©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Requirements for the Interaction of Class II Major Histocompatibility Complex Molecules and Invariant Chain with Calnexin (*)

(Received for publication, September 27, 1994; and in revised form, November 22, 1994)

Balasubramanian Arunachalam Peter Cresswell (§)

From the Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Molecular chaperones are believed to retain misfolded and incompletely assembled oligomeric proteins in the endoplasmic reticulum (ER). Here, we have further analyzed the association of one such chaperone, calnexin, with human major histocompatibility complex class II alpha and beta subunits and the invariant chain. Calnexin associates with transport-competent invariant chain trimers (p33 or p41), as well as ER-retained trimers (p35/33 or p43/41), suggesting that ER retention of the latter is not because of calnexin association. Neither the replacement of the transmembrane segment of the DRbeta subunit with a glycosyl phosphatidylinositol anchor nor deglycosylation of any of these proteins with tunicamycin or endoglycosidase H treatment abolished calnexin association. Using a cell-permeabilization system, we were unable to observe association of newly synthesized glycopeptides with calnexin, arguing that calnexin may not act like a simple lectin for association with glycoproteins. The results indicate that neither transmembrane regions nor N-linked glycans are exclusively responsible for calnexin association. Based on our data and the observations of others, we suggest that these features may have varying significance for different glycoproteins in determining their interaction with calnexin.


INTRODUCTION

Proteins synthesized within the endoplasmic reticulum (ER) (^1)undergo folding, assembly, and a variety of modifications before they are secreted or transported to various compartments within cells. Proper conformational maturation of such proteins is vital for their function (1, 2, 3) and is facilitated by an oxidative environment, favoring disulfide bond formation, and the presence in the ER of molecular chaperones(4, 5, 6) . Misfolded proteins and incompletely assembled oligomeric proteins are often retained and degraded in the ER(4) . Several monomeric and multimeric proteins in the ER have been shown to transiently associate with resident ER proteins, such as BiP/GRP78 and GRP94, collectively known as chaperones (reviewed in (6) and (7) ). Association of different proteins with such chaperones is transient, and their dissociation coincides with the folding, assembly, and transport of proteins(8) . The chaperones are believed to exert a control on the interactions within and between polypeptides, thus limiting the formation of incorrect structures. Moreover, they are thought to retain improperly folded and assembled proteins in the ER. One of the newly emerging chaperones, calnexin (also known as IP90 or p88), has been found in association with unassembled Ig, T cell receptor, and major histocompatibility complex (MHC) class I and class II subunits(9, 10, 11, 12, 13, 14) , newly synthesized viral glycoproteins(15) , and monomeric secretory glycoproteins(16) . Calnexin is a nonglycosylated type I integral membrane protein with an apparent molecular mass of 90 kDa by SDS-PAGE(10, 11, 12, 17) .

Recently, we reported the association of calnexin with MHC class II alpha and beta chains, the associated invariant chain, and partially assembled alphabeta-invariant chain complexes(14) . There are four forms of invariant chain (p33, p35, p41, and p43) in humans generated by the alternative initiation of translation (p33 versus p35 and p41 versus p43) and alternative splicing (p33 versus p41 and p35 versus p43)(18, 19) . In the absence of class II, p33 and p41 forms, which lack the N-terminal 16 residues, exit the ER, whereas the longer forms (p35 and p43) are retained(20, 21, 22) . Similarly, class II alpha and beta chains expressed alone remain in the ER. In normal circumstances, cells expressing all four isoforms of invariant chain generate mixed trimers that are retained in the ER(22, 23) . Each invariant chain subunit is thought to bind to one MHC class II alphabeta dimer, forming a transport-efficient nonamer complex through a series of intermediates(24, 25) . Addition of the alpha and beta chains is believed to mask the invariant chain ER retention signal, resulting in transport of the complex through the Golgi apparatus into the endocytic pathway. Calnexin associates with these chains individually and remains associated until the complete nonamer is formed(14) .

