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Effect of adenovirus E3/19K protein on cellular processes in the endoplasmic reticulum Lomas, Cyprien 1999

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E F F E C T O F A D E N O V I R U S E3/19K PROTEESf O N C E L L U L A R PROCESSES IN THE E N D O P L A S M I C R E T I C U L U M by CYPRJEN LOMAS B . S c , The University of British C o l u m b i a ,  1987  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES Faculty of Science Department o f Z o o l o g y  W e accept this this as c o n f o r m i n g to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A M a y 1999 © C y p r i e n Pierre-Etienne L o m a s ,  1999  In presenting this thesis  in partial fulfilment  of  the  requirements  for  an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. 1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  publication of this thesis for financial gain shall not be allowed without permission.  • Department The University of British Columbia Vancouver, Canada  DE-6 (2/88)  copying  or  my written  Abstract  The human adenovirus has developed several methods to evade the immune system. One mechanism by which it accomplishes this involves the endoplasmic reticulum (ER) retained adenovirus E3/19K protein. This protein interferes with antigen presentation by binding and retaining Major Histocompatibility Complex Class I (MHC CI I) proteins in the ER.  E3/19K binds all human and all but one mouse M H C CI I molecules tested to date. Differences in mouse M H C CI I sequences were exploited to determine the structures involved in binding. Human 293 cells transfected with the mouse H-2 alleles K , K , K , D , D , L were infected d  b  k  d  b  d  with adenovirus 2. It was found that M H C CI I alleles could be grouped into three categories. The H-2 allelic proteins K  d  and K  b  were found to be binders; K  k  and D non binders and D d  b  and L slow binders. d  Examination of a cell line transfected with the slow-binding H-2D protein revealed that D is b  b  expressed at a reduced level at the cell surface. To determine the cause of this, cells were subjected to conditions previously used to restore defective cell surface expression of MHC CI I including culture at reduced temperature, addition of excess P m and exposure to gamma 2  interferon. All these methods were unsuccessful in increasing cell surface expression of D . b  Rather than being due to a missing co-factor or unstable conformation, the accumulation of the allelic proteins in the ER in this transfectant was due to an undetermined mechanism.  Because E3/19K binding quickly stabilised a mature M H C CI I conformation in the presence of tunicamycin it was suggested that it bound M H C  ii  CI I like a chaperone. It was found that  E3/19K binding to MHC  CI I did not block the association of endogenous ER resident  chaperones calnexin and TAP.  Peptide binding to the MHC  CI I-E3/19K complex could also  occur. These experiments showed that E3/19K did not associate with MHC  CI I through the  peptide binding groove and did not disrupt the interaction between the MHC  CI I and the  processing machinery found in the ER, namely calnexin and TAP, and therefore does not retain MHC  molecules by making them conformationally immature.  This study shows E3/19K works independently of chaperones. E3/19K may be a tool to trap MHC  CI I - chaperone complexes at a specific point in their maturation.  ni  Table of Contents  Abstract  11  List of Tables  v i  List of Figures  y  Nomenclature  ii  x  Acknowledgements  X 1  1. Introduction  1  1.1. Immunity  •  1  1.2. Pluripotent Stem Cells  2  1.3. B Cells  2  1.4. T Cells  2  1.5. M H C  4  1.6. Cellular Mechanics  11  1.6.1. Peptides 1.6.2. Proteasome  U ...  H  1.7. Antigen Presentation  13  1.7.1. Nature of the ER  14  1.7.2. Chaperones  15  1.7.3. Other chaperones  •  1.7.4. Glycosylation  15 17  iv  1.8. The Golgi complex and membrane traffic  18  1.9. T Cell Receptor/Ligand Interactions  19  1.10. Viruses  22  1.11. Adenovirus  25  1.11.1. E3 region  29  1.11.2. Overview of proteins and effects of the E3 region  31  Objectives  34  2. Materials and Methods  36  3. Comparison of Differential Binding of E3/19K to M H C Class I allelic proteins  46  4. Efforts to increase the cell surface expression  76  5. E3/19K binding to M H C CI I does not exclude association with calnexin  93  6. Discussion  119  7. Conclusion  127  References  128  Appendix 1  147  Appendix 2  148  v  List of Tables T a b l e 1. S u m m a r y o f the effects o f E 3 region proteins  30  T a b l e 2. Reagents used  37  T a b l e 3. S u m m a r y o f E 3 / 1 9 K association w i t h H - 2 a l l e l i c proteins  66  T a b l e 4. A c o m p a r i s o n o f the c e l l surface expression o f the H - 2 D protein at 2 6 ° C and 3 7 ° C w i t h and without excess P m  86  2  T a b l e 5. T h e effect o f y - I F N o n M H C C I I c e l l surface e x p r e s s i o n  89  T a b l e 6. D e t e r m i n a t i o n o f M o l e c u l a r W e i g h t ( M W ) o f proteins i n s u c r o s e gradient fractions  160  vi  List of Figures  F i g u r e 1. T h e M H C C I I regions for different species  5  F i g u r e 2. T h e M H C C I I gene  7  F i g u r e 3. C r y s t a l structure o f M H C C I I  9  F i g u r e 4. T h e interaction between M H C C I I and the T C R  21  F i g u r e 5. T h e antigen presentation p a t h w a y  24  F i g u r e 6. T h e adenovirus g e n o m e  27  F i g u r e 7: T h e E 3 r e g i o n o f adenovirus  29  F i g u r e 8 T h e E 3 / 1 9 K protein  32  F i g u r e 9. F A C S analysis o f 293 cells and 293 transfectants  .  449  F i g u r e 10. T h e rate o f transport o f H - 2 M H C C I I allelic proteins transfected into 2 9 3 cells  Figure 11.  53  T h e effect o f A d 2 i n f e c t i o n assessed b y the c o - i m m u n o p r e c i p i t a t i o n o f  E 3 / 1 9 K w i t h H - 2 M H C C I I i n 2 9 3 cells  vii  59  F i g u r e 12. T h e effect o f A d 2 infection o n M H C C I I expression i n 293 cells and 293 transfectants  63  F i g u r e 13. O v e r v i e w o f maturation paths for the M H C C I I c o m p l e x  74  F i g u r e 14. Intracellular accumulation o f H - 2 D i n 293 cells  80  b  F i g u r e 15.  T h e effect o f g r o w t h at 2 6 ° C o n the intracellular accumulation o f H 2 D  b  in  2 9 3 D cells  82  b  F i g u r e 16.  T h e effect o f infection w i t h a v a c c i n i a virus h u m a n - ( 3 m construct o n 2  Pm 2  expression  85  F i g u r e 17. T h e effect o f g r o w t h at 2 6 ° C and infection w i t h a v a c c i n i a virus human-(3 m 2  construct o n escape o f intracellular stores o f H - 2 D protein f r o m the E R b  F i g u r e 18.  87  T h e effect o f i n c r e a s i n g levels o f t u n i c a m y c i n o n M H C C I I i n 293 and  293.12 cells  97  F i g u r e 19. T i m e course o f maturation o f M H C C I I i n 293 and 2 9 3 . 1 2 cells  viii  99  Figure 20. Calnexin association with MHC CI I precipitated from 293 and 293.12 cells 102  Figure 21. Calnexin association with W6/32 and TAP precipitates from 293 and 293.12 cells  Figure 22. Precipitation of M H C  105  CI I in enriched suspension using a biotinylated  peptide  110  Figure 23. Separation of 293 lysates over a 5-20% sucrose gradient  151  Figure 24. Precipitation of M H C CI I and E3/19K from 293 and 293.12 cell lysates separated over a 5-20% sucrose gradient  ix  154  Nomenclature 293 293.12 621 Ad  human cell line derived from embryonic kidney carcinoma 293 cell line transfected with E3/19K 293 cell line transfected with truncated E3/19K adenovirus  Pm BiP BSA DMSO DNA DSP DTT E3/19K ECL EDTA EGF Endo H ER ER FACS Gal  beta-2-microglobulin heat shock protein in the ER bovine serum albumin dimethyl sulfoxide deoxyribonucleic acid dithiobis (succinimidylpropionate) dithiothreitol 19 kilodalton adenovirus protein coded in early region 3 enhanced chemiluminescence ethylenediaminetetraacetic acid epidermal growth factor endoglycosidase H endoplasmic reticulum endoplasmic reticulum fluorescence activated cell sorter galactose  ylFN H-2 HEPES HLA HRPO Ig IR Kd, Kb, etc kDa mAb MHC MW NRS PMSF R425, R426 SA-HRPO SDS TAP TCA TcR UPR W6/32  gamma interferon mouse M H C CI I region The mouse versions are K, D and L 2-(2-hydroxyyethyl)-l-piperadineethanesulfonic acid human M H C CI I region H L A A, B, and C horse radish peroxidase immunoglobulin immune response Alleles d and b of the mouse H-2K gene kilodalton monoclonal antibody Major Histocompatibility Complex molecular weight normal rabbit sera phenylmethylsufonylfluoride rabbit anti M H C CI antisera streptavidin horse radish peroxidase sodium dodecyl sulfate transporters associated with antigen processing trichloroacetic acid T cell receptor unfolded protein response monoclonal that detects mature CI I H L A  2  x  Acknowledgements I would like to thank my supervisor, Dr. Wilfred Jefferies for his tireless assistance and endless patience in guiding both my research and many other aspects of my life. His influence will remain with me for a lifetime. I am also grateful for the environment that he created both in and outside the lab. Many lifelong friendships were started here. A special thanks is in order for the members of my committee who invaluable contribution to this document was greatly appreciated. I must thank the members of the lab without whose contribution and assistance this work would never have been completed. In particular, special thanks go to Reinhard Gabathuler and Daphne Blew. These two individuals offered continual help and assistance. I would also like to thank the members of the department whose daily antics and thoughtful comments kept me both amused and focused. These people include Roger Lippe, Ian Haidl, Gregor Reid, Gerry Kolaitis, Michael Food, Kathy Shimizu, Nancy Carpenter, Greg Lizee, Jacqueline Tiong and Brandie Thorlakson. Special thanks to Alex Moise, Joseph Yang, and Liz Frey. I am indebted to the Department of Zoology for their financial support in the form of T A ships and for the opportunities to teach. In particular, the guidance of Ellen Rosenberg, Brian Oates and Carol Pollock was appreciated. Finally I am grateful for the support of my friends and family who supported my quirkiness throughout my degree. Keith MacCrimmon was always a phone call away. Nick Heap was especially introspective at all the right moments. Elizabeth Richards helped keep everything in perspective. My mother and father supported me in countless ways. Above all, the contribution, support and guidance of Renee leNobel were invaluable in the completion of this work.  xi  1.  Introduction  The human body with its immune system is like a heavily fortified medieval donjon. While a great number of attacks on the integrity of the body are thwarted on a continual basis, breaches of the immune system by pathogens can result in discomfort, disease and worse. Persistent viral infections result when viruses escape detection by the immune system. Many viruses have evolved a set of mechanisms that effectively target susceptible components of the immune system (1). These viruses can propagate undetected and unchallenged by the host immune system. Persistent viral infections offer a good model to examine the immune system. Investigation of the strategies used by viruses to escape the immune system provides a useful method for understanding the components involved in antigen presentation and the interactions between them. This study investigates the role of the endoplasmic reticulum (ER) resident E3/19K protein of human adenovirus in the immune system, protein processing and maturation through the ER and Golgi, antigen presentation and viral clearing.  1.1.  Immunity An immune system is present in all vertebrates and many invertebrates. It is a  mechanism employed by organisms to protect themselves from pathogens and other foreign materials. This system also serves as a method to detect self from non-self. While in many lower organisms this differentiation of self versus non-self is a simple mechanical differentiation between 'in and out', in higher vertebrates the immune system is more sophisticated with multi-faceted methods for preserving the integrity of the living body (2). The immune system in humans and mice has both specific and non-specific methods for eliminating foreign substances. Non-specific mechanisms include physical barriers, temperature levels and pH to make living conditions hostile to potential pathogens. 1  Additionally, lysozymes, interferons, complement and natural killer (NK) cells are present and non-specifically respond to foreign objects. A second, more sophisticated method available to the body to defend itself are the specific responses to foreign material. These are known as the acquired immune response. Some features of the acquired immune response include immunological memory, the ability to respond to a high number and diverse set of antigens and the ability to recognize foreign antigens in the context of self antigens (3).  1.2. Pluripotent Stem Cells The acquired immune response is driven by the action of lymphocytes. Pluripotent stem cells are the progenitors of two lines of cells, the lymphoid and the myeloid. The lymphoid line can be further divided into several cell types including B and T cells. B Cells have characterizing immunoglobulins present on their surface; T Cells are distinguished by the presence of a T Cell Receptor and Cluster Differentiation marker CD3.  1.3.  B Cells B Cells are specialized for immunoglobulin (antibody) production. Antibodies can be  raised against almost any type of foreign antigen. Antibodies are very specific and one antibody recognizes one epitope. Immunoglobulins consist of a heavy (H) and light (L) chain. Different combinations of H and L chains can be assembled to generate a large diversity of antibodies specific for different antigens. The diversity of antigens against which antibodies can be raised is considerable.  1.4.  T Cells T Cells recognize fragments of proteins bound to self M H C molecules (4).  other) proteins are degraded into peptides. 2  Viral (and  These peptides are bound by major  histocompatibility complex (MHC) molecules and recognized by T cell receptors (TCR) at the surface of infected cells (5).  Both M H C  CI I and CI II molecules present foreign (and  endogenous) antigens to the TCR of circulating T cells (6). The T Cell population can be further divided in C D 4 and C D 8 populations. C D 4 cells are involved in the recognition of +  +  +  M H C CI II with bound peptide; C D 8 cells are involved in the recognition of M H C CI I with +  bound peptide. Immunoglobulins and the TCR are structurally related to one another and are part of the immunoglobulin gene superfamily. Additional members of this family also include M H C CI I, CI II, CD4, CD8, (3 m, MRC-OX2, ICAM-1, ICAM-2 and many others. These proteins have 2  a characteristic 110 residue domain and a cysteine bridge (7). It is likely that these proteins evolved from a limited number of original genes. The great diversity present in both immunoglobulins and the TCR is the result of gene rearrangements. This diversity is produced by rearrangements between V, D and J segments. RAG-1 and RAG-2 genes are responsible for this genetic rearrangement (8). As the primary methods for clearing viral infections include removing infected cells from the host before the viral cycle has completed, detection of infected cells is very important. T Cells effect the killing, recognizing the processed foreign proteins presented at the cell surface of an infected cell by a M H C CI I protein (9). In cases where the immune system is unable to clear the viral infection, persistent infection occurs. The effects of the persistent infection will not become apparent until the general health of the victim declines, often as the patient succumbs to old age or another illness (such as HIV). Some of the viruses that cause persistent infections include Herpes Simplex Virus (HSV), Cytomegalovirus (CMV), Adenovirus (Ad), and Human Immunodeficiency Virus (HIV).  Each has particular and  differing strategies to achieve a persistent infection due to 'stealth' (10).  3  1.5. MHC The major histocopatibility complex (MHC) is one target of persistent viruses. The M H C region is found in all vertebrates and many invertebrates (11). Precursors to the MHC are also found in some invertebrates. The region codes for more than one thousand proteins including many that play key roles in the immune system. These proteins include MHC CI I , CI II, complement, TAP 1 and 2. The M H C region is on chromosome 17 in humans and chromosome 6 in mice. There are three M H C CI I loci in both humans (HLA-A, B and C) and mice (H-2 K , D and L) (12). Polymorphism at the M H C CI I locus is extensive. With as many as one hundred alleles at each loci, the diversity found within the population is very high. M H C CI I molecules have been studied extensively, with CI I regions studied in vertebrates ranging from xenopus to human (figure 1).  4  Figure 1.  The M H C CI I regions for different species  M H C regions for m a n y different species have been i d e n t i f i e d based o n s i m i l a r i t i e s between species ( 1 2 ) . M H C C I I genes are represented by w h i t e boxes; M H C C I II genes by b l a c k boxes.  DP  DQ  BC  DK  " f t  D  Rhesus m o n k e y  Chimpanzee  8  RbLA-  Cb  LA-  G-  -U—D a  D  P-9  Sheep  OLA  AC Dog  FLA  1—G—-fl-  Rabbit  B "Qo"  G u i n e a Q\q  [] [HK -LHHf OOHHHF  CPLA—IIHH K  A  A  B  E  DML  O  Rat  Syrian hamster  Si~£i  Hm-l  Smh-  Fowl  Xfmopus  XLA  .  if  5  -  Qo-2  Th  M H C C I I is f o u n d noncovalently associated w i t h b e t a - 2 - m i c r o g l o b u l i n (P m) 2  cell surface o f most nucleated c e l l s in the b o d y .  M H C C I I m o l e c u l e s c o n s i s t o f three  e x t r a c e l l u l a r d o m a i n s (alpha 1-3), a transmembrane r e g i o n a n d a c y t o p l a s m i c t a i l . e x t r a c e l l u l a r d o m a i n consists o f about 9 0 a m i n o acids a n d is a l s o a m e m b e r superfamily.  at the  Each  o f the  Ig  T h e M H C C I I m o l e c u l e is g l y c o s y l a t e d at a s p a r a g i n e 86 i n h u m a n s w i t h a  second (and sometimes third) g l y c o s y l a t i o n site i n m i c e . T h e mature M H C C I I m o l e c u l e is about 46-55 kilodaltons ( k D a ) . T h e p o l y m o r p h i c regions o f the M H C C I I m o l e c u l e are found m a i n l y i n the alpha 1 and alpha 2 regions. T h e genetic o r g a n i z a t i o n o f the M H C C I I r e g i o n reveals m a n y exons and introns. E a c h o f the three alpha regions and the transmembrane region are encoded b y a separate exon and the cytoplasmic tail is c o m p r i s e d o f three exons (figure 2).  6  Figure 2. The M H C CI I gene T h e M H C C I I gene is made up o f large e x o n a n d i n t r o n regions (12).  Similarities  between the exons (black boxes) and introns o f mouse (K<» and L A n o n classical Q 6 ) , human H L A 12.4 and fj m are depicted b e l o w . 2  5'UT  5'UT  {  «1  El"  E2  1  «1  *3  / \ \  3'UT  ~ ? r \ C ? n \ s ' \ t  1 3  £3  TH t  <r2  U  E  £6 E7 E8  5  S3—I—fi—S I  5'UT Q 6 (27.1) 5'UT HLA 12.4  1  o1  °1  a3  x3  CY  CT  TM  5'UT . 1  /5,m  TH  ..3'UT  1—— 5 kb  7  S i n c e antigens are presented by M H C m o l e c u l e s , e x t e n s i v e p o l y m o r p h i s m a l l o w s for great variability in the number and types o f antigens that can be presented w i t h i n a population. S o m e a l l e l e l i c forms o f M H C C I I proteins present specific antigens far m o r e efficiently than others.  I n d i v i d u a l s w i t h certain alleles are more susceptible to s o m e disease because their  M H C C I 1 is s i m p l y u n a b l e to present the antigen to their i m m u n e s y s t e m .  For example,  certain a u t o i m m u n e diseases have been linked to specific H L A alleles. S t r u c t u r a l l y , the M H C C I I molecule consists o f two antiparallel alpha helices (alpha 1 and 2) resting o n top o f t w o antiparallel beta pleated sheets (alpha 3) (13).  T h e helices f o r m a  25 A l o n g by 10 A w i d e g r o o v e . T h e highly conserved 12 k D a protein 3 m is non-covalently 2  associated w i t h the alpha 3 d o m a i n . T h e crystal structure for M H C C I I has been determined for m a n y a l l e l i c proteins.  The  ribbon structure is demonstrated i n figure 3. Peptide is b o u n d i n the g r o o v e between the alpha 1 and a l p h a 2 r e g i o n s ( f i g 3a). Peptide hypervariable regions o f the M H C C I I m o l e c u l e are f o u n d near the peptide b i n d i n g g r o o v e . T h e resolved crystal structures have demonstrated the existence o f pockets i n s i d e the g r o o v e into w h i c h the R - g r o u p s o f b o u n d peptides fit t i g h t l y . Peptide sequence constrains w h i c h peptides fit into the groove. Different a l l e l i c forms o f M H C C I I b i n d sets o f peptides l i m i t e d i n sequence by k e y ' a n c h o r ' positions. Peptides d e r i v e d f r o m v i r a l (and e n d o g e n o u s ) proteins are tightly b o u n d i n the peptide b i n d i n g g r o o v e .  F i g u r e 3b  illustrates the peptide b i n d i n g g r o o v e f r o m the top. T h i s a s s o c i a t i o n is a requisite step i n the assembly o f a stable M H C C I I m o l e c u l e (14).  8  Figure 3.  Crystal structure of M H C CI I  T h e crystal structure o f M H C C I I reveals the presence a large peptide b i n d i n g groove at the top.  In a) the peptide b i n d i n g groove is demonstrated l y i n g between a l and a 2 regions.  A l s o s h o w n is the close association o f (3 m. In b) and c) the peptide b i n d i n g groove is seen from above. T h e p h y s i c a l constraints o f the g r o o v e p e r m i t l i m i t e d sets o f peptides to b i n d . T h e constraints on the peptide include pockets w i t h i n the g r o o v e for a m i n o a c i d R group side chains and w e l l c o n s e r v e d anchor positions (13). 2  9  1.6.  Cellular Mechanics  1.6.1.  Peptides T h e ability o f the i m m u n e system to respond to millions o f potential pathogens is due to  the large number o f different peptides that c a n be b o u n d b y the M H C C I I m o l e c u l e .  Some  investigators have suggested that potentially m i l l i o n s o f different peptides c a n o c c u p y the peptide b i n d i n g g r o o v e o f M H C C I I. T h e T C R found on the surface o f T cells recognises both the p o l y m o r p h i c r e g i o n o f the M H C C I I m o l e c u l e and the peptide contained w i t h i n the groove.  Recent studies demonstrate direct contact between the T C R and the peptide occurs  (15). A study i n w h i c h self peptides eluted f r o m M H C C I I were sequenced identified many motifs o f peptides associated w i t h a single allelic protein (16). Other studies that examined the stability o f M H C C I I m o l e c u l e s associated w i t h peptides s h o w e d that there is a hierarchy o f peptide sequences that confer stability ( 1 7 ) .  T h e peptide specificity o f different M H C C I I  a l l e l i c p r o t e i n s i s i n part d e t e r m i n e d b y the s i d e c h a i n s o f the a m i n o a c i d s l i n i n g the p o l y m o r p h i c peptide g r o o v e o f the M H C C I I m o l e c u l e . Different allelic proteins o f M H C C I I have preferences f o r peptides r a n g i n g f r o m 8 to 11 a m i n o acids i n length ( 1 8 ) . A m i n o a c i d side chains o f b o u n d peptides e x t e n d i n g i n t o the g r o o v e dictate that certain peptide residue p o s i t i o n s are r e g a r d e d as a n c h o r r e s i d u e s ( 1 9 , 2 0 ) .  F o r e x a m p l e , the H - 2 p r o t e i n K  b  preferentially binds octapeptides w i t h a tyrosine o r phenylalanine at position 5 a n d a M e t o r He at p o s i t i o n 8 ( 1 8 ) .  1.6.2.  Proteasome Peptides that b i n d to M H C C I I m o l e c u l e s are generated w i t h i n the c y t o p l a s m b y the  proteasome.  T h e proteasome is a barrel shaped structure made up o f many distinct subunits.  11  Viral and endogenous proteins are tagged with ubiquitin and targeted for degradation by the proteasome. The  proteasome is well-conserved throughout  many species ranging from  Archaebacterium (21) through to humans and serves as a general cellular method to remove abnormal, short lived and otherwise redundant proteins. The proteolytic process starts with the tagging of the doomed protein with ubiquitin to mark the protein for hydrolysis by the 26S proteasome (22). The proteasome is also able to function without ubiquitin. The proteasome consists of a 20S Multi Catalytic Protease complex (MCP) catalytic core with additional subunits. The 20S core is approximately 650 kDa (23), composed of 13-15 subunits of similar size (24) stacked in a barrel shape and is found in the cytosol and the nucleus of all eukaryotic cells. It has multiple catalytic sites and comprises up to 1% of the total protein of a mammalian cell.  