Distinct differences in association of MHC class I with endoplasmic reticulum proteins in wild-type, and ß2-microglobulin- and TAP-deficient cell lines

Kajsa M. Paulsson1, Ping Wang1,2, Per O. Anderson1, Shangwu Chen1, Ralf F. Pettersson3 and Suling Li1,4

1 Tumor Immunology, Lund University, Solvegatan 21, 22362 Lund, Sweden
2 Immunology Group, Department of Pediatric Gastroenterology, St Bartholomew's and the Royal London School of Medicine and Dentistry, London, UK
3 Ludwig Institute for Cancer Research, Box 240, 17177 Stockholm, Sweden
4 Department of Biological Sciences, Brunel University, Uxbridge, Middlesex UB8 3PH, UK

Correspondence to: S. Li


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study we have compared the interaction of human MHC class I molecules with IgG heavy chain (HC) binding protein (BiP), calnexin, calreticulin, tapasin and TAP in ß 2 -microglobulin 2 m)- or TAP-deficient cells, as well as in wild-type B-LCL cells. Distinct differences between the association of HC and these endoplasmic reticulum (ER) proteins were found in the three cell lines. In the absence of ß 2 m (Daudi cells), HC associated with both BiP and calnexin. A prominent portion of HC was complexed simultaneously to both chaperones, as indicated by co-precipitation with either anti-calnexin or anti-class I antisera. In the presence of ß 2 m, but absence of TAP (T2 cells), HC could be co-precipitated with calnexin, whereas no detectable interaction with BiP could be demonstrated. This suggests that calnexin interacts with HC at a later stage than BiP. In B-LCL cells, HC–ß 2 m associated with calreticulin and tapasin, whereas no interaction with calnexin and BiP was observed. In the absence of ß 2 m, HC were rapidly degraded in the ER, while the ER retained HC were stabilized in the presence of ß 2 m, even in the absence of TAP. The dissociation of class I molecules from TAP in B-LCL cells correlated with the kinetics of appearance of class I molecules on the cell surface, suggesting that TAP retains peptide-free class I molecules in the ER. Taken together, our results suggest the model that BiP and calnexin sequentially control the folding of MHC class I, before MHC class I molecules associate with the loading complex.

Keywords: ß 2 -microglobulin, assembly, chaperones, MHC class I


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mature MHC class I molecules, which present peptides to CD8 + T cells, consist of three subunits: two polypeptide chains [heavy chain (HC) and ß 2 -microglobulin (ß 2 m)] in intimate association with a short peptide ( 1 , 2 ). The folding and assembly of class I molecules begins immediately after the translocation of their subunits into the lumen of the endoplasmic reticulum (ER) ( 1 , 3 , 4 ). The fully assembled complexes rapidly exit the ER and are transported to the cell surface via the intermediate compartment and the Golgi complex ( 1 ). Deficiency in ß 2 m prevents assembly and transport of functional class I molecules, while absence of TAP or tapasin results in a defect in peptide translocation from the cytosol to the ER lumen ( 512 ). Deficiency in either the ß 2 m or TAP complex results in the accumulation of HC in a pre-Golgi compartment ( 1 , 3 , 8 , 9 ). Thus, proper folding and assembly in the ER are essential steps for the expression of functional class I molecules on the cell surface.