Chaperones associate with a variety of newly synthesized proteins perhaps by recognizing some common feature(s). Recently, BiP has been shown to preferentially bind peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions(26) . Attempts have been made to find the feature that is recognized by calnexin. Using various truncated mutants of MHC class I heavy chain, Margolese et al.(27) showed that replacement of the transmembrane segment and cytoplasmic domains of class I heavy chain with a glycosyl phosphatidylinositol anchor resulted in loss of calnexin association. However, the same group observed that a soluble, truncated mutant of T-cell antigen receptor (TCR) alpha chain lacking a transmembrane region retained a trace level of association with calnexin. Ou et al.(16) reported that preventing the glycosylation of secretory glycoproteins by tunicamycin treatment of cells abolished most calnexin association. Recently, Hammond et al.(15) reported that altering the glycosylation state of influenza hemagglutinin and vesicular stomatitis virus G protein, either by tunicamycin treatment or by inhibiting the trimming of N-linked glycans by glucosidase inhibitors, resulted in complete loss of calnexin association, suggesting that calnexin may interact with the N-linked carbohydrates of newly synthesized glycoproteins. Here, we have analyzed this question using MHC class II alpha and beta subunits and the associated invariant chain and report that neither the transmembrane region nor N-linked glycosylation of these proteins is exclusively responsible for their association with calnexin. However, both play a significant role. Based on our data and existing information in the literature, we propose that various features on glycoproteins are recognized by the calnexin. The transmembrane region, glycosylation state, or unidentified feature(s) may have varying significance for different proteins in establishing a detectable interaction, explaining the conflicting observations with different proteins.


MATERIALS AND METHODS

Cell Lines

HeLa cells, the human B-lymphoblastoid cell line Swei, and the MHC class II-negative TxB hybrid line 174 X CEM.T2 (T2) were maintained at 37 °C in Iscove's modified Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% fetal calf serum (FCS; Hyclone, Logan, UT) and 20 µg/ml gentamicin.

Antibodies and Plasmids

The anti-calnexin monoclonal antibody AF8 was the generous gift of Dr. M. Brenner(12) . The monoclonal antibodies PIN.1 (anti-invariant chain, (24) ), DA6.147 (anti-HLA-DRalpha chain, (28) ), and XD5.A11 (anti-class II beta chain, (29) ) have been described. Rabbit antiserum R114B3 specific for the chicken hepatic lectin (CHL) was obtained from Dr. K. Drickamer (30) . cDNAs encoding both the p33 and p35 forms of human invariant chain(31) , p41 and p43 forms of human invariant chain, HLA-DRalpha chain (32) , HLA-DRbeta(1) chain(33) , and CHL (34) were obtained from the respective groups. cDNAs encoding only p33 and p41 forms of human invariant chain were generated by mutating the ATG initiating p35 and p43 translation into CTG on p35/33 and p43/41 cDNAs, respectively, by the Kunkel method (35) using the Bio-Rad Muta-gene kit (Bio-Rad). All the cDNAs were cloned into the pAR2529 vector under the control of the phage T7 RNA polymerase promoter (36) and are referred pAR.p35/33, pAR.p33, pAR.43/41, pAR.p41, pAR.CHL, pAR.DRalpha, and pAR.DRbeta. The cDNA construct (GPI-DRbeta(1)) coding for a chimeric protein comprising the first two extracellular domains of DRbeta1 fused with the glycosyl phosphatidylinositol (GPI) linker derived from human placental alkaline phosphatase was a gift from Drs. Ed Collins and D. C. Wiley and was cloned in pcDNAI/Neo (Invitrogen) plasmid under the control of the phage T7 RNA polymerase promoter.

Peptides and Peptide Iodination

The glycosylation substrate (RRYQNSTEL) was previously described(37) . It was synthesized by Quality Controlled Biochemicals (Hopkinton, MA). The peptide was purified by high pressure liquid chromatography and shown to be single species by mass spectrometry. Iodination (I) of the peptide was performed as described(38) .

Transient Expression of Proteins

Invariant chain isoforms, CHL, DRalpha, DRbeta1, and GPI-DRbeta1 were transiently expressed in HeLa cells using a vaccinia-based expression system. Cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase and then transfected with plasmids carrying cDNA for the proteins of interest under the control of phage T7 promoter. Replication of virus in cells results in the synthesis of T7 RNA polymerase, which in turn enhances the synthesis of mRNA from DNA that is under T7 promoter. Transient expression of specific proteins using recombinant vaccinia was achieved by following the protocol described earlier(21) . In brief, subconfluent HeLa monolayer cultures were infected with vTF7-3 suspended in Opti-MEM at a multiplicity of infection of 20 for 30 min at 37 °C. Excess virus was removed, and the infected cells were transfected with cDNAs in Opti-MEM using Lipofectin (Life Technologies, Inc.). 3 h after infection, cells were cultured in medium containing 10% FCS.

