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Structure-function correlates in human apolipoprotein A-1 McLeod, Roger Stephen 1992

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STRUCTURE-FUNCTION CORRELATES IN HUMAN APOLIPOPROTEIN A-I by ROGER STEPHEN McLEOD B.Sc.(Hons.), University of British Columbia, 1977  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pathology)  We accept this thesis as conforming to the required Signature(s) removed to protect privacy  THE August 1992 ©Roger Stephen McLeod, 1992  In  presenting this  degree at the  thesis  in  partial  fulfilment  of the  requirements  I further agree that permission for extensive  copying of this thesis for scholarly purposes may be granted or  advanced  University of British Columbia, I agree that the Library shall make it  freely available for reference and study. department  for an  by  his  or  her  representatives.  It  is  by the  understood  that  head of my copying  or  publication of this thesis for financial gain shall not be allowed without my written permISsiOn.  Signature(s) removed to protect privacy  (Signature)  Department of  /ti  The University of British Columbia Vancouver, Canada  Date  /_ 1  DE-6 (2/88)  ABSTRACT Apolipoprotein (apo) A-I is the major protein component of human high density lipoprotein (HDL). The plasma level of HDL or apo A-I is inversely related to the risk of developing atherosclerosis and the protective effect of HDL is believed to be related to its ability to promote the movement of cholesterol from peripheral cells to the liver. Apo A-I is involved in the remodelling of HDL that accompanies this  reverse” cholesterol transport but the elements of its structure that are responsible  for its function are not well understood. In this thesis, in vitro mutagenesis and eucaryotic expression were used to study the structure and function of recombinant human apo A-I. Four expression systems were developed and were utilized to express the wild-type and mutant cDNA constructs. In vitro translation studies in rabbit reticulocyte lysate established that the cDNA encodes the precursor, preproapo A-I. This precursor was proteolytically processed to proapo A-I on addition of microsomal membranes, simulating in vivo translocation and processing on the membrane of the endoplasmic reticulum (ER). Apo A-I was synthesized and secreted constitutively from the precursor cDNA by three eucaryotic cell, types: transformed simian kidney (COS), baby hamster kidney (BHK) and chinese hamster ovary (CHO) cells. The apo A-I secreted by COS and BHK cells was proapo A-I, while CHO cells secreted mature apo A-I, indicating that the latter possess propeptide proteolytic activity. Low level propeptide hydrolysis was also detected during long term culture collections from BHK cells. In defined, serum free culture conditions, much of the apo A-I synthesized was eventually degraded. Long term collections of medium were found to contain the following levels of apo A-I: COS lOng/mI, BHK  lOOng/ml and CHO  l3Ong/ml per 24 hours.  Proapo A-I secreted from COS cells was readily integrated into the HDL density fraction of fetal bovine serum. The majority of the apo A-I secreted by CHO cells was lipid-free or lipid-poor but retained its ability to integrate into liposomes in vitro. A large portion of the apo A-I within COS cells retained its signal peptide sequence following translation, indicating that ER processing of preproapo A-I was inefficient. It was concluded that COS cells were a poor model for large scale apo A-I expression and CHO cells were the best model system for this purpose. The role of the apo A-I propeptide was investigated in BHK cells expressing apo A-I. The 11  results indicated that the propeptide was required for efficient cellular transport and secretion of apo A I. Removal of the propeptide from the cDNA sequence had no effect on the rate of apo A-I synthesis or on the fidelity of signal peptide hydrolysis, but the altered protein remained in the cells in large vesicular structures which had some morphologic features of the ER. This change also appeared to reduce the rate of apo A-I degradation. The observations suggested that non-hepatic cells expressing apo A-I degrade a substantial portion of this protein. Furthermore, removing the propeptide caused much of the apo A-I to remain in the cell, perhaps by preventing the movement of the protein out of the ER. The functional roles of the middle and C-terminal regions of the apo A-I sequence were also investigated by generating mutants in these regions. Deletion of Lys 107 (a naturally occurring mutation with functional abnormalities) had minimal influence on the ability of these proteins to bind to liposomes, although the resulting complexes were more heterogeneous by density gradient centrifugation. Deletion of one amphipathic helix from the C-terminus of apo A-I altered its ability to form stable lipidprotein complexes when compared to wild-type. While recombinant wild-type apo A-I was approximately 80% as effective a lecithin:cholesterol acyltransferase (LCAT) activator, the Lys’° 7 and a -helix deletion mutants were extremely poor LCAT activators. In conclusion, the results indicate that the propeptide portion of apo A-I is involved in the cellular transport of apo A-I and might regulate the movement of proapo A-I between the ER and the Golgi apparatus. Furthermore, low affinity amphipathic helices in the middle hinge region and the C terminal region of the apo A-I sequence may play a significant role in the LCAT activating mechanism. These elements do not appear to be major structural determinants of initial phospholipid binding but may be involved in subsequent transformations of HDL. Extension of these studies will provide important insight into the mechanisms underlying the anti-atherogenic properties of HDL.  111  TABLE OF CONTENTS Page ii iv viii xii xiii xiv xvii  ABSTRACT TABLE OF CONTENTS ABBREVIATIONS AMINO ACID DESIGNATIONS LIST OF TABLES LIST OF FIGURES ACKNOWLEDGEMENTS CHAPTER 1 1.  INTRODUCTION  1  Li.  High Density Lipoproteins and HDL Remodelling Processes  3  1.2.  High Density Lipoprotein (HDL) and Atherosclerosis  4  1.3.  HDL and Reverse Cholesterol Transport  5  1.4. 1.4.1. 1.4.2.  Apolipoprotein A-I Apo A-I Gene Structure Apo A-I Gene Expression and Secretion  7 7 9  1.5. 1.5.1. 1.5.2. 1.5.3.  Functions of Apo A-I Apo A-I and HDL Structure Apo A-I and the Interaction of HDL with Cells Apo A-I and Lecithin:cholesterol Acyltransferase (LCAT)  12 12 13 15  1.6. 1.6.1. 1.6.2. 1.6.3. 1.6.4.  Structural Elements of Apo A-I The Apo A-I Signal Peptide The Apo A-I Propeptide The Ampliipathic Helix Motif Apo A-I Secondary Structure: Epitope Mapping  16 16 17 19 23  1.7.  Naturally Occuring Structural Variants of Human Apo A-I  26  1.8.  Expression of Recombinant Apo A-I  30  1.9.  Scope of Thesis: Specific Aims  32  CHAPTER 2 2.  MATERIALS AND METHODS  2.1.  Materials  34  2.2.  Growth and Transformation of E. Co1i  35  iv  2.3. 2.3.1. 2.3.2. 2.3.3.  Purification of DNA Small Scale Plasmid Preparation Large Scale Plasmid Preparation Preparation of M13 Phage DNA Preparation of Uracil-Containing ssDNA from M13 Preparation of M13 Phage DNA for Sequencing  36 36 36 37 37 38  2.4. 2.4.1. 2.4.2. 2.4.3.  Oligonucleotide-directed Mutagenesis In vitro Mutagenesis Identification of Putative Mutants DNA Sequence Analysis  38 38 39 40  2.5. 2.5.1. 2.5.2. 2.5.3.  Construction of Expression Plasmids Isolation of cDNA Fragments Modification of Fragment Ends Ligation into Expression Plasmids  41 41 41 42  2.6.  In vitro Transcription  43  2.7.  In vitro Translation  44  2.8.  Eucaryotic Cell Culture  44  2.9.  Transient Transfection of COS-1 Cells  45  2.10. 2.10.1. 2.10.2. 2. 10.2.2. 2.10.3. 2.10.4.  Isolation of Stable Eucaryotic Cells Expressing Apo A-I Calcium Phosphate Transfection Selection of Stably Transfected Cells BHK Cells CHO-K1 Cells Screening of Clones for A-I Secretion Amino-Terminal Amino Acid Sequence Analysis  45 45 46 46 46 47 47  2.11. 2.11.1. 2.11.2. 2.11.3.  Metabolic Labelling Studies Determination of Synthesis Rate Long Term Labelling Studies Determination of Apo A-I Degradation and Secretion  48 48 48 49  2.12.  Determination of  49  2.13.  Isolation of Apo A-I by Immunoabsorption  49  2.14. 2.14.1. 2.14.2. 2.14.2. 1.  Electrophoretic Analysis DNA Fragment Separation on Agarose Gels Protein Analysis SDS-Polyacrylamide Gels Isoelectric Focusing Gel Electrophoresis Two-dimensional Polyacrylamide Gel Electrophoresis Immunoblot Analysis  50 50 50 50 51 51 51  2.15.  Indirect Immunofluorescence Microscopy  52  2.16.  Immunogold Electron Microscopy  53  S]Methionine Incorporated into Protein and Apo A-I  V  2.17. 2.18. 2.18.1 2.18.2 2.18.3  Quantitation of Apo A-I by Competitive ELISA Analysis of Apo A-I Function Preparation of Single Bilayer Vesicles Assessment of Lipid Binding Characteristics Measurement of LCAT Cofactor Activity  53 54 54 54 55  CHAPTER 3  3.  DEVELOPMENT OF APO A-I EXPRESSION SYSTEMS  3.1.  Sequencing of the Full Length Apo A-I cDNA  56  3.2.  In vitro Transcription and Translation of the Full Length cDNA  59  3.3. 3.3.1. 3.3.2.  Expression of Wild-type Apo A-I (Apo A-Iwt) in COS Cells Properties of Apo A-I Secreted by COS Cells Immunofluorescence Localization of A-Iwt in COS Cells  62 68 73  3.4. 3.4.1. 3.4.2.  Baby Hamster Kidney (BHK) Cell Expression of Apo A-Iwt BHK-AIwt Cells Produce proapo A-I Characterization of Apo A-I Synthesis and Secretion  75 75 77  3.5.  CHO-Ki Cell Expression of Apo A-Iwt  82  3.6.  DISCUSSION  85  CHAPTER 4 4.  THE APO A-I PROPEPTIDE AND INTRACELLULAR TRAFFICKING  4.1  OVERVIEW  91  4.2 4.2.1 4.2.2. 4.2.3. 4.2.4.  RESULTS Transient Expression of Apo A-IdPRO in COS Cells Stable Expression of Apo A-IdPRO in BHK cells Comparison of Apo Al Synthesis and Secretion Rates Intracellular Localization of Apo A-I Accumulations  91 91 97 99 102  4.3.  DISCUSSION  105  CHAPTER 5 5.  7 AND THE C-TERMINAL AMPHIPATHIC HELIX IN LCAT ACTIVATION LYS’°  5.1  OVERVIEW  111  5.2 5.2.1. 5.2.2. 5.2.3. 5.2.4. 5.2.5.  RESULTS Production and Identification of Mutant cDNAs Transfected COS Cells Produce the Respective Mutant Proteins Development of CHO Cell Lines Expressing Apo A-I Mutants Role of Lys 107 in Apo A-I Cellular Transport and Secretion Functional Characteristics of Apo A-I Mutants  111 111 112 115 117 118  vi  Lipid Binding Properties of Apo A-I Mutants LCAT Activation by Apo A-I Mutants  DISCUSSION  5.3  118 125 128  CHAPTER 6 6.  CONCLUSIONS  6.1 6.2  Summary of Major Findings Perspectives for Future Study  130 133  REFERENCES  136  vii  ABBREVIATIONS Apo  apolipoprotein  ATP  adenosine triphosphate  BHK  baby hamster kidney  bp  base pair  BSA  bovine serum albumin  CAD  coronary artery disease  cDNA  complementary DNA  CE  cholesteryl ester  CETP  cholesteryl ester transfer protein  CHO  chinese hamster ovary  Chylo  chylomicron  CIP  calf intestinal phosphatase  CMV  cytomegalovirus  ConA  concanavalin A  COS  transformed simian kidney cell  Dl or d(Pro ) 41 -Asr? 220  deletion of helix Prc? 41 -Asr? 20  DAB  3,3-diaminobenzidine  dAT?  deoxyadenosine triphosphate  dCTP  deoxycytidine triphosphate  ddATP  dideoxyadenosine triphosphate  ddCTP  dideoxycytidine triphosphate  ddGTP  dideoxyguanosine triphosphate  ddTTP  dideoxythymidine triphosphate  DEAE  diethylaminoethyl  DEPC  diethylpyrocarbonate  dGTP  deoxyguanosine triphosphate  DHFR  dihyrofolate reductase viii  107 ,dLys dK 107  deletion of lysine residue at position 107  DMEM  Dulbeccos modified minimal essential medium  DMPC  dimyristoyl phosphatidylcholine  DMSO  dimethylsulfoxide  DNA  deoxyribonucleic acid  DNase  deoxyribonuclease  dNTP  deoxynucleotide triphosphate mixture  dPRO  propeptide deletion  dsDNA  double-stranded DNA  DTT  dithiothreitol  dTTP  deoxythymidine triphosphate  dut  deoxyuracil triphosphatase  EDTA  ethylenediamine tetra-acetic acid  ELISA  enzyme-linked immunosorbent assay  ER  endoplasmic reticulum  FBS  fetal bovine serum  FITC  fluorescein isothiocyanate  G418  Geneticin  GGE  gradient gel electrophoresis  HDL  high density lipoprotein  HDL-C  high density lipoprotein cholesterol  HEPES  N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid  hGH  human growth hormone  HRP  horse radish peroxidase  HTGL  hepatic triacylglycerol lipase  IEF  isoelectric focussing  IgG  immunoglobulin G  IPTG  isopropyl-8 -D-thiogalactopyranoside ix  IVS  intervening sequence (intron)  kD  kilodáltons  LB  Luria-Bertani  LCAT  lecithin:cholesterol acyltransferase  LDL  low density lipoprotein  107 Lys  lysine residue at position 107  mMT-I  mouse metallothionein-I  MOl  multiplicity of infection  mRNA  messenger RNA  O.D.  optical density  PAGE  polyacrylamide gel electrophoresis  PBS  phosphate buffered saline  PEG  polyethylene glycol  p1  isoelectric point  PMSF  phenylmethylsulfonyl fluoride  PNK  polynucleotide kinase  poly A  polyadenylation  rATP  adenosine triphosphate  RCT  reverse cholesterol transport  rCTP  cytidine triphosphate  RF  replicative form  RFLP  restriction fragment length polymorphism  rGTP  guanosine triphosphate  RNA  ribonucleic acid  RNase  ribonuclease  RNAsin  placental ribonuclease inhibitor  rUTP  uridine triphosphate  SDS  sodium dodecylsulfate x  SSC  saline sodium citrate  ssDNA  single-stranded DNA half-time  TAE  Tris-acetate-EDTA buffer  TBE  Tris-borate-EDTA buffer  TBS  Tris buffered saline  TCA  trichloroacetic acid  TE  10mM Tris-HC1, pH8.0/ 0.1mM EDTA  TG  triacyiglycerol, triglyceride  Tris  tris(hydroxymethyl)aminomethane  TRITC  tetramethylrhodamine isothiocyanate  UC  unesterified cholesterol  UFP  universal forward primer  ung  uracil N-glycosylase  URP  universal reverse primer  VLDL  very low density lipoprotein  WGA  wheat germ agglutinin  wt  wild-type  X-GAL  5-bromo-4,4-chloro-3-indoyl-3 -D-galactoside  YT  yeast tryptone  xi  AMINO ACID DESIGNATIONS  Amino Acid  Three Letter  Single Letter  Glycine  Gly  G  Alanine  Ala  A  Threonine  Thr  T  Serine  Ser  S  Tyrosine  Tyr  Y  Tryptophan  Trp  W  Aspartic acid  Asp  D  Glutamic acid  Glu  E  Glutamine  Gln  Q  Asparagine  Asp  N  Phenylalanine  Phe  F  Arginine  Arg  R  Leucine  Leu  L  Proline  Pro  P  Methionine  Met  M  Histidine  His  H  Lysine  Lys  K  Valine  Val  V  Isoleucine  Ile  I  Cysteine  Cys  C  xii  LIST OF TABLES Table  Description  Page  I  Properties and composition of lipoprotein classes  2  II  The plasma apolipoproteins  3  III  Variant forms of apolipoprotein A-I  27  IV  Apo A-I mutagenic oligonucleotide primers and their properties  38  V  Apo A-I cDNA sequencing primers  41  VI  Amino terminal sequence analysis of apo A-I secreted from cell line BHK-AIwtB5  77  VII  Effect of growth conditions on apo Al synthesis and secretion in BHK-AIwtB5  79  VIII  Secretion of apo A-I recombinants by CHO cell lines  117  XIII  LIST OF FIGURES  Figure  Description  Page  1  Generalized structure of the plasma lipoproteins  1  2  Schematic diagram of the process of reverse cholesterol transport  6  3  Location and structural organization of the human apo A-I gene  7  4  Schematic diagram of the charge and size heterogeneity of apo A-I at different stages of proteolytic processing  9  5  Diagramatic representation of the proteolytic processing of the apo A-I precursor  11  6  Hydropathy plot of the apo A-I precursor  19  7  Helical wheel projection of the consensus sequence for the 22 residue repeat structure of human apo A-I  21  8  Schematic diagram of the hinged domain hypothesis for the interaction of apo A-I with HDL  24  9  Proposed supersecondary structure of human apo A-I  25  10  DNA sequence analysis of the apo A-I cDNA pBL13AI  57  11  In vitro transcription and translation of the full length apo A-I cDNA  60  12  Expression of the apo A-I cDNA in transiently transfected COS cells  64  13  Immunoblot analysis of apo A-I expression in COS cells  67  14  Apo A-I produced by transfected COS cells is associated with HDL  69  15  Apo A-I secretion from transfected COS cells is stimulated by FBS  71  16  Apo A-I produced in COS cells is subject to intracellular phosphorylation  72  17  Immunofluorescence analysis of transfected COS cells indicates that apo A-I is retained in the ER  74  xiv  Figure  Description  Page  18  BHK cells expressing apo A-Iwt produce a single molecular species with the electrophoretic properties of proapo A-I  76  19  FBS stimulates the secretion of apo A-I by BHK-AIwtB5  78  20  Long-term pulse-chase analysis suggests that degradation competes with secretion of apo A-I in BHK-AIwtB5  80  21  Zinc sulfate induction of apo A-I synthesis results in the accumulation of apo A-I immunofluorescence in BHK AIwtB5  81  22  CHO cells expressing apo A-Iwt secrete mature apo A-I  83  23  Removal of the propeptide from the apo A-I sequence delays its secretion from transfected COS cells  92  24  Apo A-IdPRO is retained in the ER of the transfected COS cell  96  25  BHK clones contain proapo A-I (p-Al) or mature apo A-I (rn-Al)  98  26  Apo A-I and protein synthetic rates in BHK lines p-Al and rn-Al  100  27  Apo A-I secretion and accumulation in BHK lines p-Al and rn-Al  101  28  Time course of apo A-I degradation and secretion in BHK cell clones  103  29  Immunofluorescence localization of apo A-I in transfected BHK cell lines  104  30  Immunogold localization of apo A-I in ultrathin cryosections of transfected BHK cell lines  106  31  Transfected COS cells express apo A-I mutants with the appropriate molecular characteristics  113  32  CHO cells express apo A-I mutants of the expected charge and size  116  33  The apo A-IdK’° 7 mutant protein is secreted and degraded more rapidly than apo A-Iwt in transfected COS cells  119  34  Apo A-IdK 107 incorporates into extracellular lipoprotein in cell culture  121  xv  Figure  Description  Page  35  Cells expressing apo A-IdK 107 contain apo A-I accumulations in both the ER and the Golgi apparatus  122  36  Apo A-I and mutants are secreted from CHO cells into serum free medium in lipid-poor form  123  37  Recombinant apo A-I produced by CHO cells activates LCAT to approximately the same extent as the purified plasma protein  126  38  The ability of apo A-I mutants to activate LCAT is markedly diminished  127  39  Proposed model for the role of the apo A-I propeptide in BHK cell transport  132  xvi  ACKNOWLEDGEMENTS  The invaluable assistance of members of the Hayden, Gillam and Pritchard laboratories was of paramount importance at various phases of this work. In particular, I would like to thanl Carolyn Robbins, Linda Peritz and Jeff Hewitt for teaching me the aspects of molecular biology which were required to initiate the work. Tom Hobman, Marita Lundstrom and Paul Sunga were instrumental in my intoduction to cell biology and occasionally provided a warm place to unwind. Alan Burns provided expertise in cryoelectron microscopy and Dr. Ruedi Abersold performed the amino terminal sequence analysis. I would like to thank Karmin 0 and John Hill for their important contributions to my life, in science and outside. I acknowledge all members of the Pritchard laboratory: Haydn, Jiri, Linda, Xingbo, Lida, Liz, Janine and Stan for providing the framework in which to operate. Most importantly, I acknowledge the tolerance and personal support of my family: Shirley, Clinton arid Keith who have provided stability and sustenance through late nights and short weekends. This thesis is dedicated to the memory of my father, Gerald Wesley McLeod, and my grandmother, Lena Springfeld.  xvii  1. INTRODUCTION The plasma lipoproteins are microemulsions composed of a non-polar lipid core surrounded by a polar surface monolayer which maintains the solubility of the complex in the aqueous environment of the plasma (Figure 1). The core components are mainly triglycerides (TG) and cholesteryl esters (CE), while the surface layer is composed of unesterified cholesterol (UC), phospholipids (PL) and associated proteins, the apolipoproteins.  PL I  iUG  ////i  TG, EC Apo  Figure 1. Generalized structure of the plasma lipoproteins. PL = phospholipid, UC = unesterified cholesterol, TG = triacylglycerol, EC = esterified cholesterol, Apo = apolipoprotein. Lipoproteins differ in the quantity of the various components and have been classified on the basis of their size and density. These properties form the basis for the separation techniques employed to isolate these macromolecules from the plasma (Table I). The major classes can also be separated by electrophoresis in agarose gels (Noble, 1968). In this system, chylomicrons remain at the application point, VLDL migrates in the prqB region, LDL migrates in the fi region and HDL has x -mobility. The larger lipoproteins (chylo and VLDL) contain the bulk of the plasma TG, whereas the smaller classes (principally LDL and HDL) are relatively TG poor and carry the majority of the plasma cholesterol. In normal individuals, LDL carries approximately 60-75% of the plasma cholesterol, and the remaining 2540% is carried in the HDL density class. However, the distribution of components among the lipoproteins is subject to dynamic processes and the ultimate lipid composition of each lipoprotein is 1  governed by its apolipoprotein content and by the action of enzymes which alter the lipid moiety of these macromolecular structures. Table I. Properties and composition of lipoprotein classes Size, hydrated density and mass composition of the major lipoprotein classes from normal individuals. Adapted from Havel and Kane (1989).  Class  Density (g/ml)  Surface Components (wt%)  Diameter (nm)  Core Components  (%)  UC  PL  Apo  TG  CE  Chylo  0.93  75-1200  2  7  2  86  3  VLDL  0.93-1.006  30-80  7  18  8  55  12  IDL  1.006-1.019  25-35  9  19  19  23  29  LDL  1.019-1.063  18-25  8  22  22  6  42  2 HDL  1.063-1.125  9-12  5  33  40  5  17  3 HDL  1.125-1.210  5-9  4  25  55  3  13  The apolipoproteins are a family of polypeptides characterized by their ability to solubilize lipids spontaneously in an aqueous environment. Macheboeuf (1929) showed that plasma lipids were associated with proteins in water-soluble macromolecular complexes. These proteins have been obtained in pure form by extracting the lipid from the isolated lipoprotein and separating the individual protein species by protein fractionation techniques. The apolipoproteins range in size from 8,000 to 550,000 Daltons (Table II) but share some structural characteristics and the ability to interact with lipids. The apolipoprotein component of each lipoprotein particle governs its interaction with cells and with other proteins. Since protein structure is genetically determined while lipoprotein size and composition is largely governed by physicochemical considerations, the apolipoproteins provide an important link to our understanding of the genetic control of lipoprotein metabolism. The elucidation of specific functional roles of the individual apoproteins in lipoprotein metabolism remains a major challenge of lipoprotein biochemistry.  2  Table II. The plasma apoilpoproteins. Plasma concentration (mean value for normolipidemic donors) and polypeptide molecular weight of the plasma apolipoproteins. Adapted from Havel and Kane (1989).  Apolipoprotein  Plasma Concentration (gIL)  Molecular Weight (kilodaltons)  A-I  1.3  29.0  A-lI  0.4  17.4  A-IV  0.15  44.5  B-48  0  241  B-100  0.8  513  C-I  0.06  6.6  C-lI  0.03  8.9  C-Ill  0.12  8.8  D  0.10  19.0  E  0.05  34.1  1.1. High Density Lipoproteins and HDL Remodelling Processes HDL is the most protein-rich of the plasma lipoproteins. Fifty percent of HDL mass is apoprotein, of which 70% is apolipoprotein (apo) A-I. HDL can be separated into two major subfractions by ultracentrifugation, HDL 2 and HDI. While this distinction may not reflect true metabolic pools of HDL, the two are sufficiently distinct to warrant individual consideration. HDL 2 and HDL differ in density and in size as a result of differences in lipid and protein composition. However, additional properties of the HDL subfractions differ due to the relative quantities of the individual apolipoproteins. These differences may more accurately reflect the metabolic heterogeneity of HDLs. Thus, subpopulations of HDL can be separated by size criteria using nondenaturing gradient gel electrophoresis (GGE) or by immunoaffinity chromatography on columns with coupled antibody to a specific apolipoprotein. Using the latter technique, lipoproteins containing apo A-I  only (Lp-AI)  and  lipoproteins containing apo A-I and apo A-Il (Lp-A-I!A-II) can be isolated (Cheung et al, 1987). The HDLs are modified during a series of enzymatic conversion and lipid transfer processes. The details of this complex remodelling have only recently been elucidated. High density lipoproteins enter the circulation as  precursors from  two separate sources: the liver and the intestine. A nascent form  of HDL is liver-derived and is protein-enriched and lipid-poor compared with the mature HDL.  3  Intestinally-derived chylomicrons are partially catabolized in the circulation and release surface remnants, containing PL, UC and apo A-I, which are also precursors of HDL. These two nascent species acquire additional lipid and protein components by exchange processes in the circulation and can be isolated in the HDL density range. Precursors from either source acquire UC from other lipoproteins or from cell membranes. A small portion of the UC-enriched HDL has pre-3 electrophoretic mobility on agarose gel electrophoresis and appears to be a major portion of the Lp-A-I isolated by immunoaffinity tecirniques. The cholesterol in this fraction is subsequently esterified by the enzyme lecithin:cholesterol acyltransferase (LCAT, EC to produce lipoprotein of HDL 3 density and  electrophoretic  mobility. Cholesterol esterification within the lipoprotein leaves the surface relatively deficient in UC as the product CE moves from the particle surface to its core. The UC-depleted HDL then acquires additional UC from other lipoproteins or from cells by diffusion along the concentration gradient. The particle enlarges and becomes lighter as the lipid-to-protein ratio increases, eventually attaining HDL 2 density. The HDL remodelling cycle is completed by the action of hepatic triglyceride lipase (HTGL) and cholesterol ester transfer protein (CETP). These activities remove surface phospholipid and core cholesteryl ester (CE), respectively, to regenerate HDJ. The CE may be transferred to other plasma lipoproteins or may be taken up directly by hepatocytes and catabolized. Under most conditions CE is transferred to lower density lipoproteins in exchange for TG. HTGL possesses phospholipase activity, responsible for hydrolysis of HDL phospholipid, and TG hydrolase activity, contributing to a reduction in particle diameter. HTGL and CETP may act sequentially or, alternatively, the modifications may occur simultaneously on the hepatocyte surface.  1.2. High Density Lipoprotein (HDL) and Atherosclerosis Atherosclerosis is the progressive narrowing of large arteries due to intimal thickening and lipid accumulation. It is the major cause of mortality and morbidity in developed nations. Atherosclerotic lesions develop as a result of complex and poorly understood interaction of genetic and environmental influences (Sing and Moll, 1990). Hemodynamic, thrombogenic and metabolic factors contribute to the atherogenic process (Fuster et al, 1992) and the importance of each is supported by both epidemiologic and experimental data. In animal models of atherogenesis, high levels of plasma cholesterol play an 4  important role in the development and progression of this disease in response to an initiating biochemical or physical insult to the vessel wall (Ross, 1986). What is still poorly defined, however, is the sequence of molecular events that initiate the injury and the factors which might prevent or reverse the progressive accumulation of lipid that characterizes the disease. Epidemiologic studies have attempted to identify the causal and modifying factors in atherogenesis. Studies of free-living populations and of populations selected for increased atherosclerotic risk have indicated that the plasma cholesterol level, and specifically the portion associated with low density lipoprotein (LDL), are directly correlated with atherogenic risk. However, the strength of this correlation decreases with advancing age. Conversely, the portion of plasma cholesterol associated with HDL (HDL-C) is inversely correlated with atherosclerotic risk (Miller and Miller, 1975; Gordon et at, 1977) and the strength of this relationship increases with age, ie. as we age our HDL-C level becomes a more powerful predictor of atherosclerotic risk. Prospective intervention trials (Manninen et at, 1988) have shown that raising the level of HDL-C, with pharmacologic agents or by lifestyle modification, has a protective effect. Paradoxically, the analysis of genetic disorders affecting HDL level has indicated that low levels of this lipoprotein may or may not be associated with increased atherogenic risk (Schaeffer, 1984) although derangements of cholesterol metabolism are observed.  1.3. HDL and Reverse Cholesterol Transport The molecular processes through which high levels of HDL reduce atherogenic risk are largely speculative. HDL may protect against vascular lipid deposition by mediating the transport of excess cellular cholesterol from peripheral tissues to the liver by the process termed “reverse cholesterol transport”. While all cells are capable of cholesterol synthesis, only the hepatocyte is capable of cholesterol degradation. There is, therefore, a need to move cholesterol from the peripheral cell to the hepatocyte and this movement is driven by the HDL remodelling process as indicated in Figure 2. Reverse cholesterol transport (RCT) was originally described by Glomset (1970) and is composed of three distinct elements: (i)  Fluid phase transport of UC between cell membrane and lipoprotein surface by diffusion along a concentration gradient. This process may be facilitated by high affinity 5  binding sites for HDL on the surface of peripheral cells (Oram et at, 1984). (ii)  Development of a cholesterol concentration gradient by reduction of UC on the accessible lipoprotein surface. This is achieved through esterification of UC to CE by the enzyme LCAT (Fielding and Fielding, 1982).  (iii)  Transport of CE to the liver in lipoproteins for catabolism and excretion into bile. This may involve the direct interaction of HDL with liver cells or may require interlipoprotein lipid transfer processes (Barter et at, 1987) and hepatic uptake of the acceptor lipoproteins.  CELL MEMBRANE CETP  FC  I  CE  LCAT CE  J EFFLUX  LIVER  A-I  -+  ESTERIFICATION  -  TRANSFER  -  CLEARANCE  Figure 2. Schematic diagram of the process of reverse cholesterol transport. FC = free (unesterified) cholesterol, CE = cholesteryl ester, LAT = lecithin: cholesterol acyliransferase, A-I apo A-I, nHDL = nascent high density lipoprotein, HDL = high density lipoprotein, LDL = low density lipoprotein, VLDL= very low density lipoprotein. After Fielding and Fielding (1982). Recent evidence has suggested that apolipoprotein A-I may be involved in all three aspects of RCT. In addition, the expanding knowledge of the molecular genetics of this apoprotein has indicated that abnormalities of its gene locus may be associated with some forms of hyperlipidemia and atherosclerosis (Karathanasis et al, 1983; Rees et at, 1985; Ordovas et al, 1986).  6  1.4. Apolipoprotein A-I 1.4.1. Apo A-I Gene Structure The mature plasma apo A-I protein is a single polypeptide chain of 243 amino acids with a molecular weight of approximately 28,000 daltons. The gene for apo A-I is one member of the soluble  apolipoprotein gene family which includes apolipoproteins A-Il, A-IV, C-I, C-lI, C-Ill and E. The genes for these apoproteins have regions of homology and similar genomic structure with similar distribution of intron-exon borders, suggesting evolution from a common ancestral gene (Li et at, 1988). The human apo A-I gene has been cloned and sequenced (Law et at, 1983; Karathanasis et at, 1983b; Cheung and Chan, 1983; Sharpe et at, 1984) and the cDNA sequence has verified the amino acid sequence obtained earlier from the purified plasma protein (Brewer et at, 1978).  A  C-Ill  A-I  A-IV  I  5•  13.  