Changes in the conformation of class I molecules from free HC, immature HC–ß 2 m heterodimers to peptide-bound class I complexes have been detected by conformation-specific antibodies ( 13 ). Folding and assembly of class I molecules are catalyzed by ER chaperones, the general role of which is to assist in the folding and oligomeric assembly of glycoproteins, to mediate ER retention of incompletely folded and oligomerized proteins, and to prevent premature degradation ( 1318 ). Several resident proteins of the ER have been found to associate with MHC class I molecules during the maturation process including the chaperones IgG HC binding protein (BiP), calnexin, calreticulin, tapasin as well as TAP ( 1927 ). The heterodimeric TAP1–TAP2 complex specifically associates with ß 2 m-assembled HC via tapasin ( 19 , 24 , 26 , 27 ). Following binding of peptide, the mature class I molecule is released from TAP. Association of HC–ß 2 m heterodimer with TAP may stabilize the complex and facilitate peptide loading ( 19 , 24 , 26 , 27 ). Calnexin binds transiently to folding and assembly intermediates of a diverse array of newly synthesized membrane and secretory glycoproteins, and a distinct folding stage of these proteins is accompanied by the dissociation from calnexin ( 14 , 15 , 2830 ). Calnexin is thought to primarily interact with binds to monoglucosylated N -linked oligosaccharides on folding intermediates in the ER ( 3135 ). Calnexin rapidly and quantitatively binds to newly translocated HC of class I ( 14 , 15 , 36 , 37 ). In cells lacking either ß 2 m or TAP, calnexin does not dissociate from class I HC, or does so slowly ( 21 , 22 , 37 , 38 ). In human cells, the assembly of ß 2 m and HC releases calnexin ( 21 , 22 , 38 ). Calnexin has been proposed to retain unassembled class I molecules in the ER ( 21 , 22 , 37 , 38 ). It was also shown that calnexin facilitates the folding and assembly of class I molecules of both human and mouse ( 15 ). Like calnexin, calreticulin is a lectin, also binding to class I and several other glycoproteins ( 9 ). However, it has been shown that calnexin and calreticulin bind to distinct maturation stages of MHC class I molecules ( 23 ). The immature HC–ß 2 m heterodimers then interact with TAP via tapasin, followed by peptide loading, release of the stable, mature, class I molecules from TAP and transport to the cell surface ( 19 , 24 , 26 , 27 ). By cross-linking, another ER chaperone, BiP, was also found to associate with free class I HC in ß 2 m-deficient human cells ( 22 ). BiP binds preferentially to linear arrays of hydrophobic amino acids ( 39 , 40 ) and is transiently bound to many newly synthesized proteins, and in a prolonged fashion to misfolded proteins, or unassembled subunits ( 41 , 42 ). Therefore, it has been suggested that in human cells either BiP or calnexin may facilitate assembly of newly synthesized HC with ß 2 m, after which the heterodimers dissociate from the chaperones and associate with TAP ( 22 ). This suggestion was supported by the in vitro dissociation of HC from BiP and calnexin after addition of ß 2 m to the cell lysate ( 22 ) or transfection of ß 2 m cDNA into a cell line lacking ß 2 m ( 22 , 38 ).

In this study, we have characterized the association of class I HC with different ER proteins including BiP, calnexin, calreticulin, tapasin and TAP in the ß 2 m-deficient Daudi cell line, in the TAP-deficient T2 cell line, as well as in a wild-type cell line represented by the Epstein–Barr virus-transformed lymphoid B-LCL cell line. In Daudi cells, we found that BiP and calnexin interact simultaneously with the same class I HC molecules, while in T2 cells only calnexin, but not BiP, associated with HC. In the absence of a cross-linker, neither BiP nor calnexin were found associated with HC in the B-LCL cells. Both calreticulin and tapasin interact with HC in LCL cells but not in Daudi cells. Our findings suggest a sequential interaction of class I HC with BiP and calnexin, then HC–ß 2 m dimer binds to the loading complex, followed by transport of assembled MHC class I molecules from the ER to the cell surface.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells
T2 and Daudi cells were obtained from the ATCC (Rockville, MD). The B lymphoblastoid cell line WW1 (B-LCL) was kindly provided by Dr Maria Masucci (Karolinska Institute, Stockholm, Sweden). The cell lines were cultured in RPMI 1640 medium (Gibco/BRL, Gaithersburg, MD), supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine, at 37°C in a 5% CO 2 atmosphere.

