©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calnexin Influences Folding of Human Class I Histocompatibility Proteins but Not Their Assembly with -Microglobulin (*)

(Received for publication, March 27, 1995; and in revised form, June 8, 1995)

Matthew Tector Russell D. Salter (§)

From the Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Class I major histocompatibility complex heavy chains bind to calnexin before associating with beta(2)-microglobulin (beta(2)m) and peptides. Calnexin has been shown to retain in the endoplasmic reticulum those class I heavy chains which have not assembled properly and, thus, to serve as a quality control mechanism. In addition, calnexin may direct the folding of class I subunits or their subsequent assembly. We asked whether calnexin plays a role in the initial folding of HLA-B*0702 heavy chains by assessing disulfide bond formation in vivo. Our results show that class I heavy chains form intrachain disulfide bonds very soon after translation, and that calnexin is bound to both reduced and oxidized forms during this process. When a cell-permeable reducing agent, dithiothreitol, was added to cells, disulfide bond formation in newly synthesized heavy chains was substantially blocked, as was their association with calnexin. The reducing agent appeared to affect calnexin directly, since binding was similarly abolished to a subset of proteins which do not contain internal disulfide bonds. Addition of the glucosidase inhibitor castanospermine to cells, shown previously to disrupt calnexin binding to ligands, slowed formation of disulfide bonds but did not decrease the amount of assembled heavy chain-beta(2)m complexes that formed. Our data suggest that calnexin can promote disulfide bond formation in class I heavy chains but does not directly facilitate subsequent binding to beta(2)m.


INTRODUCTION

Class I MHC (^1)molecules display intracellularly derived peptides at the plasma membrane enabling the immune system to recognize virally infected and tumor cells. These molecules are composed of integral membrane glycoproteins called heavy chains, a second soluble polypeptide called beta(2)-microglobulin (beta(2)m), and peptides of 8-10 amino acids in length (reviewed in (1) ). Class I heterotrimers form in an early biosynthetic compartment and, once assembled, are transported through the Golgi to the plasma membrane.

Assembly and transport of class I molecules is a highly regulated process and has been studied intensively. Newly synthesized class I heavy chains bind calnexin(2, 3) , a protein found in the rough endoplasmic reticulum (ER). Heavy chain-beta(2)m dimers then form and associate with TAP transporters(4, 5) , which have been shown to translocate peptides from the cytosol into the lumen of the ER(6, 7) . Upon peptide binding, class I molecules dissociate from TAP and continue through the secretory pathway.

One function of calnexin is to retain incompletely assembled multimeric proteins, preventing their premature release from the ER(3, 8, 9, 10, 11, 12) . In addition, however, calnexin may bind to individual polypeptides that have not folded properly(13, 14, 15, 16, 17, 18) . This raises the possibility that calnexin may promote folding and disulfide bond formation of many polypeptides(19) . Such a role has been directly demonstrated for VSV-G protein, which binds calnexin transiently during its biosynthesis. When its binding to calnexin is blocked, VSV-G does not fold properly, but instead remains in the ER in association with grp78(20) . Together these studies demonstrate that calnexin has multiple functions in the biogenesis of monomeric and multimeric proteins.

Mouse and human class I heavy chains associate with calnexin before binding beta(2)m. However, mouse class I proteins remain bound to calnexin after binding beta(2)m(2, 21) , whereas human class I heavy chains appear to dissociate from calnexin either before or while binding beta(2)m(8, 21) . The reason for this difference between class I molecules of the two species is unclear, but in any case it is possible that the chaperone both promotes folding of class I heavy chains and facilitates their binding to beta(2)m. We have addressed this issue by analyzing the formation of intrachain disulfide bonds, an indicator of heavy chain folding, and subsequent assembly with beta(2)m under conditions where calnexin is either active or inactive. We find that calnexin promotes heavy chain folding, but not apparently association with beta(2)m.


MATERIALS AND METHODS

Cell Lines

The HLA-A,B-deficient B-cell line CIR transfected with B*0702 was obtained from Dr. Peter Cresswell. Cells were grown in RPMI 1640 (Irvine Scientific, Irvine, CA) containing 10% transferrin supplemented calf serum (HyClone, Logan, UT).

