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J. Biol. Chem., Vol. 277, Issue 4, 2750-2755, January 25, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/4/2750    most recent M110300200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Faubert, A. Articles by Thibodeau, J. Search for Related Content PubMed PubMed Citation Articles by Faubert, A. Articles by Thibodeau, J. Social Bookmarking                      What's this?

Functional Analysis of Tryptophans 62 and 120 on HLA-DM^*

Amélie Faubert^§, Angela Samaan^¶, and Jacques Thibodeau

From the  Laboratoire d'Immunologie Moléculaire, Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal, Québec, H3C 3J7, Canada and ^¶ INSERM U462, Hopital Saint-Louis, 75475, Paris, France

Received for publication, October 26, 2001, and in revised form, November 15, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the endocytic pathway of antigen-presenting cells, HLA-DM catalyzes the exchange between class II-associated invariant chain peptide (CLIP) and antigenic peptides onto major histocompatibility complex class II molecules. At low pH of lysosomal compartments, both HLA-DM and HLA-DR undergo conformational changes, and it was recently postulated that two partially exposed tryptophans on HLA-DM might be involved in the interaction between the two molecules. To define contact regions on HLA-DM, we have conducted site-directed mutagenesis on those two hydrophobic residues. The HLA-DM W62A,W120A (DM_W62A/W120A) double mutant was expressed in HLA-DR⁺ HeLa cells expressing invariant chain, and the activity of this DM molecule was assessed. Flow cytometry analysis of cell surface DR-CLIP complexes revealed that DM_W62A/W120A removes CLIP as efficiently as its wild-type counterpart. DM_W62A/W120A was found in the endocytic pathway by immunofluorescence, and DM-DR complexes were immunoprecipitated from these cells at pH 5. Finally, mutations W62A and W120A on HLA-DM did not affect the association with HLA-DO. The complex egresses the endoplasmic reticulum and accumulates in endocytic vesicles. Moreover, DO and DM_W62A/W120A were co-immunoprecipitated at pH 7. We conclude that the 62 and 120 tryptophan residues are not required for the activity of DM, nor are they directly implicated in the interaction with DR or DO.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Major histocompatibility complex class II molecules are primordial for activation of CD4+ T cells and for immunological response against pathogens (1). Three major histocompatibility complex II / heterodimers associate with a trimer of invariant chain (Ii)¹ to form a nanomeric complex in the endoplasmic reticulum (ER) (2). This association allows the proper folding and trafficking of the major histocompatibility complex II molecules (3-5). Gradual proteolysis of Ii occurs in the endocytic compartments by the sequential action of proteases including cathepsins (6). A residual peptide of Ii (CLIP) remains in the peptide groove of the class II molecule and stabilizes the heterodimer by preventing collapse of the groove (7, 8). For most allotypes, CLIP must be actively removed to allow binding of antigenic peptides. HLA-DM, a nonclassical intracellular class II molecule, plays a central role in the efficiency of antigen presentation as it catalyzes CLIP release from HLA-DR and stabilizes the class II in a chaperone-like fashion (9-14). In fact, mice lacking the H2-Ma gene express a high level of surface class II-CLIP complexes (15-17).

The precise molecular mechanism of action of HLA-DM remains to be established. X-ray diffraction studies on HLA-DM crystals revealed a closed peptide-binding groove and an overall quaternary structure similar to classical class II molecules (18, 19). Time-resolved fluorescence anisotropy and far-UV circular dichroism spectrum analysis using soluble HLA-DM revealed that its structure is quite rigid and that it is not subjected to gross pH-dependent conformational change. However, many other experiments by the same groups suggest that protonation in the endocytic pathway results in minor, reversible structural changes exposing hydrophobic regions of the DM heterodimer (20, 21). For example, fluorescence spectroscopy studies revealed that hydrophobic tryptophan residues are buried in native DM and would become more solvent-exposed at endosomal pH (20). Accordingly, Ullrich et al. (21) used 8-anilino-1-naphthalenesulfonic acid (ANS), a fluorescent dye binding to hydrophobic protein patches, to demonstrate subtle pH-induced change in DM. Since the interaction of DM with DR reduces ANS binding to both molecules, it was postulated that the surface of contact comprises these pH-sensitive regions (21, 22).

