. Experiments emphasizing this point were perfprmed on lysozyme and its 'loop' peptide, corresponding to amino acid residues 60-83 and containing a disulphide bridge

(Arnon and Sela 1969; Arnon et al., 1971). Antisera raised against native lysozyme reacted with the free 'loop' peptide and reciprocally, antisera prepared against the 'loop' peptide reacted with native lysozyme and the native peptide, but not with the reduced open chain peptide. It appeared therefore, that the antigenic determinant contained within the loop peptide was dependent upon the intact conformation of the loop. Such experiments created the environment that anti-peptide antisera weire unlikely to recognize their native parent proteins, as the free peptides were likely to assume conformations different from the corresponding regions in the proteins.

These ideas pertained until the demonstration by Walter et al (1980) that antisera to synthetic peptides corresponding

-81- to the amino and carboxyl termini of the SV40 virus large T antigen, were capable of immunoprecipitating native large T antigen. The novelty of this report lay in that sequence selection was based on theoretical considerations alone and that neither the three dimensional structure nor the antigenic determinants of large T antigen were known. This investigation and others which followed (Vyas et al., 1980; Audibert et al.,

1981), illustrated that no prior knowledge of the tertiary structure of a protein was necessary for the production of anti peptide antibodies with specificity towards its native form.

Consequently, the use of antibodies generated against synthetic peptides has now become widespread.

3.1.2 Selection Of Peptides For Immunization

The potential utility of antibodies which recognize selected regions of a protein is manifest. Their production however, is not without its difficulties. The most important and often the most difficult consideration in the production of anti-peptide antisera is the selection of a sequence of amino acids from a protein that will elicit antibodies that recognize its native and/or denatured form. Several attempts have been made to develop a set of criteria that could serve this aim. In short, there are no universal rules for the selection of immunogenic sequences, but there are guidelines that can improve the likelihood of producing anti-peptide antibodies with anti protein activity. Among these are the selection of sequences from the polypeptide termini, which are often conformationally less restricted regions of proteins. In an analysis of 103

-82- oligopeptide immunogens reported from between 1980 and 1983, all peptides corresponding to amino and carboxyl termini (10 and 24 peptides respectively) produced antisera with reactivity towards the native parent protein (Palfreyman et al., 1984). As antibodies recognize determinants on the surface of proteins, which are sterically accessible and predominantly hydrophilic, sequences from hydrophilic regions of proteins are often selected for immunization. For proteins of known primary structure, regions of high hydrophilicity can be determined by hydropathy analysis using a variety of algorithms (table 3.1).

This is useful when applied to integral membrane proteins as potential membrane spanning regions can be identified and omitted from selection. More recently, the regions of proteins with high flexibility (estimated from temperature factors derived from high-resolution X-ray crystal structures) were reported to coincide with known antigenic determinants (Westhof et al., 1984). In the case of the Tobacco Mosaic Virus coat protein, seven known epitopes recognized by antibodies against the native protein corresponded to flexibility peaks.

Furthermore, anti-peptide antibodies raised to selected sequences of myohaemerythrin gave the best anti-protein activity when these corresponded to sequences from regions of high local flexibility (Taiper et al., 1984). Karplus and Schultz (1985) have developed an algorithm for predicting the flexible regions of polypeptides of known sequence (table 3.1). The flexibility and hydropathicity profiles of connexin 32 are illustrated in

Fig. 3.1. It is noteworthy that the four hydrophobic segments of connexin 32, proposed to span the lipid bilayer (M1-M4), co-

-83- co E s W h HYDROPATHY AND FLEXIBILITY INDICES OF NATURAL AMINO ACIDS « O E W W PL « H O O H H < X < Q P 2 § z o h m M C O C in VO r- CO r-H 03 o r-H CM O C n i 0 0 03 r-H r-H i—H t-H iH lO i—H r* H r CN O rH — i TJ 03 TJ co w DC £ w x: p o M XI O O PC Pd Oi 6- DC 2 > P pH Z CL < >-• O CL CL 0 0 a O O o , I— • •o cr> m c CO O -P «H «c P > M 43 Q H r +3 ' Z P O H DC CL >H CO s &4 0 0 d o o 0 I . i - •rH •rH H • d T3 d d M iH 43 (C U M 0 0 w C| 0 C| 0 d a u u 0 0 Ci U 4 8 - (d 03 in 4-H p o Cij oo > d o CO E-* < DC >-• T3 fH S W XJ a PL Q XJ H CO 43 Ci 0 o 3 CL d CO N 0 d H - H • H ■ *44 *44 44 I—I 43 43 0 c Cl 0 o 0 u u w d Oi 0 X u 0 c

0 •rH -rH 144 H • •H •iH •rH H • -rH •*H d 'O r H • •rH d T3 d T3 TS *44 *44 *44 O *44 H 43 43 t-H 43 43 43 * < 43 0 Cl Ci > 03 o d u 0 d > 0 0 0 O 0 0 1 > rH o 0 iCl Ci d 0 Ci 0 d CO d d X u d O 01 0 0 u o 0 d H • -rH H - r Ci •rH H - H - H * H - • H - •o d T3 d H - *44 d d e* 05 d *14 x 43 43 iH 43 r4 - - o Cl 0 0 d O d d 0 d 0 d 0 O 0 0 0 X 0 0 3d 03 o 0 d 0 h d d d 03 Cl E 03 03 • d d •rH *r4 •rH H • •rH •rH •rH •rH d T d d 73 •rH •o 43 d 43 *44 *44 Cl 0 0 0 Cl 0 E 0 u d 0 d 03 Cl o 0 O 0 PL 0 u 0 d d 03 d 0 u d Oi C| > E d 0 0 o 0 o d O O1 0 Ci 0 3 • Fig.3.1

(TOP) Flexibility Profile of Connexin 32 According to

Karplus and Schultz (1985^.

(BOTTOM) Hydrooathicitv Profile of Connexin 3 2 According to

Kvte and Doolittle (1982).

(adapted from Nicholson and Zhang (1988).

Bars along bottom scale indicate regions of predicted beta- turns. Hydropathicity and flexibility coefficients have been plotted with respect to amino acid sequence (Paul, 1986). Note that there are four prominent predicted hydrophobic areas which co-align with four prominent predicted rigid areas (M1-M4). UCI V * V U I Y IT C IU H P O R D Y H

ROPHO8CI . Y IT 8IC O H P O 0R Y H RIGIDITY FLEXIBILITY 0.9 0 1 1 1 . . . . 0 2 1 8 * - - - _2i . e_*2Li 0 8 2 0 6 2 - 0 4 2 0 2 2 0 0 2 ' 0 8 1 0 6 1 0 4 1 0 2 1 0 0 1 0 8 0 6 0 4 0 2 i i _j 0 5 _ * « » j . i j » . « ■ 2M3 M2 mio cd Number Acid ino Am m o cd Number Acid Ami no

10 - I

_ 0

5 8 - i 0 5 1 _ i —. ga g > .— i— i i_a a _ L * ■ ■ 200 M4 i ...

!50 i.3.1 3 Fig. __ 0 0 3 s align with four regions of predicted rigidity. Novotny and

Haber (1986) however, have proposed that antigenicity does not correlate with chain mobility (flexibility), but rather with the surface exposure of amino acid residues such that peptides corresponding to regions which protrude into the solvent serve as good immunogens. Finally, Parker et al (1986) determined that hydrophilicity of amino acid side chains correlated best with antigenicity when considered in concert with other factors such as chain flexibility and surface exposure.

With the aim of generating a panel of defined immuno

logical reagents applicable to both biochemical and functional

studies (i.e. with specificity towards both native and denatured protein), a panel of anti-peptide antibodies were generated to

selected sequences of rat liver connexin 32. Sequence selection was based on the two-dimensional topographical model of connexin

32 in the junctional membrane, constructed by a combination of

structural prediction (Paul, 1986) and biochemistry (Zimmer et

al., 1987). Sequences were chosen from predicted non-membrane

embedded regions of connexin 32 including amino and carboxyl

termini (Fig. 3.2). Owing to the relatively small size of these

non-membrane embedded domains, sequence selection was

comparatively straight forward. However, for larger membrane

proteins with extensive non-membrane embedded domains (e.g. the 2+ . ryanodine receptor, Ca -ATPase), application of the guidelines

outlined above reduces the area of choice to those regions

predicted to contain antigenic sequences. The following

sections describe the generation, characterization and

-86- Fig.3.2 Topographical localization of synthetic peptides?

Diagrammatic representation of connexin 32 in association with the lipid bilayer according to Zimmer et al (1987) and showing the full amino acid sequence (Paul, 1986). Alignments of synthetic peptides are shown. qJ2^>QOL1->C0 oh u H- I LL i < O o'- 2 %i 1« jj/ V CC**">LLI

< \ ■/ V / i i/>l . u _ < U _ _ i L L . > _ ! > - > J \ / ' ' \ / \ y J UJ CC' 03 H .

^ o i - /

°-U. ODlLiT<

n X q;Z>0

+ 2 Z ^ h ''3

C\J CO O) LJL

- 87- properties of a panel of anti-peptide antibodies with specificity towards connexin 32. The remainder of this thesis describes their use in a variety of biochemical studies.

3.2 Results

3.2.1 Production Of Anti-Peptide Antisera

Polyclonal antisera were generated in rabbits against synthetic peptides, corresponding to selected sequences of connexin 32, conjugated to KLH (Fig. 3.2). The identity of these peptides, their sequences and residue assignments are shown in table 3.2.

A total of 12 peptides, accounting for approximately 72% of the predicted non-membrane associated regions of connexin 32, were synthesized. Rabbits were immunized with peptide-KLH conjugates as described in the materials and methods (sections

2.1.2 and 2.1.3). A minimum of two rabbits were immunized per peptide and with particular peptides (GAP 8 and GAP 8-M) as many as four rabbits were used. An antiserum generated against the

N-terminal peptide GAP 3 has been described previously (Zervos et al. 1985, Zimmer et al. 1987) and has been superceded by antisera raised against the larger peptide GAP 10. Peptides GAP

5 and GAP 6, derived from sequences within the same putative extracellular loop, were injected as mono-peptide conjugates and as a dipeptide conjugate mixture (GAP 5+6). Peptides GAP 7-M and GAP 8-M were modified derivatives of their respective parent peptides, with amino-terminal extensions of three amino acids.

Finally, two discontinuous or 'designer peptides7 corresponding

-88- to sequences within the presumptive intracellular loop were also

synthesized. Peptide DES 1 was constructed from two lysine-rich segments and peptide DES 2 from three segments, rich in histidines.

3.2.2 Affinity Purification of Anti-Peptide Antisera

For their eventual use in structural and functional studies, all experimentally useful antisera were affinity purified against their respective peptides. The antibody elution conditions, and recoveries are summarized in table 3.3.

The optimal conditions for releasing anti-peptide lgG from affinity columns varied with antisera. For example, the

release of GAP 7-M lgG from its corresponding peptide was achieved using conditions of extreme pH, but always resulted in

the loss of immunoreactivity. The recovery of immunoreactive

lgG during affinity purification was often very low and

decreased with the continued use of the affinity columns. This

suggested that a fraction of the anti-peptide lgG population within the antisera was binding irreversibly to the affinity matrix. The recovery values for different antisera was

therefore not a true reflection of the anti-peptide antibody

titre within the crude serum. A typical affinity purification

experiment is illustrated in Fig. 3.3 for the DES 1 antiserum,

producing a reagent that recognised native and denatured forms

of connexin 32 (see below).

3.2.3 Characterization Of Antisera

Anti-peptide antisera were assessed for reactivity

-89- Table 3.2 properties of antipeptioe antisera generated to connexin 32 0 9 - - >1 p TJ •H I > 0 •H 73 E a p 0 \ d u P tn I d Di O d § 0 d •rH O H M •H U CM P d o M 0 p d d P d CO 0 0 •H 0 p p -H O 0 73 d O «H d P E •<—*•H p CM CN « Jm (X d ro CO >1 fl) C d rH CX 0 0 73 •H • p 0 d 0 P /•~N d d di •H a ✓“n d 0 •H «H p P 0) c 0 -H TJ 0 •H Pm o •H P 0) 0 X| i •H p d B M 01 d •rt p P a P B d d c x P u P d d •iH cx in q P o d -H co m p • >1 < • p • Cm d 7 3 p CM p d 00 Cm CO d 0 0 *H P 0 d rH 0 B P d 0 •H SC 0 SC E 73 U 0 0 •H P a e = = •H cx in O - d P d d co in p • P P Cn •H P 0 co d w • d 0 0 d M E d d ■H •H 0 o o <—1 O rH 0 aj «— O) p H *H P \ *H P X c x d Cn a d P CX »—I P (X 1—| p 0 d u M *H d d d d •H CO 00 73 •H •H P 73 2: o O 52 O £ d CM 0 0 P d d o rH O 0 > W a d •H a o X <-* CM S rH CO •H M ss o 1—( *»» X sc >1 0) d o d O M P d p H 0 •H X •H P d 0 P o cx 1 73 O P p 0 d M rH d d 0 0 o CX O d d 0 >1 •H 0 0 u >1 rH P 2 X p *«H d P d d *H r H p 0 d H • • (0 d u • 73 d W 0 0 d CO 0 0 -rH >1 H d d • -H «-H 73 d •iH 0 P d O •H E •H d •H P P X| X| P rH 73 P •H 0 0 u U M X d d P p P d 0 0 O rH I 0 0 d d 0 Xt > CO rH rH H i-* CT> •H O 2

- 91 - Fig.3,3 Affinity purification of DES 1 antibodies

A two fold concentrated gamma-globulin fraction (3.5ml) from rabbits injected with DES 1-KLH, was recycled through a DES l-

Sepharose affinity column. The elution profile and the activity present in each peak, determined by Western blotting, is shown.

Fractions (RLGJ: rat liver gap junctions and PM: liver plasma membranes) were analysed by 10% SDS-PAGE and the immunoreplicas probed with 1/10 dilution of the PBS eluted material, the total material eluted by 2M KC1, and the 0.1M propionic acid eluted material at 11 .ug/ml (diluted 1/100). Activity towards connexin

32 monomeric and dimeric forms (Mr 27 & 47 KDa respectively) was restricted to the 0.1M propionic acid eluted material.

Approximately 0.5mg of anti-DES 1 lgG was purified in this preparation. PROTEIN (E280) 0-3- 0-2 20 0 10 - 1 - - PBS I LJ PM RLGJ 15 -92 -92 - RCIN NUMBER FRACTION M C 01 POINC ACID PROPIONIC 0.1M KCL 2M LJ M LJ PM RLGJ PM RLGJ \ I 30 D 45 r kDa Mr. -115 0 3 r 8 4 - 60 towards their respective unconjugated peptides and towards connexin 32. The results of some of these experiments are summarised in table 3-2- All antisera generated contained reactivity towards their respective native peptides as assessed by ELISA. Seven antisera (including anti-GAP 3) displayed reactivity towards connexin 32 and its oligomers, as demonstrated by Western blotting (Fig. 3.4). The absence of immunological reactivity towards connexin 32 (assessed by

Western blotting and immunocytochemical approaches) in certain antisera containing anti-peptide activity, may possibly be attributed to the production of immunoglobulins recognizing conformational determinants on the peptide not assumed by the corresponding region in the parent protein. The anti-peptide reactivities of antisera with specificity towards connexin 3 2 are shown in Fig. 3.5. These dot blots illustrate the specificity of the anti-peptide antisera towards their antigen.

Little or no cross-reactivity was observed to non-immunizing peptides with the exception of GAP 11 antibodies, which also cross-reacted with peptide GAP 6. However, since the entire sequence of GAP 6 is contained within the peptide GAP 11, this observed cross-reactivity might have been expected.

Interestingly, GAP (5+6) antibodies reacted with peptide GAP 6 only, containing little or no reactivity towards peptides GAP 5 and GAP 11. Taken together, these observations might imply that whereas the GAP 11 antiserum portrays sequence-related immunological activity, the specificity of the GAP (5+6 antiserum appears to be directed towards a conformational determinant on the peptide GAP 6, not adopted within peptide GAP

-93- Fig.3.4 Interaction of anti-peptide antisera with SDS-

denatured connexin 32.

Rat liver gap junctions were analysed by 12.5% SDS-PAGE and

Western blotting. CBB; 2.5ug of a gap junction fraction stained with Coomassie brilliant blue. Similar quantities were electroblotted onto nitrocellose and probed with the antisera as shown. Antiserum dilutions of 1/25-1/50 were used.

Antisera recognized connexin 32 (Mr =28 KDa in 12.5% gels) and its multimeric forms i.e. dimer = 54 KDa and presumptive trimer

80 KDa. P.S. : pre-immune serum. All pre-immune sera gave negative results, and peptides (native) were capable of competitively inhibiting reactivity to connexin 32 (not shown). ? / £ * & 4 Mr. KDa c> C5- cT

200 -

9 2 . 5 -

6 9 - 4 54K 46 - I

30 - • + m -

21 -

14.3 -

- 94 - Fig.3.5 Interaction of anti-peptide antisera with free peptides

Uncoupled and native peptides were absorbed onto nitrocellulose strips in two loadings of lOOng and l.Oug each. These were probed with the crude antisera shown, at a dilution of 1/100, 125 and visualized by autoradiography with I-protein A. All antisera used had previously demonstrated positive reactivity towards connexin 3 2 by Western blotting. ANTIBODY

PEPTIDE O) ^ /\ vj) ^

O Q > Q > G >

GAP-3

GAP-5

GAP-6

GAP 7-M

GAP 8-M

GAP-9

DES-1

GAP-10

GAP-11

-95 - 11. Alternatively, the native conformation of the peptide GAP

11 may render the region corresponding to GAP 6 inaccessible to antibody binding. Similarly, the entire sequence of GAP 3 (with the exception of Gly 21) is contained within the peptide GAP 10.

Antisera raised to GAP 10 however, in contrast to the GAP 11/GAP

6 phenomenon, failed to cross-react with GAP 3. This absence of cross-reactivity suggests that antibodies elicited by GAP 10 recognize a determinant containing amino acid residues 1-5 (see table 3.2).

