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Association of the B cell antigen receptor with protein chaperones Foy, Shaun Patrick 1997

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ASSOCIATION OF THE B C E L L ANTIGEN RECEPTOR WITH PROTEIN CHAPERONES by S H A U N PATRICK FOY B.Sc. (hons), The University of British Columbia, 1993 A THESIS SUBMITTED FN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH C O L U M B I A October 1997 © Shaun Patrick Foy, 1997 In present ing this thesis in partial fulf i lment of the requ i rements for an a d v a n c e d d e g r e e at the University of British C o l u m b i a , I agree that the Library shall make it freely available for re ference and study. I further agree that pe rmiss ion for extensive c o p y i n g of this thesis for scholar ly p u r p o s e s may b e granted by the h e a d of m y depar tment or by his o r her representat ives. It is u n d e r s t o o d that c o p y i n g o r pub l ica t ion of this thesis for financial gain shall not b e a l l o w e d wi thout my writ ten p e r m i s s i o n . D e p a r t m e n t of ~^<?g> ^ ^ ^ J ' T h e University of British C o l u m b i a V a n c o u v e r , C a n a d a Date Qcfo-W )^ /W^ D E - 6 (2/88) Association of the B cell antigen receptor with protein chaperones Shaun P. Foy The multimeric B cell antigen receptor (BCR) consists of four different receptor subunits: immunoglobulin heavy chain (Igu), light chain (X), Iga and Igp. These four chains must assemble correctly in the endoplasmic reticulum (ER) before the receptor can be transported to the cell surface. Recent evidence in lymphoid cells suggests that during receptor assembly the mlgM part (Igu. + light chain) of the BCR interacts with chaperone proteins in the ER. In this study we have examined the proteimprotein interactions between three chaperones, GRP78 (BiP), GRP94 and calnexin with the wildtype B C R and with a B C R containing an altered u heavy chain. By generating cell lines that express various intermediate assembled forms of the B C R (Igu+A,+Iga+Ig(3, Igu+Ji+Iga, Igu+k, Igu, X, Iga, or Igp), we have been able to identify the protein chaperones associated with each receptor subunit. Our data showed that, when expressed by itself in non-lymphoid cells, Iga associated with GRP94 and that the level of association increased dramatically when other BCR chains were co-expressed. Iga was found associated with BiP and calnexin in lymphoid cells, however we believe these interactions were likely mediated through other BCR chains present. We could find no evidence of Igp interactions with any of the other chaperone proteins tested. Interestingly, our results also showed that, when expressed alone, X light chain associated with calnexin. Consistent with the literature, u heavy chain was found associated with all three protein chaperones tested. Our results are consistent with the model suggesting an ordered and sequential interaction between assembling B C R components and the GRP78 and GRP94 protein chaperones during early stages during receptor assembly. Recent evidence suggesting the membrane spanning region of Igu is particularly important for interacting with protein chaperones led us to examine an altered form of u heavy chain that escapes ER retention in lymphoid cells. We found that this mutant did not escape ER retention in non-lymphoid cells nor did it bind chaperones differently from wildtype. This observations suggests that the presence of B cell specific proteins may mediate the transport of the mutant protein to the cell surface in lymphoid cells. ii Table of Contents Abstract ii Table of Contents iii List of Tables iv List of Figures v Acknowledgments vii Dedication viii Introduction 1 Materials and Methods 15 Results 30 Discussion 58 References 69 Appendix 75 iii List of Tables Table 1 - Oligonucleotides used to create site-directed mutations in the genomic (i heavy chain gene 16 Table 2 - Oligonucleotides used for sequencing membrane region of constructed pLpAu33m plasmid 18 Table 3 - Synthetic peptides used as antigens for synthesis of anti-Igoc and anti-Igp antibodies 24 Table 4 - Summary of anti-peptide antibodies 31 Table 5 - Summary of anti-IgP antibodies produced by rabbit SF2 32 iv List of Figures Figure 1 - GroEL Model 5 Figure 2 - Structure of the BCR 9 Figure 3 - Schematic of u heavy chain transmembrane region 13 Figure 4 - Construction of the pLpAu33m plasmid 21 Figure 5 - Screen of anti-IgP SF2.B 16 antibody 33 Figure 6 - Antibody controls for Western blot analysis and immunoprecipitations 34 Figure 7 - Association of BCR chains with protein chaperones in lymphoid cells 36 Figure 8 - Expression of protein chaperones in lymphoid and non-lymphoid cells 37 Figure 9 - Expression of BCR proteins in transfected non-lymphoid cells 38 Figure 10 - Oligomers present in transfected non-lymphoid cells 39 Figure 11 - Expression of protein chaperones in transfected non-lymphoid cells 41 Figure 12 - Association of u heavy chain with protein chaperones in transfected non-lymphoid cells 42 Figure 13 - Association of X light chain with protein chaperones in transfected non-lymphoid cells 43 Figure 14 - Association of Iga with protein chaperones in transfected non-lymphoid cells 44 Figure 15 - Stability of u heavy chain in transfected non-lymphoid cells 47 v Figure 16 - Stability of A, light chain in transfected non-lymphoid cells 48 Figure 17 - Stability of Iga in transfected non-lymphoid cells 49 Figure 18 - Expression of mutant p heavy chain and other BCR proteins in transfected non-lymphoid cells 52 Figure 19 - Expression of protein chaperones in pLpAu33m transfected non-lymphoid cells 53 Figure 20 - Altered u heavy chain is retained internally in transfected non-lymphoid cells.... 54 Figure 21 - Oligomers present in non-lymphoid cells transfected with an altered p. heavy chain 55 Figure 22 - Association of an altered u heavy chain with protein chaperones in transfected non-lymphoid cells 56 Figure 23 - Stability of an altered p heavy chain in transfected non-lymphoid cells 57 Figure 24 - Schematic of u33 alteration 65 vi Acknowledgments Many people have helped me throughout my graduate career, both at U B C and elsewhere. I doubt I could have completed this degree without their assistance, guidance and support. First, I would like to thank my supervisor, Linda Matsuuchi, who has been incredibly patient and supportive throughout my degree and who gave me an opportunity for which I will always be grateful. Thanks to my fellow lab members who suffered from my bouts of hyperactivity that followed sugar and caffeine binges - something that made me intolerable. Very special thanks to Sharon Hourihane, Lorie Joyce, HeeJoo Moon and May Dang who provided superb technical support. My fellow student, Jessica Escribano, has been very supportive and helpful throughout the years. Corey Ming-Lum and Lorna Santos helped to establish the transfected cell lines used in the project. Karen Chua helped to screen the anti-peptide antibodies. Thanks to Colm Condon for being a great source of amusement and for the help with the anti-Igp antibody. I would like to thank the members of Hugh Brock's, Wilf Jefferies', Don Moerman's, Terry Snutch's and Bi l l Milsom's labs for all their help. In particular, I would like to thank Ian Haidl and Jacob Hodgson for the many hours they spent with me discussing the nature of science and life in general. Greg Mullen has always been very willing to provide technical expertise, both in molecular biology and immuno-microscopy. Don Nelson, DNA guru, helped me to overcome the molecular biologist's version of a runner's wall. Michael Harris has been a great source of extra-laboratory events, both directing me to the best donut hiding spots in the department and, occasionally, stealing a few extras for me when I couldn't find them myself. I would like to thank Alistair Blachford and Lance Bailey for answering my continuous stream of questions and for providing me with a greater appreciation of life as a computer weenie. Thanks to the members of my research committee, Mike Gold, Reinhard Gabathuler, Terry Snutch and Linda Matsuuchi, for their direction and feedback. Finally, I would like to thank my family. My parents have supported me through the past nine years of university, both emotionally and financially - thanks mom and dad, I couldn't have done it without you. My loving partner, Kirsty, has always been patient and supportive of my goals -Kirsty, I would have been miserable without you. vii I dedicate this thesis to my grandfather, John Luke. viii Introduction Receptors of the immune system play a crucial role in protecting the body from infection by foreign pathogens. When localized at the plasma membrane, these receptors mediate cellular responses that ultimately bring about the killing and removal of the invading pathogens by regulating the lymphocyte development. Due to the essential role these receptors play in protecting the body, cells have developed a protective system that ensures receptors are functional when expressed at the cell surface. This system works at the level of the endoplasmic reticulum (ER) to identify and retain improperly folded proteins and incompletely assembled protein complexes. Expression of improperly folded receptor subunits or incompletely assembled receptor complexes could have profound affects on lymphocyte development, potentially causing extreme harm to the body. Once the retained proteins are properly folded and assembled, they are released from the ER and allowed to move through the secretory pathway to the cell surface. Proteins that do not acquire their proper folding state or assembly state are transported into the cytosol and then delivered to proteasomes for degradation (Wiertz et al, 1996). This type of retention that is based on the state of protein folding or receptor assembly has been termed both 'quality control' (Hurtley and Helenius, 1989) and 'architectural editing' (Klausner, 1989). This protective system monitors not only to immune receptors, such as the T cell antigen receptor (TCR) (Samelson et al, 1985; Clevers et al, 1988), M H C class I (Ploegh et al, 1981) and the B cell antigen receptor (BCR) (Venkitaraman et al, 1991; Matsuuchi et al, 1992), but also numerous other multisubunit protein complexes including the insulin receptor, transferrin receptor and acetylcholine receptor (Hurtley and Helenius, 1989). The mechanisms giving rise to this type of quality control have not been clearly defined, although it appears this system relies heavily on the association of folding proteins and assembling protein complexes with a network of resident ER proteins known as protein chaperones. 1 Protein Chaperones Protein chaperones are a group of proteins that act to promote correct folding of nascent polypeptides and assembly of multisubunit complexes. While many protein chaperones were initially identified for their protective role during stress-induced situations, researchers have more recently identified two important roles for protein chaperones in normal cellular activities. First, protein chaperones bind both nascent polypeptides and subunits of partially assembled protein complexes and prevent them from forming aggregates. Preventing aggregates from forming allows both protein folding and protein complex assembly to occur more quickly. Second, protein chaperones bind terminally denatured proteins and target them for degradation (Ellis, 1994). Thus protein chaperones ensure cells only express properly folded and properly assembled proteins and that non-functional proteins are targeted for degradation. This is a particularly difficult task given the large number of different proteins, and therefore a wide variation of protein sequences, which must be monitored by these quality control proteins. By recognizing conformational determinants rather than specific amino acid sequences, protein chaperones can bind a wide variety of proteins (Hammond and Helenius, 1995). Some of the conformational determinants identified by protein chaperones include exposed hydrophobic elements, free exposed sulfhydryl groups, partially trimmed glycans (Hammond and Helenius, 1995). Interestingly, the mechanisms for retaining terminally misfolded proteins appear to be quite different than those associated with incompletely folded polypeptides and incompletely assembled protein complexes (Hurtley and Helenius, 1989); terminally misfolded proteins tend to form aggregates and have stable interactions with protein chaperones. This research focuses on the quality control mechanisms that regulate both the folding of nascent polypeptides and assembly of protein complexes. Early in vitro studies demonstrated that denatured ribonuclease could spontaneously self-assemble in the absence of other proteins and without expending energy. Thus, it was concluded that the information contained within a polypeptide's primary amino acid sequence is 2 sufficient for a protein to fold to its native state (Anfinsen, 1973). In contrast to folding in vitro, several factors make it significantly harder for a protein to acquire its native state in vivo. First, a high concentration of protein in the ER, estimated at 100 mg/ml, means there are many available proteins to aggregate with nascent polypeptides. Second, rapid protein synthesis results in a large number of proteins entering the ER over a short period of time. For example, lymphoid cells can synthesize over 200,000 immunoglobulin (Ig) molecules every minute, with each Ig molecule taking only 2-4 minutes to produce (Wiest et al., 1995). To avoid protein aggregation and enable nascent polypeptides to efficiently fold and assemble into complexes, quality control systems make use of protein chaperones. These specialized proteins protect nascent polypeptides from their surrounding environment, producing conditions favourable for rapid protein folding and assembly of protein complexes. In addition to assisting folding, an important function of protein chaperones is to prevent improperly folded proteins and incompletely assembled protein complexes from being expressed at the cell surface. It appears that protein chaperones retain their target proteins by binding them. But, how are protein chaperones themselves retained? While protein chaperones identify their target proteins through conformational determinants, such as exposed hydrophobic patches and glycans, they themselves are identified by the presence of a more obvious motifs generally consisting of a linear sequence of amino acids. Two examples of these motifs are the H/KDEL motif (for review see Nilsson and Warren, 1994) and the di-lysine motif (for review see Teasdale and Jackson, 1996). The H/KDEL motif consists of either a glutamate (H) or a lysine (K) followed by an aspartic acid (D), glutamic acid (E) and a leucine (L). This motif is commonly found on soluble proteins that reside