Metabolic Labeling and Immunoprecipitations

Cells (10^6-10^7) expressing specific proteins were deprived of methionine by incubation in methionine-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 3% dialyzed FCS for 1 h and then pulsed with [S]methionine (ICN Biochemicals, Costa Mesa, CA) (0.25-0.5 mCi) for 15-30 min. For chasing, the cells were washed after labeling and cultured in medium containing 10% FCS and 15-fold excess of non-radioactive methionine for different periods of time. Cells were extracted in 150 mM NaCl, 10 mM Tris, pH 7.4 (TS), containing 1% digitonin (Wako, Richmond, VA), 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mMN-p-tosyl-L-lysine chloromethyl ketone, and 5 mM iodoacetamide (IAA). Post-nuclear supernatants were precleared overnight with normal rabbit serum and protein G-Sepharose (Pharmacia Biotech. Inc.) and were then precipitated with specific antibody and protein G-Sepharose. Pellets were washed three times with TS, 0.1% digitonin.

Elution and Reprecipitation of Immunoprecipitates

Proteins bound to specific antibody in the first immunoprecipitation were eluted under denaturing conditions by adding 100 µl of TS containing 2% SDS with or without 2 mM dithiothreitol (DTT) and boiling for 5 min. The supernatants were diluted with 1 ml of TS, 1% Triton X-100, 10 mM IAA, incubated for 45 min at 25 °C, and then reprecipitated with antibody and protein G-Sepharose as described above. The precipitates were washed with TS, 0.1% Triton X-100 and analyzed by SDS-PAGE. Native invariant chain was eluted from anti-calnexin immunoprecipitates by suspending the immunoprecipitates in 0.5 ml of 0.13 M NaCl, 0.02 M bicine, pH 8.2 (BS), containing 1% polyoxyethylene 9 lauryl ether (CE(9)). After 15 min at 25 °C, the beads were pelleted and re-eluted with 0.5 ml of 1% CE(9)/BS. The supernatants were pooled and cross-linked by adding dithiobis (succinimidyl propionate) (DSP) (Sigma) to 200 µg/ml, quenched with glycine at a final concentration of 10 mM, and then precipitated as above.

Endoglycosidase H Treatment

Calnexin and the associated glycoproteins in a digitonin extract of cells were precipitated with the anti-calnexin antibody (AF8) and protein G-Sepharose. Non-calnexin-associated proteins were subsequently precipitated from the same extract with specific antibody and protein G-Sepharose. These protein G-Sepharose beads were suspended in 150 µl of 20 mM sodium phosphate buffer, pH 6.5, with or without 4.5 milliunits of endoglycosidase H. After 16 h of incubation at 37 °C, pellets and supernatants were analyzed for the presence of specific protein. In brief, proteins were eluted from washed beads under reducing condition and precipitated with a conformation-independent antibody specific for the protein expressed as described above. Similarly, protein from the supernatant was precipitated, and all proteins were analyzed by SDS-PAGE (12% acrylamide).

Chemical Cross-linking in Cell Extracts

Metabolically labeled cells were extracted in 1% CE(9)/BS containing 200 µg/ml DSP for 30 min at 4 °C. The cross-linking reaction was quenched with glycine at a final concentration of 10 mM, and then proteinase inhibitors were added. Precipitation was carried out as mentioned earlier, and samples were analyzed under reducing (10.5%) or non-reducing (6%) one-dimensional SDS-PAGE gels.

Electrophoresis

SDS-PAGE was performed as described(23) . ^14C-Labeled molecular weight markers (Amersham Corp.) were used.

Translocation of Radiolabeled Peptides into the ER

The plasma membrane of Swei cells were permeabilized using activated streptolysin O (Murex, Norcross, GA), as previously described(39, 40) . In brief, cells were washed with cold serum-free medium and incubated with activated streptolysin O for 3 min. Permeabilized cells were resuspended in 1 ml of freshly prepared intracellular transport buffer (50 mM Hepes, 78 mM KCl, 4 mM MgCl(2), 8.37 mM CaCl(2), 10 mM EGTA, 1 mM dithiothreitol, pH 7.0) and incubated for 10 min at 37 °C with the I-labeled peptide derived from human histone at 50 nM final concentration. The peptide with an amino acid sequence RRYQNSTEL contains an N-glycosylation acceptor site. Cells were then washed once in 1 ml of fresh intracellular transport buffer and extracted in 1 ml of 1% digitonin/TS. The extract was precipitated with concanavalin A-Sepharose beads (Sigma) or using different antibodies followed by Protein G-Sepharose beads. The pellets were washed three times in 1 ml of 0.1% digitonin/TS and counted in a counter (LKB Compugamma CS, Turku, Finland).