B PstI  J  PstI IVS-1  PJS-2  50 I  I  IVS-3  100 I  150 I  200 I  Amino Acid  V////, V////JV////t’////4’////4’////F/A’////,  Exon3  Exon4  Figure 3. Location and structural organization of the human apo A-I gene. A- Chromosomal organization of the apo A-I/C-III/A-IV gene cluster on chromosome 11. B- Approximate positions of the intervening sequences (IVS) within the apo A-I gene. PstI indicates the location of the restriction sites for this enzyme which flank the 2.2 Kbp gene. C- Organization of amphipathic helical repeats within the apo A-I coding region indicating the derivation from exon 3 or exon 4 of the gene. Large rectangles indicate 22 amino acid residue helix, small rectangles are 11 residue repeats. (Redrawn from Segrest et at, 1992). cDNA clones have provided significant information on the structural and evolutionary aspects of  7  apo A-I. The cDNA sequence specifies a mRNA of 950 base pairs (bp) including 35 bp of 5 flanking sequence and 54 bp of 3 untranslated sequence preceding a poly A tail. The coding region spans 893 bp (see Figure 3) of which repeating sequences of 66 bp occupy the 3 half of the message and encode 22 amino acid repeats of  -helical structure in the apoprotein. The repetitive nature of this region has been  the subject of intense speculation regarding evolutionary origins. Karathanasis and colleagues (1983b) have suggested that the sequence arose out of intragenic duplication events. Based on computer analysis, Fitch et at (1984) have proposed a smaller repeating sequence (33 bp) with duplication and unequal crossover events leading to the observed 22 amino acid repeat structure. This structural motif is common to many of the soluble apolipoproteins. Comparison of apo A-I genomic DNA and cDNA clones (Shoulders et at, 1982; Karathanasis et at, 1983b; Sharpe et al, 1984) has provided insight into the genomic organization. The apo A-I gene has been mapped to the long arm (q) of chromosome 11 where it is clustered with apolipoproteins C-Ill (Bruns et at, 1984) and A-TV. The apo A-I gene is 1863 bp in length and contains three intervening sequences (IVS) which interupt coding region at sites which are similar in apos A-I, A-IT, C-Il, C-Ill and E (Li et at, 1988). In apo A-I, exon 1 contains the 5-untranslated region of the mRNA and is interrupted by the first intervening sequence (IVS-1, see Figure 3B). Exon 2 contains a small portion of 5untranslated sequence and the majority of the signal peptide coding region. This is interrupted near the signal peptide hydrolysis site by the second intervening sequence (IVS-2). Exon 3 encodes the signal peptidase recognition site, and the N-terminus of the protein to residue 43 of the mature protein. The third intervening sequence (IVS-3) interrupts this codon and the remaining protein coding sequence of exon 4 which comprises the remaining x -helical tandem repeats. A number of studies have attempted to identify a genetic marker for the presence of atherosclerosis, and apo A-I has been a candidate gene. Restriction fragment length polymorphisms (RFLP) within the A-I/C-III/A-IV gene cluster have been identified in some populations, and their relationship to hyperlipidemia and/or atherosclerosis has been tested. In general, the results of these studies have been conflicting or misleading. Reliable markers in the vicinity of the apo A-I gene have not been consistently identified in the affected groups (Breslow, 1992). Most gene polymorphisms would not be expected to affect apo A-I structure or function, since they are found more frequently in the regions 8  flanking the gene or within intervening sequences. Only rarely have mutations been described in which apo A-I structure is altered (see Section 1.7).  1.4.2. Apo A-I Gene Expression and Secretion In humans, apo A-I mRNA is found only in the liver and the small intestine (Li et at, 1988). Both the hepatocyte and the enterocyte have been shown to synthesize and secrete apo A-I protein (Zannis et at, 1983). Intracellular processing of the primary translation product in these tissues has been demonstrated and the successive stages can be monitored by changes in the p1 of the product (Figure 4). Zarmis and Breslow (1985) have established a nomenclature for cellular and plasma apo A-I species based on isoelectric point (p1): A-I , p1= 5.85; A-I4, p1= 5.74; A-I 2 , p1= 5.65; A-I4, p1= 5.52; and A4  I4,  p1 = 5.40. The predominant isoform in normal plasma is A-I . 4  123456  4 SDS  . .  ()  Preproapo A-I  0  Proapo A-I  .  Mature Apo A-I  ....  .  .  .  .  Lymph  Plasma  Figure 4. Schematic diagram of the charge and size heterogeneity of apo A-I at different stages of proteolytic processing. Solid circles represent the species found in the biological system indicated on the right. Dotted circles indicate the position of mature plasma apo A-I marker. Symbol size is proportional to the quantity of each isomorphic form. Arrows at left indicate the direction of migration in either isoelectric focussing (IEF) or sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The numbers at the top of the diagram refer to the subscript of the apo A-I isoform as referred to in the text. (Modified from Bojanovsky et al, 1985)  9  Apo A-I is synthesized as a preproapolipoprotein (Figure 5) containing an amino terminal 18 residue prepeptide, or signal sequence, which directs the protein to the endoplasmic reticulum (ER). Nterminal prepeptides are a common structural feature of many proteins which are secreted from eucaryotic cells (Nothwehr and Gordon, 1990). Other proteins contain signal sequences which are a distance from the N-terminus (internal signal sequences) and some secretory proteins lack any prepeptide (Meusch et at, 1990). As preproapo A-I is synthesized on the ribosome, the emerging signal peptide is recognized by a cytosolic factor, signal recognition particle (SRP). SRP binds the peptide chain, stopping further translation instil SRP anchors the ribosome to a specific ER membrane receptor. Translation then continues on the bound ribosome and the growing polypeptide is translocated across the ER membrane into the lumen. Coincident with this translocation, the signal peptide is hydrolysed by signal peptidase which is found on the luminal membrane of the ER. This processing has been delineated in studies of N-terminal signal sequences based on the original work of Blobel and Dobberstein (1975) who isolated translocation and proteolytic activity in the microsomal pellet of subcellular fractionations. The early secretory targetting of preproapo A-I has also been described in rabbit reticulocytes (Zannis et at, 1983). Preproapo A-I has p1>5.85 and is approximately 2000 Daltons larger than the mature plasma protein. The proapo A-I (A-I ) product of signal peptide hydrolysis can be identified 2 during in vitro translation in the presence of microsomal membranes and in secretions from hepatocytes. The function of the apo A-I propeptide is not known although functions of propeptides in other plasma proteins have been delineated (Peters, 1987). Some propeptides mediate the correct folding of the polypeptide chain (eg., insulin). In other proteins, the propeptide serves to direct the post-translational modification of the primasy translation product (eg., y -carboxylation of clotting factors). Propeptides can also be used to target proteins to a specific cellular compartment or as a control element which regulates the synthesis or secretion of the mature protein (eg., neuroendocrine hormones). A common feature of propeptides is the amino acid sequence at the hydrolysis site, which is often preceeded by two basic amino acid residues, Arg-Arg or Lys-Arg. The enzymes which catalyse the hydrolysis are poorly characterized, although a recent report suggests that similar endopeptidases may catalyse the hydrolysis of more than one proprotein (Wise et at, 1990). The apo A-I propeptide is 10  unusual, in that the hydrolysis site is preceded by two Gin residues (Figure 5).  Preproapo A-I [MetLysAla  SerGinAla  -24  -7  ArgHisPheTrpGInGIn  Asp  Gin  -6  +1  243  ArgHisPheTrpGInGIn  Asp  Gin  —6  ÷1  243  Asp  Gin  +1  243  -1  Jr  Signal Peptidase Proapo A-I  I  -1  A-I Specific Propeptidase Mature Apo A-I  Jr I  I  Figure 5. Diagramatic representation of the proteolytic processing of the apo A-I precursor showing the amino acid sequnece in the region of proteolysis. Positive and negative integer labels indicate the amino acid sequence with respect to the first residue (+1) of the mature protein. The macromolecular nature of secreted apo A-I is not clear at present. Proapo A-I appears to be secreted in a lipid-free or lipid-poor state and is then incorporated into HDL outside the cell (Vance and Vance, 1990). Such a process would, by necessity, require remodelling of the lipoprotein to accommodate the additional surface apoprotein. Although apo A-I appears to be secreted as the proprotein, this form accounts for less than 10% of circulating apo A-I. Propeptide hydrolysis may occur at or shortly after incorporation into HDL. An extracellular endopeptidase (propeptidase) catalysing this reaction has been identified in lymph and plasma (Scanu, 1987) and it has been suggested that this modification may regulate apo A-I metabolism or HDL biogenesis in the plasma compartment (Edeistein et al, 1983, Bojanovsky et al, 1985). A similar converting activity has been described for the  11  apolipoprotein A-IT precursor (Gordon ci at, 1984). The extracellular conversion of proapo A-I (A-I ) to 2 mature apo Al (A-I4) is a slow process which has been demonstrated in vivo (Bojanovsky et at, 1985) and in vitro (Edeistein et at, 1983). Processing activity is Ca dependent (EDTA sensitive) and has unusual proteolytic specificity for the Glrf 1 -Asp’ peptide bond (Edelstein ci at, 1983). The enzyme is not a serine protease, based on serine-specific inhibitor studies. While some investigators have suggested that hydrolysis of the propeptide may be required for the secreted apo A-I to associate with HDL (Schmitz ci at, 1983), the timing of propeptide hydrolysis and lipoprotein integration is still largely speculative. Evidence from cell culture systems (Mallory ci at, 1987; Forte ci at, 1990) which do not normally express apo A-I but do possess proapo A-I endopeptidase activity, suggests that the apo A-I propeptidase may not be absolutely specific for proapo A-I. Additional post-traislational modifications of apo A-I have also been identified. Covalent phosphorylation (Beg ci at, 1989) and fatty acylation (Hoeg ci at, 1986) have been demonstrated, although the function of these modifications is unclear. It has been suggested that these additions may be required for correct intracellular trafficking or efficient secretion. Apo A-I is not glycosylated, unlike many secreted proteins, including apolipoproteins B, E and C-Ill. Isomorphic forms A-I4 and A-I4 on IEF gels probably represent deamidation products of the mature A-I , which apparently arise over time 4 in the circulation (Bojanovsky ci at, 1985). The origin and fate of apo A-I4, a minor component of circulating A-I, are unclear, but this peptide may be a deamidation product of proapo A-I (A-I4).  1.5. Functions of Apo A-I 1.5.1. Apo A-I and HDL Structure The functions of apo A-I are tightly associated with the role of HDL in RCT. Apo A-I serves an important structural function, maintaining the solubility of the polar lipids that are carried in the HDL density class. In addition, however, apo A-I performs metabolic functions which are central to the remodelling of HDL and RCT. The importance of apo A-I structure to HDL metabolism has been underscored by studies of human apo A-I in transgenic mice. Mouse and human apo A-I are structurally distinct since antibodies have been isolated which do not cross-react between the two species and cDNA probes for the two 12  genes do not cross-hybridize. Normal mouse plasma contains a single, homogeneous HDL population in contrast to human plasma which contains two major subpopulations (HDL 2 and HDL) which differ in both size and density. When transgenic mice were established which expressed human apo A-I, the plasma levels of HDL-C and apo A-I increased approximately two-fold (Walsh et at, 1989; Rubin et at, 1991a). This increase was largely due to expression of the human transgene product. The quantity of the endogenous mouse apo A-I protein was markedly reduced despite normal mRNA levels. From these studies, it appears that the human gene product is more abundant than mouse apo A-I because of more efficient mRNA translation or protein secretion in these animals. More importantly, the species of HDL in plasma become polydisperse, and the pattern resembles that found in human plasma. The transgenic human apo A-I mouse has also reinforced the notion of the protective role of apo A-I in atherogenesis. Rubin et at (1991b) assessed the development of atherosclerotic lesions iii a susceptible mouse strain (C57BL/6) with or without the human apo A-I transgene. On an atherogenic diet, these mice without the transgene rapidly developed fatty streak lesions, while those mice expressing the human apo A-I developed significantly fewer lesions. The relative contributions of HDL level and HDL composition in this model have yet to be established. Chajek-Shaul et al (1991) have suggested, based on kinetic studies, that the presence of human apo A-I might prevent the selective hepatic uptake of CE from HDL in these mice. They propose that elements of human apo A-I structure prohibit direct HDL-CE removal by particle uptake, necessitating transfer to LDL and whole particle uptake as the route of CE removal. Therefore, in the presence of human apo A-I, CE can only be removed from circulation if CETP is also present. Since mice do not have CETP, the esters accumulate in HDL in the transgenic animal model. Thus, a more appropriate model for human HDL metabolism might be an animal possessing human transgenes for both CETP and apo A-I.  1.5.2. Apo A-I and the Interaction of HDL with Cells The first component of reverse cholesterol transport is the desorption of excess cellular cholesterol onto HDL from cells. HDL has been shown to interact with cells and apo A-I has been implicated as the ligand for the interaction with an HDL binding protein. Biesbroeck el at (1983) and Oram et at (1984) characterized high affinity, saturable binding of HDL to cultured cells which was 13  biochemically distinct fiom binding to the LDL receptor. Binding was increased by cholesterol loading of the cells and evidence was obtained that apo A-I was the ligand for binding (Fidge and Nestel, 1985; Brinton et at, 1985). Tyrosine residue modification (Brinton et at, 1985; Chacko, 1985) markedly diminished binding, suggesting that this  amino  acid residue might mediate the interaction. Subsequently,  the binding site was demonstrated in many cell types, including human liver (Schouten et at, 1990; Tozuka and Fidge, 1989; Hoeg et at, 1985), but the apoprotein ligand specificity is broader than initially anticipated, recognizing apo A-IT and apo A-TV in addition to apo A-I (Dvorin et at, 1986; Tozuka and Fidge, 1989). The binding component appears to be a protein of 100-110 kilodaltons (Graham and Oram, 1987; Tozuka and Fidge, 1989; Bond et at, 1991) which mediates the translocation of intracellular membrane UC to the plasma membrane (Oram et at, 1991). Binding of HDL does not affect the rate of plasma membrane UC desorption (Karlin et at, 1987). Therefore, while the putative receptor does appear to mediate cellular cholesterol movement, it does not facilitate the direct movement of membrane cholesterol into HDL. Schmitz and colleagues (1985) have suggested that a retroendocytotic process is involved in the interaction of HDL with cells, whereby the lipoprotein is bound, internalized, CE-depleted or UC enriched, and then resecreted without protein degradation. This is in contrast to the receptor-mediated endocytosis of LDL, where the lipoprotein is degraded following internalization. A retroendocytotic mechanism for HDL uptake and resecretion has also received support from additional studies in other laboratories (Rahim et at, 1991; Kambouris et at, 1990; DeLamatre et at, 1990). While neither hypothesis is yet confirmed, elements of both may be involved in the actual mechanism. There is evidence that a plasma membrane protein is responsible for the interaction with HDL. This binding mediates changes in cellular cholesterol metabolism. Following HDL binding, cellular UC may trarislocate from intracellular membranes to the cell membrane (Oram et at, 1991) or the CE of the HDL may be selectively taken up by the cell (Schmitz et at, 1985). The direction and type of lipid transfer might depend on the cell type and its cholesterol status. The biologic activity of this binding protein (the 110 kD HDL binding protein) has been recently demonstrated by cloning and expression of the cDNA (Oram et at, 1992). However, the requirement for a distinct mechanism for interaction of HDL with cells has not received universal support. Reichl and Miller (1989) have calculated that, in man, 14  the majority of the CE generated within HDL is delivered to the liver only after transfer to lipoproteins of lower density. Therefore, the interaction of HDL with cells need not occur for either the peripheral uptake of UC or for the liver delivery of EC. However, even in the absence of a specific cell binding site, HDL is the best acceptor of excess cellular UC.  1.5.3. Apo A-I and Lecithin:cholesterol Acyltransferase (LCAT) Lecithin:cholesterol acyltransferase is the plasma enzyme responsible for generating the majority of plasma cholesterol esters via transfer of sn-2 fatty acid from phosphatidyicholine to the 3-position hydroxyl group of cholesterol. Apo A-I has long been recognized as a potent activator of this enzymic reaction (Fielding et al, 1972; Soutar et at, 1975; Albers et at, 1979; Matz and Jonas, 1982). Despite extensive investigation, the exact molecular mechanism of apoprotein activation of LCAT is still incomplete. Several investigators (Albers et at, 1979; Fielding and Fielding, 1972; McLeod et at, 1986) have established that apo A-I is the principal activator of cholesterol esterification by LCAT under in vitro  conditions which resemble those in vivo. Indeed, when apo A-I containing lipoproteins are removed from plasma by immunoaffmity chromatography, much of the cholesterol esterifying activity is also removed (Fielding and Fielding, 1980; Cheung et al, 1986). This implies a physical association between A-I and LCAT and reflects their functional relationship. While apo A-I and LCAT do not. appear to interact directly, LCAT associates largely with lipoproteins that contain apo A-I. In certain pathologic states (Pritchard et at, 1986) and with some in vitro manipulations, LCAT can be activated by other apolipoproteins. Apo E, apo A-TV and apo C-I have been shown to activate LCAT in the absence of apo A-I (Soutar et at, 1975; Albers et at, 1979; Steinmetz and Utermann, 1985; Steinmetz et at, 1985; McLeod et al, 1986). In the presence of suboptimal levels of apo A-I, apo A-Il can also activate the enzyme despite being inhibitory at optimal apo A-I concentrations (Chen and Albers, 1986; Nishida et at, 1986)[ The observation that other apoproteins can activate LCAT is of particular interest since several HDL deficiency states have been described where cholesterol esterification proceeds normally, or at only partially reduced rates, despite extremely low levels of apo A-I (Schaeffer et at, 1984). Our understanding of LCAT activation by apo A-I is incomplete, reflecting our lack of complete 15  understanding of the mechanism of LCAT catalysis. The application of a molecular genetic approach to the study of LCAT protein structure will no doubt provide insight into the mechanism of apo A-I activation. Studies using chemical modification techniques (Jauhiainen and Dolphin, 1986; Park et at, 1987) and the application of predictive algorithms to the LCAT primary sequence (Yang et at, 1987) have provided preliminary evidence for the reaction mechanism, the active site and the secondary structure of the enzyme. The active site contains a reactive serine residue at position 181 of the primary sequence. This region has considerable sequence homology with the serine esterase class of enzymes including the lipases. This residue hydrolyses the sn-2-position fatty acid of phosphatidylcholine to form an enzyme-oxyester intermediate. The proposed mechanism (Jauhiainen and Dolphin, 1986) indicated that intraenzyme transfer of the acyl group to a free sulthydryl group at Cys-31 and/or Cys-184 results in a thioester intermediate which ultimately transfers the acyl group to cholesterol. However, the proposed role for the free sulfhydryl residues in the mechanism has been recently called into question by molecular genetic studies in which the reactive Cys residues were replaced without affect on transacylation (Francone and Fielding, 1991). In the absence of the cholesterol acceptor, the oxyester intermediate formed in the first stage can be hydrolysed by water to form unesterified free fatty acid and regenerate enzyme-OH. Analysis of the cDNA clone for the human LCAT (McLean et at, 1986) has revealed that only one region of the enzyme has amphipathic character analogous to the soluble apolipoproteins. However, LCAT can associate with phospholipid surfaces. The question then arises: If LCAT can interact with phospholipid in the absence of apo A-I, what other role might the apolipoprotein play that makes it unique?  1.6. Structural Elements of Apo A-I 1.6.1. The Apo A-I Signal Peptide Apo A-I, like all apolipoproteins, contains an N-terminal signal sequence which targets the protein for translocation across the ER membrane and into the secretory pathway (see Section 1.4.2). The structure of the apo A-I signal peptide conforms to the general features documented for a number of eucaryotic proteins (Nothwehr and Gordon, 1990). These include a positively charged N-terminus, a 16  hydrophobic interior segment (see Figure 6), and small  amino  acid side chains at positions -1 (Ala) and  -3 (Ser) from the cleavage site. The apo A-I signal sequence is 18 residues in length, which is within the range described for many of these peptides (15 to 50 amino acids).  1.6.2. The Apo A-I Propeptide Three of the soluble apolipoproteins (A-I, A-IT, and C-IT) have propeptide sequences, for which a function has yet to be identified. These segments are retained following translocation across the membrane of the endoplasmic reticulum, but are removed as the protein attains its mature plasma form. The recognition sequences may be apolipoprotein-specific since proteolysis occurs at an unusual site and appears to require a specific endoprotease in each case. The apo A-I propeptide ends with a Gln-Gln dipeptide and the propeptidase hydrolyses the Gin-Asp peptide bond between the propeptide and the mature N-terminus. The propeptides of many other proproteins end in a pair of positively charged residues which appear to constitute a recognition site (see Section 1.4.2). The physiological role of apo A-I propeptidase activity (Edelstein et at, 1983) is not yet known, nor are the tissue and species distribution of this hydrolytic activity. This enzyme has been found in plasma and in lymph where the hydrolysis is presumed to take place. Alternatively, the small amount of proapo A-I found in plasma may represent a minor component of apo A-I that escapes a cleavage event which would mainly occur within the cell. This suggestion is supported by observations in hepatocyte-derived cultures (HepG2) which secrete partially processed (50%) apo A-I (Forte et  at,  1987). In addition, the medium of CHO cell  cultures expressing the human apo A-I cDNA contain as much as 90% mature apo A-I and only a minor portion as proapo A-I (Mallory et  at,  1987). These experimental models suggest that the hydrolysis might  occur within cells. In vivo, propeptide hydrolysis appears to be sensitive to secondary structure at the N-terminus of the protein. Von Eckardstein et at (1989) have studied natural mutations of the amino terminus of mature apo A-I and found that some mutations altered the quantity of circulating proapo A-I. They found that subjects with the Pro 3 to Arg variant had normal plasma levels of HDL-C and apo A-I but that the ratio of proapo A-I to mature apo A-I in plasma was increased. Similarly, the Pro 3 to His mutation also affected the rate of hydrolysis, but Pro 4 to Arg did not. Analysis of the predicted 17  secondary structure in this region suggested that the apo A-I propeptide forms part of an  -helix and  3 has high probability of being a 8 -turn residue (see Figure 6). It appears, therefore, that critical that Pro positioning of a,8-turn residue near the Glrf 1 -Asp 1 hydrolysis site may be an important element of recognition by the propeptidase. The presence or absence of the propeptide segment has no clear effect on the extracellular functions of apo A-I (Fennewald e at, 1988). While some evidence has suggested that proapo A-I might bind to lipoprotein less avidly than the mature form (Zannis et at, 1983; Rosseneu et at, 1984), studies of proapo A-I binding to HDL (Edeistein et a!, 1983) have indicated that the formation of the complex is required for the conversion to the mature form. The crucial function of the propeptide may be intracellular. Gordon et al (1986) have suggested that the propeptide may play a role in appropriate folding of the polypeptide chain, or may act as a targeting signal to compartmentalize apo A-I within the secretory pathway. Removal of the propeptide from the apo A-I cDNA delayed ER translocation during cell-free translation (Folz and Gordon, 1987; Stoffel and Binczek, 1988) but did not affect the fidelity of signal peptide hydrolysis. Two modes of secretion are generally recognized for eucaryotic proteins, and the selection is based on cell type and elements of protein structure (Kelly, 1985). Endocrine and exocrine cells are capable of regulated secretion in which protein products are stored in secretory granules for secretion on a specific stimulus. Most other cells secrete by a constitutive pathway in which no storage occurs; proteins are secreted immediately following their synthesis. Secretion of apo A-I from hepatocytes (Vance and Vance, 1990) and probably from enterocytes appears to occur by the constitutive pathway. When the apo A-I cDNA was expressed in the neuroendocrine cell line AtT-20 (Fennewald et at, 1988), cellular transport was not affected by the presence or absence of the propeptide. Neither the secretion rate nor the selection of regulated versus constitutive secretory pathway were altered. However, in this expression model apo A-I was secreted primarily via the regulated pathway, perhaps because the multiple amphipathic x -helices in apo A-I can target the protein to the secretory granules (Kizer and Tropsha, 1991). Detailed analyses of the role of the apo A-I propeptide on constitutive secretion have not been conducted.  18  1.6.3. The Amphipathic Helix Motif Primary sequence analysis and the application of structure prediction algorithms have indicated that many proteins contain regions of amphipathic structure which appear to mediate the interaction of proteims with hydrophobic compounds (Segrest et at, 1990). The amphipathic helix motif is a prominant feature of all of the apolipoproteins (Segrest et at, 1992). These structural elements are responsible for the lipid binding properties of this protein class. Much of our understanding of apolipoprotein function is derived from studies of model peptides designed to duplicate their secondary structure, most notably from study of the amphipathic helix (Segrest et at, 1974). In its present form (Segrest et at, 1990), the model proposes that the hydrophobicity and the cx -helical potential of a given peptide, as defmed by its primary sequence, are independent determinants of its structure. Both determinants are required for interaction with phospholipid surfaces, including lipoproteins. Experimental evidence from other laboratories (Ponsin et at, 1986) also supports this contention.  NATIVE APOLIPOPROTEIN Al  >< D  z  C-) I 0 0 >I  RESIDUE NUMBER  Figure 6. Hydropathy plot of the apo A-I precursor, preproapo A-I. Hydropathic index was calculated by the method of Kyte and Doolittle (1982) using Sequence (Delaney Software, Vancouver, B.C.) and plotted against residue number. Points below the line of average hydropathy indicate a residue likely to reside in an hydrophilic environment, residues above the line indicate probable location in a hydrophobic environment. Open bars indicate the positions of the pre- and pro- peptides of the N-terminus. Inverted triangles indicate the positions of high -turn probability as predicted by Gamier et at (1978). Using the algorithm of Gamier et al (1978), apolipoprotein A-I contains many hydrophobic regions (Figure 6) with 66% of its residues in  -helix. All members of the soluble apolipoprotein class  have similar structural features. A common property of the apolipoprotein class is the ability to bind to  19  phospholipid emulsions. This is also a prerequisite for LCAT activation although not all apolipoproteins activate LCAT equally. The depth of penetration of the apolipoprotein into the phospholipid monolayer (Fukushima et al, 1980) may be a critical determining factor. Alternatively, apolipoproteins may promote LCAT catalysis by enhancing the transfer of the enzyme between substrate lipoprotein particles following transacylation (Nishida et al, 1986), that is, to increase the enzyme “off-rate”. Apo A-I may promote transacylation by LCAT more effectively than other apolipoproteins because it has regions which enhance both binding to and release from phospholipid substrates. As yet, the mechanism of LCAT activation is not clearly established. Some aspects of LCAT activation were identified through analysis of fragments of the purified plasma apo A-I protein. Cyanogen bromide cleavage analysis was used to isolate an LCAT activating region from purified plasma apo A-I. Four fragments result from this treatment, only two of which are capable of activating the enzyme (Soutar et al, 1975). The most effective fragment contained the majority of the C-terminus of apo A-I. This region contains the majority of the 6 repeating sequences of 22  amino  -helix. Residues 99 to 230 specify  acids each (22mer). The secondary structure of each repeat in this  region is cx -helix with opposing poiar and nonpolar faces, ie an amphipathic helix (Segrest et al, 1974). Eleven amino acid residues complete three turns in an  -helix and represents the monomeric unit (Fitch  et al, 1984). The 22mer repeat structures found in the apolipoproteins are derived from this monomer unit. A wheel diagram of the predicted structure of a single 22mer (see Figure 7) indicates that negatively charged residues occupy one face of the protein and uncharged residues define an opposing face. The midline of this structure is occupied by predominantly positively charged residues. Functionally, these elements of the apo A-I sequence are believed to interact: (i) with the hydrophobic lipoprotein core via the nonpolar helical face; (ii) with the aqueous environment via the negatively charged polar face; (iii) with phospholipid headgroups via the positively charged helical midline residues (Sparrow and Gotto, 1982). Proton magnetic resonance indicates that binding of apo A-I to lipoproteins and analogous structures involves intercalation of  -helical regions with the phospholipid monolayer (Brouillette et al,  1982). 20  Figure 7. Helical wheel projection of the consensus sequence for the 22 residue repeat structure of human apo A-I. (Modified from Segrest et cii, 1992). The consensus sequence of the repeating unit (see Figure 7) is not totally conserved among all 22mers. Compared to the amino acids at most positions of the 22mer, the 13th residue of the consensus sequence is least well conserved. This residue might, therefore, have additional functions in specific areas of the apo A-I molecule. The importance of the amphipathic helix in apo A-I function is underscored by studies of synthetic peptides containing this motif. Studies of peptides synthesized with the native apo A-I sequence (Sparrow et cii, 1980) have indicated that residues 148-185 were involved in LCAT activation (achieving 20% of native apo A-I) and that residues 164-185 are involved in lipid binding. Residues 227-243 of the A-I sequence did not bind phospholipid and that polypeptides of multiple length spaiming residue 197 to the carboxy terminus bound phospholipid but did not activate LCAT. Fukushima et at (1980) showed that residues 121-164, which form two lipid binding domains, activated LAT up to 30% of the native protein. Therefore, peptides spanning one helical repeat can activate LCAT but two repeat sequences are more potent activators. Furthermore, while lipid binding is essential for activation by synthetic peptides and native fragments, it is not sufficient for the full activation observed with the native protein. 21  These early conclusions must be viewed with caution, however, since recent evidence indicates that the DMPC substrates used in many studies do not accurately reflect activation of physiologic LCAT substrates (Anantharamaiah et al, 1990). Synthetic peptides modelling the amphipathic helix but unrelated to apo A-I in sequence are also capable of activating LCAT. The cofactor activity of these peptides correlates with their ability to form c -helix (Fukushima et at, 1980). Yokoyama et al (1980) described a 22 amino acid peptide with amphipathic potential that stimulated the phospholipase A 2 activity of LCAT (to 50% of apo A-I) but was substantially less effective at stimulating cholesterol esterification (18% of apo A-I). Another peptide (LAP-20) containing a single lipid-binding domain (Pownall et at, 1980) activated cholesterol esterification in DMPC substrates by up to 65% of the level obtained with native apo A-I. Both of these studies indicated that activation was due to interaction of the activator with the phospholipid substrate, rather than via an enzyme-activator interaction. Yokoyama suggested that in the absence of activator phospholipid fatty acid side chains might be oriented toward the aqueous surface of the particle and that reorientation of sn-2 groups into the hydrophobic region of the phospholipid monolayer might occur during activation. Therefore, early synthetic peptide studies indicated that LCAT activation is mediated by tx -helix interaction with substrate phospholipids, rather than with the enzyme. The separate contributions of hydrophobicily and  -helix have been analysed by inserting a  proline residue at various positions in the LAP-20 peptide (Ponsin et al, 1986). The introduction of this helix-breaker” alters phospholipid binding and LCAT activation by the peptide without affecting the overall hydrophobicity. The extent of binding and activation depended upon the site of substitution, with most pronounced reduction observed when Pro was introduced near the middle of the sequence. The authors suggested, based on these observations, that helical segments of at least 15 uninterrupted residues were required for lipid binding and LCAT activation. Segrest and colleagues have used synthetic peptides extensively to investigate the mechanism of LDAT activation and have concluded that secondary structure is more important than amino acid sequence in LCAT activation. Synthetic peptides homologous to apo A-I sequence were no more potent LCAT activators than non-homologous amphipathic helical peptides. However, no synthetic peptide modeling a single amphipathic helix (22iner) exceeded 30% of the activation of the isolated native 22  protein. Dimers of 22mer repeats approach the LAT activation properties of apo A-I more closely than the monomeric unit (Anantharamaiah et at, 1990). These studies also indicated that a single position in the helix appeared to have an additional role. In dimer studies, Glu in the variable position 13 of each monomer unit was the most effective activator. In the apo A-I sequence, only amphipathic helical repeats 2 and 3 (residues 66-121) contain Glu at position 13. They have proposed, therefore, that this region and, in particular, the Glu residues at positions 91/92 and/or 110/111 of apo A-I are involved in LCAT activation. Investigation of other specific residues in the A-I sequence has been somewhat limited. Jonas et at (1985) investigated the role of lysine residues in LCAT activation using chemical modification techniques. These authors concluded that while modification of lysine did reduce the ability to stimulate cholesterol esterification, the effect was evident  only  when modification altered the residue charge.  