Association of an ATPase complex results in a 26S proteasome which degrades  ubiquitinated proteins in the presence of ATP (25). In another configuration of the proteasome, the two subunits, LMP also found. They are encoded in the MHC  2 and LMP  7, are  region, are upregulated by y-IFN and have been  shown to participate in a proteasome involved in the generation of peptides bound by MHC  CI  I molecules (26). Proteasome-generated peptides are presented to immature MHC  CI I molecules present  in the ER by the MHC-encoded transporter associated with antigen presentation (TAP) proteins (27). Encoded in the MHC  CI II region, TAP 1 and 2 form a heterodimer that pumps peptides  across the ER membrane in an ATP-dependent fashion. These two proteins are non-covalently associated in the ER.  The TAP proteins are members of the ATP binding cassette (ABC)  transporter protein family and show homology across species. The binding of TAP proteins with empty MHC  CI I heavy (H) chains and P m 2  in the ER (28-30) is required for  MHC  loading with peptides (31). Cells with deficiency in the TAP proteins show reduced peptide 12  l o a d i n g and antigen presentation (32-35). Studies have demonstrated that stringency o f these transporters is not h i g h for peptides is not h i g h .  Peptides must be longer than 7 residues and peptides longer than 12 residues are  transported w i t h l o w e r e f f i c i e n c y (36, 37).  S o m e s p e c i f i c i t y i n peptides transported by T A P  has been, s h o w n . In rats, different alleles o f T A P transporters favour different sets o f peptides (38). L i k e most plasma membrane  and secretory proteins, M H C C I I m o l e c u l e s  are  synthesized on E R b o u n d ribosomes and transported across the E R membrane to the lumen o f the E R . P r o c e s s i n g occurs i n the l u m e n o f the E R and continues w i t h transport from the E R to the c i s , m e d i a l and trans sections o f the G o l g i to the c e l l surface.  In g e n e r a l , proteins are  transported out o f the E R w h e n they have a c h i e v e d a correct c o n f o r m a t i o n for exit. T r e a t i n g c e l l s w i t h r e d u c i n g agents or otherwise altering the conditions f o u n d i n the E R can delay or even b l o c k transport o f proteins out o f the E R (39, 4 0 ) . S e v e r a l studies have s h o w n that i n the case o f M H C C I I  m o l e c u l e s , e x i t i n g the E R c a n be the rate l i m i t i n g step for c e l l surface  expression.  1.7.  Antigen Presentation T h e maturation o f the M H C C I I into a trimolecular c o m p l e x m a d e up o f heavy ( H )  c h a i n , P m and b o u n d peptide i n v o l v e s several specific proteins i n the E R . Peptide is supplied 2  b y T A P 1 and T A P 2. R e c e n t findings suggest that a 48 k D a glycoprotein c a l l e d tapasin is also associated w i t h the M H C C I I - P m - T A P c o m p l e x (41). C a l n e x i n functions i n a chaperone like 2  manner for glycoproteins such as transferrin (42) and is required for association o f M H C C I I w i t h T A P (43).  T h e c a l c i u m - b i n d i n g calreticulin protein also associates w i t h nascent proteins  f o l l o w i n g the association o f c a l n e x i n . B o t h c a l n e x i n and calreticulin i m p r o v e the chances o f a mature conformation b y preventing aggregation and premature d i s u l p h i d e b o n d formation (44). 13  T h e interaction o f many different intermediates results i n antigen presentation by M H C C I I at the c e l l surface. T h e c y t o k i n e g a m m a Interferon ( y - I F N ) can upregulate m a n y aspects o f  antigen presentation. In c e l l lines with l o w M H C CI I expression, treatment w i t h y - I F N results in increased c e l l surface expression (45). Studies in M H C C I I deficient cells demonstrate that the a d d i t i o n o f y - I F N upregulates the association o f 3 m w i t h M H C C I I H chains and the 2  subsequent c e l l surface e x p r e s s i o n ( 4 6 ) .  In a d d i t i o n , y - I F N upregulates  the proteasome  subunits e n c o d e d i n the M H C region a n d M H C C I I H c h a i n p r o d u c t i o n (47).  1.7.1.  Nature of the ER L i k e other m e m b r a n e and c e l l surface proteins, the i n i t i a l a s s e m b l y o f the M H C C I I  c o m p l e x o c c u r s i n the l u m e n o f the E R .  T h e e n d o p l a s m i c r e t i c u l u m is an interconnected  n e t w o r k o f m e m b r a n e s f o r m i n g a tubular s y s t e m w i t h i n the c e l l that is c o n t i n u o u s w i t h the nuclear m e m b r a n e . Proteins destined for secretion o r c e l l surface e x p r e s s i o n are processed in the E R s o o n after translation. These proteins contain a N terminal signal sequence consisting o f about 2 0 residues that mediates transport across the E R m e m b r a n e . O n c e inside, the signal is q u i c k l y c l e a v e d and the nascent protein associates w i t h E R resident proteins that b i n d to and further process the protein. F o r m e m b r a n e b o u n d proteins, a s e c o n d signal sequence found on the p r o t e i n stops t r a n s l o c a t i o n across the m e m b r a n e a n d c a u s e s this r e g i o n to b e c o m e embedded i n the E R , s e r v i n g as the transmembrane anchor (48). P r o c e s s i n g i n the E R is not yet c o m p l e t e l y u n d e r s t o o d . p r o c e s s i n g e n z y m e s that act i n c o n c e r t o n nascent proteins.  T h e r e are m a n y types o f  T h e s e i n c l u d e s i g n a l peptidase,  p r o t e i n d i s u l p h i d e i s o m e r a s e ( P D I ) , p r o t e i n p r o l y l i s o m e r a s e ( P P I ) , E R G I C 53 (49), o l i g o s a c c h a r y l transferase,  a l p h a g l u c o s i d a s e I, a l p h a g l u c o s i d a s e I I ,  E R alpha  mannosidase, thiol dependent reductase E R p 5 7 , c a l n e x i n , calreticulin a n d B i P .  14  1,2-  Different sets  o f m o l e c u l e s associate together and m o d i f y the n e w l y f o r m e d E R p r o t e i n .  Modifications  i n c l u d e the a d d i t i o n o f o l i g o s a c c h a r i d e s , o l i g o m e r i z a t i o n and the f o r m a t i o n o f d i s u l p h i d e bridges. W h e n complete, a protein with a mature and correct conformation c a n exit the E R and proceed to further processing through the cis G o l g i .  1.7.2.  Chaperones P r o p e r assembly o f E R proteins i n v o l v e s E R chaperone proteins.  Chaperones can be  defined as proteins that interact w i t h i m m a t u r e or m i s f o l d e d proteins and either help them to achieve a proper c o n f o r m a t i o n or direct t h e m to be degraded.  S o m e w e l l k n o w n chaperones  include the stress proteins (50) i n c l u d i n g B i P ( 5 1 , 52) and c a l n e x i n . B i P is i n v o l v e d i n both translocation o f nascent proteins into the E R and i n the b i n d i n g and retaining o f misfolded proteins i n the E R , whereas c a l n e x i n b i n d s and retains nascent g l y c o p r o t e i n s i n the E R until they achieve a mature conformation (53, 54). T h e chaperone c a l n e x i n (55) associates w i t h nascent g l y c o p r o t e i n s i n the E R i n a l e c t i n - l i k e m a n n e r b y b i n d i n g to the i n t e r m e d i a t e G l C j M a n G l c N A c 9  2  (56, 57).  Calnexin  a s s o c i a t i o n stabilises intermediate structures p r e v e n t i n g t h e i r r a p i d d e g r a d a t i o n (58) and enhances efficient assembly.  1.7.3.  Other chaperones In addition to interacting w i t h c a l n e x i n , it is clear that m e m b r a n e proteins bound for the  cell surface (and other glycoproteins) must also interact w i t h other chaperones.  These include  chaperone m o l e c u l e s s u c h as p r o t e i n d i s u l p h i d e i s o m e r a s e , g l u c o s y l a s e s a n d c i s - p r o l y l isomerase.  A l s o i n v o l v e d directly w i t h M H C C I I molecules and antigen presentation are the  T A P proteins and tapasin and B i P . C a l n e x i n associates w i t h i m m a t u r e M H C C I I (and other m e m b r a n e glycoproteins) i n  15  the ER. Calnexin binds immature glycoproteins through a lectin-like domain (56). The lectin action (and subsequent association) of calnexin requires a mono-glucosylated oligosaccharide. The glycosylation/de-glycosylation cycle is an effective means of generating and re-generating substrate for calnexin binding and re-binding, causing incorrectly folded or immature proteins to be retained by calnexin (59). The binding step will apply steric constraints to the bound protein, and during this step inter- and intra-chain disulphide bonds can be formed. Glucosylation of glycoproteins regulates transport of glycoproteins out of the ER (60).  Once the calnexin-bound protein  achieves correct conformation, the final glucose residue is removed and association with calnexin terminates (57). With M H C CI I , the correct conformation is achieved with the addition of the peptide and P m forming a tri-molecular complex (61). 2  Studies with reagents that inhibit some of the above mentioned chaperones demonstrate that disruption of regular maturation results in incorrectly folded or immature proteins. These proteins are either retained in the ER or they are tagged for degradation. They are not further modified in the ER and are not further processed through the Golgi. Recent studies using lactacystin to inhibit the proteasome have suggested that such tagged proteins are exported back to the cytoplasm for degradation (62). Studies with daudi cells that do not produce correctly folded M H C CI I because they are missing P m protein showed the accumulation of heavy (H) 2  chains in the ER when treated with a proteasome inhibitor (63). Use of the reducing agent dithiothreitol (DTT) and permeabilised cells showed that disulphide bridge formation in nascent proteins is a fairly early reaction and is mediated by protein disulphide isomerase. Preventing disulphide bridge formation prevents proper folding of M H C CI I, but is reversible upon removal of D T T (64). While formation of the disulphide bridges occurs during protein folding, it is likely that this step does not influence folding, but rather promotes rapid reshuffling of incorrect disulphide pairings and stabilising of a correct 16  one (65, 66) E x c e s s u n f o l d e d protein i n the E R can trigger the u n f o l d e d protein response ( U P R ) . T h e U P R is an upregulation o f E R l u m e n a l proteins i n c l u d i n g B i P and P D I in response to m a l f o l d e d proteins i n the E R .  Stresses that c a n cause the U P R i n c l u d e i n h i b i t i o n o f  g l y c o s y l a t i o n by starvation or the addition o f drugs, addition o f r e d u c i n g agents, expression o f f o l d i n g mutants, o r a d d i t i o n o f c a l c i u m ionophores that reduce c a l c i u m stores i n the E R . In yeast, a transmembrane serine/threonine kinase has been identified that is i n v o l v e d i n the U P R , p o s s i b l y transmitting the U P R signal to the nucleus (67).  It has also been found that protein  o v e r l o a d o f the E R c a n trigger N F - k B i n a m a n n e r independent o f the U P R , p o s s i b l y b y c a u s i n g the release o f C a  2 +  f r o m the E R (68).  T h i s may be a generalised cellular antiviral  response.  1.7.4.  Glycosylation N a s c e n t proteins are g l y c o s y l a t e d i n the E R . A m o n g s t the early a c t i n g E R chaperone  proteins are the g l y c o s y l a t i o n mediators.  A n oligosaccharide a s s e m b l e d o n the l i p i d carrier  d o l i c h o l phosphate is transferred to a nascent g l y c o p r o t e i n . T h i s o l i g o s a c c h a r i d e consists o f t w o G l c N A c , 9 m a n n o s e and three g l u c o s e residues ( G l c M a n G l c N A c ) and is able to 3  g  efficiently associate c o t r a n s l a t i o n a l l y at m o s t A s n - X - S e r / T h r sites.  2  S t e r i c h i n d r a n c e and  accessibility o f the site s e e m to influence transfer o f the o l i g o s a c c h a r i d e to the glycoprotein; different g l y c o s y l a t i o n sites o n the same protein e x h i b i t different but consistent g l y c o s y l a t i o n patterns (69, 7 0 ) .  O n c e a p r o t e i n has f o l d e d , the sites o f g l y c o p r o t e i n attachment m a y no  longer be accessible to the c e l l u l a r g l y c o s y l a t i o n machinery. U p o n transfer,  this o l i g o s a c c h a r i d e is a l m o s t i m m e d i a t e l y set u p o n b y E R resident  proteins. G l u c o s i d a s e I acts almost immediately on the outermost g l u c o s e o f the N - l i n k e d core g l y c a n , r e m o v i n g it.  T h e s e c o n d g l u c o s e is then s e q u e n t i a l l y r e m o v e d b y g l u c o s i d a s e II.  17  G l u c o s i d a s e II also subsequently removes the t h i r d glucose, l e a v i n g a h i g h mannose f o r m o f the sugar.  Replacement o f the third glucose residue is mediated b y U D P - g l u c o s e . g l y c o p r o t e i n  glucosyllransferase  (60)  resulting  in  a  mono-glucosylated  glycan.  A  glucosylation/de-glucosylation c y c l e (71) seems to be one m e t h o d e m p l o y e d to effect quality control w i t h i n the E R (57, 72). Studies  with  t u n i c a m y c i n , w h i c h b l o c k s the  s y n t h e s i s o f the  dolichol  linked  o l i g o s a c c h a r i d e , s h o w that g l y c o s y l a t i o n is an e s s e n t i a l step i n the p r o c e s s i n g o f a glycoprotein.  F a i l u r e to become g l y c o s y l a t e d results i n i m m a t u r e proteins that collect i n the  E R , associated w i t h B i P (73). T h e a b o v e studies s h o w that each step i n the maturation o f a g l y c o p r o t e i n is essential for any subsequent steps.  Studies w i t h castanospermine reveal that inhibiting the glucosidases  has a s i m i l a r effect, resulting in r a p i d degradation o f u n a s s e m b l e d M H C C I I (74).  Indeed,  n e w l y f o r m e d proteins are subject to q u a l i t y c o n t r o l w i t h those that are m i s f o l d e d b e i n g retained and degraded (75). There is some evidence that degraded proteins exit the E R and are degraded i n the c y t o s o l by the proteasome machinery (76).  1.8.  The Golgi complex and membrane traffic P r o t e i n s are further m o d i f i e d as they travel f r o m the E R i n t o the c i s G o l g i .  They  continue through to the trans face o f the G o l g i and are packaged i n vesicles or make their w a y to the c e l l surface. W h i l e the 100 year anniversary o f the d i s c o v e r y o f the G o l g i apparatus was recendy celebrated (77), m u c h o f the structure, function and o r g a n i z a t i o n o f the G o l g i have yet to be determined. T h e G o l g i is the location o f hundreds o f e n z y m e s . M u c h debate has centered o n the nature o f g l y c o p r o t e i n m a t u r a t i o n .  W h a t is agreed  u p o n is that proteins w i t h s i g n a l sequences are subject to the 'export' m e c h a n i s m o f the cell (78).  Proteins are exported from the c y t o s o l to the l u m e n o f the E R where they have access to  the exterior o f the c e l l through the G o l g i . Proteins m a k e their w a y through the m e d i a l and trans  18  G o l g i before b e i n g packaged i n vesicles that can transport them to the c e l l surface where they are either released outside the c e l l or they form part o f the outer layer o f the p l a s m a membrane. W h a t is less clear are the rules g o v e r n i n g the transport o f material between the various compartments o f the E R - G o l g i system. T h e bulk flow theory suggests that there is a bulk flow from the E R through the G o l g i to the cell surface (79, 80). A n y resident i n the E R that is not tagged w i t h a retention signal w i l l eventually make its w a y through the E R , the G o l g i c o m p l e x and t h r o u g h to the c e l l surface.  R e c e n t l y , investigators e l u c i d a t i n g the nature o f v e s i c u l a r  b u d d i n g i n yeast have c h a l l e n g e d the 'bulk f l o w m o d e l ' , p o s t u a l t i n g that proteins ( k n o w n as cargo) associated w i t h membrane b o u n d vesicles are transported through to the G o l g i from the E R i n response to s o m e p o s i t i v e signal (81-83). A sugar m a y mediate this positive signal (84) and E R G I C - 5 3 (that has some lectin-like properties) is the protein m e d i a t i n g this (49, 85).  .9.  T Cell Receptor/Ligand Interactions O n c e an M H C C I I m o l e c u l e has matured through the E R - G o l g i p r o c e s s i n g m a c h i n e r y , it presents b o u n d peptide at the c e l l surface for r e c o g n i t i o n b y T c e l l s .  CD 8  +  cytotoxic T  l y m p h o c y t e s ( C T L ) are i n v o l v e d i n v i r a l clearance. R e c o g n i t i o n a n d subsequent destruction o f virally infected cells is effected by T C e l l Receptors ( T C R ) that must r e c o g n i z e foreign antigens i n the context o f a host o r ' s e l f p r o t e i n (86).  T h e antigen that stimulates a response is the  b o u n d peptide fragment d e r i v e d f r o m foreign protein. Studies have s h o w n that C T L responses may be raised against a single i m m u n o d o m i n a n t epitope (87, 88).  T h e crystal structure o f the  T C R interacting w i t h the M H C C I I m o l e c u l e has a l l o w e d i n s i g h t into the interaction. T h e M H C C I I peptide g r o o v e a l l o w s b i n d i n g o f the peptide i n a s i n g l e orientation; m u c h o f the peptide is h i d d e n f r o m the T C R .  Studies w i t h mutants suggest that the T C R b i n d s diagonally  across the a l p h a helices (89), a v o i d i n g the p r o t r u d i n g N - t e r m i n a l s o f e a c h o f the a l and a2 alpha helices a n d m a x i m i s i n g contact w i t h the p e p t i d e - M H C C I I c o m p l e x (13, 90).  19  Recent elucidation o f the c r y s t a l structure o f a T C R interacting w i t h the p o l y m o r p h i c region o f M H C C I I and peptide shows the intimacy (and specificity) o f the contact between the  t w o proteins.  F i g u r e 4 reveals the tight association o f the a and (3 chains o f the T C R with the  a l and a.2 regions o f M H C C I I and the b o u n d peptide.  2 0  Figure 4. The interaction between M H C  CI I and the T C R  T h i s figure demonstrates the tight association between the a and (3 chains o f the T C R ( s h o w n in p i n k and blue respectively) and the M H C CI I protein (depicted in light green) with b o u n d peptide ( y e l l o w ) . T h e peptide in the peptide g r o o v e is s a n d w i c h e d between the T C R and the a l and al  sections o f the M H C C I molecule. C l o s e association o f (3 m (dark green) 2  w i t h the a 3 regionis also observed (15).  21  1.10.  Viruses M o s t v i r a l infections are cleared i n the procedures m e n t i o n e d above.  In general, the  viral life c y c l e starts with infection, f o l l o w e d b y transcription o f early viral genes that hijack the cellular machinery.  T h e host m a c h i n e r y is then u s e d to r e p l i c a t e the v i r a l genome and  s y n t h e s i z e v i r a l proteins.  P r o t e i n s and the genetic m a t e r i a l are p a c k a g e d to produce more  viruses w h i c h are released into the surrounding area. In short, a v i r u s turns its host cell into a v i r u s - m a k i n g factory. Different c e l l types are m o r e or less susceptible to infection b y particular viruses. N o n p e r m i s s i v e c e l l types d o not r e a d i l y s u p p o r t i n f e c t i o n a n d p r o p a g a t i o n o f v i r u s whereas permissive c e l l types readily s u c c u m b to v i r a l infection and contribute to the viral life cycle. T h e i m m u n e system attempts to destroy v i r a l l y infected c e l l s p r i o r to the release o f more virus.  C T L s target infected c e l l s and cause their i m m i n e n t d e s t r u c t i o n . T C e l l s r e c o g n i z e  antigens w i t h i n the context o f a l l e l i c f o r m s o f M H C m o l e c u l e s . restriction (4).  22  T h i s is k n o w n as a l l e l i c  Figure 5.  The antigen presentation pathway  The steps occurring in antigen processing are detailed here, a) Proteins are synthesised within the cell. Proteins tagged with ubiquitin are degraded by the multicatalytic protealytic complex or proteasome. The proteasome is a large (650 kD), well conserved found within the cytoplams. Peptides of 7 to 12 residues are transported into the ER through the action of the ATP dependant TAP proteins. b) MHC CI I proteins are co-translationally translocated into the ER where the associate with the chaperone calnexin. The H chain also associates with L chain (P2 ) d other chaperones such as Erp57 and calreticulin to name a few. Tapasin mediates the association of the immature MHC CI I H chain-(P m) complex with TAP. Peptide loading by TAP proteins results in a mature conformation accompanied with release by calnexin. The tri-molecular complex is subsequently expressed on the cell surface where it can be recognised by the TCR of passing T Cells. (91) m  2  23  a n  24  1.11.  Adenovirus T h e r e exist at least 7 0 different serotypes o f human adenovirus w h i c h are d i v i d e d into 6  groups termed A through F .  T h e pathological effects o f adenovirus are quite variable, but in  general, they can cause acute disease o f the eye, respiratory tract and gastrointestinal tract (92). W h i l e adenovirus rarely causes a c h r o n i c medical c o n d i t i o n i n healthy i n d i v i d u a l s , the virus has the ability to evade the host i m m u n e system and cause a persistent i n f e c t i o n . In several studies, infection b y viruses i n general and adenovirus i n particular tended to result i n a sub clinical effect (93).  In a d d i t i o n , adenoviruses m a y become persistent and shed i n feces for years post  infection i n o t h e r w i s e healthy i n d i v i d u a l s (94).  A d e n o v i r u s e s c a n e x i s t i n a latent state and  cause d i s s e m i n a t e d disease i n i m m u n o c o m p r o m i s e d patients d u e to r e a c t i v a t i o n (95).  For  e x a m p l e , it has been demonstrated that over 1 0 % o f patients w i t h acquired immunodeficiency s y n d r o m e ( A I D S ) shed adenoviruses i n their urine (94). It has b e e n suggested that i n f e c t i o n and persistence o f adenoviruses in early c h i l d h o o d may result i n respiratory illness i n a d u l t h o o d (96). T h e a d e n o v i r u s is a n o n - e n v e l o p e d icosahedral structure m a d e up o f an outer protein c a p s i d a n d a n i n n e r core c o m p o s e d o f a tightly p a c k e d D N A - p r o t e i n c o r e (97).  T h e protein  c a p s i d is c o m p o s e d o f 2 4 0 hexons and 12 pen ton bases that are noncovalently attached to fibre proteins.  E a c h penton is s u r r o u n d e d b y f i v e h e x o n s a n d is f o u n d at e a c h o f the twelve  ' c o r n e r s ' o f the v i r i o n w i t h an attached p r o t r u d i n g fiber p r o t e i n . T h e c o r e consists o f v i r u s D N A and core proteins. T h e v i r i o n is approximately 6 0 0 - 7 0 0 A i n diameter (97). T h e capsid enters host cells b y association o f the fiber proteins w i t h specific receptors on the c e l l surface (98) f o l l o w e d b y import o f the v i r i o n into the c e l l . T h e v i r i o n is transported to the nucleus w h e r e the c a p s i d is stripped a w a y and the D N A is transcribed either i n an 'early' or a 'late' phase. replication.  T h e e a r l y phase precedes v i r a l r e p l i c a t i o n ; the late phase f o l l o w s viral  E a r l y genes c o d e proteins that are able to d i s r u p t n o r m a l c e l l u l a r processes,  25  i n c l u d i n g those that i n v o l v e presenting peptides d e r i v e d f r o m f o r e i g n proteins at the c e l l surface.  L a t e phase proteins have functions that aid packaging the large amounts o f viral D N A  and viral capsids into new v i r i o n s . T h e g e n o m e o f a d e n o v i r u s consists o f about 3 6 , 0 0 0 base pairs o f linear, d o u b l e stranded D N A . It contains m u l t i p l e o v e r l a p p i n g s p l i c e d m R N A s generated from a single m R N A precursor ( F i g u r e 6). T h e early genes are d i v i d e d into 6 major transcriptional units, c o n s i s t i n g o f the E l A , E 1 B , E 2 , E 3 , E 4 and L l .  T h e immediate early region consists o f E l A  gene products i n c l u d i n g a 2 8 9 a m i n o a c i d (aa) protein and a 243aa protein that act as transacting regulators enhancer-dependent  o f other genes.  early r e g i o n genes  (99)  and  as  suppressors  E 1 B gene p r o d u c t s are p o o r l y u n d e r s t o o d .  o f certain  E 2 r e g i o n gene  products participate i n viral D N A replication. T h e E 3 region codes for several gene products that enhance the a b i l i t y o f a v i r a l l y infected c e l l to evade the i m m u n e system. T h e E 4 region also codes for a trans-activating protein and little is k n o w n about the functions o f the L l gene products.  