RESULTS AND DISCUSSION

Association of Human Invariant Chain Isoforms with Calnexin

Recently, Jackson et al.(41) showed that association of calnexin with murine class I heavy chain or peptide-deficient heavy chain-beta2m heterodimers slowed their intracellular transport. Rajagopalan et al., using full-length and truncated versions of calnexin, demonstrated that the intracellular localization of associated CD3 (42) or class I heavy chain (43) is determined by the associated calnexin. These groups suggested that calnexin acts as an ER-retention molecule for incompletely folded or misfolded proteins in the ER. An initial question we asked was whether calnexin acts as an ER-retention molecule for invariant chain by its specific association with the ER-retained forms p35 and p43 and not with the transport-efficient forms p33 and p41(22) . To determine whether each form of invariant chain can associate with calnexin, we expressed them individually using the vaccinia virus-based expression system in HeLa cells. Cells were radiolabeled 8 h post-infection with [S]methionine for 30 min. They were extracted in 1% digitonin/TS and immunoprecipitated with anti-calnexin (AF8) (Fig. 1, lanes1 and 3) and anti-invariant chain (PIN.1) (Fig. 1, lane2) antibodies. Bound proteins were stripped from protein G beads under denaturing conditions using DTT and SDS and reprecipitated with control antibodies (Fig. 1, lane1) or conformation-independent anti-invariant chain antibodies (Fig. 1, lanes2 and 3). While there is no band seen with the control antibody (Fig. 1, lane1), anti-invariant chain antibody brought down the invariant chain (Fig. 1, lane2), showing the specificity of the technique. Proteins eluted from the anti-calnexin antibody included invariant chain, which could be reprecipitated with specific antibody (Fig. 1, lane3). The presence of bands corresponding to invariant chain in lane3 of all of the panels clearly demonstrates that all of the isoforms of invariant chain associate with calnexin. The ratio of calnexin-bound invariant chain remains more or less the same with all the isoforms (data not shown). If calnexin were the major retention molecule keeping the p35 and p43 in the ER, its association with the transport-efficient p33 and p41 forms would not be expected. The association of calnexin with all of the forms suggests that calnexin is not the major ER-retention factor for the invariant chain. p33 and p41 forms of invariant chain presumably attain a transport-competent conformation, resulting in their release from calnexin. The presence of the additional 16 residues in the N-terminal domain of p35 and p43 perhaps prevents these forms from attaining a transport-competent conformation. Alternatively, a secondary mechanism involving the interaction of the added N-terminal sequence with a cytosolic molecule may be responsible for the retention of p35 and p43.


Figure 1: Association of invariant chain isoforms with calnexin. HeLa cells transiently expressing invariant chain isoforms (p33, p35/33, p41, p43/41) were pulsed with [S]methionine for 30 min. The cells were extracted in 1% digitonin/TS and immunoprecipitated with anti-calnexin (lanes1 and 3) or anti-invariant chain (lane2) antibody. Proteins were eluted from the protein G-Sepharose beads with TS, 2% SDS, and 2 mM DTT, diluted with TS, 1% Triton X-100, and 10 mM IAA, and reprecipitated with control (lane1) or conformation-independent anti-invariant chain antibodies (lanes2 and 3). Precipitates were subjected to SDS-PAGE (10.5%) under reducing conditions. The position of molecular mass markers (kDa) are indicated on the left. Discrete bands representing p43 and p41 are not seen due to lack of resolution in 10.5% gels.



Cross-linking of Invariant Chain and Its Associated Proteins

We previously showed that the invariant chain forms trimers that may contain different isoforms(22) . In cross-linking experiments, we observed two prominent bands containing invariant chain. One had a molecular weight consistent with a trimer (I(3)) and the other a potential hexamer (I(6)) of invariant chain(22) . It was also possible that this higher molecular weight species could result from calnexin association. We tested this using T2 cells, a mutant cell line expressing invariant chain but not MHC class II, and the results are shown in Fig. 2. Cells were metabolically labeled with [S]methionine for 15 min and extracted in 1% CE(9)/BS containing DSP. The precleared extract was immunoprecipitated with anti-calnexin (lanes1 and 3) or anti-invariant chain (lanes2 and 4) antibodies. The primary precipitates were eluted under non-reducing condition using SDS, and the supernatants reprecipitated with control (lane1), anti-invariant chain (lanes2 and 3), or anti-calnexin (lane4) antibodies. Samples were analyzed in 6 and 10.5% gels under non-reducing and reducing conditions, respectively. When the primary and secondary precipitations were performed with anti-invariant chain antibodies, two bands were observed under non-reducing conditions (upperpanel, lane2). They had molecular masses of approximately 90 and 180 kDa, respectively. When the anti-calnexin antibody was used for primary (lane3) or for secondary precipitations (lane4), only the 180-kDa band was observed. We and others (16, 22, 44) have observed that metabolically labeling calnexin is difficult due to its low turnover rate, which is why the invariant chain bands and not the calnexin band can be seen in lane4 in the reducing gel. The results argue that the higher molecular weight band represents an I(3)-calnexin complex. We obtained similar results using HeLa cells transiently expressing invariant chain isoforms (data not shown).


