©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Unique Expression of Major Histocompatibility Complex Class I Proteins in the Absence of Glucose Trimming and Calnexin Association (*)

(Received for publication, August 1, 1995; and in revised form, September 27, 1995)

Jeffrey P. Balow Jocelyn D. Weissman Kelly P. Kearse (§)

From the Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892-1260

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recent evidence indicates that efficient expression of major histocompatibility complex (MHC) complexes requires their interaction with the resident endoplasmic reticulum (ER) chaperone calnexin, which for certain proteins functions as a lectin specific for monoglucosylated glycans. In the current report, we studied the expression of MHC class I proteins in BW wild type thymoma cells (BW WT) and glucosidase II-deficient BW PHAR2.7 cells. Consistent with a requirement for glucose (Glc) trimming for interaction of class I proteins with calnexin, we found that nascent H-2K^k proteins associated with calnexin in untreated BW WT cells, but not in BW WT cells treated with the glucosidase inhibitor castanospermine (cas), or in untreated glucosidase II-deficient BW PHAR2.7 cells. Suprisingly, we found that H-2K^k expression occurred with similar efficiency in BW PHAR2.7 cells as in BW WT cells and that formation of nascent H-2K^k complexes was perturbed by cas treatment in BW WT cells but not in BW PHAR2.7 cells. Finally, it was noted that expression of the molecular chaperone Bip was markedly increased in BW PHAR2.7 cells relative to BW WT cells, which is suggested to play a role in regulating the expression of H-2K^k complexes in BW PHAR2.7 cells. The current study demonstrates that Glc trimming is required for efficient interaction of nascent H-2K^k proteins with calnexin; that expression of MHC class I proteins can, under certain conditions, proceed effectively in the absence of Glc trimming and calnexin association; and that Bip expression is markedly increased under conditions where diglucosylated glycans persist on nascent glycoproteins within the ER. These data are consistent with the hypothesis that alternative oligomerization pathways exist for class I proteins within the quality control system of the ER that have differential requirements for removal of Glc residues from nascent glycan chains.


INTRODUCTION

Most major histocompatibility complex (MHC) (^1)class I molecules expressed at the cell surface consist of heavy chains (HC) noncovalently associated with beta(2)-microglobulin proteins and processed peptides(1, 2) . The molecular chaperone calnexin plays an important role in the assembly of nascent HCbulletbeta(2)bulletpeptide complexes within the endoplasmic reticulum (ER) and moderates their transport to the Golgi compartment(3, 4, 5, 6) . Assembly of nascent murine MHC class I complexes is believed to occur by: (i) association of newly synthesized HC with calnexin to form HC-calnexin protein complexes, (ii) interaction of beta(2)-microglobulin proteins with calnexinbulletHC proteins to form calnexinbulletHCbulletbeta(2) complexes; and finally (iii) addition of peptide to calnexin-associated HCbulletbeta(2) proteins to form HCbulletbeta(2)bulletpeptide complexes, which rapidly dissociate from calnexin and exit the ER(7, 8, 9) .

A growing body of evidence signifies that removal of glucose (Glc) residues from nascent oligosaccharide chains is important for association of certain glycoproteins with calnexin, including clonotypic alphabeta chains of the T cell antigen receptor complex(10) , viral gene products(11, 12) , and many unidentified proteins(10) . Removal of Glc residues from immature Glc(3)Man(9)GlcNAc(2) (Glc = glucose, Man = mannose; and GlcNAc = N-acetylglucosamine) glycan chains is accomplished by the sequential action of ER glucosidase I and glucosidase II enzymes, which remove the outermost and two proximal Glc residues, respectively(13) . Since calnexin associates with glycoproteins containing monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycan chains(12, 14) , both glucosidase I and glucosidase II activities are necessary to create glycan substrates for calnexin binding. Oligosaccharide chains are not strictly required for calnexin association, however, as several nonglycosylated molecules interact stably with calnexin, including recombinant multidrug resistance P glycoprotein lacking N-linked addition sites (15) and the CD3 subunit of the T cell antigen receptor complex(16) .