On the basis of these results, Wiley and co-workers (19) proposed that two partially exposed tryptophans (62, 120), located on the same lateral surface of DM, could be critical for DR binding. The model predicts a major interaction between tryptophan 62 of DM and the protruding phenylalanine 51 of DR, resulting in the breakage of multiple hydrogen bonds at the end of the groove and in the release of unstably associated peptides. At least part of this model was recently confirmed when mutation of DRF51 was shown to abolish the interaction with DM (23).

To confirm the possible interaction between DRF51 and DMW62, we have mutated the exposed tryptophan on HLA-DM and tested the ability of this mutant to release CLIP from HLA-DR. Our results show that this mutation on DM does not affect DR contact, CLIP release, or the localization of the protein within the cell. Similar conclusions were drawn after mutating the second exposed tryptophan at position 120. Finally, those amino acids substitutions on DM did not affect its interaction with HLA-DO, an intracellular nonclassical class II molecule that binds to DM and regulates its activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmids-- DM cDNA was digested by Ecl136II-ClaI from pMCFR-PAC and subcloned into EcoRV-ClaI of pBluescript (pBS) (24). The DM cDNA has been described previously (25). DM and DM cDNAs from pBluescript were subcloned successively on the same pBudCE4-A vector (as previously described) (25). The Ii p35 cDNA was obtained by mutagenesis of the second ATG codon of Ii (kindly provided by Rafick Sékaly) and subcloned in the BamHI site of SRpuro. Details will be provided elsewhere.

DM and DM Mutagenesis-- Mutations into the DM and DM cDNA sequences were introduced by PCR overlap extension (26). Briefly, 5' PCR products were generated from pBS DM and pBS DM using mutagenic primers (DMW62AEcoRI, 5'-CTG AGC CGC GTC AGC GAA TTC GGG-3'; DMW120ANspI, 5'-GAA GCC CGC CAC ATA GCA TGC CAG CAT-3') as well as the universal (DM) or reverse (DM) primers. The 3' PCR product was generated using the complementary mutagenic primers. The two overlapping PCR products were mixed, and a final PCR was performed using the flanking primers. Fragments were subsequently subcloned into the PstI-SalI sites of pBSDM and SalI-HindIII of pBSDM, thereby replacing the wild-type fragment with the PCR product containing the appropriate mutation. The nucleotide sequence was confirmed by DNA sequencing using T7 polymerase (Amersham Biosciences). Mutant cDNAs and wild-type were introduced into pBudCE4-A as NotI-XhoI (pBSDM) and SalI-XbaI (pBSDM) fragments.

Antibodies-- Monoclonal antibody (mAb) MaP.DM1 (IgG₁) is directed against the luminal portion of HLA-DM (PharMingen International, Oakville, Canada). mAb L243 (IgG2_a) binds a specific DR conformational determinant (27). mAb BU45 (IgG₁) binds the C-terminal part of the human invariant chain (CD74) (The Binding Site, Birmingham, United Kingdom). mAb Cer-CLIP (IgG₁) is directed against the N-terminal segment of CLIP (PharMingen International) (24). Goat anti-mouse and goat anti-rabbit IgG coupled to Alexa Fluor®488 were obtained from Molecular Probes, Inc. (Eugene, OR). Biotinylated goat anti-mouse was from BIO/CAN scientific (Mississauga, Canada), and Texas Red-coupled streptavidin was from Amersham Biosciences. The anti-DO serum was produced in C₃H mice (H-2^k) by repeated intraperitoneal injections of DAP fibroblasts transfected with DR and DR₁₈/DO cDNAs (28). Rabbit antisera against the cytoplasmic tail of HLA-DO or HLA-DM have been described previously (25). The CD107a-specific monoclonal antibody H4A3 (IgG₁) reacts with the heavy glycosylated 100-kDa lysosome-associated membrane protein (Lamp-1) (Developmental Studies Hybridoma Bank, NICHD, University of Iowa, Iowa City, IA)