Figure 2: Association of calnexin with invariant chain trimers. T2 cells were pulsed with [S]methionine for 15 min, and the cells were extracted in 1% CE(9)/BS with 200 µg/ml DSP. The reaction was quenched with glycine, and proteins were immunoprecipitated with anti-calnexin (lanes1 and 3) or anti-invariant chain (lanes2 and 4) antibodies. The proteins were stripped from the protein G-Sepharose beads using 2% SDS and reprecipitated with control (lane1) or anti-invariant chain (lanes2 and 3) or anti-calnexin (lane4) antibodies. Precipitates were subjected to non-reducing (6%, upperpanel) or reducing (10.5%, lowerpanel) SDS-PAGE. The lower and upperarrow heads indicate positions of I(3) and I(3)-calnexin, respectively. Molecular mass markers (kDa) are indicated on the left.



We followed different strategies to determine whether the 180-kDa band is solely the result of an I(3)-calnexin complex or if it contains some genuine invariant chain hexamers. Initially, we attempted to preclear calnexin from cross-linked extracts by sequential immunoprecipitation using anti-calnexin antibodies. However, we were unable to completely deplete the sample of the 180-kDa band, perhaps because the antibody did not recognize all of the calnexin-containing complex (data not shown). Another approach was based on the observation that calnexin-invariant chain association is retained in digitonin and not in CE(9) or Triton X-100(14) . Here, we labeled invariant chain-positive, class II-negative T2 cells for 15 min and extracted them in 1% digitonin/TS. Proteins were precipitated with anti-calnexin antibody (Fig. 3, lanes1 and 2) and dissociated from calnexin using CE(9). They were then cross-linked with DSP. Dissociated materials were reprecipitated with control (Fig. 3, lane1) or anti-invariant chain (Fig. 3, lane2) antibodies. Samples were analyzed in 6 and 10.5% gels under non-reducing and reducing conditions, respectively. This procedure yields less material than stripping with SDS. However, unlike in Fig. 2, only the 90-kDa trimer band and no 180-kDa band was observed (Fig. 3, lane2) under non-reducing conditions. This suggests that no hexameric invariant chain associates with calnexin. In addition, preliminary experiments in which invariant chain multimers were studied in a calnexin-negative mutant cell line showed only the 90-kDa trimer band (data not shown). We therefore believe that the invariant chain does not form genuine hexamers.


Figure 3: Dissociation of calnexin from I(3) results in loss of I(6)-like complex. T2 cells were pulsed with [S]methionine for 15 min, and the cells were extracted in 1% digitonin/TS. Proteins were precipitated with anti-calnexin antibody; the proteins in their native conformation dissociated from calnexin using 1% CE(9)/BS and then cross-linked with DSP. Dissociated proteins were reprecipitated with control (lane1) or anti-invariant chain (lane2) antibodies. Precipitates were subjected to non-reducing (6%, upperpanel) or reducing (10.5%, lowerpanel) SDS-PAGE. The arrowhead indicates position of I(3). The position of molecular mass markers (kDa) are indicated on the left.