To further our understanding of the role of Glc trimming and calnexin association in the assembly and expression of immune receptor molecules, we studied the expression of MHC class I proteins in BW wild type thymoma cells (BW WT) and the glucosidase II-deficient BW variant cell line, PHAR2.7. We found that expression of H-2D^k proteins was markedly reduced on BW PHAR2.7 cells relative to BW WT cells, and, unexpectedly, that expression of H-2K^k proteins was equivalent on both cell types. Our results show that Glc trimming and calnexin association are required for efficient formation of nascent H-2K^k complexes in BW WT cells but not BW PHAR2.7 cells, indicating that alternative folding pathways exist within the ER quality control system for assembly of MHC class I protein complexes. Lastly, Bip expression was observed to be significantly increased in BW PHAR2.7 cells relative to BW WT cells, which is implicated to play a role in regulating H-2K^k expression in BW PHAR2.7 cells.


EXPERIMENTAL PROCEDURES

Cells and Reagents

BW5147 cells (17) and BW PHAR2.7 cells (18) were maintained by weekly passage in RPMI 1640 medium containing 5% fetal calf serum at 37 °C in 5% CO(2). Castanospermine (cas) was purchased from Calbiochem and was used at a 50-100 µg/ml.

Metabolic Labeling, Cell Lysis, and Immunoprecipitation

Metabolic labeling with [S]methionine (TranS-label) (ICN) was performed as described previously(10) . Cells were solubilized in either 1% Nonidet P-40 lysis buffer or 1% digitonin lysis buffer and lysates immunoprecipitated with the appropriate antibodies preabsorbed to protein A-Sepharose beads as described previously(10) . The following monoclonal antibodies (mAb) were used in this study: 15-5-5, anti-D^k (Pharmingen)(19) ; 36-7-5, anti-K^k (Pharmingen); CTKk anti-K^k (Caltag, San Francisco, CA). The following antibodies were used: SPA-827 anti-grp-78 Ab (Stressgen Biotechnologies Corp, Victoria, BC, Canada)(20) ; rabbit antisera directed against the COOH terminus of human and canine calnexin(3) , kindly provided by Dr. David B. Williams (University of Toronto, Ontario, Canada) and also purchased from Stressgen Biotechnologies Corp. (Victoria, BC, Canada), Ab SPA-860.

Glycosidase Digestion, Gel Electrophoresis, and Immunoblotting

Digestion with Endo H (Genzyme) was performed as described previously(10) . Digestion with PNGase F (Glyko, Novato, CA) was performed according to the manufacturer's instructions. One- and two-dimensional gel electrophoreses were carried out as described previously(10) . For immunoblotting, samples were probed with a 1:200 dilution of antisera in phosphate-buffered saline containing 5% milk and 0.02% NaN(3), followed by I-labeled protein A (10 µCi/ml) (ICN).

Flow Cytometry

Cell surface staining of H-2D^k and H-2K^k proteins was performed by incubating cells with primary anti-H-2D^k and anti-H-2K^k Abs and, after washing, with secondary goat anti-mouse Ig (GAM) Ab conjugated to fluorescein isothiocyanate (Pharmingen). Cells were analyzed on a FACSSCAN flow cytometer (Becton Dickinson). All fluorescence data were collected using logarithmic amplification on 50,000 viable cells as determined by forward light scatter intensity and propidium iodide exclusion.


RESULTS

Surface Expression of MHC Class I Proteins on BW WT and BW PHAR2.7 Cells

Because glucosidase II-deficient BW PHAR2.7 cells are impaired in the generation of monoglucosylated glycans important in calnexin binding(14, 18) , we predicted that expression of both H-2D^k and H-2K^k proteins would be markedly decreased on BW PHAR2.7 cells relative to BW WT cells. As shown in Fig. 1A, H-2D^k expression was markedly reduced on BW PHAR2.7 cells relative to BW WT cells, but, suprisingly, H-2K^k expression was comparable on both cell types (Fig. 1A, 3rd column). These findings were confirmed by immunoprecipitation studies of surface-labeled BW WT and BW PHAR2.7 cells (Fig. 1B).


