©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calnexin Recognizes Carbohydrate and Protein Determinants of Class I Major Histocompatibility Complex Molecules (*)

(Received for publication, September 27, 1994; and in revised form, November 21, 1994)

Qing Zhang 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

Proper folding of nascent polypeptides is essential for their function and is monitored by intracellular ``quality control'' elements. The molecular chaperone calnexin participates in this process by retaining in the endoplasmic reticulum a variety of unfolded proteins, including class I major histocompatibility complex molecules. We transfected human B cell lines with genes encoding either wild-type HLA-A2 heavy chains or mutant heavy chains lacking sites for glycosylation or deficient in binding to beta(2)-microglobulin (beta(2)m). In CIR cells, calnexin did not associate detectably with wild-type heavy chains but bound strongly to mutant heavy chains unable to bind beta(2)m. Removal of the glycosylation addition site by further mutagenesis prevented binding of mutant heavy chains to calnexin. In Daudi cells, deficient in synthesis of beta(2)m, wild-type HLA-A2 heavy chains, but not a nonglycosylated mutant, bound calnexin. Castanospermine, which blocks trimming of glucose residues from asparagine-linked glycans, inhibited association of calnexin with heavy chains encoded by a second class I gene, HLA-B*0702. Although initiation of calnexin binding appears to depend on the presence of oligosaccharide on the substrate, removal of the glycan from calnexin-associated heavy chains by digestion with endoglycosidase H did not disrupt the interaction. These results suggest that calnexin first recognizes carbohydrate on substrate proteins and then binds more stably to peptide determinants, which disappear upon folding.


INTRODUCTION

The molecular chaperone calnexin binds incompletely folded proteins and prevents their premature export from the endoplasmic reticulum (ER)(^1)(1, 2, 3) . Calnexin may also facilitate directly folding and disulfide bond formation in nascent polypeptides(4) . Not all proteins interact with calnexin, however, and it is known that some proteins that normally interact with calnexin can fold properly in vivo in its absence(5) . This supports the proposal that calnexin functions primarily as a quality control mechanism in the ER.

As many of its substrates are glycoproteins, it has been suggested that calnexin recognizes carbohydrate. Tunicamycin, which inhibits transfer of the core saccharide to asparagine residues, blocks binding of many proteins to calnexin(4) . Castanospermine and other drugs that inhibit trimming of glucose residues from high mannose glycans also block calnexin binding, leading to the suggestion that calnexin recognizes monoglucosylated glycans on substrates(6) . This implies that calnexin may be a lectin, although it does not appear to have homology to known lectins. Alternatively, calnexin may recognize determinants of proteins that disappear upon further glycan modification, as previously proposed(6) .

Our experiments addressed the structural features of class I major histocompatibility complex (MHC) heavy chains, which are important for recognition by calnexin. Previous studies demonstrated that calnexin binds to class I MHC heavy chains prior to binding beta(2)-microglobulin (beta(2)m) in the ER and then dissociates before class I MHC molecules enter the Golgi. We found that calnexin bound only to non-beta(2)m-associated heavy chains, which were glycosylated at asparagine 86. Our results suggest that calnexin recognizes both oligosaccharide and unfolded regions of proteins.


MATERIALS AND METHODS

Cell Lines

The B cell lines CIR (HLA-A negative, HLA-B*3503 low, HLA-C*0401 normal) and Daudi (beta(2)m negative) were grown in RPMI 1640 (Irvine Scientific, Irvine, CA) containing 10% calf serum (Hyclone, Logan, UT). HLA-B*0702-CIR transfectants were provided by Dr. Peter Cresswell. HLA-A2 transfectants were grown in medium containing 300 µg/ml hygromycin (Sigma).

Reagents

Castanospermine, protein A-Sepharose beads, and fluorescein isothiocyanate-conjugated goat anti-mouse antibody were purchased from Sigma. Endoglycosidase H was from New England BioLabs (Beverly, MA). TranS-label ([S]methionine and [S]cysteine) and cysteine- and methionine-free RPMI 1640 were from ICN (Costa Mesa, CA).