Role of Protein Glycosylation in Calnexin Association

Calnexin associates rapidly with newly synthesized DRalpha, beta, and invariant chains. Invariant chain is a type II membrane protein with two N-linked glycans, and DRalpha and beta chains are type I membrane proteins with two and one N-linked glycans, respectively. We expressed individually these proteins and another type II membrane protein, CHL, in HeLa cells and treated the cells with tunicamycin to determine how critical N-linked glycosylation is for their association with calnexin. Here, precipitation and reprecipitation of proteins were carried out as in Fig. 1, and the results are shown in Fig. 4. Cells were extracted and precipitated with anti-calnexin antibody (lanes1 and 3) followed by control antibody (lane1) or a conformation-independent antibody specific for the protein expressed (lane3). In lane2, the cell extract was both initially precipitated and reprecipitated with a conformation-independent antibody specific for the protein expressed. Precipitation with anti-calnexin antibody followed by antibody specific for protein expressed (lane3) demonstrated the association of the proteins with calnexin. With all of the proteins tested, in the absence of tunicamycin treatment, 10-30% of the total were found in association with calnexin (compare lanes2 and 3). This may be an underestimate due to overexpression of the proteins relative to calnexin, inhibition of host protein synthesis by vaccinia virus, and/or an inherent problem with the technique and/or reagents used in the experiments. In wild-type B cell lines, up to 70% of newly synthesized DRalpha chains could be found in association with calnexin(14) , which may suggest that calnexin is limiting in the vaccinia virus expression system used here. Tunicamycin treatment resulted in a shift in the molecular weight of all four proteins, showing the complete absence of N-linked glycans (Fig. 4). Completely unglycosylated invariant chain, CHL, DRalpha, and DRbeta chain still remain associated with calnexin, although the amount of calnexin-associated protein was significantly reduced for CHL and DRbeta (lanes3). Ou et al.(16) observed that the calnexin association of a number of undefined glycoproteins was virtually abolished in tunicamycin-treated hepatoma cells. Hammond et al.(15) reported that the removal of N-linked glycans from the viral proteins influenza hemagglutinin and vesicular stomatitis virus glycoprotein by tunicamycin treatment resulted in complete loss of calnexin association with these proteins. Moreover, they observed that treating cells with inhibitors of glucosidases I and II also resulted in complete loss of calnexin association and proposed that calnexin preferentially associates with glycoproteins bearing a glycosylation intermediate. Recently, Kearse et al.(45) made similar observations for TCR alpha and beta chains.


Figure 4: Association of glycosylated and deglycosylated proteins with calnexin. HeLa cells transiently expressing p33 form of invariant chain, chicken hepatic lectin, or DRalpha or DRbeta1 chain were untreated or treated with tunicamycin (10-20 µg/ml) for 2 h before and during labeling (30 min) with [S]methionine. Cells were extracted in 1% digitonin/TS and precipitated with anti-calnexin (lanes1 and 3) antibodies or antibodies specific for the proteins expressed (lane2). Proteins were eluted from protein G-Sepharose beads as shown for Fig. 1and reprecipitated with control antibodies (lane1) or antibodies specific for the proteins expressed (lanes2 and 3). Precipitates were subjected to reducing (10.5%) SDS-PAGE. The position of molecular mass markers (kDa) are indicated on the left.



The experiments shown in Fig. 4showed that unglycosylated class II MHC proteins do associate with calnexin. We also performed experiments to show that removal of carbohydrate from glycoproteins already associated with calnexin does not dissociate the complex. The results are shown in Fig. 5. Calnexin and associated glycoproteins, bound to anti-calnexin antibody on Protein G-Sepharose beads, were treated or mock-treated with the enzyme endoglycosidase H (leftpanel). Proteins remaining associated with calnexin after endoglycosidase H treatment or mock treatment were eluted from the complex and re-precipitated with specific antibodies (leftpanel). Similarly, we looked for the specific proteins that might have dissociated from calnexin in the supernatants of endoglycosidase H-treated samples. Invariant chain, DRalpha, DRbeta1, and GPI-DRbeta1 were found in the endoglycosidase H-treated pellets (leftpanel). No released proteins were found in the supernatants (data not shown). The reduction in apparent molecular weight of the associated proteins in the endoglycosidase H-treated samples compared with the untreated samples demonstrates the complete removal of carbohydrate from the proteins examined. In parallel, proteins in the calnexin-depleted extracts were precipitated with specific antibodies and treated or mock-treated with endoglycosidase H. We again looked for expressed proteins both in the pellet (rightpanel) and supernatant (data not shown). The majority of the proteins were found in the pellets. These experiments clearly show that despite removal of the N-linked glycans, the proteins remain associated with calnexin. Similar observations have been made by others studying MHC class I-calnexin association. (^2)This is consistent with the observations shown in Fig. 4and argues that the N-linked glycans of the proteins tested here are not essential in the association with calnexin. There is a small amount of endoglycosidase H-undigested GPI-DRbeta1 both in anti-calnexin (leftpanel) and in specific antibody precipitates (rightpanel), suggesting that in some of the molecules the N-linked glycans are inaccessible. Conventionally, endoglycosidase H treatment is performed in the presence of SDS to ensure complete denaturation and accessibility. Digestion in a non-denaturing buffer may explain the lack of complete removal in these particular cases. The reduction in apparent molecular weight after endoglycosidase H treatment in these experiments and in experiments conducted under denaturing conditions is comparable (data not shown). The retention of the calnexin-protein interaction in these experiments also shows that the presence of digitonin, CHAPS, or cholate, normally used to retain the association of glycoproteins with calnexin upon solubilization, is not required to maintain the association.