Figure 1: Surface expression of H-2D^k and H-2K^k proteins on BW WT cells and BW PHAR2.7 cells. A, surface expression of H-2D^k and H-2K^k complexes was examined by indirect staining with anti-H-2D^k(15-5-5) and anti-H-2K^k(36-7-5) mAbs, followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG2a Ab molecules (GAM). Control staining was performed with FITC-GAM Ab only. The relative expression of MHC class I proteins on BW PHAR2.7 cells relative to BW WT cells was 0.35 for H-2D^k proteins and 1.1 for H-2K^k proteins. The data shown are representative of at least five independent experiments; variation between experiments was less than 10%. B, BW WT and BW PHAR2.7 cells were surface-labeled by biotinylation, solubilized in 1% Nonidet P-40, and lysates were precipitated with anti-H-2D^k (15-5-5) and anti-H-2K^k(36-7-5) mAbs preadsorbed to protein A-Sepharose. Immunoprecipitated material was analyzed on one-dimensional SDS-PAGE gels under reducing conditions and visualized by chemiluminescence techniques. The positions of H-2D^k and H-2K^k proteins are indicated.



To study the requirement for glucosidase activity for expression of MHC class I complexes on BW WT cells, experiments were performed using the glucosidase inhibitor castanospermine (cas)(13) . As shown in Fig. 2, surface expression of H-2D^k complexes on BW WT cells was markedly reduced by overnight culture in cas (Fig. 2). Not suprisingly, surface density of H-2D^k complexes was similar on untreated and cas-treated BW PHAR2.7 cells (Fig. 2), as expression of H-2D^k complexes was already decreased on glucosidase II-deficient BW PHAR2.7 cells. Most importantly, these data show that cas treatment dramatically reduced expression of H-2K^k complexes on BW WT cells, but not BW PHAR2.7 cells (Fig. 2). Reduced expression of MHC class I complexes on cas-treated BW WT cells was specific in that expression of other surface molecules (CD45 proteins, thymic shared antigen proteins, and peanut agglutinin lectin-binding proteins) was unaffected by cas treatment, and no change in class I expression was observed following culture with the mannosidase inibitor deoxymannojirimycin (data not shown). These results show that impaired glucosidase activity is correlated with decreased expression of H-2D^k complexes on both BW WT and BW PHAR2.7 cells and that BW WT and BW PHAR2.7 cells exhibit differential requirements for glucosidase activity in the surface expression of H-2K^k complexes.


Figure 2: Effect of cas treatment on surface expression of H-2D^k and H-2K^k proteins on BW WT cells and BW PHAR2.7 cells. Surface expression of H-2D^k and H-2K^k complexes was examined by cell surface staining as described in Fig. 1A. Where indicated, cells were cultured overnight at 37 °C in the presence of the glucosidase inhibitor castanospermine (Cas) at a concentration of 100 µg/ml. The data shown are representative of at least three independent experiments; variation between experiments was less than 10%. Viability was identical in media and cas-treated groups (data not shown).



Intracellular Transport of Nascent H-2K^k Complexes in BW WT and BW PHAR2.7 Cells

Because of our curious results regarding H-2K^k expression on BW PHAR2.7 cells, we performed a series of studies to examine the molecular requirements for formation of nascent H-2K^k complexes in BW WT and BW PHAR2.7 cells. Although the anti-H-2D^k Abs used in our studies efficiently recognized surface H-2D^k proteins on BW WT and BW PHAR2.7 cells, they failed to effectively immunoprecipitate newly synthesized H-2D^k proteins from either cell type; therefore, our studies focused on H-2K^k molecules. In our first set of experiments, the intracellular transport of nascent H-2K^k complexes was examined. For these studies, BW WT and BW PHAR2.7 cells were metabolically pulse-labeled with [S]methionine, chased in unlabeled medium, and solubilized in 1% Nonidet P-40; lysates were immunoprecipitated with anti-H-2K^k Abs, and precipitates were digested with Endoglycosidase H (Endo H), specific for removal of immature glycan chains. Nascent H-2K^k glycoproteins synthesized in BW PHAR2.7 cells migrated with slightly decreased mobility compared to H-2K^k proteins made in BW WT cells, reflecting persistence of Glc residues on glycan chains of H-2K^k proteins in BW PHAR2.7 cells (Fig. 3; compare pulse groups). As demonstrated, significant amounts of nascent H-2K^k complexes were formed in both BW WT and BW PHAR2.7 cells during the 30-min pulse period (Fig. 3). H-2K^k complexes efficiently exited the ER and transited through the Golgi compartment in both BW WT and BW PHAR2.7 cells as demonstrated by their acquisition of Endo H resistance during the chase period (Fig. 3). Note that maturation of oligosaccharide chains can occur under conditions of impaired glucosidase activity via the alternative processing pathway for N-linked oligosaccharides, which involves deglucosylation of glycan chains by endomannosidase enzymes(21, 22) .