However, since changes in the secondary structure of the apoprotein were also apparent, it was impossible to conclude that lysine, in itself, was involved in the activation process. Similar studies in our laboratory (R. McLeod and M. Bergseth, unpublished observations) have indicated that this type of chemical modification reduces the ability of apo A-I to bind to DMPC vesicles. A recent report has suggested that in vthv glycation of lysine residues also reduces its ability to activate LCAT (Gugliucci and Stahl, 1991). At present, however, chemical modification studies of the role of lysine residues in apo A-I activation must be regarded as inconclusive. In vitro mutagenesis techniques have recently been used to assess the role of the amphipathic helix in LCAT activation. Bruhn and Stoffel (1991) deleted two adjacent helices (d41, residues 146 to 186) from apo A-I and showed that LCAT activation by the mutant protein was reduced by only  10%.  They indicated, however, that this alteration might, in addition, destabilize the protein structure in vivo, since protein expression was reduced by40% in their E. coti  system.  1.6.4. Apo A-I Secondary Structure: Epitope Mapping The secondary and  tertiary  structures of proteins are critical determinants of their ultimate  function. Several investigators have employed monoclonal antibodies to define structural epitopes of apo A-I. Continuous epitopes, defined as those that contain linear segments of primary sequence, are rare in 23  apo A-I and when present are near putative  -turns in the sequence (Marcel et at, 1991). The N-  terminus of apo A-I appears to exist in a compact conformation. Many of the epitope-specific monoclonal antibodies which have been generated in a number of laboratories are directed towards the middle of the molecule within a single  -helix (residues 98 to 121). Since mobile, accessible domains are  known to be particularly antigenic, this region of apo A-I has been suggested to represent a mobile, “hinge” domain (Marcel et at, 1991; Cheung et at, 1987; Figure 8). Curtiss and colleagues (Curtiss and Edgington, 1985; Curtiss and Smith, 1988) recently developed monoclonal antibodies to apo A-I, three of which blocked activation of LCAT (Banka et at, 1991). The extent of inhibition was related to the ability of the antibody to bind to a specific apo A-I epitope. Other antibodies in this series bound avidly to distant epitopes but had no affect on activation. The results suggested that a specific region of apo A-I was involved in LAT activation and they were able to identify residues 95-121 as a critical element for cofactor activity using peptide competition studies. These studies provided further evidence for the existence of a distinct mobile hinge domain in the third helix of apo A-I (residues 99-121) and for involvement of this region in LCAT activation.  TANDEM AMPt4IPAThIC HELIXES  OF APOLIPOPROTEIN A-I  AUPHI4’AThIC HtI.IX-PHOSPHOL,pu, MONOLAYER SHELL  TANDEM AMPHIPAThIC 45jj5ES OF APOLIPOPROTEIN A-I  HEUX-PHOSPHOLIPID MONOLAYSR SHELL  :5  HINGED DOMAIN  NEUTRAL LIPID CORE  2  ORES  TANDEM AMPHIPATWC HELIXES OF APOLIPOPROTEIN A-I  SHELL  Figure 8. Schematic diagram of the hinged domain hypothesis for the interaction of apo A-I with HDL. The hypothesis suggests that amphipathic helices of apo A-I may be in an exposed or buried confor mation depending on the size of the HDL. From Cheung et at (1987), with permission of the publisher.  24  Anantharamaiah et at (1990) have used peptide dimers to activate LCAT and have suggested that Glu residues within the hinge region might stabilize this domain via charge-charge interaction. They proposed that Glu pairs at positions 91/92 and at 110/111 might provide hinge stability. However, direct assessment of this hypothesis in the intact protein has not been performed. Preliminary studies (Bruhn and Stoffel, 1991) have indicated that G1u 111 has little influence on LCAT activation, since substitution of this residue reduced activation of the enzyme by only  15%. If ion pairing is involved in hinge stability,  one might expect that Lys residues in the same region might play a role. The effect of altering Lys residues in the hinge domain region has not yet been reported. Using an improved modelling technique, Segrest and colleagues (1992) have identified some differences among the amphipathic helices of apo A-I. The majority of the helices are of class A and are predicted to penetrate the phospholipid layer to a greater depth than class Y helices. Class Y helices are much more prevalent in apo A-IV, and appear to penetrate the lipoprotein surface to a lesser extent than class A helices. Only two regions of apo A-I contain helices of class Y, the C-terminus and the Nterminal side of the mobile hinge domain (see Figure 9). Therefore, these structural elements may contribute to the unique functional properties of apo A-I.  COOH  220  Figure 9. Proposed supersecondary structure of human apo A-I. The position of amphipathic helices (Segrest et at, 1992) have been superimposed upon the structural model derived from epitope mapping (Marcel et at, 1991). Rectangles represent amphipathic helices of 11 (small) or 22 (large) amino acid length. Hatched regions are class Y, open regions are class A amphipathic helix. P indicates the Sei , 201 site of covalent phosphorylation. Arrow indicates Lys residue at position 107. The residues marking the ends of helix repeats are indicated by residue number from amino (NH ) to carboxy terminus (COOH) 2 of the mature protein. Triangles indicate the positions of proline residues. The putative hinge domain is between residues 99-143. 25  1.7. Naturally Occuring Structural Variants of Human Apo A-I The importance of apo A-I in cholesterol homeostasis has been demonstrated in pathologic states in which the protein is absent or near absent. The combined deficiency of apo A-I and apo C-Ill (Karathanasis et at, 1984) is characterized by profound reductions in HDL and severe atherosclerosis. The defect has been elucidated at the molecular level (Karathanasis e a!, 1987) and is the result of inversion of a 6 Kbp segment containing portions of both the apo A-I and apo C-Ill genes and intergenic sequence. Since these two genes are transcribed in opposite orientations, both produce abnormal mRNA. Neither apo A-I nor apo C-Ill is found in the plasma of subjects with this mutation. Tangier disease is an HDL deficiency state originally thought to be due to an apo A-I structural defect (Kay et at, 1982). Subsequent studies have shown that the apo A-I gene in Tangier disease specifies the normal protein sequence (Rees et at, 1984; Makrides et at, 1988), but the marked reduction in plasma apo A-I (1-5% of normal mass) in Tangier disease is associated with a larger than normal proportion  ( 50%) of proapo A-I (Zaniis et al, 1982). The conversion of proapo A-I to the mature  plasma protein occurs at the normal rate in Tangier plasma (Bojanovski et at, 1984) but the HDL particle appears to be catabolized rapidly. Perhaps the most striking observation, yet to be explained, is the persistence of the proapoprotein form in Tangier plasma despite the absence of the mature form. This might reflect the presence of separate apo A-I pools in vivo, with the proapo A-I pool largely spared by the defect in this disease. The cause of Tangier disease remains undefined despite several decades of study. Structural variants of apo A-I have been identified in which the deletion, insertion, transposition or substitution of a small segment of DNA results in the synthesis of a protein with altered structure. Several of these variants have been identified opportunistically or through population studies (Menzel et al, 1982; Menzel et al, 1984; Utermairn et at, 1982;  von  Eckardstein et a!, 1989). In many cases both the  amino acid alterations and the nucleotide changes have been defined. The identification of these variants was based on the isoelectric focussing pattern of the apo A-I, and therefore all variants involve deletion or substitution of a charged amino acid. The variants identified and characterized to date are shown in Table III. Mutations of Lys at position 107 and of Pro residues throughout the protein appear to be the most frequent. 26  Table III. Variant forms of apolipoprotein A-I. DEFECT  FAMILIES AFFECTED  Pro Arg — 3 —’ His 3 Pro —’ Arg 4 Pro  1 2  Arg O_ Leu t — Arg 6 GI9  1  —÷ Thr 37 A1a —’ Stop 84 Gin  Many 1  —’ Glu 89 Asp Asp 03’ Asn 1 1 07_, o Lys  1  Lys Met —’ 107 1 1O.._ Lys Giu t —* Lys Giu  2 1  G1u —* Gly 1 1 43_, Arg Pro  2  del 1 46, P.ig Gin ° 16 —* Val 147 G1u t 58, Giu Ala 1 65, Arg Pro  1  G1u 69 Gin 1 173 Cys Arg 177 His Arg —’ Lys 198 Glu  A-I  Mueflster—3C  A—I  MueIlster—3B  A—I  von Eckardstein etal, 1989 Menzel et ai, 1984 Menzeletal, 1984; von Eckardstein etai, 1989 Ladias et ai, 1990 Nichols et ai, 1988  Baltimore  A-I  7  REFERENCE  NAME  iowa  Matsunagaetal, 1991 Matsunagaetal, 1991  A—I Muenster—3A A—I Marburçj’ A—I Muenster—2  A-I Fukuolca A-I Norway  A—I  ciessen  A-I  Seattle  von Eckardstein et ai, 1990 Mahleyetal, 1984 von Eckardstein etal, 1989  4  A-I  Mi  von Eckardstein et ai, 1990 Weisgraberetal, 1983 Jabs et al, 1986  400  4  Mahieyetal, 1984; von Eckardstein etal, 1990; Strobi et al, 1988 Funke et al, 1991  -’ FS 202 A-I  213 Gly Asp  Familial amyloidic poiyneuropathy 10% of Japanese controls HDL deficiency, A-I deficiency, atherosclerosis  von Eckardstein etai, 1990 Menzel et al, 1984 Utermann etal, 1982; Menzel et al, 1982; RaIl et al, 1984; von Eckardstein et al, 1990 von Eckardstein etai, 1990 Takadaetal, 1991 Mahleyetal, 1984; RaHet al 1986 von Eckardstein etal, 1990 Utermannetai, 1984 Deeb et al, 1991  1  NOTES  Dominant decreased A-I  Abnormal HDL catabolism Hypoalpha  Fish-eye Disease variant, homozygous, low HDL, A-I with Cys  Mahieyetai, 1984  The frequency of genetic variation in the human apo A-I protein (0.8/1  amino  acids) is higher  (von Eckardstein et cii, 1990) than in hemoglobin (0.2 per 1 residues) which suggests that variants in apo A-I protein sequence are less subject to negative selection. Within the N-terminus and the z -helical regions of the apo A-I coding region, more charge-shift variants of the protein are found than would be expected from random nucleotide substitution. The N-terminal mutants may arise due to variability at the junction of exon 2 and intron 2. Inter-species comparison of apo A-I sequences has indicated that the amino acid sequence of the  -helix region is poorly conserved across species and this region is also a  27  frequent site of human variation. However, few of the variants appear to alter substantially the secondary structure in this region. Mutations which are predicted to alter the orientation of the amphipathic helix, for example those altering Lys 107 and Pro , appear to alter function also. Residues 66-98 are highly 165 conserved among species and no genetic variation in this region has been described in man. Despite the consistency of the inter- and intra-species distribution of variation in the apo A-I sequence, the techniques used for detection of mutants may have under-represented some regions of the protein where the alteration conserved charge or when the alteration is sufficiently deleterious to cause absolute apo A I deficiency. Apo A-I structural variants have rarely been associated with disorders of lipoprotein metabolism. One exception is the apo AJMiiano variant, in which affected subjects have reduced HDL levels (10-20% of normal). These individuals have no evidence of accelerated atherosclerosis. The abnormal protein forms apo A-I homodimers and apo A-I/A-Il heterodimers via the variant Cys 173 residue. Wild type apo A-I does not contain cysteine and apo AlMIIano is reported to have reduced LCAT activating ability (Mahley et at, 1984, Jonas et at, 1991). A second apo A-I variant containing Cys residues has been described (Funke et at, 1991) in a subject without personal or family history of CAD. The mutation affected plasma LCAT activity, which was reduced to 1/3 of normal. The defective apo A-I was distributed in LDL and d> 1.21 g/ml fractions following ultracentrifugation, indicating abnormal lipid binding properties. The defect was shown to be a frameshift mutation (apo A-1 -FS) caused by loss of C from codon 202 leading to premature 202 termination after 229 amino acids. Codons 203-229 are altered from the wild-type sequence and shift the p1 by +7 charge units from the normal protein. The frameshift also results in the introduction of several Cys codons. The defect was detected in both homozygous and heterozygous individuals and affected HDL and apo A-I levels in both. As with the apo Aliano the presence of Cys residues caused formation of stable homodimers and apo A-Il heterodimers. Since the region of apo A-I affected by the mutation is highly conserved among species (von Eckardstein et at, 1990), the authors have suggested that this region may be linked to LCAT activation. Apo  Al e 5 attie  (Deeb et at, 1991) is unique in that reductions of HDL and apo A-I are dominant  in the heterozygous state. It is the only apo A-I variant involving a large coding region deletion. The loss 28  of 45 bp from exon 4 removes 15 residues of a single  ). It was suggested 60 146 to Arg -helix motif (Gin  that the mutant protein alters HDL structure and causes hypercatabolism. The changes also appear to reduce the affinity of LCAT and other apolipoproteins for the HDL. In all other subjects with variant forms of apo A-I, modest reductions in the level of HDL are the most severe consequence of the abnormal protein. However, as previously noted, the overall impact of the mutation may not be expressed, since most of the variants have been identified only in the heterozygous form. One notable exception is the Norwegian variant, Glu - Lys, which has been 136 identified in the homozygous state (Mahley et al, 1984). The levels of LDL are markedly decreased in this condition but HDL-C is near normal. The full influence of the remaining mutations on lipoprotein metabolism awaits the identification and subsequent characterization of homozygous individuals, or the functional characterization of mutants generated in vitro. Functional studies of the purified mutant apo A-I proteins have been limited due to the difficulties encountered isolating large quantities of pure mutant protein from plasma of heterozygous subjects. Only two of the variant apo A-I have consistently abnormal ability to activate LCAT. Deletion of Lys 107 reduced the activation by about 50% compared to native protein (Rail et al, 1984), an effect ascribed to reduced lipid association properties (Ponsin et al, 1985). Models of secondary structure predict that the deletion turns the polar face of the helix by 903, which may account for its reduced function. Apo AI, -’Arg) also demonstrated reduced ability to activate LCAT (Rall et al, 143 iessen (Pro 3 1983). However, a more detailed analysis of Pro’ -.Arg, comparing the activation of the normal and 43 variant protein (isolated from the same plasma sample of heterozygous subjects), was inconclusive (Jonas et al, 1991). In this comprehensive study, only Lys -’O function was significantly reduced from normal, 107 forming HDL-like recombinants with altered structure which resisted normal transformation to larger species on incubation with LDL. LCAT activation by the variant was also reduced compared to normal but the large variation among different normal preparations reduced the impact of the findings. Apo  AJFukuoka  -. Lys) was shown to activate LAT normally (Takada et al, 1990). The 110 (G1u  structural changes predicted for this mutation are minimal. Despite the change of 2 charge units, the amino acid substitution is not predicted to alter the orientation of the polar and nonpolar faces of the amphipathic helix. The predicted structure is therefore consistent with the functional observations and 29  tends to support a more important functional role for Lys . The abnormal apo AINorway (Glu 107 — Lys) 36 has been identified in homozygous individuals, in which LCAT activity was normal (Rail et al, 1986) suggesting that this Glu residue is also not involved in the activation mechanism. While observations of apo A-I variants have provided valuable insight into apo A-I function, their scarcity has limited the amount of information acquired. The application of molecular genetic techniques to apolipoproteins in recent years has provided the basis on which structure-function correlates can be more systematically investigated.  1.8. Expression of Recombinant Apo A-I  Expression of apo A-I in vitro has been the aim of many laboratories since the first cDNAs were cloned in the early 1980s. Zannis et a! (1983), using rabbit reticulocytes, demonstrated that the primary translation product of the apo A-I cDNA is preproapo A-I and that, in the presence of dog pancreatic microsomal membranes, preproapo A-I was processed to proapo A-I. Lamon-Fava et a! (1987) have suggested that the ability to express apo A-I may be sufficient to permit a cell to produce lipoprotein and tested the hypothesis in 3T3 cells transfected with the apo A-I cDNA. They showed that these cells secreted proapo A-I, some of which was isolated in HDL, with properties similar to HepG2 cell secretions. Unlike HepG2 cells, the quantities of HDL produced were extremely low unless exogenous lipid was provided in the medium. In addition, HDL associated apo A-I was a minor component of the total product. The majority (75%) of the apo A-I produced in the absence of lipid was found in the lipoprotein-free (d> 1.25 g/ml) fraction. Similarly, C127 (Rhogani and Zannis, 1988) and L6E9 myogenic cells (Ruiz-Opazo and Zannis, 1988) transfected with the human apo A-I gene also secreted proapo A-I. Direct secretion of the mature protein was established in cells after deletion of the propeptide coding sequence from the gene (Rhogani and Zannis, 1988). This alteration had no apparent affect on the rate of secretion or on the fidelity of signal peptide processing. Mallory et a! (1987) described the most practical and efficient apo A-I expression in eucaryotic cells. They established CHO-Ki cell clones which expressed the human protein from a genomic DNA sequence under control of the human metallothionein-Il promoter. Many of the lines expressed as much as 1 mg of apo A-I per litre in serumfree culture. One clone produced as much as 10 mg/L. In addition, the apo A-I was fully processed to 30  the mature plasma isoform in all cases. They suggested that the apo A-I propeptidase was synthesized and secreted from CHO-K1 cells and that the proteolysis occured extracellularly. For large scale preparation of recombinant proteins, procaryotic expression systems, in general, produce larger quantities of protein with much less effort and expense. However, given that these cells do not possess the ability for post-translational processing of mammalian gene products, these expression systems are applicable only to those mammalian proteins which do not require these modifications. Apo A-I would appear to be a candidate for procaryotic expression, since it is not glycosylated and it does not contain cysteine residues, which can also limit expression levels in these systems. Attempts have been made to produce apo A-I as a recombinant in E. coli, with extremely limited success. Most authors have reported that the protein is rapidly degraded. A /9 -galactosidase fusion protein (Lorenzetti et at, 1986) has been described, but its half-life was only 45mins. The degradation appeared to affect specifically the apo A-I portion of the fusion product. No apo A-I could be detected without linkage to the bacterial product. Monaco et at (1987) described a Protein-A/apo A-I fusion protein which exhibited biological function. This product bound to mammalian cells and was selectively displaced by HDL. Large quantites could be isolated from cell lysates (40-50 mg/L) but the protein formed pentamer aggregates which proved difficult to disrupt. Isacchi et al (1989) have carefully investigated the role of the N-terminus of apo A-I in its degradation by E. coli. They found that by inserting the codons for the first 8 amino acids of mature apo A-I into the 5’ end of bacterial proteins, the quantity of protein expressed was markedly diminished. Alterations were then made in the nucleotide sequence which retained the amino acid sequence. Some of these changes increased the level of expression of the bacterial protein and also improved the yield of unfused apo A-I when incorporated into the original sequence. They also found that the propeptide segment of apo A-I was important for optimal expression in E. coli. In all of their studies, the level of apo A-I expression was related to the steady state level of the respective mRNA. Only one report has described the assessment of LCAT activation by apo A-I produced in bacteria. Bruhn and Stoffel (1991) expressed proapo A-I in E. coli by remoing the eucaryotic signal peptide and attaching bacterial codons for Met-Gly onto the N-terminus of proapo A-I. The quantity of apo A-I produced appeared to be substantial  ( 5mg/L). LCAT activation by the recombinant proapo A  I produced in this study was indistinguishable from the purified plasma protein. 31  Yeast expression of apo A-I has also been investigated by at least one laboratory (Segrest, personal communication 1989). This system has the advantages of procaryotic systems with respect to high level expression and low cost. In addition, since yeast are primitive eucaryotic cells, they have the capacity to modify mammalian proteins following translation. However, there appears to be some question as to whether the recognition systems in yeast are the same as in higher eucaryotic cells. Regardless of these theoretical considerations, no reports of yeast expression of apo A-I have yet appeared. It appears that yeast may be incapable of apo A-I secretion and, like E. coli, degrade apolipoproteins rapidly.  1.9. Scope of Thesis: Specific Aims It is clear that while a great deal is known of the structure, metabolism and role of apo A-I in lipoprotein metabolism, surprisingly little information is available regarding the molecular mechanisms involved in its functions. Our knowledge at present is largely confined to LCAT activation properties and controversial studies of receptor interaction. The evidence has been derived mainly from studies of the purified native protein and its fragments, or from the study of model peptides containing the amphipathic helix. Chemical modification of the native protein has provided only limited information due to the inability of this approach to affect a specific subset of amino acid residues and the potential conformational changes induced by the modifying agents. Phenotypically expressed inborn errors of metabolism have provided information on functional domains of many other proteins. However, this approach has been limited by the rarity of mutations of the apo A-I gene in the homozygous state. This has made purification of the mutant protein in sufficient quantity an arduous task. Thus, the purpose of this thesis was to utilize recombinant DNA methodology to introduce specific changes within the apo A-I cDNA and to express the mutant proteins in cell culture. The structure-function relationships of apo A-I could then be addressed by analysing their function in vitro. Specifically, I set out to: (a)  Develop expression systems for the production of human apo A-I in eucaryotic cell culture and to validate the expression of the wild-type protein.  (b)  Delete the propeptide of apo A-I and determine the functional consequences on cellular 32  processing and secretion. (c)  Produce the apo A-I Lys -.O mutant in vitro and determine its LCAT activating 107 properties.  (d)  Modify the apo A-I cDNA by deletion of residues encompassing a complete c -helix in the lipid binding domain and determine the effect of this deletion on LCAT activation.  33  2. MATERIALS AND METHODS 2.1. Materials  The cDNA encoding the apo A-I precursor (Seilhamer et al, 1984) was kindly provided by Dr. Beatriz Ley-Wilson of the Gladstone Foundation Laboratories, San Francisco, Ca. Restriction and modification enzymes for manipulation of DNA sequences were purchased from Bethesda Research Laboratories (BRL, Burlington, Ont.), Pharmacia-LKB Biotechnology (Baie dUrfe, Que.), or Boehringer Mannheim Corporation (BMC, Laval, Que.). Enzymes and reagents for DNA sequencing were from United States Biochemical (Cleveland, Ohio). Bacterial strains DH5a and DH5a -F’ were also purchased from BRL, while the dut ung strain CJ236 was supplied by Biorad Laboratories (Mississauga, Ont.). Geneclean, for purification of double stranded DNA fragments, is produced by BiolOl (La Jolla, Ca). a  -  Sj-methionine, y -r 5 P]-ATP and rH]-cholesterol were obtained from Dupont Canada 2 Sl-dATP, r 5 r (Mississauga, Ont.) or Amersham Canada Ltd (Oakville, Ont.). Oligonucleotides were prepared in the OligonUcleotide Synthesis Laboratory, Department of Biochemistry, UBC and purified by denaturing polyacrylarnide gel electrophoresis and reverse-phase chromatography (Sep-Pak C, Waters) as described (Atkinson and Smith, 1984). The expression vector pCMV5 (Thomsen, 1984; Andersson, 1988) was a gift from Dr. David Russell, Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas. The pNUT vector (Funk, 1990; Palmiter, 1987) and recipient BHK cells were provided by Dr. Ross MacGillivray, Department of Biochemistry, UBC. pSPT19 was purchased from Pharmacia. COS-1, an SV4O-transformed African Green monkey cell line (Gluzman, 1981; ATCC CRL-1650) and chinese hamster ovary cells (strain CHO-Ki, ATCC CCL61) were obtained from the American Type Culture Collection (Rockville, Md). Tissue culture reagents including fetal bovine serum (FBS) were supplied by Gibco-BRL (Mississauga, Ont.). Methotrexate (Cyanamid Canada, Inc., Montreal, Que,) for selection medium was obtained in sterile saline from the University Hospital Pharmacy. Reagents for in vitro transcription and translation (SP6 polymerase, rabbit reticulocyte lysate and canine pancreatic microsomes) were purchased from Promega Corp (Madison, Wi). All fluorescence and gold conjugates used for immunocytochemical studies were from Sigma Chemical Co (St. Louis, Mo). Monoclonal mouse anti-human apo A-I (6B8) was a gift of Drs. Ross Milne and Yves Marcel, 34  Clinical Research Institute, Montreal, Quebec. Polyclonal sheep antibodies to human apo A-I were obtained from BMC. Recombinant Protein G reagents were produced by Genex Corp (Gaithersburg, Md). Electrophoresis grade reagents for gel elctrophoresis were obtained from Biorad or from BRL. All other chemicals were of reagent grade or better and were purchased from Sigma or from BDH Inc (Vancouver, BC).  2.2. Growth and Transformation of E. Coli E. coli strains DH5ft and DH5x -F were maintained in LB (lOg/L tryptone, 5g/L yeast extract, lOg/L NaC1) and YT (8g/L tryptone, 5g/L yeast extract, 5g/L NaC1), respectively. Strain CJ236 was maintained on agar plates containing minimal medium (Maniatis, 1982) and grown in YT broth. The F-pillus was maintained by including chioramphenicol (3 g/ml) as recommended by the supplier. Colonies maintained on the appropriate agar were viable for 2-4 weeks. Frozen bacterial stocks were prepared in 20% glycerol from late log-phase broth cultures and were stored at -70 C. Competent E. coil were prepared from fresh lOOml cultures at mid-log phase (O.D. 550  =  0.4-  0.6) by the calcium chloride method (Maniatis et at, 1982). Bacteria were cooled on ice, pelleted by low speed centrifugation (5000 x g, 5mins) and gently resuspended in 25m1s of cold 10mM Tris-HC1 pH 8.0, 50mM CaCl 2 in an ice bath. After 30mins the cells were again pelleted by low speed centrifugation and resuspended in l0mls of ice-cold Tris/CaC1 . The competent cells were used immediately or were mixed 2 with glycerol (to 20%, v/v), flash frozen in liquid nitrogen and placed at -70 C in 0.3m1 aliquots. Transformation was accomplished by gently mixing competent cells (0.3mls) with 5-4Ong of plasmid DNA in an ice bath. After 30mins, the mixture was agitated briefly and heat shocked at 42 C for 3mins. Transformants were diluted to 1.Oml with LB and incubated at 3 C for 1 hour with gentle agitation. Mixtures were plated onto LB-agar plates containing 10( g/ml ampicillin and incubated inverted at 37 C for 16-24 hours. All expression plasmids used in these studies contained the  -lactamase  gene allowing growth of transformants in the presence of ampicillin. Recombinants in M13 were identified by blue-white color selection (Messing, 1983). Transformed DH5 -F were mixed with fresh DH5 -F’ overnight culture (2 1) and diluted to 2.5mls 35  with YT top agar (6g/L agar, 8g/L tryptone, 5g/L yeast extract, 5g/L NaC1) containing 3g/ml X-GAL (5-bromo-4,4-chloro-3-indoyl-3 -D-galactoside) and 0.7mM IPTG (isopropyl.9 -D-thiogalactopyranoside). This mixture was prepared at 45) C and was plated immediately onto YT agar. Once solidified, the plates were inverted at 3? C for 12-16 hours at which time plaques were visible. Recombinant phage give rise to clear plaques on the bacterial lawn whereas those without DNA insertions are blue.  2.3. Purification of DNA 2.3.1. Small Scale Plasmid Preparation Two ml aliquots of LB broth (containing antibiotic) were innoculated with a single bacterial colony and were incubated at 3? C for 12-16 hours with vigorous agitation (250-280 rpm). Bacteria were pelleted by microcentrifugation (12,000 x g, 1mm) and resuspended in 10 1 of 25mM Tris-HCI pH 8.0, 50mM glucose, 10mM EDTA. After 5mm at room temperature the cells were lysed with 20 1 of 0.2N NaOH, 1% SDS and placed on ice. After Smins the suspension was neutralized with 150i 1 of SM KAcetate pH 4.8 on ice. Cellular debris was removed by microcentrifugation (5mm) and the supernatant 40 1) was extracted with an equal volume of phenol:chloroform (1:1, v:v). After brief centrifugation to separate phases, the upper aqueous layer was recovered and plasmid precipitated as the potassium salt with 2 volumes of cold ethanol. The pellet after microcentrifugation was washed with 70% ethanol to remove coprecipitating salts. Finally, the pellet was dried in vacuo and dissolved in 5 1 of TE (10mM Tris-HCI pH 8.0, 0.1 mM EDTA) containing 2 g/ml DNase-free pancreatic ribonuclease.  2.3.2. Large Scale Plasmid Preparation Plasmid preparations of suitable quality for eucaryotic cell transfection were purified according to a protocol published by Promega Biotec (Technical Bulletin 009). An aliquot of fresh overnight culture (from a small scale plasmid preparation) was used to innoculate 10-l5mls of selective broth. Once an O.D. 550 of approximately 0.6 was attained (4-6 hours incubation at 3? C), the entire culture was added to 2SOmls of warm selective broth in a 1L flask. This mixture was incubated overnight with vigorous agitation. Cells were recovered by cold centrifugation (2800 x g, l5mins) and were resuspended in 6mls of 25mM Tris-HCI pH 8.0, 10mM EDTA, 15% sucrose, 2mg/ml lysozyme on ice. 36  After 20mins, the cells were lysed with l2mls of 0.2N NaOH, 1% SDS and mixed by gentle inversion. The lysate was neutralized with 7.5mls cold 5M KAcetate and placed on ice for a further 20mins. The viscous suspension was iransfered to silanized glass tubes (Corex) and cellular debris was removed by centrifugation (25,000 x g, l5mins). The clear supernatant was recovered arid RNA in was digested with 5Qu g ribonuclease for 20mins at 37 C. The DNA solution was extracted twice with equal volumes of phenol:chloroform and total nucleic acid was precipitated from the upper phase with 2 volumes of 95% ethanol at -70 C. The pellet was recovered by centrifugation and dissolved in 1.6 mls water. 0.4mls of 4M NaC1 and 2mls of 13% polyethyleneglycol (PEG-8000) were added and plasmid precipitated on ice for at least one hour. The pellet was recovered by centrifugation (12,000 x g, lOmins) and washed with 70% ethanol. The final product was dissolved in 20C I of TE and quantitated by ultraviolet absorbance (Maniatis, 1982).  