Figure 5: Endoglycosidase H (Endo H) treatment of calnexin-glycoprotein complexes does not cause their dissociation. HeLa cells transiently expressing invariant chain (p43/41) or DRalpha, DRbeta1, or GPI-DRbeta1 chain were labeled with [S]methionine and extracted in 1% digitonin/TS. Calnexin and the associated glycoproteins were precipitated with anti-calnexin antibodies (leftpanel). Expressed proteins in the calnexin-depleted extracts were precipitated with specific antibodies (rightpanel). Both sets of immunoprecipitates on protein G-Sepharose beads were suspended in 20 mM sodium phosphate buffer, pH 6.5, and treated or mock treated with endoglycosidase H enzyme for 16 h at 37 °C. Presence of expressed proteins both in the pellet and supernatant of processed materials were analyzed as described under ``Materials and Methods.'' The position of molecular mass markers (kDa) are indicated on the left.



To further strengthen the argument that glycosylation of proteins is not critical for their association with calnexin, we followed a different strategy using a permeabilized cell system that we and others recently reported(39, 40) . A I-labeled nonamer peptide that has an N-linked glycosylation acceptor site was introduced in the cytoplasm of streptolysin O-permeabilized Swei cells. The labeled peptides were translocated into the ER through the transporters associated with antigen processing (TAP1 and TAP2). Glycosylation of this peptide allowed its binding with concanavalin A-Sepharose beads (Fig. 6). While concanavalin A-Sepharose beads clearly bound the radioactive glycopeptide, precipitation of calnexin did not bring down any detectable associated radioactive glycopeptide (Fig. 6). This argues against the view that calnexin acts like a simple lectin for association with glycoproteins. Other groups have made similar arguments and speculated that the glycosylation machinery, e.g. the oligosaccharyl transferase or the processing glucosidases, may mediate the interaction between calnexin and glycoproteins(46) . However, in the above experiments, we cannot rule out the possibility that trimming of core saccharide unit on glycopeptides inefficiently generates peptides with monoglucosylated oligosaccharides, which are suggested to be substrates for calnexin (15) .


Figure 6: Lack of association of glycopeptides with calnexin. Swei cells (10^7) were permeabilized with streptolysin O and incubated with labeled peptide (50 nM) for 10 min at 37 °C. Cells were washed and extracted in 1% digitonin/TS and precipitated with concanavalin A (ConA)-Sepharose beads, control (PIN.1), or anti-calnexin (AF8) antibodies. Radioactivity of the precipitate was counted in a counter, and the cpm is shown on the y axis.



Recently, Rajagopalan et al.(42) demonstrated that CD3 chain associates with full-length or truncated forms of calnexin when co-expressed in COS cells. Its ER-retention or transport to the Golgi, lysosomes, and cell surface was shown to be determined by the form of associated calnexin. Interestingly, CD3 chain is naturally unglycosylated. Similarly, viral proteins whose glycosylation acceptor sites were mutationally eliminated have also been found in association with calnexin. (^3)Recently, Kearse et al.(45) observed association of many proteins with calnexin in glucoside II-deficient mutant cell line and in castanospermine-treated wild-type cells, even though association of TCR alpha and beta with calnexin was considerably reduced. Taken together, our results from tunicamycin, endoglycosidase H, and cell-permeabilization experiments and the observations of others suggest that the carbohydrate moieties of many glycoproteins are not essential for calnexin association.

Role of the Transmembrane Region of HLA-DRbeta1 Chain for Its Association with Calnexin