Antibodies
mAb W6/32, specific to ß 2 m-associated human class I molecules ( 43 ), was obtained from the ATCC (Rockville, MD). Rabbit antisera against human TAP1 and tapasin were described previously ( 6 ) A rabbit polyclonal anti-calnexin antiserum ( 34 ) was kindly provided by Dr Ari Helenius (Yale University School of Medicine, CT). In some experiments, a rabbit antiserum made in our laboratory against the same C-terminal calnexin peptide ( 34 ) was also used. The rabbit antiserum, R425, specific to both free and assembled HLA HC, has been described previously ( 44 ). Rabbit anti-calreticulin antiserum was obtained from Affinity Bioreagents (Golden, CO). Anti-BiP mAb was from StressGen (Victoria, Canada).

Metabolic labeling, immunoprecipitaion and immunoblotting
Cells were washed twice with PBS and incubated for 15 min at 37°C in methionine-free RPMI 1640 medium containing 3% dialyzed FBS. Then, 0.2 mCi/ml of [ 35 S]methionine (Amersham, Little Chalfont, UK) was added and the incubation was continued for 5–60 min. Where indicated, pulse-labeled cells were chased for different periods of time at 37°C in complete medium. At the end of the chase periods, cells were washed 3 times with ice-cold PBS and lysed either in 1% digitonin (repurified from digitonin purchased from Sigma, St Louis, MO), or 1% NP-40 lysis buffer containing 0.15 M NaCl, 25 mM Tris–HCl, pH 7.5, 1.5 mg/ml iodoacetamide and a mixture of protease inhibitors (2 mM PMSF, 10 µg/ml leupeptin, 30 µg/ml aprotinin and 10 µg/ml pepstatin). The cleared lysates were added to antibodies previously bound to Protein A–Sepharose beads (Pharmacia, Uppsala, Sweden). After washing, the immunoprecipitates were analyzed by SDS–PAGE as previously described ( 44 ). The radioactive bands were quantitated by a Phosphoimager (Fuji, Tokyo, Japan). Mean values from three separate experiments were converted to d.p.m. according to the standard.

For sequential immunoprecipitation, the first round immunoprecipitates were disrupted by incubating in 0.5 ml of 0.2% SDS at 37°C for 1 h. The samples were then diluted to 5 ml in 1% NP-40 lysis buffer and the second antiserum was added for a second round of immunoprecipitation.

For Western immunoblotting, aliquots of cell lysates, or immunoprecipitates, were loaded onto a 10% SDS–PAGE. Proteins were transferred onto a nitrocellulose filter, which was probed with the anti-BiP antibody at a dilution of 1:500, anti-tapasin antiserum and anti-calreticulin antiserum at 1:1000 dilution. Detection was performed according to Kaltoft et al . ( 45 ).

Immunoprecipitation of cell-surface-expressed class I molecules was performed in the following way. After a 5-min pulse and chases for different periods, the cells were washed once with ice-cold PBS, and incubated with 5 µg/ml W6/32 antibody for 10 min on ice. Cells were then washed 3 times with ice-cold PBS to remove unbound antibodies and were finally lysed in 1% NP-40 lysis buffer. Lysates were incubated with Protein A–Sepharose beads followed by immunoprecipitation and analysis by SDS–PAGE.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Differences in class I HC association with calnexin and BiP in Daudi, T2 and LCL cells
It has been reported that calnexin in human cells only associates with ß 2 m-free class I HC ( 22 , 38 ). To examine the influence of ß 2 m and TAP on the association of calnexin with HC, digitonin lysed, metabolically labeled Daudi, T2 and LCL cells were immunoprecipitated by anti-calnexin antiserum followed by SDS–PAGE analysis. From Daudi cell lysates, the anti-calnexin antiserum precipitated, in addition to the 90-kDa calnexin itself, HC and an additional 80-kDa protein ( Fig. 1A Go , lane 1). In T2 cells, HC co-precipitated with calnexin, while the 80-kDa protein was not detected ( Fig. 1A Go , lane 3). The identity of HC was confirmed by re-precipitation with the broadly reactive anti-HLA anti-serum, R425 ( Fig. 1C Go , lanes 1 and 3). The 80-kDa protein was identified as BiP by immunoblotting of the anti-calnexin immunoprecipitates with a monoclonal specific to BiP ( Fig. 1B Go , lane 1) and co-migration with a BiP marker (data not shown). From LCL cells, neither HC nor BiP co-precipitated with the anti-calnexin antiserum ( Fig. 1A Go , lane 2) and neither protein was detected by immunoblotting of the precipitates with the anti-BiP ( Fig. 1B Go , lane 2) or the R425 anti-class I antibody ( Fig. 1C Go , lane 2) respectively. The same results were also obtained with three other B-LCL cell lines (data not shown). Figure 2 Go shows that similar amounts of BiP (and cross-reacting gp94) were expressed in all three cell lines. This excludes the possibility that the co-precipitation of BiP and calnexin in the Daudi cells was due to a direct interaction between the two proteins. The lack of co-precipitation of calnexin and BiP from LCL and T2 cells also showed that the co-precipitation was not mediated by any other cellular proteins. Instead we conclude that calnexin, BiP and HC form a heterotrimeric complex in Daudi cells, i.e. that both chaperones are able to interact with the same class I HC molecule.