Antibodies

Monoclonal antibody W6/32 (ATCC) recognizes an epitope on all HLA-A, -B, and -C heavy chains after assembly with beta(2)m, which is not present on free heavy chains(7) . Monoclonal antibody BB7.2 (from Dr. Peter Cresswell) binds to HLA-A2 and -A69 complexes but not to the free heavy chains(8) . Calnexin-reactive monoclonal antibody AF8 was obtained from Dr. Michael Brenner(9) . UCSF#2, a serum obtained by immunization of a rabbit with carrier-conjugated peptide from the cytoplasmic domain of HLA-A2 (residues 316-340), recognizes assembled and unassembled class I heavy chains (10) and was provided by Dr. Bruce Koppelman. Anti-56-69 serum reacts with denatured HLA-A2 and HLA-B17 heavy chains (11) and was obtained from Dr. Peter Parham. Anti-invariant chain antibody (PIN1) was from Dr Peter Cresswell(12) . Antisera reactive with human beta(2)m and mouse immunoglobulin were purchased from Sigma.

Site-directed Mutagenesis and Transfections

A cDNA clone encoding HLA-A*0201 subcloned into the HindIII and SalI sites of M13mp18 was obtained from Dr. Peter Parham (13) . Mutants with substitutions of Ser to Ala at position 88 (S88A) and Gln to Lys at position 242 (Q242K) of the protein were introduced by site directed mutagenesis using the method of Kunkel(14) . A mutant bearing both substitutions (S88A/Q242K) was generated by a second round of mutagenesis using the S88A single mutant. Sequences of the mutants were confirmed by dideoxy sequencing. SalI- and HindIII-purified inserts were then subcloned into corresponding sites in the vector pREP10 (Invitrogen Corp, San Diego, CA). Stable transfectants were isolated by electroporation and selection in hygromycin(10) .

Metabolic Labeling and Gel Electrophoresis

Cells were washed in Met-free Cys-free RPMI 1640 (deficient medium) and then incubated for 1 h at 37 °C. Cells were labeled with 150 µCi of TranS-label. Ten volumes of 10% RPMI 1640 with cysteine and methionine (chase medium) were added, and aliquots were removed at times indicated and stored on ice. Following one wash in 0.01 M Tris, 0.15 M NaCl, pH 7.4 (Tris-buffered saline), at 4 °C, cells were lysed in Tris-buffered saline containing 1% CHAPS (v/v), 0.1 mMN-p-tosyl-L-lysine chloromethyl ketone, and 1 mM phenylmethylsulfonyl fluoride for 20 min on ice. Lysates were centrifuged at 13,000 times g for 5 min and precleared at 4 °C by the addition of 30 µl of a 10% suspension of formalin-fixed Staph A for 1 h. Samples were split and incubated with 1 µl of antiserum or 50 µl of hybridoma supernatant for 60 min at 4 °C. Antigen-antibody complexes were isolated with 20 µl of 50% suspension of protein A-Sepharose beads. After washing in 0.5% CHAPS-Tris-buffered saline, beads were eluted with reducing gel buffer for 5 min at 95 °C.

Gel Electrophoresis

Samples were separated on isoelectric focussing tube gels with pH 4.5-8.0 gradients. SDS-PAGE samples were reduced and separated on 12% slab gels as described(10) . For two-dimensional gel electrophoresis, isoelectric focussing gels were placed upon SDS-PAGE slabs. Fluorography and scanning densitometry was performed as described(15) .

Endo H Treatment

Beads bound to antigen-antibody complexes were resuspended in 0.5 ml of phosphate-buffered saline, pH 7.0. Endo H (0.3 units) was then added for 16 h at 37 °C. Mock-treated samples were incubated at either 4 or 37 °C. Beads containing antibody-antigen complexes were pelleted and then eluted with reducing sample buffer.


RESULTS

Binding of Calnexin to a Temperature-sensitive Mutant of HLA-A2

Previous studies have shown that class I heavy chains in Daudi cells lacking beta(2)m have prolonged association with calnexin, whereas in human cells with both beta(2)m and heavy chains, no binding to calnexin was evident(2, 16) . We showed that HLA-A2 during its initial folding in CIR cells does not associate measurably with calnexin. (^2)These data suggest that HLA-A2 heavy chains fold very rapidly and either do not bind calnexin or dissociate too rapidly for binding to be detected.