Antibodies

Antiserum UCSF#2 reacts with the cytoplasmic tail of class I HLA proteins and was provided by Drs. Frances Brodsky and Bruce Koppelman. Anti-H serum reactive with non-beta(2)m-associated class I HLA heavy chains was provided by Dr. Hidde Ploegh(22) . Monoclonal antibodies W6/32 (ATCC) and 4E (from Dr. Walter Storkus) react with beta(2)m-associated heavy chains(23, 24) . Anti-calnexin monoclonal antibody AF8 was provided by Dr. Michael Brenner(3) . Anti-invariant chain antibody (PIN1) was from Dr. Peter Cresswell(25) . Antisera reactive with human beta(2)m, human IgG, and mouse IgG were from Sigma.

Radiolabeling and Disulfide Bond Analysis

Cells were washed and resuspended at 8 10^6 cells/ml in RPMI 1640 lacking methionine and cysteine. After 1 h at 37 °C, 0.15 mCi of TranS-label (ICN, Costa Mesa, CA) was added to cells at 300 µCi/ml and incubated for 3 min. Incorporation of radiolabel was stopped by adding 6-fold excess RPMI 1640 containing 10% bovine serum. In some experiments (indicated in text), dithiothreitol (DTT) was then added. Aliquots removed after chase periods were added to equal volumes of TBS (10 mM Tris, pH 7.4, 150 mM NaCl) and 20 mM thiol alkylating agent N-ethylmaleimide (Sigma) on ice. Cells were lysed in TBS containing 0.1 mM tosyl lysine chloromethylketone, 1 mM phenylmethylsulfonyl fluoride, and 1% CHAPS detergent, or in 1% Triton X-100 as indicated. Debris was pelleted by centrifugation at 13,000 g for 5 min at 4 °C, and supernatants precleared by adding rabbit anti-human IgG and formalin-fixed Staphylococcus aureus for 90 min at 4 °C. Specific proteins were isolated by adding antibodies to the cleared lysates for 1 h, and antigen-antibody complexes collected by incubation with Sepharose beads cross-linked to protein A (Sigma). Protein A-Sepharose beads were first coated with anti-mouse Ig before use with antibody AF8. N-Ethylmaleimide was present throughout the lysis and immunoprecipitation steps to prevent artifactual disulfide formation. Samples were eluted with sample buffer and separated on SDS-PAGE.

Quantitation of Images from Fluorographs

Exposed films were scanned on a Millipore Omni Media XRS scanner, and integrated optical density values calculated.


RESULTS

Reduced and Oxidized Forms of B*0702 Heavy Chains Are Bound to Calnexin

Class I MHC heavy chains have two intrachain disulfide bonds located in the alpha2 and alpha3 domains, which appear to form rapidly after insertion of the protein into the ER membrane. Polypeptides containing disulfide bonds often have altered migration on non-reducing SDS-PAGE, which can be easily visualized and used as a indicator of folding. To determine when calnexin binds to B*0702 heavy chains relative to folding, cells were radiolabeled, and then further incubated in varying concentrations of DTT(26, 27) . In the absence of DTT, at 0 min of chase, UCSF#2 bound two species of heavy chains, an upper band representing about 20% of the total protein (Fig. 1), and a lower band, which upon reduction of gel samples migrated with the upper band. (^2)This demonstrates that the upper band is not fully oxidized. After 5 min of chase, the upper band is no longer visible, suggesting that it progresses to the faster migrating oxidized form. Both reduced and oxidized forms of B*0702 could be isolated with AF8, indicating they were bound to calnexin (Fig. 1). This suggests that intrachain disulfide bonds in B*0702 heavy chains form after binding calnexin. Addition of DTT to cells immediately after labeling slowed (1 mM) or blocked (3 mM) conversion to the oxidized form, which allowed better visualization of the reduced band in some lanes. However DTT also appeared to disrupt the interaction of calnexin with substrates, an effect most evident at 5-min chase times.