in the ER. Soluble proteins containing the H/KDEL motif are bound by the membrane associated K D E L receptor. Once bound by the receptor, soluble proteins are either retained in the ER or, if in the Golgi, returned to the ER by a recycling mechanism. In contrast to the H/KDEL motif, the di-lysine motif is found only on membrane proteins. This motif consists of a K K X X sequence of amino 3 acids where any two amino acids (XX) follow two lysine residues (KK). The dilysine motif is located on the cytoplasmic tails of many membrane bound proteins that are retained in the ER. Not surprisingly, the K D E L receptor itself contains this motif. The di-lysine motif interacts with a group of cytosolic proteins, referred to as coatamer proteins, which line the cytosolic surface of the ER and form coats on budding vesicles (Cosson and Letoumeur, 1994; Letourneur et al, 1994). It appears these interactions cause di-lysine containing proteins to be localized in the ER. Quality Control in Prokaryotes A well studied protein chaperone in Escherichia coli, GroEL provides an excellent example of how protein chaperones may assist folding proteins (Hartl, 1994). The GroEL complex consists of 14 identical subunits, each of approximately 54 kDa. Crystallographic evidence suggests these subunits form two identical ring like structures, with one ring stacked on top of the other (Braig et al, 1994). This 'double-donut' configuration results in the formation of a protein complex containing a central cavity. Mutational analysis suggests polypeptides bind residues located on the interior of this central cavity, with interactions occurring between hydrophobic residues of GroEL and exposed hydrophobic patches on folding proteins (Fenton et al, 1994). Binding occurs in an ATP-dependent fashion, with eventual release of the nascent polypeptides caused by binding of a second protein complex, GroES. GroES is a itself a heptameric ring structure, consisting of identical 10 kDa subunits. GroES binding to the GroEL complex is thought to cause release of the folded polypeptide by displacing it because mutational analysis shows GroES binding is abrogated in GroEL mutants in which highly conserved hydrophobic residues are altered (Fenton et ai, 1994). The GroEL approach to facilitating folding of nascent polypeptides is very effective, taking advantage of structural characteristics such as exposed hydrophobic patches for binding rather than specific signal sequences such as linear sequences of amino acids. This approach allows GroEL to identify a broad range of proteins since many proteins will have exposed hydrophobic patches during the 4 early stages of protein folding. This approach to protein folding is not unique to prokaryotic cells as it appears to be used by eukaryotic cells as well. Figure 1 - Model for GroEL mediated folding of unfolded polypeptides. GroES is not illustrated in the above figure but is proposed to act as a cap for GroEL to enclose the unfolded polypeptide in a protective environment so it can fold. GroES is not illustrated above. This figure has been adapted from Alberts et al. (1996). Quality Control in Eukaryotes The approaches used by prokaryotic quality control proteins appear to be used by eukaryotic proteins as well. A good example is the eukaryotic protein chaperone BiP, also referred to as the heavy chain binding protein and GRP78. BiP, like GroEL, identifies its target proteins through the presence of exposed patches of hydrophobic amino acids (Flynn et al., 1991; Blond-Elguindi et al, 1993). The high levels BiP present in the lumen of the ER, making up approximately 5% of the total protein in the ER lumen (Gething and Sambrook, 1992), suggests it plays an important for normal cellular activities. BiP activity appears to be regulated through protein modifications. When not bound to proteins, BiP is present as stable aggregates that are both phosphorylated and ADP ribosylated (Freiden et al, 1992). These aggregates are 5 disrupted by the presence of ATP which causes an increase in abundance of active, unmodified BiP monomers that can bind target proteins. Once bound to target proteins, BiP dissociates in an ATP dependent fashion, activity that is regulated by an intrinsic ATPase activity (Gaut and Hendershot, 1993). Because there is no crystallographic data on BiP, it is not as clear how BiP mediates protein folding or assembly of multisubunit proteins. By analogy to GroEL, BiP may promote protein folding by covering up exposed hydrophobic patches and protecting them from the unfavourable conditions of the surrounding environment. Calnexin is a second, quite different example of a eukaryotic protein chaperone. Unlike BiP and GroEL, the 65 kDa protein chaperone is a transmembrane protein that appears to target both monoglucosylated glycans and transmembrane regions of proteins. The relative importance of each of these motifs seems to vary depending on the target protein. For example, calnexin binding is inhibited in cells treated with tunicamycin, a glycosylation inhibitor, suggesting calnexin interaction is glycan dependent (Ou et al., 1993). Later work indicated the interaction is with monoglucosylated N-linked glycans, such as Glc,Man 5 . 9GlcNAc 2 (Kearse et al., 1994; Hebert et al., 1995; Ora and Helenius, 1995). However, calnexin binding has also been observed with certain proteins that lack glycans (Rajagopalan et al, 1994; Kim and Arvan, 1995; Loo and Clarke, 1994; Arunachalam and Cresswell, 1995) suggesting calnexin may have several different means of binding target proteins. Models explaining glycan-dependent calnexin binding suggest unfolded proteins are particularly susceptible to the addition of monoglucosylated glycans thus making them targets for calnexin binding. Since monoglucosylated glycans are only added to partially folded proteins, calnexin does not bind proteins that are completely folded (Hammond et al., 1994; Hebert et al., 1995; Sousa and Parodi, 1995). Calnexin binding appears to be important for retaining incompletely assembled complexes of membrane proteins present in the ER. When a calnexin mutant that lacks a cytoplasmic tail is overexpressed in COS cells, both the altered calnexin and an associated protein are identified in a lysosomal-like compartment (Rajagopalan et al, 1994) suggesting the 6 truncated calnexin is no longer able to retain proteins in the ER. Further evidence suggesting calnexin associates with partially folded proteins and partially assembled complexes (Ou et al., 1993) is consistent with a role for calnexin in mediating the assembly of multisubunit proteins (Hochstenbach et al, 1992; Rajagopalan etal, 1994). BCR Function B lymphocytes express receptors on the cell surface that recognize specific antigen. Each lymphocyte expresses a group of receptors with the same specificity, but with specificity different from other lymphocytes. The difference in binding specificity results in a repertoire of B cells capable of binding to many different antigens. The B C R plays two important roles in the immune response. First, the B C R plays an important role in regulating B lymphocyte development. When the BCR is crosslinked by multivalent antigen it activates a number of protein tyrosine kinases (Gold et al, 1990; Lane et al, 1990; Brunswick et al, 1991; Campbell etal, 1991) that consequently activate key intracellular signaling pathways including the phospholipase C y pathway, Ras/Gap/Map kinase pathway and the phosphotidyl inositol 3 kinase pathway (Gold and Matsuuchi, 1995). In immature B lymphocytes - cells exposed only to self antigen - this signal causes programmed cell death (apoptosis) preventing an immune response against the body's own antigens (Nossal, 1983; Ralph, 1979). However in mature B lymphocytes, this signal may cause cell activation, leading to lymphocyte proliferation and differentiation into either antibody secreting cells or memory cells (DeFranco, 1987). These different responses enable the immune system to establish a repertoire of cells that recognize foreign antigen and not self antigen. The second function of the B C R is to act as a high affinity endocytic receptor that interacts specifically with foreign antigen. Bound antigen is taken up into the endocytic pathway where the antigens are degraded into peptides. These peptides associate with M H C class II molecules in a compartment that is intermediate to Golgi and endosomal compartments. The peptide is then expressed on the cell surface in the context of M H C molecules for recognition by T helper cells. 7 B C R Structure Structurally, the BCR is made up of two basic units: mlg and Iga/Igp. In the mouse, mlgM consists of two covalently associated u heavy chains (-72 kDa), each of which is covalently associated with one light chain protein (-25 kDa). As figure 2 illustrates, both membrane and secreted u heavy chain proteins consist of a variable domain (VH) followed by a constant region comprised of four domains (CHI-4). In contrast to secreted u heavy chain, membrane u heavy chain has a different 41 amino acid (aa) carboxyterminal region that consists of a 26 aa transmembrane domain, 3 aa cytoplasmic tail, and a 12 aa lumenal portion that extends from the cell membrane. As with heavy chain, associated light chain also has a variable region (VL) and a constant region (CL). Together, the variable regions of the heavy (VH) and light (VL) chain form the antigen binding region. The second structural unit, the Iga/Igp heterodimer, consists of two covalently associated glycoproteins, -32 kDa Iga and -37-39 kDa IgP (Reth, 1992), products of the genes Mb-1 and B29 respectively (Hombach et al, 1990; Hermanson et al, 1988; Campbell etal., 1991). Both Iga and IgP have transmembrane regions of 22 aa and long cytoplasmic tails: 61 aa (Iga) and 48 aa (IgP). mlgM and the Iga/IgP heterodimer alone appear to form the basic components of the BCR and are sufficient to allow surface expression of a partially functional BCR (Venkitaraman et al, 1991; Matsuuchi et al, 1992). Other B C R -associated proteins include the prohibitin-related p32, p37 and p41 (Terashima et al, 1994). The significance and function of these components have yet to be determined although they may be involved in any of the different BCR activities including receptor assembly and transport, signal transduction and antigen internalization. 8 A) B) mlgM slgM Figure 2 - Schematic of murine IgM. (A) Membrane IgM (mlgM) and the Iga/Igp heterodimer are the basic components of the B cell antigen receptor. (B) Secreted IgM (slgM) is identical to mlgM with the exception of the c-terminus. slgM has a different c-terminus than the 41 aa c-terminus present on mlgM. Quality Control and the B C R Quality control systems ensure BCR molecules are fully assembled before being expressed at the cell surface. For surface expression of the BCR, a cell must express each of the four different BCR proteins (Venkitaraman et al., 1991; Matsuuchi et al, 1992). In the absence of any one of the four B C R chains, all the components of the BCR are retained internally, presumably in the ER. It is likely that these partially assembled subunits are actively retained by protein chaperones. This is supported by several observations, in both lymphoid cells and non-lymphoid cells. First, protein chaperones associate with immune complexes of both BCR heavy chain proteins and light chain proteins under a variety of different conditions, including in different cell types and with different isotypes of both heavy chain proteins and light chain proteins. Second, analysis of mutant proteins that have abnormal trafficking indicates there is a difference in protein chaperone binding of the altered BCR components. The details of these observations are described in below. 9 BiP BiP binds both heavy chain and light chain proteins when either protein is expressed alone (Haas and Wabl, 1983; Bole et al, 1986; Melnick et al, 1992). BiP binding is specific for exposed hydrophobic amino acids on target proteins (Flynn et al, 1991; Blond-Elguindi et al, 1993). BiP appears to bind the CH, portion of \i heavy chain. When the CH[ region is deleted, the altered heavy chain protein is expressed abnormally at the cell surface (Cherayil et al, 1993). A role for BiP in the assembly of BCR components is supported by several observations. First, significantly less BiP is associated with heavy chain in cell lines that co-express heavy chain and light chain proteins than in cell lines expressing heavy chain alone (Haas and Wabl, 1983; Bole et al, 1986). Second, fully assembled H 2 L 2 molecules do not associate with BiP while partially assembled complexes do (Haas and Wabl, 1983). Third, a yeast mutant of the BiP homologue, KAR2, shows a dramatic reduction in the oligomerization of a multisubunit marker protein, a phenotype that can be reversed by the addition of wildtype K A R 2 (Schonberger etal, 1991). These observations are consistent with an important role for BiP in the assembly of protein complexes. GRP94 Like BiP, GRP94 also binds both heavy chain and light chain proteins. In cell lines expressing heavy chain alone or light chain alone, low levels of GRP94 co-immunoprecipitate both proteins following chemical crosslinking (Melnick et al, 1994; Melnick et al, 1992). For X light chain, GRP94 binding appears specific for later folding intermediates, species that are partially or fully oxidized (Melnick et al, 1994). This is in contrast to BiP which associates with both early and late folding intermediates. These observations are consistent the sequential binding of X light chain, first by BiP and then later by GRP94. In addition to being specific for particular folding intermediates, it appears that GRP94 is also specific for particular assembly intermediates. Cells that coexpress X light chain and heavy chain proteins show slightly less 10 GRP94 associated than cells expressing either light chain or heavy chain alone (Melnick et al., 1994). The actual role GRP94 plays in regulating folding of nascent polypeptides and assembly of protein complexes is not known. The presence of common substrates and similar control elements (Liu and Lee, 1991) suggests it may play a role similar to BiP. GRP170 As with the other GRPs examined, GRP170 also associates with free heavy chain and light chain proteins (Nilsson and Warren, 1994). Co-immunoprecipitation experiments demonstrate that GRP170 associates with both membrane and secreted forms of heavy chain proteins. As well, these studies demonstrate GRP170 also associates with light chain proteins. The association of 78 kDa and 94 kDa bands with GRP170 immune complexes suggests GRP170 may form complexes with other protein chaperones, presumably while assisting folding of nascent polypeptides. Consistent with a role as a protein chaperone involved in folding and assembly of nascent polypeptides, GRP170 is localized in a pre-Golgi