To further investigate binding of class I HLA heavy chains to calnexin, we studied a mutant (Q242K) of HLA-A2 with substitution of lysine for glutamine at position 242, a residue in the interface between the alpha3 domain and beta(2)m(17) . The mutation weakens association of the heavy chain with beta(2)m and inhibits beta(2)m binding and subsequent transport at 37 °C(10) . The class I HLA phenotype of CIR cells transfected with Q242K is thus similar to Daudi cells, as heavy chains do not assemble with beta(2)m and are retained in the ER.

Q242K heavy chains co-isolate with calnexin at 37 °C, in contrast to wild-type HLA-A2 molecules (Fig. 1). This indicates that the mutation both inhibits association with beta(2)m and results in strong binding to calnexin. Q242K heavy chains remain bound to calnexin after 1 h, whereas class II MHC-associated invariant chains dissociate more rapidly, suggesting that Q242K heavy chains are stabilized by calnexin.


Figure 1: Substitution at position 242 in the alpha3 domain of HLA-A*0201 results in prolonged association with calnexin. CIR cells transfected with Q242K (A) and wild-type (B) genes were radiolabeled for 30 min (A) or 10 min (B) and then incubated in chase medium for the times indicated at 37 °C. After lysis, proteins were isolated with antibodies and separated on SDS-PAGE. In A, bands migrating below Q242K in the AF8-precipitated lanes correspond to invariant chain.



Binding of HLA-A2 Heavy Chains to Calnexin in Daudi Cells

Experiments with Q242K suggest that our inability to detect binding of wild-type HLA-A2 heavy chains to calnexin in CIR transfectants is due to rapid folding and assembly with beta(2)m. To further test this possibility, we transfected Daudi cells with HLA-A*0201. Transfectants were radiolabeled, and calnexin or class I proteins were isolated with antibodies AF8 or UCSF#2, respectively. Two-dimensional gel electrophoresis was used to separate endogenous from transgene-encoded heavy chains. Fig. 2demonstrates that both endogenous and HLA-A2 heavy chains are calnexin-associated. Specific antibodies were used to confirm that HLA-A2 heavy chains migrate at the indicated position (data not shown).


Figure 2: HLA-A*0201 encoded heavy chains in Daudi cells associate with calnexin. Transfected or untransfected Daudi cells were radiolabeled for 2 h. After lysis, proteins were isolated with anti-calnexin (AF8) or anti-H chain (UCSF#2) antibodies and analyzed on isoelectric focussing gels, with separation from acidic (left) to basic (right) followed by SDS-PAGE. Endogenous Daudi heavy chains are indicated by largertriangles. Smallertriangles mark A2 heavy chains in A2-Daudi or the corresponding position in Daudi samples. Invariant chains co-precipitating with calnexin are labeled I.



Role for the N-Linked Glycan of HLA-A2 in Binding to Calnexin

As calnexin associates with unfolded proteins, it is likely that it has a binding site for polypeptide determinants. In addition, there is evidence that calnexin recognizes N-linked glycans on newly synthesized proteins. To test this possibility, a substitution at position 88 of alanine for serine was introduced into HLA-A*0201. This mutation alters a glycosylation recognition sequence in the alpha1 domain and prevents attachment of core saccharide to asparagine 86, the single site of glycosylation in class I HLA heavy chains. The mutated gene (S88A) was transfected into CIR and Daudi cells. In addition, the S88A mutation was combined with Q242K and the double mutant (S88A/Q242K) transfected into CIR cells.

In transfected CIR cells, S88A assembled with beta(2)m, although somewhat less efficiently than the wild-type molecule, and was expressed at the cell surface at approximately 50% of wild-type levels (data not shown). No binding to calnexin was apparent (Fig. 3), as observed for the wild-type molecule. The double mutant, S88A/Q242K, which is not glycosylated and does not assemble with beta(2)m at 37 °C, was synthesized in transfected CIR cells, but in contrast to singly mutated Q242K, it did not associate with calnexin (Fig. 3B).