26  Figure 6.  The adenovirus genome  T h e a d e n o v i r u s g e n o m e is made up o f e a r l y a n d late t r a n s c r i b i n g units. i l l u s t r a t i o n , the d o u b l e line represents the d s D N A .  In this  A r r o w s represent transcription products  and numbers refer to proteins. R i g h t w a r d transcription products are depicted by the r; leftward by the 1. (100).  III4  III  pVU  V  pVI  IOO.JJK  II (hexon)  14  pVUl  * 1 L5  L3 L2  E1B 289R 243R 123R  E3  495R  \25K. airC 6 7 K  Ll  15SR  gp19K. 11.6K. 7 5 K 10.4K. 145K, 14.7K  I—  ia6K  20  55.52K  30  50  40  60  ro  80  90  . i .... i  100  E2A U3K.  14.6K. U 2 K  7.1K. 133K. 34.1K  IV«,  17.1K. 3 3 K  E4 80K terminal protein 140K D N A polymerase  72K s s O N A binding  E2B  27  T h e a d e n o v i r u s has d e v e l o p e d s e v e r a l m e c h a n i s m s to e v a d e the i m m u n e s y s t e m . Perhaps the best characterised is its ability to downregulate the c e l l surface expression o f M H C CI I  m o l e c u l e s and therefore evade the i m m u n e s u r v e i l l a n c e m e c h a n i s m . V i r a l  a c h i e v e this result by different means.  sub-groups  A d 12 b l o c k s t r a n s c r i p t i o n o f M H C CI I m R N A ,  c a u s i n g a repression o f c e l l surface e x p r e s s i o n (101).  A d 2 and 5 have a 25 k D a protein  ( E 3 / 1 9 K ) that b i n d s the M H C C I I m o l e c u l e a n d causes its retention inside the infected c e l l . Other mechanisms utilised b y adenovirus include d o w n r e g u l a t i o n o f the E G F - R e c e p l o r s at the c e l l surface and protection f r o m T N F - i n d u c e d c e l l destruction.  1.11.1.  E3 region W h i l e the E 3 r e g i o n has been s h o w n to be non-essential to adenovirus replication i n  cultured cells (102), it is the r e g i o n that codes for several proteins that may confer an ability to evade the i m m u n e system o f the host (100). W h i l e the differences i n sequence between the E 3 r e g i o n o f a d e n o v i r u s o f different serotypes is s i g n i f i c a n t , there is consensus  i n the open  reading frames ( O R F s ) generating s o m e o f the proteins. I n A d 2 a n d A d 5, variable s p l i c i n g o f a c o m m o n m R N A precursor gives rise to at least 6 different O R F s .  28  Figure 7: The E3 region of adenovirus T h e E 3 r e g i o n encodes several proteins that a i d i n e v a s i o n o f the i m m u n e system. S o l i d a r r o w s s h o w m R N A ; dotted lines s h o w introns.  H a t c h e d bars s h o w identified peptides  and grey bars are those proposed to exist. (100).  E3 A 3.6 K  6.7 K  ED"  291  785  E3B  gp19 K  C S S t\\\\\\SS 1022 .205 1204 1681  I 3 72 ' ' 768  a c  3308  1 1.6 K I860  d  1  2163  740-  e  7.5 K m m 2166 2361  10.4 K 14.5 K 2173  2490 2495  2891  2157  14.5 K 2495  2891  14.7 K S.WWN 2886  951  2680!  12.5 K 291  612  29  3270  1.11.2.  Overview of proteins and effects of the E3 region Several gene products o f the E 3 region have been studied. E 3 / 6 . 7 has been shown not  to participate i n d o w n r e g u l a t i n g the c e l l surface l e v e l o f M H C C I I m o l e c u l e s i n concert w i t h E3/19K.  E 3 / 1 9 K is the focus o f this study and the features s h a l l be discussed i n detail b e l o w .  T h e protein E 3 / 1 4 . 7 K has been s h o w n to prevent c y t o l y s i s o f a d e n o v i r u s - i n f e c t e d cultured cells b y t u m o u r necrosis factor (103, 104). E 3 / 1 0 . 4 downregulates the E G F receptor (105).  Table 1. Summary of the effects of E3 region proteins E3/19K  B l o c k s surface expression o f M H C C I I (106)  E3/14.7K  P r e v e n t s l y s i s o f infected c e l l s b y T u m o u r N e c r o s i s F a c t o r ( T N F ) (103)  E3/10.4K  Downregulation o f Epidermal G r o w t h Factor ( E G F ) receptor (107) - a l s o w o r k s i n c o n j u n c t i o n w i t h 14.5 to prevent T N F c y t o l y s i s (108)  E3/14.5K  Downregulation o f Epidermal G r o w t h Factor ( E G F ) receptor (107) - a l s o w o r k s i n c o n j u n c t i o n w i t h 10.4 to prevent T N F c y t o l y s i s (108)  E3/11.6K  A d e n o v i r u s Death Protein-induces (109)  E3/6.7K  N o p u b l i s h e d function  apoptosis  T h e E 3 / 1 9 K protein is conserved across several serotypes. T h e E 3 / 1 9 K protein o f A d 2 is a 142 a m i n o a c i d T y p e 1 E R resident transmembrane g l y c o p r o t e i n . T h e protein consists o f a 15 a m i n o a c i d (aa) c y t o p l a s m i c tail, a transmembrane r e g i o n , a c o n s e r v e d 2 0 a m i n o acid spacer r e g i o n (110), a n d then a variable region. T h i s l u m e n a l section o f the protein is glycosylated at positions 12 a n d 6 1 ( 1 1 1 ) . A t positions 11 a n d 28 are the first pair o f cysteines that participate  30  i n a d i s u l p h i d e b o n d ; the s e c o n d spans f r o m C y s 2 2 to C y s 8 3 . T h e s e d i s u l p h i d e bonds are conserved across several serotypes (112) and confer stability and contribute to the structure o f E3/19K.  T h e c o n s e r v e d spacer and cysteine residues have been s h o w n to be important for  b i n d i n g to M H C C I I molecules (110, 113). Other studies have s h o w n that truncated E 3 / 1 9 K molecules l a c k i n g the cytoplasmic tail and some o f the transmembrane regions retain the ability to b i n d M H C C I I molecules (114).  Site-directed mutagenesis o f this protein has revealed that  almost every stretch o f the l u m e n a l d o m a i n is required for association o f this protein w i t h M H C C I I (110).  31  Figure 8 The E3/19K protein T h e adenovirus E 3 1 9 K protein (114).  The E3/19K protein is cotranslationally translocated into the ER. Upon transcription, its signal sequence is cleaved. Some processing occurs and it migrates as far as the cis Golgi before returning to the RER. ER retention is mediated by a short stretch on its cytoplasmic tail; truncation of the tail results in E3/19K at the cell surface (114). cotranslationally with nascent MHC  E3/19K associates  CI I molecules. By virtue of the ER retention site, it  retains M H C CI I molecules in the ER preventing travel through the Golgi to the cell surface. The E3/19K molecule has been shown to associate with rodent, human, and simian MHC CI I molecules (115, 116). E3/19K shows varying affinities for different M H C  CI I molecules  (117, 118). While it binds to every human MHC CI I molecule tested to date, it is unable to bind to at least one mouse H-2 protein. During viral infections, the E3/19K protein of adenovirus type-2 (Ad2) binds to major histocompatibility complex (MHC) Class I proteins in the endoplasmic reticulum (ER) where these molecules are assembled. This prevents MHC CI I transport to the cell surface where they can present adenovirus peptides to cytotoxic T lymphocytes (CTL) (100, 119).  Despite studies showing the E3 region not to be essential for virus growth in cultured cells (102), data supporting the role of the E3/19K protein in persistent infections is provided by experiments in which this protein is deleted from an otherwise normal virus and shown to result in the early clearance of the virus in Ad2 infections in mice (120). It is likely that E3/19K is involved in permitting adenovirus infection to spread and become persistent in humans (96). Lack of a good animal model has hampered study in this area.  33  Objectives W h i l e adenovirus m a y have m a n y strategies to evade the i m m u n e s y s t e m , the best studied mechanism is the retention o f M H C C I I proteins w i t h i n the E R a w a y from detection by r o v i n g T cells. T h i s study e x p l o i t e d the properties o f the adenovirus E 3 / 1 9 K protein to perturb the antigen p r o c e s s i n g c o m p o n e n t o f the i m m u n e s y s t e m .  A s E 3 / 1 9 K is an E R retained  protein, study o f this protein a l l o w e d detailed analysis o f many functions o f the E R i n c l u d i n g antigen presentation and p r o t e i n maturation.  A d d i t i o n a l l y , the role o f chaperones i n quality  control and large functional c o m p l e x e s was studied. T h e objectives o f this study were to use the effects o f the E 3 / 1 9 K protein to study antigen processing o c c u r r i n g w i t h i n the c e l l . B l o c k i n g o f antigen presentation revealed details o f p r o t e i n p r o c e s s i n g a n d m a t u r a t i o n i n the E R . T h e i d e n t i f i c a t i o n o f and the interactions between several E R resident proteins was revealed. T h e exact nature o f the b l o c k i n g o f antigen presentation was e x p l o r e d b y e x a m i n i n g the role o f E 3 / 1 9 K i n the E R . E 3 / 1 9 K was b o u n d to m a n y different M H C C I I proteins i n order to establish a m i n i m u m conformation or sequence o f residues for b i n d i n g . Instead the nature o f b i n d i n g was s h o w n to be i n f l u e n c e d b y other factors i n c l u d i n g the inherent a b i l i t y o f a M H C C I I m o l e c u l e to be processed by the endogenous E R maturation p a t h w a y .  A d d i t i o n a l l y , it was  p o s s i b l e to g r o u p M H C C I I a l l e l i c proteins into three categories: b i n d e r s (those that b o u n d E 3 / 1 9 K ) , non-binders and a new category called s l o w - b i n d e r s . S l o w - b i n d e r s b i n d M H C C I I quite strongly but require a longer p e r i o d o f association to achieve this b i n d i n g . Investigation o f one s l o w b i n d e r revealed the retention o f large amounts o f protein i n the E R with comparatively s m a l l amounts at the c e l l surface. Attempts to reverse the retention led to the examination o f m a n y factors w h i c h influence protein maturation i n the E R and s h o w e d this process was m o r e c o m p l i c a t e d than o r i g i n a l l y suspected.  T h i s i n t u r n suggested that  E 3 / 1 9 K retention o f M H C C I I m a y i n v o l v e more interactions than s i m p l y M H C C I I - E 3 / 1 9 K association.  34  R e g u l a r m a t u r a t i o n o f M H C C I I w i t h and w i t h o u t E 3 1 9 K and the association o f this c o m p l e x w i t h E R resident chaperones was e x a m i n e d .  E 3 / 1 9 K r a p i d l y p r o m o t e d a stable  conformation under adverse conditions acting like a chaperone. E 3 / 1 9 K association w i t h M H C C I I d i d not b l o c k the i n t e r a c t i o n o f endogenous chaperones s u g g e s t i n g that the s i z e o f c o m p l e x e s i n the E R w e r e q u i t e large.  O t h e r m e c h a n i s m s s u c h as the u n f o l d e d protein  response m a y be i n v o l v e d i n the E 3 / 1 9 K effect on infected cells. Peptide b i n d i n g b y M H C C I I is a step in antigen presentation.  T h e effect o f E 3 / 1 9 K  b i n d i n g to M H C C I I o n peptide b i n d i n g was examined. E x p l i c i t peptide b i n d i n g to M H C C I I i n the presence o f E 3 / 1 9 K was demonstrated.  T h i s s h o w e d that both peptide and E 3 / 1 9 K  b o u n d M H C C I I c o n c u r r e n t l y , r e d u c i n g the l i k e l i h o o d that the a s s o c i a t i o n was through the peptide b i n d i n g g r o o v e . D e m o n s t r a t i o n o f m u l t i p l e proteins b i n d i n g to M H C C I I s u g g e s t e d that large c o m p l e x e s o f proteins w e r e f o r m e d i n the E R . E 3 / 1 9 K w i t h these c o m p l e x e s also o c c u r r e d . S t e r i c h i n d r a n c e b e t w e e n m e m b e r s o f these large c o m p l e x e s seems l i k e l y .  T o examine i f  E 3 / 1 9 K and other m o l e c u l e s o c c u r r e d as multimers, c o m p l e x e s were precipitated and separated o n a s u c r o s e d e n s i t y gradient.  T h e s e f i n d i n g s l e n d further c r e d e n c e to the i d e a that other  effects o f E 3 / 1 9 K r e m a i n to be discovered.  35  2.  Materials and Methods  2.1  Cellular Methods  2.1.1  Tissue Culture The cell lines used in these studies are listed i n table 2.  2 9 3 c e l l s or transfected 2 9 3 c e l l s . transformed w i t h A d 5 ( 1 2 1 ) .  M o s t experiments used either  2 9 3 c e l l s are adherent h u m a n e m b r y o n i c k i d n e y cells  A d h e r e n t cells were g r o w n to 8 5 - 9 5 % c o n f l u e n c e in D M E M  s u p p l e m e n t e d w i t h L - G l u (2 raM), 10 m M H E P E S ( p H 7.2), and 1 0 % F e t a l C a l f S e r u m . A n t i b i o t i c s were o m i t t e d f r o m c u l t u r e m e d i a .  C e l l s w e r e passaged u s i n g 0 . 0 5 % t r y p s i n  ( w t / v o l ) in phosphate buffered saline ( P B S ) w i t h 1 m M E D T A at 3 7 ° C , d i l u t i n g cells 1:10 i n fresh culture media. Non-adherent cells were g r o w n i n flasks w i t h the m e d i a supplemented as above.  L i v i n g c e l l concentration was m o n i t o r e d b y trypan b l u e e x c l u s i o n and cultures were  g r o w n to a concentration o f l x l O  6  to 5 x l 0  6  c e l l s / m l before passaging at a 1:10 d i l u t i o n .  With  the exception noted b e l o w , a l l c e l l s were i n c u b a t e d at 3 7 ° C i n a h u m i d i f i e d 5 % C 0 / 9 5 % air 2  environment. Large scale cultures were g r o w n i n sealed B e l c o spinner flasks at 3 7 ° C i n incubator not supplemented w i t h C 0 . C e l l spinners were set at 6 0 - 7 0 r p m . M e d i a was supplemented w i t h 2  Ca  2 +  .  C e l l c o n c e n t r a t i o n was m o n i t o r e d as a b o v e w i t h t r y p a n b l u e a n d was m a i n t a i n e d  between 1 x 1 0 and 5 x l O 6  7  cells/ml.  36  Table 2.  Reagents used  Cell Lines 293  human embryonic kidney (ATCC C R L 1573)  293.12  293 transfected with E3/19K  A549  human lung carcinoma (ATCC C C L 185)  293p39  Low passage 293  Monoclonal Antibodies (TCS) W6/32  mouse anti H L A A, B, C (ATCC HB 95)  PA 2.1  mouse anti H L A A2, Aw69 (ATCC HB 117)  OKT6  mouse anti transferrin receptor  34.5.8s + (34-l-2s)  mouse anti D (ATCC HB 102)  15.5.5s + (H100.27.55)  mouse anti D (ATCC HB 24)  34.1.2s  mouse anti K (ATCC HB 79)  16.3.IN  mouse anti K (ATCC HB 25)  28.14.8s  mouse anti D , L (ATCC HB 27)  d  k  d  k  b  d  Antisera R418  Rabbit anti E3/19K (122)  R426 R425  Rabbit anti M H C CI I H chains (123)  and calnexin  Rabbit anti calnexin(124)  anti transporter  Rabbit anti TAP (125)  Anti human (3 m  Rabbit anti human P m (126)  2  2  Lysis Buffers 0.6% Chaps  0.6% Chaps, PBS  1% NP40  1% NP40, 120 mM  1% N-Octyl Glucoside  1% N-Octyl Glucoside, 50mM N a P 0 pH 7.0  PBS + 1% SDS  1% SDS, PBS w/o Mg + or Ca+  NaCl, 4mM  MgCl , 20 m M Tris 2  4  2  Media Hams F12 DMEM alpha M E M  37  2.1.2.  Transfection 293 cells were transfected using Lipofectin (GibcoBRL). Genomic D N A in PBR322  was purified using the Promega MaxiPrep kit. 10 [Lg D N A was combined in 10 fold excess with a neomycin resistance marker and transfected following the manufacturers instructions. Cells were allowed to grow 36-48 hours before the selection agent, G418 (neomycin) (GibcoBRL) was added at a final concentration of 1 mg/ml. Dead cells were removed with frequent media changes. Surviving colonies were isolated with cloning rings. Individual clones were grown up and tested for gene expression.  2.1.3.  Viruses Adenovirus 2 (Ad2) stock was obtained from Dr. Frank Graham through Roger Lippe.  Stocks were grown in bulk on low passage 293 cells (121) in complete alpha MEM, harvested and stored at -80°C in 10% glycerol/PBS^at 2xl0 plaque forming units per ml (PFU). P F U 9  were calculated using viral stock serially diluted onto A549 cells and overlaid with 2x F l l media and 1% Agarose. Plaques were scored 7 days after infection. Prior to use, virus stock was gently thawed at 4°C. Vaccinia virus containing the human P m gene was generously 2  provided by Dr. J. Yewdell (National Institutes of Health, Bethesda, MD).  Prior to infection cells were washed in P B S . Cells were infected at a multiplicity of ++  infection (MOI) of 5. Virus was added to the cells and allowed to attach for one hour. Media was added back after the first hour and infection was allowed to proceed for approximately 18 hours.  38  2.1.4.  Peptides Synthetic peptides were purchased from the Alberta Peptide Institute. Peptides were  biotinylated at the N terminal using NHS-Ester biotin (Sigma). Peptides dissolved at 1 mM in NaPhosphate buffer (pH 7.5) were mixed with lOOx excess NHS-Biotin Ester for two hours. Unreacted NHS-ester was neutralised with Tris-HCl (pH 7.4) to a final concentration of 10 mM. Peptide (biotinylated and non-biotinylated) was added to growing cell cultures 24 hours prior to labelling at concentrations ranging from 5nM to 5p.M.  2.1.5.  Fluorescence activated cell sorting (FACS) Adherent cells were pre-treated with versene or 0.05% trypsin and washed in FACS  buffer to remove them from the plate. Cells were suspended at l x l O cells/ml in D M E M 6  supplemented with 200 mM L-Glutamine, 20 mM Hepes (pH 7.2) and 20 mM NaAzide (hereafter referred to as F A C S buffer).  Cell suspensions and all manipulations were  performed at 4°C for the duration of the antibody labelling. Suspensions were washed twice with FACS buffer and incubated with primary antibody (200 pi of monoclonal TCS or 3 pi of ascites) for 45 minutes. Unbound primary antibody was washed out with two more FACS buffer washes and the samples were incubated with 100 jxl of 10 mg/ml stock fluorescein isothiocyanate (FITC) labelled secondary antibody (Jackson) for 45 minutes.  Excess  secondary antibody was washed out with two more FACS buffer washes followed by a wash with PBS with C a  2 +  or M g (PBS"). The cells were fixed in a 1.5% paraformaldehyde/PBS +  and stored in the dark at 4°C until analyzed. Analysis was performed on a Becton Dickson (BD) FACScan. 5000 gated events were collected and subsequently analyzed using the Lysis II software from BD.  39  An alternate protocol involved substituting FITC coupled secondary antibody with biotinylated secondary antibody. A third incubation using FITC coupled streptavidin was also added.  2.1.6.  Animals Rabbits used for antisera generation were housed in the University of British Columbia  south campus arrimal facility. They were kept under the care of Willem Schoorl according to the guidelines of the Canadian Council on Animal Care. Rabbits were initially injected with antigen in the lymph nodes and subsequently bled to raise antisera. When necessary they were sacrificed by C 0 asphyxiation. 2  2.2. 2.2.1.  Protein Techniques Antibodies Monoclonal antibodies (mAbs) were used as Tissue Culture Supernatant (TCS) but in  some cases ascites fluid was available. Rabbit antisera to E3/19K (R418) and to M H C CI I (R425 and R426) were donated by Dr. Sune Kvist. Anti calnexin antisera (124) was a gift from Dr. Bergeron, McGill University, Montreal, Canada and mouse T A P 1 and T A P 2 antisera were obtained from Dr. G. Butcher, AFRC, Cambridge, UK. Antisera to E3/19K was raised in rabbits using synthetic peptide coupled to KLH.  2.2.2.  Metabolic Labelling An 85-95% confluent 65 mm plate with approximately Ixl0 -2xl0 cells received 300 6  6  p.Ci S in 2 mis during labelling. Cell cultures were washed in methionine and cysteine free 3 5  D M E M (Gibco) one hour prior to labelling and subsequently labelled with 150 \iCUm\ Pro-Mix 40  ( S-Met-Cys) (Amersham) for periods ranging from 5 min to one hour. In pulse chase experiments, the labelling media was replaced with standard media for the chase. All labelling was performed under standard cell culture conditions. Cells were lysed with a variety of lysis buffers (see table 2) supplemented with PMSF (40 p.g/ml) on ice. . Efficiency of labelling was determined by counting small aliquots (3 pi) of lysate following T C A precipitation of labelled proteins.  2.2.3.  Immunoprecipitation  Lysates were spun at 10,500 g for 30 minutes. Volumes of lysates were normalised to account for differences in labelling and aliquots ranging from 200 pi to 1ml were used for immunoprecipitation. All immunoprecipitation steps were performed at 4°C. Samples were precleared by incubation with 3 pi Normal Rabbit Sera (NRS) for 45 minutes, followed by a 45 minute incubation with 45 pi of a 1:1 Lysis Buffer:Prot-A-Sepharose (Pharmacia) slurry. Specific antibodies were added to pre-cleared samples. In general, 100 pi of TCS from monoclonal cell lines or 1 pi of ascites fluid or sera was incubated with the samples for 45 minutes. Following specific incubation, 45 pi of a Prot-A-Sepharose slurry was added and allowed to incubate for a further 45 minutes. Beads were washed several times with buffers of varying salt concentration (three times with buffer B: 0.2% NP40, 150 mM NaCl, 2mM EDTA, 10 mM Tris, two times with buffer C: 0.2% NP40, 500 mM NaCl, 2 mM  EDTA, 10 mM Tris, once with Buffer D: lOmM  Tris) and finally sucked dry. Beads were then either stored at -80°C or boiled at 95°C for 5 minutes with bromomix (60% sucrose, bromophenolblue 0.1%, Tris-HCl pH 8.8 with freshly made 0.5M dithiothreitol)-20% SDS mixture.  41  Samples were then blocked with 0.5mM  iodoacetimide, and separated with SDS polyacrylamide gels electrophoresis (SDS-PAGE).  2.2.4.  SDS-PAGE  Immunoprecipitated samples were separated on SDS-PAGE. Large gels were prepared as gradient gels with 10-15% polyacrylamide topped with a 5 % stacking gel. 25 |il samples were loaded and run O/N at 18 mAmps (350 V). Gels of 10, 12 and 15% were also used. Upon complete migration of the dye front across the gel, gels were fixed (10% Acetic Acid, 3 0 % v/v Methanol) for 30 minutes and then enhanced in 20mM NaSalicylate (known as enhance). Gels were dried and placed on Kodak X-AR radiographic film at -80°C for periods ranging from one day to several months. Alternatively, samples were separated on the BioRad Minigel (10 x 7 cm) system. Gels were made as above and samples separated according to manufacturers instructions.  2.2.5.  Western Blotting As an alternative to fixing after PAGE, western blotting was performed. In addition to  1 4  C labelled molecular weight (MW) standards, biotinylated standards were also present on the  gels. Following complete migration of the dye front in the electric field, the gel equilibrated in western transfer buffer () for 30 - 90 minutes. The gel was then sandwiched against a sheet of Immobilon P polyvinylidene difluoride (PVDF) membrane, submerged in western transfer buffer and blotted in an electric field for time periods between one and twelve hours. Alternatively, the BioRad mini western apparatus was used according to manufacturers instructions. After transfer, membranes were washed 2 times in western wash buffer (0.1% BSA, 0.05% Tween 20, 0.01M thimerasol in PBS), and blocked for one hour in B S A (2.5%) fortified western wash buffer. Membranes were washed 3 more times and exposed to antisera. Antisera was added at dilutions of 1:10 to 1:5000 in 45 mis total volume. Alternatively, the glass plate techniquewas used to conserve antisera. Briefly, the membrane was placed on a  42  glass plate with 100 pi of diluted antisera. Antisera was distributed over the membrane with a glass spreader every 30 minutes for two hours creating a thin film of antisera between the plate and the membrane. Following the two hour incubation, the primary antibody was washed away and the secondary goat anti rabbit coupled with biotin (Jackson) was added at a dilution of 1:10,000 for 45 minutes. Membranes were then washed several times and incubated with HRPO coupled to streptavidin.  Subsequent detection was performed using Enhanced  Chemiluminescence (Amersham ECL) system. Membranes were exposed to Kodak X-AR film for periods ranging from 5 seconds to overnight. Subsequently they were stored damp at 4°C for future use.  2.2.6.  Tunicamycin Tunicamycin was used to block N-linked glycosylation prior to pulse-chase  experiments. Cells were incubated with varying concentrations of tunicamycin in regular media for one hour. Media was replaced with labelling media (prior to addition of  S-Met-  Cys) supplemented with tunicamycin for one hour. Cells were then labelled in the presence of tunicamycin. Subsequent treatment of the cells is described below in section 2.2.7.  2.2.7.  Cross linking Cross  linking  was performed  during lysis  with  the addition  of DSP  [dithiobis(succinimidylpropionate)] (Pierce) to the lysis buffer. Cells were prepared, labelled and lysed as above with the exception that the lysis buffer contained DSP (200 Jig/ml). The lysis buffer used with DSP was PBS with 1% SDS. Crosslinking was stopped with 100 pi of 1M Tris-HCl pH 7.5 prior to spinning the lysate down.  43  2.2.8.  Endoglycosidase H (Endo H) digestions Endo H (Boehringer Mannheim) was prepared according to manufacturers instructions  with the addition of PMSF (800 (xl NaCitrate, 200 pi Endo H, 1 u.1 PMSF). Precipitates treated with Endo H were washed as above (2.2.7) except that in the final wash with Buffer D, beads were resuspended and divided into two aliquots prior to final drying. The first aliquot was incubated with 12.5 ul Endo H for 12 hours at 37°C. A second 12.5 ul was added for an additional 12 hours. The second aliquot was incubated in the same mixture without Endo H. Following a 24 hour incubation, the Endo H was washed out with Buffer D and the samples were prepared for SDS-PAGE as above.  