Figure 3: Intracellular transport of nascent H-2K^k complexes in BW WT and BW PHAR2.7 cells. A, BW WT and BW PHAR2.7 cells were metabolically pulse-labeled with [S]methionine for 30 min, chased for the time period indicated, solubilized in 1% Nonidet P-40 detergent, and lysates immunoprecipitated with anti-H-2K^k mAb -(5) . Precipitates were either mock-treated or digested with Endo H and analyzed on SDS-PAGE gels under reducing conditions. The positions of H-2K^k proteins and beta(2)-microglobulin proteins are indicated. K = K^k proteins resistant to digestion with Endo H; K = K^k proteins sensitive to digestion with Endo H.



These data show that formation of nascent H-2K^k complexes proceeds with similar efficiency in BW WT and BW PHAR2.7 cells and that H-2K^k complexes are effectively transported from the ER to the Golgi compartment in both cell types, two findings which are in agreement with our results regarding surface expression of H-2K^k proteins on BW WT and BW PHAR2.7 cells (Fig. 1, A and B).

Requirement for Glc Trimming for Association of Nascent H-2K^k Proteins with Calnexin

As calnexin association is believed to be an important step in the assembly of nascent MHC class I protein complexes(3, 4, 5, 6, 14) , we next examined the association of newly synthesized H-2K^k molecules with calnexin in BW WT and BW PHAR2.7 cells. For these studies, cells were metabolically labeled, solubilized in 1% digitonin, and lysates precipitated with anti-calnexin and anti-H-2K^k Abs. Immunoprecipitated material was analyzed on two-dimensional NEPHGE/SDS-PAGE gels. As shown in Fig. 4A, numerous proteins coprecipitated with calnexin molecules in BW WT lysates, including nascent H-2K^k proteins (Fig. 4A, left, top panel, arrow). Identity of H-2K^k proteins in calnexin precipitates was verified by mixing experiments and release/recapture studies (data not shown). As is evident, markedly fewer proteins were present in anti-calnexin precipitates of BW PHAR2.7 lysates relative to BW WT lysates (Fig. 4A, right, top panel). Most importantly, nascent H-2K^k proteins were not associated with calnexin in BW PHAR2.7 lysates (Fig. 4A, right, top panel, arrow), despite the abundant existence of H-2K^k proteins within the lysate (Fig. 4A, right, bottom panel, arrow). Association of newly synthesized H-2K^k proteins with calnexin molecules in BW WT lysates was inhibited by treatment with the glucosidase inhibitor cas (Fig. 4B, arrow), as was the association of numerous unidentified protein species with calnexin (Fig. 4B). These data demonstrate that Glc trimming is required for effective interaction of calnexin with nascent H-2K^k proteins in BW thymoma cells and provide evidence that efficient formation of H-2K^k complexes in BW PHAR2.7 cells does not result from their association with calnexin.


Figure 4: Association of nascent H-2K^k proteins with calnexin in BW WT and BW PHAR2.7 cells. A, digitonin lysates of radiolabeled BW WT and BW PHAR2.7 cells were immunoprecipitated with anti-calnexin Ab or anti-H-2K^k mAb(37-6-5), and precipitates were analyzed on two-dimensional NEPHGE/SDS-PAGE gels under reducing conditions. The position of radiolabeled H-2K^k proteins is indicated. B, radiolabeled lysates of untreated and cas-treated BW WT cells were immunoprecipitated with anti-calnexin Ab, and precipitates were analyzed as in A. The position of H-2K^k proteins is indicated.