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Fig. 1. Interaction of calnexin with class I HC in ß 2 m- or TAP-deficient cell lines and in wild-type B-LCL cells. Daudi, LCL or T2 cells were metabolically labeled with [ 35 S]methionine for 1 h followed by solubilization with 1% digitonin. The lysates were precipitated either with a calnexin-specific antiserum (A, lanes 1–3) or with normal rabbit serum (lanes 4–6) and analyzed on a 10% SDS gel followed by autoradiography. The identity of the 80-kDa (BiP) and 42-kDa (HC) bands was determined by immunoblotting with an anti-BiP mAb (B) or by a second round of precipitation with an antibody, R425, broadly reactive to HLA molecules (C), as described in Methods.

 


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Fig. 2. Comparison of the amount of BiP in T2, B-LCL and Daudi cells. T2, LCL and Daudi cells were lysed in 1% NP-40, and the lysates were diluted as indicated, followed by fractionation on a 10% SDS gel and immunoblotting with an anti-BiP mAb. This antibody reacts with the two ER proteins gp94 and BiP ( 55 ).

 
To further examine BiP association with class I HC in Daudi, T2 and LCL cells, HC were precipitated from lysates prepared from [ 35 S]methionine-labeled, digitonin-solubilized cells by using the R425 antiserum. Two out of the most prominent protein species co-precipitating with HC from Daudi cells were BiP and calnexin ( Fig. 3A Go , lane 1), the identities of which were confirmed by blotting the immunoprecipitates with the anti-BiP antibody ( Fig. 3B Go , lane 1) or re-precipitating with the anti-calnexin antibody ( Fig. 3C Go , lane 1). In addition, some unidentified proteins were also precipitated. From the T2 cell lysate, the R425 antiserum precipitated only a small amount of calnexin ( Fig. 3A Go , lane 3). Neither co-precipitating BiP nor calnexin were evident from the LCL cell lysate ( Fig. 3A–CGo , lane 2). These results suggest that a large fraction of free HC is associated with BiP in Daudi cells. Part of these complexes also contain calnexin ( Fig. 1A Go ). However, due to many variables, we wish to emphasize that these results do not allow quantitative estimations regarding the fraction of HC associated with BiP or calnexin, or both.



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Fig. 3. Association of calnexin and BiP with class I HC as demonstrated by co-precipitation by an anti-class I HC antibody. Daudi, LCL and T2 cells were labeled for 1 h with [ 35 S]metionine, lysed with 1% digitonin and subjected to immunoprecipitation with the anti-class I antibody R425 (see Methods), followed by analysis on a 10% SDS gel. Western blot analysis of the R425 precipitate with anti-BiP antibody identified the 80-kDa band as BiP (B) and re-precipitation with anti-calnexin antiserum identified the 90-kDa band as calnexin (C). The positions of calnexin, BiP and class I HC are as indicated.