compartment. The mechanism of GRP170 binding is not known nor has its role in protein folding or assembly of multisubunit proteins been examined. Calnexin The membrane bound protein chaperone calnexin associates with heavy chain proteins (Hochstenbach etal, 1992; Grupp etal, 1995). As previously described, calnexin binds both N-linked glycans and the transmembrane regions of particular proteins. Alterations in the membrane spanning region of u heavy chain that cause it to be abnormally expressed at the cell surface (Shaw etal, 1990; Stevens etal, 1994) may be correlated with a decrease in calnexin binding (Grupp et al, 1995). This suggests calnexin may play a role in the normal retention of incompletely assembled BCR molecules by interacting with the membrane spanning region of u heavy chain. However, the importance of this interaction is unclear since cells treated with a 11 glycosylation inhibitor, castanospermin, still express BCR complexes on their cell surface despite an apparent absence of calnexin binding (Wu et al, 1997). u heavy chain transmembrane: a target site for quality control u heavy chain has structural characteristics that suggest its transmembrane region may be important for proteimprotein interactions. The -78 kDa a heavy chain protein has a large ectodomain, a very short cytoplasmic domain of only 3 aa and a putative 26 aa transmembrane domain that is postulated to be an a-helix (Roger and Wall, 1984). There is a high degree of amino acid sequence conservation in the transmembrane region both among the various immunoglobulin isotypes and between u heavy chain isotypes of may different species (Rabbits etal, 1981; Word etal, 1983; Bernstein etal, 1984; Kokabu etal, 1988; Dorain and Gillies, 1989). Interestingly, this sequence conservation is preferential to one side of the postulated a-helix (figure 3). Since there are no known specific sequence requirements to anchor a membrane protein in a lipid bilayer (Davis and Model, 1985), it is likely that the high level of conservation within the transmembrane region is functionally significant, perhaps playing a role in surface transport, receptor internalization, or interaction with signaling components. Additionally, many of the amino acids in the proposed transmembrane region contain hydroxyl groups (Roger and Wall, 1984). By altering two of these polar residues (tyrosine and serine) to two non-polar residues (valine and valine), mlgM is no longer retained in the absence of Iga/IgP (Shaw et al, 1990; Stevens et al, 1994; Grupp et al, 1995) suggesting these residues are targeted by protein chaperones. Since hydrophilic residues are not commonly exposed in the hydrophobic environment of a lipid bilayer, it is likely that these sites are normally masked by protein-protein interactions. Proteins that may interact at these sites could include the Iga/IgP heterodimer, the newly identified p32, p37, or p41 proteins (Terashima et al, 1994) or some other unidentified molecule. o 12 A) B) V 2 1 © 3 2 © 35.® \ „ A 2 8 © cytosol Figure 3 - Schematic of the u heavy chain transmembrane region. (A) The transmembrane region of u heavy chain drawn as an a-helix. The 13 t h residue of the m l domain of \i heavy chain is at the lumenal end of the transmembrane region and the 38 t h residue is at the cytosolic end. (B) The a -helix of the membrane spanning region is drawn as though flattened into a circle. Circled residues are identical in the transmembrane regions of at least 7 of the 8 murine heavy chain isotypes (Blum, 1992). The single amino acid code used is A, alanine; C, cysteine; D, aspartate; E, glutamate; F, phenylalanine; G , glycine; H , histidine; I, isoleucine; K, lysine; L , leucine; M , methionine; N , asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V , valine; W, tryptophan; Y, tyrosine. The purpose of this study is to examine the interactions between protein chaperones and the B cell antigen receptor. In particular, we are interested in determining which protein chaperones are associated with the different BCR components; whether binding of protein chaperones depends on the assembly state of the receptor; whether the membrane spanning region of u heavy chain is particularly important for binding protein chaperones; and whether binding of protein chaperones affects the stability of bound proteins. We hypothesize that: l)-protein chaperone binding will depend on the assembly intermediates expressed by a cell, 2)-mutations of u heavy chain that cause altered trafficking will result in a decrease in protein chaperone binding, and 3)-there will be a correlation between protein chaperone binding and protein stability. We plan to address these questions in a non-lymphoid cell line, AtT20, that has been used previously to study the BCR (Matsuuchi etal., 1992). Using this cell line, we will be able 13 to express individual BCR chains, wildtype and mutant, and determine whether a specific chain can interact with a particular protein chaperone. As well, it can be used to create different cell lines that express various combinations of the BCR and, therefore, contain different complements of BCR assembly intermediates. The long term goal of this project is understand the mechanisms that regulate the retention and assembly of protein complexes in the ER. Since the chaperone proteins that associate with the BCR subunits also associate with the subunits of many other protein complexes, it is likely that the mechanisms leading to ER retention of the B C R are also similar to those of other protein complexes. 14 Materials and Methods Materials Cell Lines The AtT20 mouse pituitary cell line (Sabol, 1980) was maintained in DMEM+high glucose (Stem Cell Technologies, Vancouver, BC) containing 500 units/ml penicillin, 500 mg/ml streptomycin, 2 mM L-glutamine and supplemented with 10% fetal bovine serum (Gibco B R L , Burlington, ON). Cells were maintained at 37°C in an atmosphere of 10% C 0 2 in a Forma Scientific model 3326 incubator (Marietta, OH). Transfected AtT20 cells were maintained under identical conditions and supplemented with 0.4 mg/ml Geneticin, G418 Sulfate (Gibco BRL) . The WEHI231 B lymphoma cell line (Warner et al, 1979) was maintained in RPMI 1640 (Stem Cell Technologies) containing 500 units/ml penicillin, 500 mg/ml streptomycin, 2 mM L-glutamine and supplemented with 10% fetal bovine serum (Intergen, Purchase, NY) and 50 uM (3-mercaptoethanol (Sigma, St. Louis, MO). WEHI231 cells were maintained at 37°C in an atmosphere of 5% CO2. Plasmids Mammalian expression vectors containing murine genes encoding membrane immunoglobulin a. heavy chain (pRSVumem#3), XI light chain (pRSVM-140), Igp (pLpA-B29PCR#l) and Iga (pCMV MB-1 gA+B) were as described previously (Matsuuchi et al, 1992). pRSVumem#3 contained a functionally rearranged 2.1 kb u. membrane cDNA clone (Bothwell et al, 1981). pRSVAl-140 contained a rearranged 4.6 kb XI light chain genomic clone (Bernard et al, 1978). pLpA-B29PCR#l contained a 1.2 kb cDNA clone of B29 (Matsuuchi et al, 1992). pCMVMB-lgA+B contained a genomic/cDNA chimera of mb-1 (Travis et al, 1991; Matsuuchi et al, 1992). The pRSV and pLpA expression vectors contained the Rous Sarcoma Virus (RSV) long terminal repeat (LTR) promoter/enhancer elements whereas the p C M V vector contained the cytomegalovirus promoter/enhancer element. Al l plasmids contained the SV40 15 poly A addition region and splice site. These particular combinations of RSV, C M V and SV40 regulatory elements produce high levels of protein expression in transfected AtT20 cells (Matsuuchi and Kelly, 1991). The Pu33 plasmid, an altered version of the Pu. plasmid (Grosschedl et al, 1984), was a generous gift from Dr. Jon Blum and Dr. Anthony DeFranco (University of California San Francisco, CA). It contains a functionally rearranged murine genomic u heavy chain gene with both the membrane and secreted exons. The i24V/d36T mutation was created in the membrane exon of the genomic Pu plasmid by in vitro mutagenesis using the oligonucleotides described in Table 1 (Blum, 1991). The mutation was created in the highly conserved transmembrane region to produce an altered u heavy chain gene with a valine inserted near the N-terminal side of the transmembrane region and a threonine deleted near the C-terminal side of the transmembrane region. The sP65um plasmid contains a functionally rearranged u membrane cDNA clone (Bothwell et al, 1981) in the EcoRI site of the sP65 plasmid (Promega, Madison, WI). Name "Position Residue New Residue Oligonucleotide Sequence Y 3 1 V / S 3 2 V 31/32 tyr/ser val/val 5 '-CTGAGCCTCTTCGTG GTCACCACCGTC ACC-3' i 2 4 V / d 3 6 T 24/36 no residue val 5 '-ACCTTC ATCGTCG TACTCTTCCTCCTG-3' thr deletion 5'-CAGCACCACCGT C CTGTTCAAGGTAG-3' Table 1 - Oligonucleotides used to create site-directed mutations in the genomic u heavy chain gene (taken from Blum, 1991). "The position of the residue is relative to the first amino acid of the C-terminal portion of u heavy chain that is unique to the membrane form of u heavy chain. 16 Drug resistance was conferred to transfected cell lines by plasmids coding for G418 resistance (pSV2neo). Resistance is acquired by expression of an aminoglycoside phosphotransferase gene in pSV2neo which deactivates G418 by phosphorylation (Southern and Berg, 1982; Gritz and Davies, 1983; Palmer et al, 1987), preventing it from interfering with protein synthesis and causing cell death. As well, all plasmids contained the bacterial gene p-lactamase gene which provides ampicillin resistance to the transformed bacteria. Antibodies Polyclonal rabbit anti-mouse IgM antibodies (anti-u), u heavy chain specific, were from either Jackson Immunochemicals, distributed by BioCan (Mississauga, ON), or ICN (Mississauga, ON). Polyclonal rabbit anti-mouse X light chain antibody (anti-X), raised against purified mouse X light chain, was from Bethyl Laboratories, distributed by Cedarlane Laboratories (Hornby, ON). Polyclonal rabbit anti-mouse Iga antibody (anti-Igoc), specific for a 34 amino acid peptide from the carboxy terminal region of Iga (amino acids 187-220), was as previously described (Gold et al, 1991). Polyclonal rabbit anti-mouse IgP antibodies (anti-IgP), specific for a 24 amino acid peptide from the amino terminal region of Igp (amino acids 76-96), were as described later in the Methods section. Polyclonal rabbit anti-rat GRP78 (BiP) antibody (anti-BiP), specific for a 7 amino acid peptide from the carboxy terminal region of GRP78, was from StressGen (Victoria, BC). Monoclonal rat anti-chicken GRP94 antibody (anti-GRP94), raised against purified chicken GRP94, was from StressGen. Polyclonal rabbit anti-canine calnexin antibody (anti-calnexin), specific for an 18 amino acid peptide from the carboxy terminal region of canine calnexin, was from StressGen. Polyclonal goat anti-mouse IgM antibody linked to fluorescein isothiocyanate (anti-u:FITC), |x heavy chain specific, was from Jackson Immunochemicals. Polyclonal goat anti-mouse IgM antibody linked to biotin (anti-u:biotin), p heavy chain specific, was from Jackson Immunochemicals. Polyclonal rabbit anti-mouse X light chain antibody linked to biotin (anti-A,:biotin), X light chain specific, was from 17 Cortex Biochem (San Leandro, CA). Polyclonal goat anti-rat IgG linked to horseradish peroxidase (anti-IgG:HRP), specific for H and L chains, was from ICN. Polyclonal rabbit anti-mouse actin (anti-actin) was from ICN. Rabbit non-immune serum (NIS-rabbit) and rat non-immune serum (NIS-rat) were from Jackson Immunochemicals. Strepavidin linked to fluorescein isothiocyanate (SA:FITC), biotin specific, was from Amersham Life Science (Oakville, ON). Protein A linked to horseradish peroxidase (proteinA:HRP) was from Amersham Life Science. Protein A and Protein G coupled to Sepharose CL-4B (proteinA:seph and proteinG:seph) were from Sigma. Enzymes Restriction endonucleases BsmI, Xhol , Bgl l l , Hindlll and Sail were all from New England Biolabs (Mississauga, ON). DNA ligase was from Boehringer Manheim (Laval, QU). Endoglycosidase H (Endo-H) and N-glycosidase F (PNGaseF) were also from Boehringer Manheim. Oligonucleotides Oligonucleotides were used for DNA sequencing. Oligonucleotides outlined in table 2 were synthesized by the Nucleic Acid and Protein Synthesis unit at the University of British Columbia (Vancouver, BC). Oligonucleotides were synthesized on an Applied Biosystems (ABI) machine using a standard protocol. Name Site Type Sequence C M U M 1 S ml exon sense 5' GTGAATGCTGAGGAGG AA-3' C H 4 S CH4 exon sense 5' • ACAGCATCCTGACTGTGA-3' C H 4 I S CH4-ml intron sense 5' -GACCAGTCAATACTCGCT-3' Table 2 - Oligonucleotides used for sequencing membrane region of constructed pLpA|i33m plasmid. 18 Methods: Tissue Culture Adherent AtT20 cells were grown in 10 cm tissue culture dishes (Falcon, Franklin Lakes, NJ) distributed by V W R Scientific (Edmonton, AB) . Tissue culture media was replaced every 3-4 days. Cells were grown to 90% confluency before being moved to new dishes. Cells were removed from tissue culture dishes by rinsing with 10 ml PBS and then adding 1 ml of 0.25% trypsin (Gibco BRL) and letting stand for 5 min. Trypsinized cells were lifted off the surface of the tissue culture dishes by adding 9 ml of tissue culture media. Cells were split 1:5 into new dishes containing 8 ml of tissue culture media. Non-adherent WEHI231 cells were also grown in 10 cm tissue culture dishes. WEHI231 cells were split 1:5 every 3-4 days by pipetting cells into new dishes containing 8 ml of tissue culture media. Tissue culture cells were frozen in liquid nitrogen for long-term storage. Adherent cells were removed from tissue culture plates with 1 ml trypsin whereas WEHI231 cells were removed from plates by the pressure of pipetting media up and down on plates. Cells were placed in Falcon 2097 conical polypropylene tubes and concentrated by centrifugation at 1500 r.p.m. for 5 min in an EEC Centra-8R centrifuge that was cooled to 4°C. Media was removed from the tubes and the cells were then suspended in normal media containing 10% dimethyl sulphoxide, DMSO (Sigma). Suspended cells were placed in 2 ml Nalgene cryogenic vials (Rochester, NY) and cooled on ice for 30 min. Vials were then frozen at -70°C for at least 24 hr prior to storage in liquid nitrogen. When cells were removed from liquid nitrogen, they were warmed rapidly in a 37°C waterbath. Cells were removed from cryogenic vials and placed in Falcon 2097 tubes containing 10 ml media. Tubes were centrifuged at 1500 r.p.m. for 5 min in an TECCentra-8R centrifuge which was cooled to 4°C to remove all DMSO. Media was removed from the tubes and the cells were then suspended in normal media and incubated at 37 "C for growth at 10% C 0 2 . 