Requirement for Glucosidase Activity for Formation of Nascent H-2K^k Complexes in BW WT and BW PHAR2.7 Cells

Because our results in BW PHAR2.7 cells indicated that Glc trimming and calnexin association are not required for effective assembly of nascent H-2K^k complexes, the effect of glucosidase inhibition on formation of nascent H-2K^k complexes in BW WT cells was examined. Significantly fewer H-2K^k complexes existed in anti-H-2K^k precipitates of cas-treated BW WT cells than in untreated BW WT cells (Fig. 5A). In contrast, formation of nascent H-2K^k complexes in BW PHAR2.7 cells was not perturbed by cas treatment (Fig. 5B). Indeed, equivalent amounts of H-2K^k complexes were isolated from lysates of untreated and cas-treated BW PHAR2.7 cells (Fig. 5B). It should be appreciated that nascent H-2K^k proteins synthesized in cas-treated BW PHAR2.7 cells showed a slight shift in molecular weight relative to H-2K^k proteins made in untreated BW PHAR2.7 cells (Fig. 5B, compare pulse groups), demonstrating that cas treatment was effective in these experiments.


Figure 5: Effect of total glucosidase blockade on formation of nascent H-2K^k complexes in BW WT and BW PHAR2.7 cells. Untreated and cas-treated BW WT and BW PHAR2.7 cells were metabolically pulse-labeled for 30 min with [S]methionine, chased for 45 min, solubilized in 1% Nonidet P-40, and lysates were immunoprecipitated with anti-H-2K^k-specific mAb(36-7-5). The presence of inhibitor was maintained throughout the experiment. Precipitates were either mock-treated or digested with glycosidases and analyzed on one-dimensional SDS-PAGE gels under reducing conditions. Note that protein synthesis was identical in untreated and cas-treated cell groups ((10) ; data not shown). The positions of H-2K^k proteins and beta(2)-microglobulin proteins are indicated. In the experiment shown in A, precipitates were digested with PNGase F which cleaves both immature and mature oligosaccharides (K = deglycosylated K^k polypeptides); in the experiment presented in B, precipitates were digested with Endo H, which cleaves only immature oligosaccharides (K = Endo H-sensitive K^k protein).



We conclude from these data that removal of Glc residues from core glycan chains and calnexin association is required for effective formation of H-2K^k complexes in BW WT cells but not BW PHAR2.7 cells. Impaired formation of nascent H-2K^k complexes in cas-treated BW WT cells provides a molecular basis for reduced surface expression of H-2K^k proteins on BW WT cells cultured overnight in cas (Fig. 2). Moreover, since cas impaired formation of nascent H-2K^k complexes in BW WT cells and other T cell types, including thymocytes and T hybridoma cells, (^2)but not BW PHAR2.7 cells, we further conclude that unique assembly mechanisms exist in BW PHAR2.7 cells for assembly of nascent H-2Kk complexes in the absence of Glc trimming and calnexin association.

Increased Expression of Bip Chaperone Proteins in BW PHAR2.7 Cells Relative to BW WT Cells

Besides calnexin, the only molecular chaperone reported to associate with murine class I chains is Bip, albeit weakly(9) . As shown in Fig. 6A, expression of Bip proteins was significantly increased in BW PHAR2.7 cells relative to BW WT cells (Fig. 6A). Up-regulation of Bip in BW PHAR2.7 cells was specific in that equivalent amounts of calnexin were expressed in BW WT and BW PHAR2.7 cells (Fig. 6A), and expression of an uncharacterized 44-kDa protein was similar in BW WT and BW PHAR2.7 cells (Fig. 6A, *). To examine association of nascent H-2K^k proteins with Bip, radiolabeled lysates of BW WT and BW PHAR2.7 cells were precipitated with anti-Bip Ab and precipitates were examined on two-dimensional NEPHGE/SDS-PAGE gels. In agreement with our immunoblotting data, synthesis of Bip proteins appeared increased in BW PHAR2.7 cells relative to BW WT cells (Fig. 6B; arrowhead). Importantly, small, yet detectable, amounts of nascent H-2K^k molecules were observed to coprecipitate with Bip proteins in BW PHAR2.7 lysates but not BW WT lysates (Fig. 6B; arrow). It should be realized that precipitations were not optimal in these studies as we were unable to quantitatively precipitate Bip proteins from BW PHAR2.7 lysates; thus, these data represent a minimal estimate of the amount of nascent H-2K^k proteins associated with Bip in BW PHAR2.7 cells. These results demonstrate that Bip expression is markedly increased in BW PHAR2.7 cells relative to BW WT cells and provide evidence that nascent H-2K^k proteins made in BW PHAR2.7 cells associate with Bip molecules.