2.3.3. Preparation of M13 Bacteriophage DNA Preparation of Uracil-Conlaining ssDNA from M13 Template ssDNA for mutagenesis (Kunkel, 1985) was prepared by infection of E. coli strain CJ236 (dut lung-; Mutagene, Biorad Laboratories) with M13 phage containing the apo A-I cDNA. Briefly, J236 were grown in 2 x YT to O.D. 550  =  0.3 and infected with 2 x 1 p.f.u. of M13 phage (to  give an M.O.I. = 0.2). After 6 hours growth, the cells were removed by centrifugation (5000 x g, l5mins) and the clear supernatant transfered to a fresh tube. The uracil-enriched bacteriophage were titred on both CJ236 and DH5 -F, and routinely showed iO -1 higher titre on J236. This indicated that uracil was incorporated into the phage with high efficiency. Phage particles were precipitated on ice for at least one hour following addition of 0.25 volume of 15% PEG in 2.5M NaC1. The precipitate was recovered by centrifugation and resolubilized in TE for 1 hour on ice. Insoluble debris was removed by centrifugation and the clear supernatant was extracted twice with phenol and once with phenol:chloroform. DNA was precipitated on ice from the upper phase by addition of 0.1 volume of 3M NaAcetate pH 5.2 and 2 volumes ethanol. After one hour, the DNA was pelleted by centrifugation, washed with 70% ethanol and dried in air. The final residue was dissolved in 20Ci I of TE and quantitated by UV absorbance.  37 Preparation of M13 Phage DNA for Sequencing YT broth cultures were innoculated with fresh DH5 -F overnight culture and a single M13 plaque. After incubation for 6 hours at 37) C, bacteria were removed by microcentrifugation and retained for purification of RF DNA (see Small Scale Plasmid Preparation). Phage were precipitated from 1.3mIs of culture supernate by addition of 0.3mls of 20% PEG, 2.5M NaCI and incubation at room temperature. After 15mm, phage were recovered by microcentrifugation and resuspended in 20.i I of 20mM Tris-HC1 pH 7.5, 20mM NaCI, 1mM EDTA. The mixture was extracted with phenol:chloroform, and ssDNA was precipitated overnight at -7 C from 0.3M NaAcetate solution with two volumes of ethanol. The pellet was recovered by centrifugation and ethanol precipitation was repeated. The final DNA pellet was washed with 70% ethanol, dried in vacuo and dissolved in 2C 1 of TE.  2.4. Oligonucleotide-directed Mutagenesis 2.4.1. In vthv Mutagenesis Directed mutagenesis was performed using a single oligonucleotide primer by the method of Kunkel (1987) as described by the supplier (Mutagene, Biorad Laboratories). Briefly, 200pmoles of mutagenic oligonucleotide were 5’-phosphorylated with T4 polynucleotide kinase (PNK) for one hour at 37) C. Residual PNK was inactivated at 68 C (lOmins) and l0pmoles of the phosphorylated primer was mixed with ljt g of uracil-containing template ssDNA in 20mM Tris-HCI pH 7.4, 2mM MgC1 , 50mM 2 NaC1. The mixture was heated to the denaturation temperature (see Table IV) and primer and template arinealled by slow cooling to 30 C or less. Table IV. Apo A-I Mutagenic Oligonucleotide Primers and Their Properties. DENAT = denaturation temperature used prior to annealling M13 phage DNA with mutagenic primer. HYB temperature used during filter hybridization to screen putative mutant phage preparations. DELETION  PRIMER SEQUENCE  DENAT  HYB  dPRO  5-ACGGGGAGCCAGGCTGATGAACCCCCCCAG-3  85> C  73> C  107 dK  5-TI’CCAGAAGTGGCAGGAG-3  7(PC  N/D  5’-TCCGCCAAGGCCTGCFGACCCAGTGAGGCGCCC-3’  100> C  75> C  ) 241 ° -Asn 22 D1(dPro  Mutagenic primers were extended on the uracil template with T4 DNA polymerase in the presence of T4 DNA ligase in 23mM Tris-HCI pH 7.4, 5mM MgC1 , 1.5mM DTT, 0.75mM ATP and 2  38  400i M dNTPs. Reactions were assembled on ice and sequentially incubated for 5mins on ice, 5mins at room temperature and finally for 2 hours at 37° C. Aliquots of the final reaction were used to transform competent DH5x -F, plated in YT top agar and placed at 37°C to allow plaques to form overnight. Control reactions were performed simultaneously including all reagents with the exception of the mutagenic primer (unprimed). Experiments were evaluated according to plaque number in the primed and unprimed reactions. If the plaque ratio (primed: unprimed) was >5, phage were screened for the presence of the mutation.  2.4.2. Identification of Putative Mutants Plaques from mutagenesis experiments were recovered as agar plugs with a sterile pipet and dispersed in lml of TE. Fifty i 1 of the resulting suspension (including control wild-type phage) was used to infect fresh DH5z -F’ overnight culture (2 1) and diluted to 2mls with YT. After 6 hour culture at 370  C, phage and RF DNA were isolated as described above. The dPRO and Dl mutants were identified by selective hybridization of the mutagenic  oligonucleotide to the bacteriophage DNA. Culture supernates containing the phage were applied to nylon membranes (Nytran) using a vaccuum manifold apparatus. The membrane was removed with forceps and placed in a plastic hybridization bag containing prehybridization solution: 6x SSC (salinesodium citrate, lx SSC = 0.15M sodium chloride, 20mM sodium citrate, pH 7.0), 5x Denhardt’s reagent (lx Denhardt’s= 0.2 gIL Ficoll, 0.2 gIL polyvinylpyrrolidine, 0.2 gIL bovine serum albumin) and 0.1% sodium dodecylsulfate (SDS). After 2 hours at  550  C, this solution was replaced with hybridization  solution (6x SSC, 5x Denhardt’s). Oligonucleotide probes were prepared by incorporation of  3 P1 t  phosphate onto the 5’ end of the mutagenic oligonucleotide, using the T4 polynucleotide kinase reaction in the presence of  [32  PIATP (Sambrook et al, 1989). The labelled probe was subsequently added to the  hybridization bag and incubated for 6-16 hours at 55°C in a stationary water bath. Unbound oligonucleotide was removed by extensive washing in 6x SSC (2x lOmins each at room temperature and lx lOmins at 55° C) and an autoradiogram was exposed for 30-120 mins between  enhancing screens.  The  filter was then washed in 6x SSC at increasing temperature (3-5°C increments) and an autoradiogram was developed following each wash. The selective temperature was determined as that at which wild-type 39  phage no longer retained the labelled probe but where a strong signal was observed in slots of some of the putative mutant phage. Putative mutants for the Lys 107 deletion were identified by restriction endonuclease analysis of the M13 RF DNA. The loss of the dK 107 codon removed a single MboII site and altered the restriction fragment pattern generated by this enzyme. Putative mutants identified by either analysis were confirmed by DNA sequencing.  2.4.3. DNA Sequence Aiialysis The sequence of M13 inserts were determined by enzymatic chain termination sequencing (Sanger, 1977) of the single stranded phage. Primers for sequencing (Table V) were selected to generate chain terminated fragments which could be readily separated on 6-8% polyacrylamide gels. Primers were spaced approximately 200 nucleotides apart so that useful sequence was obtained with short electrophoresis times. The enzyme used for sequencing was modified T7 polymerase (Sequenase, United States Biochemical). Reactions were performed according to the manufacturers recommendations. Briefly, 1 g of M13 phage DNA was mixed with 0.Spmol of sequencing primer in 40mM Tris-HCI pH 7.5, 20mM MgC1 , 50mM NaC1. The mixture was heated to 63 C for 2mins in a 200ml water bath and 2 allowed to cool slowly to 2S C. The annealed primer was then simultaneously extended and labelled with Sequenase in the presence of 1.M dGTP, dTTP, dCTP and Ci of  S]-dATP (Smins at room 5 -r  temperature). Chain termination was then achieved by removing 3. I of the labelling reaction mixture into 2. 1 of a prewarmed solution containing 8( 1 dNTPs and ddGTP  (“  i  M of one of the dideoxynucleotides:  G” reaction), ddATP (“A” reaction), ddTTP (“T” reaction) or ddCTP (“C” reaction).  Termination reactions were performed at 37°C for Smins and stopped by addition of Stop Solution (95% formamide, 20mM EDTA, 0.05% Bromophenol Blue, 0.05% xylene cyanol FF). To obtain sequence information close to the primer, the dNTP concentration in the labelling reaction was reduced to 300nM. In some reactions, where GC base pairing was abundant, dITP was utilized in the reaction mixture according to the manufacturers recommendations. Labelled products (2- 1) were separated on 6% or 8% polyacrylamide gels containing 1 x TBE (89mM Tris, 89mM boric acid, 2mM EDTA) and 8M urea at 32W constant power. At the 40  completion of the separation the gel was dried onto Whatman 3MM chromatography paper and exposed to autoradiographic film (Kodak XRP) for 16-96 hours.  Table V. Apo A-I cDNA Sequencing Primers PRIMER  SEQUENCE  UFP  5-GTAAAACGACGGCCAGT-3’  URP  5-CAGGAAACAGCTATGAC-3  Si  5-ATCGAGTGAAGGACCTGGCC-3’  204-223  S2  5-CCCAGGAGTTCfGGGATAAC-3’  383-402  S3  5-CACTGGGCGAGGAGATGCGC-3  606-625  S4  5-CCGCGCTCGAGGACCFCCGC-3  804-823  Al cDNA  2.5. Construction of Expression Plasmids 2.5.1. Isolation of cDNA Fragments Fragments of the original apo A-I cDNA were obtained by restriction enzyme digestion of the double stranded plasmid. Digestions were performed under the ionic conditions recommended by the enzyme supplier. The apo A-I cDNA and mutant cDNAs generated in M13 were cut from the RF DNA with EcoRI. The resulting apo A-I cDNA fragment DNA  ( 1 Kbp) was separated from the larger M13 vector  ( 7 Kbp) by agarose gel electrophoresis in 1 x TAE and gel containing the DNA was excised with  a scalpel. The agarose was dissolved at 50 C in the presence of 65-75% saturated sodium iodide and the DNA was recovered by binding to Glassmilk (Geneclean, BiolOl) on ice. The suspension was pelleted by brief microcentrifugation and washed three times with 50-fold volume excess of buffered ethanol solution (NEWwash BiolOl). After the last wash, all traces of the wash solution were removed and the DNA eluted into 5-1.i 1 of TE.  2.5.2. Modification of Fragment Ends For blunt-end ligations the purified cDNA fragments were treated with the Klenow fragment of DNA polymerase I to fill the 5’ protruding termini. Two units of Kienow polymerase were mixed with lg of DNA in 50mM Tris-FIC1 pH 7.2, 10mM MgSO , 1QuM DTT, 5Qug/ml BSA, 125tM 4 41  dNTPs. The reaction was placed at 3? C for 30 mins, after which the DNA was purified on Geneclean. To reduce background recircularization of vectors during ligation reactions, the digested vector was treated with calf intestinal phosphatase (CIP, Boehringer Mannheim). Linearized vector (12,u g) was brought to 5 1 in 50mM Tris-HC1 pH 9.0, 1mM MgC1 , 10i M ZnSO 2 , 1mM spermidine for 4 this reaction. For fragments with protruding 5’ termini, two successive incubations with CIP (0.01 unit) were performed (30mins each at 3? C). Fragments with blunt or recessed 5’ termini were incubated at 3? C for l5mins and at 56 C for l5mins following each CIP addition. Reactions were terminated at 68 C for l5mins following addition of EDTA (to 1mM), NaC1 (to 100mM) and SDS (to 0.5%). The mixture was extracted twice with phenol:chloroform and once with chloroform prior to fragment isolation on Geneclean.  2.5.3. Ligation into Expression Plasmids Vector and insert concentrations were estimated by their ethidium bromide staining intensity on agarose electrophoresis gels and used to determine ligation requirements. For vector-insert pairs with overlapping compatible ends  (“  sticky’), the insert and vector were mixed at a molar ratio of 2:1. For  blunt end ligations the ratio was increased to 4:1. In general, 25-bOng of vector DNA was used per ligation reaction. The final reaction volume was adjusted to 5p 1 in 50mM Tris-HC1 pH 7.6, 10mM , 1mM ATP, 1mM DTT, 5% PEG. T4 DNA ligase was added to 0.1 unit per p1 for sticky ends or 2 MgC1 to 1.0 unit/p 1 for blunt ends. All ligations were performed in a water bath at 12-15 C for 12-16 hours and 5 l- 1 of the mixture was used to transform competent E. coil. p In vitro transcripts were synthesized using pSPT19 (Pharmacia-LKB Bioteclmology). This vector contains promoters for the SP6 and T7 phage RNA polymerases in opposing orientations flanking the polylinker region. Linearization of the plasmid outside of the polylinker region generates a template DNA suitable for “run-off” transcription from either promoter using purified polymerase (Krieg and Melton, 1984). The resulting transcript is suitable for translation by rabbit reticulocyte lysate (Pelham and Jackson, 1976), providing analytical quantities of protein from the cDNA. The apo A-I cDNA was most efficiently expressed in this system using the SP6 polymerase and cDNA insertion at the pSPT19 Smal site. The appropriate orientation was established by restriction enzyme analysis. 42  Transient expression of apo A-I was studied with the eucaryotic expression vector pCMV5 (Thomsen, 1984; Andersson, 1988). pCMV5 contains the promoter-regulatory region of the human cytomegalovirus (CMV) major intermediate early gene and the 3’ untranslated region of human growth hormone (hGH) which provides transcript termination and polyadenylation signals. This plasmid attains high copy number in SV4O transformed cells since the vector contains the SV4O origin of replication. Native and mutant apo A-I cDNA were ligated at the unique EcoRI restriction site, which placed the cDNA under the transcriptional control of the CMV promoter. Transformed DH were selected by ampicillin resistance and the appropriate orientation for the CMV promoter established by restriction analysis. Stable cell lines expressing human apo A-I and its mutants were developed using the eucaryotic expression vector pNUT (Palmiter, 1987). This vector contains a mutant form of the gene for dihydrofolate reductase (DHFR) which allows for immediate selection of cells containing stably integrated plasmid DNA by their survival in high concentrations of methotrexate (Funk, 1990). Insertion at the Smal site of the vector placed the apo A-I cDNA under transcriptional control of the mouse metallothionein (mMT-I) promoter. Transformants in DH5 were identified on ampicillin plates and the appropriate orientation was established by restriction endonuclease analysis.  2.6. In vitro Transcription Plasmid was linearized for “run-ofP’ transcription outside of the promoter-insert region by digestion with HindIII and ethanol precipitation. All water used for transcription was deionized and diethylpyrocarbonate (DEPC) treated prior to use. Transcription mixtures were assembled at room temperature and containing 2,u g of linearized pSPT19-AI plasmid. Each 100t 1 reaction contained 40mM Tris-HC1 pH 7.5, 6mM MgC1 , 2mM spermidine, 10mM NaC1, 10mM DTT, 100 units RNAsin (placental 2 ribonuclease inhibitor, Bethesda Research Laboratories) and 5OQu M each ribonucleoside triphosphate (rATP, rUTP, rCTP, rGTP). Transcription was initiated by addition of 20 units of purified SP6 RNA polymerase (Promega Corp) and incubation for 2 hours at 373 C. In some experiments transcripts were capped during synthesis by including the cap analogue 7 mG(5’)ppp(5)G (50 M, Pharmacia LKB Biotechnology) and reducing the rGTP concentration to 50i M. Under these conditions 7 mG was 43  preferentially incorporated at the 5 end of the in vitro transcript and the translation efficiency was improved (Alexander, 1987). Once the incubation was complete, total nucleic acid was isolated by phenol:chloroform extraction and ethanol precipitation from 0.3M sodium acetate solution. mRNA was stored as the precipitate in 70% ethanol at -7O C. In some experiments template DNA was removed prior to phenol:chloroform extraction by digestion with DNase I (2 units, Pharmacia-LKB) for l5mins, permitting the quantitation of RNA by UV absorbance. This was not necessary, however, for efficient translation of the transcript.  2.7. In vitro Translalion Rabbit reticulocyte lysate, prepared from phenyihydrazine treated New Zealand White rabbits (Promega Corp), was used to translate “run-ofP’ transcripts derived from the apo A-I cDNA. Immediately prior to the translation reactions, the RNA was recovered by centrifugation, dried in vacuo and solubilized in DEPC-treated water. Translation was initiated by addition of containing 2Qu M mixed  amino  0.5u g of in vitro transcript to 5( 1 lysate mixture  1 acids (without methionine) and 51’ Ci n S]methionine (Amersham Canada  Ltd, Translation grade, SJ.204, 1.5mCi/1’ mol). Incubations were of 30mins duration at 3O C and were terminated by freezing. Some incubations included canine pancreatic microsomes (Promega Corp), prepared by the method of Walter and Blobel (1983), at 0.5-1.0  g of microsomal protein per assay. This  preparation contains the eucaryotic enzyme systems of the endoplasmic reticulum and provides the in vitro capabilities for signal peptide hydrolysis and membrane translocation.  2.8. Eucaryotic Cell Culture COS-1 and BHK cells were maintained in T25 flasks in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% fetal bovine serum (FBS) (Gibco-BRL). CHO-Ki cells were cultured in an equivolume mixture of DMEM and Ham’s F12 nutrient mixture (Gibco-BRL) containing 10% FBS. All FBS was mycoplasma and virus free as supplied and was heat inactivated at  D 56  C for 3Omins  before use as growth supplement. Cells were passaged as they reached confluence (every 3-4 days) by trypsinization and 6-10 fold dilution in growth medium. All eucaryotic cells were maintained in a 44  humidified incubator with 5% carbon dioxide atmosphere. Media of stably transfected cells contained selection agents as indicated.  2.9. Transient Transfection of COS-1 Cells Transient transfections were performed using the DEAE-dextran technique according to published protocols (Kriegler, 1990). Briefly, COS-1 cells were seeded into 35mm dishes following trypsinization and 20-fold dilution in DMEM/5% FBS. Upon attaining approximately 80% confluence (24-36 hours) the medium was removed and the cells were washed twice with Transfection Buffer (25 mM Tris-HC1, pH 7.4, 140 mM NaCI, 1 mM CaC1 , 3 mM KC1, 0.5 mM MgC1 2 , 0.9 mM Na 2 2 HPO ). 4 Plasmid DNA was diluted to 5 i g/ml in Transfection Buffer containing 500 j g/ml DEAE-dextran (M  =  500,000; Pharmacia-LKB Biotechnology). 0.5m1 of this mixture was added to each monolayer and  incubated for 30 minutes at 37) C. The DNA solution was then removed, replaced with DMEM/5% FBS containing 80 JA M chioroquine, and incubated at 37) C for 3 hours. Chioroquine medium was removed and the cells were treated with DMEM/10% dimethylsulfoxide (DMSO) for 3 mins at room temperature. DMSO was removed with two washes of warm Transfection Buffer and growth medium was replaced. The cells were allowed to recover and initiate exogenous gene expression for 40-48 hours prior to assessment of apo A-I expression. For endogenous labelling experiments, transfections were initiated in multiple 10 cm dishes and cells harvested 18-24 hours after the DMSO shock. These cells were then pooled, plated into 35mm experimental dishes and allowed to adhere to the substratum for 20-24 hours.  2.10. Isolation of Stable Eucaryotic Cells Expressing Apo A-I 2.10.1. Calcium Phosphate Transfection Plasmid DNA was introduced into recipient cells by the calcium phosphate coprecipitation method (Kriegler, 1990). Parent cells (in 10cm dishes) were grown to 90% confluence in the appropriate growth medium prior to transfection. For transfection of BHK cells only pNUT-AI plasmids were used. However, since CHO-K1 cells were found to be relatively resistant to methotrexate, co-transfections were performed with Al-containing plasmid and pRc/CMV (Invitrogen, La Jolla, Ca) at a weight ratio 45  of 10:1. pRc/CMV provided the fl-lactamase gene for neomycin resistance. Plasmid DNA was diluted to lug/mi in HBS (5g/l HEPES, pH 7.05, 8g/l NaC1, 0.37g/l KC1, 0.lg/l Na 2 HPO , lg/l glucose). 4 Calcium phosphate was added to a concentration of 125mM to initiate coprecipitation of plasmid. One ml of this mixture was added to lOmls of fresh growth medium and incubated overnight on the cell monolayer. Transfection medium was subsequently removed and replaced with growth medium for a 24 hour recovery period prior to initiating the selection procedure.  2.10.2. Selection of Stably Transfected Cells BHK Cells BHK clones with integrated plasmid DNA sequences were selected during 10-14 days culture with DMEM/5% FBS containing 500 j M methotrexate (Funk WD, 1990). Selection medium was changed daily for the first 4 days and every 3-4 days, thereafter. Macroscopic colonies were visible at that time and were then transferred with a 1 ml glass pipet to individual 20mm culture wells (Linbro, Flow Laboratories, Mississauga, Ont.). The colony was dispersed in 1 ml selecting medium to encourage monolayer growth. Once the monolayer was established and confluent, selection medium was removed and replaced with serum-free maintenance medium (Optimem, Gibco-BRL). This was collected after 2448 hours and clones secreting apo Al were identified (see below). Clones secreting maximal quantities of Al were expanded to larger cultures in the presence of methotrexate. Pure cell populations secreting apo A-I were identified by immunofluorescence microscopy (see below) and were stored as frozen stocks in 20% FBS, 10% DMSO in liquid nitrogen. One cell line derived from each of the transfection experiments was selected for further study. Studies of apo A-I processing and secretion were performed after at least 48 hours culture in the absence of methotrexate. CHO-Ki Cells Following calcium phosphate transfection, the DNA solution was removed and replaced with growth medium for a 24 hour recovery period. The neomycin analogue Geneticin (G418, Gibco-BRL) was used for selection at 5OQu g/ml. This level was shown to reduce the survival of the parent CHO-Ki cells to virtually zero after 14 days. Selection medium was replaced every 3-4 days and macroscopic 46  colonies were visible in transfected dishes after approximately 10 days. Colonies were harvested into multiwell dishes and expanded under selection conditions as described above for BHK clones. However, since the level of apo A-I accumulation per cell was very low, the homogeneity of the cell populations could not be assessed by immunofluorescence microscopy.  2.10.3. Screening of Clones for apo A-I Secretion The serum-free media from confluent monolayers were harvested and non-adherant cells and debris were removed by centrifugation (12,000 x g, 10mm). Aliquots of media were applied to nitrocellulose membranes using a vaccuum manifold. Purified plasma apo A-I (McLeod et at, 1986) and medium from cells transfected with empty pNUT vector were also applied as positive and negative controls, respectively. Umeacted binding sites on the nitrocellulose were blocked with 5% non-fat milk powder in PBS (10 mM Tris-HC1, pH7.4, 150 mM NaCl). Apo A-I was identified by incubation with polyclonal antibody to human apo A-I followed detection with protein G conjugated to horse-radish peroxidase (HRP). Membranes were washed extensively (4 x Smins) with 0.02% Tween-20 in PBS following each incubation. The enzymatic color reaction used to visualize the immune complex contained  0.05% 3,3-diaminobenzidine (DAB), 0.03% CoC1 2 and 0.006% hydrogen peroxide in PBS. The reaction was stopped by extensive washing with tap water and relative quantities of apo A-I were determined by scanning densitometry of the dried membrane.  2.10.4. Amino-Terminal Amino Acid Sequence Analysis BHK cells expressing apo A-I were grown to 70% confluence in 20cm culture dishes in DMEM/10% FBS and subsequently depleted of serum by maintenance in Optimem (Gibco-BRL Mississauga, Ont.) without FBS. After 24 hours the medium was removed to waste and replaced with fresh Optimem containing 50 M ZnSO . This medium, which contained the secreted recombinant apo 4 A-I, was collected after 24 hours and brought to 100 I.U./ml aprotinin, 0.1mM leupeptin and 1mM phenylmethylsulfonyl fluoride (PMSF). Phosphatidyicholine-cholesterol vesicles (4:1 molar ratio)(Batzri and Korn, 1973) were added and the mixture was incubated overnight at 373 C. During this period apo A I bound to the vesicles which were recovered by ultracentrifugation at d = 1.25 g/ml (40,000 rpm, 48 47  hours). The top 2mls were recovered from the ultracentrifuge tube by tube slicing, and proteins were precipitated from 15% (wlv) trichloroacetic acid. After centrifugation the protein pellet was delipidated with ethanol:ether (3:1) and then dissolved in SDS-PAGE Sample Buffer at 9fF C and resolved by SDS PAGE electrophoresis (see below). Proteins were blotted to Immobilon-P membranes (Millipore) and visualized by staining with Coomassie Blue. The apo A-I band was cut from the membrane and subjected to automated sequence analysis according to established techniques (Abersold et al, 1986; Matsudaira, 1987).  2.11. Metabolic Labelling Studies 2.11.1. Determination of Synthesis Rate Protein and apo A-I synthetic rates were measured by short-term incubation in the presence of  I S]methionine. Monolayer cultures (35mm Falcon dishes) were methionine depleted (DMEM minus  methionine, Gibco-BRL) for 20 mins at 37 C and subsequently labelled for 0-30 mills in the same medium containing 6( Ci/mi  [ Sjmethionine (700 Ci/mmol, New England Nuclear). At the time  indicated, cells were washed free of labelling medium and the monolayer was harvested in Cell Lysis Buffer (50mM Tris-HC1 pH8.0, 62.5mM EDTA, 1% Nonidet P-40, 0.4% sodium deoxycholate, 1mM PMSF). 2.11.2. Long Term Labelling Studies Cellular retention and secretion of apo A-I were measured by long term continuous incorporation of  [ Simethionine. Near confluent monolayer cultures (35mm dishes, Falcon) were  equilibrated to lOQu M methionine by incubation for 20 mm in Optimem containing 10% FBS, lOQu M zinc sulfate and 100 I.U./ml aprotinin. Equilibration medium was removed and  [ S]Methionine was  added to a radiochemical concentration of 25-3Qu Ci/ml. After incubation for the indicated time, medium was recovered and cells harvested in Cell Lysis Buffer. Apo A-I secretion rates were determined by immunoprecipitation from medium of these long term continuously labelled cultures. Immediately after harvest, media and cells were cleared of intact cells or insoluble debris by 5 mm centrifugation at 12,000 x g.  48  2.11.3. Determination of Apo A-I Degradation and Secretion Rates of intracellular degradation were measured utilizing an  S]methionine pulse-chase  protocol. Monolayers in 35mm dishes were washed free of growth medium and depleted of methionine by incubation in methionine-free DMEM (DMEM-Met, 20mm, 37’ C). The endogenous methionine pool was then labelled with DMEM-Met containing 100-200 pCi/mi r Sjmethionine for 30 mins at 37J C. 5 After removal of the labelling medium, the cells were rinsed with DMEM-Met, and the chase incubation initiated in Optimem supplemented with 2mM methionine, 10 M zinc sulfate and 10% FBS. At the indicated time, medium and cells were recovered as described above and cleared of intact cells by centrifugation for 5mm at 12,000 x g.  2.12. Determination of r S]Methionine Incorporated into Protein and Apo A-I 5 Incorporation of methionine into cellular protein was determined by precipitation of radiolabelled cell lysate with 10% trichloroacetic acid (TCA) overnight on ice. The resulting pellet was recovered by centrifugation (12,000 x g, 10 mm) and washed twice with ice-cold 10% TCA and once with acetone. Pellets were then dried in air, dissolved in 2 x SDS sample buffer (0.1M Tris-HC1, pH 6.8, 2% SDS, 40% glycerol) and analysed by liquid scintillation spectrometry. Quantitations were performed on triplicate dishes. Incorporation of methionine into apo A-I was determined following isolation from cell lysate by immunoprecipitation and SDS-PAGE as indicated below. l-2p g of purified apo A-I was added to the Sjmethionine labelled immunoisolate for SDS-PAGE fractionation. Following electrophoresis the apo A-I band was located by Coomassie staining or by overlay of the autoradiogram on the corresponding dried gel. The gel fragment containing apo A-I was excised and dissolved by heating to 6ff C in 0.25mls of 30% hydrogen peroxide. Radioactivity was the quantitated by liquid scintillation spectrometry in Smls ACS (Amersham Canada Ltd). Each quantitation was performed on triplicate 35mm dishes.  2.13. Isolation of Apo A-I by Immunoabsorption Apo A-I in media or cell lysates was concentrated and partially purified by solid phase immunoabsorption. Monospecific polyclonal antibodies to human apo A-I were pre-absorbed onto 49  agarose-immobilized protein G (GammaBind G Agarose, Genex Corp) at  40  C. After 30 minutes, a  sample of medium or cell lysate was added, and the suspension rotated end over end for 2 hours at room temperature or overnight at  43  C. Agarose bound immune complexes were pelleted by  centrifugation (12,000 x g, 10 mins) and unabsorbed material removed by two washes with imi of TBS (10mM Tris-HCI, pH 7.4, 150mM NaC1). Antigen-antibody complexes were released by heating the beads in 2 x SDS sample buffer at 90 C for 10 mins. Sepharose beads were removed by centrifugation (2 mm, 12,000 x g) and the supernatant recovered for further analysis.  2.14. Electrophoretic Analyses 2.14.1. DNA Fragment Separation on Agarose Gels DNA fragments were separated in submersed horizontal agarose gels containing 1 x TAE (40mM Tris, 20mM NaAcetate, 1mM EDTA, pH 7.2) and l g/ml ethidium bromide. The agarose content varied between 0.8 and 2.0% (w/v) depending on the expected size of the fragment of interest. Samples were adjusted to 8% sucrose, 20mM EDTA, 0.05% bromophenol blue and 0.05% xylene cyanol to facilitate application to sample wells. The latter two components served as reference markers to monitor the progress of the separation. Electrophoresis was performed under constant voltage conditions (80-100 volts).  2.14.2. Protein Analysis SDS-Polyacrylamide Gels Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in discontinuous Tris-glycine buffer as described (Laemmli, 1970). Gels were cast in 0.75mm slabs (Biorad Mini Protean II) containing 12% acrylamide resolving gel and 4% acrylamide stacking gel. Samples were adjusted to 50mM Tris-HC1 pH 6.8, 1% SDS, 20% glycerol, 0.005% bromophenol blue and 5% /3  -  mercaptoethanol and were boiled for Smins prior to application to sample wells. Separation was accomplished at constant voltage (100V) until the tracking dye had migrated to the bottom of the resolving gel. Gels were stained (where indicated) with 0.25% Coomassie Blue R250 in 45% methanol, 10% acetic acid and destained in the same solvent. 50  For autoradiography, gels were fixed in destaining solution, equilibrated with Amplify (Amersham Canada Ltd.) and dried onto Whatman 3MM chromatography paper. Autoradiograms were exposed on X-Omat AR film (Eastman-Kodak) for 16-96 hours.  [14  Cjmethylated proteins (Amersham  Canada Ltd., Oakville, Ont.) were used as molecular mass markers for interpretation of autoradiograms. Isoelectric Focusing Gel Electrophoresis Isoelectric focussing (IEF) was performed in 5% polyacrylamide slab gels containing 8M urea and 2.5% (w/v) Ampholines (Pharmacia-LKB, pH 4.0-5.5) as described by Hill and Pritchard (1991). For autoradiography, the gels were fixed for at least one hour and equilibrated with Amplify as described above for SDS-PAGE slabs. For immunoblot analysis, proteins were electrophoretically transferred to nitrocellulose using 0.7% acetic acid as transfer buffer (see below). Two-dimensional Polyacrylaniide Gel Electrophoresis Two-dimensional gel electrophoresis was performed as described by O’Farrell (1975). The first dimension was carried out in 1mm capillaries containing 8 M urea and 2.5% (w/v) Ampholines (pH 4-5.5) for 4-6 hours at 250V. The gel was then extruded from the capillary and equilibrated with SDS sample buffer for 5-15 mins. Excess buffer was removed and the gel was placed on top of the second dimension 12% SDS-PAGE slab gel described above. Separation in the second dimension was achieved at 100V until the tracking dye had reached the bottom of the gel. The gel was then processed for autoradiography. Immunoblot Analysis Proteins separated by polyacrylamide gel electrophoresis were transferred to nitrocellulose membranes from slab gels by electroblotting (Towbin, 1979). Transfer was achieved at 100V in 1 hour in a cooled buffer chamber. Transfer buffer for SDS-PAGE was 20% methanol in 25mM Tris, 192mM glycine, pH 8.3. Transfer buffer for IEF gels was 0.7% acetic acid and utilized reverse polarity during the transfer process. Unoccupied nitrocellulose sites were blocked with 5% non-fat powdered milk in PBS (30mins). Apo A-I was identified on the membrane by incubation with polyclonal antibodies (1:500 51  dilution in 0.5% milk powder/PBS) for 1 hour. The filters were then washed extensively with 0.02% Tween-20 in PBS and incubated with protein G HRP-conjugate for 30mins. After washing, the location of the immune complex was determined with 0.5g/L DAB, 0.3g/L CoCI , 0.006% (v/v) hydrogen 2 peroxide in PBS. The color reaction was terminated in running tap water. All incubations were performed at ambient temperature.  2.15. Indirect Immunofluorescence Microscopy Cell monolayers were harvested by trypsinization and transferred to poly-L-lysine coated cover slips in appropriate growth medium. After 24 hours nonadherent cells were removed and the medium was replenished. Clonal cells expressing apo A-I under control of the mMT-I promoter were stimulated by addition of zinc sulfate (10 M). After 4 hours, the cells were washed extensively with PBS and fixed. Transiently transfected COS cells were harvested 24 hours after the DMSO shock and plated onto coverslips in growth medium. After 16-24 hours non-adherant cells were removed by PBS wash and the coverslips were fixed. Fixation was achieved with 4% paraformaldehyde (lOmins, 37 C). The fixative was then removed by PBS wash and cells were permeabilized with 1% (w/v) Triton X-100. The latter treatment allowed access of the antibody to intracellular structures. Cellular apo A-I was visualized by indirect fluorescence labelling. Murine monoclonal antibody to apo A-I (6B8) was used as the primary antibody and FITC-labelled goat anti-mouse IgG (Sigma Chemical Co.) was utilized as detection antibody. Both were used at working concentration of 1% (v/v) in PBS. Incubations were 30mins duration at ambient temperature, separated by PBS washes. Control preparations, omitting the primary antibody, were also performed and displayed negligable fluorescence. Some coverslips were counterstained with TRITC coupled wheat germ agglutinin (WGA) or concanavalin A (ConA) following the immunofluorescence detection. This permitted localization of Golgi or endoplasmic reticulum, respectively (Kaariainen, 1983). Coverslips were mounted in veronal buffered (pH 8.6) 50% glycerol and viewed under a Zeiss 1M35 fluorescence microscope equipped with filters for differential visualization of FITC and TRITC.  