3.2.4 Immunochemistry

To assess the anti-protein reactivity of these site- directed antisera, experiments were performed to determine if the anti-peptide antibodies recognized connexin 32 when organized as a channel within the gap junction. Liver sections were labelled with affinity purified anti-peptide reagents and visualized indirectly as described in the materials and methods

(section 2.2.5). A variety of embedding and fixation procedures were tested, but optimal labelling was achieved using frozen, hydrated and unfixed liver (Fig. 3.6). Some success was also achieved using paraformaldehyde fixed, wax-embedded liver tissue

(Figs. 3.7 & 3.8). A characteristic punctate staining pattern was observed using antibodies directed against putative cytoplasmic regions of connexin 32 only, consistent with the concept that the extracellular domains of connexin 32 are not generally accessible to antibodies in intact gap junctions. The punctate pattern of staining is characteristic of immunolocalization to junctional membranes. This is seen more

-96- Fig.3.6 Indirect iitununofluorescent histochemical localization to

frozen liver sections.

Sections were probed with affinity purified antibodies as shown and antibody localization visualized by FITC-goat anti-rabbit conjugate. The punctate staining, at points of cdll-cell contact, is characteristic of labelling of gap junctions.

Bottom right shows bright field. Mag. x 60. r a f t

- 97 - Fig.3,7 Indirect immunoperoxidase localization in wax-

embedded f paraformaldehyde fixed liver.

Sections were probed with affinity purified GAP 9 and DES 1 antibodies and visualized by peroxide conjugated second antibody. These antibodies, raised to putative cytoplasmic domains, display a punctate pattern of staining, as observed in frozen sections. The absence of staining at intracellular membranes, and membranes facing the sinusoids and bile canaliculi, is evident. Mag. x 60. •* LIVER clearly in Fig. 3.8, which illustrates fluorescent antibody localization to discrete regions of the hepatocyte's lateral plasma membrane at points of cell-cell contact. Little or no labelling of intracellular structures or membranes which border the blood sinusoids at the space of Disse and the bile canaliculi was observed.

To demonstrate that the observed immunolocalization was to morphologically identifiable gap junctions, and not to other

junctional complexes, Lowicryl-embedded liver sections were stained with affinity purified GAP 9 antibodies, followed by a secondary antibody-colloidal gold conjugate (Fig. 3.9).

Electron microscopy identified gap junctions that were specifically decorated with gold particles. Intracellular organelles and the plasma membrane flanking the gap junction were not labelled.

3.2.5, Inhibition Of Intercellular Communication

In collaboration with Professor A.E. Warner (University

College London), the efficacy of the anti-peptide antibodies as

functional inhibitors of intercellular communication was

assessed by an in vitro dye coupling assay (Warner et al. 1984).

Primary cell cultures (see below) were co^microinjected with

affinity purified antibody and the fluorescent dye Lucifer

Yellow (Stewart 1978). The spread of dye, indicating the level

of intercellular communication was then observed. In double

blind experiments, DES 1 antibodies were observed to block dye

transfer between murine embryonic hepatocytes, but failed to do

-99- Fig.3:8 Indirect immunofluorescent localization in wax-

embedded f paraformaldehyde fixed liver.

Sections were labelled with affinity purified GAP 9 antibodies and localization visualized with FITC-goat anti rabbit secondary antibody. Both photographs are 6 x lum assembled projections, obtained by confocal microscopy at different areas on the Same section. Discrete labelling at points of cell-cell contact was observed characteristic of localization to gap junctions. Mag. x 100. LIVER GAP-9

-100 - Fig.3.9 Immunocrold localization in ultrathin liver sections.

Ultrathin liver sections, obtained from Lowicryl embedded tissue, were probed with affinity purified GAP 9 antibodies, and immunolocalization visualized by 5nm gold conjugated goat anti rabbit secondary antibody labelling. Gold particles were specifically localized to, or near the gap junction (GJ) and not to other intracellular organelles (M = mitochondrion and E.R. = endoplasmic reticulum) I Q.

< M r ? . : o ■V#?^ -? * hij.'rf . i. V-W»»' ^ •>'_' ■». ..'V ...^ . I

M kbSs h oc LU > l*. r'-* . .■*'>*'

-101 - so between cells derived from chick anterior mesenchyme. It appears therefore that DES 1 antibodies abrogate gap junction permeability in a species specific manner. Although preliminary, these experiments provide an excellent platform for future investigations utilizing these site-directed reagents to define the regions of connexin 32 controlling channel permeability.

3.3 Discussion

The specificity of antibody-antigen interaction has been widely exploited in biology. The advent of monoclonal and anti peptide antibody technology has given rise to more refined and definitive antisera. Although monoclonal reagents possess a homogeneous specificity, the site and nature of the epitope on the protein antigen is often unknown. However, with polyclonal anti-peptide antibodies the site on the parent protein to which antibodies are directed is pre-determined.

Anti-peptide antibodies are generally applicable to all biochemical techniques Which use conventional anti-protein antibodies. In spite of their defined specificity, these site- directed reagents provide several additional advantages over conventional reagents. For example, the characterization and purification of unidentified gene products, made possible by the availability of cDNA deduced protein sequences, has been reported (e.g. the mos gene product, Papkoff et al., 1981). Iji addition, site-specific antibodies can identify, and distinguish, members of a genetically related family of proteins

-102- depending upon whether the antibody is directed towards a region

of sequence similarity or diversity respectively. Conventional anti-protein antisera are incapable of performing the useful

tasks outlined above due to the potential heterogeneity and undefined specificities of the immunoglobulins elicited.

Finally, anti-peptide antibodies have also identified the

scorpion toxin binding site on the alpha-subunit of the rat brain sodium channel by immunoaffinity isolation of the toxin- bound peptide fragment (Tejedor and Catteral, 1988).

In view of the potential utility of these powerful site- directed reagents, a panel of anti-peptide antibodies were generated to different intra- and extracellular domains of connexin 32. In the present study, 6 antibodies, with activity towards connexin 32, have been obtained from the use of 11 peptide immunogens; a success rate of approx. 55%. Within this panel of 6 site-directed antibodies are reagents with

specificities to each of the putative non-membrane embedded domains of connexin 32 (see Fig. 3.2). Furthermore, antibodies directed towards putative cytoplasmic domains (DES 1 and GAP 9

antibodies) identified gap junctions by indirect immuno-

fluorescent microscopy in non-denatured tissue. In addition,

DES 1 antibodies blocked intercellular communication between

living cells containing functional gap junctions. Taken together, these observations suggest that within this panel of

anti-peptide antibodies are reagents that not only recognise the denatured form of connexin 32, but also recognise its native configuration organised within the connexon. In view of the

-103- range of specificities and properties of the antibodies generated from the use of 11 peptide immunogens, a comparatively high rate of success has been achieved. Although good fortune always plays a sizeable part in the generation of any antibody, the success achieved may be accountable to the immunization strategy employed in the present study. The use of two different crosslinking agents to produce peptide-KLH conjugates, coupled to the injection of conjugate mixtures (see methods, section 2.1.3) into the rabbits, may have improved the likelihood of eliciting antibodies with specificity towards the parent protein. The concepts which form the basis of this rationale shall now be discussed.

In recent years, the concept of antigenic structure and the definition of epitopes on native proteins has come under revision. Whereas the surface of a protein antigen was thought to consist of a limited number of either 'sequential' or

'assembled' determinants, the contemporary consensus, which has developed largely from the advent of hybridoma technology, views the surface of a protein antigen as a complex array of potentially overlapping antigenic determinants, which in aggregate, approach a continuum (Benjamin et al., 1984). The composition of lgG specificities within a polyclonal antiserum

is dictated by the structural differences between the antigen

and host's self-proteins, and by the host regulatory mechanisms,

and is not necessarily an inherent property of the protein molecule reflecting limited antigenic sites. The best descriptions of epitopes on native proteins are the five

-104- published crystal structures of monoclonal Fab fragment-antigen complexes. The X-ray crystallographic analysis has been performed for three complexes of Fab-lysosyme (from chicken egg white) and two of Fab-neuraminidase (from influenza virus) (Amit et al., 1986; Sheriff et al., 1987; Padlan et al., 1989 and

Tulip et al., 1990). All epitopes contain between 15-22 assembled amino acid residues in contact with a similar number of residues on the antibody 'paratope' comprising of a buried

o 2 surface area of 650-900 A . There are 75-120 hydrogen bonds between antibody and antigen accompanied by salt links and hydrophobic interactions. The individual contributions of interactions to the overall binding energy are not known, but molecular modelling has suggested that a subset of 5-6 residues may contribute most of the binding energy, forming an 'energetic epitope', with surrounding residues allowing structural complementarity (Novotny, 1991). The 'energetic epitope' is also assembled from non-seguential amino acids. In view of the overwhelming data which has accrued to provide this perception of antigenic determinants of native proteins, it becomes difficult to accommodate the old view that epitopes on native proteins are comprised of segments of approximately 6 amino acids. It does not follow however, that the anti-peptide approach engenders the production of antisera with specificity towards sequential determinants only, and not to assembled determinants. The production of ahti-peptide antisera requires the use of carrier proteins which are often conjugated to many peptide molecules (e.g. a molar ratio of 20:1 was estimated for the GAP 7-M-KLH conjugate). As a result, the surface of the

-105- carrier protein may be extensively covered by synthetic peptides that adopt a variety of conformations induced by their own primary sequence, peptide-peptide interactions and peptide-carrier interactions. If some of these structures mimick the native structure of the corresponding region on the parent protein, it is quite plausible that elicited antisera will contain, amongst others, immunoglobulins which recognize assembled epitopes on the native parent protein composed of structural/energetic determinants from the peptide-corresponding region. These antibodies would still be site-directed, but they may well bind with lower affinity to their parent protein than antibodies in coventional anti-protein antisera with specificity for the same region. The concept of 'molecular mimickry' is well established within the immunological sphere of auto-immune disease (Ebringer, 1978). An observation which supports this hypothesis was made by Bahraoui et al (1986). An antiserum generated towards a synthetic peptide-BSA conjugate corresponding to a sequence from scorpion toxin II, reacted with the native toxin and the peptide-BSA conjugate, but not with the free peptide. This implies an important role for the carrier protein. In the present study however, all the anti-peptide antibodies directed towards connexin 32 reacted with their respective unconjugated peptides.

In a study investigating the importance of molecular flexibility to the reactiveness of anti-peptide antibodies towards native antigen, anti-peptide antibodies were generated to sequences of myohaemerythrin selected from highly mobile and

-106- well ordered regions of the protein, as predicted by X-ray crystallography (Tainer et al., 1984). Antibodies directed towards predicted mobile regions reacted strongly with native myohaemerythrin whereas antibodies to predicted ordered regions did not. It was concluded that molecular flexibility was a major factor contributing to the anti-peptide antibody recognition of native antigen. Furthermore, Tainer et al (1984) suggested that the flexible regions of proteins are conformationally dynamic (as are the peptides corresponding to these regions) and these interact with their corresponding antibody in a two-stage process. The initial interaction with antibody is followed by an induction of a new conformation suitable for antibody binding. Anti-peptide antibodies therefore act as a 'sink', trapping particular local conformations in mobile regions of native protein antigens.

Dyson et al (1988) have proposed a model explaining the recognition of native parent proteins by anti-peptide antibodies, which unifies a variety of ideas. Firstly, it is proposed that the best peptide immunogens have a high propensity to assume secondary structure when free in aqueous solution, detectable by NMR and other measurements. This secondary structure is stabilized in the protein environment of the carrier protein or the B-cell receptor, and is likely to mimick the corresponding region in the parent protein. As the exact three-dimensional structure of the peptide-carrier complex and the corresponding region in the parent protein are unlikely to be identical (even if the secondary structures are the same),

-107- then the local flexibility in the parent protein contributes significantly in optimizing the anti-peptide antibody-protein interaction.

Considering this scenario, the production of anti peptide antibodies with specificity for native structure can best be achieved by generating conjugates that mimick the three- dimensional structure of the parent protein. Thus, by careful selection of the peptide for immunization, the use of more than one appropriate crosslinking agent for peptide-carrier conjugation thereby improving the likelihood of mimicking the native structure, and the exposure of the host animal to mixtures of conjugates, may all contribute towards eliciting useful site-directed reagents. CHAPTER 4

TOPOGRAPHY OF CONNEXIN 32 IN THE GAP JUNCTION

4.1 Introduction

The translocation of ionic and polar molecules across the plasma membrane is catalysed by specialized oligomeric protein channels. These ion channels provide a polar pathway for the passage of ions through the low dielectric lipid by forming an aqueous pore lined by polar amino acids. The pore, or 'channel proper', is constructed from amphipathic peptides organised such that their hydrophobic amino acids side chains interact with the fatty acyl chains of the phospholipid, stabilizing the polar channel pore generated by the interactions of hydrophilic amino acid side chains.

Ion channels increase the flux of ions across 17 membranes by a factor of approx. 10 (Eisenberg 1990), while maintaining a remarkable degree of selectivity towards the permeating ion species (e.g. the selectivity of potassium over sodium by postassium channels). Ion selectivity without resistance to ionic motion represent the central properties underlying channel design. Such features of channel design are evident at both the gross and fine levels of channel structure. Fdr example the level of channel selectivity can be correlated to the number of subunits (or 'structural units') forming the oligomeric channel (Unwin 1989). That is, the fewer the number of subunits, the smaller the pore,

-109- allowing construction of selectivity filters (Fig. 4.1).

There are several examples of ion channels which display this structural theme. For instance, selective channels such as the voltage-gated sodium channel (Noda et al., 1984; Barchi,

1988) and the calcium selective dihydropyridine and ryanodine receptors (Tanabe et al., 1987; Wagenknecht et al., 1989) form tetrameric-type channels. Non selective channels such as gap junction connexons (and possibly the synaptic vesicle- associated channels) are hexameric (Makowski et al., 1977;

Unwin and Zampighi., 1980; Rehm et al., 1986;). Channels of

'intermediate selectivity' (i.e. cation or anion selective) include the related neurotransmitter-gated receptors and the glycine receptor, which are thought to be constructed from heteropentamers.

Aspects of fine structure have been obtained by a combination of approaches including, the physiological characterization of channel behaviour, the biochemical characterization of the composite subunits, and the use of recombinant DNA techniques. The cloning and analysis of the nicotinic acetylcholine receptor subunit genes coupled to the physiological characterization of the receptor, using channel specific non-competitive blockers of permeance, has allowed the construction of molecular models which identify the peptide components constructing the channel pore. However, due to the non-availability of specific inhibitors of intercellular communication, the elucidation of aspects of connexon fine structure by physiological methods has been

-110- Fig. 4.1 Oligomeric Structure of Ion Channels

Ion channels are thought to be constructed of oligomeric/ pseudo-oligomeric integral membranes proteins, displaying a cyclic symmetry in which the central axis of symmetry delineates the polar ion pathway across the membrane. These channels are composed of identical or heterologous-related subunits which may reside within a single polypeptide chain.

(Adapted from Unwin, 1989.) CD

- m - limited. Models of connexon structure have been constructed

from low-resolution diffraction data (Makowski et al., 19^7;

Unwin & Zampighi, 1980? Unwin & Ennis, 1984) and a low- resolution model for the organisation of the connexins in the plasma membrane has been proposed (Zimmer et al., 1987? Milks et al., 1988). The generation of site directed antibodies, described in chapter 3, provides a defined panel of potential channel blockers, which in the long term, may provide functional information about connexon structure.

This chapter describes the use of these characterised site-directed antibodies in analysing the membrane topography of connexin 32, with a view to extending and refining the working model proposed by Zimmer et al (1987). Two

independent immunological approaches were adopted, both of which utilized highly enriched gap junction subcellullar

fractions prepared by the alkali-extraction procedure

(Hertzberg 1984). Central to the experimental design is the

assumption that these subcellular fractions contain gap

junction double membrane structures with physical properties

identical to gap junctions in situ; that is, with cytoplasmic

faces accessible to large macro-molecules, but with

inaccessible extracellular 'gap' faces. Thus the regions of

connexin 32 which reside in the lipid bilayer and in the

'gap' of the double membrane structure are sterically

hindered from the interactions of immunoglobulins and

proteases.

The following sections describe and discuss the

-112- observations obtained by using the site-directed antibodies in immunolocalization experiments with 'intact' (double membrane) and 'split' (single membrane) gap junctions fractions (Fig. 4.2? Manjunath et al., 1984). These antibodies were also Used to analyse and characterize the products obtained from protease-treated intact and split junctions.