Cell Lines and Transfections-- HeLa DR1 (DR plus DR 0101) cells were kindly provided by Dr. R. P. Sékaly. HeLa DO cells have already been described (25). In order to enrich for DO-expressing cells, the population was treated with  interferon (PharMingen International) and sorted on magnetic beads for CLIP expression (Dynal ASA, Olso, Norway). Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen), 10% fetal bovine serum (Wisent, St.-Bruno, Canada) with appropriate selective agents (see below). Stable transfections were done with Fugene6 (Roche Molecular Biochemicals) using 1 µg of DNA per 10⁵ cells. Selective agents were added to a final concentration of 500 µg/ml G-418 (Invitrogen), 400 µg/ml puromycin (Sigma), 50 units/ml hygromycin (Cederlane Laboratories, Hornby, Canada) or 100 µg/ml Zeocin^TM (Cayla, Toulouse, France).

Flow Cytometry Analysis-- Intracellular staining was done as previously described (25). Briefly, saponin-treated cells were incubated with the appropriate primary antibody, and then they were incubated with goat anti-mouse IgG (H + L) coupled to Alexa Fluor^®488. Cells were then analyzed by flow cytometry on a FACScalibur® (Becton Dickinson, Mississauga, Canada).

Fluorescence Microscopy-- 10⁴ cells were plated on coverslips in 24-well plates and cultured for 3 days before staining (25). Cells were analyzed by fluorescence microscopy on a Zeiss axioscope microscope (Carl Zeiss, Thornwood, NY). Photographs were taken with a Zeiss microscope camera MC 100 on Eastman Kodak Co. elite chrome 400 films.

Immunoprecipitations and Western Blotting-- Cells (4 × 10⁶) were trypsinized, washed in phosphate-buffered saline, and lysed in 1% Triton X-100 at pH 7 or in CHAPS 1% at pH 5 as described previously (29). After centrifugation, supernatants were harvested and incubated with protein G coupled to CNBr-activated Sepharose 4B (Amersham Biosciences) and bound to the appropriate antibody (L243 mAb or mouse serum anti-DO). Following washes in lysis buffer, samples were resuspended in nonreducing buffers and subjected to SDS-PAGE. After transfer to nylon membranes (Amersham Biosciences), proteins were blotted with the rabbit anti-DO or anti-DM sera. Secondary antibody (peroxidase-coupled goat anti-rabbit; BIO/CAN Scientific) was used at a 1:1000 dilution for 2 h, and the signal was detected using ECL (Amersham Biosciences).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The crystal structure of DM reveals two partially exposed tryptophan residues that are thought to interact with HLA-DR and to participate in CLIP removal (19). Tryptophans 62 and 120 are located on the same lateral face of HLA-DM in the 1 and 2 domains, respectively (Fig. 1). These amino acids are highly conserved throughout evolution, emphasizing their potential importance.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Position of mutations on HLA-DM. Highlighted tryptophans 62 and 120 (W62 and W120, respectively) are located on the same lateral face of HLA-DM in the 1 and 2 domains, respectively. The GenBankTM accession numbers are NM006120 for DM (red) and NM002118 for DM (orange) (10). Molecular modeling was done with Protein Data Bank reference number 1HDM and analyzed with the Swiss PDB Viewer (19).