52  2.16. Immunogold Electron Microscopy BHK cells expressing apo A-I were harvested with a rubber policeman into PBS, 4 hours after addition of 10 M zinc sulfate. These samples were prepared for cryoultramicrotomy and immunolabelling according to established procedures (Tokuyasu, 1973; 1978; 1983; 1984; Griffiths, 1984). Briefly, cells were washed with PBS by centrifugation and fixed for 30 mins in 1% glutaraldehyde in 200mM HEPES, pH 7.4. The cell pellet was recovered by centrifugation and washed free of fixative with 200mM HEPES containing 10% sucrose and 0.1% sodium azide. Samples were cryoprotected in 2.3M sucrose for 30 mins and then frozen in liquid nitrogen. Ultrathin (80-100 nm) cryosections were cut at -85’ C (using a diamond knife in an RMC MT6000-XL ultramicrotome with a CR2000 cryochamber), collected on 2.3M sucrose droplets and transferred to Formvar-coated grids. Cryosections were immunolabelled with antibody 6B8 (1:100 dilution) or non-immune mouse serum (bug/mi) for 30 mills, followed by secondary labelling with goat-anti-mouse IgG conjugated to lOnm colloidal gold 520 (O.D.  =  0.1; GAM-lO, Sigma Chemical Co., St. Louis, Mo. Cat. No. G-3641). Immunolabelled  cryosections were embedded, contrasted with 1.8% methylcellulose and 0.3% uranyl acetate, and examined on a Phillips EM400 transmission electron microscope.  2.17. Quantitation of Apo A-I by Competitive ELISA Apo A-I was quantitated in cell culture medium and in gradient ultracentrifugation fractions by measuring the remaining unbound antibody to apo A-I after incubation with sample to equilibrium (Wong, L; personal communication). Apo A-I sample or HDL standard was mixed with antibody (diluted in PBS/0.05% Tween—20) and immune complexes formed overnight at ambient temperature. Simultaneously, wells of microtitre dishes were coated with 5Ong HDL in PBS. The coated plates were washed with PBS/0.05% Tween—20 and lOQu I of the antigen-antibody mixture was added to each well. After 30mins, the solution was removed and the wells were washed. During this incubation, IgG which had not been bound by sample apo A-I was adsorbed to the coating HDL. Detecting antibody (donkey anti-sheep IgG, HRP conjugate) was then added. After 30mins, additional washes were performed and substrate solution (0.04% o-phenylenediamine, 0.01% hydrogen peroxide in 0.1M sodium citrate pH 5.0) was added. Color development was complete within 5-l5mins (maximum absorbance had reached 53  490 O.D.  =  1-L5). The reaction was stopped by adding sulfuric acid to 2N. Absorbance was measured at  490nm in a Biorad Model 3550 microplate reader and apo A-I concentration determined by interpolation from the linear portion of the standard curve (generally between 25-150ng/ml).  2.18. Analysis of Apo A-I Function 2.18.1. Preparation of Single Bilayer Vesicles Phosphatidyicholine and cholesterol were mixed at 4:1 molar ratio for preparation of vesicles (Batzri and Korn, 1973) for use as lipid binding and LCAT activation substrates. 1.3mg of egg yolk phosphatidyicholirie (Te III-E, Sigma Chemical Co., St. Louis, Mo.), 0.15mg cholesterol (CH-S, Sigma) and l2uCi of 3 f7(n)H Jcholesterol (Amershain, 5-15 Ci/mmol, lmCi/ml) were mixed and dried under a stream of nitrogen gas at room temperature. The residue was redissolved in 12.i 1 of 99% ethanol and rapidly injected through a 25 guage needle into lOmis of TEN buffer (10mM Tris-HCI, pH 7.4, 5mM EDTA, O.15M NaC1). The resulting preparation was concentrated to 2.5mls in an Amicon stirred cell ultrafiltration device (PM-30 membrane) and stored at 4°C for up to 5 days.  2.18.2. Assessment of Lipid Binding Characteristics Recombinant apo A-I preparations were concentrated in dialysis membranes immersed in carboxymethylcellulose (Aquacide I, Calbiochem Corp., La Jolla, Ca.). Substrate lipid vesicles were mixed with recombinant apo A-I (molar ratio 200: 50: 0.005, PC: UC: A-I) in a final volume of 0.75mls and incubated at 37 C for 30mins. The mixture was then layered onto the top of a 6m1 linear density gradient of potassium bromide (KBr) in saline (1.006 to 1.250 g/ml). Centrifugation was performed at 15C, 40,000rpm in an SW41Ti rotor (Beckman Instruments Inc., Palo Alto, Ca.) and was terminated without braking after 44-48 hours. Gradients were fractionated into  0.Sml aliquots from the tube  bottom. Apo A-I was measured in each fraction by competitive enzyme linked immunosorbent assay (ELISA) and the vesicle lipids were located within the gradient by liquid scintillation counting. The density of each fraction was measured by diluting 10Oil aliquots to 5.Omls with water and measuring the conductivity of the resulting solution. Fraction densities were extrapolated from a standard curve derived from density solutions which had been measured gravimetrically and by conductivity. 54  2.18.3. Measurement of LCAT Cofactor Activity Substrate vesicles (3(u 1 containing 4.6mnoles of UC) were mixed with apo Al in a fmal volume of 3OQu 1 and incubated at 37° C for 30m ins to allow apo A-I to bind to the substrate. Fatty acid depleted bovine serum albumin (Sigma) and j3 -mercaptoethanol were added to 2% (w/v) and 10mM, respectively. The reaction was initiated by addition of 2il  100 ng) of recombinant LCAT which was  expressed in serum free culture medium by transfected BHK cells (J. Hill et al, in press). 100.i 1 aliquots from each incubation were removed after 0, 30, 60, and l20mins at 37) C and were terminated by the addition of 2mls of chloroform:methanol (2:1, v.v). Lipids were extracted by the method of Foich et at (1957), and the chloroform phase was recovered following low speed centrifugation. This was dried under a stream of air and the residue was dissolved in 100l of chloroform containing 2(ig each FC and CE (as cholesterol oleate). Lipids were applied to the origin of silica gel G thin layer chromatography plates and were developed in petroleum ether: ether: acetic acid (70:12:1). FC and CE bands were identified with iodine vapour, excised from the plate and quantitated by liquid scintillation spectrometry in toluene based scintillant (Omnifluor, NEN-Dupont, Mississauga, Ont.). LCAT activity was calculated from the slope of the time course describing the conversion of FC to CE and was expressed as nmoles FC esterified per hour per ml of rLCAT.  55  3. DEVELOPMENT OF APO A-I EXPRESSION SYSTEMS The goal of the early phase of this work was to establish cell culture systems for the expression of recombinant human apo A-I. Initially, nucleotide sequence analysis of the cDNA was performed to confirm that the coding region for the apo A-I precursor was intact. In vitro transcription and translation studies were then used to establish that the cDNA produced immunoreactive apo A-I. Eucaryotic expression vectors were constructed and utilized to assess the molecular characteristics of the protein produced in transiently expressing cell cultures. Cell lines expressing apo A-I protein were established which had stably integrated the cDNA into their genome. These cell lines were compared for their capacity to produce apo A-I under defined culture conditions. Finally, the biologic properties of the recombinant protein were assessed.  3.1. Sequencing of the Full Length Apo Al cDNA Nucleotide sequence analysis was used to verify the published sequence of the apo A-I cDNA clone pBL13AI which contains  lKbp of apo A-I sequence in the vector pBR329 (Sielhamer et al,  1984). The strategy used for M13 subcloning and sequence analysis is illustrated in Figure bA in which subclones were selected to provide sufficient overlap to verify the entire coding region in the original cDNA. Since this sequencing indicated concordance with the published sequence in the translated region, bidirectional sequencing over the entire length of the cDNA was not performed. However, the EcoRI cloning site was found at nucleotide 60 of the published sequence (Figure lOB). The same sequence was obtained in three separate M13 clones derived from the original plasmid and did not, therefore, appear to be an artifact of subcloning performed in our laboratory. This cDNA lacks 13 bp of the 5untranslated region of the apo A-I cDNA and was therefore less than full length although it contained the complete translated sequence. It appears that the clone provided was actually pBL14AI, which was also described in the same laboratoiy (Seilhamer et al, 1984). The absence of this DNA segment in the flanking region had no deleterious effect on subsequent DNA manipulations, nor on the ability to express authentic apo A-I protein in vitro. Our sequence data predicted that insertion of the apo A-I cDNA at the Smal or EcoRI site of pSPT19 would produce transcript which contained the ATG codon of the apo A-I precursor as the  only  translation initiation site. 56  A Eco RI  Taq I  1  I 185  SstI  Sst I  I I 495531  Taq I  Taq I  I 790  856  Eco RI 961 100 bp  Figure 1OA. DNA sequence analysis of the apo A-I cDNA pBL13AI. Location of restriction enzyme sites used to generate M13 subclones from the full length cDNA. Numbers represent nucleotide position in the published sequence (Seilhamer et al, 1984). Arrows indicate the direction and quantity of sequence information obtained from each subclone.  57  B 1  A ATT OAA AAA AAA AAG AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA  49  GAG AGA CTG CGA GAA GGA GGT COO CCA CGG 000 TTC AGG ATG AAA GCT Met Lys Ala  97  GCG GTG OTG ACO TTG GCC GTG CTC TTO CTG ACG GGG AGC CAG GOP CGG Ala Val Leu Thr Lou Ala Val Lou Phe Leu Thr Gly Ser Gin Ala Arg  145  CAT TTC TGG CAG CAA GAT GAA CCC 000 OAG AGC 000 TGG GAT CGA GTG His Phe Trp Gin Gin Asp Glu Pro Pro Gin Ser Pro Trp Asp Arg Val  193  AAG GAC CTG GCC ACT GTG TAC GTG GAT GTG CTC AAA GAC AGC GGO AGA Lys Asp Lou Ala Thr Val Tyr Val Asp Val Leu Lys Asp Ser Gly Arg  241  GAO TAT GTG TCC CAG TTT GAA GGC TCC GCC TTG GGA AAA CAG CTA AAO Asp Tyr Val Ser Gin Phe Glu Gly Her Ala Lou Gly Lys Gin Leu Asn  289  CTA AAG CTC CTT GAC AAC TGG GAC AGC GTG ACC TCC ACC TTC AGC AAG LGu Lys Leu Leu Asp Asn Trp Asp Ser Val Thr Her Thr Phe Ser Lys  337  CTG CGC GAA CAG CTC GGC CCT GTG ACC CAG GAG TTC TGG GAT AAC OTG Lou Arg Glu Gin Leu Gly Pro Vol Thr Gin Glu Phe Trp Asp Asn Lou  385  GAA AAG GAG ACA GAG GGC OTG AGG CAG GAG ATG AGO AAG GAT CTG GAG Glu Lys Glu Thr Glu Gly Lou Arq Gin Glu Met Ser Lys Asp Leu Glu  433  GAG GTG AAG GOC AAG GTG CAG 000 TAO OTG GAO GAO TTO OAG AAG AAG Glu Val Lys Ala Lys Val Gin Pro Tyr Lou Asp Asp Phe Gin Lys Lys  481  TGG OAG GAG GAG ATG GAG OTO TAO OGO OAG AAG GTG GAG COG OTG OGO Trp Gin Glu Glu Met Giu Lou Tyr Arg Gin Lys Val Glu Pro Lou Arg  529  GOA GAG OTO OAA GAG GGO GOG OGO CAG AAG OTG OAO GAG OTG OAA GAG Ala Glu Lou Gin Glu Gly Aia Arg Gin Lys Lou His Glu Lou Gin Glu  577  AAG OTG AGO OOA OTG GGO GAG GAG ATG OGO GAO OGO GOG OGO GOO OAT Lys Lou Her Pro Lou Gly Glu Glu Met Arg Asp Arg Aia Arg Aia His  625  GTG GAO GOG OTG OGO AOG OAT OTG GOC 000 TAO AGO GAO GAG OTG OGO Val Asp Ala Lou Arg Thr His Lou Ala Pro Tyr Her Asp Glu Lou Arg  673  CAG OGO TTG GOC GOG OGO OTT GAG GOT OTO AAG GAG AAO GGO GGO GOO Gin Arg Lou Ala Ala Arg Lou Glu Aia Lou Lys Glu Asn Gly Gly Ala  721  AGA OTG GOO GAG TAO OAO GOC AAG GOO AOO GAG OAT OTG AGO AOG OTO Arg Lou Ala Glu Tyr His Ala Lys Ala Thr Glu His Lou Ser Thr Lou  769  AGO GAG AAG GOO AAG 000 GOG OTO GAG GAO OTO OGO OAA GGO OTG OTG Her Glu Lys Ala Lys Pro Ala Lou Glu Asp Lou Arg Gin Gly Lou Lou  817  000 GTG OTG GAG AGO TTO AAG GPO AGO TTO OTG AGO GOT OTO GAG GAG Pro Val Lou Glu Ser Phe Lys Val Ser Phe Lou Ser Ala Lou Glu Glu  865  TAO ACT AAG AAG OTO AAO ACO CAG TGA GGO GOO OGC OGO OGO 000 OCT Tyr Thr Lys Lys Lou Asn Thr Gln  9i3  TOO OGG TGO TOA GAA TAA AOG TTT OOA AAG TGT TAA AAA AAA AAA AAA  961  GAA PrO  Figure lOB. DNA sequence analysis of the apo A-I cDNA pBL13AI. Nucleotide and translated amino acid sequence of pBL13AI. Numbers refer to the nucleotide position in the sequence of the original publication (Seilhamer et cii, 1984). Sequence data obtained in this study begins at position 60 (see text for details).  58  3.2. In vitro Transcription and Translation of the Full Length cDNA Vectors for coupled transcription-translation were prepared to confirm that the apo A-I cDNA contained the appropriate signals for eucaryotic translation and microsomal translocation. The cDNA was cloned into the vector pSPT19 to facilitate in vitro transcription in either orientation. The cDNA was inserted at either the EcoRI or the Smal restriction site of the vector and clones containing the apo A-I in either orientation were isolated. In many instances low quantities of heterogeneous transcript were obtained on addition of RNA polymerase. Large quantities of homogeneous transcript were synthesized when the apo A-I cDNA was placed at the Smal site of pSPT19 and in the orientation for the SP6 polymerase promoter. Approximately 1.5-2k g of DNase I resistant nucleic acid was obtained per  g of  DNA template under run-off transcription conditions (Figure hA). Addition of this product to template dependent rabbit reticulocyre lysate reactions generated a single protein species with apparent molecular mass of  31 kiodaltons (kD) on SDS-PAGE (Figure 11B, lane 2). This was consistent with the  predicted molecular mass for preproapo A-I. In some reactions the cap analogue 7 mG(5)ppp(5)G was included during transcription which substantially reduced the quantity of transcript synthesized. Subsequent addition of this material to reticulocyte lysate indicated that the capped mRNA was more efficiently translated than uncapped mRNA (data not shown). This observation was in agreement with previous reports of more efficient translation of capped transcripts in the wheat germ translation system (Krieg and Melton, 1984).  -  Translation reactions were performed in the presence and the absence of dog pancreatic microsomes. In the presence of microsomes the primary translation product was partially converted to a species of lower molecular mass  30 kD, Figure 11B, lane 3). This observation was consistent with  translocation of the preproapo A-I across the microsomal membrane and proteolysis of the signal peptide, demonstrated using liver mRNA (Zannis et at, 1983). Both the larger and the smaller apo A-I species were precipitated with monospecific antibodies to human plasma apo A-I (Figure 11B, lanes 5-8) indicating that both the primary translation product and the smaller translocated form were authentic apo A-I. The amino acid sequence of the apo A-I signal peptide (derived from the cDNA sequence) suggested that preproapo A-I and its translocated product could be differentiated by charge and size 59  B  A  12345678 mRNA  —  +  +  —  M icrosomes  —  —  +  —  Immunoprecipitate  —  —  —  +  1234  +  —  +  +  +  +  —  +  +  +  +  +  2 1  Kbp 92.5-  0.5  66.2+  Mr  4531I  ——  —  —  2 1.514.4-  Figure 11. In viiro transcription and translation of the full length apo A-I cDNA. A- Hindlil linearized pSPT19-AI (lane 1, 2i g) was incubated with SP6 RNA polymerase (lane 2, 20 units) for 2 hours at 373 C. An aliquot of the reaction was removed and treated with DNase I to remove template DNA (lane 3). Aliquots of each reaction were fractionated by gel electrophoresis in 1% agarose and visualized by ethidium bromide staining. B- Translation in template-dependent rabbit reticulocyte lysate. Each reaction (2. 1 final volume) was incubated for 30mins at 311 C in the presence (+) or absence (-) of template mRNA (0.5 g) and dog pancreatic microsomes as indicated. In lanes 1 to 3, 0.u 1 of the reaction was applied directly to SDS PAGE gel containing 12.5% acrylamide. In lanes 4, 6 and 8, 1 1 of the reaction was subjected to immunoabsorption with polyclonal anti-human apo A-I prior to SDS-PAGE fractionation. In lanes 5 and 7, immunoisolates were prepared from 0.5 1 of each reaction. The arrow indicates the mobility of purified plasma apo A-I.  60  C IEF (+  SDS  92.566.2  92.566.2  45-  45-  31-  31-  -  -  U 21.5-  21.514.4-  •1 WITH MICROSOMES  WITHOUT MICROSOMES  1 of each Figure 11C- Two dimensional electrophoretic separation of in vitro translation products. translation reaction was mixed with 1j g of purified plasma apo A-I and separated by two-dimensional electrophoresis according to O’Farrel (1975). The gels were stained with Coomassie Blue to identify the position of mature apo A-I (dotted circle) and then processed for autoradiography to identify the translation products. The position of the stained molecular mass markers was determined by overlay of the autoradiographic film on the stained gel.  61  characteristics. The products of reticulocyte translation in the presence or absence of microsomes were analysed by two-dimensional gel electrophoresis according to O’Farrell (1975). Preproapo A-I was the only species predicted from translation in the absence of microsomes. The product of this incubation displayed a more basic isoelectric point (p1) than purified plasma apo A-I, in addition to a higher molecular weight  ( 31 kD vs 27 kD, Figure 11C). Additional species with p1 between preproapo A-I  and mature apo A-I were also evident. The origin of these charge-shifted forms is not clear. Twodimensional electrophoretic analysis of products synthesized in the presence of microsomes showed that the smaller apo A-I species (M 30 kD) had p1 between preproapo A-I and mature plasma apo A-I. Precursor and product species were present in approximately equal proportions indicating that the signal peptide was only partially hydrolysed. Complete hydrolysis of the signal sequence was not achieved even with additional microsomes (up to 2j.t g protein). Since the apo A-I signal peptide contains a single charged residue (Ly ), the charge differences we observed were consistent with the processing of the 23 preproapo A-I to proapo A-I on translocation across the ER membrane. Folz and Gordon (1987) have shown previously that the proapo A-I product is sequestered within the microsome. The presence of a net charge on the propeptide (÷ 2) and on the signal peptide (+1) allows preproapo A-I, proapo A-I and mature apo A-I to be differentiated on the basis of charge and size criteria by two-dimensional gel electrophoresis. In contrast to preproapo A-I, proapo A-I was not heterogeneous in the IEF dimension, which may indicate that amino acid residues in the signal peptide are more susceptible to in vitro modification. Our findings provide evidence that apo A-I cDNA expressed in vitro from its cDNA is compartmentalized within the ER as proapo A-I and are consistent with those obtained with human liver mRNA (Zannis et al, 1983). In order to produce quantities of recombinant protein for functional analysis, COS, BHK and CHO cells were evaluated as host cell cultures for the expression of human apo A-I.  3.3. Expression of Wild-type Apo Al (Apo A.Iwt) in COS Cells Observations in the rabbit reticulocyte lysate established that apo A-I was synthesized from the cDNA as the preproapolipoprotein and was converted to proapo A-I during ER translocation. To 62  establish that, subsequent to ER translocation, the apo A-I protein was secreted from cultured eucaryotic cells which do not normally express apo A-I, trafficking and secretion were analysed following transient transfection of COS cells. Forty hours after transfection,  [ Simethionine was incorporated into cellular  protein in order to follow the cellular processing and secretion of newly synthesized apo A-I in a subsequent unlabelled methionine chase incubation. Apo A-I was isolated by immunoabsorption from cells and medium and subjected to SDS-PAGE analysis (Figure 12A). A single apo A-I species (M 3OkD) was found in the medium, consistent with secretion of proapo A-I. However, cell lysates contained a species of higher molecular mass  ( 3lkD) in addition to proapo A-I. This larger apo A-I  was observed in cell lysates throughout the chase interval, and the autoradiographic intensity of this band decreased with time. The intensity of the intracellular proapo A-I signal also decreased, in parallel with the larger form. The 31 kD band was identified as preproapo A-I on the basis of charge-size properties using two-dimensional electrophoretic analysis (Figure 12B). The presence of preproapo A-I in transfected cell lysates was unexpected since signal peptide hydrolysis normally occurs as a cotranslational event. Radiolabelled apo A-I was quantified at each time point by liquid scintillation counting of the immunoabsorbed material. Approximately one-third of the cellular apo A-I, radiolabelled during the pulse period, was not recovered from either the cells or the medium after 24 hours chase, suggesting that this portion had been degraded. Apo A-I was a major product of the cell culture, approximately 3% of total cellular and 33% of total secreted protein (TCA precipitable) radioactivity during the chase period. The cellular half-life of apo A-I synthesized de novo was estimated to be 12 hours (Figure 12C) from this experiment. To determine if the cells produced sufficient quantity of apo A-I for structure-function analysis, the apo A-I mass in the medium and cell lysates of the transiently transfected cells was estimated by western blot analysis. Media and cell lysates were collected from 35mm culture dishes 40 hours after transfection with the apo A-I cDNA and the apo A-I product was concentrated by immunoabsorption. Immunodetection was used to estimate both the cellular and the secreted apo A-I concentration following SDS-PAGE separation and electroblotting (Figure 13). Control cultures transfected with empty pCMV5 vector contained neither cellular nor medium apo A-I, establishing that COS cells do not 63  A MEDIUM  0  HOURS 2 4 6 8 24  92.566.2-  Mr  31  _  21.514.4-  CELLS 92.566.2-  Mr  31  -  __  —  21.514.4-  Figure 12A. Expression of the apo A-I cDNA in transiently transfected COS cells. COS cells were transfected with pCMV5-AIwt and the expressed product was identified in S]labelled cultures. A- Apo A-I species were isolated by immunoabsorption after the indicated chase interval, separated by SDS PAGE and visualized by autoradiography. The arrow indicates the position of plasma apo A-I which was added prior to electrophoresis as a marker. 64  B IEF SDSf  31  Kd-’  pCMV5-Alwt  Figure 12B. COS cell lysate contains prepro- and pro- apo A-I. Immunoisolate from [ SI labelled transfected cell lysate was mixed with purified plasma apo A-I (dotted circle), separated by twodimensional gel electrophoresis and visualized by autoradiography. Horizontal and vertical arrows indicate the direction of IEF and SDS-PAGE, respectively. The position of plasma apo A-I was determined by Coomassie Blue staining. 65  z II 0  UJLJ  O_o  c  0 LU ‘4-  0-c  CHASE TIME (hrs)  Figure 12C. Quantitative pulse-chase analysis of apo A-I expression in transfected COS cells. Apo A-I was isolated by immunoabsorption after the indicated chase following pulse-labelling with [35 S]methionine. Radioactivity was quantitated as the proportion of the total apo A-I radiolabel recovered per dish in CELLS or MEDIUM as determined by liquid scintillation counting. TOTAL represents the sum of cell lysate and medium apo A-I radioactivity expressed as the percent of the lysate value at t=O.  66  92.566.245-  Mr  31-  2 1.514.4-  1  5 10 50 100 C M C M  ng MATURE Al  +  Figure 13. Immunoblot analysis of apo A-I expression in COS cells. Apo A-I was concentrated by immunoabsorption from cells (C) and medium (M) following transfection with pCMV5 (-) or with pCMV5-AIwt (+). Samples were resolved by SDS-PAGE and visualized by immunoblotting as described in MATERIALS AND METHODS. Purified plasma apo A-I was used as a standard as indicated. Mr indicates the relative mobility of molecular mass markers in kilodaltons.  67  express endogenous, immunoreactive apo A-I. Cells transfected with vector containing the apo A-I cDNA produced approximately 500ng of apo A-I per 35mm dish (2mls medium). However, only 5-10% of the apo A-I mass was secreted during the 40 hour collection period. Immunoreactive partial degradation products were not evident on the blots. COS cells secreted only small quantities of apo A-I (approximately 5-10 ng/ml/24 hours), although analysis of cell lysates indicated that substantially more was synthesized than was secreted. The intracellular pool contained both preproapo A-I and proapo A-I in approximately equal proportions. Retention of the signal peptide on a considerable portion of apo A-I suggested that ER translocation was inefficient in the transfected cell. Since significant quantities of proapo A-I were also retained by the cells, later secretory events were also inefficient in these cells. The pulse-chase studies suggested that a substantial portion of the apo A-I labelled during the pulse period was later degraded, although large amounts of apo A-I protein were detectable at this time by immunoblot analysis. This suggested that at least two pools of apo A-I exist in the transfected cell, with the apo A-I synthesized 40 hours after transfection subject to more rapid degradation than the apo A-I synthesized prior to that time.  3.3.1. Properties of Apo A-I Secreted by COS Cells 1 S]methionine labelling studies The functional properties of secreted apo A-I were assessed in n of transiently transfected COS cell cultures. The ability of the radiolabelled recombinant to integrate into existing lipoproteins was determined. Since apo A-I is believed to enter plasma as free apolipoprotein, rapid association with existing HDL is physiologically relevant. Radiolabelled apo A-I was chased from the cells into standard growth medium containing 10% FBS which was then fractionated by density gradient ultracentrifugation.  S]Methionine label was found in a symmetrical peak in fractions of  density 1.10-1.20 g/ml with maximum radioactivity at 1.15 g/ml (Figure 14A). Immunoabsorption and SDS-PAGE analysis of the gradient fractions showed that this label was associated exclusively with apo A-I (Figure 14B). No apo A-I was detected in the fractions of higher or lower density. Thus, the recombinant proapo A-I produced by transfected COS cells incorporates exclusively into lipoproteins of the HDL density class. Under these culture conditions, lipid-free apo A-I was not detected. In preliminary experiments, the secreted product was also analysed by non-denaturing gradient gel 68  A 1.300  1.250  4  1.200  a)  .9?  Co 0—  Lx  p  1.150  2  1.100  1 .050  1.000 10  B  FRACTION  1  SDS-PAGE  Figure 14. Apo A-I produced by transfected COS cells is associated with HDL in the culture medium. Cells transfected with pCMV5-AIwt were pulse-labeled with r S]methionine and chased with medium 5 containing 10% FBS. The medium was concentrated by ultrafiltration and fractionated by density gradient ultracentrifugation. A- Radioactivity (solid line) was found in gradient fractions (dotted line) with density 1.12-1.18 g/ml. B- Gradient fractions were immunoabsorbed with antibody to human apo A-I and resolved by SDS PAGE. Label was found in a single protein species with M 3OkD. 69  electrophoresis (not shown) which indicated that apo A-I was associated with lipoprotein with the size characteristics of human plasma HDL. Therefore, transfected COS cells secrete recombinant human apo A-I that associates readily with high density lipoproteins. We attempted to determine if such a complex could also form in cultures without addition of serum. Very little apo A-I secretion could be detected by immunoabsorption of the medium under these conditions arid it was not possible to characterize the complexes. However, the level of  [ S]methionine  incorporated into cellular apo A-I was not different in serum free medium, despite the lower level of apo A-I secretion. We hypothesized that FBS provided an essential component for apo A-I secretion and investigated this possibility further by assessing the quantitative effect of FBS on apo A-I secretion (Figure 15). Secreted apo A-I radiolabel increased in response to the concentration of FBS in the medium in a dose-dependent manner. Near maximal effect was achieved at very low levels of FBS, as ten-fold stimulation of apo A-I secretion was observed at  only  0.5% (v/v) FBS. Further increases in FBS  had only modest stimulative effect. At extremely high levels of FBS (20%, v/v), preproapo A-I was detectable in the medium by SDS-PAGE. The individual lipoprotein components of human serum were tested for their influence on apo A-I secretion. Apo A-I secretion was stimulated by human LDL in a similar manner as by FBS, and the effect was observed with as little as 7 g of LDL-C was added per ml medium. The stimulatory effect was maximal at approximately 700ig/m1. VLDL and the intravenous lipid emulsion Liposyn (Abbott Laboratories, Montreal, Que.) also stimulated apo A-I secretion but to a lesser extent. In contrast to the the effect of other lipoproteins, HDL reduced apo A-I secretion by approximately 25% compared to control dishes without additive. Post-translational phosphorylation of apo Al has been reported in primary human hepatocytes and in the hepatoma line HepG2 (Beg el at, 1989). The authors have suggested that a phosphorylation dephosphorylation process may be involved in secretion of apo A-I from these cells. Since we had observed impaired secretion of apo A-I from the COS cell, we investigated the possibility that these cells might be unable to phosphorylate or to dephosphorylate apo A-I. Endogenous labelling of transfected cells with r Pjorthophosphate resulted in time-dependent intracellular phosphorylation of apo A-I 2 (Figure 16). The molecular mass on SDS-PAGE indicated that preproapo A-I was the labelled species. 70  15-  -  (30 LLJC  (I)— _0  <-5 5  0  0  1  0.5  5  10  20  FBS Content (Volume %)  Figure .15. Apo A-I secretion from (ransfected COS cells is stimulated by FBS. COS cells were transfected with pCMV5-AIwt and pulse-labeled with Simethionine. Secretion of apo A-I from the cells into medium containing the indicated concentration of FBS was determined after 4 hours chase incubation. Apo A-I was recovered from the medium by immunoabsorption and quantitated by liquid scintillation counting. Results are expressed as the radioactivity ratio in the presence and absence of additive and represent single dishes from a representative experiment.  71  HOURS MEDIUM  0  2  4  6  S 35 24  6904 6.0-  Mr  30.0I  14,3-  L  CELLS 69.0-  Mr  4 6.0-  30.0I  4 3-  Figure 16. Apo A-I produced in COS cells is subject to intracellular phosphorylation. COS cells were transfected with pCMV5-AIwt and radiolabeled for 2 hours with Pjorthophosphate in phosphate-free medium, as described in MATERIALS AND METHODS. After replacing the medium with DMEM/1O% FBS and incubating for the indicated chase interval, apo A-I was concentrated by immunoabsorption from medium or cell lysate and analysed by SDS-PAGE with autoradiography. Numbers along the top of the autoradiograms indicate the chase time in hours. Control lane S) is the immunoabsorbed material from the medium of a transfected culture radiolabeled with [ S]methionine. This sample was used to demonstrate that apo A-I was secreted during the chase. The arrow indicates the position of mature plasma apo A-I.  r2  72  Additional banding was detected at  46kD (and larger) molecular mass in the cells after 24 hours. It  was not clear whether these species were phosphorylated aggregates of apo A-I, or were non-specifically absorbed phosphorylated proteins. Under conditions where we had previously demonstrated maximal apo A-I secretion (DMEM/1O% FBS), immunoreactive material from the medium did not contain  2r Piphosphate-labelled apo A-I, although dishes labelled in parallel with [ S]methionine indicated that apo A-I was secreted (see Figure 16). The observations in COS cells were consistent with the observations of Beg et at, indicating that proapo A-I is subject to phosphorylation and dephosphorylation processes during secretion. However, these observations do not rule out the possibility that apo A-I phosphorylation may play a role in processes other than secretion. Abnormal phosphate modification of apo A-I in COS cells does not appear to explain the reduced rate of secretion.  3.3.2. Immunofluorescence Localization of A-Iwt in Transfected COS Cells The biochemical observations in COS cells expressing human apo A-I suggested that large amounts of the recombinant protein were retained rather than secreted. The intracellular location of apo A-I in these cells was investigated by indirect immunofluorescence microscopy to gain insight into the mechanism of retention. Intense apo A-I reactivity was found throughout the transfected cells, concentrated near the nucleus with diminishing intensity toward the cell membrane (Figure 17A). Using TRITC-ConA as a marker for the high mannose sugars of the ER (Kaariainen et at, 1983), it was observed that the apo A-I immunoreactivity and the ER were similarly distributed in the transfected cells (Figure 17B). Both labelling procedures suggested that the ER had expanded considerably in many of the cells expressing apo A-I. In some experiments, however, the fluorescence intensity and its location were variable. The apo A-I fluorescence in the ER region was less intense and a juxtanuclear staining pattern was more prominent, indicating that the Golgi apparatus was the site of apo A-I accumulation. Thus, cellular transport of apo A-I in transiently transfected COS cells was heterogeneous, not only from one experiment to another, but also from cell to cell within the same experiment.  