4•2 Results

4.2.1 Immunological Characterisation of Connexin 32

Proteolytic Products

4.2.1a Intact (Doiible Membrane) Gap Junctions

Rat liver gap junctions were treated with four proteases of differing specificities? trypsin, (hydrolysing

Lys and Arg) chymotrypsin (hydrolysing at hydrophobic and aromatic amino acids), endoproteinase Lys-C (hydrolysing at the C-terminus of Lys) and proteinase K, (a broad specificity protease), and the peptide products analysed by SDS-PAGE and characterized by Western blotting using the panel of anti peptide antisera described in chapter 3. The results of these experiments are shown in figures 4.3, to 4.5. In accordance with the observations of Zimmer et al (1987), treatment of intact gap junctions with each of the four proteases generated two major peptides resolved in 17.5% polyacrylamide gels. With endoproteinase Lys-C, chymotrypsin and proteinase K, fragments of 10 KDa and 17 KDa were generated but with trypsin however, fragments of 10 KDa and

13 KDa were produced (Fig's 4.4 & 4.5).

-113- Fig. 4.2 Intact and Split Gap Junctions

The extracellular faces of the two apposed junctional membranes of the intact gap junction structure are not believed to be accessible to molecules of molecular mass exceeding 10 KDa. The interactions between apposed connexons are undermined by treatment of gap junctions with

8M Urea at alkaline pH (Manjunath et al., 1984), generating gap junction-derived single membrane with accessible cytoplasmic and extracellular faces. f J I V I V 1 It )f If > 8M UREA

INTACT

‘SPLIT’

Fig.4.2

- 114 - Fig. 4.3 Endoproteinase Lvs-C and Chymotrypsin Digestion of

Intact Gap Junction^

Gap junction fractions were subjected to extensive proteolysis with endoproteinase Lys-C (top) and chymotrypsin

(bottom). Samples were analysed by 17.5% SDS-PAGE and electroblotted onto nitrocellulose membranes which were exposed to the various antibodies as shown. CONTROL: undigested gap junctions labelled with GAP 9 antibodies. M and D refer to the undigested monomeric (32 KDa) and dimeric

(64 KDa) forms of connexin 32. WG J: a polyclonal antiserum generated to whole gap junctions with undefined epitope specificity towards connexin 32. In both digests, peptides of 10 KDa and 17 KDa were resolved which were labelled differentially by the panel of antibodies. The approx. 8 KDa signal observed in the endoproteinase Lys-C digest by labelling with Gap 11 antibodies represents, most probably, a post-solubilization breakdown product of the

17 KDa peptide. ENDO. LYS-C

CHYMOTRYPSIN

cT ox ^

D r

I M «■»

/,*§ -17K

10K — m

- 115 - Fig. 4.4 Proteinase k and Trypsin Digestion of Intact Gap

Junctions

Gap junctions were subjected to extensive proteolytic treatment with proteinase K (top) and trypsin (bottom) as described in the materials and method. Samples were analysed by 17.5% SDS-PAGE and characterized by Western blotting, labelling with antibodies as shown. (CONTROL): undigested gap junctions labelled with DES 1 antibodies. M and D refer to the undigested monomeric and dimeric (Mr/s 32 and 64

KDa respectively) forms of connexin 32. (WGJ): a polyclonal antiserum raised against whole gap junction fractions with undefined specificity towards connexin 32. The effect of neglecting to stop proteolysis prior to sample solubilization is shown with respect to the proteinase K digest. (-) = solubilization without prior inhibition of proteolysis, (+) = the inclusion of a cocktail of proteolytic inhibitors (see materials and methods) prior to solubilization. In the absence of proteolytic inhibitors, a single peptide of Mr 8

KDa approx. was generated and labelled by all antibodies barring DES 1 and GAP 9 antibodies. Inclusion of proteolytic inhibitors resulted in the emergence of two peptides of 10

KDa and 17 KDa, analogous to those generated by chymotryptic and endo' Lys-C digests. The 8 KDa fragment corresponds to, two co-migrating post-solubilization breakdown products of the 10 and 17 KDa peptides. PROTEINASE K

-17K 10K -8K

TRYPSIN

- 116 - Fig.4.5 Further Tryptic Digest Of Gap Junctions and the

Evidence for an Intramolecular Disulphide Bridge

(TOP): Tryptic digest of intact gap junctions. (CBB);

Coomassie brilliant blue 17.5% SDS-PAGE profile of the 10 and 13 KDa peptide products. A similar sample, flanked by molecular weight standards, were transferred to nitrocellulose and the sample lane cut down the middle. The two blots generated were exposed to different antibodies 125 (anti-GAP 10 and anti-GAP 11) and visualized by I-labelled second antibody coupled to autoradiography. The two closely migrating peptides are clearly labelled differentially by the two site-directed antibodies.

(BOTTOM): (A) Rat liver gap junctions were analysed by SDS-

PAGE in 12.5% separating gels under reducing and non-reducing conditions. These samples were electroblotted onto nitrocellulose and stained with GAP 9 antibodies by Western blotting. In both samples, monomeric (28 KDa) and dimeric

(54 KDa) forms of connexin 32 were visualized implying that intersubunit disulphide bridges (i.e. adjoining connexins) are not present, and are not artifactually formed during the preparation of gap junction fractions, in agreement with

Manjanath and Page (1986). Note the increased mobility*of the non-reduced monomer which is indicative of an intramolecular disulphide crosslink(s). (B) Rat liver gap junctions were trypsinized and the products analysed by SDS-

PAGE in 17.5% separating gels, under reducing and non reducing conditions (see text and Fig. 4.12). IHYPIIU UIGtSI

-O' V £ > / & B i f f * r D M 23k • • 26k

-<13k 10k

GAP 9 GAP 7 -M GAP 11 Antibodies to peptides GAP 3, GAP 7-M and GAP 10 exclusively labelled the 10 KDa fragment generated in all proteolytic digests, indicating that the sequences recognized by these anti-peptide antibodies were present in this fragment. This 10 KDa proteolytic fragment therefore corresponds to the N-terminal portion (approx. the first 30%) of connexin 32. Antibodies raised against whole rat liver gap junctions (WGJ? of undefined epitope specificity) and to peptide GAP 11, recognized exclusively the 17 KDa fragment generated by proteolytic treatment with endoproteinase Lys-C, chymotrypsin and proteinase K. However, only GAP 11 antibodies recognized the 13 KDa tryptic fragment, suggesting that sequences in connexin 32 to which WGJ antibodies bind were hydrolysed by trypsin. Taken together, these results show that sequences corresponding to peptide GAP 11 are located within the 17 and 13 KDa proteolytic fragments, which constitute the C-terminal portion (approx. the latter 60%) of connexin 32. Finally, antibodies generated to peptides DES 1 and GAP 9 didnot label either of the peptide fragments generated from the proteolytic treatment of gap junction fractions. It is therefore concluded that the sequences required for binding of these two anti-peptide antibodies

(located within the putative intracellular loop and at the extreme of the carboxyl terminus) were partially or completely hydrolysed during the proteolytic treatment. The conclusions of these experiments are summarized diagrammatically in Fig. 4.6.

-118- Fig. 4.6 Diagrammatic Peptide Maps for Connexin 32

(A) Diagrammatic representation of the recoverable and

putative non-recoverable peptides generated by the extensive

proteolysis of gap junctions with endoproteinase Lys-C,

chymotrypsin and proteinase K.

(B) Diagrammatic representation of the recoverable and

putative non-recoverable peptides by the extensive trypsinisation of gap junctions. Fig 4.6 Diagrammatic Peptide Maps of Connexin 32 Generated from Proteolytic Digestion of Gap Junctions o- O CO C\J -119- o- CD o o CO o csj D CD CD "•4—' cd CD Q_ a: ‘c "O D J < CL < CL < CL “D < < h- < < o O T- CO O c cz CD O CD (f) CD 03 C I i o ~ CD “3 TJ JD < Ct ‘c < c: 03 C CD a >> c O CD CO CD

V-» *C CD CL o cr D T JD < < c CD O O CO CD CD >> >> I JD LL •4—< O CO o c 0 O > CD _ * 03 5 CD E 0 c 0 1

4.2.1b 'Split' (Single Membrane) Gap Junctions

To render access to the predicted extracellular

'loop' domains of connexin 32 (Fig. 4.2), gap junctions were

'split' or 'transformed' into single membranes. These fractions were treated with chymotrypsin and proteinase K, and the products analysed by SDS-PAGE and characterized by

Western blotting. Despite several attempts, the results obtained (not shown) were identical to those shown in Figs.

4.3 and 4.4, with two proteolytic fragments of 10 and 17 KDa

identified by the same anti-peptide antibodies (see Fig.4.6).

This result showed that no additional cleavage had occurred within these extracellular domains, despite rendering them

accessible to proteolytic attack.

To investigate further the inability to obtain proteolytic cleavage within these putative extracellular

loops, additional steps were adopted to procure protease

accessibility to target amino acids. The relatively high

cysteine content of the extracellular domains of connexin 32

entertains the possibility for extensive intra- or interloop

disulphide crosslinking (evidence for the latter is presented

in the following section), which could reduce accessibility

of putative cleavage sites to the relevant proteases. To

circumvent this problem, gap junction fractions were 'split'

in the presence qf lOOraM DTT, and subsequently alkylated

with lOraM N-ethylmaleimide prior to proteolysis and analysis.

These additional treatments, however, did not alter the size

nor the immuno-specificity of the peptides generated by

-120- chymotryptic digests (data not shown) demonstrating the highly protease-resistant properties of these domains of connexin 32.

4.2.2 Anti-Peptide Antibody Immunolocalisation to Intact

and Split Gap functions

To ascertain the topographical locations of the sequences of connexin 32 selected for the preparation of the site-directed antibodies, immunolocalisation experiments were performed with intact and urea-split gap junctions. The results of these investigations are illustrated in Figs.4.7^

4.10. The electron micrographs show different field views to illustrate the pattern of labelling. Intact and split junctions were labelled with affinity purified reagents (with the exception of GAP 10 antibodies which were used as a 1/10 diluted serum) and the antibody distribution visualized by colloidal gold-labelled goat anti-rabbit second antibody

Antibodies to peptide DES 1 (Fig. 4.7) immunolocalized to both outer faces of intact (double membrane) gap junctions presumed to be the cytoplasmic faces of the two apposed junctional membranes. The highly specific localization of this antibody was further emphazised by the pattern of labelling obtained with 'split' single functional membranes. In over 90% of labelled single membranes, gold particles were observed to decorate exclusively the convex

(cytoplasmic) face of the junctional membrane, providing direct evidence for the accessibility and the topographical

-121- Fig. 4.7 Immunolocalization of DES 1 Antibodies to Intact

and Split Gap Junctions

The electron micrographs show various fields from the same

labelling experiment in which affinity purified DES 1 antibodies were incubated with a 'urea-split' preparation of

rat liver gap junctions. Antibody localization was

identified by lOnm colloidal gold-conjugated goat anti-rabbit

second antibody labelling. Areas were selected to show

labelling of intact (arrows) and split (arrow heads) gap

junctions. Intact junctions are labelled on both outer faces whereas split junctions (which may vesicularize-top micrograph) label at their convex face only. Note the close association of the gold particles to the membrane surface.

Bars = O.lum, inset bar = 0.2um. DES-1

• -c.

: ' C <

% % it \

«• \ # / # # fc>

/ *

* *< •a- j| \ T w * *

* •* t.g •-1 m • • • disposition of the DES 1 sequences. Antibodies to peptide

GAP 9 produced essentially similar staining patterns as those observed for DES 1 antibodies (Fig. 4.8). Gold particles specifically decorated both outer (cytoplasmic) faces of intact junctions and exclusively the convex (cytoplasmic) face of split junctions. This staining pattern also provides direct evidence for the accessibility and the topographical disposition of the GAP 9 associated sequence. An important feature of the two staining patterns produced by the GAP 9 and DES 1 antibodies, is that the gold particles decorating

DES 1 antibody labelled junctional membranes appear closely associated with the membrane. In contrast however, the gold particles decorating GAP 9 antibody labelled junctional membranes are clearly displaced from the membrane surface by a mean distance of approximately 22nm±5nm. Since the distance between the antigen and the gold particle of an antigen-specific lgG-gold conjugated second antibody complex, is not thought to exceed 14nm (assuming a'stoichiometry of

1:1:1; Rohde et al«, 1984), this observation infers that the extreme region of the carboxyl terminus may be displaced from the surface of the membrane by as much as 8nm. This is value comparable to the centre to centre spacing of a connexon lattice.

Antibodies to peptide GAP 10 were observed to stain both outer surfaces of intact junctions (Fig. 4.9), a property not observed when antibodies to peptide GAP 3 were used (Zimmer et al., 1987). Labelling of split junctions was

-123- Fig. 4.8 Immunolocalisation of Gap 9 Antibodies to Intact

and Split Gap Junctions

Urea-split gap junctions were labelled with GAP 9 antibodies

and subsequently with 5nm colloidal gold-conjugated goat

anti-rabbit second antibody.

(TOP): Microscopic field showing labelling of intact gap

junctions on both outer surfaces.

(BOTTOM): Areas with split junctions clearly demonstrate a

labelling bias towards the convex faces of the single

junctional membranes. Note the clear displacement of the

gold particles from the membrane surface. Since these

antibodies are directed towards the extreme of the carboxyl

terminus, this labelling pattern may be indicative of an

extended carboxyl terminal 'tail'. Bars = 0.2ura. GAP Fig. 4.9 Immunolocalisation of Gap 10 Antibodies to Intact

and Split Gap Junctions

(TOP): Intact gap junctions labelled with GAP 10 antibodies and 5nm colloidal gold-conjugated goat anti-rabbit second antibody. The heptalaminar configurations are clearly labelled on both outer faces.

(BOTTOM): Urea-split gap junctions labelled as described above. Bars = 0.2um. G A P - 1 0

hk >T ^ ^

^ T>J #v fe*

T v * : » ' * k’ V ' ** / / * * *•

‘ ‘ N - C " i »V . * A * ' • r ■

t { it t k $ feA-

-125- evident, but the specificity towards the convex face of single junctional membranes was less clear cut than in experiments with GAP 9 and DES 1 antibodies. In approximately 50% of labelled single membranes, gold particles were observed on both sides, although in general, labelling was biased towards the convex faces. Antibodies to

GAP 7-M, in contrast to the antibodies described above, were npt observed to label intact junctions except at the 'ends' of double membrane structures (where membranes are often parted) and in regions where the integrity of the junctions was poor (Fig. 4.10). Labelling of 'split' junctions was also observed but at a very low level. In regions where labelling was seen, gold particles were confined to the concave (extracellular) aspects of the single junctional membranes. Similar results were obtained with affinity purified GAP 11 antibodies (data not shown). These results indicate that sequences selected for peptides GAP 7-M and GAP

11 have limited accessibility in intact gap junctions, but become more accessible when junctions are 'split', thereby exposing the extracellular surfaces of the membranes.

Fig. 4.11 shows that when gap junctions were exposed to a non-immune lgG fraction, little or no gold particles were associated with either gap junction single or double membrane structures.

4.2.3 Evidence for a Disulphide Bridge(s) Connecting the

Extracellular Loops of Connexin 32.

A general feature of the putative extracellular

-126 Fig. 4.10 Immunolocalization of Gap 7-M Antibodies to Intact

and Split Gap Junctions.

A urea-split gap junction sample labelled with GAP 7-M antibodies and 5-10nm colloidal gold-conjugated goat anti rabbit second antibody.

(TOP): Micrographs of intact junctions in the sample, which are not labelled at their outer surfaces, except at the ends of the junctions where the membranes are often parted. This is seen more clearly in the bottom right micrograph.

Labelling was also observed in regions where the integrity of the junctions was poor.

(BOTTOM LEFT): A region of split junctions showing a generally low-level of labelling, but predominantly at the concave face of the single junctional membrane. Bars =

0.2um.

Fig. 4.11 Intact Gap Junctions Labelled with Pre^Immune Serum

Intact (TOP) and split (BOTTOM) gap junction fractions were

labelled with a pre-immune serum from rabbits immunized with a GAP 10-KLH conjugate mixture, followed by second antibody

exposure with 30nm colloidal gold-conjugated goat anti-rabbit

antibody. A very low level of background labelling was

apparent, but mainly to non-junctional structures. N.R.S y '

f Y y _ _

il^ S . ^ V & - * E

-128- 'loops' of all connexins described so far is the presence of

6 cysteine residues? three in each loop. To investigate whether these loops are interconnected by disulphide bridges, intact gap junctions were treated with trypsin and the peptide fragments analysed by SDS-gel electrophoresis under reducing and non-reducing conditions. The results of subsequent Western blotting experiments are shown in Fig.

4.5 (bottom). Under reducing conditions, typical tryptic peptide products of 10 and 13 KDa were obtained, detected by

GAP 7-M and GAP 11 antibodies respectively. Under non reducing conditions however, a moiety of 23 KDa was observed and recognized by both antibodies, corresponding presumably to the combined 10 KDa and 13 KDa tryptic peptides. Since the dissociation of this 23 KDa peptide into 10 and 13 KDa forms was promoted by the inclusion of beta-mercaptoethanol in the solubilization buffer, the stable linkage generating the combined 23 KDa moiety is concluded to be a disulphide bridge(s) connecting the two putative extracellular loops

(see Fig. 4.12).

4.3 Discussion

One of the first stages in elucidating the structure- function relationship of an integral membrane protein is to decipher its topographical arrangement in the lipid bilayer.

This is usually achieved by a combination of structural prediction (hydropathy analysis) and experimentation. In general, the former identifies potential membrane embedded

-129- Fig. 4.12 Identification of an Interloop Disulphide Bridge.

Trypsinization of intact gap junctions and analysis of the peptide products by SDS-PAGE under reducing and non-reducing conditions was performed, and the results shown in Fig. 4.5.

A diagrammatic representation of the peptide products and their molecular guise during analysis, under reducing and non-reducing conditions, is shown opposite. Arrows indicate putative cleavage sites for trypsin, and black dots indicate the approx. location of cysteine residues. The hypothetical arrangement of disulphide crosslinks illustrated in this figure are in agreement with the results shown in Fig. 4.5, identifying an interloop disulphide bridge(s). c .2 . 2 o cS-3 .2 CD CO 1 5 ° -

— Z3 CO o o CM C/D-C

o C GC

o CO

+ o c c 13 (/) • OHM T3 (D !5 QC JD O C/D

- 130 - domains of the polypeptide, while the latter focuses on non membrane embedded regions that are accessible to biochemical reagents.

Non-membrane embedded regions of polypeptides can be identified by their accessability to proteolytic attack. For example, the controlled proteolysis of reconstituted preparations of H+-ATPase from Neurospora crassa and the analysis of both the hydrophilic and hydrophobic peptide products by microsequencing, identified both membrane embedded and cytoplasmic sequences (Hennesey and Scarborough,

1990). Alternatively, the topographical disposition of non membrane embedded regions of proteins can be elucidated by antibody localization using either monoclonal or polyclonal, anti-protein or anti-peptide antibodies. Anti-peptide reagents possess the advantageous property of having specificity towards a particular, pre-determined region of the protein under investigation. The anti-peptide antibody approach was successful in elucidating the topography of the human erythrocyte glucose transporter (Davies et al., 1990), and the extracellular disposition of the beta-adrenergic receptor amino terminus (Theveniau et al., 1989). In addition to the use of antibodies and proteases, topography can be examined by the covalent modification of target amino acids by using highly specific chemical reagents, often in a capacity for cross-linking polypeptide subunits.

As an alternative to biochemical approaches, the topography of membrane proteins has been studied by

-131- molecular biology, employing a gene fusion approach. This involves the preparation of a panel of fusion constructs comprising of a marker enzyme gene (such as alkaline phosphatase) and the gene encoding the target polypeptide under investigation. The marker enzyme is either fused to the carboxyl region of truncated constructs of the target polypeptide, in which one or more of the putative membrane- spanning segments has been deleted (Lewis et al., 1990), or alternatively, inserted into the target gene at sites between the putative membrane-spanning domains (Ehrmann et al.,

1990). In both situations, the membrane sidedness of the marker enzyme activity is assessed, providing information about the number of membrane traversions assumed by the target protein.

The dense packing of connexins within gap junctional membranes, coupled to their routine preparation, renders the topographical analysis of connexin 32 highly suitable for the immunolocalization/protease susceptibility approach. This chapter has described the use of a panel of site-directed antisera in elucidating the topography of connexin 32 in the gap junctional membrane. The following sections draw conclusions concerning the membrane topography of connexin 3 2 based on the evidence obtained from this study and from other laboratories.