2.2.9.  Sucrose density centrifugation Sucrose density centrifugation was performed as described in the literature (127).  Continuous 5 % to 2 0 % sucrose gradients were created using stock sucrose solutions. The detergents CHAPs, digitonin or N-Octyl glucoside were utilised in the lysis buffer and sucrose solutions. Gradients were made using the Pharmacia gradient maker. 300 ul of sample was placed on top of a 12 ml cushion in a Beckman SW 41 UltraClear tube and centrifuged at 4°C and 148,000g (36,800 rpm) for 28 hours in an SW41 rotor. Fractions were manually collected in 1 ml aliquots from the top of the tube. Samples were then diluted and run on SDS-PAGE.  2.2.10.  Densitometry  Densitometry was performed using the Molecular Dynamics scanner.  Gels were  prepared without enhance solution and exposed using the Phosphorlmaging (Molecular Dynamics) cassette. Computer analysis including densitometric traces were performed on the  44  resulting data.  2.2.11.  Data Imaging Data were scanned and manipulated using Adobe Photoshop and MacDraw and printed  on a Codonix printer.  45  3.  Comparison of Differential Binding of E3/19K to M H C Class I allelic  proteins 3.1  Introduction This study relates the transport rates of M H C CI I molecules to their ability to interact  with the E3/19K molecule. E3/19K binds to every human allelic protein tested so far and all but one H-2 protein. Previous studies involved many groups of researchers using cell lines from different species (128-132).  However, since the original E3/19K studies were  performed, elucidation of the steps in antigen presentation has occurred.  Many different  proteins, some of which are allele specific, may be involved. Therefore the rate of maturation for different M H C CI I proteins is likely to vary when studied in different cell lines. Alleles of mouse M H C H-2 proteins were transfected into human cells to establish a model system with a consistent cellular background.  3.2 Rationale and Goals The goals of this study were to elucidate the binding requirements of E3/19K for M H C CI I. By comparing differences between those allelic forms of M H C CI I proteins that did and did not bind, it was hoped that a set of factors influencing binding would be elucidated. These data would also shed further light on the nature of adenovirus evasion of the immune system and further understanding of M H C function.  3.3  Results Previous studies had assessed the ability of E3/19K to bind to different M H C CI I  allelic proteins in different cell lines from different species. Comparison of differences of E3/19K binding M H C CI I alleles in these experiments reflected many variables, some of  46  which related to differences between species. By transfecting many different M H C alleles into the same cell line, some of the variation due to species differences could be removed. At the same time, because of the allele specific nature of these effects, this also represents a limitation of the study; effects seen in 293 cells may be completely different from those seen in other cell lines.  3.3.1.  Transfected H-2 allelic proteins are expressed at different levels at the  surface of 293 cells In order to establish the relative level of surface expression of the H-2 allelic proteins in 293 cells, F A C S analysis was performed on the transfectants. Several clones from each transfection were analyzed, and the clone with the highest expression for each transfected allele of mouse M H C CI I genes was used in all subsequent experiments. The decision to use the clone with the highest level of expression was based on the assumptions that E3/19K had an innate ability to bind particular alleles of M H C CI I and that large amounts of mouse M H C CI I proteins expressed in 293 cells would provide more ligand for E3/19K. Limiting amounts of E3/19K seemed unlikely since in adenovirus infected cells, E3/19K is expressed in excess. Data in Figure 9a shows that these transfectants express the H-2 proteins at the surface in varying levels. The level of endogenous H L A expression in untransfected 293 cells is shown on the left (labelled HLA).  While some of the allelic proteins show low levels of  expression, all show a detectable level of expression. As a control for transport to the cell surface, in Fig 10c the cell surface levels of the transferrin receptor show that transport to the cell surface is not disrupted. To determine whether low surface expression of some M H C CI I proteins was due to competition for factors with endogenous HLA, the level of HLA-A, B and C expression was assessed using W6/32. A comparison of the W6/32 cell surface expression in the transfected and untransfected 293 cells is shown in Figure 9b.  47  These data showed that in every  transfectant except the K transfectant, the level of cell surface expression of the W6/32 epitope d  was reduced. The level of reduction of W6/32 expression indirectly allowed the monitoring of the effect of the foreign M H C CI I molecule on the endogenous M H C CI I. In both the D and b  K  b  transfectants the level of W6/32 expression was reduced to a level of 5 8 % and 5%,  respectively, of untransfected 293 cells. The K transfectant alone showed an increase in the d  level of W6/32 expression; this was due to cross reactivity of W6/32 with K  d  (133).  Comparison with levels of K expression assessed with the K specific monoclonal 34.1.2s d  d  and W6/32 expression in the non-transfected 293 cells suggests that the K  d  and endogenous  expression were additive. Reduction in cell surface expression of endogenous M H C CI I proteins could reflect a competition for limited resources such as 3 m or peptides. Mouse M H C CI I proteins have a 2  higher affinity for human P m than endogenous M H C CI I (133). Excess mouse M H C CI I 2  may leave little P m available for endogenous H L A alleles, resulting in lowered W6/32 2  expression.  The phenomenon of internal competition modulating H L A expression is  previously unreported.  48  Figure 9.  FACS analysis of 293 cells and 293 transfectants  Confluent plates of 293 cells and 293 transfectants were analyzed by FACS. Cells were harvested as described in Materials and Methods. In all cases the untransfected 293 cell data is in the first column and labelled HLA. a) Levels of cell surface expression of transfected M H C CI I was determined by incubation with allele specific antibodies as listed in table 2. Calculation of arbitrary fluorescence units (AFU) involved taking the difference between mock treated and specifically labelled FITC labelled samples. . b) Levels of endogenous HLA-A2 expression was assessed using the monoclonal antibody W6/32. c) Transferrin receptor levels were determined using the OKT9 monoclonal antibody.  49  S p e c i f i c CI I E x p r e s s i o n  Allele  W6/32  Expression  293 transfectant  0 K T 9 Expression 350/"  293 transfectant  50  3.3.2. Transport of H-2 allelic proteins to the cell surface of 293 cells To determine levels of expression of transfected M H C CI I proteins, monoclonal antibodies were used to precipitate M H C CI I from cells pulsed for 15 minutes and chased for 0, 30, 120, 240 and 480 minutes. During the course of the chase, the mobility of the precipitated M H C CI I decreases, reflecting modification of the proteins. M H C CI I proteins undergo processing from a 43 kDa protein at two hours to a 46 kD MW form present at four hours of the chase. The rate limiting step for transport to the cell surface is escape from the ER. To assess whether the M H C CI I proteins had left the ER and migrated to the cis-Golgi, Endo H digestion was performed on half of the M H C  CI I precipitates.  Glycans of  glycoproteins are modified from a high mannose form to a more complex Endo H resistant form in the cis-Golgi. The analysis shows that a portion of the population of D molecules exit the ER (as b  demonstrated by their insensitivity to Endo H) after two hours. However, a large proportion of the D molecules remains Endo H sensitive after four hours, suggesting an extremely slow b  transport rate. The half life (t ) for this protein to leave the ER is greater than four hours. In J/2  the D  d  transfectant, the D molecules achieve a mature conformation after four hours. D is d  d  present in an immature lower molecular weight form for most of the four hour chase. The higher molecular weight form appears after four hours of chase. In the samples treated with Endo H, most of the sample has disappeared from all but the 480 minute timepoint. This likely reflects an unstable D M H C CI I protein, with most of it falling apart during the 24 hour, 37°C d  Endo H incubation. The Endo H resistant form visible at the final four hour chase point suggests that the mature protein is more stable. The L molecule is processed marginally over d  the four hour chase. It appears that the epitope recognized by the monoclonal used may not be stable under the conditions of the 24 hour incubation with the Endo H buffer. 51  The K  b  molecule is partially Endo H resistant after about one hour and is therefore  rapidly transported. Analysis of the K  d  precipitation demonstrates that an epitope recognised  by the conformation specific mAb for the K  d  protein does not appear until 30 minutes post  pulse. Endo H digestion reveals that the forms observed at 30 minutes and one hour have not exited the ER; by two hours, much of this molecule is Endo H insensitive and at the end of four hours, almost no Endo H sensitive K molecules remain. A similar pattern is observed for d  K. k  Endo H digestion reveals that the K  k  protein has left the ER by two hours post pulse,  suggesting that it is even more rapidly transported than K . d  52  Figure 10.  Levels of expression and rates of transport of H-2 M H C CI I  proteins transfected into 293 cells Lysates from confluent 293 cells transfected with different mouse M H C CI I (H-2) proteins were precipitated with allele specific monoclonal antibodies as listed in table 2. Cells were pulsed with Pro-Mix (35S Cys-Met) for 30 minutes and chased for 0, 30, 60 120 and 240 minutes (lanes 1 to 5 respectively). Aliquots which have been incubated with Endo H are in lanes 6-10. Levels of expression for each allele were determined. M H C CI I proteins with different mobilities reflecting differently processed forms are labelled with arrows. Precipitates were split and alternately mock treated (lanes 1-5) or treated with Endo H (lanes 6-10) for 24 hours and then separated by SDS-PAGE. Endo H resistant proteins have reduced mobility and are labelled by the upper arrow.  53  Once expression of H-2 proteins in human 293 cells was established, the effect of the E3/19K on the different proteins was examined. Initially, it was hoped that expression of the transfected mouse H-2 alleles would be uniformly strong and that this level of expression would provide a clear picture about whether E3/19K bound or not. In transfectants with M H C CI I allelic proteins that bound E3/19K, reduced cell surface expression was expected. In transfectants with proteins that did not bind, no reduction in cell surface expression was expected. For proteins that bound less effectively, it was postulated that temporary association with E3/19K may result in slower transport to the cell surface. It should be noted that the level of expression for each allele was quite variable in the 293 cells. This variable expression probably reflects the accessory proteins and chaperones available for the transfected allele. Additionally, as shown in Fig l i b , the expression of the transfected allele did affect the cell surface expression of the endogenous M H C CI I alleles. Highly expressed transfectants such as K  b  cause a large reduction in cell surface expression of H L A A, B and C  detectable by the conformation specific monoclonal antibody W6/32. This could reflect limited P m or peptide. 2  3.3.3.  Co-precipitation of the adenovirus E3/19K protein with M H C CI I in  293 transfectants  >  To address the binding capacity of H-2 proteins to E3/19K, the transfectants were infected with Ad2 at a MOI of 5 for 18 hours and metabolically labelled with a pulse of S 35  methionine and then chased for 0-4 hours. After lysis, the H-2 allele expressed in each transfectant was immunoprecipitated and examined by SDS-PAGE and autoradiography. Controls were carried out to confirm that the infected transfectants expressed equal amounts of the E3/19K protein (appendix 1). Half of the precipitated samples were digested for 24 hours 55  at 37°C with Endo H to determine whether the M H C CI I molecules had acquired mature Nlinked glycans. Results are shown in figure 11. E3/19K is detected by co-precipitation with M H C  CI I in Ad2 infected cells. While  confirmation of the identity of E3/19K by subsequent western blotting was not performed in the mouse M H C  CI I transfectants, comparison with parallel immunoprecipitations using the  293.12 cell line helped to establish the identity of E3/19K. 293.12 cells have transfected with the EcoRI d fragment containing the E3/19K protein from Ad2 and exhibit reduced cell surface expression of M H C CI I in FACS experiments. Comparison of immunoprecipitation of MHC CI I from 293, Ad2 infected 293 cells and 293.12 cells reveals a protein corresponding to E3/19K(131). After a chase of 2 and 4 hours, the D  b  molecule co-precipitates a protein at about 25  kDa that corresponds to E3/19K. A corresponding band also appears at the 2 and 4 hour time points in the Endo H digested samples. corresponding to E3/19K has the lower MW  In the Endo H digested samples, the band consistent with digestion of the N-linked sugars  on this molecule.The three bands in the Endo H digested samples correspond to differences in sensitivity in the two carbohydrates present on E3/19K. Co-precipitating with D  molecule, suggesting that transfected D  b  b  is the P m 2  proteins are being assembled with human P m. 2  The  appearance of P m in the samples that have been exposed to 37°C for 24 hours in the Endo H 2  treatment suggests that the D molecule has adopted a stable conformation. b  The D molecule does not co-precipitate with E3/19K. The Endo H digestion of D d  d  reveals it exits the ER at the same rate as in the uninfected cells. An Endo H resistant protein is observed at 4 hours. The L molecule also does not co-precipitate E3/19K during the four hour d  chase. However, in both infected and uninfected cells, the L  56  d  molecule remains Endo H  sensitive throughout the four hour chase. The  K  b  protein co-precipitates E3/19K.  E3/19K  appears to co-precipitate  instantaneously with the K molecule appearing at all time points. At the 2 and 4 hour time b  points, the molecular weight of E3/19K has decreased marginally, suggesting carbohydrate processing. The Endo H digestion shows that two more forms of E3/19K appear after two hours; they differ in carbohydrate attachments. After two hours, a second protein at about 22 kDa appears. At four hours post pulse an additional band at the same molecular weight as the untreated precipitates appears. This band likely represents an Endo H resistant form of E3/19K. The K  molecule co-precipitates E3/19K strongly at all timepoints. K is assembled by  d  d  about 30 minutes post chase. At this point, the heavy chain and P m is observed. The K  d  2  molecule remains Endo H sensitive throughout the entire four hour chase. Once again, two forms of partially Endo H resistant E3/19K are observed co-precipitating with Endo H sensitive K molecules. These data suggest that E3/19K is cycled back and forth through more d  mature compartments such as the cis Golgi and is able to bind M H C CI I molecules in an earlier compartment and retain them there. Differences in the maturity of the carbohydrates on E3/19K bound by H-2 M H C CI I proteins suggests differing affinities for E3/19K based on its processing. The K  k  molecule does not co-precipitate with E3/19K. The K is entirely Endo H k  resistant after about four hours. To summarize results from figure 11, the E3/19K protein clearly co-precipitates with K , K , and D proteins. L does not co-precipitate E3/19K during the chase times performed b  d  b  d  in this experiment. The Endo H sensitivity of K , D , and K d  b  b  during the four hour chase  confirms that they do not exit the ER during this period and thus the E3/19K binding of these  57  molecules. The K , and D k  molecule are Endo H insensitive after four hours. Comparison  d  with Figure 10 shows differences between MHC  CI I transport in adenovirus infected and non  infected cells. E3/19K had no effect in retarding the egress of these allelic forms of MHC proteins from the ER.  The D  d  CI I  protein is able to achieve Endo H sensitivity at the same rate that  it did in non-infected cells (Figure 10), becoming Endo H resistant after 4 hours. Interaction between D  d  and E3/19K is likely to be weak or nonexistent.  58  Figure 11.  The effect of Ad2 infection on the rate of transport of transfected  M H C CI I proteins in 293 cells 293 cells and 293 transfectants were infected with Ad2. Infection was at MOI of 5 eighteen hours prior to labelling with Pro Mix ( S-Met/Cys). The cells were pulsed for 30 minutes and chased for 0, 30, 60 120 and 240 minutes (lanes 1 to 5) respectively. Aliquots which have been incubated with Endo H are in lanes 6-10. M H C CI I was precipitated with allele specific antibodies as listed in table 1. Samples were divided and alternately mock treated or treated with Endo H for 24 and then separated by SDS-PAGE. E3/19K migrates at -25 kDa 35  and is denoted by arrows. (3 m also co-precipitates and is seen on the gel at 12kDa. 2  59  3.3.4.  Adenovirus  infection  of  293  transfectants  reduces cell  surface  expression of M H C CI I proteins To assess the effect of E3/19K on cell surface expression, F A C S analysis of Ad2 infected transfectants was performed (figure 12).  While the cell surface expression of  transfected H-2 proteins is variable, when infected with Ad2, cell surface expression of many of these M H C CI I proteins is reduced. The FACS data show that in the untransfected 293 cells, infection with Ad2 causes reduction of the cell surface H L A (W6/32 epitope) to 6 2 % of uninfected expression. The difference in cell surface expression of the transferrin receptor in infected and mock infected cells is shown in figure 12b demonstrating that the effect of adenovirus infection is not blocking all transport to the cell surface. In the transfected 293 cells, Ad2 infection causes the cell surface expression of the H-2 transfectants to be reduced as well. A comparison of levels of cell surface expression of H-2 proteins in infected and uninfected cells shows which alleles are susceptible to E3/19K binding. Figure 12 shows the greatest level of reduction of cell surface expression was highest for D  b  which expressed 4 3 %  of the D that the uninfected transfectant expressed. L also showed a large reduction at 44%. At the other end of the spectrum, the proteins that were affected the least were K  k  and D , k  which showed 9 3 % and 9 5 % of uninfected expression respectively. The cell surface expression of K  d  (62%), K  b  (73%) and D  d  (81%) were moderately inhibited. These data  support the hypothesis that amongst the H-2 molecules K , D d  b  and L bind to the E3/19K d  molecule with the greatest affinity. However, it should be noted that while a FACS experiment represents 5000 individual events, the results are subject to some presorting. Sampled cells are gated by size and cells are selected to represent a healthy population. While size selection is performed prior to observing  61  results, the outcome of the experiment could reflect arbitrary sampling of a larger, more diverse population.  62  Figure 12.  The effect of Ad2 infection on M H C CI I expression in 293 cells  and 293 transfectants a) 293 cells and transfectants were alternately infected or mock infected with Ad2 at MOI of 5. Infection was started eighteen hours prior to harvest for FACS analysis. Cell suspensions were specifically labelled with M H C CI I allelic protein specific antibodies as listed in table 1. Suspensions were analyzed using flow cytometry. Arbitrary fluorescence units (AFU) are the difference between mock treated and specifically labelled FITC labelled samples for each transfectant. The first bar of each pair shows the uninfected cell surface expression; the second the level of cell surface expression in infected transfectants.  63  B uninfected i •  2 9 3 transfectant  64  ArJ2  b) The levels of the transferrin receptor at the cell surface are compared in infected and uninfected transfectants.  Transferrin Receptor Expression  • mock • infected  Transfected Allele  Results representative of these experiments are summarized in table 3. As determined by immunoprecipitation, K , K d  b  and D  b  all co-immunoprecipitate with E3/19K, but the  products of alleles K and D do not. The data for L is less conclusive. Column two shows k  d  d  the reduction of cell surface expression of these proteins as detected in F A C S analyses. For the proteins that co-immunoprecipitate E3/19K, a reduction in cell surface expression is noted. Reduction in cell surface expression results range from 43% of the level of uninfected  65  expression to 73%. For the L allelic protein, cell surface expression of 44% that of uninfected d  is observed in the presence of E3/19K (Ad infection). This is suggestive of down regulation due to association with E3/19K. Surface expression of D is unaffected by Ad infection. k  Finally, in the third column, the rate of exit from the ER assessed by the Endo H sensitivity (or the difference in Endo H sensitivity) of MHC CI I proteins from infected and non-infected cells is summarized. In this column, it is observed that there are no differences in Endo H sensitivity in the K and D proteins in infected and non-infected cells. k  Table 3.  d  Summary of E3/19K association with H-2 allelic proteins  Mouse proteins  Allelic  Binders  Slow-binders  FACS data (b)  Endo H * sensitivity  K  d  +  62%  +  K  b  +  73%  +  D  b  +/-  43%  +/-  -  44%  -  L Non-binders  Co-IP (a)  d  K  k  -  93%  -  D  d  -  81%  -  D  k  -  95%  9  * After 4 hours An overview of all the techniques employed gives an indication of the ability of each H2 transfectant to associate with E3/19K. The highest expressing clone for each transfectant was selected and tested above. Based on each technique, the H-2 proteins tested are grouped into three categories. The binders show co-immunoprecipitation (a). The slow-binders are not observed to co-immunoprecipitate, but do result in reduced cell surface expression as seen in the FACS experiment demonstrated here (b). Finally, the non-binders have not been shown to co-precipitate and also do not reduce the cell surface expression of MHC CI I.  66  3.4  Discussion The strong point of this study is that it is the first in which the binding of the H-2 , , b  d  and proteins to the Ad2 E3/19K protein in the context of a common cellular background has k  been examined. This set of experiments made the initial assumption that variable cell surface expression of transfected mouse genes was likely due in part to varying transport rates through the cell.  These transport rates likely reflect incompatibilities between human antigen  presentation proteins and mouse MHC CI I alleles. Additionally, it is possible that accessory proteins required for mature, stable M H C  CI I molecules may also be missing or of an  inadequate fit. The initial assumption made in this set of experiments was that overexpression of MHC CI I alleles would provide more ligand for potential E3/19K binding limits the conclusions that may be drawn from the data.  While overexpression of transfected proteins in these  experiments results in clear demonstrations of E3/19K binding and non-binding for some allelic proteins, in other cases it results in unstable, short lived M H C  CI I proteins. The  instability observed in Endo H digestions suggest that some of the transfected alleles may be very short lived in 293 cells. The ability of E3/19K to bind these alleles is likely low and reflects limits of the expression system rather than the ability to bind these alleles. These instability seen in these MHC CI I proteins may be due to our detection system. Perhaps the monoclonals selected do not recognize or act to destabilize transfected M H C  CI I proteins.  