In an effort to define the binding site for HLA-DR and to gain insights into the mechanism of action of HLA-DM, we mutated those two bulky aromatic tryptophans to small alanines and tested in vivo the ability of mutant proteins to remove CLIP from HLA-DR. Various combinations of mutated and wild-type DM cDNAs were transfected in HeLa cells expressing DR1 and Ii. DR1 is known to be dependent on DM for the release of CLIP at acidic pH (30). We obtained four stable cell lines: DR1 Ii DM, DR1 Ii DM_W62A, DR1 Ii DM_W120A, and DR1 Ii DM_W62A/W120A. Expression of DR was monitored at the cell surface, while DM and Ii were analyzed in permeabilized cells. As measured by flow cytometry, all cell lines express high levels of the various transfected molecules (Fig. 2). The proper folding and intracellular sorting of DM was assessed by immunofluorescence microscopy. Fig. 3 shows that wild-type HLA-DM (B) accumulates in intracellular vesicles that are also positive for the lysosomal marker Lamp-1 (G). A tyrosine-based motif on the cytoplasmic tail of DM is responsible for its accumulation in endocytic compartments (31-33). The three mutated forms of DM were also found in peripheral vesicles (Fig. 3, C-E) and co-localized with Lamp-1 (Fig. 3, H-J). Control cells lacking DM showed a weak background using the DM-specific antibody but definite Lamp-1-positive compartments (Fig. 3, A, F, and arrows). Together with the fact that the MaP.DM1 conformational antibody efficiently recognizes the DM mutants, these results show that the overall structure of DM is not affected by replacement of tryptophans 62 and 120.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Expression of HLA-DR, invariant chain, and HLA-DM in transfected HeLa cells. HeLa DR1 cells were stably transfected with the p35 form of the invariant chain cDNA, and the cell population was supertransfected with cDNAs for wild-type or mutant DM molecules. Flow cytometry analysis was performed on HeLa DR1 Ii, DR1 Ii DM, DR1 Ii DMW62A, DR1 Ii DMW120A, or DR1 Ii DMW62A/W120A using L243 mAb for surface HLA-DR staining. BU45 and Map-DM1 mAbs were used for intracellular staining of Ii and DM, respectively. Alexa 488-coupled GAM was used as secondary antibody. Open histograms represent the control fluorescence of HeLa cells incubated in the same conditions.

View larger version (43K): [in this window] [in a new window]   Fig. 3.   Mutants of HLA-DM localize in the endocytic pathway. DR1 Ii (A and F), DR1 Ii DM (B and G), DR1 Ii DMW62A (C and H), DR1 Ii DMW120A (D and I), and DR1 Ii DMW62A/W120A (E and J) cells were analyzed for DM (A-E) and for Lamp-1 (F-J). Immunofluorescence microscopy analysis was performed on permeabilized cells double-stained using a rabbit anti-DM polyclonal antibody and a mouse anti-Lamp-1 mAb. Secondary antibodies were Alexa-488 coupled to goat anti-rabbit and a biotinylated GAM followed by Texas Red-coupled streptavidin. The arrows point to cells not transfected with HLA-DM.

The activity of these mutant forms of DM was verified by monitoring the levels of DR-CLIP complexes at the cell surface of transfected cells. As demonstrated by many groups, introduction of HLA-DM in deficient cell lines favors the intracellular exchange of CLIP for more stable peptides (24, 34, 35). Consequently, CLIP expression is low at the surface of DM⁺ cells. The cell surface expression of CLIP was measured using the Cer-CLIP monoclonal antibody that is specific for the N terminus of CLIP bound to class II molecules and which does not recognize the intact invariant chain (24). As shown in Fig. 4A, control DM cells (HeLa DR1 Ii) express high levels of DR-CLIP complexes at their surface. On the other hand, cells expressing wild-type or mutant forms of DM do not express significant levels of CLIP (Fig. 4, B-E). These results suggest that DR-Ii complexes are sorted to the endocytic pathway, where Ii is degraded until a last fragment, CLIP, is actively removed from the peptide binding groove by wild-type or mutant forms of HLA-DM. To confirm the proper interaction between the DM mutants and DR, co-immunoprecipitations were carried out at acidic pH in the CHAPS detergent (36). Fig. 5 shows that DM can be co-immunoprecipitated by the DR-specific antibody only in cells expressing both molecules (Fig. 5, left three lanes). Also, the three mutant forms of DM could all be efficiently co-immunoprecipitated with HLA-DR (Fig. 5). The stronger DM signal obtained by Western blotting on the HeLa DR1 Ii DM_W62A/W120A samples most probably reflects the higher level of HLA-DR expression on these cells (mean fluorescence value = 789) as compared with the other DM⁺ transfectants (mean fluorescence values = 248-374) (Fig. 2). Taken together, these results show that the mutations W62A and W120A do not affect the folding, sorting, and activity of HLA-DM, nor its ability to interact strongly with HLA-DR.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Mutations on HLA-DM do not impair CLIP release. Surface flow cytometry analysis was performed using Cer-CLIP monoclonal antibody followed by an Alexa 488-coupled GAM (filled histograms). Open histograms represent the control fluorescence of nontransfected HeLa cells stained in the same conditions.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   HLA-DM mutants bind to HLA-DR. Transfected HeLa cells were lysed in 1% CHAPS at pH 5, and HLA-DR was immunoprecipitated using L243 mAb. The samples were analyzed on Western blot using the DM-specific rabbit serum.