4.3.1 Evidence for the Cytoplasmic Disposition of the Amino

Terminus of Connexin 32

Inspection of the hydropathy profile for the amino

-132- terminal 20 amino acid residues (Fig. 3.1) illustrates a substantial hydrophobicity associated with this region of connexin 32 as a whole. The assignment of the amino terminus to the cytoplasm was based predominantly on the absence of glycosylation in this region, despite a consensus

N-linked glycosylation signal at amino acids 2-4 (Zimmer et al., 1987). Furthermore, the failure of antibodies to GAP 3

(amino acids 6-21) to immunolocalize to intact gap junctions, coupled with its use in demonstrating the resistance of the amino terminus to proteolytic attack, suggested that the amino terminal 20 amino acids were inaccessible and possibly membrane embedded.

In the present study the binding of antibodies to GAP

10 (amino acids 1-20) to the outer surfaces of intact junctions provides direct evidence for the cytoplasmic disposition of the amino terminus in intact junctions. The conflicting results obtained in immunolocalization experiments with both amino-terminal directed reagents, suggests that GAP 10 aptibodies contain a sub-population of immunoglobulins which recognize an accessible binding site, probably towards the extreme of the amino terminus. This conclusion is supported by the identification of chymotryptic cleavage sites at Tyr 7 and Leu 10 and a proteinase A cleavage site at Leu 9 (Hertzberg et al., 1988). Taken together, these observations suggest that amino acid residues

1-10 are accessible for antibody binding and therefore susceptible to proteolytic breakdown, whereas access to

-133- residues 11-20 appears restricted. The labelling pattern observed with GAP 10 antibodies on 'split' junctions, although biased towards the convex (cytoplasmic) face of the junctional membrane, was suggestive of an alteration in the accessibility of the amino terminus to antibody binding. The labelling pattern of GAP 10 antibodies is highly similar to that observed by Milks et al (1988) using an amino terminal directed antibody (peptide B, residues 6-17). It would appear that properties peculiar to the amino terminal region of connexiri 32 have been observed in different laboratories.

For example, microsequence analysis of the 10 KDa fragment generated by chymotryptic digestion of intact junctions, identified three populations of peptide; those which sequence from the N-terminal methionine, those which sequence from Thr

8 ahd those which sequence from Ser 11 (Hertzberg et al.,

1988). This indicates that, even after prolonged exposure to chymotrypsin, a sub-population of the amino termini remain undergraded. This observation might also explain why both

GAP 3 and GAP 10 antibodies always identified the 10 KDa proteolytic fragment in Western blotting experiments. It appears therefore that the rate of proteolytic hydrolysis at the amino terminus is far slower than at other protease- susceptible regions of connexin 32.

4.3.2 Evidence for the Cytoplasmic Disposition of the

Carboxyl Terminus of Connexin 32

In a previous study, antibodies (of undefined epitope specificity) identifying the 17 KDa proteolytic fragment of

-134- connexin 32, were observed to immunolocalize to the outer faces of intact gap junctions and to the convex-cytoplasmic face of 'split' junctions (Zimmer et al., 1987), providing indirect evidence for the cytoplasmic disposition of the carboxyl terminus. In the present study two independent observations provide further compelling evidence for the cytoplasmic disposition of the carboxyl terminus. Firstly, antibodies to GAP 9 (amino acids 263-283) were never observed to label any proteolytic product of connexin 32? in particular the 17 KDa and 13 KDa carboxyl-associated fragments. Furthermore the 13 KDa tryptic fragment was not identified by WGJ antibodies, indicating that the cleavage of an additional 4 KDa was responsible for loss of antibody recognition. These observations indicate that sequences from the carboxyl terminus of connexin 32 are susceptible to proteolytic degradation in intact gap junctions, and must therefore be located at the cytoplasmic aspect of the junction. Secondly, the immunolocalization of GAP 9 antibodies to both outer aspects of intact gap junctions and exclusively to the convex-cytoplasmic face of 'split' junctions, provides direct evidence for the cytoplasmic disposition of the carboxyl terminus. On the basis of the average displacement of gold particles from the surface of

GAP 9 antibody-labelled junctional membranes, a rough estimate for the extension of the carboxyl tail into the cytoplasmic milieu has been determined to be in the order of

8nm. This value is greater than that determined from 25A resolution X-ray diffraction data (Makowski et al., 1984),

-135- which 'visualizes' cytoplasmic connexon structure protruding approx. 2nm from the membrane surface. Furthermore, since

WGJ antibodies have been shown previously to immunolocalize to both outer faces of intact junctions (Evans et al., 1988), coupled with its failure in labelling the 13 KDa proteolytic

fragment, the epitope for WGJ antibodies must also lie within the carboxyl tail. Bearing these observations in mind, the site of tryptic cleavage within the carboxyl tail responsible for the further reduction of the carboxyl-associated peptide fragment from 17 to 13 KDa, can be predicted to lie within an

Arg rich sequence between amino acids 215-224 (Fig. 4.13).

This region may also Correspond to the cleavage site(s) for endoproteinase Arg-C which generated a 23KDa fragment (Zimmer et al., 1987). In general, these results are in excellent agreement with those presented by Milks et al (1988) which also demonstrate cleavage and loss of anti-peptide antibody binding to sequences at the extreme of the carboxyl terminus.

4.3.3 Evidence that the Peptide Connecting the M2 and M3

Domains of Connexin 32 is Intracellular

The hydropathy profile of connexin 32 predicts a hydrophilic domain between amino acid residues 90 and 130 which connects the second and third membrane spanning domains

(Fig. 3.1). Further evidence for the existence and cytoplasmic disposition of this domain was presented by

Zimmer et al (1987) by demonstrating the proteolytic bifurcation of connexin 32 into an amino terminal containing

10 KDa fragment and carboxyl terminal containing 17/13 KDa

-136- Fig. 4.13 Mapping of Proteolytic Cleavage and Antibody

Binding Sites in Connexin 32

The location of protease accessible regions of connexin 32 is illustrated by the mapping of identified (numbered arrows) and putative (arrowheads) protease cleavage sites, based on the data presented by Zimmer et al (1987), Hertzberg et al

(1988) and the present study. The identified cleavage sites are for the following proteases; (1) chymotrypsin, (2) proteinase A, (3) chymotrypsin, (4) proteinase A, (5) V8 protease, (6) thermolysin, (7) V8 protease, (8) chymotrypsin,

(9) trypsin and endo' Lys-C. Arrowheads indicate potential cleavage sites for trypsin (Arg) and endo-Lys-C (Lys) which generate non-recoverable fragments and cannot therefore be verified by microsequencing. An Arg rich segment at the origin of the carboxyl-tail could represent a target site for trypsin and endoproteinase Arg-C, resulting in the formation of 13 and 23 KDa peptides respectively. The shaded regions highlight the regions of connexin 32 thought to contain sequences necessary for the binding of DES 1 and WGJ antibodies in Western blotting experiments. It is noteworthy that protease accessible regions of the polypeptide correlate

(overlap) well with those regions accessible to antibody binding in immunolocalization experiments. CL < P i— _ca D

r- h- "OQ^2H>c^ Li->WCQi“LU'K CL IL < \W# CO H I 5 <4 > GO a:>2oeiQ.>jj>u.>[> £ <-ui L. ul \ ^ y' cc-' »

o <* CD CO co O 2 CO > Q > Q X u- Ll|Q- CL O y—

EP'-h ^ S >* + '2 Z ^ H O CO

; :

- 137 - fragments. The proteolytic cleavage site(s) responsible for this bifurcation was determined by microsequence analysis of the 17/13 KDa fragments to be at Lys 124.

In the present study, antibodies to the composite peptide DES 1 (amino acids 102-112+116-124) did not bind to either 10 KDa or 17/13 KDa proteolytic fragments generated by the proteolytic digestion of intact gap junctions. This antibody however, clearly recognized the undigested monomeric and dimeric fofms of connexin 32 in Western blots. It is concluded that DES 1 antibodies probably require sequences within the region between amino acids 116 and 124 for binding, and that multiple cleavage sites for a variety of proteases are located within this region. Thus the inability of DES 1 antibodies to recognize either the 10 KDa or

17/13 KDa proteolytic fragments suggests that an approx. 0.5-

1.0 KDa fragment within this loop region is lost during proteolytic treatment (see Fig. 4.6). This conclusion is in agreement with the results of Hertzberg et al (1988) which show that the region between amino acids 108-124 is accessible to a variety of proteases. Indeed two cleavage sites for endoproteinase Glu-C have been identified at positions Glu 109 and Glu 119 which, under conditions of complete hydrolysis, would result in the loss of an approx.

1.0 KDa peptide. Furthermore, proteases with specificity for

Lys residues may also cleave at Lys 121 which could account the loss of DES 1 antibody binding. If so, DES 1 antibodies require essential sequences between amino acids 121-124 for

-138- binding to connexin 32 (Fig. 4.13).

The accessibility of this loop region of connexin 32 to proteases, in intact gap junctions, indicates that it is located in the cytoplasm. The immunolocalization of DES 1 antibodies to the outer surfaces of intact gap junctions, and exclusively to the convex (cytoplasmic) face of split junctions, provides direct and additional evidence for the accessibility and cytoplasmic disposition of sequences contained within the predicted loop peptide. The close association of gold particles with the junctional membrane is consistent with the notion that DES 1 antibodies require sequences between amino acids 121-124 for binding, with this portion of connexin 32 predicted to lie close to the junctional membrane (see Fig. 4.13). The immunolocalization staining pattern of DES 1 antibodies is in excellent agreement with the reports of Milks et al (1988) and

Goodenough et al (1988) in which antibodies directed towards the sequences 111-125 and 98-124 respectively were used. The combined information from a number of laboratories suggest that the region between amino acids 90-108 is resistant to proteolytic degradation, even though this sequence is not predicted to be membrane embedded. This region could well interact with other cytoplasmic regions of connexin 32

(possibly within the carboxyl tail) to generate a protease- resistant structure.

-139- 4.3.4 Evidence for Two Extracellular Loop Peptides

Connecting the Four Transmembrane Domains of

Connexin 32

The two-dimensional model depicting the arrangement of connexin 32 in the lipid bilayer predicts the extracellular disposition of two looped peptides connecting the first and second, as well as third and fourth membrane spanning domains. Furthermore, X-ray diffraction data has demonstrated the presence of an ordered portion of the connexon extending some 15-20A into the extracellular 'gap'

(Unwin and Ennis, 1984).

In this study, proteolytic treatment of intact junctions generated fragments containing intact sequences from these putative loops, exemplified by the binding of GAP

7-M antibodies (directed towards aminQ acids 43-57) and GAP

11 antibodies (directed towards amino acids 151-187) to the

10 and 17/13 KDa fragments respectively. This observation indicates that sequences within the putative extracellular loops are not accessible to proteases i.e. are not located in the cytoplasm. Furthermore, antibodies to GAP 7-M and GAP 11 peptides were not observed to immunolocalize to intact gap junctions. However, when gap junctions were split by treatment with 8M Urea at alkaline pH, some low-level labelling was evident on the concave (extracellular) aspect of the single junctional membranes. Consistent with the observations of Zimmer et al (1987), treatment of split junctions with proteases produced similar fragments to those

-140- obtained from proteolytic treatment of intact junctions. It appears that this ordered portion of the connexin forms a protease-resistant structure, indicative of a high level of interloop interaction which might render potential target sequences inaccessible to antibody binding. Immuno localization of anti-peptide antibodies to one or other of the extracellular loops has also been demonstrated by Milks et al (1988) and Goodenough et al (1988).

The demonstration of an interloop disulphide bridge(s) entertains further the possibility of extensive interloop interactions, giving rise to an ordered extracellular domain. Hypothetical configurations for this covalent link are illustrated in Fig. 4.14, which extends the possibility for intraloop disulphide bridges. That the six cysteine residues located within these loops participate in disulphide bond formation is supported by the observation that conserved cysteine residues in soluble proteins of known

3-D structure predominantly form disulphide bridges

(Thornton, 1981). Furthermore, Muskal et al (1990) have demonstrated that the occurrence of disulphide bonds in soluble proteins of known 3-D structure is related to the chemical microenvironment created by the side chains of amino acids flanking the participating half-cystines.

Close proximity (i.e. within 6 amino acids on either flank of the cysteine) of favourable amino acids including Y,

N, K, G, S and T, accompany the occurence of disulphide bonds. Inspection of the sequences flanking the cysteine

-141- Fig. 4.14 Favoured Hypothetical Organisation of Disulphide

Bridges

A variety of disulphide crosslinking arrangements can exist within and between the extracellular domains of connexin 32. Assuming that all six cysteine residues located within these domains participate in disulphide bridge

formation, a total of 15 different arrangements are possible;

9 for the formation of 3 interloop disulphide bonds, and 6

for the formation of 1 interloop and 2 intraloop disulphide bonds. Favouring the latter configuration (see text), 2 equally likely arrangements are shown opposite in A & B, based on the consensus derived from the disulphide arrangements observed within soluble proteins of known 3-D structure. Favoured Theoretical Arrangements of Disulphide Bonds Within the Extracellular Loops of Connexin 32

Q PG / d > © L C CP Nj 171 T 60 s A V N V E D 53 C - - C 6 4 166 C — — C 177 I Y K F F D V H V S F L S S F R R Extracellular 'Gap'

M1 M2 M3 M4

Cytoplasm

60 171 g ? n 166 PC

FC 53 F P M V 177 S I A S S S Y R K H L P Extracellular 'Gap'

M1 M2 M3 M4

Cytoplasm

142 - residues located within the extracellular loops of connexin

32, reveal a high concentration of 'disulphide promoting' amino acids (Fig. 4.15). The favoured hypothetical arrangements of disulphide bridges shown in Fig. 4.14 were based on the following considerations. Local disulphide bridges (half-cystines separated by less than 43 amino acids) are frequently observed in globular proteins, with the most frequent separation of between 10 and 14 residues (Thornton,

1981). Such 'looped' peptides often harbour amino acids with positive beta-turn potential (P, G, S, T, D and N).

These amino acids are also clustered between the cysteine residues located within the extracellular loops of connexin

32 (Fig. 4.15), particularly between Cys 53 and Cys 60, and

Cys 171 and Cys 177, favouring the configuration shown in

Fig. 4.14(B). The presence of such intra-and interloop disulphide connections causes reduced access to the half- cystines and neighbouring amino acids (Clothia, 1976), consistent with the biochemical properties of the extracellular domains of connexin 32 encountered in past and present studies.

Since the disulphide bridge connecting the 10 and 13

KDa tryptic fragments is unlikely to result from interactions between extracellular loop 1 and cysteine residues located within the carboxy tail or the M4 segment (see Fig. 4.12), this observation provides further evidence for the folding pattern depicted in the two-dimensional model, which brings cysteine residues far apart on the linear sequence into

-143- Fig. 4.15 Flanking Sequences of Putative Half-Cystines

Loop 1

Cys 53

EKSSFICNTLQPG * * * *

Cys 60

NTLQPGCNSVCYD * * * * * *

Cys 64

PGCNSVCYDHFFP * * * * *

Loop 2

cys 166

MVRLVKCEAFPCP * *

Cys 171

KCEAFPCPNTVDC *****

Cys 177

CPNTVDCFVSRPT * * * *

* Highlights the 'bend promoting' amino acids. See text for details.

— 144 — spatial juxtaposition through the formation of two extracellular loops.

4.3.5 A Model for Connexin 32 and the Organisation of the

Connexon

A variety of experimental approaches from a number of laboratories have accrued to provide a unifying endorsement of the topographical model of connexin 32, proposed by Zimmer et al (1987). In this study and those of other laboratories, data has been presented which refines various aspects of the model as illustrated in Fig. 4.13. It is important however, to develop a model which can relate to the functional properties and hence the organisation of the connexon.

Unwin (1986) proposed that membrane spanning segments of integral membrane proteins assumed alpha-helical configurations, based on the predictions from the hydropathy analysis of their primary structure. Since the polar backbone groups of amino acids, within the membrane-spanning portions, participate in intra-chain hydrogen bonding when an alpha-helical structure is assumed, a favourable lipid- spanning structural unit can be assembled from 20 to 25 hydrophobic amino acids. Recent CD spectroscopic (Cascio et al., 1990) and X-ray diffraction (Tibbits et al., 1990) approaches have estimated the alpha-helical content of liver gap junction preparations to fall between 40 and 60% (the upper value was generated by X-ray diffraction data which is a measure confined to the ordered portion of the connexon).

-145- These experimental values fall in the range predicted by the

topographical model of connexin 32, allowing for four membrane spanning alpha-helices.

Based on its amphipathic character, the putative helix M3 has been proposed to provide a face of polar amino

acids to line the interior of the pore (Milks et al., 1988).

Each connexin is thought to adopt a left handed four alpha- helical bundle motif, commonly observed in four alpha-helical polypeptides (Milks et al., 1988; Weber and Salemme 1980).

Based on structural and biochemical data, a structural model of connexin 32, in relation to the arrangement of a connexon,

is shown in Fig. 4.16. X-ray diffraction data (Makowski et

al., 1982) has demonstrated a strong reflection associated with a spacing in the connexon of approx. 4.8A; this is

shorter by 0.6& than the average connexin helical pitch.

This reflection is thought to arise from layers of side chains between membrane-spanning alpha-helices whose axes are relatively inclined, a feature typical of a four alpha- helical bundle. A recent study has estimated an average helical tilt of 20° relative to the connexon axis (Tibbitts

et al., 1990). Whereas such an alpha-helical incline might

predict an interhelix angle somewhat greater than 18° (a value which is uniquely associated with the attainment of

regular periodic interactions between all helix pairs in a

four alpha-helical bundle), it appears that larger inter

helix angles can also achieve a stable and regular

arrangement. Such inclines however, may reduce the number of

-146- Fig. 4.16 Structure of the Gap Junction

(A) Diagrammatic representation of a gap junction (cross- section) composed of two closely apposed regions of the plasma membrane separated by a 2-3nm wide extracellular

'gap'. This specialized region of the cell surface contains a high density of intercellular channels created by the combination of connexons from each membrane.

(B) Diagrammatic representation of a connexon corresponding to a hexameric ion channel of approx. 80A diameter with a

15-20A wide pore.