Alternately, some M H C CI I-P m-peptide complexes may be more susceptible to disruption by 2  the detergents used in these experiments. This set of experiments revealed many problems associated with protein trafficking and re-establishment of antigen presentation by mouse M H C proteins in human cells. Further experiments which would more finely control the many variables could include a comparison  67  of unfolded or improperly folded proteins to those detected by conformation specific monoclonal antibodies. This would allow some comparison of total levels of expression to transport of mature proteins out of the transfected cell. A wider sampling of the clones of transfectants to include those which expressed mouse MHC better assessment innate affinity between MHC  CI I at lower levels would allow a  CI I proteins and E3/19K. Finally, to broaden  the scope of the conclusions, this experiment could be repeated in other cell lines.  Within the constraints outlined above, this study has identified those allelic proteins that interact with E3/19K in our model system. This study also extends those studies done earlier with K , K d  K  d  k  and the hybrid MHC  CI I molecules containing regions of K  k  spliced to regions of  molecules (131). Furthermore, this study has identified a new sub-group of slow binding  molecules. The identification of those allelic MHC determined in three ways.  CI I proteins that bind E3/19K has been  Co-immunoprecipitation of E3/19K with MHC  CI clearly  demonstrates binding. In addition to co-immunoprecipitation, the effect of E3/19K is assessed by comparing the rate of exit of MHC  CI I proteins from the ER in infected and mock infected  transfectants. The third method to determine binding is by detecting reduction of MHC  CI I  expression at the cell surface. Taken together, these methods identified which allelic proteins were binders, non-binders and slowbinders. These data are summarised in table 3. The first method shows E3/19K clearly binds to D, K b  d  and K  b  but not K , D k  and L . The second method confirms that E3/19K does not  d  slow the exit from the ER of D  d  d  and K . k  Differences in Endo H sensitivity between infected  and mock infected transfectants are observed for D , K b  d  and K . b  L is poorly transported to d  the cell surface, but is more stable (i.e. does not fall apart during Endo H treatment) in Ad2 infected cells.  68  FACS analysis shows a reduction in cell surface expression for all transfected allelic proteins in the presence of E3/19K. While overall expression is of transfected alleles is quite variable, the percentage difference between infected and uninfected expression was compared. This comparison shows the level of reduction of M H C CI I cell surface expression is not as dramatic for the K  k  and D  d  alleles as for K , K , D , and L . While the data used here was d  b  b  d  from a single experiment, repetitions of the FACS experiments demonstrated that while overall cell surface expression varied greatly between experiments, the differences in expression due to E3/19K (between infected and non-infected) remained fairly constant, varying no more than 10%. The results above suggest that the nature of the L association with E3/19K is different d  from that of other allelic proteins. The first method employed shows that over the course of a four hour chase, L does not co-immunoprecipitate E3/19K. This could be due to the fact that d  the monoclonal antibody used interferes with E3/19K binding. It could also be because that over the course of the four hour chase, L does not fold into a conformation that promotes d  binding to E3/19K. Figure 10 shows that during a four hour experiment, only a limited amount of this protein is detected and Endo H digestion and exposure to 37°C for 24 hours suggests that what little is there is quite unstable. FACS analysis confirms the level of cell surface expression of the L  d  protein is lower than that of other allelic proteins, but is still  detectable. FACS analyses, which assay a steady state level of expression, demonstrate that L  d  does fold into a mature conformation and can be expressed on the cell surface (Figure 9a), and that this level of expression is reduced by E3/19K (figure 12). This finding allows the proposal of a new subgroup of allelic proteins that associate with E3/19K. L (and D ) illustrate the subgroup of allelic proteins that is slowly transported d  b  out of the ER and will bind E3/19K, but only once having achieved a transport competent  69  conformation. These proteins can be called "slow-binders" because the rate at which they fold this mature conformation and are able to bind E3/19K is greatly reduced. Slow-binders are characterised by their slow maturation rate and are transported out of the ER quite slowly. It is likely the length of time that they are present in the same compartment with E3/19K that enhances the opportunity to associate and be retained. The D  b  protein is another member of this "slow binding" group. One hour after the  beginning of the pulse, only trace amounts of E3/19K are co-immunoprecipitated; it is only after four hours that significant amounts of E3/19K are seen. In contrast, K  d  binding strongly  to E3/19K occurs almost immediately, or shortly after translation. The E3/19K molecule coprecipitates even at time zero suggesting that E3/19K made during the pulse can bind already pre-existing K  d  molecules. In addition, at the 30 minute timepoint, K  E3/19K to bind. This contrasts sharply with the D (acquire P m, 2  and L  d  is still available for the  molecules that appear to mature  peptide, dissociate calnexin) more slowly than K .  However, once a mature  d  conformation has been achieved, E3/19K binds the "slow-binders" with great efficiency. The conformation of D  b  that E3/19K binds is initially not present in the D  b  transfectant, but appears  with subsequent maturation. This suggests that E3/19K binding is dependant on overall  MHC  CI I conformation rather than specific residues. This view is in contrast with previous studies that have attempted to map specific residues crucial for E3/19K binding (128, 131). In cells transfected with the MHC endogenous MHC  CI I alleles classified as slow-binders, the level of  CI I (or W6/32) cell surface expression is quite low.  components that participate in the maturation of M H C  The cellular  CI I molecules (peptide, peptide  transporters, calnexin, and other chaperones) may be occupied with or competed for by the slow binding H-2 MHC  CI I and therefore have reduced availability for the endogenous HLA.  The reduction of endogenous MHC  CI I indicates that there is some limit to the number of  70  M H C CI I proteins that can be correctly folded and/or expressed by a cell. The K  k  protein is one of the allelic proteins which does not bind E3/19K. It is also  exits the ER very quickly. The t  {/2  for Endo H resistance is approximately 15 minutes; the  average for endogenous M H C CI I is 45 minutes to 1 hour. Rapid exit from the ER may reflect a lack of competition for cofactors such as P m 2  or peptide resulting in very rapid folding.  Alternately, it could reflect a reduced ability for the human chaperones present in 293 cells to recognize and retain the K  k  protein and ensure quality control. However, K  k  has limited  exposure to E3/19K (reduced period of time) and the opportunity to associate may simply not occur. The inability of K  k  to bind E3/19K may not due to lack of affinity but rather lack of  exposure.  If E3/19K binding to M H C  CI I is partly due to being in the right place for the  appropriate period of time, then the contribution of affinity between these proteins becomes less clear. In studies where E3/19K is precipitated directly, the ratio of E3/19K to MHC CI I is always much higher than when MHC CI I is precipitated. Clearly there exists a large pool of E3/19K that is not binding to M H C CI I. Conversely, the level of E3/19K expression within a cell correlates inversely to the level of cell surface M H C CI I expression (134). While affinity between M H C CI I proteins and E3/19K has been noted, an estimation of the relative strength of this affinity has never been made. Secondly, the period of time required for maturation, and hence the length of exposure to E3/19K is influenced by many other factors including but not limited to availability of correct peptide and the ability of chaperones such as calnexin, calreticulin, tapasin, TAP, glucosyltransferases, protein disulphide isomerase and others to bind to mouse M H C  CI I  proteins. Our evidence shows that for the small sample of alleles expressed in 293 cells, great  71  variations in stability were observed. Our findings are limited by the sources of variability inherent in our expression system.  This variability reflects the complexity of antigen  presentation and protein folding in general. Some specific limitations include the level of expression of the transfected allele. As mentioned above, endogenous H L A cell surface expression was reduced when mouse allelic proteins were expressed suggesting that there is a limit to the total M H C  CI I that can be  processed by a particular cell. Overexpression of transfected genes may perturb regulation of this limit. Secondly, species differences in proteins involved in antigen presentation may affect the expression of stable conformations of mouse M H C CI I proteins in human cells. However, it is likely that such limitations will be present in any study of E3/19K binding M H C  CI I  proteins and that a better picture of M H C CI I binding by E3/19K could be obtained by using multiple expression systems.  The binding of E3/19K by MHC  CI I allelic proteins was examined against a common  cellular background. 293 cells were chosen as they are amongst the best characterised cell lines for Ad 2 infection and are easily infected. The highest expressing clones were chosen to be representative for each allelic protein, with differences in levels of gene expression observed for some alleles. However, when infected with Ad 2, levels of E3/19K expression are well in excess (appendix 1); therefore limited access to E3/19K is unlikely a factor in this study. Differences in allele expression may influence proper folding and exit from the ER.  Based on the this set of experiments, a speculation about E3/19K binding can be made. E3/19K binds to those M H C  CI I molecules that it is exposed to for. M H C CI I alleles which  are normally processed through the ER remain there for the period of time necessary for peptide loading and proper folding. Chaperones such as calnexin serve effect the quality control. M H C  CI I molecules that fold quickly probably escape the attention of E3/19K. 72  Rapid folding may  reflect bypassing the quality control mechanism altogether. Reduced  affinity between chaperones and MHC functional MHC  CI I proteins and could result in empty or non-  CI I at the cell surface which in adenovirus infected cells, are not bound by  E3/19K.  Figure 13 outlines the different fates for MHC taken are represented by arrows (labelled 1, 2 or 3).  CI I. The different paths that can be The length of the arrow gives an  indication of the amount of time the process takes. The dashed arrow indicates an expedited path.  The binders are assumed to follow the first path.  They are efficiently and rapidly  transported through the ER and Golgi to the cell surface. The slow-binders take the second path. These allelic proteins are less efficiently transported and spend more time in the ER. Finally, the non-binders are very rapidly transported to the cell surface and may possibly bypass the processing machinery altogether. This results in low chance of contact with E3/19K.  73  Figure 13.  Overview of maturation paths for the M H C CI I complex  The maturation path through the ER and Golgi is illustrated. Different potential paths are shown for each of the types of M H C CI I proteins based on their ability to bind E3/19K. Binders, non-binders and slow-binders are depicted. 1) Binders travel through the ER and Golgi in the most straightforward manner. Interaction with chaperones in the ER and Golgi ensures proper processing and maturation. 2) Slow-binders interact with the same chaperones, but the rate of transport through the ER is slower. This is depicted as a circuitious route through different ER compartements. 3) Non-binders travel quickly through the entire ERGolgi maturation pathway, arriving at the cell surface very rapidly. This is depicted with a dashed line.  74  C o m p l e x of H L A c l a s s I a n d p e p t i d e  Cell surface  1 fl _  HLA heavy chain  §  P2m  Q  Calreticulin  75  @  ERp57  B  Tapasin  4. 4.1.  Efforts to increase the cell surface expression of H-2 D  b  Introduction In the previous chapter experiments with different H-2 transfected 293 cells were  discussed. Of the transfectants studied, the 293D transfectant was the most intriguing. The D protein was unusual because while the cell surface expression was quite high, a much larger b  proportion of the D  b  molecules remained inside the cell and was Endo H sensitive. This  protein was slowly transported through the ER with the majority remaining intracellular. Other proteins seemed to mature through the ER properly, indicating that mouse allelic proteins can be successfully transfected and expressed in the human 293 cells. Much of the D is still within the cell at four hours post pulse.  b  that is made  In the last chapter it was shown that in  transfected cell lines, surface expression for endogenous M H C  CI I was reduced. This  suggested that the H-2 proteins were competing for factors required for M H C CI I maturation. The limit for levels of cell surface expression in the 293D transfectant could be a shortage of b  an essential co-factor required for proper conformation. Improperly folded H-2 Db proteins could be prevented from escaping from the ER. Previous studies in cell lines exhibiting low cell surface expression revealed many strategies to restore expression to regular levels.  These studies were instrumental in  determining which components of a cell were crucial for regular expression of the MHC  CI I  molecule. In general, the deficiencies in these cells could be broken down into two or three categories. In order to determine the nature of the incomplete expression of the D protein, b  strategies previously applied to other surface expression mutants were utilised. Some of the earliest M H C that lacked P m. 2  CI I cell surface negative mutants characterised were those  The Daudi cell line, a mouse cell line lacking p^m and consequently MHC CI  76  I cell surface expression, was restored to a normal phenotype when fused with a cell line expressing P m (135). Other experiments also demonstrated the dependence of the H chain on 2  the P m for cell surface expression (136). 2  Another effective treatment for cells with reduced cell surface expression is the incubation with y-IFN (137). y IFN is part of a normal cellular antiviral response; many _  elements of the antigen presentation pathway have interferon regulatory elements in their promoters. The antiviral effects of y-IFN have been widely documented (138). The effects of y-IFN include widespread upregulation of antigen presentation elements. y-IFN reactive elements upregulate transcription of many of the factors necessary for M H C  CI I gene  upregulation. These factors include P m, M H C CI I, TAPs and LMPs. Upregulation of these 2  elements enhances the ability of infected cells to present antigens at the cell surface. In cells that present little M H C CI I at the cell surface, y-IFN increases expression. The mutant mouse cell line CMT-64 has reduced M H C  CI I expression at the cell surface due to missing  transporters (139). Similar results have been reported with other cell lines (140). Restoration of M H C CI I expression has been achieved by transfecting the cell line with T A P 1, TAP 2 and both T A P 1 and TAP 2 (47). In the absence of transfected TAPs, cell surface expression can be stimulated by treating these cells with y-IFN. Treatment with y-IFN upregulates the transporters, as well as P m and M H C CI I, resulting in cell surface expression (139). 2  Studies with the T A P 2 defective mouse cell line RMA-S and the parental line RMA demonstrated that a population of M H C  CI I molecules devoid of peptide exists. These  'empty' M H C CI I molecules are expressed at the cell surface (141). Indeed, some of the  77  earliest studies with peptides and MHC  CI I were done showing RMA-S cells becoming  sensitized to C T L killing by the addition of extracellular peptide (142). mutation in the TAP 2 peptide transporter and are unable to form a TAP required to transport a full complement of peptides into the ER.  These cells have a  1/ TAP 2 heterodimer  Many of the D  and  b  K  b  molecules in this cell line are never loaded with peptides. They are quite unstable and consequently expressed at a low levels on the cell surface. Growing cells at 26°C increases the thermodynamic stability of characteristically unstable MHC  CI I molecules devoid of peptide  and permits them to survive longer at the cell surface. The phenomenon of empty MHC  CI I  molecules on the cell surface was shown to be normally occurring when empty MHC  CI I  molecules were not only found on mutant cells growing at low temperature but also found on the surface of the non-mutated parental cell line RMA One  of the mechanisms above might be responsible for the lack of cell surface  expression of the D  4.2  (35).  b  Rationale and  protein in the transfected 293D cell line. b  Goals  The goals of this study were to examine a MHC  CI I protein that remained intracellular.  Several different strategies were employed to determine if intracellular stores of the D  b  protein  could be coerced to travel to the cell surface. These studies would shed light on the nature of antigen presentation and also protein maturation and transport. transport of cell surface bound glycoproteins may  The analysis of regular  allow elucidation of several of the  mechanisms present for quality control in protein transport through the ER and Golgi.  78  4.3  Results  4.3.1. The D protein remains intracellular in the 293D  transfectant  Endo H analysis of the 293D transfectant reveals that the D protein is well expressed. b  b  Figure 14 shows large amounts of D expressed imunoprecipitated from lysates of these cells. b  However, Endo H digestion reveals that only a small fraction of the transfected H-2 D protein is able to exit the ER. This figure shows a pulse-chase experiment. At the right side of the figure the lysates have been incubated with Endo H. Most of the lysates are Endo H sensitive; at 2 and 4 hours post chase Endo H resistant D is observed (upper arrow). b  79  Figure 14.  Intracellular accumulation of H-2D in 293 cells b  C o n f l u e n t 2 9 3 c e l l s transfected w i t h M H C C I I H - 2 D b ( 2 9 3 D b ) w e r e p u l s e d for 30 minutes and chased for 0, 3 0 , 6 0 120 a n d 240 minutes (lanes 1 to 5) respectively. M H C C I I was precipitated w i t h protein specific antibodies as listed i n table 1. S a m p l e s were d i v i d e d and alternately m o c k treated o r treated w i t h E n d o H for 24 a n d then separated b y S D S - P A G E . E n d o H sensitive proteins are identified b y the l o w e r arrow; E n d o H resistant proteins b y the upper arrow.  293D 1  b  . EndoH 2 3 4 511 2 3 4 5  80  4.3.2.  Growth at 26° C does not result in the bulk of the intracellular stores of  the D protein escaping the  ER  b  In figure 15 the effects of growth at 26° are shown. The MHC  CI I allele D  was  precipitated and half of the lysate was incubated with Endo H.Culture at 26°C results in many contaminating proteins co-immunoprecipitating with the mouse M H C  CI I protein. These  contaminants are characteristic of precipitates from temperature shocked cells; incubation at 37°C for 24 greatly reduces the levels observed suggesting that they are quite unstable. While growth at 26°C results in more Endo H resistant D  b  proteins, most of the population remains  Endo H sensitive. It was reasoned, that 293 cells may lack the peptide repertoire bound by the D  b  protein.  Only the earliest studies examining peptide transporters had suggested there  existed allele specificity to the peptide transporters (38, 143). However, if the D  b  molecules in  the 293 cell were unable to obtain the correct peptide to form a trimolecular complex, they would be unable to achieve the correct conformation to exit the ER. This would result in large amounts of the D  b  population remaining in the ER.  Alternately, D  b  complexes devoid of  peptide may be formed, but be thermodynamically unstable and hence very short lived. These short lived species would likely be too unstable to be detected outside of the ER. previously been demonstrated that unstable MHC  It had  CI I molecules lacking peptide could be  stabilised and detected at the cell surface by culturing cells at 26°C (144). Table 4 shows the FACS results from growing D  b  cells at 26°C and 37°C. The level of D  grown at 26°C is comparable to the cell surface expression grown at 37°C.  81  b  at the cell surface  Figure 15. H-2D  b  The effect of growth at 2 6 ° C on the intracellular accumulation of  in 2 9 3 D  b  cells  2 9 3 D b transfectants w e r e alternately c u l t u r e d at 2 6 ° C or 3 7 ° C for 24 hours p r i o r to l a b e l l i n g . C e l l s w e r e p u l s e d for 3 0 minutes and c h a s e d f o r t w o hours at 2 6 ° C or 37°C r e s p e c t i v e l y . C e l l s w e r e l y s e d o n i c e and lysates precipitated w i t h a D b specific m o n o c l o n a l antibody (as listed i n table 2). P r i o r to S D S - P A G E , precipitates and molecular weight ( M W ) standards were either treated or m o c k treated w i t h E n d o H for 24 hours.  293 D @ 26° and 37 ° +/- Endo H b  14 82  4.3.3.  Excess P m 2  does not  intracellular store of the D  b  promote proper folding of the  2  the  normally, the extra burden of large amounts  proteins could result in insufficient quantities of P m 2  transfected and the endogenous MHC endogenous H L A  bulk of  protein  While 293 cells show no shortage of P m  of transfected D  the  to supply both the  CI I molecules. A comparison of the levels of  (W6/32) expression in both 293 and the transfectants suggested that in  almost every case, the presence of an H-2 transfectant reduced the level of endogenous  MHC  CI I expressed at the cell surface (Figure 9). One explanation for this observation is that levels of P m 2  in the transfectants were limiting the total number of MHC  CI I molecules that could  achieve a mature conformation and make it to the cell surface. By adding more P m 2  to these  cells, it would be possible to promote more H chain-P m interactions and thereby upregulate 2  the surface expression o f D . 293D cells were infected with the P m-Vaccinia construct. b  b  2  Infected and mock infected cells were FACS analyzed (table 4). While the level of P m 2  was  increased when this method was used, the cell surface expression was unchanged. In figure 16 we demonstrate that infection with the human-p m-Vaccinia construct 2  b markedly increases the amount of P m 2  mock infected with a human P m 2  The precipitated P m 2  precipitated with D  b proteins. 293D cells infected or  vaccinia construct were precipitated with anti P m 2  antisera.  from the infected cells co-immunoprecipitates many other proteins 83  characteristic of vaccinia infections.  Figure 16.  The effect of infection with a vaccinia virus human-P m construct  on P m expression 2  293D  b  c e l l s w e r e alternately infected (+) o r m o c k infected (-) w i t h a hurnan-P m 2  construct expressed i n v a c c i n i a v i r u s ( h u - P m - V a c ) . 2  C e l l s were infected 18 hours p r i o r to  l a b e l l i n g w i t h P r o M i x ( 3 5 S - M e t / C y s ) . T h e cells were p u l s e d for 30 minutes and chased for 120 minutes. P m 2  w  a  s  precipitated w i t h rabbit anti h u m a n - P m antisera (as listed i n table 2). 2  M H C C I I and | 3 m are denoted b y the arrows. 2  +  85  Table 4. A comparison of the cell surface expression of the H-2 D protein at b  26°C and 37°C with and without excess P m  non-infected  infected percentage  (AFU)  (AFU)  26°C  44.8  64.4  144  37°C  58.9  68.1  116  percentage  132  106  I+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+l+A £+1+1+1+1+1+1+1+1+1+1+1+1+1+1+1+1+^^  $$$$$$$$$$$$$$$$$$$$$$$$ + + + + + + + + + + + x + + + + + + + + + + + ^  The 293Db transfectant was alternately cultured at 26°C and 37°C. Additionally, cells were either mock infected or infected with the hu-P m-Vaccinia as described in Materials and Methods. At 18 hours post infection, the cells were harvested and F A C S analyzed as described in Materials and Methods. Levels of cell surface expression are given in arbitrary fluorescence units (AFU). Calculation of A F U involved taking the difference between mock and specifically labelled FITC labelled samples. Percentage of cell surface expression is determined by comparing changes in A F U along rows or down columns, e.g Uninfected 293Db transfectants at 37°C express 132% the level of Db expressed at 26°C at the cell surface. 2  The result of adding excess human P m in cells cultured at both the standard 37°C and 2  26°C is shown in table 4. Addition of P m results in an increase in cell surface expression of 2  15%.  