73  Figure 17. Inimunofluorescence analysis of transfected COS cells indicates that apo A-I is retained in the ER. A- Cellular apo A-I was displayed in fixed, permeabilized cells by indirect immunofluorescence using mouse anti-human apo Al (6B8) and FITC-conjugated goat anti-mouse IgG. Intense fluorescence was found extending from the nucleus to near the cell membrane. B- The extent of ER in the cells was visualized by counterstaining the same cell preparation with TRITC-conjugated ConA. Apo A-I and ConA colocalize within the transfected cells. Bar indicates 1 m in both panels.  74  3.4. Baby Hamster Kidney (BHK) Cell Expression of Apo A-Iwt 3.4.1. BHK-AJwt Cells Produce proapo A-I COS cell expression studies indicated that low levels of apo A-I could be produced and secreted by transiently transfected non-hepatic mammalian cells in culture. Since the transfected cell population often appeared heterogeneous by immunofluorescence microscopy, a cell line which had the apo A-I cDNA stably integrated into its genome could provide a continuous source of apo A-I from a homogeneous cell population. BHK cell lines expressing the apo A-I protein from the wild-type cDNA (apo A-Iwt) were established. Approximately 40 BHK clones were established following transfection and selection and were screened by immunoblot analysis for secretion of apo A-I. One third of the cell lines secreted detectable quantities of apo A-I when maintained at confluence in serum-free culture. A single clone (BHK-AIwtB5), secreting the highest level of apo A-I under these conditions was chosen for detailed analysis. The apo A-I produced by BHK-AIwtB5 was isolated by immunoabsoiption from cells labelled with r S]methionine. The autoradiogram in Figure 18 indicates that a single species of apo A-I was 5 labelled, with an apparent molecular mass of approximately 28,000 daltons. Lysates prepared from cells of the parent BHK line or cells transfected with pNTJT vector alone contained no detectable apo A-I (data not shown). Based on its p1 with respect to the major isoform of plasma apo A-I, the apo A-I produced by BHK-AIwtB5 was identified as proapo A-I. In contrast to COS cell transfectants, hydrolysis of the signal peptide from preproapo A-I was complete in the BHK cell. Furthermore, since BHK cells produced proapo A-I from the cDNA encoding preproapo A-I, the electrophoretic analysis suggested that BHK cells lack apo A-I intracellular propeptidase activity. The apo A-I secreted by these cultures was also analysed by sequential Edman degradation of the amino terminus which indicated that two polypeptides were present in the medium. The major secreted apo A-I species was proapo A-I (Table VI). However, a portion of the secreted apo A-I (estimated to be 20%) was mature apo A-I following 24 hour collections of serum-free medium.  75  IEF SDS+  31 Kd  BHK-AlwtB5 (p-Al)  Figure 18. BHK cells expressing apo A-Iwt produce a single molecular species with the electrophoretic properties of proapo A-I (p-Al). BHK cell line AIwtB5 was pulse-labeled with [ Simethionine and harvested by cell lysis. Apo A-I was recovered by immunoabsorption, mixed with purified plasma apo A-I and resolved by two-dimensional gel electrophoresis. Horizontal and vertical arrows indicate the direction of IEF and SDS-PAGE, respectively. A single molecular species was observed, with more basic p1 than mature plasma apo A-I used as marker (dotted circle). The electrophoretic properties of apo A-I in this cell line are those of proapo A-I (p-Al).  76  Table VI. Amino terminal sequence analysis of apo A-I secreted from cell line BHK-AIwIB5. X = amino acid detected but not identified. Major component is 80% of protein present, minor component is 20%. Source  N-Terminal Sequence  Assignment  Plasma  X-Glu-Pro-Pro-Gln-Ser  Mature apo A-I  BHK-AIwtB5  (major) X-X-Phe-X-Gln-Gln-Asp  Proapo A-I  BHK-AIwtB5  (minor) X-Glu-Pro-Pro-Gln-Ser  Mature apo A-I  This indicates that BHK cells may secrete a small quantitity of propeptidase activity. Nonetheless, BHK cells secrete the major portion of apo A-I as proapo A-I when expressed from the preproapo A-I cDNA, similar to 3T3 and L6E9 cells (L,amon-Fava ci at, 1987; Ruiz-Opazo et at, 1988). Unlike CHO-Ki, these cells do not rapidly and efficiently hydrolyse the propeptide (Mallory et at, 1987).  3.4.2. Characterization of Apo A-I Synthesis and Secretion by BHK Clone AIwtB5 The vector utilized for transfection and selection of BHK-AIwtB5 placed apo A-I expression under control of the mouse metallothionein promoter. By including divalent metal cations in the culture medium, we could increase the transcription of the apo A-I gene. Northern blot analysis was performed and was corrected for nonspecific stimulation and mRNA recovery by normalizing to the fi -actin signal of the same nucleic acid preparation. Apo A-I mRNA levels increased 3.2-fold during 24 hour culture in the presence of 10 M zinc sulphate (Zn50 ). However, pulse-chase analysis of the apo A-I protein 4 (Figure 19) indicated that secretion of apo A-I into serum free medium (DMEM) was minimal in the presence or absence of ZnSO . Similar results were obtained using lower concentrations of zinc or with 4 20-10t M cadmium chloride. The addition of FBS (10%, v/v), however, stimulated secretion of apo A-I, similar to the earlier observations in transfected COS cells. Further characteristics of this effect were obtained from continuous labelling studies (Table VII). Culture in the presence of 10 M ZnSO 4 decreased the synthesis of apo A-I more than two-fold from synthesis in DMEM only. Addition of 10% FBS to DMEM increased apo A-I synthesis approximately 1.5-fold. In the presence of both 10% FBS and 10 M ZnSO , apo A-I synthesis increased 4  77  cJ) LiJJ  OLL  LU  0  40  20  60  TIME (mins)  Figure 19. FBS stimulates the secretion of apo A-I by BHK-AIwtB5. BHK-AIwtB5 cells were pulse labeled with S]methionine and the secretion of apo A-I determined in the presence or absence of 10% FBS and l0Qu M zinc sulfate after chase incubation for the indicated time. Apo A-I in cell lysates and media were isolated by immunoabsorption and quantitated by liquid scintillation counting. Results are expressed as medium apo A-I (cpm) divided by total dish apo A-I (cpm) as percent. Data points are the mean of duplicate dishes from a single experiment.  78  more than 5-fold, suggesting that ZnSO 4 increased apo A-I synthesis  only  if FBS was present. ZnSO 4 did  not appear to alter the amount of apoprotein secreted since the distribution between cells and medium was not affected by its addition to the medium. FBS, however, had an effect similar to that observed earlier in COS cells. When 10% FBS was included in the medium the portion of the apo A-I found in the medium increased from 40% to 60%.  Table VII. Effect of growth conditions on apo Al synthesis and secretion in BHK-AIwtB5. BHK cells expressing the apo A-Iwt cDNA were labelled with L S]methionine for 12 hours in the presence of the additives indicated. Apo A-I was immunoabsorbed from medium and cell lysate and radioactivity was measured by liquid scintillation counting. Values represent the mean of duplicate dishes, differing by less than 15%. GROWTH CONDITION  TOTAL APO Al (cpm/dish)  SECRETED APO AT (% of total)  DMEM Only  5568  40  4 +10tMZnSO  2160  45  +10% FBS  8384  62  +10% FBS/10zM ZnSO 4  30000  64  The time course of apo A-I secretion was then investigated in long term radiotracer pulse-chase incubations. In accord with our observations in continuous labelling studies, the highest level of apo A-I expression and secretion was observed in the presence of both ZnSO 4 and FBS. Apo A-I secretion was initially rapid (Figure 20), as 25-30% of the pulse-labelled apo A-I was recovered in the medium after 24 hours. However, less than 10% of the remaining label was secreted thereafter. As much as 50% of the initial apo A-I label was not recovered in the medium or the cell lysate, apparently degraded early in the time course. As in the earlier COS cell studies, no evidence of partial degradation products was obtained in the BHK cell experiments. Accumulation of apo A-I in the cells was monitored by immunofluorescence light microscopy. Apo A-I was barely detectable in BHK-AIwtB5 cells under standard growth conditions (Figure 21A). However, the addition of 10 M ZnSO 4 caused a profound increase in immune fluorescence decoration of the cells, 4 hours (Figure 21B) and 8 hours (Figure 21C) after the addition. The fluorescence intensity was greatest in the juxtanuclear region of the cells and apo A-I label colocalized with the Golgi 79  I (/)  LLJ+  0  0  ck UJLJ H0  0 LiJ 4-  o_o  CHASE TIME (hrs)  Figure 20. Long-term pulse-chase analysis suggests that degradation competes with secretion of apo A-I in BHK-AlwtB5. BHK-AIwtB5 cells were pulse labeled with Simethionine and the time course of apo A-I secretion was determined in the presence of 10% FBS and lOQu M zinc sulfate. Apo A-I was isolated form cell lysates and media by immunoabsorption and quantitated by liquid scintillation counting. Results are expressed as the ratio of apo A-I radioactivity in CELLS or MEDIUM over total apo A-I radioactivity recovered at that time point. In the top panel TOTAL apo A-I radioactivity at each time during the chase is expressed as percent of radioactivity at the initiation of the chase (t 0). Data was obtained from single dishes. 80  Figure 21. Zinc sulfate induction of apo A-I synthesis results in the accumulation of apo A-I immunofluorescence in BHK-AlwtB5. BHK-AIwtB5 cells were grown on coverslips and incubated with growth medium containing 1Ot M zinc sulfate. After the induction period, cells were washed, fixed and prepared for indirect immunofluorescence localization of apo A-I. A- 0 hours induction, B- 4 hours induction, C- 8 hours induction. The cellular immunofluorescence colocalized with the Golgi apparatus marker TRITC-WGA. Bar = 10tm.  81  apparatus marker WGA. This marker stains the complex carbohydrate moiety of glycoproteins of the Golgi (Kaariainen et al, 1983) but does not stain apo A-I. The biochemical and immunofluorescence evidence suggested that apo A-I was expressed and secreted more efficiently from BHK cells than from COS cells. However, even with the BHK cell, optimal secretion required the presence of FBS in the medium. Since FBS contains apolipoproteins, these conditions were not acceptable for preparation of recombinant protein for structure-function analysis. Bovine apo A-I is structurally homologous to its human counterpart (Auboiron et at, 1990; Sparrow et at, 1992) and the antibodies used were found to have low level cross-reactivity between the two species. It was necessary, therefore, to exclude FBS from cultures during recombinant apo A-I collections, at the expense of secretion efficiency. Furthermore, we found that BHK cells often adapted poorly tO these conditions, and were viable for only 24-48 hours. Twenty-four hour collections of serumfree medium (Optimem) contained approximately lOOng/ml of apo A-I. As an alternative to the BHK expression system, stable recombinant lines using CHO-Ki cells were also established, since these cells have been shown to adapt well to serum-free conditions. Since BHK cells lack efficient propeptide hydrolytic activity, BHK cells expressing apo A-I from the preproapo A-I cDNA are a useful model for investigation of the functional role of the propeptide segment in constitutive cellular transport (Chapter 4).  3.5. CHO-Ki Cell Expression of Apo A-Iw( CHO-K1 cells produce and secrete mature apo A-I when transfected with the cDNA encoding preproapo A-I (Mallory, 1987). Therefore, this cell line provides a valuable model system for the production of mature wild-type apo A-I and its mutants as recombinants. Cells were transfected with pNUT-AI plasmid and pRc/CMV (which provided neomycin resistance) and were selected in medium containing the neomycin analogue Geneticin (G418). Drug resistent colonies expressing apo A-I were identified and expanded. Apo A-I produced by the cells was isolated from cell lysates and medium by immunoabsorption following 12 hours labelling with  ( S]methionine. Apo A-I was not detected by autoradiography of extracts  from cells transfected with  empty pNUT vector (Figure 22A). Cells transfected with the apo A-I cDNA displayed apo A-I radiolabel 82  A  pNUT  AIwt  CMCM -  6 6. 2  -  -  -I--I  M r  31-p  21  5-, •  Figure 22A. CHO cells expressing apo A-Iwt secrete mature apo A-I. CHO cells were transfected with pNUT vector with or without the apo A-Iwt cDNA and selected with Geneticin (G418) for chromosomal integration. Cells expressing apo A-I were identified and cultured in the presence of [ Simethionine at 1O M for 12 hours. Medium (M) and cell lysates (C) were recovered and absorbed with anti-apo A-I. Immunoisolates were then resolved by SDS-PAGE and visualized by autoradiography. Cells transfected without the apo A-I cDNA (pNUT) contain no detectable apo A-I. The majority of the apo A-I radiolabel in cells transfected with the apo A-I cDNA (Alwt) is extracellular. 83  B  e I  -pro —m  Figure 22B. CHO cells expressing apo A-Iwt secrete mature apo A-I. Apo A-I was isolated by immunoabsorption from CHO cells transfected with the apo A-Iwt cDNA after 24 hours growth in serum free medium. Isomorphic forms of apo A-I were resolved by IEF and detected by immunoblot analysis. Proapo A-I (pro) is 25% and mature apo A-I (m) is 75% of the staining material as assessed by scanning densitometry. Cathode and anode are indicated by (-) and (+) symbols respectively.  84  in both medium and cells. Approximately 2/3 of the apo A-I which was recovered from the dish was extracellular, suggesting that secretion was more efficient than in our other expression systems. Cellular apo A-I species were analysed by isolelectric focusing with immunoblot detection which confirmed that the apo A-I produced by transfected CHO cells was fully processed to the mature protein (Figure 22B). Scanning densitometay of the immunostained filter indicated that approximately 75% of the intracellular apo A-I was fully processed to the mature apo A-I providing evidence that, in this model system, propeptide proteolysis is an intracellular event. The remaining 25% of intracellular apo A-I was proapo A-I and no preproapo A-I was evident.  3.6. DISCUSSION The first major objective of this thesis was to establish eucaryotic expression systems for the production of recombinant human apo A-I in sufficient quantity for functional studies. My approach was to evaluate each system for its ability to synthesize and secrete apo A-I and to assess the function of the recombinant as sufficient quantity became available. Four eucaryotic expression systems have been employed: in vitro translation in rabbit reticulocyte lysate, transient expression in COS cells, and stable transfection of both BHK and CHO cells. Sequencing of the apo A-I cDNA established that this fragment encoded the entire apo A-I precursor and that it did not contain upstream sequences that could initiate translation. Subsequent in vitro translation studies established that the construct contained appropriate information for translation initiation and ER translocation of apo A-I in rabbit reticulocytes. This analysis also verified that the three molecular forms of human apo A-I: preproapo A-I, proapo A-I and mature apo A-I could be differentiated by their gel electrophoretic mobility following translation and subsequent proteolytic processing. The cellular transport of apo A-I after ER translocation was initially investigated in transiently transfected COS cells. Apo A-I secretion from these cells was very slow and the majority of the apo A-I synthesized remained intracellular. Since only proapo A-I was found in the medium of these cultures, it was concluded that signal peptide hydrolysis was necessary for apo A-I secretion. Evidence was obtained for a large intracellular pool of apo A-I following transfection and for rapid degradation of newly 85  synthesized apo A-I. This suggested that more than one kinetic pool of apo A-I was present in the transfected COS cell. Furthermore, a portion of the cellular apo A-I was preproapo A-I, indicating that signal peptide processing was incomplete. Part of the explanation for this finding may be that the rate of processing is affected by the level of apo A-I expression achieved in transient transfection. Stoffel and Binczek (1991) have recently described the expression of human apo A-I in COS cells using a minigene expression construct which retained the apo A-I promoter region under the control of the CMV promoter-enhancer. A mutant construct which resisted signal peptidase action was also described. Their studies showed that, as indicated here, cleavage of the signal peptide was necessary for secretion of apo A-I. However, in contrast to the results presented here, the signal peptide of the wild type protein was completely hydrolysed when expressed in this construct. Incomplete signal peptide hydrolysis has been previously demonstrated using apo A-I minigene constructs in C127 cells (Roghani and Zaimis, 1988). Therefore, the presence of an unprocessed signal sequence does not appear to be unique to the COS cell system. The evidence from this and other studies suggests that apo A-I signal peptide retention may be related to the intracellular level of protein. A high level of apo A-I synthesis may saturate the capacity of the cell to efficiently hydrolyse the signal peptide and/or translocate the protein at the ER membrane. The slow rate of apo A-I secretion from COS cells may, in part, reflect slow transport out of the ER. However, proapo A-I is still a large proportion of the total protein secreted by these cells (30%), suggesting that transport through other compartments might also be slowed as a consequence of high level apo A-I expression, or as a consequence of the transfection itself. Although apo A-I synthesis was extensive, degradation was the eventual fate of much of the apo A-I produced. Previous reports of apo A-I expression have not addressed this alternate fate of the expressed apo A-I. Many of these expression systems did not provide sufficient apo A-I for functional analysis, perhaps because the majority was degraded intracellularly. Transient transfection could not provide sufficient material for structure function studies, since the secretion of the protein was so inefficient, especially in the absence of serum. This sytem did provide a rapid means of identifying cellular and secreted apo A-I species at the analytical level. The assembly of HDL precursor lipoproteins has not been conclusively demonstrated within cells (Vance and Vance, 1990). Only apo B containing lipoproteins have been shown to assemble  86  intracellularly and  only in  hepatocyte systems. The remaining apolipoproteins are believed to be secreted  in soluble form and assemble into lipoproteins outside of the cell. Since COS cells do not normally produce any of the soluble apolipoproteins, they represent a potential model for testing the ability of apo A-I to form lipoprotein in the absence of other apolipoproteins. Secreted proapo A-I associated exclusively with HDL indicating high affinity for this lipoprotein fraction, in agreement with other studies (Dixon et a!, 1989; Hussain et a!, 1991). In contrast to Hussain et a! (1991), however, we did not find apo A-I associated with L,DL, although this may reflect the low levels of this lipoprotein in fetal bovine serum. Our inability to isolate apo A-I complexes from serum-free medium reflects both the low quantity secreted and the large losses in our attempts to isolate them in concentrated form. The most striking finding in our attempts to stimulate apo A-I secretion from COS cells was the effect of human lipoproteins. The observed effects of the lipoproteins might be explained by their opposing influences on cellular lipid. LDL, which delivers cholesterol to cells, stimulated apo A-I secretion, whereas HDL, which promotes cholesterol efflux, reduced apo A-I secretion by the transfected cells. COS cells, in contrast to hepatocytes, appear to have an extremely limited capacity to synthesize the lipid components required for lipoprotein assembly and vesicular transport. The inability to secrete large quantities of apo A-I, particularly in serum free medium, may indicate that the cells ability to assimilate lipid has been exhausted by the high level of apo A-I synthesis. Once this has occurred, the cell may be incapable of further secretion and the apo A-I may then be degraded. Similar results might be expected from other extra-hepatic cells in culture and is supported by the observations of Lamon Fava et a! (1987) who noted that more apo A-I was associated with lipid if the cells were preincubated with a phospholipid emulsion. The covalent phosphorylation of apo A-I is of interest but its biological significance remains unclear. We could demonstrate phosphorylation in COS cells in spite of the low level of secretion. It may be that this process is related to events other than secretion, perhaps as a covalent marker for intracellular degradation (Stadtman, 1990). Cellular retention of the large majority of apo A-I radiolabel prompted an assessment of the subcellular localization of apo A-I in the transfected cells. Since apo A-I does not undergo post translational glycosylation, lectin markers for these organelles will not reflect the location of the 87  overexpressed protein but rather the location of other, glycosylated proteins within the organelle. Apo A I accumulations were found in both the ER and in the Golgi apparatus of transfected COS cells. Localization to the ER can be accounted for by the impaired translocation. It was suspected, but not verified experimentally, that the differences in staining pattern represented cell-to-cell differences in the level of apo Al expression. In general, however, the immunotluorescence pattern was consistent with the biochemical observations, suggesting marked expansion of the ER. The level of apo A-I accumulation was sufficient to impair ER function markedly, since the hydrolysis of the signal peptide was incomplete and the rate of secretion was slow. We could not exclude the possibility that the observations reflected the net consequence of two distinct transfected cell populations: one which accumulates apo A-I exclusively in the ER and is then incapable of further signal peptide hydrolysis, and a second which displays apo A-I fluorescence in the Golgi apparatus and processes and secretes the protein efficiently. This cell to cell heterogeneity may reflect either heterogeneous expression of apo A-I or varying transfection efficiencies. BHK cells stably transfected with the apo A-I cDNA provided several advantages over the transiently transfected COS cell. Selected BHK clones expressed apo A-I to high levels since gene amplification occurs in the region of the DHFR gene under the influence of methotrexate (Shimke et at, 1987). The apo A-I cDNA will coamplify under these conditions. However, even with high level expression, signal peptide hydrolysis in these cells was complete. Intracellular apo A-I was proapo A-I, indicating that propeptidase activity was inefficient or not present within these cells and this observation provided the opportunity to study the role of the propeptide on cellular transport and processing of apo A-I (see Chapter 4). Although apo A-I transcription was under the influence of the mMT-I promoter in BHK cells, induction conditions did not increase the quantity of apo A-I recovered in the medium unless FBS was present. This provided additional evidence for the role of serum factors, possibly lipids, in the regulation of apo A-I synthesis and secretion. Under optimal conditions, apo A-I was secreted rapidly for 2-4 hours, but slowed markedly thereafter, when degradation was increasingly apparent. Immunofluorescence studies indicated that the cellular apo A-I in BHK was Golgi-associated, reflecting more efficient secretion than in COS cells. These findings were supported by pulse-chase data. 88  Since the fluorescence was confined to the Golgi, we believe that the site of apo A-I degradation in these cells is at or distal to this organelle in the constitutive secretory pathway. Despite the improvements in the efficiency of cellular processing and secretion in the BHK expression system, these cells were less satisfactory for large scale production of recombinant protein since, under serum free conditions, secretion was essentially undetectable and cell viability was markedly reduced. We turned to the CHO cell as a cultured cell with well documented growth characteristics in defined serum free medium. Stably transfected cells expressing apo A-I from the cDNA encoding preproapo A-I contained predominantly mature apo A-I. Only a minor component of the intracellular apo A-I pool retained the propeptide and preproapo A-I was not detected. This is the first demonstration of intracellular apo A-I propeptide hydrolysis. Although it has previously been shown that CHO cells do catalyse the conversion process (Mallory et al, 1987), it was presumed to be extracellular. The overall expression level in CHO was lower than in BHK since the selection conditions did not promote gene amplification. CHO cells produced approximately 25-30% of the apo A-I mass of the corresponding BHK clone. However, secretion was much more efficient in CHO since approximately 2/3 of the apo A-I in each dish was found in the medium. The secretion efficiency was also evident in serum free conditions, and suggested that intracellular propeptide hydrolysis may be an additional factor promoting secretion of apo A-I from other cells (eg. hepatocytes) which are known to contain this activity. This expression system allowed for the production of apo A-I under serum free conditions for 57 days, without appreciable loss of secretion efficiency or cell viability. We therefore chose to produce wild-type and mutant apo A-I in CHO cells for further functional studies (Chapter 5). In conclusion, these studies showed that apo A-I was expressed from the cDNA as preproapo A I and was translocated across the ER membrane coincident with the prepeptide hydrolysis to form proapo A-I. Signal peptide hydrolysis was required for secretion from cultured cells expressing the apo A-Iwt cDNA. Transiently expressing COS cells synthesized relatively large quantities of apo A-I (sufficient for western blot detection), but secreted only small amounts of the protein (5-10 ng/ml/24 hours). In addition, signal peptide hydrolysis of the intracellular pooi of apo A-I was incomplete. Evidence was provided indicating that efficient secretion of apo A-I may be influenced by the provision 89  of lipoprotein to the cells. Interestingly, LDL was the most stimulatory of the lipoproreins, perhaps suggesting that lipoprotein internalization and provision of intracellular membrane lipid might enhance apo A-I secretion. When the same cDNA construct was incorporated by stable transfection into BHK cells, a cell line expressing proapo A-I was obtained. The isolation of this cell line provided a model system for investigation of the role of the apo A-I propeptide during intracellular trafficking and constitutive secretion. Finally, CHO cells which had incorporated the apo A-Iwt cDNA were also isolated and it was demonstrated that these cells, grown in serum free medium, expressed as much as 130 ng/ml/24 hours of apo A-I. These cells hydrolysed the apo A-I propeptide as an intracellular modification, thus secreting only mature apo A-I. This system was subsequently utilized for the production of mutant recombinant apo A-I (Chapter 5).  90  4. THE APO A-I PROPEPTIDE AND INTRACELLULAR TRAFFICKING 4.1. OVERVIEW  The functional role of the apo A-I propeptide in intracellular movement and secretion of the protein was investigated by expressing the human cDNA with or without the propeptide segment in eucaryotic cells. Deletion of the propeptide coding segment from the cDNA encoding preproapo A-I was achieved by oligonucleotide-directed mutagenesis and was confirmed by sequencing of the altered DNA. The modified segment was then subcloned into the eucaryotic expression vectors pCMV5 and pNUT for transient and stable transfection studies, respectively. COS cells were used as host cultures for transient transfection studies and BHK cells were used to establish stable lines expressing the respective cDNA constructs.  4.2. RESULTS 4.2.1. Transient Expression of AIdPRO in COS Cells Initial investigations of the role of the apo A-I propeptide were performed in transiently transfected COS cells. In these experiments, transfected cells were pulse-labelled with  [35  S]methionine  for 30 minutes and the subsequent fate of the apo A-I radiolabel was observed during unlabelled methionine chase incubations. Approximately one-half of the biosynthetically labelled apo A-I in cells transfected with apo A IdPRO cDNA was degraded within 12 hours of  initiating  the chase (Figure 23A, upper panel). In the  same experiment, the 1t 12 of the wild type protein was approximately 8 hours. The quantity of apo A-I radiolabel in cells transfected with the wild-type construct was lower at all time points than the corresponding value in apo A-IdPRO. The differences, however, were not of sufficient magnitude to establish convincingly that the propeptide had a significant effect on cellular expression of apo A-I. The intracellular apo A-I in AIdPRO transfectants represented a greater proportion of the total apo A-I per dish than that produced by Alwt transfected cells (Figure 23A, bottom panel). A correspondingly smaller proportion of the total apo A-IdPRO in each dish was recovered in the medium when compared with apo A-Iwt (Figure 23A, middle panel). This was the first indication that loss of the propeptide might alter secretion of apo A-I. 91  A 100—  T:TAL  c— AIdPRO  0-  MEDIUM  50-  ‘  I  I  I ‘I  I ‘I  CELLS  !‘  ::::::E::::: 100  C t4  C_.  0—  I  0  I  16  8  I  24  CHASE TIME (hrs)  Figure 23A. Removal of the propeptide from the apo A-I sequence delays its secretion from transfected COS cells. COS cells were transfected with cDNA for apo A-Iwt (AIwt) or apo A-IdPRO (AIdPRO) and pulse labeled with [35 Slmethionine. Cells and media were recovered after the indicated chase interval and apo A-I was isolated by immunoabsorption. Apo A-I radiolabel was quantitated by liquid scintillation and expressed as percent of apo A-I radioactivity per dish (TOTAL) at the initiation of the chase (t = 0), or as percent of the apo A-I cpm per dish recovered in the MEDIUM or the CELLS. Apo A-IdPRO was secreted more slowly than apo A-Iwt.  92  AIdPRO  AIwt 0  2  4  8  12  16 24  0  2  4  8  12  0  16 24  66.2-  2  4  8  12  16 24  0  2  4  8  12  16 24  66.246-  M  M  31 -  21.5-  1  1.• MEDIUM  31-  ——----  CELLS  21.5-  JL  MEDIUM  CELLS  Figure 23B. Removal of the propeptide from the apo A-I sequence delays its secretion from transfecled COS cells. Immunoisolates were analysed by SDS-PAGE with autoradiography and showed that MEDIUM apo A-I was a single species in both transfectants. In both transfection experiments, CELLS contained an apo A-I doublet. Numbers along the top of each autoradiogram represent chase time in hours. Autoradiograms were overexposed to permit visualization of apo A-I in the medium. This exposure obscures the doublet present in cell lysates of AIdPRO, which were evident at shorter exposure times. 93  C  IEF —  —ø  +  SDS+  W  31 Kd  31 Kd  0  AIwt  AIdPRO  Figure 23C. Removal of the propeptide from the apo A-I sequence delays its secretion from transfected COS cells. Pulse-labelled cell lysates were mixed with purified plasma apo A-I, resolved by twodimensional gel electrophoresis and visualized by Coomassie Blue staining and autoradiography. Horizontal and vertical arrows indicate the direction of IEF and SDS-PAGE, respectively. The position of mature plasma apo A-I marker is indicated on each autoradiogram (dotted circle). The position of a 3lkD molecular mass marker in the SDS-PAGE dimension is show to the left of each panel.  