Although these tryptophan residues are not directly involved in the binding to HLA-DR, they might very well be crucial for the binding to HLA-DO. The latter is mostly expressed in B cells and is a nonclassical class II molecule that modulates the activity of DM (37-39). The mode of action of DO is not determined, but it clearly affects the peptidic repertoire when present in physiological amounts in an APC (40-42).

To further characterize the DO-DM contact regions, wild-type and double mutant (DM_W62A/W120A) DM molecules were stably transfected in HeLa DO⁺ cells. Expression of both proteins was confirmed by intracellular staining and flow cytometry (Fig. 6). Since HLA-DO is absolutely dependent on DM association to egress the ER (43) and gain access to the endocytic pathway, we first verified the ability of mutant HLA-DM to direct the transport of DO. Immunofluorescence microscopy showed that HLA-DO reaches the endocytic pathway in the presence of either the wild-type or the DM_W62A/W120A molecule (Fig. 7). As opposed to the diffuse ER-like pattern of expression observed for DO in DM cells (Fig. 7b), defined HLA-DO-containing vesicles can be seen in DM⁺ cells (Fig. 7, d and f). The co-localization between DO and DM_W62A/W120A in the endocytic pathway (Fig. 7, e and f) strongly argues for an efficient interaction between the two molecules, allowing DO to egress the ER.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Expression of HLA-DM and HLA-DO in transfected HeLa cells. Flow cytometry analysis was performed on saponin-permeabilized cells using a mouse serum against DO (left panels) or Map-DM1 mAb (right panels) followed by Alexa 488-coupled GAM (filled histogram). Open histograms represent the control fluorescence of HeLa DM (left panels) or HeLa cells (right panels) incubated with the same antibodies.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   HLA-DM mutants bind to HLA-DO. A, immunofluorescence microscopy analysis was performed on permeabilized cells double-stained using a rabbit anti-DM (a, c, and e) and a mouse anti-DO (b, d, and f) sera. Secondary antibodies were Alexa 488-coupled goat anti-rabbit and a biotinylated GAM followed by Texas Red-coupled streptavidin. a and b, HeLa DO; c and d, HeLa DO DM; e and f, HeLa DO DMW62A/W120A. B, cells were lysed in 1% Triton X-100 at pH 7, and co-immunoprecipitations were performed using a DO-specific mouse serum. Samples were analyzed by Western blotting using the DM-specific or DO-specific rabbit sera.

The interaction between DO and DM was confirmed by co-immunoprecipitation experiments. Cells were lysed in 1% Triton X-100, and DO was immunoprecipitated using a polyclonal antibody against the cytoplasmic tail of the  chain. Western blotting with antibodies specific for DM or DO showed that wild-type DM and the DM_W62A/W120A double mutant were both efficiently co-immunoprecipitated with DO (Fig. 7B). Altogether, these results suggest that 62 and 120 tryptophans on HLA-DM are dispensable for the interaction with DR or DO.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Understanding the molecular mechanism by which HLA-DM and HLA-DM/DO catalyze peptide exchange will allow the development of new approaches for manipulating antigen loading and presentation. With this in mind, we have undertaken the mapping of amino acids involved in the interactions between these molecules. So far, the group of Mellins has characterized regions of HLA-DR that interact with HLA-DM (23, 44). Random mutagenesis on HLA-DR allowed the identification of critical residues on a lateral face encompassing both the 1 and 2 domains. From these experiments, it was proposed that DM releases unstable peptides through its "lever effect" on amino acids DR40 and 51, the last one being critical for stability around the P1 pocket of the groove (23). A role for DM in destabilizing those P1 anchors was also proposed from the results of Chou and Sadegh-Nasseri who showed that mutation of DR1G86Y rendered the class II rigid (as in a peptide-bound conformation) and reduced the binding to DM (45).