When cells cultured at 26°C are exposed to excess P m, the level of cell surface 2  expression increases by 43%. Figure 17 shows the analyses of the Endo H resistance of these transfectants. While the percentage of cell surface expression of D increases, the overall b  amount that is Endo H resistant remains much less than the Endo H sensitive population.  86  Figure 17.  The effect of growth at 26°C and infection with a vaccinia virus  h u m a n - P m on escape of intracellular stores of H-2D protein from the ER b  2 9 3 D b cells cultured at 2 6 ° C and 3 7 ° C were alternately infected or m o c k infected with a human-P m construct expressed i n v a c c i n i a virus ( h u - P m - V a c ) . C e l l s were infected 18 hours p r i o r to l a b e l l i n g w i t h P r o M i x ( 3 5 S - M e t / C y s ) . T h e c e l l s were p u l s e d f o r 3 0 minutes a n d c h a s e d f o r 120 minutes. M H C C I I H - 2 D b was precipitated w i t h a Db specific antibody (as listed i n table 1). P r i o r to S D S - P A G E , precipitates and M W standards were alternately m o c k treated or treated w i t h E n d o H for 2 4 hours. 2  2  293D +/- p m-Vac @ 26° and 37° C b  2  26<  37<  +  +  hu-f^m-Vac  + Endo H kD 43  • • - • M 29  87  4.3.5. y l F N has no effect in upregulating cell surface expression of D Previous studies had demonstrated that y-IFN had been used to upregulate cell surface expression of M H C CI I molecules in cell lines that normally had low levels. In these studies, the non-specific effects of y-IFN included upregulating many components involved in antigen presentation, with the obvious final effect of increasing cell surface expression of M H C CI I. This treatment was applied to 293 D cells to determine if some other element required for the efficient b  transport of D was regulated with the other antigen processing elements. b  Table 5 shows the results of this treatment.  293 cells tranfected with E3/19K (293.12  cells) and the 293 D transfectant cell lines were pretreated with y-IFN. While the effect of y-IFN b  can be observed on M H C CI I expression in 293 cells, treatment with y-IFN has absolutely no effect in upregulating the cell surface expression of D in the transfectants. In 293.12 cells, which b  normally exhibit reduced cell surface expression, y-IFN is able to upregulate cell surface expression to levels close to that observed in 293 cells exposed to y-IFN.  88  Table 5. The effect of y-IFN on M H C CI I cell surface expression  normal  y-IFN  (AFU)  (AFU)  293  12.8  20.4  160  293.12  3.91  19.5  498  293D  6.43  5.89  91.6  Cell Line  b  percentage  Transfectants were alternately treated or mock treated with y-IFN. Cells were cultured in the presence of y-IFN for 24 hours. FACS analysis was performed to assess the effect of yIFN on cell surface expression. Levels of cell surface expression were detected with W6/32 in 293 and 293.12 cells and 28.14.8s in 293Db cells and are given in arbitrary fluorescence units (AFU). Calculation of A F U involved taking the difference between mock and specifically labelled FITC labelled samples. Percentage of cell surface expression is determined by comparing changes in A F U along rows, e.g y-IFN treated 293D transfectants express 91.6% the level of Db at the cell surface expressed in untreated cells. b  89  4.4  Discussion Techniques documented in the literature were attempted to increase cell surface  expression in the 293D transfectant to levels closer to the overall level of expression ofD . In b  b  figure 15 upregulation of cell surface expression of H-2 D  b  does not occur when cells are  cultured at 26°C. Previously, it was thought that the regulation of cell surface expression of MHC  CI I relied on factors such as rate of transport to the cell surface and turnover of surface  molecules. The bulk flow theory predicts that proteins deposited in the ER will eventually pass through the ER, cis and trans Golgi and either out to the cell surface or into endosomes unless they are retained by an organelle specific retention or retrieval signal. The rate of transport of MHC  CI I molecules was believed to be directly related to the ability of the MHC  to achieve a stable and mature conformation and escape the ER. that affect this are often the cofactors such as 3 m 2  CI I molecule  In many mutants, the factors  (Daudi) and peptide (RMA-S, CMT-64).  Peptide is controlled by TAP 1 and TAP 2. The experiments in this chapter suggest that in this case, some other factor is responsible. Results reported in the previous chapter demonstrated that D  b  is a slow binder to  E3/19K. The length of time that this molecule spends in the same compartment as E3/19K enhances its ability to be bound by E3/19K. FACS experiments show that this protein is expressed at the cell surface in uninfected cells at a level similar to that of endogenous MHC  CI  I. These experiments also showed that when this (or other allelic proteins) are expressed at the cell surface, the level of the endogenous MHC  CI I expression goes down correspondingly.  The reduction in cell surface expression likely is related to a shortage of cofactors. Results reported in this chapter suggest that the addition of an excess of co-factors still has no effect on upregulating cell surface expression.  90  It is possible that there is feedback control of total MHC surface. When a certain number of MHC  CI I molecules at the cell  CI I molecules is expressed on the cell surface, a  signal is generated that reduces the number to follow. Rather than each allelic protein being regulated differently and by separate mechanisms, each protein expressed may have a 'set' contribution or ratio of the final cell surface population. In 293D , the level of the endogenous b  MHC  CI I does not drop to zero; the block in the maturation and transport of one allelic protein  (in this case the D ) is clearly not blocking the maturation and transport of the endogenous b  allelic proteins. It has been shown that the retention of E3/19K in the ER triggers a UPR  (68).  The  level of activation of N F - K B has been shown to be dependant on the amount of E3/19K present in the ER. It is not the binding of E3/19K with MHC appearance in the ER.  An excess of D  show that the release of C a  2 +  b  CI I that is the trigger of N F - K B ; it is its  molecules in the ER may trigger the UPR.  Others  from the ER into the cytoplasm seems to trigger this response  (68). Further studies in yeast suggest that activation of a membrane serine/threonine kinase triggers the alternate splicing of a transcription factor, HAC1, HAC1  in the nucleus (145, 146).  has an affinity for the unfolded protein response element (UPRE) and upregulates  expression of genes with this element. Some known products include heat shock proteins such as BiP. In a recent review (145) it is speculated that free BiP plays a role in the regulation of this process. Excess protein in the ER may mop up all free BiP in the ER.  In yeast, free BiP  may associate with a membrane protein called Irelp. Dissociation of BiP allows Irelp to dimerise and autophosphorylate, sending a signal through the UPR  to upregulate transcription  of more BiP. Presumably, the increased levels of Bip should establish a new equilibrium of unfolded proteins to unfolded protein binding capacity in the ER.  In these studies, the  investigators suggest that the UPR is instrumental in preventing apoptosis.  91  The regulation of the UPR  may influence the turnover of molecules at the cell surface.  The UPR  upregulates expression of heat shock protein in response to proteins that remain in  the ER.  Cell surface regulation is probably influenced by a combination of concentration,  regular turnover at a specific rate and a positive signal to either keep proteins at the cell surface or to tag them for turnover.  92  5.  E3/19K binding to M H C CI I does not exclude association with calnexin  5.1  Introduction The observation thatE3/19K very rapidly bound and stabilised a mature conformation  of MHC  CI I proteins leads to the speculation that E3/19K behaves like a chaperone.  Examination of E3/19K binding to MHC  CI I in the ER involving the ER resident chaperones  leads to the proposal that E3/19K binding in the ER will disrupt or involve the chaperones involved in the MHC  CI I processing pathway.  Chaperones are molecules that bind to immature peptide sequences. They bind to proteins before they fully mature and form either a transient or permanent association with them. Chaperones are usually associated with a protein that has not yet been transported to its final destination. The role of a chaperone protein is to bind to immature and misfolded proteins and to cooperatively act with them to allow them either to achieve their correct conformation (e.g. calnexin) or to shuttle for degradation (BiP). Many chaperones act with protein intermediates in the ER.  Ribosomes transcribing  nascent peptides are directed to the ER by way of the signal sequence;the peptide is directed into the lumen of the ER.  The newly made protein is modified by many enzymes that act in  concert to further process it. The signal sequence is removed by signal peptidase.  Transport  through the ER membrane may be enhanced by association with BiP, which acts as a ratchet pulling the peptide into the ER.  Glycans are added and modified by a series of glycosylation  enzymes. Disulphide bridges are established between cysteine residues; proline isomerisation is mediated by protein prolyl isomerase (PPI). Calnexin and calreticulin act in concert as a type of quality control mechanism; once proteins have achieved a correct conformation, they are released by calnexin and permitted to progress further through the ER-Golgi maturation pathway. More recently, other participants such as ERp57 have been shown to have a role in  93  the glycoprotein quality control mechanisms in the ER.  BiP/Kar2p has been implicated in  playing a regulatory role in the UPR of cells (147). These proteins are involved with most, if not all, glycoproteins that travel through the ER.  In addition to the concerted action of these ER resident proteins, the M H C CI I molecules  that are involved in antigen presentation are acted on by a further set of specialised chaperones. This list has grown lately as the interactions among all the participants are better defined. Some chaperones involved in the antigen presentation path include calnexin and calreticulin with specialized roles. Additionally, since M H C CI I molecules are composed of a trimolecular complex that includes a peptide fragment, the TAP proteins also have been demonstrated to act as chaperones. More recently, essential roles for tapasin (148) have been elucidated.  5.1.1.  Maturation of a Cell Surface Membrane Protein A cell surface membrane protein comes in contact with this progression of chaperones,  mentioned above, on its way to the cell surface. If it is properly folded, the interaction with the chaperone proteins will be temporary. If it is not properly folded, there exist many chaperones that will bind the improperly folded protein and feed it to the degradation machinery of the cell. Almost any path that a peptide will take through the cell will involve the participation of chaperones. It is in this manner that E3/19K also functions as a chaperone. E3/19K binds to immature peptides and seals their fate in a manner not unlike BiP. It stabilises a mature epitope and halts its progression through the cell. In the pulse chase studies with H L A proteins, the W6/32 antibody was used to follow the rate of appearance of mature M H C CI I molecules. Acquisition of the W6/32 conformation has been used as an indication of maturity (133, 149).  In contrast to the Rabbit antiserum  R426, W6/32 recognises only the M H C CI I molecules that have escaped the ER.  While the  epitope recognised by the W6/32 epitope is coincident with a mature epitope, it should be noted that the converse is not necessarily true; recognition by W6/32 does not guarantee that a M H C  94  CI I protein is mature. Calnexin is one effector of quality control in the ER, indiscriminately binding glycoproteins in the ER until they achieve a mature conformation and are fit to be transported to the cell surface. Calnexin binds M H C CI I molecules at some point in their maturation.  5.2.  Rationale and Goals The goal of this study was to determine whether the association of E3/19K with MHC  CI I complex in the ER would disrupt the association of any other chaperones in the ER. This set of experiments set out to determine if an E3/19K bound protein would still associate with calnexin. This data would give information on the physical association of participants in complexes. It would also shed light on the timing of different events occurring in protein maturation and quality control.  5.3. 5.3.1.  Results E3/19K preserves W6/32 binding of M H C CI I H L A in the presence of  tunicamycin Many studies have demonstrated that proper glycosylation of M H C CI I molecules is required for maturation of M H C  CI I molecules (150).  Growth in the presence of a  glycosylation inhibitor such as tunicamycin results in increased levels of improperly folded proteins in the presence of BiP in the ER and can trigger the UPR. When assessed with W6/32 the mature epitope is lost when 293 cells are grown in the presence of tunicamycin (figure 18). Unglycosylated M H C CI I glycoprotein is unable to exit the ER. An unglycosylated M H C CI I molecule is likely unable to associate with calnexin (56, 151) and therefore can proceed no further down the maturation pathway. When the 293.12 cells are treated with tunicamycin the MHC  CI I loses its carbohydrates but the W6/32 epitope is still detected and M H C CI I  co-precipitates with E3/19K. This finding suggested that E3/19K can bind to M H C CI I in the 95  absence of carbohydrates and is able to force it into a mature conformation. In figure 18b the effect of a tunicamycin titration on 293 and 293.12 cells is examined. In this experiment, the non-specific epitope recognized by the antisera R426 (and the identical R425) can be precipitated at all concentrations of tunicamycin. A similar result is observed in 293.12 cells. As the tunicamycin concentration increases, the mobility of the M H C CI I increases coincident with the loss of carbohydrates. Figure 18a illustrates the results of the same experiment using W6/32. When the mature conformation specific monoclonal W6/32 is used, M H C CI I ceases to be precipitated from 293 cells at ~7]ig/ml Tunicamycin. In 293.12 cells, M H C CI I can be detected at all tunicamycin concentrations. E3/19K is also observed co-immunoprecipitaring at all points. This result demonstrates that E3/19K stabilises a mature (W6/32) epitope under adverse conditions. In this case, E3/19K is able to cause conformation specific mAb to bind even in the absence of carbohydrates.  96  Figure 18. and  The effect of increasing levels of tunicamycin on M H C CI I in 293  293.12 cells  2 9 3 a n d the E 3 / 1 9 K transfected 2 9 3 . 1 2 c e l l l i n e s w e r e c u l t u r e d i n the presence o f i n c r e a s i n g concentrations o f t u n i c a m y c i n . C e l l s w e r e g r o w n i n m e d i a s u p p l e m e n t e d w i t h 0, 3.5, 7, 14 a n d 21 Lig/ml (lanes 1-5) respectively for 18 hours p r i o r to l a b e l l i n g . B o t h labelling and chase m e d i a were also supplemented w i t h t u n i c a m y c i n . C e l l s were l a b e l l e d for 30 minutes and c h a s e d f o r t w o h o u r s . I m m u n o p r e c i p i t a t i o n w a s p e r f o r m e d w i t h a) the c o n f o r m a t i o n s p e c i f i c m o n o c l o n a l W 6 / 3 2 o r b ) n o n c o n f o r m a t i o n - s p e c i f i c sera R 4 2 6 f o l l o w e d b y S D S PAGE.  Tunicamycin titration a) W6/32  1  43 kD —  b) R426 43 kD  293.12  293 2  3  4  5  1  2  3  4  5  3  4  5  mm 293.12  293 1  2  3  4  5  i 97  1  2  5.3.2.  E3/19K rapidly stabilises a mature epitope The rapidity of the formation of a mature epitope was investigated in figure 19. This  figure illustrates R426 and W6/32 precipitated products at various time points post chase. The R426 population appears immediately in both 293 and 293.12 cells. The increased mobility of E3/19K with time reflecting modification of sugars. In figure 19b an immunoprecipitation with W6/32 is shown. In 293 cells it is apparent that the rate of transport through the ER and coincident mature conformation is 2-4 hours. The first appearance of the mature W6/32 epitope starts at about one hour post chase. In the 293.12 cells a W6/32 epitope can be observed almost immediately, with some precipitate visible at 0 minutes post pulse. Once again, this gel confirms that both P m and 2  E3/19K are co-precipitated with M H C CI I molecules in 293.12 cells. This experiment demonstrates that a mature M H C CI I epitope is very rapidly stabilised in the presence of E3/19K, even though the M H C CI I molecule is not transported out of the ER.  98  Figure 19.  Time course of maturation of M H C CI I in 293 and 293.12 cells  293 and 293.12 cells were grown to 9 5 % confluence and labelled using 35S-Met/Cys Pro-Mix. Cells were pulsed for 30 minutes and chased for 0, 30, 60, 120 and 240 minutes (lanes 1-5) respectively. Lysates were precipitated with non conformation-specific sera R426 or the conformation specific monoclonal W6/32. Precipitates were separated by SDS-PAGE. M H C CI I (a), (3 m (b) and E3/19K (c) are indicated. 2  99  Because E3/19K binds to MHC  CI I so quickly and almost immediately induces it to  form a mature conformation, it was suggested that it was acting in a chaperone like manner. It is interesting that E3/19K quickly stabilizes a mature epitope. The following experiments examined whether the association of the 'chaperone' E3/19K would affect the association of other more regular ER resident chaperones such as calnexin. Specifically, an experiment was performed to determine if E3/19K and calnexin binding to MHC  5.3.3.  CI I was exclusive.  E3/19K binding to M H C CI I does not exclude calnexin association  An experiment that tested if E3/19K and calnexin could bind to MHC time is shown in figure 20. MHC  CI I at the same  CI I molecules were immunoprecipitated with either W6/32  or R426. This experiment was done as documented in Materials and Methods except the cells were lysed in the gentle CHAPs buffer. Lysis in NP40 typically abolished any association of calnexin with MHC  CI I molecules. MHC  CI I was precipitated, run on a gel and then  transferred to a membrane. The membrane was probed with anti calnexin antisera as described in Materials and Methods. Figure 20 shows the association of calnexin with the MHC  CI I-P m complex. In 293 2  cells precipitated with W6/32, no calnexin can be found associated with this complex. When precipitated with R426, calnexin is found associated with the complex at all timepoints. In the 293.12 cells, the M H C  CI I-P m-E3/19K complex does associate with calnexin at all 2  timepoints using both W6/32 and R426.  101  Figure 20.  Calnexin association with M H C CI I precipitated from 293 and  293.12 cells 2 9 3 a n d 2 9 3 . 1 2 c e l l s w e r e g r o w n to 9 5 % c o n f l u e n c e a n d l a b e l l e d u s i n g P r o - M i x . C e l l s w e r e p u l s e d for 3 0 m i n u t e s a n d c h a s e d for 0, 6 0 , 120 a n d 2 4 0 m i n u t e s (lanes 1-4 respectively). L y s a t e s w e r e precipitated w i t h either the c o n f o r m a t i o n specific m o n o c l o n a l W 6 / 3 2 o r n o n c o n f o r m a t i o n - s p e c i f i c sera R 4 2 6 . Precipitates w e r e separated b y S D S - P A G E . G e l s were blotted onto I m m o b i l o n P V D F membranes overnight ( O / N ) . M e m b r a n e s were fixed and western b l o t t e d w i t h a n t i - c a l n e x i n antisera (1:10,000) u s i n g the glass plate incubation d e s c r i b e d i n m a t e r i a l s a n d m e t h o d s . S p e c i f i c antisera w a s detected u s i n g H o r s e R a d i s h P e r o x i d a s e ( H R P O ) c o n j u g a t e d s e c o n d a r y a n t i b o d y ( J a c k s o n ) a n d the E C L m e t h o d ( A m e r s h a m ) . K o d a k X A R film was exposed for 1 to 15 minutes.  102  These data confirm that when W6/32 and R426 are used to immunoprecipitate M H C CI I molecules from 293 and 293.12 cells, different subsets of these molecules are precipitated corresponding to M H C CI I molecules of different maturity. The R426 precipitate is the total M H C CI I population including both immature and mature M H C CI I molecules; the W6/32 precipitate is the subset of those that are mature. Calnexin is not expected to be associated with the mature W6/32 epitope but will associate with the R426 epitope. That calnexin also associates with the M H C CI I-E3/19K complex is not completely unexpected as this complex is retained in the ER. The observation that E3/19K does not block this association is noteworthy.  5.3.4.  M H C CI I precipitated with anti T A P antisera also associates with  calnexin and E3/19K The role of the TAPs has been intimately linked to empty M H C CI I molecules. Empty M H C CI I molecules are retained in the ER until they receive the correct peptide, then they are able to escape the ER and 20-80 minutes later are expressed on the cell surface (152). The association of other ER resident proteins such as tapasin have also recently been observed (153). Precipitation with anti-transporter antisera should bring down those immature M H C CI I that are accepting peptides. Figure 21 illustrates 293 and 293.12 lysates precipitated with W6/32 mAb and antitransporter antisera. This experiment consisted of a pulse followed by several chase timepoints. The precipitates were separated on a gel and subsequently transferred to a membrane for western blotting analysis. This figure demonstrates once again that M H C CI I in 293.12 cells forms a complex with calnexin. When precipitated with anti M H C CI I, calnexin still associates with the E3/19K-MHC CI I-P m complex. Moreover, this figure reveals that, when precipitated with 2  103  the antitransporter antisera, c a l n e x i n c a n s t i l l be f o u n d i n the w e s t e r n b l o t o f the same precipitate.  A d d i t i o n a l l y , E 3 / 1 9 K can be observed co-imrnunoprecipitating w i t h this c o m p l e x .  T h i s E R resident c o m p l e x consists o f M H C C I I, E 3 / 1 9 K , P m , c a l n e x i n and p o s s i b l y T A P 1 2  and T A P 2. T h i s helps establish a clearer picture o f the b i n d i n g process i n the E R . It suggests that this is a fairly large protein c o m p l e x .  104  Figure 21.  Calnexin association with W6/32 and Tap precipitates from 293  and 293.12 cells 293 and 293.12 cells were grown to confluence and labelled as detailed in materials and methods. This figure shows an immunoprecipitation by W6/32 and anti transporter antisera followed by western blotting with anti calnexin antisera. Cells were pulsed 30 minutes and chased for 0, 30, 75, 150 and 240 minutes (lanes 1-5 respectively), a) Lysates were precipitated with the conformation specific monoclonal W6/32 or the anti transporter antisera. Precipitates were separated by SDS-PAGE on the BioRad minigel system according to manufacturers instructions. Co-immunoprecipitated M H C CI I and E3/19K are denoted by arrows labelled i and ii respectively. b) Subsequently, membranes were probed with anti calnexin antisera using western blotting techniques detailed in Materials and Methods. Calnexin is denoted by the arrow labelled iii. Visualisation of the anti calnexin antisera utilised secondary goat anti rabbit antisera conjugated to HRPO (Jackson) and the Amersham E C L detection system. E C L treated membranes were exposed to Kodak X A R film for 1 to 15 minutes.  105  ^  n  r - CM CO  lO 1 OT-  5.3.5.  Peptide binding to M H C CI I does not exclude E3/19K binding The repertoire of peptides able to associate with specific M H C CI I proteins has been  fairly well characterised. In addition to hundreds of peptides eluted from various M H C CI I proteins, a set of rules dictating the topography of peptides able to bind specific proteins has been formulated (154). One such peptide that binds the HLA-A2 protein is one derived from the HIV reverse transcriptase (RT) protein. The sequence of the peptide is ILKEPVHGV. While it had been demonstrated that such peptides could be eluted from M H C CI I and then identified in mass spectroscopy experiments, it had not been shown that these peptides were bound by the peptide binding groove. In an attempt to demonstrate binding of the peptide to the M H C CI I, the observation that E3/19K co-precipitated with this complex was made.  