(C) Diagrammatic illustration of the organisation of the connexin (connexin 32) in the plasma membrane, based on biochemical and structural data. The channel forming polypeptide assumes a four alpha-helical bundle stabilized by interhelix dipole interactions engendered by the anti- parrallel arrangement motif; with the M3 helix predicted to provide a polar face for the construction of an ion pathway through the lipid core. Tilting of the subunit and helix assures that the axis of the aqueous pore lies perpendicular to the plane of the membrane. Estimates for the subunit dimensions were taken from Unwin and Ennis (1984), Caspar et al (1988), Makowski et al (1985), and the present study. c Gap Mid Plane

Plasma Membrane

Cytoplasm n

-147 - side chains interacting between helices by increasing their separation. Thus, 4 alpha-helical polypeptides with larger interhelical inclines probably represent less rigid structures than polypeptides with smaller interhelical inclines adopting a similar tertiary structure.

4.3.6 Channel Construction in the Connexon and its Gating.

The assembly of the connexon from connexins has been proposed to generate an ion pathway through the plasma membrane constructed from the combined arrangement of M3 helices as depicted in Fig. 4.17(A). The channel pore is constructed from an association of M3 helices which have an overall right handed twist (which is in an opposite sense to the alpha-helical bundle; Unwin 1989). A notable feature of right handed helical assemblies generated by inter-subunit packing, is that packing constraints result in a ridge of small residues from one of the pair of interacting helices apposing a ridge of large residues from the other (Chothia,

1981). Analysis of the sequences of putative pore-lining segments from a variety of ion channel subunits illustrates this structural theme, and also highlights a periodicity of polar residues flanked by hydrophobic/bulky aromatic residues indicative of a pore lining alpha-helix (Table 4.1). These sequences indentify a potential 'channel-pore motif', extending some 10-15A, common to all channel forming subunits

(Unwin, 1989). Also shown are two putative membrane-spanning segments of the Mr 16 KDa polypeptide, originally proposed to be of gap junction origin, but now associated with vacuolar

-148- TABLE 4.1 AMINO ACID SEQUENCES OF PUTATIVE PORE-LINING A—HELICES O H O w H CO P w W E-t 2 o < Q w a W 55 H § 2 CM E > CO 2 M 2 CO > E E -P CJ M 2 W W < >H 2 > 2 2 2 >H 2 2 2 2 2 2 2222 2 2 2 2 CO 2 2 2 cd (0 X h h h CM CM 2 2 M 2 OC OCO CO CO CO 2 2 CO > -P O O > 2 < >H E 2 E >H > > 2 > 2 X X X h h CO M 2 2 2 2 > E > 2 2 H>H >H > 2 < >H >H E 2 > 2 2 > 2 2 2 2 2 W W 2 2 2 2 2 2 2 a E h h 2 0 M CO M M M E 0 0 0 CO X > > 2 2 2 2 2 < < & 2 2 < 2 2 0 a h X 2 2 E OCO CO O M E 2 X > 2 CO 2 2 2 2 2 2 O 2 2 2 E O 2 2 cd 0 c X h h h M CO M M - 2 *-3 2 M I 2 2 2 I— I > >H E 2 > > C < 2 2 2 2 2 - 2 2 2 2 2 o p X X X h 2 2 1 M | 2 2 w 2 M 2 >H E 2 2 2 2 2 O O O — a h 1 M 2 CO l w 1 * 2 M 1 2 > > > > O M 2 H I >H 0 M1 X - — — 0 c I • 1 1 -

1 -149- 2 1 1 . C < \ £ £ 2 2 CO 2 2 < 2 2 2 2 2 2 £ 2 < 2 l ° ° > > CO CO >H 2 >H < E O O CL, 2 CL, , O O 2 > 2 M M 2 2 2 O O 2 2 h h H

2 _ - J . 1 ^ 2 2 2 < M 2 2 £ C M 2 > > > 2 • > O -

_i | ' . ? i 1 2 | ? L i co Ic

--- 3 ^ 2 2 w MCM CM c < E 2 2 2 > 2 2 M H 2 rf: u 2 2 2 P 2 2 ,c 2 2 > > CU - 2 2 x o w U 0 h 2 E M 2 2 CO E > 2 M E O 2 2 2 < 2 > > < < 2 2 X O 2 h h h

1 .1 j • i • ■ 1 , ' < \ > U \ w ! . M * ! * I o l o f 2 w 2 2 C 2 E < 2 2 : 2 O o < O O i~H CO < O 2 > >< O 2 2 c >22 2 > tc 2 2 2 2 2 P o > 2 2 2 U m i M P < < << l O D h m

2 ! " 9 I > ! ^ 1 2 2 < E 2 E E 2 W CM 2 E ^3 > o < < > 2 2 2 2 0 0 0 2 rtj m >h < 2 a M 2 2 < < a 2 0 2 U 0 w u h h h h " < E E E 2 E 2 CM O 2 2 E >H t O ; 2 l 2 2 2 > M CM M > O O O 2 u 0 h h h h h

• >. .« > ■ 2 2 2 2 2 2 2 2 | o | o < > Q 2 O 2 2 > >H 2

. . Fig, 4.17 Gatina Mechanism for Ion Channels

(A) The diagram illustrates the twisting sense of alpha-

helices within the subunit bundle (top arrow) and around the

channel pore (bottom arrow). In a four alpha-helical bundle,

the sense of twisting is left-handed. The arrangement of

identical subunits in an ion channel (whether tetrameric,

pentameric or hexameric) generates a pore constructed from

combined alpha-helices which adopt a right-handed twist.

(B) Hypothetical gating mechanism of the connexon and other

ion channels proposed by Unwin (1989). In the open state the

pore is lined by small polar residues (S and T) and the

backbone CO and NH groups, contributed by the M3 helices.

Closure is accompanied by small, concerted displacements of

the subunits which interdisplace the small polar residues with large bulky (Phe) residues (for hexameric channels)

as opposed to amino acids with smaller hydrophobic side

chains such as Leu, which appear to be adopted in tetra and pentameric channels (see table 4.1), thus blocking the pore. A

9* s '9 O f p fsO

O f ^ f O O s ^

Open Closed

F ig.4.17

- 150 - H+-ATPases (Findlay et al., 1990). The authors propose that the Ml segment represents the most likely candidate for the pore-lining helix, of the putative hexameric channel, on account of its amphipathic character. Close inspection of the M3 segment sequence however, shows that it conforms more closely to the motif displayed in the sequences of other pore lining segments.

The helical conformation proposed for the M3 segment of connexins generates a polar ridge flanked by hydrophobic and/or aromatic ridges of amino acids (a highly conserved feature between connexins). These ridges are thought to be inclined by approx. 30° to the helix axis and would therefore align nearly parallel to the connexon axis by a corresponding tilt of the helix and/or the subunit (Milks et al., 1988? Unwin 1989? Fig. 4.16). Whereas the presence of ridges composed of large hydrophobic/aromatic amino acids flanking the polar ridge containing small polar residues would facilitate the formation of a sealed channel, they may also serve as a gating structure as described in Fig.

4.17(B). A concerted movement of the pore-lining alpha- helices, by an order of magnitude observed to occur (for connexins) between the open and closed states of the channel

(Unwip and Ennis 1894), would interdisplace the polar and hydrophobic ridges thereby filling the aqueous pore with hydrophobic side chains. These side chains generate a physical barrier to permeating molecules and may interact to generate a stable configuration (Unwin 1989). Alternatively,

-151- studies have been reported which suggest that the intramembranous portion of the connexin remains rigid and unaltered between conditions which affect gating (Caspar et al., 1988). Open and closed states of the channel may be controlled by a gating structure identified at the mouth of the channel (Makowski, 1985).

4.3.7 Special Considerations for the Modelling of Gap

Junction Channels

A unique feature of gap junction channels is that they provide an extended ion pathway which spans the approx.

30A wide 'gap' in addition to the two plasma membranes. Each apposed connexon is thought to contribute equally to the formation of the channel pore extension which must provide an insulated pathway across the hydrophilic environment of the extracellular space. This property of the gap junctional channel ascribes special functions to the extracellular domains of the connexins. Firstly, these domains must create a polar ridge/face with similar pore-forming properties to the putative polar ridge created by the M3 helix. Since the extracellular portion of the connexon has been shown to have a smaller diameter than at its cytoplasmic mouth (Unwin and

Ennis 1984), the diameter of the pore in this region may well be at its narrowest. Secondly, structural elements in these domains must provide the adhesive properties necessary for the connection of apposed connexons, which ultimately accounts for the adhesive property of the intercellular junction as a whole. Since splitting of heart and liver gap

-152- junctions occurs under conditions of low pH (Zimmer et al. ,

1987) or 8M area at alkaline pH (Manjunath et al., 1984), the attractive forces responsible for the stability of the junctional structure are derived from hydrophilic (ionic) and hydrogen bonds (suggesting that no covalent bonds or disulphide bridges exist between apposed connexons).

The amino acid sequences of the putative extracellular loops of connexins of known primary structure are displayed in table 4.2. These sequences illustrate highly conserved stretches of amino acids both within the connexin subclass, and across subclasses (the latter are the boxed sequences). Whereas loop 1 appears to be highly conserved across connexin subclasses, a greater demarcation between the alpha and beta subclasses is evident from the sequences of the loop 2 peptide. An important conserved feature of both loops is the presence of proline and cysteine residues which may be involved in the formation and stabilization of hairpin turns (Fig. 4.14). In a recent report (Tibbits et al., 1990), evidence was presented suggesting the presence of alpha-helical structure within the extracellular disposed portion of the connexon. Thought to arise from an extension of membrane-spanning alpha-helical structure, putative regions may be expected to display cross subclass sequence similarity at a level comparible to that exhibited by the membrane spanning segments. Possible candidates for such regions lie at the carboxy1-ends of both loop 1 and loop 2 peptides (table 4. 2), that is, regions

-153- TABLE 4.2 AMINO ACID SEQUENCES OF PUTATIVE CONNEXIN EXTRACELLULAR DOMAINS o X H p O « o o 2 W >5 E u 5 CO CO 05 CO O U H CO a D u ex, e as «J3 Q w w W 2 h • 1 M2 CM VO pa -P O < ■ 2 2 CO w P o O Q > 2 2 p P p P p U > CD CD CO CO CO CO EC O X ^ £ «C O p p O O O O O O O O X 2 > > P 1—1 M M M M O w M M w Q > 2 2 rd CM h h h h E CM pa •P o u CJ < OCO CO CO CO M o p co CO Q PPP,<222 >H p P p W W > EC p p p 2 > rd «- h 2 2 2 CM X 2 P O 2 2 < r< < CD< ( > < > > > CD CD E P PPP P P P P P H > H > H > w W w O P P CDP CD CD CD 2E 2 2 P p P P P 2 X 2 22 2 2 2 2 > G B r— h

I E ,o O 2 2 2 CD o o o w W ec 2 2 2 > > P X < 0 G G T h Q 2 2 X Eh O < < CO JO CJ P p P a o o o 2 P P P E 2 2 2 0) CM h I h Q ’sT P -P o o OCO CO P P >H H > P P H CD CD CD W 2 2 2 <3 p 05 E w > > rd r— h

2 2 Q 2 O < 2 2 W 2 2 a W > > W E DCD CD < p G G g r— h - 154 - Q X O CO < o W P W 2 E > M | p O 2 P 2 P P < > 2 0 h «—i Q VI f A w 6- pa u CM 2 W CO p 2 o 2 O 2 CO < m 05 o W 1 1

>H >H H > H > I 2 2 2 VO CMCMCMCMCMCMCMCM £ £ £ p p M P P £ p p p p !2 W o Q o o o o o o o P > >H > Oh> P H > W H > H >H> >H a a o o o o o o o o S 2a > > S o o o o o o o o H p 2 i O O a ^ ' P - P E ' P - P E - H ' t - PE P P P >HP >H <3P << P P P > > > X X X P P P P P P P P 22222222 drd rd N H (J - H rH (NJ »-H H H H H (N < > CM -p pa O h 2

2 >-)' p G B

2 2 o a a a o - t I—I H H H M o o M M M M P M > > M P < X X > H > H > H > H > H | H > H > H > H > H > PU O O CO CO CO M p p p M S >-. p p p M O O CO CO > CO P P £ £ 05Oh >-• M p p p 1 CtH 0-1 0-| pin >1 El >H>H H p > > X be: w tsc: p x 0 0 G G p 02 00 lilt JH H > H > J>H H > pa < -P 0 (% < < p p p 2 2 X 2 G B

p CO< E-* CO M >H 0 G

w e- o < p 2 2 05 o W CO w CO CO lO 2 o o 2 connected to helices M2 and M4. Thus, helices M2 and M4 may extend beyond the boundaries of the lipid bilayer and into the extracellular gap.

This chapter has described an investigation of the topography of connexin 32 in the gap junctional membrane by the use of a panel of site-directed antibodies. The data presented here is in excellent agreement with data from other laboratories and essentially endorses the model proposed by Zimmer et al (1987) and Milks et al (1988). In addition, the model has been refined in several aspects, as illustrated by Fig. 4.13, especially the demonstration of a disulphide bridge(s) linking the extracellular ldops.

It is becoming apparent that the connexins are the membrane-spanning structural units which associate to form ion channels in gap junctions. In common with a variety of other channels, the ion pathway is generated from amphipathic alpha-helices contributed by each subunit. These alpha- helices display a periodicity of sequence which appear to be characteristic of their structural role. The biochemistry of ion channels entertains many such theoretical models, which await refinement from high resolution structural data.

-155- CHAPTER 5

LIVER GAP JUNCTION POLYPEPTIDES AND HOMOLOGOUS ANTIGENS IN

BRAIN

5.1 Introduction

The biochemical characterization of the gap junction was pursued by analysing the composition of isolated subcellular fractions enriched in gap junctions. The isolation strategy involved the preparation of plasma membranes and their treatment with membrane-denaturing reagents, namely detergents and alkali. The differential solubilization of non-junctional membrane, a phenomenon attributable to the relative resistance of junctional membranes to these solubilizing reagents (Benedetti and

Emmelot, 1968), was accompanied by an enrichment of gap

junctions.

Early attempts at preparing liver gap junctions

incorporated the enzymatic treatment of plasma membranes with collagenase and hyaluronidase, prior to exposure to detergents. Treatment of plasma membranes with detergents alone gave rise to partially solubilized amorphous material

and contamination with collagen fibres (Goodenough and

Stoeckenius, 1972? Evans and Gurd, 1972; Culvenor and Evans,

1977). As a consequence, biochemical degradation of the

isolated gap junctions was occurring despite appearing to

retain their morphological integrity under the electron

-156- microscope. This proteolysis of the gap junction preparations, coupled to persisting contaminants including a

34 KDa polypeptide later identified as uricase (Hertzberg and

Gilula, 1979), represented the central problems in

identifying the principal protein components of gap

junctions.

Improvements in preparative methods allowed the

omission of the enzymatic pre-treatment of plasma membranes

and included a combined urea-Triton X-100 enrichment

procedure (Henderson et al., 1979). These gap junction preparations from mouse liver were composed predominantly of

polypeptides of Mr 26 (connexin 32) and 21 KDa (connexin 26),

and higher molecular weight components corresponding

presumably to dimers, the formation of which was promoted by boiling samples in Laemmli buffer prior to electrophoresis.

An alternative method for isolating liver gap junctions

substituted an alkali-extraction (20mM NaOH) procedure in

place of the use of non-ionic detergents, generating an

improvement in yield (Hertzberg, 1984). Polypeptides within

the size range of 26-30 KDa were identified as the principal

component of rat liver, heart, uterus and lens gap junction

preparations. Although the level of homology between the

principal liver and heart polypeptides was debatable, the Mr

26 KDa component of lens junctions appeared to be unrelated

(Hertzberg and Gilula, 1979? Kensler and Goodenough, 1980?

Hertzberg et al., 1982? Gros et al., 1983? Zervos et a l .,

1985). The first indication that gap junctions were composed

-157- of a diverse but related family of proteins arose from the generation of a defined immunological reagent, directed against a synthetic peptide corresponding to amino acid residues 6-21 of the liver 28 KDa (connexin 32) polypeptide.

This antibody cross-reacted with polypeptides of similar apparent molecular masses in heart and uterine preparations

(Zervos et al., 1985). Subsequently isolated cardiac gap junctions were shown to consist of a distinct, but related polypeptide of Mr 47 KDa (connexin 43) which degraded to 28

KDa N-terminal and 17 KDa C-terminal peptides during isolation (Nicholson et al., 1985? Manjunath et al., 1987).

Recently a refined protocol for the preparation of ventricular myocyte gap junctions has identified a major novel component of Mr 70 KDa, immunologically related to connexin 43 (Harfst et al., 1990). It appears that the periodic refinement of isolation procedures engenders the revision of our perception of gap junction bidchemistry.

This chapter addresses various aspects of the biochemistry of hepatic gap junctions isolated from rat and guinea pig, highlighting some of the technical difficulties of working with their components. The biochemical properties of these components are compared and information is presented which suggests that the biochemical characterization of hepatic gap junctions, as yet, is incomplete. In addition, the detection of homologous antigens in adult and neonatal brain is presented.

-158- 5.2 RESULTS

5.2.1 Preparation of Rat Liver and Guinea Pig Liver

Gap Junctions

Gap junctions were prepared from ret and guinea pig livers according to Hertzberg (1984). The recovery of gap junction from liver homogenates, measured as total protein, was found to be Similar and reproducible from the livers of both species using the alkali-extraction procedure (table

5.1). Fig. 5.1 illustrates a typical discontinuous sucrose gradient purification of gap junctions from alkali-extracted rat liver plasma membranes and the analysis of the density- equilibrated fractions by SDS-PAGE. When analysed by electron microscopy (Fig. 5.2) and SDS-PAGE (Fig. 5.IB), the material equilibrating at the 46/30% sucrose interface was comprised predominantly of gap junction double membrane structures and the monomeric (28 KDa) and dimeric (54 KDa) forms of connexin 32 respectively. Analysis of the material with corresponding buoyant density derived from the sub fractionation of alkali-extracted guinea pig liver plasma membranes provided similar results (Fig. 5.3). Electron microscopy showed a high proportion of dense double membrane gap junctions amidst some non-junctional membrane. Analysis of this fraction by SDS-PAGE identified major protein components of Mr 22 KDa and 28 KDa, consistent with the report of Takeda et al (1988). These polypeptides presumably correspond to connexin 26 and 32 respectively, based on their co-migration with the corresponding mouse and rat liver

-159- TABLE 5.1 RECOVERY OF GAP JUNCTIONS FROM RAT AND GUINEA PIG LIVERS LT) C - - 4 d C 4-J E . o ai u OJ OJ 03 O a O > 03 > p s- u ' O ' O 4- E c c c o a c ) > O •>“ «— 03 -r- D_ *’-> — 03 ■— - E 4- •r— Q_ 4-> C J CO - QJ 4-> 4-> -t-> -M 4-> E o - C S- C o CD to C LO 03 03 l/) o 03 i_ 03 03 C o C o 3 C 03 E 03 03 CD- U C l

O«=r -O o E 03 s -160 _ -— o -— • r— CM CO o o CM CO CD r"- CM CM o O C o O o o CO O C QJ 03 - Q C • • • • r> * - •f— > QJ to S- LO O LD CM o 03 O or CM CD CD CO LO CM • i— O •i— > to i_ Q ■a •M 4 +-> 03 03 03 > 03 £_ or 03 03 03 O O 03 CL a) . a o i_ 03 S-. OJ c to - Figr5.1 Preparation of liver gap junctions

(A) Discontinuous sucrose gradient showing the density equilibration of alkali-resistant material purified from alkali-extracted liver plasma membranes.