Figure 3: Class I HLA heavy chains mutated at the glycosylation site do not bind calnexin. Transfectants expressing mutants S88A (A) or S88A/Q242K (B) were radiolabeled for 10 min (A) or 30 min (B) and then incubated in chase medium for the indicated times. Proteins were isolated from cell lysates with antibodies and separated by SDS-PAGE. Heavy chains lacking glycans migrate faster than endogenous HLA-C*0401 and HLA-B*3503 molecules expressed at low levels in CIR cells(21) .



In transfected Daudi cells, HLA-A2 heavy chains have a prolonged association with calnexin, due apparently to a lack of beta(2)m. The nonglycosylated mutant S88A did not bind calnexin (Fig. 4), although the protein was stably expressed, and could be isolated with UCSF#2 or anti-56-69 sera. These results provide strong evidence that the N-linked glycan at position 86 is important for class I binding to calnexin.


Figure 4: HLA-A2 heavy chains mutated at the site of glycosylation do not associate with calnexin in Daudi cells. Daudi cells and transfectants were radiolabeled for 2 h before lysis and isolation of proteins with indicated antibodies. Positions of full-length endogenous heavy chains (CLASSI) and the glycan-deficient mutant heavy chains (88A) are marked.



Castanospermine Blocks Binding of HLA-B7 to Calnexin

Previous work from our laboratory using CIR transfectants showed that HLA-B7 heavy chains had a relatively stable association with calnexin(15) . We therefore asked whether calnexin required oligosaccharide for its binding to a second class I HLA molecule. HLA-B7-CIR cells were treated before pulse labeling for 3 min with castanospermine, an inhibitor of glucose trimming shown previously to inhibit calnexin-substrate interactions(6) . The drug was effective in blocking glucose trimming, causing a pronounced shift in gel mobility of invariant chain (containing two asparagine-linked glycans(18) ) and to a lesser extent in HLA-B7 (Fig. 5A). Both proteins showed decreased association with calnexin in increasing amounts of castanospermine, with 70-80% maximal inhibition observed (Fig. 5B). In this experiment, cells were lysed immediately after the labeling period and before assembly with beta(2)m occurs to any significant extent. (^3)We are currently addressing whether those heavy chains, which do not bind calnexin, are degraded rapidly or can associate with beta(2)m.


Figure 5: Castanospermine inhibits the interaction between calnexin and substrate proteins. CIR cells transfected with HLA-B*0702 were incubated for 45 min with the indicated amounts of castanospermine before labeling for 3 min. Cells were lysed, and proteins were isolated with antibodies as indicated. SDS-PAGE analysis is shown in A, with positions of HLA-B7 heavy chains (B7) and invariant chains (I) marked. In B, scanning densitometry of the results in A are shown. Circles and squares indicate invariant chain and B7 proteins, respectively. Opensymbols show calnexin-associated molecules, and closedsymbols show total amounts of each protein. Relative optical densities (R.O.D.) were calculated from individual optical density values as a percentage of the value obtained with no drug added.



It is notable that calnexin associates with slower migrating invariant chains induced by castanospermine, a result consistent with partial inhibition of trimming. There is no indication, however, that HLA-B7 molecules, which contain a single glycan, show a similar effect. This suggests that trimming of glucose residues from the glycan of class I HLA heavy chains occurs before binding calnexin.

Evidence That N-Glycan Is Not Required to Maintain Association between Calnexin and Class I Heavy Chains

We next asked whether removal of glycans disrupts the interaction between calnexin and bound proteins. Cells were radiolabeled for 2 h, and calnexin and associated proteins were isolated with AF8 and beads. Beads were next treated directly with Endo H. Antigen-antibody complexes were then dissociated and separated by SDS-PAGE. Fig. 6A shows that Endo H removed the single glycan of class I heavy chains isolated in association with calnexin from Daudi cells. Q242K heavy chains bound to calnexin from CIR transfectants were also sensitive to digestion (Fig. 6B), as was invariant chain protein from both cell lines. This demonstrates that glycans on substrate proteins are accessible after binding to calnexin, and suggest that carbohydrate may play a role in initiating rather than maintaining interaction with calnexin.