19 Construction ofpLpAfim Expression Vectors The membrane exons of genomic Pu33 were moved into pRSVumefn#3 cDNA to: l)-achieve high levels of protein expression in transfected AtT20 cells and 2)-eliminate coexpression of secreted u heavy chain which is also coded for in the Pu33 plasmid. Figure 4 outlines the steps followed to construct the new vector, pRSVu33m. The genomic Pu clone contains exons that code for both the membrane and secreted tails of u heavy chain. The membrane portion of u heavy chain is coded for by two exons, m l and m2. Both these exons were removed from the Pu33 plasmid to transfer to the altered um DNA sequence into a more appropriate expression vector. The membrane exons were removed from the genomic clone by digesting the Pu33 plasmid with BsmI and Xhol; BsmI cuts the gene at the 5' end of the ml exon and Xhol cuts 3' to the m2 exon. The 1.2 Kb BsmJTXhoI fragment was ligated into the intermediate vector, sP65um. The fragment was ligated into the um cDNA using compatible BsmI and Sail restriction sites producing a cDNA/genomic chimera. A 2.3 Kb Bglll /Hindll l fragment of the cDNA/genomic chimera in sP65u33m, containing the u CHI to m2 exons, was placed in the pRSVumem#3 plasmid to produce pRSVu33m. Plasmids were transformed into either DH5a or HB101 (Gibco BRL) competent bacteria. Recombinant plasmids were selected for by resistance to ampicillin. Plasmids were screened initially by restriction mapping. To confirm the pRSVu33m plasmid contained the correct fragment of the original Pu33 plasmid, the region of D N A containing the i24V/d36T mutation was sequenced and compared to both the published sequence of genomic u heavy chain (Rogers et al., 1980) and the mutated sequence of the Pu33 plasmid (Blum, 1991). Sequencing was carried out by the NAPS unit using an Applied Biosystems Model 373 Stretch sequencing machine. DNA Preparations Mini-lysate preps were used for rapid screening of recombinant plasmids. DH5oc bacteria were cultured in 2-3 ml of LBroth containing 50 ug/ml of ampicillin. Preparation of bacterial coded plasmid D N A was carried out using the "Focus quick mini-lysate prep" protocol (Morelle, 20 A VDJ Figure 4 - Construction of the pLpA(i33m plasmid. The p membrane alteration contained in the P|i33 plasmid was moved to the pLpA expression vector using two steps. (A) First, the 1.2 Kb Bsml/Xhol fragment containing the mutation was isolated and then ligated to a 4.9 Kb Bsml/Sall fragment of from the Sp65um plasmid to produce Sp65u33m. (B) Second, a 2.2 Kb fragment was isolated from the new Sp65u33m plasmid and ligated into a 5.2 Kb fragment from the pLpAu33c plasmid to produce pLpA|j33m. (C) The membrane spanning region of pLpAu33m was sequenced to confirm it contained the membrane exons but not secreted exon. Purified fragments from each step are illustrated to the right of the diagnostic gels. The molecular weight standards are indicated to the left of each gel. The asterisk (*) indicates the location of a very faint band in the Ffindlll lanes that did not scan well. 21 Hind III 22 1989). The protocol was revised to extend centrifugation time of the RNA/genomic DNA precipitate to 10 min. Recombinant plasmids from the mini-lysate preps were digested at 37°C, for 2-4 hr, using 5-10 units of various endonucleases (Boehringer Manheim; Gibco B R L ; New England Biolabs). Samples were then electrophoresed on 1% agarose gels, containing 25 rig/ul ethidium bromide (Sigma), to separate DNA fragments. Gels were illuminated under ultraviolet conditions, causing fluorescence of the D N A bound ethidium bromide, and Polaroid pictures were taken. Large quantities of purified plasmid D N A were prepared using a "Cleared Lysate" protocol (Clewell and Helinski, 1972) with revisions by Matsuuchi (unpublished, 1987). Bacterial cultures of 40 ml LBroth, containing 50 mg/ml ampicillin, were used to inoculate growth media containing: 1L M-9 salts, 1 ml 100 mM CaCl 2 , 1 ml 1M MgS0 4 , 20 ml 20% glucose, 1 ml 4% proline, 1 ml 4% threonine, 4 ml 1% leucine and 20 ml 20% casamino acids. HB101 bacterial cultures were supplemented with 170 mg chloramphenicol at O . D . 6 0 0 0.6-0.7 to promote plasmid amplification by inhibiting replication of the bacterial genome, whereas DH5a bacteria were grown overnight without amplification since chloramphenicol is toxic to DH5oc bacteria. Bacteria was cultured overnight at 37°C with shaking to allow for aeration. Bacteria were pelleted and lysed by freezing and thawing followed by the addition of lysozyme and 10% Triton X-100, all of which act to break down the cell wall and cell membrane. Lysates were pelleted, removing most of the genomic D N A and high molecular weight protein aggregates, and the supernatant was adjusted to a final concentration of 10 mM Tris (pH 8.0), 1 mM EDTA and 1.6 g/ml CsCl. Samples were then centrifuged, in the presence of ethidium bromide (0.1 mg/ml), at 45000 r.p.m. on a 60Ti fixed angle rotor for 48 hr in a Beckman L8-80 ultracentrifuge. Plasmid DNA, indicated by the EtBr containing bands, was removed from the gradient, extracted with CsCl saturated Butanol and dialysed extensively against 6 L TE (10 mM Tris, pH8.0; 1 mM EDTA) over two to three days at 4°C. D N A was then precipitated with ethanol, centrifuged, and resuspended in 1 ml TE. O . D . 2 6 0 / 2 g 0 was measured to determine the 23 yield, where 1 OD=50ug/ml dsDNA. Sterility of prepared DNA was ensured by addition of 200 ul of chloroform per 1 ml D N A solution. DNA Transfections Plasmid D N A containing various genes of interest were transfected into AtT20 cells by a standard CaP0 4 precipitation method (Moore et al, 1983; Burgess et al, 1987). Subconfluent 10 cm plates containing ~5 x 106 cells were transfected with a total of 100 ug of plasmid DNA (combinations of expression vectors containing ji, XI, B29, and mb-1) and 20 ug of a selectable drug marker plasmid DNA (pSV2neo) mixed together as a calcium phosphate precipitate. Stable drug resistant clones were selected based on their resistance to 0.4 mg/ml Geneticin. Clones were picked from 10 cm dishes using 9 mm teflon rings (Fisher Scientific, Nepean, ON). Teflon rings were rimmed with high vacuum grease (Dow Corning, Midland, MI) to seal the ring to the tissue culture dish. Trypsin was added to the inner part of the ring that also contained the desired clone. Clones were removed from the teflon ring by adding tissue culture media, pipetting up and down several times and pipetting into wells of a 24 well tissue culture dish (Falcon #3040). Clones were grown up from 24 well dishes to 12 (Falcon #3043) well dishes, 6 well dishes (Falcon #3046) and finally 10 cm plates (Falcon #3003). Drug resistant clones were screened for expression of BCR proteins from transfected plasmids by immunofluorescence and western blotting. Anti-peptide Antibodies Rabbit polyclonal antibodies were raised against peptides corresponding to defined regions of Iga and Igp. Peptides were synthesized by Ian Clark-Lewis at the Biomedical Research Centre, U B C , Vancouver, BC. The peptides are listed below in table 2. They were designed to represent either the cytoplasmic or lumenal portions of Iga and Igp. Peptides were coupled to keyhole limpet hemocyanin (KLH). Antigens were emulsified in complete Freund's adjuvant (Difco Laboratories, Detroit, MI) for initial injections, for subsequent injections antigens were 24 emulsified in incomplete Freund's adjuvant (Difco Laboratories). Rabbits SF1, SF5 and SF11 were injected with the IgP-1 peptide, rabbits SF2 and SF4 were injected with the IgP-2 peptide and rabbits SF3, SF6, SF9 and SF10 were injected with the Iga peptide. Al l injections were done intra-muscularly (I.M.) and were carried out at 1-2 month intervals. 7-18 days following injections, approximately 10 ml of blood was taken from each rabbit via its ear vein. Blood was stirred and kept at 4°C overnight to promote clotting. Blood serum was removed the next morning and stored at -80°C. Name From Peptide Sequence Igp-1 Igp-2 Iga C-terminus IgP N-terminus IgP N-terminus Iga D K D D G K A G M E E D H T Y E G L N I G G C F R K R G S Q Q P Q E L V S E E G R I V Q G G C P P V P L G P G Q G T T Q G G C Table 3 - Synthetic peptides used as antigens for synthesis of anti-Iga and anti-Igp antibodies. Cell Extracts Detergent extracts were prepared for all experiments in which cellular proteins were being examined. Cells were lysed with either cold M G lysis buffer (1% triton x-100, 2 mM EDTA, 10% glycerol, 137 m M NaCl, 20 mM Tris pH8.0) or with C L B (1% Triton X-100, 1.26 mM MgCl 2 , 1 mM CaCl 2 in phosphate buffered saline). Both lysis buffers contained the following concentrations of protease inhibitors to prevent protein digestion: 1 mM pepstatin A , 1 mM aprotinin, 1 mM PMSF and 1 mM leupeptin (Sigma). Nuclei were removed by centrifugation at 14000 r.p.m. for 10 min in an Eppendorf 5415C centrifuge. 0.3% SDS and 0.4% DOC were added to lysates prepared with M G lysis buffer once nuclei were removed from the lysates. B C A protein assays (Pierce, Rockford, IL) were carried out to determine the protein concentration of each sample. 25 Western Blots Western blots were used to detect the presence of specific proteins in whole cell extracts and immune complexes. For example, western blots were used to determine whether non-lymphoid cells were expressing transfected BCR chains. Lysates were prepared as previously described using M G lysis buffer. Samples were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using standard protocols (Laemmli, 1970). Proteins were separated on a mini-gel apparatus (CBS Scientific, Del Mar, CA) using polyacrylamide gels ranging in polyacrylamide concentration from 5-12%. Gels were loaded with 5-15 ug protein per lane and run at constant amperage (25 mA). Proteins contained in gels were transferred to nitrocellulose for 1 hour at constant voltage (70V) in a Transblotter (BioRad, Richmond, CA) . Filters were then blocked overnight at 4°C in tris-buffered saline (TBS) containing 5% bovine serum albumin (Boehringher Manheim). Filters were washed with tris-buffered saline containing 0.05% tween20 (TBST) four times, each time for 10 min. Filters were incubated with the primary antibody, diluted in TBST, for 1.5 hr and then washed in TBST again as described above. Filters were then incubated with either proteinA:HRP or anti-IgG:HRP (both diluted to 1:10 000 in TBST) for 30 min and washed in TBST as described above. Probed filters were developed by incubating in enhanced chemilumenescence (ECL) reagents for 1 min and exposing them to Hyperfilm-MP autoradiography film (Amersham Life Science) for a variety of lengths of time. The ECL reagent contains a reactive chemical, luminol, which produces light following oxidation by HRP and H 2 0 2 in the presence of phenol (Amersham Life Science Publication, 1993). Re-probed filters were first stripped by incubating in pH 2.5 TBS for 15 min with rotation. Stripped filters were washed twice in pH 7.5 TBS then blocked overnight in TBS containing 0.05% tween20 (TBST) and 5% bovine serum albumin. Filters were then probed as described above. 26 Multimer Assays Multimer assays were carried out to determine which assembly intermediates of the B C R were present in transfected AtT20 cell lines; this protocol detects only disulphide bonded proteins. Cell extracts were prepared with M G lysis buffer as previously described. 100 ul of 2X non-reducing phosphate sample buffer (0.003% bromophenol blue, 2% SDS, 20% sucrose) was added to 100 ul of cell extract. Samples were warmed at 37°C for 15 min. Urea was added to 8 M then samples were warmed further at 37°C until all urea had dissolved. Samples were then boiled for 5 min to dissociate any complexes. Proteins were separated by SDS-PAGE on a large gel apparatus (CBS Scientific) using phosphate gels containing 5% polyacrylamide (Melnick and Argon, 1995). Gels were loaded with 25 ug protein per lane (each lane was 6 mm wide, 1 mm deep and 10 mm high) and run at constant amperage (60 mA) for 3.5 hr. Proteins contained in gels were transferred to nitrocellulose overnight at constant voltage (10V) in a TE series Transphor electrophoresis unit (Hoefer Scientific Instruments, San Francisco, CA). Filters were blocked for 24 hr at 4°C in TBS containing 5% BSA. Filters were probed with anti-u, anti-A, or anti-Igoc antibodies and developed exactly as described above. Chaperone Association Assays Co-immunoprecipitation experiments were carried out to determine whether BCR proteins were associated with chaperone proteins. Cells extracts were prepared using C L B as previously described. Cell extracts containing either 2 mg of protein from transfected AtT20 cells or 1 mg from WEHI231 cells were immunoprecipitated with 5 ug of either anti-BiP, anti-GRP94 or anti-calnexin. Immunoprecipitations were done at 4°C for 20 hr with continuous rocking. Immune complexes were recovered by incubating samples with 50 ul of either 50% proteinA:seph or 50% proteinG:seph at room temperature for 30 min while rocking. The entire sample was then layered on top of, and centrifuged through, a sucrose pad (30% sucrose, 1/2 CLB) . The pellet was washed once with 1 ml of CLB and then once with 1 ml of water to remove proteins non-specifically bound to the immune complexes. Precipitated protein 27 complexes were dissociated by boiling the samples with 45 ul reducing sample buffer (RSB) containing 5% SDS, 10% glycerol, 5% (3-mercaptoethanol, 62.5 mM tris-acetate (pH 6.8) and .001% bromophenol blue. Proteins were separated by SDS-PAGE on gels containing 10-12% polyacrylamide. Western blots were carried out with anti-BCR chain antibodies as described above. BCR Chain Stability Assays Pulse-chase experiments were used to monitor the stability of B C R proteins in the transfected AtT20 cell lines. Cells were grown to near confluence in 6 well dishes containing ~5xl0 5 cells/well. Cells were starved in labeling media depleted of methionine and cystine (DMEM+high glucose, 500 units/ml penicillin, 500 mg/ml streptomycin, glutamine, no cystine, no methionine). Each well of starved cells was labeled for 15 min with 1 ml of labeling media containing -150 uCi 35S-translabel (ICN) and pre-warmed to 37°C. Labeled cells were lysed with M G L B , as described previously, and immunoprecipitated overnight at 4°C with 10 ul antibody; this amount of antibody was determined to be in excess of the amount required to immunoprecipitate all of the target BCR proteins. Immunoprecipitates were incubated with 50 pi of 50% protein A: seph at room temperature for 30 min. The entire sample was then layered on top of, and centrifuged through, a sucrose pad (30% sucrose, 1/2 M G L B , 0.15% SDS, 0.2% DOC), and subsequently washed in M G L B to remove proteins non-specifically bound to the protein/antibody/proteinA:seph complexes, hrrmune complexes were dissociated by boiling the samples in 25 ul of RSB. Proteins were separated by SDS-PAGE on gels containing 10-12% polyacrylamide. Gels were fixed and stained in Coomassie Blue stain (0.1% coomasie blue, 50% methanol, 10% acetic acid). Gels being exposed to autoradiography film were treated with 0.5M sodium salicylate and 10% methanol for 20 min at room temperature, for autoradiographic enhancement, dried and exposed to Hyperfilm-MP autoradiography film using standard procedures (Harlow and Lane, 1988). Gels being quantified were dried and exposed for a 72 hr period to a BioRad cassette then imaged using a 28 Molecular Dynamics model PSI-MAC phosphorimager (Sunnyvale, CA) . Captured images were quantified using Molecular Dynamics ImageQuant version 1.1 software. Numerical values were translated into the percentage of radioactivity present relative to the t=0 and then plotted on bar graphs using Cricket Graph software version 1.3.1 (Cricket Software, Malvern, PA). Immunofluorescence Cells were stained for the presence of |x heavy chain by indirect immunofluorescence as described previously (Stevens etal, 1994). Glass coverslips were coated with poly-D-Lysine (Sigma) as previously described (Matsuuchi, 1988). Transfected colonies were grown to semiconfluency in 24 well dishes on Poly-D-Lysine coated coverslips. Cells were rinsed in phosphate buffered saline (PBS) then fixed in 3% pH7.5 paraformaldehyde (BDH, Toronto, ON) for 20 min at room temperature. Cells were then rinsed in either PBS/20 mM glycine for surface staining or permeabilized in PBS/20 mM glycine/0.1% saponin for internal staining. Fixed cells were then incubated with 12 ul of anti-u:biotin (1:50) for 1 hour, at room temperature, followed by SA:FITC (1:100) for 30 min. Coverslips containing anti-u-stained cells were rinsed and placed on slides containing mounting media (Johnson et al., 1982) consisting of 90% glycerol, 10% PBS and 2.5% l,4-diazobicyclo-[2.2.2]-octane (DABCO) from Sigma. Glass coverslips were then sealed to the slide with nailpolish and allowed to dry in the dark for 30 min. Samples were viewed using a Kodak Optiphot-2 microscope attached to a BioRad MRC600 confocal laser scanning system (CLSM) equipped for epifluorescence and confocal microscopy. Images were captured digitally, cropped in Adobe Photoshop and printed from a Codonics NP-1600 dye-sublimation printer. 