 
Rate of dissociation of HC from calnexin in T2 and Daudi cells
Previously, it was shown that ß 2 m can dissociate calnexin from HC ( 18 , 19 ). These results were obtained by analyzing a ß 2 m-deficient cell line transfected with a ß 2 m cDNA. If calnexin binds exclusively to the ß 2 m-free HC, the rate of dissociation of calnexin from HC in Daudi cells should be slower than in the T2 cells. To examine this point, we carried out a pulse–chase experiment in these two cell lines. Cells were labeled for 1 h and chased for up to 2 h followed by immunoprecipitation with anti-calnexin antiserum. Contrary to the prediction, the dissociation rate of HC from calnexin in the ß 2 m-expressing T2 cells was clearly slower than in the ß 2 m-deficient Daudi cells ( Fig. 4A Go , lanes 1–5 and 6–10). BiP co-precipitated in Daudi cells with HC for the same period of time, again indicating the presence of a trimeric complex from which BiP and HC dissociate with the same kinetics ( Fig. 4A Go , lanes 6–10). Figure 4(B)Go shows the identification of BiP in anti-calnexin immunoprecipitates from Daudi cells ( Fig. 4B Go , lanes 6–10), but not from T2 cells ( Fig. 4B Go , lanes 1–5).



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Fig. 4. Pulse–chase analysis of the dissociation rate of class I HC from calnexin in T2 and Daudi cells. T2 (A, lanes 1–5) and Daudi cells (lanes 6–10) were metabolically labeled with [ 35 S]methionine for 1 h and chased in an excess of unlabeled methionine for the indicated periods of time before lysis with 1% digitonin. Aliquots were immunoprecipitated with anti-calnexin antiserum. One portion of the precipitates was analyzed by SDS–PAGE and autoradiography (A), and the other portion by immunoblotting with the anti-BiP mAb (B). The positions of calnexin, BiP and class I HC are indicated. The anti-calnexin antiserum also precipitated some unknown proteins from cellular lysates. Results are from one of three similar experiments.

 
Difference in class I HC association with tapasin and calreticulin in Daudi, T2 and LCL cells
To examine the influence of ß 2 m and TAP on the association of calreticulin and tapasin with HC, digitonin lysed, metabolically labeled Daudi, T2 and LCL cells were immunoprecipitated by anti-tapasin or calreticulin antiserum followed by SDS–PAGE analysis. Calreticulin and tapasin can be easily detected in all three cell lines ( Fig. 5 Go , lanes 1–3 and Fig. 6 Go , lanes 1–3). Both antisera could co-precipitate HC in LCL cells ( Fig. 5A Go , lane 2 and Fig. 6A Go , lane 2). Also in T2 cells, HC co-precipitated, though to a much less amount ( Fig. 5A Go , lane 3 and Fig. 6A Go , lane3), whereas no HC at all could be detected in Daudi cells ( Fig. 5A Go , lane 1 and Fig. 6A Go , lane1), indicating that only MHC class I HC–ß 2 m dimers bind to tapasin and calreticulin. TAP proteins (TAP1 and 2, each of a size ~70 kDa), were also co-precipitated in Daudi and LCL cells with anti-tapasin ( Fig. 5A Go , lanes 1–2). Anti-calreticulin precipitated tapasin in all three cell lines ( Fig. 5A Go , lanes 1–3) with an additional strong band at 90 kDa in Daudi cells ( Fig. 5A Go , lane 1). The 60-kDa protein was identified as calreticulin ( Fig. 5B Go ) and the 48-kDa protein as tapasin ( Fig. 6B Go ) by immunoblotting of immunoprecipitates with anti-tapasin or anti-calreticulin antisera respectively.



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Fig. 5. Interaction of calreticulin with class I HC in ß 2 m- or TAP-deficient cell lines, and in wild-type B-LCL cells. Daudi, LCL or T2 cells were metabolically labeled with [ 35 S]methionine for 1 h followed by solubilization with 1% digitonin. The lysates were precipitated with calreticulin-specific antiserum (A, lanes 1–3) or with normal rabbit serum (A, lanes 4–6) and analyzed on a 10% SDS gel followed by autoradiography. The 60-kDa (calreticulin) band was determined by immunoblotting with an anti-calreticulin antibody (B, lanes 1–3). Results are from one of three similar experiments.