Figure 1: Identification of HLA-B*0702 folding intermediates. After radiolabeling for 3 min, cells were incubated during a chase period of 0 or 5 min with 0, 1, or 3 mM DTT. Cells were then lysed with CHAPS and class I heavy chains or calnexin with associated proteins isolated using UCSF#2 or AF8, respectively. Samples were then separated on non-reducing SDS-PAGE. Upper and lower class I bands correspond to reduced and oxidized heavy chains, respectively. Ichain, invariant chain.



Since oxidized heavy chains are quite abundant immediately after 3 min of radiolabeling, we think it is likely that one or two disulfide bonds form co-translationally under normal circumstances. Post-translational folding can be forced by adding DTT after radiolabeling, to cause complete reduction of B*0702 heavy chains. After DTT removal, oxidation and folding proceeds, apparently with normal kinetics.^2 Under these conditions, calnexin rebinds heavy chains quite rapidly, and before they associate with beta(2)m.

Calnexin Has Intramolecular Disulfide Bond(s) Essential for Its Function

As shown above, treatment of cells with DTT induces dissociation of invariant chain and B*0702 heavy chains from calnexin. This is likely to be due to a direct effect on calnexin for the following reasons. First, both oxidized and reduced forms of class I heavy chains associate with calnexin in untreated cells (Fig. 1), demonstrating that reduced heavy chains can bind calnexin. Second, the invariant chain does not have intrachain disulfide bonds and does not migrate differently on reducing and non-reducing SDS-PAGE^2 or after pretreatment of cells with DTT (Fig. 1). Thus DTT treatment should not affect its conformation. Third, essentially all radiolabeled proteins were released from calnexin after treatment of cells with DTT, and the same effect could be obtained in vitro by adding DTT to cell lysates before isolation of calnexin.^2 Based on these observations, we suggest that calnexin has an intramolecular disulfide bond(s) essential for its function. Consistent with this hypothesis, altered migration of calnexin on non-reducing SDS-PAGE was observed after treatment of cells with DTT (Fig. 2).


Figure 2: Calnexin contains intramolecular disulfide bond essential for interaction with other proteins. Cells were radiolabeled for 5 min, then incubated in the presence of unlabeled amino acids for 0 or 30 min. Samples were then lysed in lysis buffer containing 1% Triton X-100 and 20 mMN-ethylmaleimide either immediately(-) or after (+) incubation for an additional 5 min in 3 mM DTT. AF8 was then used to isolate calnexin and samples separated on non-reducing SDS-PAGE.



Inhibiting Calnexin Function Reduces the Stability of B*0702 Heavy Chains but Has No Demonstrable Effect on Assembly with beta(2)m

Since calnexin is associated with both reduced and oxidized heavy chains, we next asked whether it is involved in catalyzing folding and disulfide bond formation. Cells were treated with castanospermine, an inhibitor of glucosidases I and II shown previously to impair binding of calnexin to various proteins(28) . Heavy chain stability, disulfide bond formation, and association with beta(2)m was then assessed. After radiolabeling, cells were lysed, and samples immunoprecipitated with anti-heavy chain (anti-H), anti-calnexin (AF8), or anti-class I complex (w6/32) antibodies. Fig. 3A shows scanning densitometry of reducing SDS-PAGE of these samples. The drug inhibits interaction between calnexin and heavy chains (AF8 panel). The stability of heavy chains overall was reduced (anti-H panel) and was evident at all time points. However, no reduction in assembled complexes was observed (w6/32 panel). The latter observation suggests that class I heavy chains are made in excess of other factors needed for assembly of class I complexes.


Figure 3: Evidence that calnexin promotes disulfide bond formation, but not assembly of HLA-B*0702 molecules. CIR cells transfected with B*0702 were incubated in the presence or absence of 400 µg/ml castanospermine (CST) for 1 h prior to radiolabeling for 3 min. After the indicated chase times, cells were lysed in CHAPS. In A, lysates were split and subjected to immunoprecipitation with AF8 (calnexin-specific), anti-H (non-beta(2)m-associated heavy chains), or w6/32 (beta(2)m-bound heavy chains). Samples were run on reducing SDS-PAGE, and appropriate bands quantitated by scanning. Integrated optical density values are shown on the y axis and were normalized relative to the highest value obtained, which was seen at 0 min with anti-H and no castanospermine added. Chase times are shown on the x axis. Incubation with castanospermine (opensymbols) or without (closedsymbols) is indicated. In B, samples were obtained as in A, but non-reducing SDS-PAGE was performed. Upper and lower class I bands correspond to reduced and oxidized forms respectively. Migration of invariant chain (Ichain) and calnexin is also marked.