(B) Analysis of this material by SDS-PAGE on 12.5% separating gels, stained with Coomassie brilliant blue. Lanes 1 & 2;

3.0ug of material equilibrating at the 46/30% (w/w) sucrose

interface (gap junction fraction) from two separate preparations. Lanes 3 & 4? 3.0ug of material equilibrating at the 30%/HC03” interface. This fraction is highly enriched with an Mr 70 KDa polypeptide which correlates with an

enrichment of 5 '-nucleotidase activity Evans & Gurd, (l972). % % A. 46 30 HCO Fig.5.2 Morphology of isolated rat liver gap junctions

Electron micrographs of thin sectioned material equilibrating at the 46/30% (w/v) sucrose interface, stained with uranyl acetate/lead citrate.

(TOP) Pentalaminar, double membrane gap junctions. Bar =

130nm.

(BOTTOM) Higher magnification reveals the heptalaminar configuration of gap junctions, making the 2-3nm 'gap' visible

(arrowheads). Bar = lOOnm. -162 - Fig.5.3 Morphological and biochemical analysis of guinea pier

liver gap junctions

(A) Electron micrograph of thin section material equilibrating at the 46/30% (w/v) sucrose interphase during the purification of alkali-resistant membranes from guinea-pig liver plasma membranes extracted with 20mM NaOH. A high proportion of electron dense, double membrane gap junctions are present amidst some non-junctional membrane. Bar = 0.5 um.

(B) Coomassie brilliant blue profile of guinea pig liver fractions at different stages of the preparation of gap junctions, after analysis by SDS-PAGE on 12.5% separating gels. HOM: 30ug of liver homogenate, P.M.: 30ug of parent plasma membranes, GPLGJ: 3.0ug of the gap junction fraction shown in (A), RLGJ: 3.0ug of rat liver gap junctions. In ascending order, the arrowheads highlight the monomers at 22 and 28 KDa and the homo- and heterodimers at 40, 47 and 54

KDa, corresponding to connexins 26 and 32 and their oligomers. 0v O B Mr kDa

-69

-46

-3 0

-21 If t r

-163 - counterparts (Takeda et al., 1988? Fig. 5.3B) and some limited immunological data. Also visible in the guinea pig gap junction SDS-PAGE gel profile were faint bands of apparent molecular mass 40, 47 and 54 KDa which presumably correspond to homo- and heterodimers of the 22 and 28 KDa polypeptide. In contrast to rat and mouse liver gap

junctions the 22 KDa polypeptide was determined (by

densitometric scanning) to be three times more abundant than

the 28 KDa component in guinea pig gap junction preparations.

5.2.2 Immunological Comparison of Rat and Guinea Pig Gap

Junction Components

Rat and guinea pig liver gap junction fractions were

analysed comparatively by SDS-PAGE and Western blotting,

utilizing the panel of polyclonal anti-peptide antibodies to

rat liver connexin 32, in order to establish a 'connexin-

relationship' between the 22 and 28 KDa guinea pig

components. The results of this analysis are shown in Fig.

5.4. All antibodies recognized the 28 KDa polypeptide of

both rat and guinea pig gap junctions and its corresponding

homo- and heterodimeric forms migrating at 54 and 47 KDa

respectively. This observation implied that both rat and

guinea pig 28 KDa components are highly homologous isoforms

of connexin 32, probably sharing extensive sequence

similarity. The predominant 22 KDa component of guinea pig

liver gap junctions was only detected by GAP 7-M and GAP 11

antibodies and to a lesser extent by WGJ antibodies. I'hus,

antibodies directed towards conserved, extracellular

-164- Fig.5.4 Immunological comparison of rat and guinea

pig liver gap junction connexins.

Rat and guinea pig liver gap junctions (3.0ug) were analysed by SDS-PAGE on 12.5% separating gels. CBB: Coomassie brilliant blue profile of thd resolved proteins. Similar gels were electrophoretically transferred to nitrocellulose membranes and stained with the various antisera at dilutions of 1/25 - 1/50, and processed for Western blotting. Black arrowheads highlight the monomeric forms of connexins 32 (28

KDa) and 26 (22 KDa). White arrowheads highlight homo- and heterodimers of connexins 32 and 26. Note how connexin 32- specific antisera (GAP 9) labels both 54 KDa homo-connexin 32 dimer and the 47 KDa hetero-connexins 32/26 dimer with an intensity of 2:1 approx. respectively. Only antisera with cross connexin class immunospecificity (GAP 7-M and GAP 11, but not GAP 3 antibodies apparently) label the 40 KDa homo- connexin 26 dimer. ^ N O 00 CVJ L D ^ t C \J C \J v w ▼ ▼

o £

CL < o o

CL CD -t cn < z 0 o 0 I— I Q- o C/D 0 z LUO 3 I ~D

CL CD < CL « I < 0 CD CD DC LU 0 > i CL h» o -* CL I- < < 0 cr

0 CL CO 0 DO o H CC<

>- o o CO

- 165 - sequences of connexin 32 cross-reacted with the 22 KDa component and its 40 KDa dimeric form, in contrast to antibodies directed towards intracellular, connexin 32- specific sequences. Amino terminal directed GAP 3 antibodies however, did not identify the 22 KDa polypeptide, despite having previously been shown to contain cross-connexin class reactivity (Zervos et al., 1985). The low level of activity towards the guinea pig 22 KDa component present in WGJ antibodies probably reflects a minor population of anti-rat

22 KDa immunoglobulins, produced as a result of immunization with intact gap junctional membranes.

5.2.3 Biochemical Properties of Connexins

5.2.3a Dimerization

The tendency of connexins to dimerize during denaturing gel electrophoresis is a well-established phenomenon associated with gap junction biochemistry

(Henderson et al., 1979). That these higher molecular mass bands originate from monomers of greater mobility is corroborated by immunological data, using highly specific anti-peptide antibodies (Fig. 5.4), and biochemical techniques (Henderson et al., 1979? Green et al., 1988? Evans et al., 1988).

When analysed by SDS-PAGE using 10% separating gels, connexin 32 derived from rat liver gap junctions migrates as monomeric 27 KDa and dimeric 47 KDa forms (Green et al.,

1988). To obtain amino-terminal sequence information the 27

-166- and 47 KDa bands were excised from preparative gels and the protein recovered by electroelution. Re-analysis of each sample by 1 0 % denaturing gel electrophoresis indicated that the electroeluted 4 7 KDa dimer had remained predominantly dimeric (Fig. 5.5B). These observations are in‘mirror* contradiction to those reported by Henderson et al (1979), although in this more extensive study of the dimerization phenomenon, the samples had been precipitated by dehydration and iodinated under highly oxidizing conditions. It appears that under the solubilizing conditions adopted in the present study, the dimeric form of connexin 32 was more stable.

Microsequence analysis of the 47 KDa electroeluted protein was performed in order to assess the biochemical composition of the mixture. The analysis indicated the availability of two different amino termini producing different sequences but with similar molar quantities, suggesting that they were derived from the same polypeptide.

The amino acid sequences obtained were manually co-aligned with the published sequences for connexins 32, 26 and 30, as shown in Fig. 5.6. A high level of sequence similarity was observed between sequence 47 KDa (A) and residue 6-20 for the beta-class connexins. Sequence 47 KDa (B) appeared related to, but distinct from, connexin sequences located between amino acids 120-140. Taken together, these suggest the presence of a third connexin expressed in liver, probably closely related to connexin 32 as inferred by the level of amino terminal sequence similarity.

-167- Fig.5.5 Dimerization of connexin 32

Electroeluted monomeric and presumptive dimeric forms of connexin 32 were re-analysed by SDS-PAGE in 10% separating gels, and silver stained.

(A) Lanes loaded with a 10% fraction of the recovered electroeluted 27 KDa monomer.

(B) Lanes loaded with a 10% fraction of recovered 47 KDa presumptive dimer. Bands at 66 KDa correspond to serial loadings of BSA to facilitate quantification of recovered electroeluted protein. AB Mr. kDa

115-

^ 66 K

48- «47K

30- <«27K

- 168 - Fig.5.6 Microsequence analysis of electroeluted 47 KDa

protein

The sequences obtained from the microsequence analysis of the

47 KDa band excised and electroeluted from 10% separating gels were manually co-aligned with sequences from the published beta-connexins. Note the high level of homology between sequence 47 KDa (A) and residues 1-20 of the beta-connexins.

A low-level of homology is evident between sequence 47 KDa (B) and beta-connexin sequences derived from the predicted intracellular loop. •H in 10 fcl rPw P er • • HICROSEQUENCE ANALYSIS OF ELECTROEI»UTED 47KDa PROTEIN 0 O w w w 2 a H 2 2 E W H 2 2 Q W h O in CN cn rH rH lO 00 cr> »H 0 in 00 iH rH rH rH o CO > cn ID iH rH H CN rH »H M < 0 to o 2 > ' ' SzN < r> XI C222 * 2 2 2 SC E-* Q M E 0 0 0 0 >< 0 — 2 s o O n3 h 2 0 bd W Q 0 CN o CN o i—l o U a « CO 0 E-< E« 2 £ E-* 0 E >■< M H W W H M 2 < CO O 0 > X 1 h -169- ■w* * CN NCN CN 0 rH oCO to 2 2 E 2 P CO 0 0 O > 0 p a M — X X h 1 s r—N o o Hr HrH rH rH 0 rH 2 CO * E- 2 2 * 0 •< < 2 2 < cu CO * o > > o * 0 >H M 0 0 1 * * 0 w 0 2 N o 0 < 0 M 0 XJ Q x: x: rd < 0 2 XJ W Q < w s<> rH H 0 0 0 E-* 2 0 2 2 E-* 0 0 0 2 2 rH M 0 M W 0 2 2 0 >-• w W 2 > :x: X: * X 2 X: XJ W o u u X X i 1 i CN * 0 0 0 0 E 0 M ’cr H E- > a Q > > W h h w E 2 E o 0 -K M rH 0 rH >H p < 2 w * is: i*: x: > w x. h h

CONSERVED AMINO ACID RESIDUES 5.2.3b Anomalous Electrophoretic Mobility

Prior to the acquisition of complete amino acid sequences, the nomenclature of connexins was based on their electrophoretic mobility in denaturing polyacrylamide gels.

Examination of the literature reveals that gap junction poly peptides of M 26, 27, 28, 30, 32 correspond to rat liver connexin 32, indicative of its anomalous mobility during SDS-

PAGE (Green et al., 1988). To ascertain whether this behav iour was exhibited by the components of guinea pig liver gap junctions, both rat and guinea pig liver preparations were analysed by SDS-PAGE using 12.5% and 17.5% separating gels.

The results of this analysis are shown in Fig. 5.7. In 12.5% gels, the monomeric and dimeric forms of connexin 32, derived from both rat and guinea pig liver gap junctions, resolve with relative mobilities of 28 and 54 KDa respectively. In

17.5% gels however, the relative mobilities of both monomers and dimers 'shifted' to 32 and 64 KDa respectively. Thus, both connexin 32 homologues display altered electrophoretic migration with a change in polycrylamide concentration. The magnitude of the observed electrophoretic shift for connexin

32 and its oligomers is in agreement with the report of Green et al (1988). Furthermore, the reduction and alkylation of the gap junctions prior to solubilization in non-reducing buffer, had no effect on the pattern of migration of connexin

32 or 26. The electrophoretic migration of connexin 26 was not observed to alter with the corresponding change in polyacrylamide^concentration. In contrast to connexin 32,

-170- Fig.5.7 Anomalous migration of connexin 32

during SDS-PAGE.

Guinea pig (GPLGJ) and rat (RLGJ) liver gap junctions were analysed by SDS-PAGE on 12.5% and 17.5% separating gels. (+): samples were reduced and alkylated prior to solubilization in non-reducing Laemmli buffer, (-): samples were untreated and solubilized in reducing Laemmli buffer. The monomeric (M) and dimeric (D) forms of connexin 32 and 26 are highlighted by the arrowheads. Note the shift in relative mobility of connexin

32 (both monomer and dimer) relative to the 30 KDa standard

(carbonic anhydrase) between 12.5 and 17.5% gels. Plots of distance migrated versus log molecular weight quantifies this shift as 2 KDa approx. for the connexin 32 monomer. No such shift in apparent molecular mass was observed for connexin 26. T T TT PG R LGJ GPLGJ 12-5 % - 171 - G GPLGJ R LGJ 17*5 % the change in R^ for connexin 26 between 12.5% and 17.5% gels was consistent with that predicted for a 26 KDa polypeptide, that is, the estimated molecular weight of connexin 26 in

12.5% and 17.5% gels was similar.

5.2.3c Differential Rates of Electrophoretic Transfer of

Connexin 32 and 26

The electrophoretic transfer of guinea pig liver gap junction connexins during Western blot analysis, was observed consistently to produce distorted relative quantities of connexin 32 and 26 on nitrocellulose membranes (see Fig.

5.4), with an apparent loss of a large proportion of connexin

26. Necessitating the inclusion of SDS in transfer buffers, longer transfer periods of low electric field strength produced more faithful nitrocellulose Jreplicas than shorter transfer periods at high electric field strength. This

implied that the rate of electrophoretic migration of connexin 26 was considerably higher than that of connexin 32, with a large proportion of molecules passing through the nitrocellulose membrane. Transfer conditions generating a

faithful reproduction of the relative quantities of connexins

32 and 26 were not determined definitively.

5.2.4 Microsequence Analysis of Guinea Pig Liver Connexins

To obtain microsequence information for the

components of guinea pig liver gap junctions, thereby

allowing a direct comparison with connexins from rat liver,

gap junction fractions were chemically modified by

-172- succinylation and carboxymethylation, analysed by SDS-PAGE, and the components of Mr 22-28 KDa excised and trypsinized.

Tryptic fragments were purified by reverse-phase HPLC and sequenced (see materials and methods). The HPLC elution profile and peptide sequences are shown in Fig. 5.8.

Surprisingly, all peptides purified possessed identical amino termini suggesting that they were isoforms of similar tryptic products differing perhaps in length, or the possession of modified amino acids, underlying the different retention times observed. Comparison of the sequences obtained with published cDNA deduced protein sequences for connexins 32, 26

and 30, revealed that the tryptic peptides were all derived

from the guinea pig liver connexin 32 homologue,

corresponding to regions within amino acid residues 263-280

of the rat liver counterpart. A high level of sequence

similarity is visible between the guinea pig sequences

obtained by chemical methods and the cDNA predicted rat liver

sequence, with the exception of a substitution at lie (cycle

4) for Ser 266. Peptides corresponding to tryptic products

derived from the guinea pig liver 22 KDa polypeptide

(connexin 26 homologue) were not recovered.

5.2.5 Immunological Analysis of Liver and Brain Homogenates

The identification of antigens related to connexin 32

in liver and brain, and across species, was performed by

Western blotting. Whole tissue homogenates, some of which

were alkali-extracted with 20mM NaOH, were analysed by SDS-

PAGE and probed with various connexin 32-directed anti-

-173- peptides by HPLC

(opposite)

Elution profile of the HPLC analysis of guinea pig liver connexin tryptic peptides. The elution was monitored at 480nm to identify peptides containing cysteine residues (alkylated with IAF; see material and methods, 2.3.1b). Peptides are identified alphabetically, and CBB refers to the elution of

Coomassie brilliant blue.

(Below)

Sequences obtained by microsequence analysis of purified peptides and alignment of peptide E with rat liver connexin

32. CYS residues are not detected by the Beckman instrument.

” "" H | Peptide Retention Time Acetonitriie Cone. Amino Acid Seq. j i

(min) % (v/v)

D 35.0 38 LRRIP

E 27 .5 30 LRRIPGTGAGLAEKSD-S-

F 36.5 40 L—IPGT-A

G 38.0 42 LRRIPGT-A i i H 39.0 43 LRR-PG

! 1 4 0 .5 44 LRR-P

CYCLE NUMBER

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Peptide E LRRIPGTGAG L A E K S D - S -

Rat Cx 32 LRRSPGTGAG L A E K S D C S A (265) (270) (275) Fig. 5.8 - 174 - o E c TT B c E TTTT ^in r r o CM I o L O U _ _ ID O CD

Retention Time (min) peptide antibodies after transfer to nitrocellulose- All

antibodies detected polypeptides of Mr 28 and 54 KDa in both mouse and chick liver homogenates, corresponding to the monomeric and dimeric forms of connexin 32 (Fig. 5-9). The

high affinity signal obtained by GAP 9 antibodies, an

antibody directed against a C-terminal sequence peculiar to

connexin 32, suggests that gap junctions in mouse and chick

livers contain a closely related homologue of connexin 32, as

observed with guinea pig hepatic gap junctions (Fig. 5.4).

The results obtained with homogenates from brain

tissues suggest the possible existence of hitherto

uncharacterized connexins. In both mouse and guinea pig

homogenates two antigens were detected, including a common

component of Mr 95 KDa. As many as three different

antibodies detected this component, with the highest affinity

signal obtained by GAP 7-M antibodies. A 30 KDa polypeptide

was detected in mouse brain homogenates by all antibodies

except those directed against the peptide GAP 9. This

observation suggests that the 30 KDa antigen is closely

related to, and possibly a homologue of connexin 32,

containing sequence polymorphism at the extreme of its

carboxyl terminus. The 30 KDa polypeptide was not present in

guinea pig homogenates, although a 22 KDa antigen was

detected by DES-1 antibodies, possibly resulting from

degradation.