Based on the crystal structure of HLA-DM, the group of Wiley had already proposed that the contact between DM Trp62 and DR Phe51 could destabilize the P1 pocket and liberate an unstable peptide. Much experimental evidence points to a critical role of such hydrophobic residues in the contact between DM and DR. For example, based on studies measuring the binding of ANS, it was concluded that DR-CLIP complexes display a larger hydrophobic surface than DR molecules associated with stable peptides. Also, there is a preferential association of the two molecules at the acidic pH of endocytic vesicles, where both molecules would expose hydrophobic residues (21). The presence of tryptophan residues in those contact regions was deduced by their preferential binding of ANS as well as from spectroscopy studies measuring variations in fluorescence emission between exposed and buried residues (21, 45).

Two of 11 tryptophans are partially exposed and located on the same lateral surface on the crystal structure of DM. These residues may be part of the hydrophobic patches that become accessible to ANS at acidic pH but that are buried in the DM-DR interface (19). While DR Phe51 could interact with DM Trp62, tryptophan DM 120 may contact those hydrophobic residues identified by Mellins and co-workers (23) in the 2 domain of DR and which may serve to increase the affinity for DM. However, our results presented here do not support such a model. Mutation of the two tryptophans on DM did not disturb the activity, the sorting, or the conformation of the protein. Still, the possibility remains that those mutations could finely tune the specificity of DM and influence the peptide repertoire of the class II molecules.

The fact that random mutagenesis on HLA-DR allowed the identification of Phe51, Leu184, and Val186 residues certainly suggests the involvement of hydrophobic residues on DM as well (23). However, replacement of residue Glu187 for a lysine in the 2 domain also decreased DM binding in these studies. The importance of this charged residue on DR prompted us to evaluate the potential role of positively charged DM Lys109, Lys115, Arg93, and Arg95 residues. However, the simultaneous mutation of these residues did not inhibit CLIP release by DM in our system (data not shown).

Altogether, our results suggest that the DM/DR interaction relies on other hydrophobic residues in the above described region or that it may implicate another interface of DM. Indeed, Fremont et al. (18) identified two other hydrophobic regions that are located on distinct faces of the H2-M heterodimer. Site-directed mutagenesis in the corresponding regions of DM should help delimiting contact sites with DR.

Finally, our results from immunofluorescence and co-immunoprecipitation studies revealed that tryptophans 62 and 120 on DM are not necessary to make contact with HLA-DO. Although DO also undergoes conformational change following acidification of the environment (46), it first interacts with DM in the ER, and the strong association is resistant to lysis in 1% Triton X-100 (43). These observations suggest that the nature of the interactions is likely to differ between DR-DM and DO-DM. The existence of a three-molecule complex between DM, DO, and DR suggests the presence of two distinct functional interfaces on DM, and random mutagenesis is under way to delineate the contact regions with HLA-DR and DO (47).

	ACKNOWLEDGEMENTS

We thank the Developmental Studies Hybridoma Bank, NICHD, University of Iowa. We thank Dr. Eric Cohen for the use of the fluorescence microscope and Serge Sénéchal for assistance with the flow cytometer. We also thank Georges Azar, Angélique Belmarre, Alexandre Brunet, Francis Deshaies, and Hayssam Khalil for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by grants (to J. T.) from the Medical Research Council (MRC) of Canada and from the Cancer Research Society Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Supported in part by a studentship from Pfizer Canada Inc.

Recipient of a fellowship from the MRC. To whom correspondence should be addressed: Laboratoire d'Immunologie Moléculaire, Dépt. de Microbiologie et d'Immunologie, Université de Montréal, CP 6128, Succ. Center-Ville, Montréal, Québec H3C 3J7, Canada. Tel.: 514-343-6279; Fax: 514-343-5701; E-mail: jacques.thibodeau@umontreal.ca.

Published, JBC Papers in Press, November 16, 2001, DOI 10.1074/jbc.M110300200

	ABBREVIATIONS

The abbreviations used are: Ii, invariant chain; ANS, 8-anilino-1-naphthalenesulfonic acid; CLIP, class II-associated invariant chain peptide; CHAPS, 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate; ER, endoplasmic reticulum; GAM, goat anti-mouse; HLA, human leukocyte antigen; mAb, monoclonal antibody.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/4/2750    most recent M110300200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Faubert, A. Articles by Thibodeau, J. Search for Related Content PubMed PubMed Citation Articles by Faubert, A. Articles by Thibodeau, J. Social Bookmarking                      What's this?