The HIV RT peptide I L K E V F V G was identified as a peptide that fit the motif of an H L A A2 binding peptide. This peptide was N-terminally biotinylated to generate a marker for binding to M H C CI I. N-terminally biotinylated peptides were incubated with M H C CI I and ultimately would be used to precipitate these complexes. The experimental procedure has been described in Materials and Methods. Figure 22 demonstrates that biotinylated peptide was able to precipitate a M H C CI I enriched lysate. It was also noted that less M H C CI I was observed when less biotinylated peptide was added. In a competition with cold peptide at lOx excess, binding to the biotinylated peptide was abrogated. Finally E3/19K could still binds peptide bound M H C CI I molecules. This demonstrated that the association of E3/19K with M H C CI I molecules did not block the peptide binding groove. In figure 22 we show an increase in the amount of acid eluted M H C CI I pulled down with biotinylated peptide corresponding to peptide concentration. A 10 fold increase in biotinylated peptide yielded an increase in the amount precipitated. In a competition with 10 fold excess cold peptide, no MHC CI I was pulled down, demonstrating the specificity of the peptide for HLA-A2. Finally cold peptide alone did not precipitate M H C CI I. 108  This experiment was performed using lysates from 293.12 cells. In 293.12 cells, the interference of E3/19K with the association of the biotinylated peptide was investigated. If E3/19K was binding to the MHC CI I molecule through the peptide binding groove, then the binding of the biotinylated peptide should be competitively inhibited. Alternately, E3/19K binding to M H C  CI I molecules might alter the geometry of the peptide binding groove  rendering it unable to hold the biotinylated peptide. This did not occur. When the 293.12 lysates were used, the biotinylated peptide continued to precipitate M H C CI I molecules. In addition, the precipitated M H C  CI I molecules co-precipitated with E3/19K. This shows that  peptide binding is not excluded by E3/19K association.  It may suggest that M H C  CI I  molecules that have associated with E3/19K may be empty facilitating peptide binding in lysate, but this remains to be conclusively shown  109  Figure 22.  Precipitation of M H C CI I in enriched suspension  using a  biotinylated peptide 293.12 cells were grown to 9 5 % confluence and labelled and labelled using 3 5 S Met/Cys Pro-Mix (Amersham). Cells were pulsed for 30 minutes and chased for two hours. Chase media was alternately supplemented with biotinylated peptide at 5 uM and 50 nM (lanes 1 and 2 respectively), a mixture of peptide (5 uM) and biotinylated peptide (50nM) (lane 3), peptide (5 uM) (lane 4), biotin (25 uM) (lane 5) or mock treated (lane 6). At the end of the chase period, cells were washed and lysed in the presence of the crosslinking reagent DSP (200 ug/ml). Lysates were precipitated with non conformation-specific anti M H C CI I antisera R426. Precipitates were subjected to a glycine acid solution to remove Prot-A-Sepharose. The supernatant was divided into two aliquots; a 450 ul aliquot and a 50 ul aliquot. The 450 ul aliquot was precipitated with streptavidin-agarose (Pierce) (lanes 1-6 on the left). The 50 ul aliquot was precipitated with R426 a second time (lanes 1-6 on the right). Precipitates were separated on SDS-PAGE.  110  Ill  5.4.  Discussion The results in this chapter demonstrated two significant points. The first was that  E3/19K in 293 cells rapidly stabilised a mature M H C CI I epitope. The implications of this are discussed below. Second, the complex involving E3/19K also involves several other ER resident proteins including calnexin and at least one and possibly both TAP proteins. When testing to determine if E3/19K could bind in the absence of carbohydrates, it was contrary to expectations to observe that in a tunicamycin treatment of 293 and 293.12 cells, the mature M H C CI I epitope was lost in 293 cells but was retained in 293.12 cells. Tunicamycin blocks the transfer of the dolichol associated carbohydrate to the asparagine residue of the M H C CI I molecule. In most cases, the lack of this carbohydrate on M H C CI I molecules prevents any further maturation through the ER and they remain intracellular (124). Some studies have even shown that tunicamycin triggers the onset of apoptosis (155). 293.12 cells stably transfected with the E3/19K gene exhibit some of the characteristics reported in Ad2 infections including reduced cell surface expression of M H C CI I molecules (156). In 293.12 cells grown in the presence of tunicamycin, E3/19K was able to bind M H C CI I even when it was lacking a carbohydrate. It was also determined that the mature conformation detected by W6/32 was stabilised in this case. This observation suggested that the binding of E3/19K to the M H C CI I molecule stabilised a mature conformation. This study makes the assumption  that the W6/32 epitope indicates a mature  conformation of M H C CI I. Previous studies have used a W6/32 conformation as an indication of a mature conformation. However, the fact that this epitope can be precipitated from 293.12 cells which have been incubated in the presence of tunicamycin suggests that there may be many similarities between the structure stabilised by E3/19K and a mature M H C CI I molecule, but that the two conformations are not identical.  It should be noted that the mature  conformation referred to in this chapter is likely a stable conformation which resembles that of a mature M H C CI I protein and as such may differ from the surface bound protein in several  112  key features. When using the conformation specific monoclonal W6/32, it was observed that in 293 cells a mature conformation could be detected about 30-60 minutes post chase. This period of time required for maturation includes the participation of many other ER resident proteins including PDI (157, 158), rotamase, calnexin, calreticulin (159, 160), BiP (161-164) and many others. In the presence of E3/19K, M H C CI I molecules were able to achieve a mature (or W6/32 reactive) conformation almost immediately in 293.12 cells. This suggested that E3/19K binds to nascent M H C CI I molecules and quickly stabilize them. M H C CI I molecules could mature faster than in the absence of E3/19K, possibly bypassing the action of the concert of ER chaperones mentioned above. When precipitated with W6/32 in 293 cells, the mature M H C CI I epitope is not associated with calnexin molecules. This result was expected as the W6/32 recognizes a mature M H C CI I molecule which should no longer be accessible to calnexin. Calnexin binds to proteins until they achieve a mature conformation; in the case of M H C CI I molecules, calnexin release seems to coincide with the addition of peptide and the achievement of the correct conformation for release from the ER. When M H C CI I molecules are precipitated with R426 from the 293 cell line, M H C CI I associates with calnexin at all time points. Once again, this can be explained by the fact that R426 recognizes a wide subset of M H C CI I molecules including those that are immature. Immature M H C CI I molecules should still be associated with calnexin until they have achieved the correct conformation to leave the ER. As in 293 cells, the R426 precipitate of 293.12 cells associates with calnexin. This is due to R426 binding both mature and immature M H C CI I molecules.  E3/19K co-  immunoprecipitates with this complex demonstrating that E3/19K binding to M H C CI I molecules does not sterically or in any other way inhibit the association with calnexin. The M H C CI I protein with the W6/32 epitope is still able to bind calnexin in the presence of E3/19K in 293.12 cells. This is not unexpected as M H C CI I should be resident in the ER.  113  Although the initial association of calnexin with glycoproteins is mediated through the carbohydrates (56), the areas that appear to be crucial for continued association are found within the transmembrane region (165) reducing the likelihood of E3/19K sterically interfering with binding. Calnexin is readily dissociated from the M H C CI I complex in detergent. Further studies probe the associations between all of the participants of this complex. This complex appears to be loosely associated and fairly large with several participants. Having established this, it was investigated whether other ER resident proteins would also be found in this complex. Earlier it had been surmised that the binding of E3/19K might involve enveloping the alpha 3 region of the M H C CI I molecule. Key association points would be the alpha 1 and alpha 2 hypervariable regions. However, it is difficult to understand how calnexin fits into this picture. Calnexin may protrude further into the lumen of the ER than E3/19K and thereby associate with the N terminal portion of the M H C CI I molecule. One paper suggests that the areas involved in M H C CI I binding are in the transmembrane region (166). The above scenario raises the question whether calnexin associates with E3/19K as well. To further elucidate the nature of this calnexin-MHC CI I-P m-E3/19K complex 2  resident in the ER, it was examined whether calnexin binding was preceded by the addition of peptide. Previously it had been shown that M H C CI I molecules formed ternary complexes with TAP and calnexin and dissociation coincided with the acquisition of a peptide (28). The immunoprecipitation of 293 and 293.12 cells with both an anti transporter antisera and W6/32 addressed this. In the 293.12 cells precipitated with W6/32 the E3/19K co-immunoprecipitates with M H C CI I molecules. Surprisingly, precipitation of 293.12 lysates with the anti-transporter antisera precipitates a large complex including E3/19K. This suggests a large complex  114  involving the peptide transporter, M H C  CI I, P m and E3/19K. The association with the 2  peptide transporter is significant because it had been proposed that the nature of E3/19K binding to the M H C  CI I molecule is through the peptide binding groove. That E3/19K and  peptide transporter binding to M H C  CI I is not exclusive suggest that E3/19K is may not be  binding in the peptide binding groove. The experiment that shows that biotinylated peptides were able to bind to and cause the immunoprecipitation of M H C  CI I molecules in a lysate demonstrates that when  immunoprecipitated with the biotinylated peptide, the E3/19K proteins still associates with M H C CI I. This is significant in that it demonstrates that E3/19K binding to M H C CI I does not occur through the peptide binding groove. In addition, the association between E3/19K and M H C CI I does not sterically interfere with peptide access to the binding groove. In an earlier chapter, the nature of E3/19K binding to MHC CI I molecules was examined. One of the proposals considered was the possibility of E3/19K binding to the M H C CI I molecule through the peptide binding cleft. Binding to the cleft could interfere with the transfer of peptides from TAP to M H C CI I, possibly preventing release from TAP. With the results of this set of experiments this possibility seems unlikely. These findings also elucidate the order of the sequence of events in the ER. It appears that calnexin binds the M H C CI I glycoprotein first. Subsequent association with the peptide transporter and loading of the peptide triggers dissociation of both calnexin and the transporter. In cells not infected with Ad , the MHC CI I molecule is released for further travel through to the cell surface. In Ad infected cells, E3/19K may interfere with the transfer of peptide to the M H C CI I molecule preventing its subsequent release. In a previous chapter it is suggested that E3/19K is only able to bind those allelic proteins that remain in the ER to be loaded with peptide. Those that travel empty to the cell surface are not bound as effectively by E3/19K (i.e. K ). In the case of a slow binder, like D , k  115  b  the length of time that this protein stays associated with the peptide transporter and calnexin in the ER enhances the effectiveness of E3/19K binding. E3/19K binding to D  b  seems to take  longer than for other proteins. Binding does not occur co-translationally. This means there is a large population of empty MHC CI I proteins that need to be loaded with peptide present in the ER. If E3/19K intercepts the MHC CI I molecules before they are loaded with peptide, does this excess of empty M H C  CI I molecules monopolise all available peptide transporters?  Previous results suggest this not to be the case. In the case of D , it seems that M H C  CI I  molecules which remain in the ER for a long time do not influence the maturation of other proteins. Recent studies implicate a role for tapasin in M H C CI I loading, stating that one TAP complex can service up to four M H C CI I molecules (167). In the last chapter it was noted that E3/19K proteins with the ER retrieval signal are returned to the ER (168). In the case of E3/19K mutants that were missing the ER retention signal, the passage of M H C CI I through the ER was slowed (114)! The ability of E3/19K to slow the passage through the ER, along with the inherent ability to stabilize mature epitopes suggests it shares some of the functions normally attributed to chaperones. Once the ER retention signal is removed, the E3/19K protein still delays the passage of M H C CI I through the ER (169). In addition, Gabathuler et al. (169) are unable to show mutant E3/19K and M H C CI I associating outside of the ER. In the absence of its ER retention signal, E3/19K association with M H C CI I molecules may be temporary. Schekman and his colleagues (83, 170-172) have speculated the existence of a positive signal required for macromolecules to be transported from the ER . The fact that the E3/19K molecule is not readily transported out in the absence of its ER retention signal may be an example of an ER protein that needs a positive signal to leave the ER. Gabathuler et al. (114) show that removal of the ER retention signal does slow the  116  passage of MHC  CI I molecules through the ER. However, they do not clearly show whether  or not the E3/19K protein without its ER retention signal (621 protein) makes its way out of the ER. MHC  It is likely that MHC  CI I is not continually bound by 621 since the level of cell surface  CI I is not reduced in F A C S experiments (173). There is no evidence to show  association in any compartment other than the ER.  It is conceivable that when the 621-MHC  CI I complex reaches a subcompartment with physical characteristics different from the ER, dissociation occurs. E3/19K has an affinity for only those MHC  CI I molecules to which it is  exposed in the same compartment. This affinity is likely not high and if the MHC  CI I  -E3/19K complex moves to a compartment such as the cis Golgi, the complex spontaneously dissociates. Finally, it is the ER retention/retrieval signal K K M P that plays a role in retrieving the E3/19K and possibly the entire complex to the ER (168, 174), keeping them in an environment favouring association of E3/19K and MHC  CI I. There is no evidence that bound MHC  CI I  are retrieved from a distal compartment, possibly because they will not progress past being TAP associated without a peptide. If the association of M H C dependant on the conditions found within the ER  CI I and E3/19K is largely  and is unable to bind outside this  compartment, then the relationship between these two proteins is similar to that of a chaperone. In the last chapter it was suggested that at the cell surface there may be a type of regulation of MHC  CI I molecules. A positive signal generated at the cell surface related to the  concentration of MHC of more MHC  CI I molecules present, may feed back to the ER and limit the maturation  CI I molecules. The speedy appearance of a 'mature epitope' within the cell may  have further implications. The overloading of molecules ready to leave the ER may send a signal to the cell surface that upregulates cell surface turnover. Alternately, the mature epitope of the MHC  CI I molecules within the ER may help avoid the inevitable UPR.  speculated that the UPR  It has been  is stimulated by the levels of free BiP in the ER (175) and vice versa  (176). Mature conformations of retained proteins may be less likely to be bound by BiP and  117  less likely to trigger the UPR. Previously it has been proposed that the UPR helps prevent apoptosis of affected cells (176, 177), but some of the signalling triggers leukotrienes that stimulate inflammation. Infiltration of an afflicted area by immune response cells may allow a body to subsequently mount a more effective immune response to an affliction. Adenovirus infected cells may be less susceptible to such a response not only because of their characteristic ability to evade the immune system but also because of their weak stimulation of the UPR (68). The complex involving E3/19K, M H C CI I and transporter does exist. Additionally, it is observed that T A P and calnexin are also associated. components yields a very large complex.  118  The involvement of all of these  6.  Discussion The effect of the adenovirus E3/19K protein on both the cellular machinery involved in  antigen presentation and the regular maturation of MHC  CI I molecules has been examined with  investigations on the role of the interactions of the many accessory proteins found in the ER, Golgi and other compartments of the cell. This study outlined the participants in the processing of cell surface proteins and helped elucidate the sequence and relative order of many of the steps required for effective antigen presentation.  This study also investigated the role of  E3/19K in the normal infection process of adenovirus and possibly other related viruses (adeno associated viruses). The first chapter (three) examined the ability of E3/19K to bind specific protein products of different H-2 proteins in mice. This was the first time that the binding ability of E3/19K has been assessed against a common background (human 293 cells). Previously, pairs of proteins in different cell lines were exposed to E3/19K and the retention compared. The K not.  d  and K  b  allelic proteins bind to E3/19K very effectively while the K  k  and D  k  proteins do  These proteins were classified into binding and non-binding groups. The D  b  and L  d  proteins were also found to bind, albeit slowly. Previously they had been shown not to bind to E3/19K. The binding of these proteins had a half life of 3-12 hours, that contrasted greatly with the t  1 / 2  for the strong binders K  d  and K . b  This new  class of binders was  designated  slow-binders. E3/19K binding to MHC  CI I is based partly on an innate affinity of specific allelic  proteins to E3/19K. More importantly, binding requires that E3/19K and the target protein occupy the same intracellular compartment for an extended period of time. Allelic proteins like K  d  bind quickly to E3/19K whereas proteins like D  119  b  bind less quickly, but equally as  effectively, to E3/19K. That E3/19K does not bind K  k  and D likely reflects the amount of k  exposure that they have to E3/19K rather than a lack of affinity. As these molecules are quickly transported to the cell surface, they are not in the same compartment with E3/19K long enough for an association to occur. Unlike in the mouse, when surveying E3/19K binding to human H L A proteins, it is found that all tested allelic proteins bind. The K  k  and D  k  allelic proteins are the only two  shown to date not to bind to E3/19K. It is noteworthy that the t  for K  m  k  to travel through the  ER and become Endo H resistant is the shortest of all allelic proteins tested. The observation that the K protein does not bind may be due to the fact that the allelic protein is transported out k  of the ER at a very rapid rate and therefore is not physically available to be bound by E3/19K. It follows that if the rate of transport out of the ER for the K  k  protein could be retarded,  E3/19K binding may be observed. The observation that the D protein binds E3/19K strongly but with a long t  m  suggests  that the factors that influence E3/19K binding are closely related to factors that influence transport from the ER and to the cell surface. Normally, M H C  CI I proteins are translated into  the ER where they immediately achieve a correct conformation through the interaction of many chaperones and ER resident proteins. Proteins that achieve a correct conformation are quickly shuttled along the ER-Golgi pathway where they are acted upon by other chaperones. Those that do not achieve a correct conformation are retained in the ER. Previous studies have shown that different allelic proteins are transported with different rates through the ER. One of the factors that influences the rate of transport out of the ER is the availability of peptides that can be bound by the M H C specific M H C  CI I molecule. The affinity of  CI I proteins for existing peptides and the competition for these peptides may  greatly influence the rate of transport of M H C CI I molecules to the cell surface. In the case of 120  D  b  the inability to bind peptides that confer a stable conformation may be the reason for slow  transport. For K , which is rapidly transported from the ER, the protein may have ready access to k  peptides that allow the correct mature conformation and the resulting transport to the cell surface. Another scenario is that K  k  may bypass the quality control mechanism of the ER and  is transported empty. Several studies have demonstrated that empty MHC  CI I molecules can  be found on the cell surface in low numbers. These molecules are unstable and are rapidly turned over at the cell surface.  Factors that influence their stability include the allelic protein  which is being expressed and the cell line (and therefore cellular machinery) in which it is expressed. In the experiment with transfected 293 cells, the K  k  protein may  bypass the  chaperones responsible for quality control and proceed to the cell surface without peptides. This would explain the quick rate of passage through the ER to the cell surface and could also account for the inability of E3/19K to form any association with this protein. Unlike the K Comparison with K  d  protein, D  b  proceeds through the ER  shows that D  b  binds E3/19K much more slowly. This may be due to its  k  with a much longer half life.  slow rate of maturation through the ER. It could also reflect a lower affinity of D  b  for E3/19K.  A molecule with lower affinity for E3/19K may still be able to bind based on the length of time in the same compartment. The greater exposure of D Conversely, the fact that K  d  b  to E3/19K offsets its lower affinity.  is rapidly transported through the ER is offset by its higher affinity  for E3/19K. The  findings of earlier researchers should be reconsidered in light of these  observations.  Previously, groups determining the affinity of E3/19K for specific allelic  proteins may actually have been determining the amount of exposure these allelic proteins had  121  to E3/19K. When D  b  was identified as a non binder, its rate of transport out of the ER limited  the binding to E3/19K. The inability to form a complex with E3/19K was dependant on cellular factors that influence the rate of transport through the ER rather than an overall affinity between the two proteins. The ability of D  b  to bind has been overlooked by other groups  partly because of the longer time required for this association. Perhaps all allelic proteins are either fast binders or slow-binders. Those previously shown not to bind may not have been exposed to the conditions that would allow them to share the compartment with E3/19K for a period long enough to promote binding. In the following chapter, factors that influence the normal maturation of MHC molecules were discussed.  The 293D transfectant expressed large amounts of D  CI I but  processed relatively small amounts to the cell surface. Much of the translated product was found within the ER where it remains for an extended period of time. The 293D cell line was subjected to conditions that have been demonstrated to restore b  defective cell surface expression. TAP 2 deficient RMA-S cells grown at 26°C instead of the regular 37°C show increased levels of MHC These MHC  CI I molecules expressed at the cell surface .  CI I molecules were devoid of peptide in the binding cleft.  The reduced  temperature was believed to lower the kinetic energy of the system and reduce the rate of dissociation of these unstable complexes at the cell surface. The experiment consisted of growing 293D at 26°C and comparing the cell surface expression to 293D grown at 37°C. D  The results showed no increase in cell surface expression in FACS analysis. To complement this experiment, level of P m 2  available to the MHC  CI I molecules in  the 293D cell line was increased. The rationale for this experiment was that if [3 m was in b  limiting supply, then increasing this supply would result in greater numbers of D  122  (and  presumably all other) MHC  CI I proteins at the cell surface. Increased P m 2  caused by the  infection of 293D with a vaccinia construct did not increase the cell surface expression. Finally, studies with the CMT-64 cell line showed that high levels of cell surface expression could be achieved by exposing these cells to y-IFN.  y l F N acted as a general  upregulator of many cellular processes including the expression of TAPs, P m and MHC 2  When the 293D was exposed to ylFN, it was found that the levels of D b  b  CI I.  at the cell surface did  not increase. Failed attempts to upregulate the levels of D intriguing property of MHC  b  at the cell surface demonstrated an  CI I expression. In a cell with normal levels of MHC  expression (endogenous HLA), levels of foreign MHC  CI I  CI I molecule could not be upregulated.  