29 Results Synthesis of anti-peptide antibodies To characterize the interactions between the BCR chains and protein chaperones, we first needed to acquire reagents against the various BCR chains. While there are commercially available reagents against u heavy chain and X light chain, there are no commercial reagents available against the ectodomain of murine Iga or against murine Igp. In several attempts to produce antibodies that reacted with peptides resembling Iga and Igp ectodomains, no rabbits produced serum that reacted against Iga peptides (table 4). Detection of Iga was thus determined using a previously characterized antibody that is specific for the cytoplasmic tail of murine Iga (Matsuuchi et al., 1992). One rabbit, SF2, did produce serum that reacted against a peptide resembling the cytoplasmic tail of IgP (table 5). SF2 rabbit serum can be inhibited from binding IgP by the immunogenic peptide demonstrating the interaction is specific (data not shown). Interestingly, the antibody reacts well against IgP in lymphoid cells but not as well against IgP present in transfected non-lymphoid cells (figure 5). Characterization of the serum suggests the greatest response against IgP is from bleeds following injections of at least 0.3 mg of conjugated peptide; serum from bleeds following injections of less peptide did not maintain a high response over time (table 5). Due to the lack of a suitable anti-Igp antibody that could detect IgP in non-lymphoid cells, we were unable to examine the interactions between IgP and protein chaperones. Antibody Control Experiments Control experiments were carried out to ensure that the antibodies used for both immunoprecipitation and Western blot experiments were binding to the expected target proteins. Western blot analysis of a non-lymphoid cell line that expressed all four B C R subunits (u, X, Iga, Igp) indicated that, relative to anti-u and anti-GRP94 controls, only low levels of background were present in Western blots using both rabbit serum and rat Igy2a 30 RABBIT PEPTIDE # BLEEDS RESPONSE? S F 1 Igp-l 11 NO S F 2 Igp-2 20 YES S F 3 Iga 10 NO S F 4 IgB-2 18 NO S F 5 Igp-l 11 NO S F 6 Iga 8 NO S F 9 Iga 7 NO S F 1 0 Iga 7 NO S F 1 1 Igp-l 7 NO Table 4 - Summary of anti-peptide antibodies. Rabbits SF1-SF6 and SF9-SF11 were injected with 0.1 - 1.0 mg of antigen that consisted of synthetic peptides that were covalently linked to K L H . Conjugated peptides included Iga, representing an N-terminal region of Iga; IgP-1 representing a C-terminal region of IgP; and IgP-2 representing an N-terminal region of IgP (for peptide sequence see Materials and Methods section). Injections were given at 1-2 month intervals. Rabbits were bled approximately 14 days following each injection. Rabbit bleeds were screened for reactivity against either Iga or IgP using lymphoid (WEHI231) and non-lymphoid (R98, ASS) extracts. 31 Name Date Amount Date Signal Injected Injected Bled Strength S F 2 . P B — — 6/14/93 -S F 2 . 1 8/27/93 1 mg 9/8/93 +/-S F 2 . 2 9/24/93 0.8 mg 10/7/93 -S F 2 . 3 10/22/93 0.8 mg 11/5/93 + S F 2 . 4 11/18/93 0.8 mg 12/3/93 + S F 2 . 5 12/17/93 0.8 mg 12/29/93 ++++ S F 2 . 6 1/28/94 0.3 mg 2/11/94 ++++ S F 2 . 7 3/11/94 0.3 mg 3/19/94 ++++ S F 2 . 8 4/13/94 0.1 mg 4/18/94 ++++ S F 2 . 9 — — 4/27/94 ++++ S F 2 . 1 0 5/11/94 0.1 mg 5/28/94 ++ S F 2 . 1 1 — — 6/28/94 + S F 2 . 1 2 7/11/94 0.1 mg 8/4/94 + S F 2 . 1 3 8/12/94 0.1 mg 8/26/94 ++ S F 2 . 1 4 9/16/94 0.1 mg 9/26/94 ++ S F 2 . 1 5 — — 11/8/94 ++ S F 2 . 1 6 11/16/94 0.1 mg 12/10/94 + S F 2 . 1 7 1/16/95 0.8 mg 1/27/95 ++ S F 2 . 1 8 2/22/95 0.8 mg 3/9/95 +++ S F 2 . 1 9 — — 5/15/95 + Table 5 - Summary of anti-IgP antibodies produced by rabbit S F 2 . The SF2 rabbit was injected with the IgP-2 peptide that was covalently linked to K L H . 0.5 - 1.0 mg of antigen was injected at 1-2 month intervals. Bleeds were taken approximately 14 days following injections. Antibodies were screened by western blotting lymphoid (WEHI231) extracts and looking for reactivity against Igp. Relative intensities of the IgP signal are indicated by (-) and (+), indicating absence or presence of reactivity. 32 A / ^ / i ~~II ~~ir PNGaseF: 1 - + " - + " - + M r 69 -43 -^-glycosylated 29 - M | | -<-deglycosylated 18 -anti-Igp (SF2.B16) Figure 5 - Screen of anti-lgp SF2.B16 antibody. 10 ug of cell extracts from IgB+ lymphoid cells (WEHI231), or 25 ug Igp+ non-lymphoid cells (R98) and IgP" non-lymphoid cells (ASS) were prepared with M G lysis buffer as described in the Materials and Methods. PNGase F treated extracts (+) and untreated extracts (-) were separated by SDS-PAGE using 12% polyacrylamide gels. Proteins were transferred to nitrocellulose and filters were probed with SF2 immune serum (SF2.B16). Molecular weight standards are indicated to the left of each filter. The arrows to the right of each filter indicate IgP before (glycosylated) and after (deglycosylated) PNGaseF treatment. 33 . # # B > ^ ° ^ 0 ^ 0 ^ < ^ M r 71 I ^ j " GRP94 4 8 _ 4 8 -28 -28 -19 -Figure 6 - Antibody controls for Western blot analysis and immunoprecipitations. (A) Cell extracts of transfected non-lymphoid cells (u+,A.+,Iga+,IgP+) were separated by SDS-PAGE and transferred to nitrocellulose for Western blot analysis. Each lane was probed separately with one of the following: PA:HRP alone; NRS then PA:HRP; anti-u then PA:HRP; anti-IgG:HRP alone; Rat IgGy 2 a then anti-IgG:HRP; anti-GRP94 then anti-IgG:HRP . (B) Transfected non-lymphoid cells (u+,X+,Iga+,IgP+) were labelled with 35S-translabel for 2 hr. Labelled proteins were immunoprecipitated with either PA:seph alone or anti-u followed by PA:seph and then separated by SDS-PAGE and examined by autoradiography. Molecular weight standards are indicated to the left of each filter. The proteins being examined are indicated by the arrow to the right of each filter. 34 antibodies (figure 6A). Similarly, coimmunoprecipitation experiments carried out on the same cell line also contained only low levels of background binding relative to the anti-p control (figure 6B). Establishing a system to examine BCR:protein chaperone interactions The main goal of our project was to establish a system in which we could examine interactions between protein chaperones and the various BCR proteins. Previous work on the association of B C R proteins with protein chaperones has not examined associations with Iga or Igp. To determine whether Iga or IgP interact with protein chaperones we immunoprecipitated the protein chaperones and western blotted for specific BCR chains. It appears Iga interacts with BiP and calnexin (figure 7B). Unfortunately, the IgP result is ambiguous due to the presence of the heavy chain proteins from the immunoprecipitating antibody that co-migrate with IgP (figure 7C). We confirmed that this approach would give us results consistent with published data (Haas and Wabl, 1983; Bole et al, 1986; Melnick et al, 1992) by demonstrating that B iP , GRP94 and calnexin interact with u heavy chain under our conditions (figure 7A). To further examine the interactions between BCR chains, assembling B C R complexes and protein chaperones, we established a system in a non-lymphoid cell line that lacked B cell specific proteins. The non-lymphoid cell line used, called AtT20, was from the mouse pituitary. These cells appear to express levels of protein chaperones comparable with the WEHI231 lymphoid control cell line (figure 8). AtT20 cells secrete large amounts of protein and, therefore, presumably possess the synthetic and secretory machinery necessary to produce and process large amounts of secretory products. AtT20 cells were co-transfected by CaP0 4 precipitation with an expression vector containing a gene that encoded a drug resistance marker and either one or several expression vectors that contained BCR genes. Clones were selected 35 M r 103-6 8 -4 4 -A 4 cf/ ll anti-Li B of* ^ M r 103-6 8 -4 4 -2 9 -H S s Iga anti-Iga M r 103-6 8 -4 4 -0> 4^ U N I •IgP 29-anti-Igp Figure 7 - Association of BCR chains with protein chaperones in lymphoid cells. Lymphoid cell (WEHI231) extracts were prepared with C L B as described in the Materials and Methods. Whole cell extracts (WXT) and extracts immunoprecipitated with either anti-BiP, anti-GRP94 or anti-calnexin antibodies were separated by SDS-PAGE on 10% polyacrylamide gels. Proteins were transferred to nitrocellulose. Filters were probed with (A) anti-u, (B) anti-Iga, or (C) anti-IgP followed by PA:HRP and then developed using E C L . Molecular weight standards are indicated to the left of each filter. The arrows to the right of each filter indicate the particular protein being probed. The asterisk (*) indicates the heavy chain from the immunoprecipitaring antibody (Note: the anti-GRP94 heavy chain is not detected by PA:HRP). 36 B 4> M , M r M r 108-7 9 -• BiP 108-7 9 -• GRP94 108-7 9 - I •Calnexin 4 7 -4 7 -4 7 -anti-BiP anti-GRP94 anti-calnexin Figure 8 - Expression of protein chaperones in lymphoid and non-lymphoid cells. Cell extracts from both lymphoid cells (WEHI231) and non-lymphoid cells (AtT20) were prepared with M G lysis buffer as described in the Materials and Methods. Proteins were separated by SDS-PAGE using 12% polyacrylamide gels and transferred to nitrocellulose. Filters were probed with (A) anti-BiP, (B) anti-GRP94, or (C) anti-calnexin and then with either PA:HRP (A and C) or anti-IgG:HRP (B). Filters were developed using E C L . Molecular weight standards are indicated to the left of each filter. The arrows to the right of each filter indicate the particular protein chaperone being probed. 37 A W > . „©• ib ^ M r 112 -69 -43 -B A /V\V/V . rt& 5h ^ ^ ^ ^ ^ s^, ^f> anti-A, anti- | i ^ ^ ^ ^ ^ ^ %cg< ^ D M r 112 -69 -43 - •Iga M r 43 -29 4 anti-Igfi •IgP •IgP 29 -anti-Iga Figure 9 - Expression of BCR proteins in transfected non-lymphoid cells. Cell extracts of both untransfected and transfected non-lymphoid cells (AtT20) were prepared using M G lysis buffer. Proteins were separated by SDS-PAGE using 10 or 12% polyacrylamide gels and transferred to nitrocellulose. Filters were probed with (A) anti-u, (B) anti-A., (C) anti-Iga or (D) anti-IgP followed by PA:HRP and then developed using E C L . Molecular weight standards are indicated to the left of each filter. The B C R chain of interest is indicated by the arrow to the right of each filter. 38 B A M r ^ ^ ^ 2 0 0 -112 * . 69 -43 -29 -anti-Li 2 L 2 2 HL H ^ ^ ^ ^ > ^ y y y > Non-Reduced Reduced IPPT anti-?t M r <^VV vff / ^ W 200 •Iga Non-Reduced Reduced IPPT anti-Iga Figure 10 - Oligomers present in transfected non-lymphoid ce l l s . (A) Cell extracts of transfected non-lymphoid cells were prepared using M G lysis buffer. Proteins were separated by SDS-PAGE, using 5% phosphate gels, and transferred to nitrocellulose. Filters were probed with anti-u followed by PA:HRP and then developed using ECL. (B,C) Transfected non-lymphoid cells were labelled with 35S-translabel for 2 hr then lysed with M G lysis buffer. Lysates were immunoprecipitated with either anti-A (B) or anti-Iga (C) followed by PA:seph. Immunoprecipitates were separated by SDS-PAGE under non-reducing and reducing conditions then exposed to film for autoradiography. Molecular weight standards are indicated to the left of each filter. The relative mobility of the different BCR oligomers are indicated by the arrows to the right of each image. 39 for their resistance to the drug Geneticin and then screened for the expression of the transfected B C R chains (figure 9, appendix 2). In particular, we established cell lines expressing either u heavy chain alone, X light chain alone, Iga alone, IgP alone or specific combinations of these proteins. It was important to determine which BCR assembly intermediates were present in the transfected cells because we believed they would influence binding of protein chaperones. Western blot analysis was carried out using 5% acrylamide gels to provide clearer resolution of higher molecular weight complexes. It is important to note that this approach only examines monomeric and disulfide bonded multimeric structures; non-disulfide linked multimeric structures cannot be determined using this approach. Cells expressing Iga alone contained predominantly monomeric Iga (figure IOC). In contrast, IgP homodimers are observed in transfected cells expressing high levels of Igp (Hourihane, personal communication). Consistent with published data, transfected cell lines expressing u heavy chain also expressed homodimers (Haas and Wabl, 1983; Bole et al, 1986). When u and X light chain were co-expressed, all possible H 2 L 2 intermediates were expressed with the exception of H 2 homodimers. X light chain multimers were difficult to detect despite using several different approaches. Since previous work has demonstrated that overexpressing B C R proteins in COS cells cause the expression of a number of protein chaperones to be upregulated (Lenny and Green, 1991), we were interested to see whether protein chaperones were also upregulated in transfected AtT20 cells. This was important because upregulation would suggest that the synthetic pathway was working at its maximum and may be overloaded. The effects of this overload could cause ambiguous results in experiments involving protein chaperones. Western blot analysis was used to determine whether AtT20 cells also upregulated expression of protein 40 M , B y <y <y <t > \#\#<^  M , 69 J 43 -, — M , 112 -69 J •Calnexin 43 - i Figure 11 - Expression of protein chaperones in transfected non-lymphoid cells. Cell extracts of both untransfected and transfected non-lymphoid cells (AtT20) were prepared using M G lysis buffer. Proteins were separated by SDS-PAGE, using 10% polyacrylamide gels, and transferred to nitrocellulose. Filters were probed with (A) anti-BiP, (B) anti-GRP94, or (C) anti-calnexin and then with either PA:HRP (A and C) or anti-IgG:HRP (B). Filters were developed using ECL. Molecular weight standards are indicated to the left of each filter. The protein chaperone of interest is indicated by the arrow to the right of each filter. 