The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Doebele, R. C.
Right arrow Articles by Mellins, E. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Doebele, R. C.
Right arrow Articles by Mellins, E. D.
The Journal of Immunology, 2003, 170: 4683-4692.
Copyright © 2003 by The American Association of Immunologists

Point Mutations in or Near the Antigen-Binding Groove of HLA-DR3 Implicate Class II-Associated Invariant Chain Peptide Affinity as a Constraint on MHC Class II Polymorphism1

Robert C. Doebele*, Achal Pashine*, Wendy Liu*, Dennis M. Zaller{dagger}, Michael Belmares{ddagger}, Robert Busch2,3,* and Elizabeth D. Mellins3,4,*

* Division of Immunology and Transplantation Biology, Department of Pediatrics, Center for Clinical Sciences Research, Stanford University School of Medicine, Stanford, CA 94305; {dagger} Merck Research Laboratories, Rahway, NJ 07065; and {ddagger} Department of Chemistry, Stanford University, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During maturation of MHC II molecules, newly synthesized and assembled complexes of MHC II {alpha}{beta} dimers with invariant chain (Ii) are targeted to endosomes, where Ii is proteolyzed, leaving remnant class II-associated Ii peptides (CLIP) in the MHC II peptide binding groove. CLIP must be released, usually with assistance from the endosomal MHC II peptide exchange factor, HLA-DM, before MHC II molecules can bind endosomal peptides. Structural factors that control rates of CLIP release remain poorly understood, although peptide side chain-MHC II specificity pocket interactions and MHC II polymorphism are important. Here we report that mutations {beta}S11F, {beta}S13Y, {beta}Q70R, {beta}K71E, {beta}K71N, and {beta}R74Q, which map to the P4 and P6 pockets of the groove of HLA-DR3 molecules, as well as {alpha}G20E adjacent to the groove, are associated with elevated CLIP in cells. Most of these mutations increase the resistance of CLIP-DR3 complexes to dissociation by SDS. In vitro, the groove mutations increase the stability of CLIP-DR3 complexes to dissociation. Dissociation rates in the presence of DM, as well as coimmunoprecipitation of some mutant DR3 molecules with DM, are also diminished. The profound phenotypes associated with some of these point mutations suggest that the need to maintain efficient CLIP release represents a constraint on naturally occurring MHC II polymorphism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Molecules of MHC class II are heterodimeric, membrane-tethered glycoproteins that bind peptides and display them at the surface of APC, where they may be recognized by Ag receptors (TCR) of CD4+ T cells. MHC II-bound peptides adopt a type II polyproline conformation and reside in an elongated groove, comprised of a {beta}-pleated sheet floor and two {alpha} helices that flank the bound peptide (Fig. 1 and Ref. 1). A conserved set of hydrogen bonds involving the peptide backbone provides sequence-independent tethering; in addition, peptide side chains, predominantly those at relative positions P1, P4, P6, and P9, interact with complementary specificity pockets in the groove. Despite the sequence specificity imposed by these interactions, the repertoire of MHC II-bound peptides comprises thousands of highly diverse peptides, derived from proteins that have access to the endocytic or secretory pathway (2).



View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 1. DR3 mutations that elevate CLIP. A ribbon diagram of the peptide-binding domain of DR3 complexed with CLIP is shown. A, Top view, with the CLIP N terminus to the left. B, Side view, with the CLIP N terminus pointing out of the plane of the paper. The {alpha}-chain is gray, and the {beta}-chain is blue. The P1 (M91 of Ii), P4 (A94), P6 (P96), and P9 (M99) anchor residues are shown as ball-and-stick models in black. The DR3-CLIP images were generated from PDB coordinates (accession code 1A6A), using WebLab ViewerLite version 3.2 software (Molecular Simulations, San Diego, CA). Mutations that elevate CLIP are shown; the font size of the labels indicates the strength of the phenotype.

 
Peptides derived from the MHC II-associated invariant chain (Ii), class II-associated Ii peptides (CLIP)5 spanning Ii residues 81–104 (LPKPPKPVSKMRMATPLLMQALPM; anchor residues underlined), bind MHC II molecules much like other peptides but play a unique role during MHC II maturation and peptide loading (3). Ii is a trimeric type II transmembrane protein that rapidly associates with three nascent MHC II {alpha}{beta} dimers in the endoplasmic reticulum. During assembly, Ii residues 91–99, which are part of a flexible, disordered region in the unbound Ii trimer, occupy the MHC II peptide binding groove (Fig. 1 and Refs. 4, 5, 6). Assembly with Ii is required for efficient egress of MHC II from the endoplasmic reticulum, and sequences in its cytoplasmic tail direct transport of the nonameric {alpha}{beta}3Ii3 complexes to late endosomal/prelysosomal compartments. There, most of Ii is removed by the proteolytic action of cathepsins, leaving CLIP, the terminal Ii degradation product, in the groove (7, 8). Release of CLIP in endosomes is required for subsequent binding of antigenic peptides. Thus, CLIP-MHC II complexes are obligate intermediates during peptide loading of newly synthesized MHC II molecules.