Analysis of rat embryonic tissue produced similar

results to those obtained with adult tissue. GAP 9, GAP 10

-175- Fig.5.9 Western blot analysis of tissue homogenates

(TOP) CBB: Coomassie brilliant blue profile of lOOug of liver protein analysed by SDS-PAGE in 12.5% separating gels.

Proteins from similar gels were electrophoretically transferred to nitrocellulose membranes and processed by

Western blotting staining with the antisera as indicated.

Serum dilutions of 1/25 - 1/50 were used.

(BOTTOM) Western blot analysis of alkali-extracted brain homogenates, as described above. 4- * / / ^ <4 n cT o°O O' c? o cT o0, cT

200 - 92 - i m | * 69 - *

46 - * D

3 0 - - «M

21 -

1 4 -

MOUSE CHICK

WHOLE BRAIN HOMOGENATES

V q> *\ 7N <* p v* V O P P °D < y

92 - m k ^ 9 5 K 95K

69 - %

46 -

<« 3 OK 30 - • 22K

21 -

14 -

i S f

MOUSE G UINEA PIG

-176 - and DES 1 antibodies detected a 28 KDa polypeptide in liver homogenates, corresponding to connexin 32 (Fig. 5.10).

However, only GAP 10 and DES 1 antibodies detected a component of similar size in brain homogenates providing further evidence for a unique connexin relative or homologue in brain possessing a divergent carboxyl terminus.

Antibodies to GAP 10 identified a further component of Mr 40 KDa in embryonic liver homogenates. This polypeptide was retained after alkali-extraction with 20mM

NaOH in contrast to the 28 KDa (connexin 32) polypeptide which was lost. Since the amino termini of connexins display partial sequence similarity, GAP 10 antibodies may portray cross-connexin class immunospecificity, analogous to GAP 3 antibodies, suggesting that this 40 KDa polypeptide is connexin related, and possibly of the alpha-class. The latter notion is consistent with an observed absence of labelling by DES 1 and GAP 9 antibodies, which are directed towards regions of sequence diversity between connexin classes.

5.3 Discussion

In recent years, many of the anomalies associated with the biochemistry of gap junctions have been clarified by the information made available through the isolation of cDNA clones coding for connexins, and the development of defined antibodies. In this chapter, some of the biochemical properties of liver connexins have been investigated and the

Capacity of anti-peptide antibodies to distinguish between,

-177- Fig.5.10 Western blot analysis of rat embryonic liver and brain homogenates.

Homogenates derived from 5 embryonic livers and brains were analysed by SDS-PAGE in 12.5% separating gels, and processed for Western blotting, labelling with affinity purified GAP 10 antibodies (20ug/ml), DES 1 antiserum (1/25) and GAP 9 antiserum 1/50. (+): homogenates extracted with 20mM NaOH, (-): untreated homogenates. Degradation of connexin 32 from 28 to 22 KDa forms is apparent in blots labelled with DES 1 antibodies. v

CD I CL < (D

0 ) LU

I Q_

k c c < LU > o

cc i r T T O CM CD CD o v CD CD CO C\J

- 178 - and identify putative novel connexins in liver and brain has been described.

5.3.1 Biochemical Properties Of Liver Connexins

The predominant connexins associated with mammalian liver are connexins 32 and 26, which migrate at 28 and 22 KDa in 12.5% denaturing polyacrylamide gels (Green et al., 1988? Takeda et al., 1988).

Altering the acrylamide concentration affected the migration of connexin 32, but apparently did not alter the migration of connexin 26 (Fig. 5.7). Furthermore, the reduction and alkylation of gap junction fractions prior to solubilization and electrophoresis did not appear to alter this property implying that the formation/persistence of disulphide bonds was not an underlying factor promoting this phenomenon. Since the major difference between connexins 32 and 26 lay in the length of the hydrophilic carboxyl 'tail', the effect of polyacrylamide concentration on the migration of connexin 32 may be mediated through this region of the polypeptide, i.e. by altering the proportion of SDS that it binds. That this phenomenon is attributable to the intrinsic properties of connexin 32 has been suggested by Green et al (1988), arguing that the occasional observations of 28 and 32 KDa monomeric, and 47 and 54 KDa dimeric forms in the same gel (Kumar and Gilula, 1986? Green et al., 1988; Warner et al., 1984) are indicative of a 'two-state' molecular guise.

A common biochemical property exhibited by connexins

-179- 32 and 26 was the formation of dimers under highly denaturing conditions. Under the solubilization conditions adopted in this study, the 54 KDa dimeric form of connexin 32 constitutes approx. 35-40% of the connexin 32 protein obtained from rat liver gap junctions (Figs. 5.2, 5.3, 5.4). In contrast, Coomassie blue profiles of the SDS-PAGE analysis of guinea pig liver gap junctions illustrate that greater than 90% of the connexin 26 protein exists as the monomeric 22 KDa form, with a minor amount of a 40 KDa homodimer (Figs. 5.3, and 5.4). Furthermore, a heterodimer of Mr 47 KDa, composed presumably of one molecule of connexin 32 and on^ molecule of connexin 26, was apparent in guinea pig liver gap junction SDS-PAGE profiles. These observations suggest that although both connexins form oligomers under denaturing conditions, the tendency of connexin 32 to oligomerize appears to be greater than for connexin 26. Alkylation of samples prior to electrophoresis did not appear to alter the oligomerization phenomenon either. As the relative levels of connexin 32 monomeric and dimeric forms alters with polyacrylamide concentration (Green et al., 1988), the oligomerization phenomenon may also reflect the chemistry within the hydrophilic carboxyl 'tail' region of the polypeptide. In this study, the dimerization of connexin 32 was promoted by heat and could be reduced significantly by periodically cooling samples on ice during solubilization (data not shown). Henessey and Scarborough (1989) have reported a similar effect by solubilizing hydrophobic peptides in denaturing buffers at 0°C, implying that the

-180- oligomerization phenomenon associated with connexins may be related to hydrophobic interactions between non-polar sequences.

5.3.2 Comparison Of Rat And Guinea Pig Liver Connexins

A number of reports have documented the absence of cross-reactivity between defined antibodies and cross-species homologues of their parent connexins. For example, an anti peptide antibody directed towards amino acid residues 314-322 of rat cardiac connexin 43, while labelling a 43 KDa protein on immunoblots of whole heart extracts from human, mouse and guinea pig, did not label the corresponding homologue in frog, chicken and trout heart extracts (El Apumari et al., 1990). The loss of cross-reactivity was interpreted as a consequence of sequence polymorphism in this region of the polypeptide. Furthermore, a monoclonal antibody (7-3H6) raised towards intact rat liver gap junctions, did not cross- react with guinea pig liver connexin 32 on Western blots, while labelling connexin 32 homologues from mouse and rabbit liver gap junctions (Takeda et al., 1988). In the present study five anti-peptide antisera raised towards different cytoplasmic and extracellular regions of rat liver connexin 32 cross-reacted with the homologue derived from guinea pig liver gap junctions (Fig. 5.4). This extensive immunological similarity is suggestive of a high level of sequence similarity between species. This was confirmed chemically by the sequencing of HPLC purified tryptic fragments, identifying a guinea pig liver connexin 32 sequence at the

-181- extreme of the carboxyl terminus. Only one amino acid substitution was evident in this region, consistent with the observed labelling by GAP 9 antibodies (Fig. 5.4). This substitution however, implies that further changes at other cytoplasmic domains may account for the loss of labelling with monoclonal antibody 7-3H6 (Takeda et al., 1988).

Labelling of guinea pig liver connexin 26 by GAP 7-M and GAP 11 antibodies only, is consistent with the reported sequence for rat liver connexin 26 (Zhang and Nicholson, 1989). This connexin contains homologous extracellular domains and divergent cytoplasmic domains in comparison to connexin 32. These observations illustrate how a panel of anti-peptide antibodies can identify and discriminate between the products of a gene family. Furthermore, antibodies to GAP 7-M, DES 1 and GAP 9 labelled a 28 KDa polypeptide in liver homogenates from mouse and chick, suggesting that these homologues of connexin 32, like that expressed in guinea pig liver, closely resemble the rat liver polypeptide.

5.3.3 Identification Of Novel Connexins

A variety of serendipitous observations made during the course of this study have suggested that further uncharacterized connexins are present in the tissues studied. Microsequence analysis of a 47 KDa polypeptide found in rat liver gap junction preparations generated sequences related to, yet distinct from the connexins characterized thus far. It would appear that liver gap junctions contain a third

-182- connexin component, although its molecular weight cannot be evaluated from this study due to the possibility of dimerization. This observation has been supported recently by the apparent isolation Of a novel liver connexin of molecular mass 31,000 Da, in the laboratory of J.P. Revel (Dr C.R. Green U.C.L., personal communication). Careful scrutiny of the literature reveals that a minor component of Mr 26 KDa is routinely observed in liver gap junction preparations (see Takeda et al., 1988. Fig. 5.4), which has been regarded as a degradation product. This polypeptide may correspond to the connexin isolated in the laboratory of J.P. Revel, which in turn may correspond to the putative novel liver connexin now identified in this study.

Immunological studies have also elucidated a 30 KDa antigen in neonatal and adult brain homogenates, labelled by three anti-peptide antibodies to rat liver connexin 32. GAP 9 antibodies however, did not label this antigen suggesting that it was either a connexin 32 homologue containing sequence polymorphism at the extreme of the carboxyl terminus, or a closely related but distinct gene product. Recently a connexin 32 cDNA was isolated from a rat brain library in this laboratory, and sequencing data has elucidated a high degree of sequence conservation, implying that loss of GAP 9 antibody labelling was unlikely (Kalopolthakis et al., unpublished data). This implies that the second scenario is more probable, supported in turn by the recent report of the isolation of a further two novel

-183- connexin cDNA clones, including a gene coding for a 37, 603 Da polypeptide derived from a mouse embryonic brain cDNA library (Willecke et al., 1990). An antigen of Mr 95 KDa was also detected in the present work in adult brain homogenates by three anti-peptide antibodies, suggesting that it too may be a member of the connexin family. Numerous studies have identified a variety of antigens using anti-peptide antibodies to connexin 43, which migrate at diverse apparent molecular weights (Dupont et al., 1988, 1989? El Aoumari et al., 1990? Harfst et al., 1990). Some of these antigens have been identified as modified forms of connexin 43 (i.e. containing various levels of phosphorylation), whereas the identity of others remains unresolved (e.g. the 70 KDa polypeptide of heart and lens). The identity of these uncharacterized antigens should be resolved in time using molecular biology approaches.

-184- CHAPTER 6

THE SUBCELLULAR DISTRIBUTION OF CONNEXIN 32 IN LIVER

6.1 Introduction

The main functional units of the liver are the cells which compose the parenchyma, namely the hepatocytes. They constitute approx. 80% of the liver weight, which accounts for the extensive use of this relatively homogenous tissue in morphological and biochemical studies. The hepatocyte plasma membrane, in common with other epithelial cells, is organized into morphologically and biochemically distinct domains, performing distinct functions (Evans, 1980? Simons & Fuller, 1985). The polarized cell surface is distinguished by apical and basolateral membranes bordered by tight junctions. The apical membrane encompasses 10-15% of the total surface area and consists of microvilli protruding into the bile-canalicular space (Fig. 6.1). The basolateral domain is sub-divided into regions comprising a microvillar, sinusoidal membrane facing the blood that continues into a flattened lateral region, characterized by the presence of intercellular junctions (Evans 1980? Maurice et al., 1985). Furthermore, subcellular fractionation approaches have identified an assymmetric distribution of catalytic activities associated with the hepatocyte plasma membrane, in addition to lipids and non- catalytic proteins (Evans, 1970? Aronson and Touster, 1974? Wisher and Evans, 1975? Toda et al., 1975? Hubbard., et al 1983? Taylor et al., 1983? Evans and Enrich, 1989).

-185- Fig.6.1 Morphology of hepatocyte plasma membrane

Transmission electron micrograph of liver tissue shows the morphology of the hepatocyte plasma membrane. LAT: lateral plasma membrane containing a gap junction (GJ), SIN: sinusoidal plasma membrane, CAN: bile canalicular membrane. TJ: tight junction. This liver section was stained with an integrin specific antibody which shows a surface membrane distribution. The micrograph was kindly donated by Stamatis Stamatoglou. - P A . »

, ■ * ■ ^ . . . -■

C P w

-vSr • T h- £ > i M a ^ t V* v I »*>..*. - * V frtfC*

jr*n. •<4iC?$X^W 'ijk ^5 •- £'

k | w c

THBSWes^r%- ' * ' Sir^R ^

•U'* V ' . Vstt

■ ■

- 186 - The isolation and biochemical characterization of liver plasma membrane fractions, and fractions derived from intracellular membranes, has been based predominantly on the distribution of marker enzymes. Consequently, procedures have been developed allowing the preparation of subcellular fractions from liver homogenates highly enriched in membranes originating from sinusoidal, lateral and canalicular surface domains, and Golgi, lysosomal and endosomal intracellular compartments. Since little is known about the fate of cdnnexin 32 between its synthesis on ribosomes and its assembly into the gap junction at the cell surface, the subcellular distribution of connexin 32 was investigated with a view to providing some insight into the movement of this connexin through the membrane networks of the hepatocyte.

6.2 Results

The distribution of connexin 32 in liver plasma membranes and various intracellular membranes was analysed by SDS-PAGE and Western blotting. Fractions isolated from a number of different preparations wgre analysed to develop a consensus semi-quantitative distribution pattern. Nitrocellulose replicas were stained with GAP 9, DES 1 and WGJ antibodies (see table 3.1 for details) to assess whether proteolytic processing events were evident. A typical staining pattern observed with GAP 9 antibodies is shown in Fig. 6.2. Comparatative assessment of the levels of connexin 32 in different membrane fractions was performed by quantitative densitometry (Fig. 6.3). Comparison of the relative intensity ratios (expressed as %, ± S.E.M. (n=6)

-187- Fig.6.2 Western blotting of liver subcellular fractions

Liver subcellular fractions (50ug/sample) were analysed by SDS- PAGE in 12.5% separating gels and processed for Western blotting, staining with affinity purified GAP 9 antibodies.

(TOP): Coomassie brilliant blue gel profiles of named fractions.

(BOTTOM): Nitrocellulose replicas of corresponding fractions stained with GAP 9 antibodies. LAT: lateral plasma membranes, SIN: sinusoidal plasma membranes, CAN: canalicular plasma membranes, LYS: lysosomal membranes, GOL: Golgi membranes, END: endosomal membranes, M-Res: membranes comprising the microsomal residue. 28 and 54 KDa correspond to the monomeric and dimeric forms of connexin 32. LIVER SUBCELLULAR FRACTIONS

2? Q)

^ - f & & / Mr kDa ^ °o O v/ G> <(/ ^

w r m • " » • m r m - 2 0 0 - 9 2 -

6 9-i

4 6 -

3 0 - i . 1 -

2 1-

5 4

GAP-9

- 188 - Fig.6.3 Subcellular distribution of connexin 32 in liver

The histogram indicates the relative distribution of connexin 32 in liver, determined by densitometric analysis of connexin 32 monomer and dimer peaks obtained from six experiments. Connexin 32 monomer signals detected by WGJ, GAP 9 and GAP 10 antibodies are shown above each fraction. M-Res: microsomal residue, GOL: Golgi, LAT: lateral plasma membrane, SIN: sinusoidal plasma membrane, CAN: canalicular plasma membrane, LYS: lysosomes. Distribution of Connexin 32 in Liver Subcellular Fractions

GAP 9

— GAP 10

C 3 0

i— — r M-Res Gol Lat Sin Can End Lys Subcellular Fractions

- 189 - of the total intensity of all monomer and dimer peaks of fractions analysed per experiment) for the various fractions analysed showed that lateral plasma membranes had by far the highest levels of connexin 32. Considerable levels of connexin 32 were also detected in Golgi membranes suggesting that this membrane complex presented a 'bottle-neck' in the distribution of connexin 32 to the cell surface. Both sinusoidal plasma membranes and lysosomal membranes contained similar and appreciable amounts of connexin 32. However, very low levels of connexin 32 were detected in canalicular and endosomal fractions. Comparatively low levels of connexin 32 were also present in the microsomal residue. This result is suggestive of a rapid transfer of connexin 32 to the Golgi complex from the endoplasmic reticulum, following synthesis on ribosomes.

Biochemical studies with isolated gap junctions have shown that connexin 32 is unglycosylated (Hertzberg and Gilula, 1979). As many, probably most proteins are covalently modified while migrating through the Golgi complex, the possibility that connexin 32 is also glycosylated during intracellular trafficking and subsequently deglycosylated prior to connexon assembly was investigated. Golgi, sinusoidal and lateral fractions were treated with endoglycosidase F (which cleaves N-linked high mannose side chains) prior to analysis by Western blotting. The enzymatic treatment however, had no detectable effect on the relative mobility of the connexin 32 monomer or dimer, indicative of an unglycosylated polypeptide (Fig. 6.4). Finally, all the antibodies used in these

-190- Fig.6.4 Treatment of subcellular fractions with endoglycosidase F

Golgi (GOL), sinusoidal (SIN) and lateral (LAT) membrane fractions were treated with endoglycosidase F (materials and methods, 2.3.3) prior to anlysis by SDS-PAGE. (+) treated membranes (50ug), (-) untreated membranes (50ug). Samples were transferred to nitrocellulose membranes and stained with GAP 9 antibodies. 28K experiments identified monomeric and dimeric forms of connexin 32 of similar mobility across the spectrum of subcellular fractions. This indicated that proteolytic processing (of a detectable magnitude) of connexin 32 during trafficking to and from the cell surface did not occur. However, connexin 32 degradation products were detected in lysosomal fractions, and included peptides of Mr 21, 17, and 10 KDa, detected by those antibodies directed towards relatively protease resistant regions of connexin 32 (Fig. 6.5).