Figure 6: Increased Bip expression in BW PHAR2.7 cells relative to BW WT cells. A, Nonidet P-40 lysates of equivalent numbers of BW WT and BW PHAR2.7 cells were precipitated with anti-calnexin Abs (top) or anti-Bip Abs (bottom) and blotted with the same Ab used for precipitation. The positions of calnexin and Bip proteins are indicated; * denotes an unknown 44-kDa protein which is reported to react with the anti-Bip Ab used in these experiments (Stressgen product sheet). Lane 1, BW WT cells; lane 2, BW PHAR2.7 cells; lane 3, Ab + lysis buffer. Identical results were obtained when whole lysates of BW WT and BW PHAR2.7 were analyzed (data not shown). B, radiolabeled digitonin lysates of BW WT and BW PHAR2.7 cells were immunoprecipitated with anti-Bip Ab and precipitates were analyzed on two-dimensional NEPHGE/SDS-PAGE gels under reducing conditions. The positions of Bip (arrowhead) and H-2K^k proteins (arrow) are indicated.




DISCUSSION

The quality control system of the ER ensures that properly folded, fully assembled protein complexes are expressed on the cell surface. The molecular chaperone calnexin is believed to play a key role in maintaining the fidelity of class I complexes expressed on the cell surface by functioning in the assembly of nascent class I HC-beta(2)-peptide complexes within the ER and regulating their transport to the Golgi compartment. The data in the current study show that: (i) removal of Glc residues from core glycans is required for efficient interaction of nascent H-2K^k proteins with calnexin; (ii) calnexin association is not essential for stable expression of H-2K^k complexes; (iii) expression of Bip chaperones is markedly increased in glucosidase II-deficient BW PHAR2.7 cells relative to BW WT cells; and (iv) H-2K^k proteins were associated with Bip proteins in BW PHAR2.7 cells but not BW WT cells.

Recent evidence indicates that initial association of oligomeric glycoproteins with calnexin requires recognition of monoglucosylated Glc(1)Man(9)GlcNAc(2) species on glycoproteins(12, 14) . In agreement with this idea, we found that nascent H-2K^k chains synthesized in BW WT thymoma cells, but not in cas-treated BW WT cells or in glucosidase II-deficient BW PHAR2.7 cells, associated with calnexin. Degradation of murine and human MHC class I proteins under conditions of impaired glucosidase activity has been reported(23, 24) . As the Abs used in our studies effectively recognize beta(2)-assembled H-2K^k chains, but not unassembled H-2K^k chains, it remains to be determined if reduced formation of H-2K^k complexes in cas-treated BW WT cells results from rapid degradation of nascent H-2K^k HC or the failure of H-2K^k HC to assemble effectively with beta(2) molecules. Regardless, our data clearly show that Glc trimming and calnexin association are not essential for stable expression of H-2K^k complexes as H-2K^k proteins are effectively expressed in glucosidase II-deficient BW PHAR2.7 thymoma cells.