41 <V> ^ ^ . i B ^ >^ -' « ^ M , M, 112 -69 -112 -69 -43 - 43 - mm 29 - 29 -anti-p: anti-Li \^ \^  D \^ \ $ ^ > ^ ^ ^ ^ ^ M r M r 112 -jj2 69 — * * * * * * — — - —p— u 69 - — V t ^ " ^ 43 43 -29 -2 9 . anti-|l anti-(l Figure 12 - Association of u heavy chain with protein chaperones in transfected non-lymphoid cells. Cell extracts of transfected non-lymphoid cells were prepared using CLB lysis buffer. (A) Whole cell extracts and cell extracts immunoprecipitated with either (B) anti-BiP, (C) anti-GRP94 or (D) anti-calnexin antibodies were separated by SDS-PAGE, using 10% polyacrylamide gels, and transferred to nitrocellulose. Filters were probed with anti-u, followed by PA:HRP and then developed using ECL. Molecular weight standards are indicated to the left of each filter, u heavy chain is indicated by the arrows to the right of each filter. The asterisk (*) indicates the heavy chain from the immunoprecipitating antibody (Note: the anti-GRP94 heavy chain is not detected by PA:HRP). 42 A ib B ^ ^ ^ <y ^ M r 112 -" l * 43 -29 -18 -anti-A a n t i ~ X M r 112 -69 -43 -29 -^ ^ ^ ^^^^^ — anti-GRP94 D •GRP94 M r 112 -69 -43 -29 -18 ->^ •>> • W W anti-A Figure 13 - Association of X light chain with protein chaperones in transfected non-lymphoid cells. Cell extracts of transfected non-lymphoid cells (AtT20) were prepared using C L B lysis buffer. (A) Whole cell extracts and cell extracts immunoprecipitated with either (B) anti-BiP, (C) anti-X, or (D) anti-calnexin antibodies were separated by SDS-PAGE, using 10% polyacrylamide gels, and transferred to nitrocellulose. Filters were developed using ECL after they were probed with either anti-A. followed by PA:HRP (A,B,D) or anti-GRP94 followed by anti-IgG:HRP. Molecular weight standards are indicated to the left of each filter. X light chain and GRP94 are indicated by the arrows to the right their respective filters. The asterisk (*) indicates the heavy chain from the immunoprecipitating antibody (Note: the anti-GRP94 heavy chain is not detected by PA:HRP). 43 V V rtc> ^ V \$o M , B V <^  M, 68 44 Iga 29 -68 44 -29 -•Iga anti-Iga anti-Iga V V ^ \ f r M r anti-Iga V V A V ^ \^ M r 68 -44 -29 •Iga 68 44 -29 -Iga anti-Iga anti-Iga Figure 14 - Association of Iga with protein chaperone in transfected non-lymphoid cells. Cell extracts of transfected non-lymphoid cells (AtT20) were prepared using CLB lysis buffer. (A) Whole cell extracts and cell extracts immunoprecipitated with either (B) anti-BiP, (C) anti-GRP94 or (D) anti-calnexin antibodies were separated by SDS-PAGE, using 10% polyacrylamide gels, and transferred to nitrocellulose. Filters were probed with anti-Iga followed by PA:HRP and then developed using ECL. Molecular weight standards are indicated to the left of each filter. Iga is indicated by the arrows to the right of each filter. The asterisk (*) indicates the heavy chain from the immunoprecipitating antibody (Note: the anti-GRP94 heavy chain is not detected by PA:HRP). 44 chaperones following expression of transfected BCR genes. It appears that BiP, GRP94 and calnexin are not upregulated in AtT20 cells following the expression of transfected B C R genes (figure 11). Ponceau S staining indicated this was not a result of loading differences among lanes (data not shown). Examining interactions between specific BCR chains and protein chaperones in lymphoid cells expressing the intact receptor is problematic since the potential always exists that the interactions being observed are indirect via another BCR chain. In order to eliminate this problem, interactions between protein chaperones and specific BCR chains in transfected cell lines expressing defined subsets of the BCR chains were examined. As in the lymphoid cell line WEHI231 (figure 7A), transfected cells expressing u heavy chain alone have u heavy chain associated with immune complexes of BiP (figure 12B), GRP94 (figure 12C) and calnexin (figure 12D). BiP and, surprisingly, calnexin are also associated with X light chain in cells expressing X light chain alone (figure 13B,D). It is quite surprising that calnexin associates with X light chain since calnexin is proposed to bind only membrane proteins and glycosylated proteins; X light chain is neither. Immune complexes of X light chain were examined for the presence of GRP94, rather than the reverse, because the GRP94 monoclonal antibody contains X light chain. As with BiP and calnexin, GRP94 and X light chain were associated in transfected AtT20 cells that expressed X light chain alone (figure 13C). In contrast to findings in lymphoid cells, cells that express Iga alone contain small amounts of Iga in GRP94 immune complexes (figure 14C) but not immune complexes of BiP (figure 14B) or calnexin (figure 14D). IgP association with protein chaperones was not examined due to the lack of a reliable antibody that detects IgP by Western blot analysis. In the absence of all four receptor chains, incompletely assembled B C R components are retained inside the cell (Venkitaraman et al., 1991; Matsuuchi et al., 1992). This retention could be explained by a model in which the BCR complex assembles in an orderly fashion and 45 during assembly, interacting sequentially with different protein chaperones that hold the complex in place until it is ready to proceed to the next step of assembly. To determine whether protein chaperones interact specifically with different assembly intermediates of the BCR, we examined associations between protein chaperones and specific B C R chains in cell lines that expressed one or more transfected BCR chains. We hypothesized that protein chaperones interact differently with the different BCR assembly intermediates. If this is true, then immune complexes of protein chaperones should contain the assembly intermediates which they specifically target. Using transfected cell lines, we have tried to enrich for particular assembly intermediates by expressing different complements of BCR chains. We expect that if protein chaperones association is limited to specific assembly (referred to as 'stage-dependent' association), then immune complexes would be enriched for particular B C R chains in those cells. Figure 12B shows BiP associated with u heavy chain in a 'stage-dependent' fashion, but GRP94 (figure 12C) and calnexin did not (figure 12D). Association of u heavy chain with BiP immune complexes in transfected cells expressing u heavy chain alone disappears almost entirely in cell lines expressing both u, heavy chain and X light chain (although it is present in darker exposures). However, association reappeared in cells that overexpress p heavy chain despite the presence of X light chain (figure 12B). In contrast, the same protein chaperones did not interact with X light chain in a stage-dependent fashion (figure 13). The level of X associated with protein chaperones appears to correlate with the levels of u heavy chain being expressed by the different transfected cell lines (figure 13, figure 12A). GRP94 (figure 14C) interacted with Iga in a stage-dependent fashion, but BiP (figure 14B) and calnexin (figure 14D) did not. It appears that the coexpression of u heavy chain X light chain with Iga resulted in more Iga being associated with GRP94 than in cells expressing Iga alone (figure 14C). To determine whether binding of protein chaperones to BCR chains correlated with an alteration in the stability of the BCR chains, BCR chain stability was examined by pulse chase experiments. Pulse chase experiments indicate how quickly radioactively labeled proteins 46 A Time (hours) 0 2 4 6 8 10 M r m Time (hours) 0 2 4 6 8 10 Time (hours) 0 2 4 6 8 10 112 -69 - •u -43 -M,,A,Iga,Ig(3 (i,A,Iga Time (hours) 0 2 4 6 8 10 112 69 43 M1 B U 100 o 80 60 < 40 "3 20 0 0 2 4 6 8 10 Time (hours) iiXlga A V Figure 15 - Stability of u heavy chain in transfected non-lymphoid cells. (A) Stable transfectants expressing various BCR chains were pulsed with 35S-translabel and chased over a timecourse of 10 hr. Cell extracts were prepared using M G lysis buffer. Labeled extracts were immunoprecipitated with anti-u and separated by SDS-PAGE on 10% polyacrylamide gels. Gels were exposed to a phosporimager cassette and images were captured electronically. (B) u heavy chain bands were isolated and quantified. Graphs were normalized such that 100% of the protein is present at time=0. 47 IgaJgP Iga Time (hours) M r 0 2 4 6 8 10 43 - 1 V 29 - m 18 -43 29 -18 -43 - " 29 -18 -•I B U o 3 i 0 2 4 6 8 10 Time (hours) (iXlga (xX. X Figure 16 - Stability of X light chain in transfected non-lymphoid cells. (A) Stable transfectants expressing various BCR chains were pulsed with 35S-translabel and chased over a timecourse of 5 hr. Cell extracts were prepared using M G lysis buffer. Labeled extracts were immunoprecipitated with anti-A. and separated by SDS-PAGE on 12% polyacrylamide gels. Gels were exposed to a phosporimager cassette and images were captured electronically. (B) X light chain bands were isolated and quantified. Graphs were normalized such that 100% of the protein is present at time=0. 48 Iga,Igp M r _0_ 69 -43 -29 -69 -\iX 4 3 -Iga 29 -Time (hours) 1 2 3 4 5 •Iga •Iga B OJ o Pi 0 2 4 6 8 Time (hours) JiWgalgp uXlga Iga 69 -Iga 43 - •Iga 29 -Figure 17 - Stability of Iga in transfected non-lymphoid cells. (A) Stable transfectants expressing various BCR chains were pulsed with S-translabel and chased over a timecourse of 5 hr. Cell extracts were prepared using M G lysis buffer. Labeled extracts were immunoprecipitated with anti-Iga and separated by SDS-PAGE on 11% polyacrylamide gels. Gels were exposed to a phosporimager cassette and images were captured electronically. (B) Iga bands were isolated and quantified. Graphs were normalized such that 100% of the protein is present at time=0. 49 disappear over a timecourse. The disappearance of the radioactively labeled proteins is usally a result of degradation or secretion from the cell. We hypothesized that if protein chaperone binding had any effect on BCR chain stability, then we would see a correlation between the levels of protein chaperone association and the rate of protein degradation. Most noticeably, u heavy chain disappears significantly faster in cells expressing u heavy chain alone than in cells expressing at least u heavy chain and A. light chain (figure 15). This observation correlates with a high level of u heavy chain associated with BiP immune complexes (figure 12B). In contrast to uheavy chain, disappearance of both A, light chain (figure 16) and Iga (figure 17) appeared to be unaffected by the co-expression of other BCR chains. A mutant u heavy chain protein with an altered trafficking phenotype Previous work on u heavy chain indicates the membrane spanning region of the protein is particularly important for mediating its retention. To further examine the role of the transmembrane region of u heavy chain in mediating interactions with protein chaperones, we examined an altered u heavy chain, u33, which is reported to have an altered trafficking phenotype. The u33 mutant has a valine residue inserted near the top of the membrane spanning region and a threonine deleted near the bottom. In lymphoid 2PK-3 cells, u33 is reported to be on the cell surface but, presumably, not in association with the Iga/Igp heterodimer since it is incapable of signaling (Blum, 1991). We hypothesized that protein chaperones would bind less effectively to p33 if the membrane region was important for protein chaperone binding. To test this hypothesis, we first moved the gene encoding the u.33 into the pRSVumem#3 expression vector from the Pu33 expression vector (figure 4). This step was necessary both to achieve high levels of u33 expression levels in AtT20 cells and to eliminate the secreted tail present in the Pu33. Transfected cell lines were established that expressed either the u33 or u33 in the presence of A, light chain (figure 18) or A, light chain and Iga (not shown). We were unable to isolate a cell line that expressed all four B C R chains. Western blot analysis demonstrated that, as compared to the non-transfected control, these cell 50 lines did not have increased amounts of BiP (figure 19A), GRP94 (figure 19B) or calnexin (figure 19C). In contrast to a previous mutant u heavy chain examined by our lab (Stevens et al, 1994), u33 did not escape to the cell surface in the absence of Iga or IgP in non-lymphoid cells. Immunfluorescence studies indicated that the transfected cells expressed u33 intracellularly, but not at the cell surface (figure 20A). The intracellular location of u.33 was confirmed by sensitivity to the enzyme Endoglycosidase H , which suggests the altered protein was retained internally, presumably before the medial Golgi (figure 20B). The intracellular localization of u33 was comparable to that of wildtype u heavy chain expressed with X light chain but without the Iga/IgP heterodimer (figure 20). This suggests the alteration did not cause u33 to escape the quality control mechanisms of the AtT20 non-lymphoid cells. Examination of assembly intermediates present in u33 transfected cell lines indicates u33 formed assembly intermediates comparable to the unaltered protein (figure 21). To determine whether the altered u heavy chain protein associated differently with protein chaperones, we examined the association of u heavy chain with immune complexes of BiP (figure 22B), GRP94 (figure 22C) and calnexin (figure 22D). The altered u, heavy chain did not associate differently with any of these protein chaperones (figure 22, figure 12). Further, pulse chase experiments indicated there was no difference in the degradation rate of the altered u heavy chain protein as compared to the wildtype protein in comparable cell lines (figure 23). 