Attempts to provide missing cofactors or to reduce the stringency of the quality control (by reducing the temperature) were unsuccessful in increasing the level of expression. This suggests that the level of the D  b  protein on the surface of 293 cells is actively regulated. It is  assumed that some other accessory protein may have a direct role in controlling the level of surface expression of MHC  CI I.  The bulk flow theory has been the prevailing theory regarding the transfer of membrane and secretory proteins through the ER.  More recently, other groups including one led by  Schekman have suggested the existence of positive signals that move cargo from the ER to the Golgi (83). Investigation of the regulation of cell surface molecules may turnover of MHC  be influenced by the  CI I molecules at the cell surface. Some investigators (114) have suggested  that the number of cell surface molecules is based entirely on the rate of transport to the surface. They suggest that turnover at the cell surface occurs at a set rate; reduced populations  123  result when any particular member of the population travels to the cell surface at a rate lower than that of the turnover. The results suggest that the level of expression of any particular species at the cell surface may be concentration dependant. Once the concentration of a particular population of M H C  CI I molecules reaches its saturation point, any increase in  expression will not result in increased representation at the cell surface. In the experiments, any excess D is retained in the ER. b  In the third section, it was observed that association of human H L A proteins with E3/19K caused rapid maturation as assessed by a conformation specific monoclonal antibody. H L A - A, B and C were very quickly recognised by W6/32 in the presence of E3/19K. Due to E3/19K retention in the ER, these molecules never achieved Endo H resistance. In short, E3/19K was binding to a misfolded and unprocessed immature M H C CI I molecule and forcing it into a conformation that resembled the mature conformation, yet preventing escape from the ER. Whether this interaction sterically interfered with the ability of other well characterised ER resident chaperones to associate with M H C  CI I molecules was studied. Of particular  interest was whether of calnexin and the TAP proteins could still associate with E3/19K bound MHC  CI I. The normal sequence of events for M H C  CI I association with ER resident  chaperones was probed with E3/19K. With experiments described in that chapter, the sequence of events for the E3/19K binding to M H C  CI I molecules can be elucidated.  In most cases, M H C  CI I is  cotranslationally translocated into the lumen of the ER where it associates with  P m. 2  Achievement of a mature conformation permits release by calnexin and egress from the ER is usually coincident with the loading of the correct peptide. With some allelic proteins, release from the ER may occur without peptide loading resulting in rapid transport to the cell surface. E3/19K associates with M H C CI I molecules in the ER before transport out of the ER. The  124  "slow-binders" suggest that this association is not necessarily co-translational for all allelic proteins as has been previously reported. E3/19K only will associate with those proteins which stay in the same compartment long enough to permit binding and E3/19K has different affinities for different allelic proteins. The exact nature of E3/19K association with MHC  CI I molecules has yet to be  determined. Differences in the binding affinity of different proteins have led some studies to map those residues in the MHC than others.  CI I molecule that may influence the binding to a greater degree  Site directed mutagenesis of E3/19K has shown that mutations in almost any  region can abrogate binding. Other studies have mapped the key regions in the MHC molecule to those hypervariable regions in the alpha 1 and alpha 2 regions.  CI I  This led  researchers in the past to suggest that E3/19K was binding in the peptide binding groove, possibly displacing peptides. Precipitation of E3/19K in a complex involving peptide transporters suggested that these two proteins were associating with MHC blocked the transfer of peptides to MHC experiments that precipitated MHC MHC  CI I non-exclusively. It is possible that E3/19K  CI I and their release from the TAP proteins. The  CI I with biotinylated peptide further demonstrated that  CI I and E3/19K did not associate with one another via the peptide binding groove. The role of E3/19K as a protein that was associating with MHC  CI I as a member of a  large group of proteins was investigated. The set of experiments demonstrating the association of several ER proteins in a complex with E3/19K indicated that this complex may be very large. Velocity sedimentation gradients were performed to assess the size of the complex. The sucrose gradients identified several subpopulations of the E3/19K protein with different densities. These subsets suggest that in addition to binding and retaining MHC  CI I in  the ER, E3/19K forms complexes with other proteins as well. E3/19K is in excess in studies that assess co-precipitation by alternately precipitating with anti-MHC CI I antibodies or anti E3/19K antisera, which suggests that much of it never associates with MHC  125  CI I.  The nature of binding of different MHC  CI I allelic proteins by E3/19K may reflect both  the rate of transport and the path of the MHC  CI I protein being bound. Proteins such as K  k  are not bound; these may be rapidly transported by bypassing the regular maturation process involving peptide loading. MHC  CI I proteins present in the ER for long periods of time are  bound effectively, for example D . b  Like K , D k  b  may not be maturing properly and is possibly  susceptible to the quality control mechanisms present in the ER and therefore unable to exit the ER. Both proximity and time promote association with E3/19K. Human MHC  CI I proteins are quickly bound by E3/19K and a mature conformation  quickly stabilises. This mature conformation may result in rescue from the default degradation pathway that degrades improperly formed proteins in the ER. mature MHC  Additionally, this build up of  CI I molecules in the ER may trigger a UPR response.  Finally, the experiments with ylFN reveal that the effect of E3/19K in the ER can be abrogated. This is significant because it makes the physiological role of E3/19K uncertain. Previously it was believed that as a result of its retention of MHC  CI I in the ER, E3/19K  reduced CTL killing of infected cells. However, the results of this research suggest that CI I will not be reduced in an antiviral response. The nature of the restoration of MHC  MHC  CI I cell  surface expression is also interesting. Many groups have shown that E3/19K is normally found in excess in the ER. MHC  The response to y-IFN is unlikely to be a simple upregulation of  CI I levels.  126  7.  Conclusion In summary, E3/19K served as a good tool to examine the nature of M H C CI I allele  specificity in viral infection and the subversion of antigen presentation by viral machinery. Secondly, E3/19K proved to be invaluable in probing the cellular machinery involved in the manufacture of proteins that are processed through the ER.  This includes all cell surface  proteins as well as those that remain associated with membranes inside the cell. It has been demonstrated that E3/19K binds to different M H C CI I allelic proteins with different affinities. 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The experiments with 293D demonstrated that b  there might exist different populations of E3/19K which differentially bound D  b  (and possibly the  other proteins). Finally, there was evidence that the complex that E3/19K formed in the ER involves calnexin and the TAPs, with the possibility of other chaperones being present in the complex. To address these questions, the size and shape of the MHC-E3/19K complexes was investigated. The use of the velocity sedimentation gradients allowed the opportunity to further divide the intracellular MHC-E3/19K of a cell into subgroups based on the distance it migrated through a sucrose gradient when subjected to centrifugal force for a set period of time. In previous experiments, lysates were solubilized in relatively harsh detergents, effectively destroying anything but the strongest of associations between molecules. By using gentle detergents before separating, protein complexes could be identified by their size. A specific protein such as MHC  CI I likely  associates with many different proteins in various complexes throughout its maturation stages through the cell. Identification of these different intermediates would help identify the stages of the maturation of the M H C  CI I molecule and would help in understanding its ability to bind to  E3/19K. It was suggested that MHC-E3/19K binding is more complex than first believed. It was further hypothesised that E3/19K exists in multimers.  This would shed some  insight into the life cycle of this protein and the optimal conditions for this protein to associate with 148  MHC  CI I.  This in turn would offer insight on the evasion of the immune system by the  adenovirus.  A2.2.  Rationale and Goals. To determine the relative size of the E3/19K complex, cell lysates were run over a  continuous sucrose gradient. In a series of experiments that utilized the gentle detergents NOctyl Glucoside, Chaps and digitonin, lysates were separated according to their size and shape. The proteins were identified with immunoprecipitation. Further experiments resulted in the characterization of attributes of these proteins in an attempt to determine the intracellular compartment they had resided at the time of labelling.  A2.3.  Results These experiments set out to determine the size of the M H C CI I complex by identifying  the fraction or fractions in which these molecules eluted. Initial results showed that M H C CI I was present in several different sucrose gradient fractions when precipitated with W6/32. The precipitated M H C CI I had a different MW  when precipitated from different fractions. In  293.12 cells the M H C CI I complex associated with E3/19K eluted in different sets of fractions than those in which the M H C CI I - P m complex (in 293 cells) alone eluted. These results 2  gave an estimate of the size of the complex consisting of E3/19K bound to M H C  CI I.  Furthermore, it was observed that E3/19K also eluted in fractions corresponding to larger complexes than the fractions containing M H C molecules.  A2.3.1.  Sucrose gradients separate cellular components by size  In figure 23, 293 cells were labelled overnight and the lysate run on a continuous 520% sucrose gradient as described in Materials and Methods. The purpose of this experiment  149  was to establish that the sucrose gradient did separate proteins based on size. This experiment allowed the determination of the characteristics and separating qualities of a sucrose gradient. The higher mass proteins travelled further through the sucrose and eluted in different fractions. This was not unexpected and indicated a good method for analyzing the data in the following gels.  150  Figure 23.  Separation of 293 lysates over a 5-20% sucrose gradient  293 cells were grown to confluence and labelled as above. Cells were labelled for two hours and lysed in N-Octyl Glucoside lysis buffer. 300 Lil of the lysate was layered on top of a 5-20% sucrose gradient and spun in a SW41 rotor at 36,800 rpm for 28 hours. Twelve 1 ml fractions were collected (lane 1 is the top fraction; lane 12 the bottom) and separated without precipitation by SDS-PAGE. Subsequent transfer to an Immobilon P V D F membrane followed, a) The membrane was exposed to Kodak X A R film for a period ranging from one day to three months (at -80°C).  151  A2.3.2.  M H C CI I complex precipitated from specific sucrose fraction  293 lysates run over 5-20% sucrose gradients were precipitated with W6/32. This yielded M H C CI I- P m complexes in fractions 3, 4 and 5 (figure 24). M H C CI I was found to 2  have migrated to other fractions as well, but P m was not observed associated in these 2  fractions. This suggested that P m does not associate with all the M H C CI I present in a cell, 2  but rather with a subset. The fraction of M H C  CI I that does not associate with P m 2  represented a distinct subpopulation not observed before. To elucidate differences between M H C  CI I complexes with and without E3/19K,  293.12 cell lysates were separated using the same technique and immunoprecipitated with W6/32. In the 293.12 cells, the MHC CI I complex (MHC CI I, P m, E3/19K) was found in 2  denser fractions (fractions 5, 6 and 7). E3/19K is observed co-immunoprecipitating in these fractions. The difference in migration, and therefore their size, of the complexes in the two cells is very likely due to E3/19K.  153  Figure 24.  Precipitation of M H C CI I and E3/19K from 293 and 293.12 cell  lysates separated over a 5-20% sucrose gradient 293 (a) and 293.12 (b) cells were grown to confluence and labelled as above. Cells were labelled for 2 hours and lysed in N-Octyl Glucoside lysis buffer. 300 Lil of the lysate was layered on top of a 5-20% sucrose gradient and spun in an SW41 rotor at 36,800 rpm for 28 hours. Eleven 1 ml fractions were collected (lane 1 is the top fraction; lane 11 the bottom). Fractions were precipitated with W6/32 or the anti E3/19K antisera R418. Precipitates were separated using SDS-PAGE.  154  CD 00  I I  I I  co *t  co oo r-  CM  DC  r-  O CD  0 TJ (0  (0  o (/) CM CM CO CO CO O) > CM >  CO CM  CD  co  CD CM  CO  CD  \5S  In the 293.12 lysates, E3/19K did not localize exclusively in those fractions with labelled M H C  CI I present. In addition to the fractions with labelled M H C  CI I and P m 2  complexes, E3/19K seemed to migrate further through the sucrose gradient ending up in fractions where M H C CI I was not observed. A band at -25 kDa and a band at ~50 kDa was also observed in these fractions (8-10). This led to speculation that the sucrose gradient was separating different sub-populations of E3/19K complexes within the 293.12 cells. The standard E3/19K complex observed in earlier experiments consisting of P m> M H C CI I, and 2  E3/19K were localised to fractions 5, 6 and 7. E3/19K complexes without observable MHC CI I, which could be multimers, were found in the fractions 8, 9 and 10. The discovery of E3/19K in different fractions suggested that they may be present in different sub-populations. These sub-populations may localise with different ER resident proteins. In some complexes, E3/19K may form multimers that do not include M H C CI I and P m. 2  The observation of different subpopulations of E3/19K suggested these subpopulations  may exhibit differing affinities and abilities to form complexes with M H C CI I. Additionally, M H C CI I with different mobilities was observed in different fractions. Variance in mobility reflects differences in the maturity of the M H C  CI I; specifically in the post translational  modifications of these proteins. It is possible that E3/19K has differing affinities for M H C CI I based on its maturity and its corresponding conformation. If E3/19K is in the presence of an M H C CI I molecule that is in a favourable conformation for binding, then a complex will form. If not, E319K may form complexes with other proteins, including itself, resulting in multimers.  157  A2.4.  Discussion This set of experiments demonstrated the M H C CI I complex can be precipitated from  different sucrose gradient fractions suggesting that the complex exists in different forms. In 293.12 cells, the complex precipitated by W6/32 consists of M H C CI I H chain, P m, peptide 2  and E3/19K. This complex does elute in a different fraction from the corresponding M H C CI I complex (without E3/19K) found in 293 cells.  In addition finding E3/19K present in  complexes with MHC-(3 m complex, populations of E3/19K are likely present in the ER in 2  other complexes. Association in these complexes is both with and more likely without MHC CI I molecules. In addition to determining which fractions of the sucrose gradient the different subpopulations of these proteins precipitate in, the sucrose gradient can be used to calculate the sedimentation coefficient (S value) of the protein complexes and the apparent molecular weight of these complexes. These S values are calculated by determining the distance migrated through a gradient of known density by a complex acted on by known centrifugal force. To convert the S value to the MW, estimation or determination of the stokes radius must be incorporated. The  S value was calculated using formulae previously published (178). The  sedimentation coefficient was given by the formula: S  where r|  respectively. P  20,w =  S  T,  [ T m  lT,  ( m  Pp-p20,w  ) / T  l20,w Pp-pT, (  ) ] m  and r| ^ is the viscosity of sucrose at temperature T or water at 20°C, 2o  Tm  and p  2 Q w  are the densities of the sucrose at temperature T or water at 20°C.  158  p is the density of the particle analyised. This can also be the reciprocal of the partial specific p  volume, 1/v. The values for the sucrose viscosity and density were obtained from standard tables (179). The partial specific volume of the protein-detergent complex was assumed to be similar to other transmembrane glycoprotein-detergent complexes. It was further assumed that these proteins bind 0.5 grams of detergent per gram of protein. S S  is given by the equation  TM  =ln(r 7 r ) / ( c o t ) 2  T  Where r and r is the distance travelled from r to r , co is the angular velocity of the rotor and 2  t is time. From the S value, the molecular weight of both the protein-detergent complex and the protein can be calculated according to the following: =(6TIN S „ R ) / ( l - v p „  M r  A  20,w  e  7  A  )  ^20,w  7  with N. being Avogadro's number and R the stokes radius of the protein. The stokes radius was estimated to be 4.4 nm (180). MW  estimates obtained using these sucrose gradients showed agreement with  previously published values for M H C Class II complexes (181). The size of the protein and the radius of the detergent with the proteins contribute to the size of the complex. Substituting the calculated S value in the formula above, the apparent molecular weight for a complex can be determined (see table 6). The M H C CI I-P m complex in 293 cells in the third and fourth 2  fractions give values of 60-75 kDa. This compares well to the literature value of 60 kDa. In 293.12 cells, the M H C CI I, P m - E3/19K complex were calculated to be in the 95-116 kDa 2  range. This value makes it is quite possible that a second E3/19K protein is present in this  159  complex. This would make the ratio of M H C CI I to (3 m to E3/19K to be 1:1:2. 2  Table 6.  Determination of Molecular Weight (MW) of proteins in sucrose  gradient fractions 2  S  3  9.19  4.18  93  56  4  9.89  5.57  123  75  5  10.6  7.06  156  95  6  11.3  8.62  191  116  7  11.9  10.42  230  140  8  12.7  12.69  281  170  Fraction  r  (1) MW  of protein and detergent  (2) MW  of protein alone  MW  (kD) (1)  MW (kD) (2)  The generalised calculations of the MW for complexes found in 12 different 1 ml fractions from samples layered on top of a continuous 5%-20% sucrose gradient and separated for 28 hours at 36,400 rpm (148,000g) are shown above. The S and MW of proteins found in different fractions was calculated using formulae in text, r is the distance migrated through the gradient. Viscosity and density of fractions (not shown) are derived from standard tables (179). The variables Stokes Radius (Re) was previously determined to be 4.5 nm (180).  The association of E3/19K with M H C CI I as a dimer is not completely unexpected. In experiments using the E3/19K-Vaccinia vector, E3/19K is overexpressed and many bands with molecular weights approximately twice that of E3/19K are observed (data not shown).  160  Additionally, in an earlier chapter that investigates the steric interactions of E3/19K with MHC CI I, it was proposed that for E3/19K to effectively associate with the specific amino acids of the M H C  CI I sequence shown to influence its binding, E3/19K may act in concert with  additional factors that perturbs the 3-D structure of the M H C CI I molecule. A novel method of membrane perturbation is proposed for E3/19K to extend far enough into the lumen of the ER for E3/19K to reach the residues on the M H C CI I molecule that affect its binding. Multimers of E3/19K could achieve this. E3/19K is also present in the denser fractions. corresponding to MW  E3/19K appeared in fractions  of 116-170kD in fractions precipitated with M H C CI I antisera. Unlike  the less dense fractions, in these fractions labelled MHC CI I is not apparent. Also present is the band that correlates to a protein approximately double the size of E3/19K. This complex could be a multimer of E3/19K. In the denser fractions, it is important to note that these complexes are still being precipitated with anti-MHC CI I antisera. While labelled MHC CI I is not visible under these conditions, it is likely still present. These fractions could be illustrating a novel interaction between multimers of E3/19K and M H C CI I that may precede labelling in the pulse/chase. E3/19K may have associated with M H C  CI I molecules prior to labelling and entered into a  very stable conformation. E3/19K may form very long-lived associations with M H C  CI I  molecules. The appearance of a band at approximately double the size of E3/19K (about 50 kDa) further suggests that the M H C  CI I, P m, E3/19K complex is larger than the one 2  normally observed with M H C CI I and P m and that multimers of E3/19K may be involved. 2  In an earlier chapter, it was observed that the D  b  protein in 293D does not bind to b  E3/19K until several hours post chase. The earlier results showed that one of the features of the D protein is that it needs a longer time to bind to E3/19K. Additionally, in 293 D b  161  b  cells,  several differently glycosylated subsets of the E3/19K population were observed. In the 293 D  b  it is the fully glycosylated version of E3/19K that first binds to the D  b  protein. Later time  points reveal that all the other subsets are found associated with D . This could represent a b  cooperative binding of E3/19K to M H C CI I. Identification of the slowbinding protein, D , allows the proposal that in the case of b  some proteins such as L , binding to E3/19K may take a very long time to occur. While the d  cell surface expression of L  d  is markedly reduced, very little co-immunoprecipitation  throughout the time course of the experiments is observed. E3/19K may be a very long lived resident of the ER, and it may bind to different subsets of M H C CI I dependant on its maturity. The existence of E3/19K in the denser fractions of the sucrose gradient could be due to its participation in a larger complex with other ER resident proteins or chaperones. This may represent portions of the total E3/19K population that hitherto go undetected in the M H C CI I co-precipitation. Both these findings and the literature report that the amount of E3/19K precipitated by anti E3/19K antisera is much greater than the E3/19K that co-precipitates with M H C CI I. There probably exists a pool of E3/19K that never associates with M H C CI I, or at least does not associate within the time constraints of the experiments reported here. The E3/19K found in the denser fractions may be this subset of the total E3/19K population. E3/19K in this fraction may also be associated with the ER resident proteins such as calnexin and others. The E3/19K complex with M H C  CI I is a little more complicated than originally  thought. Previously it had been assumed that E3/19K bound to the M H C CI I complex as a monomer. A sub population of E3/19K is present in a larger complex that may involve multimers of E3/19K. Additionally, the ER resident chaperones such as calnexin and TAP may be present. These complexes are originally observed when precipitated with anti MHC CI  162  I sera and antibodies. However, labelled M H C CI I is not observed in these fractions. This suggests that the M H C  CI I component of these complexes has not been labelled. This may  reflect an association of M H C CI I with E319K that is of longer duration than the length of the pulse-chase.  163  

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