A Potential Role for Calnexin in Heavy Chain Folding

When samples were separated on non-reducing SDS-PAGE, it was apparent that pretreatment with castanospermine inhibited the conversion of heavy chains from reduced to oxidized form (Fig. 3B). This result was obtained reproducibly in four separate experiments. The ratio of reduced to oxidized forms was determined by scanning and was used as a measure of folding efficiency independent of overall heavy chain levels. Table 1lists percentages of oxidized to total heavy chains reactive with anti-H antiserum for data shown in Fig. 3. This clearly demonstrates that castanospermine treatment blocks oxidation of class I heavy chains, probably by disrupting their interaction with calnexin. It should also be noted that values obtained for heavy chains isolated in association with calnexin are very similar to those for total non-beta(2)m-associated heavy chains (anti-H-reactive) in untreated cells. This suggests that most non-beta(2)m-associated heavy chains in the cell are bound to calnexin.



B*0702 Heavy Chains Form Disulfide Bonds before Binding beta(2)m

Based on previous kinetic studies of mouse class I molecules(29, 30) , we expected that only oxidized B*0702 heavy chains would bind beta(2)m. To test this directly, radiolabeled lysates were immunoprecipitated with either UCSF#2, which recognizes class I HLA heavy chains regardless of conformation, or 4E, specific for a subset of HLA-B molecules, including B*0702, in association with beta(2)m. Immediately after radiolabeling (0 min of chase), both oxidized and reduced heavy chains reacted with UCSF#2 (Fig. 4). At later times, the majority of heavy chains were fully oxidized, although a small fraction were not. In most experiments, conversion to the fully oxidized form was virtually complete. Class I molecules isolated with antibody 4E at the 10- and 20-min time points contained heavy chains that were fully oxidized, indicating that disulfide bonds form either before or at the same time that beta(2)m binds. At 40 min, some heavy chains migrate more slowly, in a similar position to reduced heavy chains. This altered migration results from modification of glycans^2 and is not due to reduction of beta(2)m-heavy chain complexes. Treatment of cells with 3 mM DTT blocked disulfide bond formation and beta(2)m binding. In 1 mM DTT, however, heavy chains were oxidized to a considerable degree, while beta(2)m binding was completely blocked. This suggests that heavy chains can fold in the absence of stable beta(2)m association.


Figure 4: Oxidation of HLA-B*0702 heavy chains precedes binding to beta(2)m. Cells were metabolically labeled and incubated in 0,1, or 3 mM DTT for the indicated chase periods, then lysed with CHAPS. Class I proteins were isolated with UCSF#2 (H) or with 4E (C), which binds to beta(2)m-associated B*0702 molecules.




DISCUSSION

Fully folded class I MHC molecules contain two disulfide bonds in the alpha2 and alpha3 domains, respectively. Disruption of either bond markedly reduces the efficiency of transport of class I MHC molecules to the cell surface(31, 32) . In vitro studies of mouse class I heavy chains showed that after translocation into microsomes, the alpha3 domain disulfide forms rapidly, even in the absence of beta(2)m(29) . The disulfide bond in the alpha2 domain was detected only when beta(2)m was present, suggesting that it forms after the alpha3 domain interacts with beta(2)m. A study examining heavy chain folding in vivo demonstrated that beta(2)m helps to form the second disulfide bond and that both disulfide bonds can form before stable association with beta(2)m is demonstrable(30) .