Figure 6: Endo H digestion indicates that glycans are accessible on class I HLA heavy chains bound to calnexin. Daudi cells (panelA) and Q242K CIR cells (panelB) were radiolabeled for 2 h and lysed, and proteins were isolated with either UCSF#2 or AF8 antibodies. Samples bound to protein A-beads were then incubated for 16 h without Endo H at 4 °C (lanesA), 37 °C (lanesB), or with Endo H at 37 °C (lanesC), before elution and separation on SDS-PAGE. The positions of Endo H resistant and sensitive forms of class I heavy chains and invariant chains are marked.




DISCUSSION

Calnexin is a calcium-binding transmembrane protein found in the ER, which associates with many newly synthesized, unfolded polypeptides (4) . Upon proper folding, proteins dissociate from calnexin, leave the ER, and continue through the secretory pathway. This suggests that calnexin functions to prevent export of unfolded proteins and perhaps to assist in the folding or assembly process as a chaperone. Strong evidence that calnexin functions to retain incompletely folded proteins in the ER comes from studies showing that truncation of the cytoplasmic domain of calnexin does not interfere with its binding to substrate proteins but causes mislocalization of complexes within the cell(1) . In addition, mouse class I heavy chains and beta(2)m, which form dimers and are transported in Drosophila cells, move to the plasma membrane more slowly in the presence of calnexin(5) .

We investigated the structural basis for calnexin binding to class I HLA heavy chains in CIR and Daudi cells. HLA-A2 heavy chains in CIR cells do not interact detectably with calnexin, an observation that can be explained by rapid folding and assembly with beta(2)m. Substitution at position 242 of the heavy chain, shown previously to inhibit pairing with beta(2)m(10) , resulted in strong binding to calnexin, consistent with this interpretation. Likewise, HLA-A2 heavy chains in Daudi cells, which lack beta(2)m, associate with calnexin. A mutant with substitutions at positions 88 and 242, which interfere with glycosylation and beta(2)m binding, respectively, did not bind calnexin, and the single S88A mutant did not bind calnexin detectably in Daudi transfectants. These data support the hypothesis that calnexin is sensitive to both folding and glycosylation of substrate proteins.

Drugs that block trimming of glucose residues from high mannose glycans on substrate proteins were shown by others and now by us to inhibit interaction with calnexin(6) . This led Hammond and co-workers (6, 19) to propose a model for calnexin function in which monoglucosylated substrate proteins that bind to calnexin are released upon partial folding and then are reglycosylated by the enzyme UDP-glucose:glycoprotein glycosyltransferase(6, 19) . After trimming to the monoglucosylated form, the protein would rebind calnexin and continue to cycle through the pathway. Once folding is complete, a protein is no longer a substrate for UDP-glucose:glycoprotein glycosyltransferase (20) and therefore would not bind calnexin and be retained in the ER. In this model, calnexin need serve only as a lectin, since folded proteins are not substrates for UDP-glucose:glycoprotein glycosyltransferase. Alternatively, calnexin might recognize both carbohydrate and unfolded regions of proteins.

Our data suggest that calnexin is not simply a lectin, since the glycan of class I heavy chains is accessible to Endo H digestion after binding. This implies that calnexin interacts with determinants on the protein. Furthermore, since the folding and glycosylation double mutant S88A/Q242K does not interact with calnexin, while the folding single mutant Q242K does, it seems unlikely that calnexin recognizes only determinants that disappear concomitantly with folding and further glycan processing. We propose that calnexin functions both as a lectin and as a chaperone with a binding site for peptides. Cleavage by Endo H of the glycan on substrate proteins after calnexin binding suggests that the interaction might be initiated via the glycan, and then maintained by recognition of determinants on the protein.


FOOTNOTES

*
This work was supported by American Cancer Society Grant IM-668 and by a student fellowship from 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. Tel.: 412-648-9471; Fax: 412-648-1916.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; MHC, major histocompatibility complex; Endo H, endo-beta-N-acetylglucosaminidase H; PAGE, polyacrylamide gel electrophoresis; beta(2)m, beta(2)-microglobulin; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate.

(^2)
M. Tector and R. D. Salter, submitted for publication.

(^3)
Q. Zhang, M. Tector, and R. D. Salter, unpublished observations.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.