 


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Fig. 6. Interaction of tapasin with class I HC in ß 2 m- or TAP-deficient cell lines and in wild-type B-LCL cells. Results are from one of three similar experiments. Daudi, LCL or T2 cells were metabolically labeled with [ 35 S]methionine for 1 h followed by solubilization with 1% digitonin. The lysates were precipitated with tapasin (A, lanes 1–3) and analyzed on a 10% SDS gel followed by autoradiography. The identity of the 48-kDa (tapasin) protein was determined by immunoblotting with an anti-tapasin (B, lanes 1–3). Results are from one of three similar experiments.

 
Taken together, these results indicate distinct folding stages of HC in the Daudi and T2 cells. In Daudi cells, folding is arrested at a stage where both calnexin and BiP are bound at least partly to the same HC, while in T2 cells, HC have matured to a conformation beyond the point where BiP is needed. At this stage, calnexin is still associated for a prolonged period and may facilitate the assembly of ß 2 m with HC. Calreticulin and tapasin then bind to mature HC–ß 2 m dimer during peptide loading.

Presence of ß2m protects HC from degradation in TAP-deficient cells
The trimeric complexes consisting of HC, ß 2 m and peptide constitute the mature and stable MHC class I molecules. It has previously been shown that absence of either ß 2 m, or TAP, results in the accumulation of HC in the ER ( 412 ). To compare the stability of HC in the ER of Daudi and T2 cells, we performed a pulse–chase experiment followed by immunoprecipitation with the R425 anti-serum. During the 4 h chase, HC were rapidly degraded in Daudi cells ( Fig. 7A Go , lanes 1–5 and B), while they remained stable in both T2 cells ( Fig. 7A Go , lanes 11–15 and B), and LCL cells ( Fig. 7A Go , lanes 6–10 and B) used as a control. ß 2 m could be co-precipitated from LCL and T2 cells throughout the chase ( Fig. 7A Go , lanes 6–15). Thus, the presence of ß 2 m protects HC from degradation, even in T2 cells in which the ß 2 m–HC association has been shown to be unstable ( 21 ).




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Fig. 7. Stability of class I HC in Daudi, B-LCL and T2 cells as analyzed by pulse–chase. Daudi, LCL and T2 cells were labeled with [ 35 S]methionine for 10 min and chased for the time periods indicated. At the end of the chase, the cells were lysed with 1% NP-40 and subjected to immunoprecipitation with the anti-class I antibody R425. The precipitates were analyzed on a 10% SDS gel. (A) Autoradiogram. (B) Mean values (as determined by a Phosphoimager) from three separate experiments converted to d.p.m. according to the standard.

 
TAP regulates the export of class I from the ER
The results shown above regarding the dissociation of HC from calnexin in Daudi and T2 cells, suggest that calnexin may not be the major factor controlling export of MHC class I from the ER. Previous results have shown that TAP1 is associated with ß 2 m-assembled HC in normal cells and that peptide binding to class I dissociates the class I from TAP ( 24 , 26 , 27 ). To analyze whether TAP controls the surface expression of class I, we performed a pulse–chase analysis in LCL cells to determine the rate of dissociation of class I from TAP ( Fig. 8A Go , lanes 2–6) and the kinetics of surface expression of class I ( Fig. 8B Go , lanes 1–5). Following the pulse ( Fig. 8A Go , lane 2), a large portion of HC was associated with TAP. The kinetics of the dissociation of HC from TAP ( Fig. 8A Go , lanes 3–6 and C) and their appearance on the cell surface ( Fig. 8B Go , lanes 2–5 and C) were reciprocal, suggesting that TAP may control the surface expression of class I molecules. This may be exerted through the peptide transport and loading rates.