51 M , <? <? <§r 112-6 9 -43-2 9 -anti-pi B M , ft? ft <? r # 6 9 -4 3 -2 9 - , 18-anti-X, Figure 18 - Expression of mutant u heavy chain and other BCR proteins in transfected non-lymphoid cells. Cell extracts were prepared from untransfected non-lymphoid cells and non-lymphoid cells transfected with mutant u heavy chain and various other BCR chains. Proteins were separated by SDS-PAGE using 10 or 12% polyacrylamide gels and transferred to nitrocellulose. Filters were probed with (A) anti-(i or (B) anti-X, followed by PA:HRP and then developed using ECL. Molecular weight standards are indicated to the left of each filter. The BCR chain of interest is indicated by the arrow to the right of each filter. 52 112 _ 69 - : B M r 112 _ 69- •GRP94 M r 112-69-r r # -Calnexin 43 -29 43-29-43-29. anti-BiP anti-GRP94 anti-calnexin Figure 19 - Expression of protein chaperones in u33 transfected non-lymphoid cells. Cell extracts were prepared from untransfected non-lymphoid cells and non-lymphoid cells transfected with various BCR chains using MG lysis buffer. Proteins were separated by SDS-PAGE, using 10% polyacrylamide gels, and transferred to nitrocellulose. Filters were probed with (A) anti-BiP, (B) anti-GRP94 or (C) anti-calnexin and then with either PA:HRP (A and C) or anti-IgG:HRP (B). Filters were developed using ECL. Molecular weight standards are indicated to the left of each filter. The arrows to the right of each filter indicate the particular protein chaperone being probed. 53 Igoc,Igp \i,X u33,A Untransfected Surface : f / Internal ^° > ft? ft E n d o H : ' - + 1 1 - + " - + ' M r Figure 2 0 - Altered u heavy chain is retained internally in transfected non-lymphoid cells. (A) The u heavy chain was localized in paraformaldehyde-fixed cells by incubating with anti-u:FITC for detection of surface expression or in cells permeabilized with 0.1% saponin for detection of internal p. Fluorescent images were captured using a confocal microscope and then cropped using image editing software. Bar = 20 uM for all panels. (B) Carbohydrate modification of u heavy chain was examined by EndoH digestion to determine its location in the secretory pathway. W X T were prepared using M G lysis buffer as previously described. EndoH treated (+) and untreated (-) samples were separated by SDS-PAGE, transferred to nitrocellulose and examined by western blot for the presence of u heavy chain. Molecular weight standards are indicated to the left of each filter. The arrows to the right of each filter indicate sensitive (us) and resistant (ur) u heavy chain. 54 M r 2 0 0 -9 9 -67 ^ ^ ^ « M * %Hf ' H2L2 • H 2 L H 2 •HL •H 4 4 -anti-(l Figure 21 - Oligomers present in non-lymphoid cells expressing an altered u heavy chain. M G lysis buffer was used to prepare cell extracts were prepared from non-lymphoid cells expressing either mutant u heavy chain alone or mutant u heavy chain with other BCR chains. Proteins were separated under non-reducing conditions by SDS-PAGE, using a 5% phosphate gel, and transferred to nitrocellulose. The filter was probed with Filters were probed with anti-u. Molecular weight standards are indicated to the left of the filter. The arrows to the right of the filter indicate the particular BCR intermediates present. 55 B M , M , 99 76 4 4 -anti-Li anti-Li D M , M , 9 9 -7 6 -4 4 -anti-Li anti-Ll Figure 22 - Association of an altered u heavy chain with protein chaperones in transfected non-lymphoid cells. Cell extracts were prepared from cells expressing with various wildtype B C R chains and an altered u heavy chain using CLB lysis buffer. (A) Whole cell extracts and cell extracts immunoprecipitated with either (B) anti-BiP, (C) anti-GRP94 or (D) anti-calnexin antibodies were separated by SDS-PAGE, using 10% polyacrylamide gels, and transferred to nitrocellulose. Filters were probed with anti-u followed by PA:HRP and then developed using E C L . Molecular weight standards are indicated to the left of each filter. The arrows to the right of each filter indicate u heavy chain. The asterisk (*) indicates the heavy chain from the immunoprecipitating antibody (Note that the anti-GRP94 heavy chain is not detected by PA:HRP). 56 Time (hours) B M r 112 -69 -43 -M r 112 -69 -43 -0 2 4 6 8 10 0 2 4 6 8 10 CD O T3 3 < 8 10 rm Mi 1 1 2 ~ 0 2 4 6 8 10 Time (hours) U33X. ^33 V ^ jx33 43 -M r 112 -69 -0 2 4 6 8 10 43 -Ml •»»» Figure 23 - Stability of an altered u heavy chain in transfected non-lymphoid cells. (A) Stable transfectants expressing various wildtype BCR chains and an altered u heavy chain were pulsed with 35S-translabel and chased over a timecourse of 10 hr. Cell extracts were prepared using M G lysis buffer. Labeled extracts were immunoprecipitated with anti-u and separated by SDS-PAGE on 10% polyacrylamide gels. Gels were exposed to a phosporimager cassette and images were captured electronically. (B) u heavy chain bands were isolated and quantified. Graphs were normalized such that 100% of the protein is present at time=0. 57 Discussion Protein chaperones assist nascent polypeptides to correctly fold and protein complexes to properly assemble. Protein chaperones may achieve these goals by binding target proteins and protecting them from the conditions of their surrounding environment. This could allow nascent polypeptides to expose hydrophobic patches while they fold and assemble, something that might otherwise result in aggregation. The purpose of this work was several fold. First, we wanted to determine which protein chaperones could bind each BCR chain. This was particularly important because only heavy chain and light chain proteins had been examined in the past and not Iga or Igp. This was also important to validate the non-lymphoid system by showing protein interactions that are observed in lymphoid cells can also occur in a non-lymphoid system. Second, we wanted to determine whether protein chaperones bind preferentially with certain assembly intermediates. Since little is known about the role of protein chaperones in B C R assembly, this would provide insight to how protein chaperones may interact with the BCR. Third, we wanted establish a system in which the interactions between altered BCR proteins and protein chaperones could be examined. We have chosen to use a non-lymphoid cell line, AtT20. Using these cells, we were able to express specific BCR proteins and examine their associations with protein chaperones in the absence of other BCR components. This approach provides a clearer picture of which B C R components are actually bound by protein chaperones. Protein chaperone binding in lymphoid cells Since protein chaperone binding of Iga and IgP has never before been examined, we were interested in determining whether they were associated with protein chaperones in lymphoid cells. In both lymphoid and non-lymphoid cells, Iga and IgP are retained in the absence of other BCR components. One explanation for this retention is that protein chaperones bind Iga 58 and IgP and retain them in the ER until they associate with the heavy and light chain proteins. We hypothesized that if protein chaperones interacted with Iga and Igp during B C R assembly, then Iga and IgP should be present in immune complexes of these protein chaperones. Western blot analysis was used to examine BiP, GRP94 and calnexin immune complexes from WEHI231 lymphoid cells. Immune complexes of both BiP and calnexin contained Iga (figure 7B). IgP was not detected in any of the immune complexes, perhaps because its presence was obscured by the heavy chain of the immunoprecipitating antibody that migrates to the same location (figure 7C). As a control to demonstrate that our approach produced the same results as published data, we examined the association of u heavy chain with immune complexes of BiP, GRP94 and calnexin. Consistent with the literature, u, heavy chain was found associated with all three protein chaperones (figure 7A). This approach is problematic. Since all four chains of the B C R are expressed by WEHI231 lymphoid cells, it is not clear whether association between specific B C R chains and protein chaperones occurs directly or whether association occurs indirectly, mediated by another BCR protein or some other unrelated protein. Protein chaperone binding in non-lymphoid cells To further examine the association of protein chaperones with assembling B C R components, we used a non-lymphoid system that lacked normal B C R expression. This system served several purposes. First, it enabled us to determine which BCR subunits were capable of associating with protein chaperones by eliminating the possibility of indirect association via other B C R subunits. Second, the system was used to determine whether the assembly state of the B C R affected protein chaperone binding. By defining these interactions, we have established a baseline from which comparisons can be drawn when examining the affect of particular B C R mutants on protein chaperone binding. For example, this system can be used to 59 determine whether specific amino acid alterations cause a difference in the binding of a given protein chaperone. Expression of BiP, GRP94 and calnexin in non-lymphoid AtT20 cells Since we wanted to examine association of BiP, GRP94 and calnexin with B C R proteins, we needed to first determine whether these proteins were expressed by AtT20 cells. We hypothesized that non-lymphoid AtT20 cells would express these proteins if they were essential to a fundamental biological process such as protein folding. BiP, GRP94 and calnexin were all present in AtT20 cells (figure 8). A comparison with lymphoid WEHI231 cells suggests the levels of these protein chaperones present in AtT20 cells is comparable to the levels present in WEHI231 cells. Presumably, this is because the ER functions at optimal ratios of protein chaperones to folding proteins and assembling protein complexes. If a cell expresses more protein, it likely produces more protein chaperones to assist the additional proteins in folding and assembly. Work on non-lymphoid COS cells supports this model, demonstrating BiP expression levels are upregulated when u heavy chain is overexpressed (Lenny and Green, 1991). Because of the findings in COS cells, we were interested to see whether AtT20 cells also upregulated protein chaperones following the expression of different BCR genes. Western blot analysis suggests transfected AtT20 cells do not upregulate BiP, GRP94 or calnexin (figure 11). The different responses of COS cells and AtT20 cells are likely due to the different functions of the two cell types. In contrast to COS cells, AtT20 cells are endocrine cells that normally synthesize and secrete large amounts of protein. It is likely that AtT20 cells have an enhanced synthetic pathway to handle this heavy load, whereas COS cells may be operating at capacity and must upregulate protein chaperones to assist in the folding of additional polypeptides. Since both our system and the COS system used the same viral promoter for expressing transfected BCR genes, it is unlikely there is a gross difference in the levels of u heavy chain expressed. 60 Different oligomeric forms of the BCR are present in the different transfected cell lines Since we are interested in determining whether protein chaperone association is specific for different B C R assembly intermediates, we needed to determine what B C R oligomers were present in the cell lines expressing the different B C R chains. We hypothesized that cell lines expressing different BCR proteins would possess a different complement of B C R assembly intermediates. Using a combination of Western blot analysis and co-immunoprecipitation experiments, we identified the prominent disulfide bonded species of both mlgM and of the Iga/Igp heterodimer. It appears that cells expressing both u heavy chain (H) and X light chain (L) contain H , L , H L , H 2 L and H 2 L 2 mlgM assembly intermediates. L 2 and H 2 homodimers are observed only in cells expressing u. heavy chain alone (H2) or X light chain alone (L 2 ) . It is possible that these species are energetically difficult to produce or that they are particularly susceptible to degradation. As with heavy and light chain, Iga and Igp prefer to form hetero-complexes when expressed together. Although IgP2 homodimers are observed when IgP is expressed alone at high levels (figure 7, Hourihane), we only found Iga present as monomers when expressed by itself. Association of BCR chains with protein chaperones in different transfected cell lines Although previous work has examined the association of protein chaperones with heavy and light chain proteins, no previous work has examined their association with Iga or Igp. Since Iga and IgP are retained in the absence of the remaining BCR proteins, it is clear that they also interact with quality control proteins. Given this, we hypothesized that Iga and Igp associate with protein chaperones. Although we could not examine Igp due to the lack of an appropriate reagent, we were able to examine the association of Iga with protein chaperones. Immune complexes of BiP, GRP94 and calnexin were examined by Western blot analysis for the presence of Iga (lane 3, figure 12B-D). Immune complexes of GRP94 contained low levels of Iga but immune complexes of BiP or calnexin did not. This observation is in contrast to the finding in lymphoid cells that Iga associated with BiP and calnexin but not GRP94 (figure 61 7B). One possible interpretation of this data is that GRP94 interacts with Iga when it is expressed alone. In contrast, BiP and calnexin association may occur when Iga is expressed with other B C R chains. This suggestion is supported by the increased association of BiP and calnexin in non-lymphoid cells that co-express u. heavy chain and X light chain with Iga. X light chain and p heavy chain association with immune complexes were also examined. Association of u heavy chain with immune complexes was consistent with published data. However, quite surprisingly, X light chain was found associated with immune complexes of calnexin (figure 12D). This was unexpected because X light chain is neither a glycoprotein nor a membrane protein and therefore does not contain a recognized binding motif for calnexin. The most likely interpretation of this result is that X light chain is associated with another, non-BCR protein that forms a complex with calnexin. Since several protein chaperones have been observed as hetero-complexes, it may be possible that X light chain association with calnexin is indirect, mediated through another protein chaperone. Indeed, it would be interesting to further examine immune complexes of X light chain and calnexin to determine what proteins mediate this interaction. Previous work showed early folding intermediates of X light chain and