MHC polymorphism clearly influences MHC II interactions with CLIP. Although inactivation of empty DR molecules is now known to complicate estimations of peptide binding affinity using competition assays (9), early studies comparing CLIP binding to different MHC II alleles revealed large, allele-dependent variations in apparent CLIP affinity (10). For human as well as murine MHC II alleles with high CLIP affinity, CLIP can be found among peptides associated with total cellular class II (2, 11, 12, 13), but CLIP release in normal APC is efficient enough to allow a majority of MHC II molecules to be loaded with other peptides. An endosomal peptide exchange factor, HLA-DM, accelerates release of CLIP, which, for most MHC II alleles, would otherwise be the rate-limiting step for endosomal peptide loading (14). In mutant APC lines or in mice lacking HLA-DM expression, MHC II molecules accumulate CLIP to varying degrees, depending on the MHC II allele studied (15, 16, 17, 18, 19, 20).

Even though the crystal structure of CLIP bound to a MHC II molecule, HLA-DR3, has been determined, and despite the functional importance of CLIP release for Ag presentation, the structural factors that influence CLIP binding to MHC II molecules have not been fully explored. Analyses of CLIP binding to different MHC II alleles (10) and peptide substitution experiments (21, 22, 23, 24) support the notion that CLIP interacts with MHC II molecules much like other antigenic peptides. Here we report on the isolation and characterization of point mutations that increase retention of CLIP on HLA-DR3 MHC II molecules by stabilizing CLIP in the Ag binding groove.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

Wild-type and mutant EBV-B cell lines used in this study are described in Table I. The numbered cell lines (e.g., 8.1.6) are clonal cell lines, whereas most of the mutational analysis uses polyclonal transfectants expressing a particular mutant transgene (e.g., {alpha}G20E). In the clonal lines, it is formally possible that other genetic or epigenetic changes, in addition to particular identified mutations, contribute to the phenotype. The retroviral packaging line, {phi}NXA (25), and culture conditions (26) have been previously described.


View this table:
[in this window]
[in a new window]
  		Table I. Expression of MHC II and DM by mutant EBV-B cell lines

 
Vectors and transfections

cDNAs for DRA and DRB1*0301 were inserted into the retroviral vector, pBMN-IRES-Neo. Random mutant libraries were generated by amplification of inserts using error-prone PCR conditions with Taq polymerase; single mutant frequencies were ~23% (26). Site-directed mutants were generated by overlap extension PCR using high fidelity Pfu polymerase. All PCR products were cloned into the retroviral vector. Vectors, random colonies from the mutant library, and PCR-amplified cDNA inserts rescued from the high-CLIP clones were sequenced at the Stanford Protein and Nucleic Acid facility (Stanford, CA).

For production of retroviral particles, vectors were transfected into {phi}NX-A cells using calcium phosphate precipitation. To infect 9.4.3 or 9.22.3 cells, 48-h supernatants containing retrovirus were used. Homogeneous DR-expressing populations were obtained by G418 selection. Cells with high (top 0.1%) levels of surface CLIP were subjected to single-cell FACS, using the anti-CLIP mAb CerCLIP.1 (27). For generation of 5.2.4 transfectants, 5.2.4 was transduced first with wild-type DRA. After a second round of transduction with mutant DRB vectors, DR-expressing populations were enriched by magnetic bead sorting, followed by FACS of DR{alpha}{beta}-expressing populations using the anti-DR mAb L243 (28). Details of all protocols have been described (26).

Phenotyping of mutant cells

To measure CLIP and total DR levels at the cell surface, cells were incubated with mAbs to the CLIP N terminus (CerCLIP.1), DR{alpha} (L243), or DR{beta} (ISCR3; Ref. 29). Indirect immunofluorescence staining was performed using goat anti-mouse IgG, and median fluorescence intensities were measured by FACS, as described (30).