As calnexin has been shown to associate with class I molecules shortly after their synthesis, we asked in the present study whether calnexin influenced either disulfide bond formation or assembly of beta(2)m with class I heavy chains in human cells. First we established that formation of disulfide bonds could be measured in B*0702 heavy chains from CIR transfectants. Heavy chain oxidation was very rapid and occurred before detectable binding to beta(2)m. Both reduced and oxidized heavy chains could be co-purified with calnexin. This suggested either that calnexin bound to both reduced and oxidized heavy chains or that reduced heavy chains bound calnexin and remained associated while becoming oxidized.

To distinguish between these possibilities, we asked whether heavy chains became oxidized at the same rate when calnexin binding was blocked by using the drug castanospermine, which inhibits trimming of glucose residues from N-glycans. Previous studies demonstrated that calnexin binds to monoglucosylated N-glycans on proteins and that calnexin binding can be inhibited by drugs which alter normal processing of glycans(28, 33) . Treatment with castanospermine decreased the total amount of non-beta(2)m-associated heavy chains and also significantly diminished the percentage of heavy chains which were oxidized. This shows that calnexin participates in folding and disulfide bond formation of class I heavy chains, but it does not appear to be absolutely required. Furthermore, these results strongly suggest that calnexin binds to reduced heavy chains and remains associated until heavy chain folding is complete.

Several alternative explanations for our results were considered. Class I heavy chains might form aggregates that constrain folding in the absence of calnexin. No evidence for this was seen on non-reducing SDS-PAGE. Mislocalization of class I heavy chains might occur without calnexin, but this seems unlikely since cells were lysed immediately after 3 min of labeling. Rapid degradation of heavy chains after castanospermine treatment was expected, as it was observed in an earlier study using CMT-cK^d1 cells(34) . In our experiments, incubation in the drug decreased heavy chain levels by about 20%. However, the altered ratio of reduced to oxidized heavy chains noted after calnexin binding is inhibited is independent of overall heavy chain levels and suggests that calnexin directly facilitates heavy chain folding.

Since calnexin has a prolonged association with heavy chains in cells lacking beta(2)m(35) , it seemed possible that calnexin might directly promote heavy chain binding to beta(2)m. Our results suggest that this is not the case, but rather that calnexin helps heavy chains fold properly and thereby increases the pool of fully conformed heavy chains available to bind beta(2)m. Heavy chain folding and disulfide bonding can occur independently of calnexin, however, since oxidized heavy chains formed after treatment with castanospermine, albeit less efficiently (Fig. 3B). Furthermore, the overall amount of class I subunits that assembled and could bind w6/32 was unaffected, presumably because other components required for class I assembly, such as beta(2)m and peptides, further limit the process.

Our data demonstrate that calnexin has one or more disulfide bonds essential for function. In support of this, we observed that addition of DTT to cells rapidly disrupted interactions between calnexin and a variety of proteins, including the invariant chain, which has no internal disulfide bonds, and both reduced and oxidized class I heavy chains (Fig. 1). These results are consistent with the hypothesis that calnexin, which is a monomer, contains intramolecular disulfide bonds. A similar conclusion was reached recently by Helenius and co-workers (36) . It should be noted that binding of the antibody AF8 appeared not to be affected by DTT treatment, ruling out the possibility of a conformational change in calnexin causing the antibody epitope to be lost. In contrast to our data, two studies have demonstrated increased binding of individual proteins to calnexin after DTT treatment, namely gp80 in MDCK cells and thyroglobulin in bovine thyrocytes(14, 37) . There is not a clear explanation for these disparate results, but they may be a reflection of the cell types used. We would also suggest that DTT treatment might cause aggregation of certain proteins in the absence of essential chaperones, as shown previously(38) . Such aggregates might include many unfolded proteins, including calnexin, and lead to an apparent increase in association between calnexin and other molecules in the aggregate.


FOOTNOTES

*
This study was supported by American Cancer Society Grant IM-668 and by a student fellowship from the Pathology Education and Research Foundation at the University of Pittsburgh. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, 733 Scaife Hall, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. Fax: 412-648-1916; Tel.: 412-648-9471.

(^1)
The abbreviations used are: MHC, major histocompatibility complex; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; beta(2)m, beta(2)-microglobulin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol.

(^2)
M. Tector and R. D. Salter, unpublished data.


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