Maintenance of class I expression is important for immune surveillance function within the body. The data in the current study show that alternative folding pathways exist within the ER that sustain expression of class I protein complexes under conditions where Glc trimming and calnexin association are impaired. Unlike H-2K^k molecules, H-D^k proteins were not stably expressed on BW PHAR2.7 cells, indicating that assembly mechanisms exist within the ER of BW PHAR2.7 cells that function effectively for the oligomerization of H-2K proteins but not H-2D proteins. Regarding this issue, we have considered the possibility that stable expression of H-2K^k proteins in BW PHAR2.7 cells results from mutations introduced during their selection procedure (18) that allow H-2K^k polypeptides made in BW PHAR 2.7 cells to uniquely assemble with beta(2) proteins and processed peptide in the absence of Glc trimming and calnexin association. While the current studies do not exclude this possibility, preliminary sequencing data suggest that H-2 K^k molecules expressed in BW WT and BW PHAR2.7 cells are identical. (^3)

The molecular basis for proficient expression of H-2K^k complexes in BW PHAR2.7 cells is unclear, but as suggested in this report may involve interaction with Bip proteins. Unlike human class I molecules which stably interact with Bip proteins(9, 25) , association with murine class I molecules with Bip is controversial as Bip associates weakly with endogenous mouse class I proteins (9) and fails to interact with mouse gene products expressed in human cell lines(25) . Indeed, no association between Bip and H-2K^k proteins in BW WT thymoma cells was observed in our studies (Fig. 5B). It is well documented that Bip expression increases during periods of ER stress and is stimulated by treatment with compounds that interfere with glycan addition or processing of oligosaccharide side chains(26, 27) . The current report shows that expression of Bip proteins is markedly elevated in glucosidase II-deficient BW PHAR2.7 cells relative to BW WT cells, suggesting that persistence of diglucosylated glycans on nascent glycoproteins results in the accumulation of numerous malfolded proteins within the ER, which, in turn, results in increased Bip synthesis via feedback regulatory mechanisms(28) . Thus, it is conceivable that quantitatively increased levels of Bip proteins in BW PHAR2.7 cells represent the molecular basis for effective expression of H-2K^k complexes in BW PHAR2.7 cells. As we were unable to determine if H-2K^k proteins associated with Bip in BW PHAR2.7 cells represent intermediates in the assembly of H-2K^k-beta(2)-peptide complexes or end-stage H-2K^k proteins targeted for degradation, the fate of Bip-associated H-2K^k proteins in BW PHAR2.7 cells is unclear. Alternatively, it is reasonable to speculate that stable expression of H-2K^k proteins in BW PHAR2.7 cells results from their association with an unknown chaperone protein(s) that, like Bip, functions independently of glycan processing and is up-regulated during periods of ER stress. We were unsuccessful in our attempts to sustain expression of H-2K^k complexes in BW WT cells by extended culture in cas, despite the fact that Bip expression was increased under these conditions (data not shown). Whether these findings reflect difficulties in approximating ER stress conditions in BW WT and BW PHAR2.7 cells by drug treatment or denote true differences between the ER environments of BW WT and BW PHAR2.7 cells remains to be determined. Finally, it should be noted that Scott and Dawson recently reported expression of human MHC class I proteins in a calnexin-deficient cell line(29) , showing that calnexin is not absolutely required for the transport and surface expression of human MHC class I molecules.


FOOTNOTES

*
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. Tel.: 301-496-5857; Fax: 301-496-0887.

(^1)
The abbreviations used are: MHC, major histocompatibility complex; Bip, binding protein; Endo H, endoglycosidase H; cas, castanospermine; ER, endoplasmic reticulum; Glc, glucose; GlcNAc, N-acetylglucosamine; HC, heavy chain; Man, mannose; mAb, monoclonal antibody; NEPHGE, nonequilibrium pH gradient gel electrophoresis; PAGE, polyacrylamide gel electrophoresis.

(^2)
J. P. Balow and K. P. Kearse, unpublished observations.

(^3)
J. Weissman and K. P. Kearse, unpublished observations.


ACKNOWLEDGEMENTS

We wish to thank Drs. Richard Hodes, Jeroen Van Leeuwen, David Segal, and Dinah Singer for critical reading of the manuscript; Drs. Dinah Singer, Hidde Ploegh, and David B. Williams for helpful discussion; Dr. David B. Williams for the gift of anti-calnexin Ab and Dr. Rosalind Kornfeld for the gift of BW WT and BW PHAR2.7 cells.


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