6.3 Discussion

Subcellular fractionation approaches have been used successfully to elucidate various aspects of hepatocyte biochemistry. The development of methods for dissecting the hepatocyte plasma membrane into its constituent domains (Wisher and Evans 1975; Evans, 1980; Shears et al., 1989; Ali et al., 1990) and the isolation of intracellular organellar membranes, including those derived from the endocytic compartment (Evans and Flint; 1985), lysosomes (Wattiaux et al., 1978) and Golgi apparatus (Evans, 1985), has enabled the biochemical characterization of liver membranes and elucidated many of their functions. For example, five bile-canalicular plasma membrane marker enzymes: leucine aminopeptidase, 57-nucleotidase, alkaline phosphodiesterase, alkaline phosphatase and ecto-Ca 2+ ATPase were enriched 115-153-fold (relative to the activity present in liver homogenates) in bile canalicular plasma membrane fractions (Ali et al., 1990). In addition, no (Na++ K+)-ATPase activity, a basolateral marker enzyme, was detected

-192- Fig:. 6.5 Connexin 32 degradation products in lysosomes

Lysosomal membranes (50ug) were analysed by SDS-PAGE in 12.5% separating gels and propessed for Western blotting staining with the antibodies shown. See text. M and D correspond to the monomeric and dimeric forms of connexin 32. LYSOSOMES

< p ^ i n v - ( f < y o

. >

21 K

17K J k -<10K in this fraction. Use of these fractions has allowed the distribution of various signal transducing proteins to be assessed. For instance, inositol tris and tetrakisphosphates (Shears et al., 1988) and G-proteins (Ali et al., 1989) were detected in all liver plasma membrane domains and endosomes, but were highly enriched (on a specific activity basis) in bile canalicular membranes. In contrast, polypeptides belonging to the ras superfamily of small GTP-binding proteins were most highly enriched in endosomal membranes (Ali and Evans, 1990a). Furthermore, six glycosyl-phophatidylinositol anchored proteins were demonstrated to be priority-targeted to the bile- canalicular domain, two of which were directed to this domain via the 'late7 endocytic compartment (Ali and Evans, 1990b). Although the presence of these proteins and enzymes in the membrane facing the bile canaliculus remains unclear, these studies have demonstrated hepatocyte biochemical surface polarity and elucidated various trafficking routes through intracellular compartments for proteins destined to specific domains of hepatocyte plasma membrane. In this targeting context, the connexins are required to be directed to the lateral plasma membrane where the gap junctions are resident. Furthermore, a central requirement in understanding the assembly of gap junctions is the identification of the membrane locus where connexins oligomerize to form connexons i.e. whether inside the cell, or at the cell surface.

In the present study, the analysis of the relative distribution of connexin 32 in various liver subcellular

-194- fractions has shown that this polypeptide is present at highest relative levels in lateral plasma membranes, as expected. Its presence, in minor quantities, in sinusoidal, canalicular and endosomal fractions, could be accountable to the cross- contamination of these subcellular fractions with Golgi (sinusoidal & endosomal fractions) and lateral (canalicular fraction) membranes, as inferred from the size of the S.E.M. calculated for these fractions. The level of connexin 32 in sinusoidal membranes however, could reflect the lateral diffusion of unassembled precursor connexin from the lateral domain across to the sinusoidal domain (Fig. 6.6). The biogenesis of connexin 32 (presumably at the rough endoplasmic reticulum) may be accompanied by a rapid transfer to the Golgi apparatus, as indicated by the low levels of connexin 32 in membranes comprising the microsomal residue fraction (M-Res). Conversely, the high levels of connexin 32 in Golgi fractions suggests that transfer through, and/or from the Golgi to the cell surface is slower than the rate of synthesis of connexin 32, resulting in an accumulation. Since no detectable levels of glycosylation was evident on this polypeptide, the possibility arises that transfer from the rough endoplasmic reticulum to the Golgi complex may occur rapidly towards the trans Golgi network (Griffiths and Simmons, 1986), encountering only briefly the cis and medial stacked Golgi compartments. Alternatively, the migration of connexin 3 2 through the Golgi complex may be accompanied by other chemical modifications including phosphorylation, sulphation or acylation. One further possibility is that the accumulation of connexin 32 in the Golgi

-195- Fig.6.6 Trafficking routes of connexin 32 in liver

Based on the distribution of connexin 32 in liver, a potential trafficking pathway for connexin 3 2 to and from the lateral plasma membrane is shown diagrammatically. Possible Routes of Connexin 32 Trafficking to Gap Junctions in Hepatocytes - 196 - . p < : . ? < complex may signify the formation of connexons as a functional pre-requisite for targeting to the cell surface. Although no N- linked glycosylation was detected, the possible modification of connexin 3 2 by 0-1inked glycosylation was not investigated in the present study. The absence of N-linked glycosylation, despite the presence of a consensus signal at amino acids 2-5, correlates with the membrane topography of connexin 32. While migrating through the Golgi apparatus, only the sequences comprising the extracellular loops are exposed to the lumenal sialyl and galactosyl transferases, indicating that N-linked glycosylation sites are not present in these loops.

The detection of the connexin 32 monomer, in addition to breakdown products, in lysosomal fractions is also indicative of an accumulation in this degradative compartment. This observation may reflect the rapid turnover of gap junctions manifested in the short half-life of connexin 32 (approx. 5.5 hours; Fallon & Goodenough, 1981). Little is known about the disassembly of gap junctions, other than it must be rapid to account for the short half lives of the connexins. In Fig. 3.9, an intracellular vesicular structure which appeared to be derived from the gap junction was heavily labelled by affinity purified GAP 9 antibodies. This structure displays a double membrane morphology and appears to be derived from a region of the gap junction with diminished integrity. This electron micrograph (Fig. 3.9) of an annular gap junction located below the plasma membrane may illustrate a disassembly event in the turnover of gap junctions. If indeed gap junctions are

-197- internalized as intact double membrane structures, such intracellular vesicles, destined to mature into lysosomes, would comprise a dense microsomal fraction, lighter than mitochondria but probably too dense to co-isolate as part of the endosomal fractions. The detection of connexin 32 physiological breakdown products in lysosomal fractions is the first biochemical evidence of gap junction disassembly. The failure of GAP 9 antibodies to detect these breakdown products indicates that the extreme of the carboxyl terminus of connexin 32 is primarily degraded.

-198- CHAPTER 7

CONCLUDING REMARKS

Our understanding of the gap junction has progressed rapidly since the isolation of the cDNA coding for connexin 32 (Paul 1986; Kumar & Gilula, 1986). The subsequent isolation of further connexin cDNA transcripts providing access to entire amino acid sequences, and the preparation of antibodies of defined specificity, constitute technical milestones in gap junction biochemistry which should secure a continuation of this progress.

The predominant component of rat liver gap junctions, and the best characterized of all gap junction proteins, is cbnnexin 32. As the hydropathy profiles of all connexins characterized thus far suggest a common structural motif, the present study focused predominantly on connexin 32 as a model connexin. Existing structural data, derived from electron diffraction analyses of rat liver gap junctions, has allowed the construction of low resolution models of the connexon; depicting gross-level channel structure. This data provides valuable information regarding the dimensions of the connexon channel and its composite subunits, but does not provide the fine details of connexin topography or the identification of functionally important domains including the pore-lining sequences. Until such time as structural data at atomic resolution becomes available, the elucidation of aspects of connexon fine structure requires the application of

-199- alternative approaches such as those adopted in the present

work.

By the use of biochemical approaches and structural

prediction, a low resolution 2-D topographical model of

connexin 32 in the junctional membrane was postulated (Zimmer

et al., 1987) and used as a platform for the present study.

By incorporating the use of a panel of anti-peptide

antibodies directed towards different putative intra- and

extracellular sequences of connexin 32, a refined extension

of these biochemical approaches was pursued. The central aim

of the present study was to validate and extend aspects of

this working model, providing additional data for future

experimentation.

7.1 Gap Junction Specific Anti-Peptide Antibodies

In the present study, antibodies were generated

against synthetic peptides corresponding to selected, non membrane embedded, intra- and extracellular domains of

connexin 32. From a total of 11 peptide immunogens used in

the present study, 6 antibodies with specificities towards

different domains of connexin 32 were obtained. Some of

these antibodies contained specificity towards the native

configuration of connexin 32, exemplified by blocking

intercellular communication when microinjected into cultured

cells. These reagents should prove invaluable tools for

elucidating the functionally important domains of connexin

32, especially those controlling channel permeability.

- 200 - In spite of numerous other reports conveying the use of anti-peptide antibodies in functional studies (see Chapter

3), a school of thought still prevails which doubts the functional application of these site-directed reagents.

Arguing that anti-peptide antibodies can never recognize native structures (i.e. assembled epitopes) but only denatured regions of the parent protein (i.e. sequential determinants), the sites on parent proteins to which anti peptide antibodies bind are therefore devoid of any biological significance (Laver et al., 1990). These ideas do not entertain the concept of molecular mimickry in the context of the synthetic peptide-carrier complex creating an

'artificial epitope' similar in 3-D structure to the corresponding region within the parent protein. This controversy could be resolved when crystals of anti-peptide

Fab-native protein complexes are analysed by X-ray diffraction. In the meantime, alternative approaches to generating anti-peptide antibodies have attempted to refine the creation of artificial epitopes. By expressing the selected peptide of interest in the form of a genetic insert at the carboxyl terminus of two different recipient proteins, one hybrid protein can act as an immunogen while the other is used to monitor anti-peptide antibody activity (Martineau et al., 1991). In this approach the recipient protein acts as a carrier providing a microenvironment suitable for the peptide to assume its intrinsic secondary structure.

- 201 - 7.2 Topography Of Connexin 32

The membrane disposition of anti-peptide antibodies immunolocalised to isolated 'intact' and 'split' gap junctions have demonstrated directly that the amino and carboxyl termini of connexin 3 2 are located in the cytoplasm.

In addition, a hydrophilic 'loop' peptide connecting the second and third transmembrane alpha-helices is also cytoplasmically disposed. Regions of connexin 32 accessible to proteolytic attack include this 'loop' peptide, the amino terminus (Hertzberg et al., 1988) and the carboxyl 'tail'.

The protease accessibility of these regions are a further indication of their cytoplasmic disposition. In contrast, predicted extracellular regions of connexin 32 wei^e highly protease resistant (even when made accessible to proteases), and antibodies directed against these regions were not observed to label the cytoplasmic face of isolated gap junctions.

The evidence in the present study is therefore consistent with the '4 membrane-spanning' alpha-helical arrangement proposed by Zimmer et al (1987), predicted by the hydropathy profile of connexin 32 and reinforced by the studies of Hertzberg et al (1988) and Milks et al (1988).

Furthermore, this arrangement complies with rules of orientation for the first transmembrane helix of integral membrane proteins, formulated by Hartmann et al (1989), in which the distribution of charges in the non-membrane embedded regions flanking the first helix dictate its

- 202 - orientation, with the cytoplasmic disposition of the more positive flanking sequence. Similar structural arrangements are shared by other putative channel forming polypeptides including the ligand-gated receptor ion channel subunits and the synaptophysins (Fig. 7.1). However, 2-D topographical models of the highly selective voltage-gated ion channel subunits, constructed by the same complementation of structural prediction and biochemistry, reveal an alternative structural motif incorporating 6 membrane-spanning alpha- helices. Greater channel selectivity may necessitate the

incorporation, into channel design, of a greater number of transmembrane alpha-helices per structural unit (subunit), in addition to reducing the number of subunits forming the channel.

Of the four membrane-spanning alpha-helices of the

connexins, the third helix (M3) has been predicted to contribute to one sixth of the channel pore, based upon its

amphipatic character and a periodicity of sequence shared with putative pore-lining helices of other channel formers

(Milks et al., 1988; Unwin, 1989; Betz, 1990). Although biochemical data supporting this prediction has yet to be

reported, the recent identification of the pore forming

region of the voltage-gated channel subunits (Hartmann et

al., 1991; Yool & Schwartz, 1991; Yellen et al., 1991), has

confirmed earlier structural predictions (Guy & Seetharmulu,

1986). This example provides great encouragement for gap

junction biochemists and other channel workers, for as

-203- Fig.7.1 Membrane Topographies of Various Channel Forming

Polypeptides

Three classes of channel forming polypeptides are depicted diagrammatically. Highly selective channels such as the voltage-gated K+ and Na+ channels are composed of four structural units, each containing six membrane-spanning alpha helices. Channels of intermediate selectivity such as the neurotransmitter-gated cation channels are composed of subunits with four membrane-spanning alpha helices. Broad selectivity channel such as connexons are also composed of subunits containing four alpha helices, but a hexameric quarternary structure may underly the reduced discrimination between permeating molecules. O 8-c.E Q-Q-0 to 0 (0z CM 0 O d ) CD X 0 0)0 C O O CD c 0 0 £-73 03 Z c £ ‘-p JZ O — f~ Q- o c \j ^ O-o 0) c a> _cz cd CL o t-|is 0 !=; o 9 - ^ S o c CO t h 73 £ CLL. D cr Q-S*03 r~ O 0 __ “3 X I CM o cr o i S t o i CL c 5,6 o 03 (D co rZ) CD C X 2 CM CO 0 CO I c X c O + o © O ■g c CO X)

= c

0 c CO c 00 JZ co co + ° < M 0 3 O

- 204 - integral membrane proteins may still pose problems to crystallographers, they appear to comply with the same folding and packing rules displayed by soluble proteins.

Furthermore, constraints on permissible structures imposed by the lipid environment, coupled to the elucidation of topogenic signals for membrane insertion, render this class of protein highly suitable for structure prediction and engineering (Von Heijne and Manoil, 1990).

7.3 The Extracellular Loops Of Connexins

Based on contemporary models of gap junction structure, the extracellular domains of connexins perform a unique bifunctional role. In common with the ligand-gated ion channels they possess a receptor binding site(s), underlying the homophilic recognition of an apposed cOnnexin, but in addition also generate an extension of the ion channel into the extracellular 'gap', which in a complete intercellular channel, allows the insulated migration of ions and small molecules across the gap junction.

Relatively little is known about the organisation of these very important structural domains of connexins, reflecting the difficulties encountered by biochemists attempting to elucidate their structure (Zimmer et alf, 1987;

Chapter 4). The demonstration in the present study of a disulphide bridge(s) interconnecting the extracellular loops of connexin 32, prompted the structural analysis of these domains. The constraints on folding conformations imposed by

-205- potential disulphide connections between the six highly conserved cysteine residues resident in these loops, warranted the prediction of likely disulphide arrangements

(Fig. 4.14). The two preferred configurations illustrated were based on consensus data derived from disulphide bridge-

containing soluble proteins of known 3-D structure.

Furthermore, both of these configurations comply with the

'EGF motif' present in the cysteine-rich domain of the

epidermal growth factor and a number of other serum proteins.

It should be noted that the arrangement of cysteine in the

extracellular domains of connexins does not comply with

consensus metal-chelating sequences. Future experimentation

shall be aimed at mapping the interloop disulphide bridge(s)

and verifying the existence and location of the putative

intraloop disulphide bonds.

The functional role of these covalent crosslinks

remains unclear, but work on the beta-adrenergic receptor has

shown an extracellular disulphide bridge to be essential for

ligand binding (Moxham et al., 1986). It is plausible that

ligand binding induces conformational stress forces on the

receptor binding domains which require the additional

stability conferred by disulphide bonds. With a two-fold

functional role, the extracellular domains of connexins must

be stable ordered structures (as indicated by structural and

biochemical data), able to maintain an insulated pore

extending across the junction while under the likely stress

forces induced by connexon-connexon interaction. It is

-206- noteworthy that the integrins and other receptors belonging to the immunoglobulin superfamily possess disulphide bonds within their extracellular domains, possibly indicative of a general requirement for added stability in receptor binding domains.

7.4 Future Prospects

The refinement and further extension of existing models of connexon structure, pursued by a combined biochemical and molecular biological approach, depends upon the development of defined model systems, e.g. cells expressing a transfected connexin cDNA. This will allow the assessment on channel properties of modifying key amino acid residues by site-directed mutagenesis or covalent modification. Such approaches will help decipher those sequences in connexins comprising the channel pore, controlling channel porosity, and underlying connexon- connexon interaction. The functional domains of connexin 32 are highlighted in Fig. 7.2

Despite the rapid progress made in elucidating aspects of gap junction biochemistry in recent years, several fundamental questions remain unresolved. It has become apparent that cells may express more than one connexin, but it is not known whether the functional channels present in these cells are composed of hetero- or homohexameric connexons. Furthermore, little is known about the site and mechanisms of connexon assembly, namely whether hexamers

-207- Fig. 7.2 Functional Topography of Connexin 32

The diagram illustrates the arrangement of connexin 3 2 in the

junctional membrane, co-aligned with an apposing molecule, generating 1/6 of the channel wall extending across entire double membrane structure. The various putative functional domains of connexin 32 are also highlighted. Functional Topography of Connexin 32

Regulatory (phosphorylation) Domain

Channel-Guarding Cytoplasm Domain

w Plasma Membrane ^ m Pore-lining m ceii 1 i Domain

Recognition Intercellular "Gap” Adhesion Domains Pore-extension Scaffold

H Plasma Membrane J i Cell 2 I

Cytoplasm

Fig-7.2 - 208 - (connexons) are formed within intracellular membranes, or exclusively at the cell surface. In the present study, the subcellular distribution of connexin 32 in liver was analysed as a prelude to investigating aspects of connexon assembly.

The apparent accumulation of connexin 32 in Golgi membranes suggests that this organelle could provide an intracellular site at which connexons are assembled prior to targeting to the lateral plasma membrane. Experiments aimed at elucidating details of connexon assembly in liver subcellular

fractions are currently in progress. In addition, the

intracellular locus where disulphide bridge formation occurs between the extracellular loops of connexin 32 is also under

investigation.

In common with the pattern of characterization of several multi-gene families, the description of connexins by the isolation of cDNA transcripts has accelerated well ahead of their functional characterization by physiological/ biochemical approaches. Connexins are being identified that

are expressed in several tissues, but failure to identify gap

junction plaques using specific anti-peptide antibodies

renders their functional significance open to question. Do connexins fulfill a wider biological role other than the

phenomena attributable to gap junction-mediated intercellular communication? Preliminary data has implicated a role for 2+ connexins in the Mg -depleted ATP-mduced permeabilization

of immunocompetent cells (Beyer & Steinberg, 1990). Whether

such a phenomenon is based upon the presence of functional

-209- connexons (half channels) at the cell surface can be investigated using the defined molecular tools which are now available to the cell biologist. Furthermore, channel activity of similar magnitude to gap junction channels has been reported between the secretory vesicle lumen and the cell exterior (Breckenridge and Aimers, 1987). Representing the only other channel known to span two membranes, these

'fusion pores' are of on unknown structure and composition but may comprise of connexin or connexin-like subunits.

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