94  Immunoisolates from cell lysates and media were also analysed by SDS-PAGE and autoradiography. As indicated in Chapter 3, two apo A-I species, differing in molecular mass by approximately 2000 daltons, were evident in both the AIwt and AIdPRO transfected cells. Only the smaller species was detected in the medium of either transfectant (Figure 23B). The band intensity in the cell lysates was consistent with secretion or degradation of apo A-I from the cellular pool during the chase interval. However, since the intensity of the apo A-I band in the medium did not increase correspondingly with time, the results suggested that degradation was much more predominant than secretion. This was also evident in the quatitative analysis (Figure 23A). Apo A-IdPRO accumulated in the medium more slowly than did apo A-Iwt and suggested that apo A-I secretion was impaired by the absence of the propeptide segment. The intracellular apo A-I species in these studies were further resolved by two-dimensional gel electrophoretic analysis of radiolabelLed cell Lysate (Figure 23C). The smaller apo A-I species in cells trarisfected with AIdPRO comigrated with mature plasma apo A-I used as electrophoretic marker. The larger, more basic form of apo A-IdPRO displayed the size and charge characteristics expected for pre(dPRO)apo A-I. This species had higher molecular mass and was more basic than the mature apo A I. This suggested that, like the AIwt expression product analysed in parallel transfections, AIdPRO transfected cells contained some primary translation product from which the signal peptide was incompletely hydrolysed. Thus, in COS cells transfected with either apo A-I cDNA, a significant portion of the apo A-I produced retains its signal sequence, suggesting a functional abnormality of the ER in the transfected cells. Immunofluorescence analysis was used to compare the cellular location of apo A-IdPRO and apo A-Iwt accumulations. As noted in earlier experiments (see Figure 17), cells expressing apo A-Iwt exhibited low level immunofluorescence throughout the cytoplasm, but greater intensity was found in the juxtariuclear region (Figure 24A). The low level fluorescence pattern was coincident with the lectin marker CoriA (Figure 24B), suggesting that at least a portion of the expressed apo A-I was retained in the ER. Cells expressing apo A-IdPRO displayed much more intense fluorescence and a consistently reticular distribution (Figure 24C). The relative fluorescence intensity suggested that AIdPRO cells contained more apo A-I than AIwt cells. The immunofluorescence in AIdPRO colocalized with the 95  Figure 24. Apo A-IdPRO is retained in the ER of the transfected COS cell. COS cells were transfected with pCMV5-AIwt (A,B) or with pCMV5-AIdPRO (C,D) and processed for intracellular apo A-I immunofluorescence (A,C). Preparations were counterstained with TRITC-labeled ConA (B,D) to demonstrate the extent of endoplasmic reticulum. Apo A-Iwt was found predominantly in the juxtanuclear region (A) and did not colocalize with the ER marker. Apo A-IdPRO was distributed throughout the cytoplasm (C) and colocalized with the ER marker. Bar represents lQu m.  96  scence analysis suggested that apo ConA marker for the ER (Figure 24D). Therefore, the immunofluore AIwt and that the site of retention was the AIdPRO is retained to a greater extent in COS cells than apo ed that secretion of apoAIdPRO ER. The findings were consistent with the biochemical data and indicat dPRO cDNA demonstrated was slower than apo A-Iwt. However, cells transfected with wild type or r the observed changes were due considerable ER functional impairment. We could not ascertain whethe to the loss of the propeptide, or due to the method of transfection.  4.2.2. Stable expression of apo A-hIPRO in BHK cells apo A-I propeptide Observations in the transient transfections suggested that the loss of the er, the results of expression of might reduce the efficiency of its cellular transport and secretion. Howev ed cell function. To separate the wild type protein in this experimental model suggested seriously impair re, we established BHK the influences of the experimental model and the influences of apo A-I structu cell lines which stably expressed the human apo A-IdPRO sequence. ate transfection with Approximately 30 BilK clones were generated following calcium phosph A-I by immunoblot analysis. pNUT-A-IdPRO. These clones were screened for the ability to secrete apo t level of apo A-I secretion was One third of all clones secreted detectable levels of apo A-I. The highes is. The expression of apo A-I by found in line BHK-AJdPRO-C4 which was selected for detailed analys the cDNA encoding this cell line was compared to BHK-AIwt-B5, which expresses apo A-I from preproapo A-I. y established by The molecular species of apo A-I produced by BHK-AIdPRO-C4 was initiall electrophoretic analysis of immunoabsorbed material from  1’ Simethionine pulse-labelled cells. The  apo A-I species with M of autoradiogram in Figure 25A indicated that each cell line produced a single nt transfectants which approximately 28,000 daltons. This was in striking contrast to COS cell transie cells (rn-AT in Figure 25) contained two distinct molecular species. The apo A-I in BHK-AIdPRO-C4 Two-dimensional appeared slightly smaller than in line BHK-AIwt-B5 (p-Al in Figure 25). the apo A-I produced in electrophoretic analysis by the OFarrell technique (Figure 25B) indicated (hat the cDNA sequence, the apo each case was also homogeneous with respect to charge. As predicted from the mature plasma apo A-I (Figure A-I expressed by clone BHK-AIdPRO-C4 (m-AI) co-migrated with 97  IEF  B  A  SDS+  925 662  —  45  M  31  31-  31-  21.5-  p-Al  p-Al  rn-Al  rn-Al  Figure 25. BHK clones contain proapo A-I (p-A!) or mature apo A-I (rn-Al). BHK clones expressing the precursor cDNA with (pNUT-AIwt) or without (pNUT-AIdPRO) the propeptide segment were pulselabelled with [ S] methionine. Apo A-I was immunoabsorbed from cell lysates, mixed with purified plasma apo A-I and fractionated by SDS-PAGE in 15% polyacrylamide (panel A) or by two dimensional gel electrophoresis (panel B). Gels were stained with Coommassie Blue to identify the mature plasma apo A-I (dotted circle) and processed for autoradiography to visualize the endogenously labelled BHK cell product. The apo A-I synthesized by the cells containing the full-length precursor cDNA (p-Al) migrates as proapo A-I. Cells expressing the apo AIdPRO cDNA synthesize apo A-I comigrating with the mature form isolated from plasma (rn-Al). The position of the mature apo A-I marker was oriented by overlay of the autoradiogram on the stained gel.  98  25B). Amino-terminal sequence analysis was performed on the protein purified from serum-free culture medium of this cell line. The first six residues of m-AI protein were identical to the apo A-I purified from human plasma. No other amino terminal sequences were detected. These results established that removing the propeptide coding sequence does not significantly affect the rate or fidelity of signal peptide hydrolysis and that BHK-AIdPRO-C4 secretes only mature apo A-I. This cell line could therefore be used to investigate the consequences of propeptide deletion on apo A-I trafficking and secretion in BHK cells. Northern blot analysis indicated that cells expressing the AIdPRO construct contained 2-2.5 fold more apo A-I rnRNA than cells expressing the wild-type cDNA. For clarity, BHK cells expressing proapo A-I (clone BHK-AIwt-B5) were called p-AT, while BHK cells expressing mature apo A-I (clone BHK-AIdPRO-C4) were called m-AJ. This nomenclature was used to identify both the cell line and the apo A-I species it produced.  4.2.3. Comparison of Apo Al Synthesis and Secretion Rates in BHK clones p-Al and rn-A!. Apo A-I and protein synthetic rates were measured in p-Al and rn-Al cells from short term labelling with r S]methionine. Radiolabel incorporation into apo A-I and TCA precipitable protein of 5 cell lysates was approximately linear over the 30 mm study period in the cell lines (Figure 26). The initial rates of apo A-I synthesis did not differ between p-Al and m-AI (Figure 26A), nor did total protein synthetic rate (Figure 26B). However, the mass of apo A-I in the cell lysates was much lower for p-Al (379 ± 28 ng/dish, n= 3) than for m-AI (645 ± 14 ng!dish, n 3). Therefore, the presence or absence of the propeptide segment affects the cellular level of apo A-I in the BHK clones without affecting the rate of synthesis. The quantity of apo A-I radiolabel secreted into the medium during these short term experiments was too low for reliable estimation. To ascertain whether differences in the rate of apo A-I secretion might account for the differences in cellular apo A-I mass, we determined the levels of cellular and medium  [ Simethionine  labelled apo A-I during twelve hour labelling experiments. Apo A-I secretion into the medium was linear and the rate did not differ between p-Al and rn-Al (Figure 27A). Therefore, the observed differences in cellular apo A-I mass were not due to marked differences in apo A-I secretion. However, as with the mass measurements, more radiolabelled apo A-I accumulated in rn-Al cells than in p-AT cells (Figure 99  6  4 I (1)  2 0 4  0  cL  C)  b x  0 0  20  10  30  TIME (mns)  Figure 26. Apo A-I and protein synthetic rates in BilK lines p-Al and rn-Al. Cell lines rn-Al (•) or p Al O) were incubated for the indicated time with [35 Slmethionine and harvested by cell lysis. Incorporation of radiolabel into apo A-I (A) was measured following immunoabsorption and SDS-PAGE as described in MATERIALS AND METHODS. Incorporation of S into total protein (B) was measured by TCA precipitation. Data points represent mean ± S.D. of triplicate dishes. 100  32EDIUM,,,,/  TI ME (hours)  Figure 27. Apo A-I secretion and accumulation in BHK lines p-Al (0) and rn-Al (•). BHK cells expressing p-AT or rn-Al were continuously labelled with Simethionine (lOOuM). Apo A-I radioactivity secreted into the medium (A) or retained by the cells (B) was determined by liquid scintillation counting after immunoabsorption and SDS-PAGE resolution as described in MATERIALS AN]) METHODS. Radiolabel in total cell protein (C) was determined by TCA precipitation. Each data point represents mean ± S.D. of triplicate dishes.  101  27B). The difference between the cell lines was selective, since radiolabel in total cell protein followed the same time course in the two cell lines (Figure 27C). Since the initial rates of synthesis were identical and secretion rates were not different, the observed differences in cellular apo A-I might be accounted for by selective degradation of proapo A-I in line p-Al. Degradation and secretion were examined in 12 hour chase incubations after 30 mm pulse incorporation of  [3 S]methionine.  Figure 28 shows the distribution of apo A-I radioactivity between cells  and medium during the chase period. Proapo A-I was released into the medium more rapidly than mature apo A-I in the early portion of the chase (Figure 28A, upper panel). After two hours chase there were minimal differences in the absolute quantity of  [35  S]apo A-I recovered in the medium of the two  cell lines, but since less radioactivity had incorporated into p-Al during the pulse, the portion secreted was greater than in rn-Al (80% vs 20%). Medium apo A-I radiolabel decreased in both cell lines after 4 hours chase. The cellular residence time of p-Al (30-40mins) was much shorter than m-AI (approximately 3 hours; Figure 28A, lower panel) due to more rapid secretion of p-Al early in the chase. However, the recovery of apo A-I as a percentage of total apo A-I radioactivity was lower (by 15-20%) from p-Al than from rn-Al during the first four hours, suggesting that p-Al was degraded more rapidly. At chase times longer than four hours, the unaccounted portion of radiolabel (degraded) did not differ between the two cell lines. No partial degradation products were detected by autoradiographic assessment of cells or medium of either cell line following SDS-PAGE (Figure 28B). However, proteolysis may have released products which were too small for recognition by the polyclonal antibody used. The more rapid disappearance of proapo A-I compared with mature apo A-I suggested that the susceptibility to intracellular degradation might be related to the presence of the propeptide. Alternatively, changes in the distribution of apo A-I among subcellular compartments, as a consequence of the propeptide deletion, may have delayed degradation of the rn-Al protein.  4.2.4. Intracellular Localization of Apo AT Accumulations in BHK Clones. Immunofluorescence analysis was used to localize the apo A-I accumulations within p-Al and m Al cell lines. The distributions of proapo A-I (in cell line p-Al) and mature apo A-I (in cell line rn-AT) differed markedly (Figure 29). In p-Al cells, apo A-I fluorescence decorated the juxtanuclear region 102  A.  B.  p-Al  rn-Al  kDa  MEDIUM -45 17  -31  21.5  S  (-)  CHASE (hrs)  2  CELLS  0 1  2 4 8 12  0 1  2 4 8 12  92.5 66.2 — -  TIME (hours)  — —  45 31  -  -  21.5  S  Figure 28. Time course of apo A-I degradation and secretion in BHK cell clones expressing apo A-I cDNAs. Cells were pulse-labelled with [35 Sjmethionine and chase incubated with medium containing 2mM methionine. After the indicated chase time, cells and medium were harvested and apo A-I was isolated by immunoabsorption. A- Immunoisolates were mixed with purified plasma apo A-I, resolved by SDS-PAGE and the apo A-I band excised from the gel and counted. Each point represents the mean ± S.D. of triplicate dishes. Where error bars are not visible, they are within the margins of the symbol. B- Aliquots were resolved by SDS-PAGE and detected by autoradiography. Chase time is indicated above or below the lane between the autoradiographic panels. S = Apparent molecular mass markers, in kilodaltons (kDa). p-Al O) and rn-Al •) indicate BHK cells expressing proapo A-I or mature apo A-I, respectively. 103  Figure 29. Immunofluorescence localization of apo A-I in transfected BHK cell lines. Apo A-I expression was induced with 100 i M zinc sulfate for four hours prior to cell fixation. Cells were permeabilized with Triton X-100 and incubated sequentially with mouse anti-human apo A-I (6B8) and FITC-goat anti-mouse IgG. Golgi zones were stained with TRITC-conjugated WGA (panel B) and ER was visualized with TRITC-ConA (panel D). Preparations A and B were transfected with pNUT-AI (cell line p-Al). Preparations C and D were transfected with pNUT-AIdPRO (cell line rn-Al). A and C were photographed for visualization of FITC (apo A-I), B and D represent TRITC-conjugated lectin. Bar represents lQum. 104  (Figure 29A), and co-localized with the lectin marker WGA (Figure 29B). This lectin identifies the terminal carbohydrate residues which are added to glycoproteins in the Golgi apparatus. Conversely, in rn-Al cells, apo A-I fluorescence was found in more numerous, intense foci near the cell periphery (Figure 29C). These structures stained with the ER marker Con A (Figure 29D) which identifies marmose-rich structures. However, the characteristic reticular distribution of the ER was not observed in these cells. The cytologic observations provided further evidence that removing the propeptide from apo A-I altered the rate of its intracellular transport, perhaps by slowing its movement from the ER to the Golgi. To delineate further the subcellular location of apo A-I in the BHK clones, the cells were processed for immunogold electron microscopy. Cryo-ultramicrotomy with immunogold labelling demonstrated that proapo A-I was present in the Golgi regions of line p-Al (Figure 30A). Gold label, indicating the presence of apo A-I, was found in the stacked membrane structures characteristic of the dictyosome. No unusual structures were observed in p-AT cells viewed at low magnification (Figure 30A, inset). In contrast, rn-AT cells contained numerous electron dense structures which were visible at low magnification (Figure 30B, inset) and were distributed in a pattern similar to the apo A-I deposits observed at the light microscopic level by immunofluorescence. At higher magnification (Figure 30B), these structures were found to contain immunogold label and were surrounded by a membrane. Control sections prepared from either cell line and incubated with non-immune serum showed negligable gold labelling. While neither the immunofluorescence nor the immunogold studies alone are conclusive, taken together, the light and electron microscopic findings suggest that the organelles containing apo A-I in line rn-AT may derive from the ER.  4.3. DISCUSSION The synthesis and secretion of recombinant human apo A-I has been studied in a number of eucaryotic cell systems (Mallory et at, 1987; Fennewald et al, 1988; Lamon-Fava et at, 1987; Ruiz-Opazo et at, 1988; Roghani and Zannis, 1988; Forte et at, 1990). Secretion levels have been low in many cell types, particularly under serum free conditions. However, the intracellular location and mechanism of 105  I.  F-.  Figure 30A. Immunogold localization of apo A-I in ultrathin cryosections of transfected BHK cell lines. Apo A-I expression was induced by 4 hour incubation in 1O M zinc sulfate prior to glutaraldehyde fixation. Ultrathin cryosections were prepared and apo A-I was labelled by sequential incubation of the grids with mouse monoclonal antibody 6B8 and goat-anti-mouse IgG conjugated with lOmn colloidal gold. Control sections incubated with normal mouse serum in place of 6B8 showed negligible gold labeling in either p-AT or rn-Al cells. The micrograph indicates 10 mu gold accumulations in Golgi (G) zones of cell line p-A! (pNUT-A1). Inset shows low magnification of a single p-AT cell. Arrowheads indicate the location of lOnm gold deposits. Arrow shows the stacked membrane structures of the Golgi apparatus. Bar corresponds to 0.5 m in high magnification view and u m in low magnification inset.  106  aj  Figure 308. Immunogold localization of apo A-I in ultrathin cryosections of transfected BHK cell lines. Large, membrane bound vesicles in the periphery of cells of line rn-A! (pNUT-AIdPRO) labelled extensively with lOimi gold (arrowheads). The lack of gold particles over the mitochondria (M) confirms the specificity of the immunolabelling for the unusual vesicular structures. Arrow indicates the membrane surrounding the apo A-I accumulations. Inset shows low magnification view of a single rn-AT cell. Bar corresponds to 0.5w m in high magnification view and i m in low magnification inset.  107  accumulation have not been addressed. Except in CHO cells (Mallory et a!, 1987; Forte et at, 1990), the eucaryotic cells expressing the full length cDNA secrete proapo A-I. This is consistent with the generally accepted view that the physiologic conversion of proapo A-I to mature apo A-I occurs in the plasma (Scanu, 1987). However, the function of the propeptide and its role in apolipoprotein metabolism have not been established. In COS cells transfected with the apo AIdPRO cDNA, I found that the altered gene product was secreted more slowly and was also somewhat more resistant to degradation. Apo A-IdPRO was retained by the COS cells to a greater extent than apo A-Iwt, and the intracellular structures containing apo A-IdPRO resembled the ER by lectin staining criteria. However, these observations were equivocal since even cells expressing the wild-type protein demonstrated ER abnormalities. I then demonstrated that BHK cells expressing apo A-I do not have evidence of the ER functional impairment found in COS cell transfectants. These cells completely hydiolysed the signal peptide of apo A-IdPRO at the predicted site, releasing mature apo A-I into the ER lumen cotranslationally. These cells did not hydrolyse the propeptide intracellularly as assessed in pulse-labelling studies. However, serum free collection.s of the apo A-I produced by p-Al cells did contain detectable mature apo A-I (20%) by N-terminal sequence analysis. The BHK cell was used to model the synthesis, trafficking and secretion of proapo A-I by eucaryotic cells which constitutively secrete apo A-I. Trafficking and secretion of apo A-Iwt and apo A-IdPRO has been previously investigated in AtT-20 cells (Fennewald et at, 1988). However, the accumulation of mature apo A-I in cells expressing the AIdPRO construct (rn-Al) was not reported. My data indicates that the absence of the propeptide alters the rate of cellular transport of apo A-I in BHK cells. The studies described here also demonstrated some differences in the level of degradation of proapo A-I compared with apo AIdPRO in BHK cells. Cellular proapo A-I was found in the Golgi apparatus suggesting that processing by this organelle may precede degradation. Post-translational modifications of this apoprotein have been described (Hoeg et at, 1986; Beg et al, 1989) which may play a role in apo A-I secretion. Based on observations in BHK cells, secretion may be a minor fate of apo A-I expressed by non-hepatic cells. An important, and perhaps, principal fate may be degradation. The accumulation of apo A-IdPRO in cellular structures that differ morphologically from the 108  Golgi apparatus, and which retain some properties of the ER, suggests that the deletion of the propeptide sequence might affect the rate of transport from the ER to the Golgi. Although I did not use ultrastructural cytological markers to determine the subcellular origin of these membrane bound vesicles, the light microscopic findings suggest that the subcellular structures in which apo A-I accumulates in rn Al are derived from the ER. Therefore, I hypothesize that when apo A-I is synthesized as proapo A-I, it is transported through the ER into the Golgi apparatus along the constitutive secretory pathway, where it is secreted or is degraded. Engineered deletion of the propeptide, however, disrupts this transport causing much of the apo A-I to remain in a pre-Golgi location and may slow the entry of apo A-I into the cellular compartment in which degradation occurs. If the membrane bound structures that I observed in BHK line rn-Al are indeed derived from the ER, they may represent remnants of the organelle that have been disrupted by accumulation of large quantities of apo A-I. The exact mechanism by which the propeptide sequence may direct intracellular trafficking of apo A-I is not known. However, it is possible that the secondaiy structure of this region serves to mask a lipid binding domain near the amino terminus, thus reducing the potential for the apo A-I to bind to the membranes of intracellular organelles. Alternatively, the propeptide may act as a recognition sequence for a process which is sensitive to the lipid status of the cell. When cellular lipid is limiting, for instance in a non-hepatic cell grown in lipid poor medium, much of the protein may be degraded. Since the absence of the propetide sequence appears to delay transport early in the secretory pathway, I predict that the hydrolysis of the propeptide sequence might occur during the latter stages of secretion, but prior to exit from the cell. This is consistent with evidence from CHO cells where cellular processing has been demonstrated (Mallory et a!, 1987; Forte et at, 1990). However, the specificity of this hydrolysis for the apo A-I propeptide must be questioned since CHO cells should have no need for such an activity. I have also observed that BHK cells may also possess extremely low levels of propeptide hydrolytic activity, although as in other eucaryotic cells (Lamon-Fava et at, 1987; Ruiz-Opazo et at, 1988; Roghani and Zannis, 1988) the majority of the apo A-I expressed form the precursor cDNA is secreted as proapo A-I. The role of the propetide in the processing of the apo A-I gene product has been previously studied by Gordon and coworkers. Initial cell-free translation studies (Folz and Gordon, 1987) suggested that removal of the apo A-I propeptide altered the interaction of preproapo A-I with eucaryotic signal 109  peptidase. However, subsequent studies in the AtT-20 cell (Fennewald el al, 1988) indicated that the apo A-I propeptide does  not  play a functional role in secretion of this apolipoprotein. This model system is  distinctly different from the BHK cells used in our investigations since the majority of the apo A-I in AtT-20 cells accumulated in post-Golgi dense core granules from which secretion could be stimulated by cAMP. The BilK cells described in our study are not capable of regulated secretion and thus secrete apo A-I constitutively. Recently, Kizer and Tropsha (1991) described a structural motif which directs proteins to the secretory granules of AtT-20 cells. Their study appears to identify an amphipathic helix motif in the Nterminal sequence of proteins which are targetted to the dense core granules of this cell line. This structure is extremely common in the apo A-I sequence, as predicted by the algorithms of Segrest and colleagues (1990; 1992). If these structural elements direct proteins to the regulated secretory pathway of the AtT-20 cell, it is not surprising that regulated secretion would predominate in this model. The preponderance of amphipathic helices in apo A-I would direct apo A-I in a maimer independent of the presence or absence of the propeptide. Conversely, our studies show that in the absence of a regulated secretory pathway, the apo A-I propeptide has a marked effect on cellular transport under conditions where apo A-I is overexpressed. Apo A-I movement from the ER to the Golgi is limited by deletion of this segment. This is physiologically relevant since secretion of apo A-I by hepatocytes and enterocytes is also constitutive and expression level may increase in these cells with physiologic changes. In summary, I have established eucaryotic cell lines which synthesize human apo A-I with or without its propeptide segment. Removal of the propeptide did not affect the fidelity of signal peptide hydrolysis but did reduce the rate of apo A-I secretion from the cell, causing much of the protein to remain within vesicular structures which appear, by some histologic criteria, to be derived from the ER. This suggests that the propeptide may be required for efficient transport from the ER to the Golgi apparatus. Proapo A-I was degraded in BHK cell cultures and deletion of the propeptide segment marginally reduced its susceptibility to degradation. This may reflect direct involvement of the propeptide in the degradation mechanism or may result from limited entry of apo A-IdPRO into the compartment in which degradation occurs. I conclude, therefore, that the apo A-I propeptide may regulate the intracellular transport and degradation of apo A-I in the constitutive secretory pathway. 110  5. LYS 107 AJND THE C-TERMINAL AMPHIPATHIC HELIX IN LCAT ACTIVATION BY APO A-I 5.1. Overview  Synthetic peptide studies have suggested that the amphipathic helices in apo A-I play an important role in its function. In addition, the potential importance of the “mobile hinge domain” of the apo A-I polypeptide has been suggested on the basis of LCAT inhibition studies using monoclonal antibodies directed against this epitope. Chemical modification of Lys residues in purified normal apo A I alter LCAT activation if the charge on the side chain is altered, although the conditions of modification may profoundly affect protein structure. LCAT activation was diminished in only one naturally occuring apo A-I mutant. This mutant lacks a single lysine residue (Lys ), within the hinge domain. The 107 available evidence supports the hypothesis that the mobile hinge region, and perhaps Lys , is involved 107 in the LCAT activation mechanism. However, this conclusion must be reserved, at the present time, because the most convincing studies, using the purified protein from plasma, may have utilized preparations contaminated with wild-type sequence. The approach chosen was to evaluate the functional importance of Lys 107 deletion by producing the protein as a recombinant in serum free eucaryotic cell culture (CHO cells) and assessing its ability to activate recombinant LCAT in vitro. Since the alteration of this one residue is predicted to alter the orientation of single class Y amphipathic helix in a polypeptide that contains two of these structural elements, the functional consequence of deleting an entire class Y amphipathic helix in a region of the molecule outside of the proposed hinge domain (mutant Dl, amino acid residues 220-241) was also investigated. Deletion mutagenesis of the helical region containing the hinge domain was also attempted. However, three separate mutagenic strategies have so far failed to generate this mutation.  5.2. RESULTS 5.2.1. Production and Identification of Mutant cDNAs  Mutagenesis was performed using the oligonucleotide primed site-directed approach. Potential mutants for the Lys 107 deletion were identified by restriction enzyme analysis. The deletion of the Lys 107 codon removes a single MboII restriction site from the apo A-I cDNA. Candidate plaques from the mutagenesis experiment were selected for sequence analysis on the basis of an altered MboII restriction 111  fragment pattern when compared to the wild-type cDNA. DNA sequence analysis of the resulting dK 107  mutant confirmed the directed deletion. Candidate plaques for the Dl mutation (deletion of Pro ° to Asr? 2 41 inclusive) were selected for sequencing based on high stringency hybridization with the mutagenic oligonucleotide. The presence of this deletion was also confirmed by DNA sequence analysis. Each cDNA was excised from the appropriate M13 RF DNA and subcloned into eucaryotic expression vectors.  5.2.2. Transfected COS Cells Produce the Respective Mutant Proteins COS cell transfection and pulse-labelling were used to identify the apo A-I produced from each  mutant cDNA. Apo A-I was concentrated by immunoabsorption from the labelled cell lysate and the resulting species were resolved electrophoretically and visualized by autoradiography. Cells transfected with expression vector without cDNA insert contained no apo A-I (Figure 31A, lane 1). Apo A-I expressed from the dK 107 construct produced an apo A-I doublet (lane 3) which was indistinguishable from the wild-type product (lane 2). This was expected since the loss of a single amino acid should not alter SDS-PAGE mobility and since I had previously observed signal peptide retention in COS transfectants. In both apo A-IdK 107 and apo A-Iwt, the larger polypeptide of the doublet represents apo A-I which has retained its signal peptide. The presence of the Lys 107 deletion was evident by isoelectric focusing of the labelled product (Figure, 31B). The wild-type pattern contains two bands (lane 1), separated by one charge unit which was predicted from the amino acid composition of the signal sequence. Since the apo A-I signal peptide contains a single basic residue (Lys at position -23), signal peptide hydrolysis results in a one charge unit shift to more acidic p1. Similarly, the molecular forms of apo A-I in the dK 107 mutant were also separated by one charge unit (lane 2). In addition both were shifted to more acidic p1 with respect to the corresponding wild type polypeptide, reflecting the loss of positive charge. However, the difference in mobility between mutant and wild-type appeared to be somewhat less than one charge unit (based upon the shift observed during signal peptide hydrolysis). Mixing the wild type and mutant preparations in one lane (Figure 31B, lane 3) confirmed that the loss of 7 Ly° does not result in the equivalent change in p1. 112  A  SDS /  31 Kd-  ‘  -  123  B  JEF —  -  123  ±)  Figure 31. Transfected COS cells express apo A-I mutants with the appropriate molecular characteristics. COS cells were transfected with the appropriate cDNA construct and were pulse labeled with [35 S]methionine as described in IvL&TERIALS AND METHODS. Labelled protein was harvested by cell lysis and apo A-I concentrated by immunoabsorption. Labelled species were resolved by polyacrylamide gel electrophoresis. . Apo A-I was not detected in cells transfected 107 A- SDS-PAGE separation of cells expressing apo A-IdK 107 (lane 3) resolved as doublets indicating with empty vector (lane 1). Apo A-Iwt (lane 2) and apo A-IdK incomplete signal peptide hydrolysis. The positions of a molecular mass standard (3lkD) and the anode (+) and cathode (-) are indicated. 107 (lane 2) are shifted cathodically . Isomorphic species of apo A-IdK 107 B- IEF analysis of apo A-IdK from the respective apo A-Iwt species (lane 1), as expected due to the loss of a positively charged amino 107 and Ly 23 do not possess acid. Mixing of apo A-Iwt and apo A-IdK 107 (lane 3) suggests that Lys equivalent charge (see text for discussion). The arrow indicates the position of mature apo A-I in the pH gradient. 113  C 1234 69-p 46-  Mr  30  14.3-  CM  Figure 31C. Transfected COS cells express apo A-I mutants with the appropriate molecular characteristics. SDS-PAGE analysis of mutant apo A-ID1. Apo A-ID1 (lane 2) and apo A-Iwt (lane 1) are both isolated as an intracellular doublet (C). Only the smaller form is found in the medium (M) after 2 hour chase incubation of either transfected cell population (lanes 3 and 4). Lane 3 = apo A-Iwt; lane 4 = apo A-ID1. Molecular mass (Mr) marker postions are shown at the left of the autoradiogram in kilodaltons.  114  23 and Lys 107 affecting the This may reflect differences in secondary structure in the vicinity of Lyc ultimate p1. The expression of apo A-ID 1 was also demonstrated in transfected COS cells. Biosynthetically labelled apo A-ID1 appeared as an intracellular doublet on SDS-PAGE (Figure 31C, lane 2). As with all other COS cell transfections, only the smaller form was found in the medium (lane 4), after 2 hour chase incubation. By this criterion, the larger species is preproapo A-ID 1 and the smaller species is proapo A ID1. Both preproapo A-ID1 and proapo A-ID1 were approximately 2 kD smaller than the corresponding wild-type species (lanes 1 and 3, respectively), reflecting the 22 amino acid deletion. Therefore, DNA sequencing of the mutant cDNAs and immunologic assessment of the proteins expressed in COS cells showed that the mutations had been introduced correctly.  107 and Apo AID1 5.2.3. Development of CHO Cell Lines Expressing Apo A-IdK The quantity of protein produced and the efficiency of secretion of apo A-I by COS cells were inadequate for most functional studies, based on observations with apo A-Iwt. Therefore, CHO cell lines expressing the apo A-IdK 107 and apo A-ID1 proteins were established. This cell type efficiently secreted the wild-type protein under serum free conditions (see Chapter 3.5) However, the conditions used for selection did not promote amplification of the apo A-I cDNA. Therefore, although secretion was more efficient in CHO, the overall level of apo A-I expression was lower than in BHK. In addition, the CHO clones responded poorly to culture conditions designed to induce expression (1O M zinc sulfate). Cells became rounded and refractory at approximately 5 M zinc sulfate but were microscopically unaffected when the concentration was reduced to 1z M. Unfortunately, the lower concentration did not improve apo A-I production. CHO cells lines expressing the dK 107 and Dl mutants were selected with Geneticin and screened for secretion of apo A-I by ELISA. Cells expressing the double mutants 107 dK /dPRO and Dl/dPRO were also generated. To establish the identity of the apo A-I species produced, medium and cell lysates were isolated following 24 hours growth under serum free conditions. The samples were resolved by 1FF or SDS-PAGE electrophoresis and detected by western blot analysis (Figure 32). As indicated in the preliminary COS cell experiments, the cells expressing the mutant cDNA 115  B  A pH 6  0  wt  S  dPRO  7 dK’° 107 dPRO dK  Dl  wt Dl dPRC 92.566.2-  proAlmAl-  .  —  -PrOMdK  3121.5—  -  pH4®  CMC  MC  14.4-  MCM  Figure 32. CHO cells express apo A-I mutants of the expected charge and size. Selected CHO cell clones were grown to near confluence and then changed to serum free medium. After a 24 hour purging incubation to remove bovine serum proteins, 48 hour medium collections were performed and cell lysates were prepared. Apo A-I was concentrated by immunoabsorption and separated by gel electrophoresis. A- IEF analysis of cell lysate (C) and medium (M) preparations between pH 4 (+) and pH 6 (-). proAl 107 indicate the positions of the apo A-I species with the propeptide. Note that these species and proAldK 107 indicate the postions of the respective are present only in the cell lysate (C) lanes, mAT and mAIdK mature polypeptide forms. S = purified plasma apo A-I standard. ) 41 -Asi 20 B- SDS-PAGE analysis of medium preparations from cells expressing the apo A-ID1 (Pr deletion mutant (Dl) and the apo A-ID1/dPRO double mutant (DIdPRO). Numbers to the left indicate the mobility of molecular mass markers (in kilodaltons). 116  produced apo A-I protein with the charge shift appropriate for the loss of the single lysine residue. Proapo A-IdK’° 7 was detected only within the cells and only mature apo A-IdI<’° 7 was found in the medium of these cultures (Figure 32A). In cells transfected with the dK 107 /dPRO construct, only mature apo A-IdK 107 was found in cells or medium. The absence of the propeptide segment appeared to reduce the accumulation of apo A-I in the medium, but this was not a consistent finding with all mutant cell lines (see Table VIII). Since only the mature form of apo A-IdK 107 was found in the medium of CHO cells expressing the cDNA which included the propeptide, this cell line must also contain the apo A-I propeptidase activity. Western blot analysis of cells expressing the Dl and D1dPRO mutants were also consistent with the analysis of COS transfectants. SDS-PAGE analysis of the secreted product showed that mutant  protein was of lower molecular mass than apo A-Iwt (Figure 32B). IEF analysis (data not shown) indicated that cells expressing apo A-ID 1 contained some proapo A-ID 1 but secreted only mature apo A-ID1. Thus, apo A-I propeptidase was also present in this cell line. In all mutant cell lines studied, the quantity of mutant apo A-I secreted was markedly lower than apo A-Iwt cell lines (see Table VIII). Table VIII. Secretion of apo A-I recombinants by CHO cell lines. Cell lines expressing apo A-I and mutants were grown to near confluence and changed to serum free medium which was then collected after 24 hours. The first 24 hour collection was discarded. After an additional 48 hours the medium was collected and the apo A-I quantified by competitive ELISA as described in Materials and Methods. Values are mean ± S.D. of the number of determinations in parentheses. CHO Clone  Apo A-I Secreted (ng/ml/24 hours)  WI  137± 58 (5)  dPRO  27± 18 (6)  107 dK  17± 6(6)  /dPRO 107 dK  11± 3(5)  Dl  8± 2(5)  D1/dPRO  45± 1 (2)  5.2.4. Role of Lys 107 in Apo A-I Cellular Transport and Secretion The consequences of the dK 107 deletion on cellular transport and secretion of apo A-I were investigated in transiently transfected COS cells. Quantitative analysis of immunoabsorbed radioactivity 117  from pulse-chase studies (Figure 33A) indicated that the dK 107 mutant protein was degraded more rapidly in COS cells than the wild-type apo A-I. Less than half of the apo A-I labelled during the pulse period was recovered as apo A-I after 2 hours (TOTAL, upper panel). However, the portion of the apo A-I that reached the medium during the chase was greater than apo A-Iwt (Figure 33B). SDS-PAGE analysis of the samples suggested that signal peptide hydrolysis was impaired in cells expressing dK 107 and occurred less efficiently in the mutant than in the wild-type transfected cells. Proapo A-IdK’° 7 appeared to leave the cells more rapidly than apo A-Iwt (Figure 33B). Degradation of apo A-I from the cultures was extensive, as observed in the previous transient expression studies. The lipoprotein binding properties of apo A-IdK 107 were evaluated in the COS cell expression system. Apo A-IdK 107 was isolated from the medium in the HDL density range by gradient ultracentrifugation (Figure 34). Lipoproteins containing apo A-Iwt or apo A-IdK 107 were found in the same density fractions. Thus, this mutant protein has a high affrnity for pre-existing HDL and this ability did not differ from the apo A-Iwt expressed in the same culture system. However, I was unable to obtain sufficient material to examine the secretion of lipoprotein particles containing apo A-IdK 107 from serum free cultures. Immunofluorescence analysis of COS cells expressing the dK 107 mutant were consistent with the observations in pulse-labelling. Apo A-I fluorescence was observed at low intensity in a reticular pattern with increasing intensity in the juxtanuclear region (Figure 35A). The latter colocalized with the Golgi marker WGA (Figure 35B). This suggested that apo A-IdK 107 might reach the Golgi apparatus more rapidly than apo A-Iwt, and was consistent with the more rapid secretion of 5 107 observed r S japo A-IdK during the pulse-chase studies.  5.2.5. Functional Characteristics of Apo A-I Mutants Lipid Binding Properties of Apo A-I Mutants Recombinant apo A-I and its mutants produced by CHO cells were used to analyse lipid binding properties in cell culture and in vüro. Lipoprotein complexes containing apo A-I from serum free culture media were concentrated by ultrafiltration and analysed by density gradient ultracentrifugation (Figure 36, left hand panels). A nadir of apo A-I concentration was observed in each gradient at 118  1oo  TOTAL  (1)  II  LLJ+  a  oe  •OA(wt 0—0 AIdK 107 MEDIUM  0_c  CELLS UJO ‘4-  0_o  4  CHASE TIME (hrs)  107 mutant protein is secreted and degraded more rapidly than apo A-Iwt in Figure 33A. The apo A-IdK transfected COS cells. COS cells were transfected with the appropriate cDNA construct and the transfected cells subjected to pulse-chase analysis as indicated in MATERIALS AND METHODS. At the indicated time, medium was collected and cells were recovered by lysis. Apo A-I was immunoabsorbed from each fraction and analysed by liquid scintillation spectrometry. Each data point represents a single dish from a typical experiment.  119  10T Aid K  AIwt 02  4  812  1624  02  4  0  8121624  2  4  8  12  18 24  0  2  4  8  12  16 24  F 56.2  M  ::  66.246-  -  Mr  ___  31  21.521.5  L  MEDIUM  CELLS  J  1 MEDIUM  CELLS  107 mutant protein is secreted and degraded more rapidly than apo A-Iwt in Figure 33B. The apo A-IdK transfected COS cells. Immunoisolates from the pulse-chase experiment in Figure 33A were fractionated by SDS-PAGE and visualized by autoradiography. Numbers along the top of each autoradiogram indicate the chase time in hours. Molecular mass (Mr) markers (in kilodaltons) are indicated on the left. Autoradiograms were overexposed to allow visualization of apo A-I in the medium. Intense bands in the cell lysate at this exposure obscure the doublet in some lanes.  120  1.300  1 .250 O),—  4  1 .200  cC  o Q)  .150  wo  1.100  1 .050  1 .000  FRACTION  AIwt  a_’ —-  107 A1dK  Figure 34. Apo A-IdK 107 incorporates into extracellular lipoprotein in cell culture. COS cells were transfected with apo A-I cDNA and pulse-labelled as described in MATERIALS AND METHODS. Apo A-I was collected in DMEM/10% FBS during a 12 hour chase incubation and concentrated by ultrafiltration. The concentrated samples were then applied to a linear salt gradient and separated by ultracentrifugation (48 hours at 40,000 rpm in SW41Ti rotor). Centrifuge tubes were fractionated from bottom (fraction 1) to top and radiolabel quantitated in each fraction by liquid scintillation spectrometry. Aliquots of each fraction were subjected to immunoabsorption, SDS-PAGE and autoradiography to 107 are both found in fractions of establish the location of apo A-I radiolabel. Apo A-Iwt and apo A-IdK HDL density.  121  1’  Figure 35. Cells expressing apo A-IdK 107 contain apo A-I accumulations in both the ER and the Golgi 107 were processed for apo A-I immunofluorescence as apparatus. COS cells expressing apo A-IdK described in MATERIALS AND METHODS. Apo A-I (A) was found in a reticular distribution but showed increasing intensity in the juxtanuclear region of the cell. The most intense region of immunofluorescence colocalized with the TRITC-WGA marker for the Golgi apparatus (B). Bar indicates 1C m.  122  WITHOUT LPs  WITH LPs  0 U  0 U  C  C  o L V  -4-i  WQ  c’-’  o 1  C.)  I  I ,  —  —  1  FRACTION  Figure 36. Apo A-I and mutants are secreted from CHO cells into serum free medium in lipid-poor form but retain the ability to associate with exogenous liposomes. Recombinant apo A-I and its mutants were collected from CHO cell cultures under serum free conditions and were concentrated by ultrafiltration. Complexes formed under these conditions were fractionated by density gradient ultracentrifugation (left panels, WITHOUT LPs). An equivalent portion of each collection was mixed with lecithin:r H] cholesterol vesicles prior to ultracentrifugation (right panels, WITH LPs). Each gradient was fractionated from bottom (fraction 1) to top and apo A-I was measured in individual fractions by competitive ELISA. H] in each fraction was measured by liquid scintillation and density determined by conductivity as described in MATERIALS AND METHODS. Each point in the lower two panels is the mean ± S.D. of six gradients. Apo A-I was measured in duplicate for each gradient fraction and data values represent the mean in ng per fraction.  r  123  1’  approximately 1.15 g/ml. 50-60% of the apo A-I mass was found in the higher density (d> 1.15 g/ml) region of the gradient indicating that most of the apo A-I was secreted in the lipid-poor or lipid-free state. The wild-type and mutant proteins were similarly distributed among the gradient, fractions, indicating that all proteins were secreted from CHO cell culture in the lipid-poor or lipid-free state. The ability of the secreted recombinant apo A-I preparations to bind to exogenous liposomes (LPs) was determined under in vitro conditions. Medium was recovered from CHO cultures and concentrated by ultrafiltration. Egg yolk phosphatidylcholine:cholesterol vesicles (4:1, molar ratio) were added and the mixture was incubated to allow apo A-I to form complexes which were then fractionated by density gradient ultracentrifugation (Figure 36, right hand panels) for comparison with the original culture concentrate (left hand panels). Liposomes incubated without culture supernate were found only in the lowest density fractions of the gradient (lower right panel). The addition of liposomes to apo A Iwt sample shifted the position of wild-type apo A-I in the gradient from the highest to the lowest density fractions (wt, left panel compared to right panel). More than 85% of the apo A-Iwt was recovered with the liposomes at the lower density limit while less than 15% of apo A-Iwt remained at high density following addition of liposomes. Therefore, apo A-I formed complexes with added lipid and these complexes were stable under conditions of density gradient ultracentrifugation. A similar pattern was observed on addition of liposomes to apo A-IdK . More than 80% of the 107 mutant apo A-I protein was found at lower density following incubation with LPs (dK , right panel). 107 However, the density of the lipid-protein complexes was not as uniform as with apo A-Iwt. Apo A-IdK 107 complexes were detected in the fractions ranging from 1.050-1.090 g/ml. 20% of the mutant apo A-I remained in higher density fractions. The results suggested that dK 107 formed lipid-protein complexes with the added liposomes, but that these structures were more heterogeneous in density or were less stable during ultracentrifugation, than those formed by apo A-Iwt. The apo A-ID1 mutant also retained the ability to form complexes with added liposomes. However, approximately 30% of the mutant protein was isolated at d> 1.15 g/ml following incubation with liposomes (upper right panel). The remaining 70% was recovered as a homogeneous population at the lower density limit. This suggested that apo A-ID 1 might have reduced ability to bind to the liposome or may have bound in a manner that was less stable to ultracentrifugation conditions. 124  107 does not substantially alter the ability of the protein to interact In conclusion, deletion of Lys with phospholipid complexes but may produce more heterogeneous species than apo A-Iwt. Complexes formed with dK 107 were more heterogeneous than those formed with either apo A-Iwt or apo A-ID 1. Furthermore, it appears that deletion of the C-terminal  -helix from apo A-I (mutant Dl) might have  reduced its ability to associate into stable lipid-protein complexes. While the apo A-I proteins secreted from CHO cells may or may not retain some cellular lipid, these proteins still possess the ability to bind to exogenous lipid substrates. LCAT Activation by Apo A-I Mutants  The functional consequences of apo A-I structural changes were tested in LCAT activation studies using recombinant LCAT as enzyme source and lecithin:cholesterol vesicles as substrates. The addition of l g of purified plasma apo A-I (pAl) to a reaction containing enzyme and substrate increased the initial rate of cholesterol esterification 95-fold (9.92 vs 0.10 nmoles/hr/ml, n=4; Figure 37). Similarly, recombinant apo A-I (rAl) increased the reaction rate 75-fold (to 7.69 nmoles/hr/ml, n=4). The interassay variation, however, was large (more than 30%), reflecting variability in liposome substrate preparations. If recombinant and plasma apo A-I preparations were analysed simultaneously and the activation was expressed as the ratio of rAT to pAl (rAI:pAI, Figure 37 right panel), the interassay  coefficient of variation was approximately 12% (n = 4). Recombinant apo A-Iwt was 80% as efficient as an LAT activator as the purified plasma protein (rAI:pAI = 0.796 ± 0.099). This difference may reflect the presence of a small quantity of cellular lipid in association with the recombinant protein, thus reducing the effective concentration of apo A-I on the LAT substrate particles during the assay. The ability of the dK 107 and Dl mutants to activate cholesterol esterification were compared with apo A-Iwt and expressed as rAI:pAI ratio (Figure 38). Neither mutant protein was an effective LCAT activator. The mean activation ratio by dK 107 (ratio  =  0.040 ± 0.025, n=4) and by Dl (0.036 ±  0.007, n=3) mutants were not significantly different from zero (ratio  =  0.010 ± 0.012, n=6; p>O.OS in  both cases) in these experiments. The results suggested that alterations of the class Y amphipathic helices of human apo A-I seriously reduce the ability of the protein to activate LCAT.  125  >-—  15  1 .5  10  1 .0  > U)  C-)  _jc: •0.5  BL  pAl  rAl  rAl:pAl  Figure 37. Recombinant apo A-I produced by CHO cells activates LCAT to approximately the same extent as the purified plasma protein. Activation of cholesterol esterification by apo A-I was measured in assays containing recombinant enzyme and lecithin:cholesterol vesicles as described in MATERIALS AND METHODS. Recombinant (rAl) or purified plasma (pA!) apo A-I (1 g) was mixed with vesicles, incubated 3Omins to allow complex to formation and then incubated in the presence of enzyme. Activity (left panel) is expressed as nmoles CE formed per hr per ml rLAT. In the right panel, the activity determined in reactions containing recombinant apo A-Iwt (rAT) and plasma apo A-I (pAl) is expressed as a ratio. BL indicates the activity observed in the absence of apo A-I. Each histogram is mean ± S.D. (n=4).  126  0.8-  0.6F  >---50 E  0.4-  F  C)  -J  0.2-  00  BL  WI  107 dK  Dl  Figure 38. The ability of apo A-I mutants to activate LCAT is markedly diminished. LCAT activation by the recombinant apo A-I (1j g) was measured and expressed as the ratio of activation achieved by purified plasma apo A-I (pAl) in parallel incubations. Histograms are mean ± S.D. of 3 different apo A , Dl) was not 107 I preparations measured in separate assays. Activation by the mutant proteins (dK significantly different from incubations without any source of apo A-I (BL). 127  5.3. DISCUSSION Segrest and colleagues have recently (1990, 1992) reviewed the amphipathic helix as a structural motif in the plasma apolipoproteins. They have identified two distinct classes of amphipathic helices in apo A-I with different predicted properties based on computer modelling techniques (Segrest et at, 1992). Class A helices are common to all of the apolipoproteins and appear to be responsible for lipid binding. A second helix motif is also present in apo A-I and in apo A-IV (Class Y). This motif is predicted by computer modelling to penetrate phospholipid surfaces to a lesser extent than Class A helix (Segrest et al, 1992) and may  not participate in  high affinity lipid association. The class Y helices in apo  A-I are found in the vicinity of the putative hinge domain and near the C-terminus (see Figure 9). The apo A-I mutations generated and expressed in vitro in my studies involved both of these regions. Only one naturally occurring apo A-I variant has been described in which a substantial portion of helix is lost (Gln , Deeb et at, 1991). Even in the heterozygous state, this abnormality was 60 146 to Arg’ associated with abnormal HDL metabolism. This deletion affects a region of class A helix and suggests that loss of structural elements responsible for lipid binding might have a more profound influence on lipoprotein metabolism than any of the point mutations. Deletion of Lys 107 is the most common mutation of the apo A-I protein (von Eckardstein et at, 1990). Preliminary studies using mutant protein purified from patient’s plasma suggested that this residue, or the secondary structure of the protein which is maintained by this residue, are important for LCAT activation and perhaps for reverse cholesterol transport (Rall et at, 1984). The structural alteration predicted in the mutant protein is a reorientation of the  -helix in the hinge region. Such a  change may be responsible for the changes observed in function (Ponsin et at, 1985). However, no consistent lipoprotein abnormality is associated with dK 107 in vivo. Further analysis of the mutant protein isolated from plasma has been consistent with the preliminary studies (Jonas et at, 1991). However, possible contamination of the preparation with wild-type protein, and large variation among functional properties of purified wild-type preparations have been cited as factors hampering more definitive analysis of these natural mutations. The ability to produce mutant apo A-I as a recombinant has avoided the contamination with wild-type protein which occurs with plasma purified preparations and has allowed me to analyse 128  functional properties of two apo A-I mutants. The findings presented here indicate that the deletion of 107 may have even more pronounced consequences on LAT activation than previously demonstrated. Lys Furthermore, analysis of a novel mutant protein (apo A-ID1), from which a single class Y amphipathic helix had been removed, indicated that changes in this region had a similar effect on apo A-I function. Neither alteration abolished the ability to bind to LCAT substrate particles, but both markedly decreased the ability to promote cholesterol esterification by LCAT. Due to limitations in the quantity of recombinant protein available to date, activation has been assessed only at a single concentration of apo A-I. There appear to be some differences in the stability of the complexes formed with the mutant proteins as assessed by gradient centrifugation. I cannot rule out that the reduced activation of cholesterol esterification is due to the formation of unsuitable LCAT substrates. However, based on the observations to date, I hypothesize that high lipid affinity (class A) amphipathic helices are important to maintain the general structure of the lipoprotein, while class Y helices may play a more important role in HDL remodelling processes. The transformations of HDL composition will necessitate changes in apo A-I secondary structure at the lipoprotein surface. My studies suggest that deletion of Lys 107 induces sufficient structural change to block LCAT activation, perhaps by preventing conformational changes in the hinge region. This possibilty should be investigated in greater detail, including complete kinetic analysis of LCAT activation by the mutant proteins.  129  6.  CONCLUSIONS  6.1. Summary of Major Findings The studies described in this thesis were designed to investigate the role of structural elements of apo A-I in the intracellular transport and extracellular functions of this apolipoprotein. Four expression systems were used in the course of this work to produce the wild-type protein. Three mutants have been generated at the cDNA level and were expressed in at least two of these systems. In vitro translation sudies were used to establish that the cDNA encoded authentic apo A-I  protein which was translocated and proteolytically processed at the endoplasmic reticulum membrane. Under the conditions employed, I was unable to demonstrate complete translocation and signal peptide hydrolysis in vitro. When the protein was expressed in non-hepatic eucaryotic cells, apo A-I was synthesized efficiently during transient overexpression of the cDNA. Secretion of the protein was not directly related to the level of synthesis in standard growth conditions and was not detectable in serum free conditions. A substantial portion of the protein was degraded or retained by the cells in these cultures. COS cells and BHK cells trarisfected with the precursor cDNA secreted only proapo A-I, and signal peptide hydrolysis was necessary for secretion in these systems. In COS cells, a portion of the intracellular apo A-I was preproapo A-I, indicating that cotranslational proteolytic processing was incomplete. Proapo A-I was readily integrated into lipoproteins of high density (mean d = 1.15g/ml) in the culture medium of the transfected cells. Mature apo A-I was secreted from CHO cells expressing the cDNA for the precursor protein, demonstrating that these cells possess apo A-I propeptidase activity. Approximately 75% of the intracellular apo A-I was the mature protein in serum free cultures, indicating that the hydrolysis occurred within the cell. The apo A-I secreted from CHO cultures was found mostly in lipid-free or lipid-poor form (d> 1.15g/ml) under these culture conditions. However, the expressed protein had the ability to bind avidly to exogenous lipid complexes in vitro. The level of apo A-I expression from CHO cells was lower than from BHK cells but secretion from these cells was far more efficient, In addition, BHK cells containing the precursor cDNA degraded apo A-I in culture. The production of apo A-I from the precursor cDNA was estimated in serum free 130  cultures of cells selected for maximal secretion under these conditions: COS lOOng/ml/24hr, CHO  =  =  lOng/ml/24hr, BHK  =  l3Ong/mJ/24hr. Therefore, it was concluded that the CHO cell is the most  suitable host cell for expression of apo A-I for structure-function analysis. In agreement with studies in liver cells, I observed intracellular phosphorylation of apo A-I in COS cells. Secreted proapo A-I did not retain the  [32  P]phosphate. I have provided no additional  information on the functional role of this post-translational modification. Perhaps the most important observation in this work was that the apo A-I propeptide was required for efficient cellular transport in BHK cells. In the absence of this hexapeptide, the majority of the apo A-I was retained as the mature protein in the cell, although a portion of the protein was also secreted. The deletion had no effect on the fidelity of signal peptide hydrolysis. However, in its absence, the rate of apo A-I secretion and, to a lesser extent, degradation were reduced. Microscopic observations indicated that apo A-IdPRO accumulated in vesicular structures which had some properties of the ER. Based on observations ii the BHK cell, it is proposed (Figure 39) that the normal translocated product (p-Al) is transported from the ER to the Golgi in transport vesicles where it is either secreted or degraded. Degradation is a major fate of the apo A-I produced in BHK cells, but may be a minor pathway in cells capable of higher levels of lipid synthesis, eg. hepatocytes. The non-physiologic removal of the propeptide (in BHK clone rn-Al) caused apo A-I to accumulate to high levels in the ER when expression was stimulated. The potent lipid binding and self-association properties of apo A-I may promote aggregation within the ER and segmentation of the reticulum into the vesicular structures observed in immunogold cryosections. Since  only  small amounts of the protein would reach the Golgi  under these conditions, the apo A-I accumulations in the ER would be relatively protected from intracellular degradation. Studies of FBS and lipoprotein stimulation of apo A-I secretion from COS and BHK cells expressing the cDNA have suggested that the lipid status of the cell might determine the rate of apo A-I secretion. At low levels of lipid, apo A-I may be degraded within the cell. Such a process has been shown to govern apo B secretion from hepatocytes (Boren et al, 1991) but has not been demonstrated for apo A-I. Clearly non-hepatic cells have lesser ability to assimilate lipid and may, therefore, be more markedly affected by culture in serum free conditions. 131  p-Al  rn-Al ER  I  Transport Vesicles  O  Golgi  /N  Degradation Secretion  Secretion  Figure 39. Proposed model for the role of the apo A-I propeptide in BHK cell transport. p-AI= BHK cells secreting proapo A-I, rn-A! = cells expressing mature apo A-I from the apo AIdPRO cDNA. The thickness of the arrows in the figure indicates the approximate quantity of apo A-I committed to that transport pathway. This thesis has also investigated the functional role of regions of the middle portion of apo A-I and the C-terminus. Lysine 107 lies within the proposed mobile hinge domain in the middle of the apo A-I sequence. Lysine 107 is also the most frequent site of naturally occurring mutations of apo A-I. This region is predicted by Segrest (1992) to be less tightly associated with phospholipid (Class Y aniphipathic helix) than the high affinity amphipathic helix of Class A. Only one other Class Y amphipathic helix is present in apo A-I, the most C-terminal 22mer (residues 220-241). This region is highly conserved (von Eckardstein et al, 1990) and the only natural mutation lacking this region (apo A-1 - FS) has reduced endogenous LCAT activity (Funke et al, 1991). 202 I have shown that deletion of Lys , or of the complete C-terminal helix, causes some changes 107 in the ability of the protein to assemble model lipoproteins in vitro although there is no apparent effect 107 mutant to interact with natural lipoproteins in cell culture. The loss of the on the ability of the dK  132  entire C-terminal amphipathic helix had a greater effect on liposome binding than did the loss of Lys’° . 7 However, in preliminary studies, both the 107 Lys 0 and the Dl mutant showed markedly reduced ability -. to activate LCAT. Conversely, the recombinant wild-type protein was 80% as effective an LCAT activator as the purified plasma product. These results suggest that low affinity amphipathic helical domains of Class Y may play an important role in the LCAT activation mechanism. The sequence of apo A-I in the vicinity of Lys 107 may govern the transformations of HDL involving the Class Y helix in the hinge domain. This study provides the first in vitro evidence for the involvement of a specific C-terminal helical region in LCAT activation. There is at present no evidence for direct interaction between apo A-I and LAT during cholesterol esterification. However, helix domains of apo A-I or LCAT might form a hydrophobic pocket to permit access of the enzyme to the hydrophobic core of the lipoprotein. This has been suggested by Au-Young and Fielding (1992) for CETP and other proteins which require access to the lipoprotein core for their action. Their work has suggested that the consensus sequence Phe-Leu-X Leu-X-X-X-N (where N is a hydrophobic  amino  acid) may be a binding site for CE or TG. In apo A-I,  the sequence Phe 229 -Leu-Ser-Ala-Leu-Glu-Glu-Tyr resembles this proposed consensus and is in the region deleted in mutant DL Perhaps cooperation between apo A-I helices forms a CE binding site or hydrophobic pocket for transport of the CE product of the transacylation reaction to the lipoprotein core.  6.2. Perspectives for Future Study Several lines of investigation should be followed to address some of the key issues raised in this thesis. First, cell fractionation studies should be inititiated in BHK cell line rn-Al to establish the origin and fate of the vesicular structures containing apo A-I. Further application of these techniques could also address the site of apo A-I degradation in this cell type. To assess adequately the functional consequences of the mutations described herein, a more efficient system for the expression of apo A-I protein must be obtained. Eucaryotic expression systems employing liver-derived cell cultures (eg., the rat hepatoma cell, McA-RH7777) may be a useful alternative to the non-hepatic cell cultures used to date, since in these cells the effects of lipid availability 133  can be assessed. The proposed model of propeptide function should be validated in this, more physiologically relevant, model system. Additional studies of the apo A-I specific propeptidase should be used to determine the location of this enzyme activity in CHO cells and in HepG2 cells, where it is known to be active. The BHK cell lines expressing apo A-I with and without its propeptide could be used to address basic features of the cell biolo’ of protein transport. The absence of the propeptide reduces the rate of degradation of apo A-I. This could be because the propeptide is a degradation signal, although this seems unlikely since hepatocytes appear to secrete proapo A-I. In addition, proapo A-I does not have altered extracellular function, so there does not appear to be a need to degrade this form if it could be secreted. A second possibility is that the propeptide provides a structural element required for intracellular transport of the protein. Chaperone proteins have been shown to be involved in protein movement between cellular compartments (Rothman, 1989). This is accomplished by preventing premature or incorrect folding of the nascent polypeptide chain. The apo A-I propeptide may regulate a specific protein-protein interaction which facilitates its transport from the ER to the Golgi apparatus. In the absence of the propeptide, apo A-I may not be transported efficiently and could aggregate in the ER, resulting in the large vesicular structures found in BHK line rn-Al. One aspect of this work, and of any studies involving mutagenesis, is the role of the structural changes on protein conformation. In the absence of definitive physical measurements (circular dichroism, X-ray crystallography, etc.), ii is not possible to exclude improper folding as the underlying mechanism for any functional abnormality. These studies can only be carried out once large (milligram to gram) quantities of each mutant protein are available. Lectin markers were used in these studies to identify the subcellular structures containing apo A-I. These assignments have assumed that the lectins are organelle-specific (Virtanen et cii, 1980; Kaarianen et cii, 1983) and that apo A-I transport is vectorial. These studies should be extended to include enzyme markers for the ER in immunolluorescence studies or in subcellular fractionation experiments. In addition, the possibility that apo A-IdPRO is subject to rapid retrograde transport (Rothman and Orci, 1992), from the Golgi to the ER, should be addressed. The latter trafficking process would appear indistiguishable from slow ER exit in the studies I have performed to date. 134  An additional line of investigation which could be extended is the role of post-translational modification of apo A-I in its function. The site of apo A-I phosphorylation has been localized to Sei 201 (Beg et at, 1989), which is in the highly conserved region of the sequence. Studies in this thesis have shown that phosphorylation also occurs in COS cells which transiently express the protein. Site-directed mutants at Sei 201 could provide important information on the role of this modification in cellular transport and secretion. In addition, the generation of the apo A-i -’ FS mutant in vitro and analysis of 202 phosphorylation in this variant might also provide important insight into the mechanism and role of phosphorylation in cellular transport. Since this mutant also possesses altered C-terminal structure, the role of this region in LCAT activation and the pathophysiology of this natural mutation could also be obtained from analysis of the protein in vitro. Studies of the site and the function of covalent acylation of apo A-I (Hoeg et ci, 1986) could also be investigated in this expression system. Finally, a more extensive analysis of the functional consequences of Lys 107 deletion and sequential deletions of amphipathic helix must be completed. 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