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Fig. 8. Determination of the dissociation rate of HC from TAP and kinetics of surface expression of class I HC. (A) To determine the rate of dissociation of class I HC from TAP, LCL cells were pulse-labeled for 1 h with [ 35 S]methionine and chased for the time periods indicated. The cells were lysed with 1% digitonin and subjected to immunoprecipitation with anti-TAP antiserum, followed by analysis on a 10% SDS gel (lanes 2–6). As a control, TAP was precipitated from similarly pulse-labeled Daudi (lane 1) and T2 (lane 7) cells. The positions of HC, TAP1, TAP2 and tapasin are indicated. (B) To analyze the kinetics of the appearance of class I on the cell surface, LCL-cells were pulse-labeled for 5 min with [ 35 S]methionine and chased for the time periods indicated. The cells were then incubated on ice with the W6/32 antibody for 15 min, washed 3 times with PBS and lysed in 1% NP-40. The immunocomplexes were precipitated by Protein A–Sepharose and analyzed on a 10% SDS gel. The positions of the class I HC, ß 2 m and the cross-reacting actin are indicated. (C) Mean values (as determined by a Phosphoimager) from three separate experiments converted to d.p.m. according to the standard.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The main steps of assembly of MHC class I molecules in the ER include: (i) newly synthesized class I HC in the state of folding to which ß 2 m is not yet associated, (ii) immature HC–ß 2 m complexes devoid of antigenic peptide, and (iii) stably assembled molecules consisting of HC, ß 2 m and peptide ( 1 , 4 , 6 , 21 ). In two of the three cell lines used in this study, Daudi and T2, maturation of class I molecules is blocked in the first two steps respectively. In Daudi cells, only free HC exist ( 4648 ), whereas in T2 cells immature HC are unstably associated with ß 2 m ( 1 , 21 ). In this study, the third stage is represented by an Epstein–Barr virus-transformed human B-lymphoid cell line (B-LCL-WW1). In most previous studies, the assembly of MHC class I molecules has been studied in these or comparable cells separately, while we set out to compare this process in the three cell lines under as similar conditions as possible with special reference to the association of ER chaperones and TAP with class I HC. Different MHC class I alleles exhibit different interaction patterns with the ER chaperones included in the loading complex ( 49 , 50 ). One important aim of this study was to be able to discriminate the class I-associated proteins before the association of class I with the loading complex in the ER of the three cell lines by co-immunoprecipitation of digitonin-solubilized cell lysates using antibodies to class I, and also to different chaperones including BiP, calnexin and calreticulin respectively. By this approach, we demonstrated that in the absence of ß 2 m, free class I HC associate with both BiP and calnexin. In contrast, HC in T2 cells were not detectably associated with BiP, but exclusively with calnexin. In LCL cells, there was no detectable HC association with either BiP or calnexin, confirming previous results ( 22 ). Instead, HC were complexed to calreticulin and tapasin. The distinct pattern of association of MHC class I molecules with BiP, calnexin, calreticulin and tapasin in the three cell lines indicates their sequential action during folding into functional class I molecules in the ER, and their exit from this compartment.

By cross-linking, newly synthesized, immature, HC were found to complex with either BiP or calnexin in C1R cells (ß 2 m ) transfected with B7 ( 22 ). The authors proposed different functions for BiP and calnexin. BiP would associate with misfolded HC retaining them in the ER and marking them for destruction, while association with calnexin would lead to the assembly with ß 2 m and production of functional HC. The authors did not find evidence indicating simultaneous interaction of BiP and calnexin with the same HC molecule. In contrast to that report, one important finding of our study was the demonstration, without cross-linking, of the simultaneous association of BiP and calnexin with HC. The reason for this difference is probably due to the choice of detergent and also to the different cell lines used—digitonin and Daudi cells in our case compared to NP-40 and CIR cells in their case. BiP has been shown to interact in a productive folding pathway for Ig ( 51 ). The folding mechanism differs from calnexin and calreticulin. The results suggest that BiP and calnexin may cooperate during the folding process leading to mature MHC class I. Previously, the vesicular stomatitis virus G glycoprotein was reported to interact both with BiP and calnexin during folding. Pulse–chase experiments suggested that the interaction with BiP may precede the interaction with calnexin ( 52 ). On the other hand, evidence has been presented suggesting that calnexin may interact with folding thyroglobulin prior to BiP ( 53 ). Based on our results from the Daudi and T2 cells, it seems that BiP may interact with HC prior to calnexin, although the co-precipitation experiments clearly indicated that both chaperones may also interact simultaneously with the same HC molecule. In Daudi cells, co-precipitation of BiP with the anti-HC antibody was much more prominent than that of calnexin (Fig, 3AGo , lane 1). Moreover, we only detected calnexin, but not BiP, associated with HC in T2 cells ( Fig. 1A Go , lane 3), and this association was more prolonged in T2 than in Daudi cells ( Fig. 4A Go ). It was also found that the presence of calnexin facilitates the association of ß 2 m and class I HC in both mouse and human cells ( 15 ). This finding supports our suggestion that calnexin functions at a later folding stage of class I HC than BiP.