early assembly intermediates of mlg are targets for protein chaperones (Haas and Wabl, 1983; Bole et al, 1986). It appears that protein chaperones bind target proteins in an ordered fashion. For example, BiP binds immature folding species of X light chain while GRP94 only binds the more mature folding species (Melnick et al, 1994). This observation suggests protein chaperones interact with target proteins in a coordinated fashion, with each protein chaperone binding at a different stage of folding. It is not clear, however, whether protein chaperones bind different BCR assembly intermediates in the same coordinated fashion. The idea of coordinated interaction with different assembly intermediates is supported by the observation that BiP binds unassembled mlg molecules and not assembled molecules. Continued retention of these molecules in the absence of BiP binding suggests other protein chaperones are 62 binding, ones that are specific for this intermediate stage rather than the earlier stages like BiP and GRP94. Presumably, quality control mechanisms are still exerting their effects on the partially assembled B C R molecule. We hypothesized that protein chaperones interact with specific B C R assembly intermediates. Using western blot analysis, we examined immune complexes of BiP, GRP94 and calnexin in cell lines that we expected would be enriched for different B C R assembly intermediates. We found that, consistent with published data, BiP binding of u, heavy chain increased dramatically in cell lines expressing u. heavy chain alone. However, when A, light chain was coexpressed with jx heavy chain, BiP association disappeared. This observation suggests BiP associated with particular assembly intermediates present in the cell line expressing u heavy chain alone, but not with the assembly intermediates in the cell line expressing u heavy chain and A, light chain. This 'stage-dependent' association was also observed with GRP94 and Iga. When u heavy chain and A, light chain are coexpressed with Iga, there is an increase in the Iga present in GRP94 immune complexes. Again, this suggests that there is stage specific association of a protein chaperone with assembling B C R components. Association of protein chaperones correlates with protein degradation Proteins that remain incompletely folded and that are unable to form complete protein complexes are degraded by the cell. Since our system relied heavily on creating an artificial block in receptor assembly, we were interested in seeing whether the BCR chains in our different cell lines were being degraded faster in cell lines expressing all four B C R chains. We hypothesized that if B C R chains were unstable as incompletely folded subunits then these proteins would degrade much more rapidly in cell lines lacking one of the four B C R subunits than in cell lines containing all four. Pulse chase analysis was used to examine protein stability. A, light chain and Iga were relatively stable when expressed alone and in the presence of other B C R chains. In contrast, u heavy chain by itself was much less stable than when it was coexpressed with other BCR chains. It is possible that the H 2 intermediate (which is the only 63 unique assembly intermediate in the cell line expressing u heavy chain alone) becomes terminally associated with BiP, thus resulting in faster degradation. This is consistent with the correlation observed between the u heavy chain instability and an increase in u heavy chain associated with BiP immune complexes. Protein chaperone association with a mutant u heavy chain There has been much work that has indirectly examined the role of the membrane spanning region of u heavy chain in both interactions with the cells quality control mechanisms and with the Iga/IgP heterodimer. Chimeric proteins containing the extracellular region of u heavy chain and the transmembrane/intracellular regions of either CD8 or M H C class I are not able to carry out the normal signal transduction activities of the BCR. Additionally, these chimeric molecules are not associated with Iga or Igp. These observations suggest the membrane spanning region of u heavy chain is necessary for associating with Iga/IgP and for interacting with quality control mechanisms. Subsequent work has demonstrated that specific residues in the membrane spanning region can also abrogate normal ER retention of u heavy chain, allowing it to move to the cell surface in the absence of Iga/Igp. One such mutant is u33. This mutant has a valine residue inserted near the top of the transmembrane region (n-terminus) and a threonine deletion near the bottom (c-terminus). This alteration should cause a 100° rotation and 15 A downward shift of the postulated a-helix (figure 24). 64 A) B) Figure 24 - Schematic of p.33 alteration. Transmembrane regions of (A) wildtype a heavy chain and (B) mutant u heavy chain, u33. Hypothetically, the u.33 mutation causes a 100° rotational shift in the postulated oc-helix and a 15 A downward shift in the plasma membrane. Ii33 forms oligomers comparable to wildtype u heavy chain Since p heavy chain consists of two identical disulfide associated heavy chain proteins, we thought that altering the membrane spanning region of p heavy chain might interfere with the formation of dimers. We hypothesized that if this region of p heavy chain was particularly important for the formation of u:u dimers then we would not observe assembled H 2 L 2 intermediates. Western blot analysis showed that the altered u heavy chains form complete H 2 L 2 assembly intermediates. Clearly, this particular mutant of n heavy chain does not interfere with the formation of H 2 L 2 intermediates as they are present in levels comparable to the wildtype protein. u33 is retained intracellularly like wildtype p heavy chain Since p.33 appears to escape ER retention in lymphoid 2PK-3 cells, we were interested to see if they could also escape retention in our non-lymphoid system. We hypothesized that if the membrane spanning region was an important site for protein chaperone binding, then the u33 transmembrane alteration may abrogate this normal interaction and allow the altered protein to be expressed at the cell surface in the absence of the normally required Igoc/IgP heterodimer. 65 Using confocal microscopy, we examined the cellular location of the altered u heavy chain by immunofluorescence and found the protein was not expressed at the cell surface. Biochemical analysis suggests the protein may actually be located in a pre cis-Golgi compartment like the wildtype protein. This observation suggests the membrane alteration had no effect on the proteins association with quality control proteins. It is unclear why this mutant escapes ER retention in B cells but not in AtT20 cells. Since the B cell line examined contained Iga and Igp, one explanation may be that Iga and IgP were transiently associated with the altered mlgM, assisting it out of the ER but not associating with it at the cell surface. An alternative explanation for the differences in the two cell types may be explained by the presence of a cell specific protein chaperone. This may be a chaperone that is circumvented in B cells or one that acts generally and binds to u33 in non-lymphoid cells. n33 associates with protein chaperones at levels comparable to wildtype JJL heavy chain Since previous work indicates that the membrane spanning region of u heavy chain is particularly important for interacting with quality control proteins, we hypothesized that if the membrane spanning region of u heavy chain was important for interacting with quality control proteins then we would observe a decrease in the levels of protein chaperones associated with the u heavy chain when this region was altered. Using Western blot analysis, we examined the levels of altered u heavy chain associated with immune complexes of BiP, GRP94 or calnexin. Compared to the wildtype protein, we found that there were no gross differences between the levels of altered a heavy chain associated with the proteins chaperones. As well, it appears there is no difference in protein chaperone association with assembly intermediates containing u33. Pulse chase analysis indicated that the u33 protein is no more susceptible to degradation than the wildtype protein. It is quite clear from these observations that the u.33 alteration has no effect on the protein's ability to be recognized and interact with quality control proteins present in the ER. 66 Previous studies indicate Iga and IgP appear to interact with the same portion of u heavy chain as quality control proteins in the ER. This is significant when one considers a model for BCR assembly and interaction with protein chaperones. In assembly of B C R molecules, mlgM is rapidly synthesized. Once synthesized, human mlgM binds Igp and then Iga to complete the B C R molecule (Brouns et al, 1995). Our data suggests Iga may actually bind mlgM in the absence of Igp (appendix 3). Consistent with this is an increase in BiP and calnexin association that is not observed in cell lines expressing Iga alone. It is interesting to speculate that Iga and Igp may bind to partially assembled mlg molecules, displacing quality control proteins and allowing the mlg molecule to move to the cell surface in association with Iga/Igp. In the absence of Iga and IgP, mlgM is retained internally. It is interesting to speculate that Igp may be a rate limiting molecule, rather than Iga as suggested by Brouns et al. (1995). In this case, the association observed in cell lines expressing u, X and Iga is due to association of protein chaperones with an arrested assembly state. This model seems plausible given the apparent lack of Igp associated with protein chaperones in WEHI231 cells. One might imagine that if association of Iga were the rate limiting step for receptor assembly, then (H2L2)(IgP) should interact with a retention protein while awaiting Iga association. Clearly, this is almost entirely speculative. Indeed, IgP may be associated with another protein chaperone that is yet to be identified. It is unclear how protein chaperones find their target proteins given the large number of proteins that are synthesized in the ER in a relatively short frame of time. High levels of BiP protein expression is an excellent example of one mechanism protein chaperones can use to increase the probability they will find a target protein. With BiP making up nearly 5% of the total protein in the lumen of the ER, it seems improbable that any protein could escape the ER without encountering many BiP molecules. However, not all protein chaperones are as abundant as BiP. Recently, an alternative mechanism has been proposed that suggests protein chaperones may increase their chances of interacting with nascent polypeptides by forming a 67 immobile network along the lumenal surface of the ER membrane (for review see Hammond and Helenius, 1995). By forming an immobile network, protein chaperones would achieve two goals. First, they would have a site at which they could concentrate, increasing the chances of interacting with nascent polypeptides being inserted into the ER. Second, the immobile nature of the network would facilitate retention of target proteins. Such a system would result in rapid association and retention with nascent polypeptides. Once the polypeptide was fully folded or assembled into a complex, it would be released from the immobile network and allowed to travel out of the ER to the cell surface. Although this work has focused entirely on protein chaperones binding as a means of retaining proteins, it is tempting to speculate about the mechanisms that may underlie B C R retention by protein chaperones. One interesting possibility is that BCR trafficking is regulated by 'linking' unassembled subunits to a recycling pathway and assembled complexes to a transport pathway. In the case of retention, this link to a recycling pathways may be mediated by protein chaperones. Protein chaperones would interact with coatamer proteins (COPI) that recycle from the cis-Golgi to the ER. Thus, protein chaperone binding would cause any associated proteins to be recycled between the cis-Golgi and the ER. In the case of transport, Iga or IgB may link the B C R to a pathway in much the same way that protein chaperones would for retention. However, instead of interacting with recycling coatamer proteins, Iga or IgP may mediate interactions with coatamer proteins involved in anterograde transport (COPII). By interacting with COPE proteins, Iga and IgP cause the B C R to be dragged out of the ER to the cell surface. Thus, Iga and IgP may be required to displace protein chaperones and to link the B C R to the anterograde transport pathway. 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Immunol. 158, 2762-2770. 74 aa - amino acid BCR - B cell antigen receptor BiP - heavy chain binding protein CLB - calnexin lysis buffer CLSM - confocal laser scanning system CMV - cytomegalovirus CO2 - carbon dioxide d - day DABCO - l,4-diazobicyclo-[2.2.2]-octane DOC - deoxycholate ECL - enhanced chemiluminescence Endo-H - Endoglycosidase H ER - endoplasmic reticulum FCS -fetal calf serum GRP - glucose response protein hr - hour kB - kilobase kDa - kilodalton KLH - keyhole limpet hemocyanin LTR - long terminal repeat mA - milliamp MG lysis buffer - Mike Gold lysis buffer MHC - major histocompatibility complex min - minute Mr - relative molecular mass NIS - non-immune serum NRPSB - non-reducing phosphate sample buffer NRSB - non-reducing sample buffer OD - optical density PNGaseF - N-glycosidase F RSB - reducing sample buffer RSV - rouse sarcoma virus SDS - sodium dodecyl sulphate SDS-PAGE - SDS polyacrylamide electrophoresis TBS - tris-buffered saline TBST - TBS with 0.05% tween20 TCR - T cell antigen receptor V - volt Appendix 1 - Abbreviations. 75 Name Cell line Wildtype Altered A * Ig- cc Ig-P u , A , I g a , I g ( 3 R 9 8 + + + + R 1 4 2 + + + 1 - U 7 I 1 2 + + l - u . 2 7 + I 1 - X.2\ + I g a l - m b l . 9 + IgP u 3 3 , A , , I g a D 1 6 2 - u 3 3 . l l + + + + u 3 3 , A . 1 - U 3 3 . 2 0 + + u 3 3 4 - U 3 3 . 5 + Appendix 2 - Summary of transfected cell lines. R 9 8 , R 1 4 2 and D 1 6 cells were previously established by Linda and Sharon. 7 6 M , 108 -69 _ 4 3 -anti-Li Appendix 3 - Iga associates with p heavy chain in CLB lysates. Cell extracts from transfected non-lymphoid cells were prepared with C L B as described in the Materials and Methods. Extracts were immunoprecipitated with anti-Iga. Immune complexes were separated by SDS-PAGE using 12% polyacrylamide gels and transferred to nitrocellulose. Filters were probed with anti-u and then with either PA:HRP. Filters were developed using E C L . Molecular weight standards are indicated to the left of each filter. The arrows to the right of each filter indicate u heavy chain. 77 

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