SDS stability was measured as described (30). Briefly, IGEPAL CA-630 cell extracts (with or without boiling) were resolved by nonreducing SDS-PAGE and immunoblotted with mAbs to the DR{alpha} cytoplasmic tail (DA6.147; Ref. 31), DR{beta} (HB10.a; Ref. 32), and CLIP (CerCLIP.1). Band intensities were quantified, using a GS-710 densitometer and Quantity One software (Bio-Rad, Hercules, CA). In some experiments, cell extracts were incubated with two rounds of CerCLIP-coated protein A-Sepharose beads (Sigma-Aldrich, St. Louis, MO), each for 1 h at 4°C, and cleared cell extracts were analyzed for SDS-stable DR dimers. CerCLIP-reactive material was eluted from the beads by boiling, analyzed by SDS-PAGE, and immunoblotted with mAb DA6.147.

Purification and characterization of mutant DR molecules

DR molecules were purified from IGEPAL CA-630 cell extracts of 8.1.6, 8.39.7, or sorted 5.2.4 transfectants, using L243 affinity chromatography as described (15, 26). DR purity was assessed by silver staining and Western blotting. Conformational integrity of DR in each preparation was compared by a L243 sandwich ELISA (33). Soluble DM molecules were purified from supernatants of transfected Drosophila cells, as described (34, 35).

CLIP dissociation was measured as described (26, 34). Briefly, DR molecules were loaded overnight in buffer containing 1% n-octyl-{beta}-D-glucopyranoside (OG) at pH 5 with CLIP81–104, labeled with long chain biotin either at the {alpha}-amino group (bio-CLIP) or at the {epsilon}-amino group of a lysine residue substituted at C-terminal position 104 (CLIP-bio). Free peptide was removed using Sephadex G-50 superfine spin columns. Complexes were diluted to {approx}10 nM in citrate-phosphate buffer, pH 5.0, containing 0.5% IGEPAL CA-630 or 1% OG, a >=100-fold excess of unlabeled CLIP, and protease inhibitors. Where indicated, 0.25 µM soluble DM was added. After incubation at 37°C for various times, the amount of remaining complex was determined by Ab (LB3.1 in Ref. 36 or L243) capture in microtiter plates and detection with streptavidin-Eu3+ and time-resolved fluorescence.

Coimmunoprecipitation of mutant DR molecules with DM was measured as described (26). Briefly, 1–2 µl of 1% CHAPS lysates of Drosophila cells expressing full length DM (35) containing {approx}90 ng DM/µl lysate were incubated (1–2 h) with wild-type or mutant DR3 molecules (100–500 ng) in pH 5.0 buffer containing 1% CHAPS. Reaction mixtures were immunoprecipitated with an anti-DM antiserum (11323; D. Zaller, unpublished data), and DM-associated DR3 molecules were detected by Western blotting using DA6.147 (DR{alpha} specific), or HB10.a (DR{beta} specific). Blots were reprobed with the DM{beta}-specific mAb, 47G.S4 (37), or with the DM{alpha}-specific mAb, 5C1 (38). The input DR was compared by Western blotting with each assay. Band intensities were quantified using a GS-710 densitometer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DR3 molecules with a {beta}R74Q mutation accumulate CLIP

In previous studies, we have used chemical mutagenesis of HLA-DR3+ EBV-transformed B cell lines, followed by immunoselection with anti-DR3 mAbs and complement, to identify mutations that affect expression and maturation of DR3, as well as recognition by Abs and T cells (39). The mutant cell line, 8.39.7, identified in this screen, harbored a point mutation in the DRB1 gene, changing Arg74 to Gln ({beta}R74Q; see Table I for a summary of mutant cell lines). This mutation diminished binding of several DR3-specific mAbs and reduced recognition by all DR3-restricted T cell lines tested. Position {beta}R74 maps to the P4 pocket of the DR3 Ag-binding groove and could affect T cell recognition directly or influence the bound peptide repertoire (Fig. 1). However, our previous study did not address a third, not mutually exclusive possibility, i.e., that the {beta}R74Q mutation might change the outcome of endosomal peptide editing by altering interactions of DR with Ag-processing cofactors such as DM or Ii.