In human ß 2 m-deficient cells, the addition of ß 2 m to the cell lysates has been shown to increase the dissociation of HC from calnexin ( 21 , 22 ). Here, using pulse–chase, we compared the dissociation of HC from calnexin in Daudi and T2 cells. The results suggested that the presence of ß 2 m per se in T2 cells did not facilitate the dissociation of HC from calnexin in comparison to Daudi cells. In contrast, the rate of dissociation was faster in Daudi than in T2 cells. This difference was not due to the presence of TAP, because HC are not associated with TAP in the absence of ß 2 m in Daudi cells ( Fig. 8A Go , lane 1). The differences in association of HC with BiP and calnexin, and in the stability of ER-retained HC in Daudi and T2 cells are most probably due to the absence or presence of ß 2 m, even though the association of ß 2 m and HC in T2 cells is unstable ( 21 ).

With the anti-class I antibody R425, which reacts with all forms of class I molecules, we could show that only a portion of the class I molecules was associated with calnexin in T2 cells, suggesting that calnexin is not the major factor for the retention of immature class I molecules in the ER of human cells, as suggested previously ( 22 , 38 ). Although accumulation of class I HC in the ER is a common feature in Daudi and T2 cells, the mechanism of retention may be different due to the difference in association to chaperones or other molecules. Also, evidence for the recycling of immature class I molecules between the ER and the intermediate compartment has been presented ( 54 ). In normal cells, it has been shown that TAP1 is associated with most of the ß 2 m–HC-assembled class I molecules in the ER ( 24 , 26 ). The correlation between the dissociation of class I molecules from TAP and the concomitant surface expression of HC ( Fig. 8 Go ) suggests that the TAP complex may control the export of class I from the ER to the cell surface. The dissociation is probably regulated by peptide loading, as previously suggested ( 6 , 24 , 26 ).

Based on the results presented here, and on those from other laboratories ( 1517 , 19 , 2126 , 37 , 38 ), we propose the following model for the sequential interaction of BiP and calnexin, before the association of MHC class I molecules with the loading complex ( Fig. 9 Go ). First, BiP binds to the nascent, folding, HC followed by binding to calnexin. Some of HC interact with both chaperones simultaneously. Binding of ß 2 m to HC releases BiP, while calnexin may still remain bound. When a stable HC–ß 2 m complex has been formed, calnexin is released and HC–ß 2 m binds to the loading complex. Finally, peptide loading results in the release of MHC class I from the loading complex, followed by transport of mature class I molecules out to the cell surface.



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Fig. 9. Processing of MHC class I in the ER. Cnx, calnexin. The shaded circle in `5' indicates the optimal peptide.

 


    Acknowledgments
 
We thank Dr S. Kvist for the antisera. This study was supported by grants from Crafoodska stiftelsen (990589), the Swedish Cancer Society (3975-B99-03XAB), the Medical Faculty, Lund University and the Foundation for Strategic Research (SFF, from Infection & Vaccinology, 36/98 and from Inflammation Research Program/99).


    Abbreviations
 
ß2m ß2-microglobulin
BiP IgG heavy chain binding protein
ER endoplasmic reticulum
HC heavy chain

    Notes
 
Transmitting editor: E. Möller

Received 15 February 2001, accepted 14 May 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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