Recently, Margolese et al.(27) mapped the major site of calnexin interaction with MHC class I heavy chains to within a region comprised of transmembrane-spanning segment and three flanking amino acids of the class I heavy chain. They suggested that calnexin, being a membrane-integrated chaperone, utilizes this unique feature for its association with membrane-bound ligands that are unavailable to the soluble chaperone, BiP. The same group using soluble TCR alpha chain did not observe complete loss of calnexin association, however. To study the role of MHC class II transmembrane regions for calnexin association, we used a DRbeta1 construct encoding a protein whose transmembrane segment is replaced with GPI linker derived from human placental alkaline phosphatase. The fusion protein (GPI-DRbeta1) was expressed in HeLa cells and examined for its association with calnexin. The results are shown in Fig. 7. GPI-DRbeta1-calnexin association was still observed but was much less efficient than that of the wild-type DRbeta1 chain (see Fig. 4, lane3). This suggests that while the transmembrane segment of DRbeta1, like its glycosylation, is not an absolute requirement for its association with calnexin, it plays an important role. However, there are numerous soluble proteins that have been found to associate with calnexin(16) . Also, certain transmembrane glycoproteins like CD5 monomer, CD28 homodimer, and transferrin receptor (12, 14) have not been found in association with calnexin, arguing against an absolute requirement for a transmembrane segment in calnexin association.


Figure 7: Association of GPI-DRbeta1 with calnexin. HeLa cells transiently expressing GPI-DRbeta1 were treated with tunicamycin (10 µg/ml) or under control conditions for 2 h before and during labeling (30 min) with [S]methionine. The cells were extracted in 1% digitonin/TS and immunoprecipitated with anti-calnexin (lanes1 and 3) or anti-class II beta chain (lane2) antibody. Proteins were eluted from the protein G-Sepharose beads with TS, 2% SDS, and 2 mM DTT, diluted with TS, 1% Triton X-100, and 10 mM IAA, and reprecipitated with control (lane1) or conformation-independent anti-class II beta antibodies (lanes2 and 3). Precipitates were subjected to reducing (10.5%) SDS-PAGE. The position of molecular mass markers (kDa) are indicated on the left.



Deglycosylation of GPI-DRbeta1 by tunicamycin (Fig. 7, rightpanel) or by endoglycosidase H digestion (Fig. 5, leftpanel) did not abolish the calnexin association. This rules out the possibility that the calnexin must recognize both the N-linked glycans and transmembrane segments of glycoproteins for its association. Removal of either of them individually does not abolish the association. It is well documented that for many glycoproteins, N-linked glycans are not critical for achieving a functional conformation (reviewed in Refs. 47 and 48). Soluble MHC class II molecules (49) and soluble invariant chain (^4)synthesized without glycosylation in a prokaryotic expression system were found to form heterodimers and homotrimers, respectively. The MHC class II heterodimers were also able to bind and present peptides to specific T cells. This suggests that neither their transmembrane regions nor glycosylation are essential for these molecules to achieve a functional conformation. Based on our results and information available from other laboratories, we suggest that neither N-linked glycans nor the transmembrane segment of glycoproteins are critical for calnexin association. There are two possibilities. 1) Calnexin may mediate lectin-like as well as protein-protein interactions with glycoproteins, and the degree of dependence on these interactions for detectable association may vary between proteins. In a similar way, the cell surface receptor CD23 has been shown to recognize both a sugar moiety and the protein backbone structure of its ligand, CD21, during binding(50) . 2) Calnexin, like other chaperones such as GroEL, and members of the Hsp 60 family (51, 52) may recognize an intermediate structure in the protein-folding process that exposes certain features. The degree of dependence on glycosylation and on the transmembrane region for attaining a calnexin-recognizable conformation may vary between proteins, explaining the conflicting observations with different glycoproteins.


FOOTNOTES

*
This work was supported by the Howard Hughes Medical Institute and National Institutes of Health Award AI23081. 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: Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, 310 Cedar St., New Haven, CT 06510. Tel.: 203-785-5176; Fax: 203-789-1059.

(^1)
The abbreviations used are: ER, endoplasmic reticulum; CHL, chicken hepatic lectin; GPI, glycosyl phosphatidylinositol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; IAA, iodoacetamide; MHC, major histocompatibility complex; PAGE, polyacrylamide gel electrophoresis; DSP, dithiobis (succinimidyl propionate); TCR, T-cell antigen receptor; FCS, fetal calf serum; DTT, dithiothreitol.

(^2)
D. Williams, personal communication.

(^3)
J. Rose, personal communication.

(^4)
D. C. Wiley, personal communication.


ACKNOWLEDGEMENTS

We are grateful to Dr. Michael Brenner for the generous gift of the AF8 antibody, Drs. Ed Collins and Don C. Wiley for providing GPI-DRbeta1 plasmid construct, Dr. K. Drickamer for providing cDNA-encoding chicken hepatic lectin and antiserum R114B3. We also thank Matthew Androlewicz for technical advice, Craig Hammond for comments on the manuscript, Michael J. Surman for technical assistance, and Nancy Dometios for secretarial help.


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