To examine removal of Ii fragments, a key outcome of DM-mediated endosomal peptide editing, we first measured cell surface CLIP levels by flow cytometry (Fig. 2A). Staining with the mAb, CerCLIP.1, directed against the N terminus of CLIP (27), was consistently {approx}2-fold higher in 8.39.7 cells ({beta}R74Q), compared with the wild-type progenitor cell line, but remained far below the levels seen in DM0 mutant 9.5.3, which has wild-type DR3 molecules but accumulates CLIP on up to 80% of its DR molecules due to a lack of DMB expression (40, 41). HLA-DR levels, measured using two different anti-DR mAbs, also were slightly elevated in {beta}Q74R mutant cells, but not enough to account for the increase in CLIP. These results indicated that the {beta}R74Q mutation was associated with increased retention of CLIP on HLA-DR molecules in vivo. By mass spectrometry, peptides associated with mutant ({beta}R74Q) DR3 molecules in 8.39.7 were a mixture of CLIP (in amounts intermediate between DM0 mutant 9.5.3 and wild-type 8.1.6 progenitor cells) and an altered repertoire of non-CLIP peptides compared with wild-type (data not shown). The {beta}R74Q mutant expressed ~2.5-fold higher levels of DM protein than did wild-type progenitor cells (data not shown); therefore, the increased CLIP retention and the altered non-CLIP repertoire were not due to a coincidental lack of DM expression. After CLIP release, the mutant DR3 molecules appeared to undergo DM editing, because {beta}R74Q molecules had normal SDS stability (Figs. 2B and 5B); this is a useful marker of DM-dependent peptide exchange for the DR3 allele (41). Thus, the altered non-CLIP peptide repertoire probably reflected a direct influence of the {beta}R74Q mutation on the P4 pocket, rather than diminished DM editing as a result of delayed CLIP release.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 2. Phenotype of 8.39.7 EBV-B cells carrying a DRB1*0301 R74Q mutation. A, Comparison of CLIP and DR levels at the cell surface. The wild-type (wt) progenitor EBV-B cell line (8.1.6) and its mutant derivatives lacking DMB expression (9.5.3) or carrying the {beta}R74Q mutation (8.39.7) were labeled using mAbs to the CLIP N terminus (CerCLIP.1), DR{alpha} (L243) or DR{beta} (ISCR3), and analyzed by FACS. Results are shown as background-subtracted median fluorescence intensities (secondary Ab alone; range, 2–11 fluorescence U), normalized to the highest fluorescence intensity for each mAb in each experiment (=100%; CerCLIP.1, range 652-2325 U; ISCR3, 1026–2784 U; L243, 911-3550 U). Error bars, SDs of 4–10 independent experiments. *, Significant (p < 0.01) differences from 8.1.6 progenitor cells by one-way ANOVA with Dunnett’s multiple comparison test. B, Normal SDS stability of mutant DR3 molecules in 8.39.7. Western blots of wild-type and mutant cell extracts, probed with a mAb to DR{beta} (HB10.a). Similar results were obtained in four independent experiments and also with a mAb to DR{alpha} (DA6.147; not shown). C, The {beta}R74Q mutation increases SDS stability of DR3-CLIP complexes. Similar experiment as in B, except that blots were probed with the anti-CLIP mAb CerCLIP.1. One representative experiment of three is shown. D, Mutant DR3-{beta}R74Q molecules from 8.39.7 cells show delayed CLIP dissociation in vitro. Left, Dissociation curves for duplicate samples in 0.5% IGEPAL CA-630 or 1% OG from one of two experiments; right, summary of half-lives for both experiments (average ± range). The DR molecules used in these experiments contain {approx}20% DRw52a molecules (DRA*0101/DRB3*0101), which do not bind CLIP detectably (10 ). Initial peptide loading gave {approx}600,000–800,000 counts above 3,000–5,000 background counts.

 


View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 5. SDS stability phenotypes of DR3 mutant molecules. A, Western blots of IGEPAL CA-630 extracts of indicated cell lines, including DM-null 9.5.3, DRA-null 9.22.3, DRB1-null 9.4.3, mutant 8.39.7, and mutant transfectants, were probed with a mAb to CLIP (CerCLIP.1). Similar results were obtained in three to four independent experiments and also with a mAb to the DR3-CLIP complex (not shown). B and C, Western blots of IGEPAL CA-630 extracts of indicated cells without (B) or with (C) sample boiling and probed with anti-DR{alpha} mAb, DA6.147. C, Mutant transfectants show levels of total cellular DR comparable to those of wild-type transfectants, with the exception of {alpha}G20E. Results represent three to four experiments. D and E, IGEPAL CA-630 lysates from indicated cells were subjected to two rounds of preclearing with CerCLIP.1 to remove CLIP-class II complexes. D, CerCLIP-associated material was eluted from beads by boiling and analyzed by SDS-PAGE and immunoblotting with mAb DA6.147. E, Precleared, nonboiled lysates were immunoblotted with DA6.147. Results represent two experiments. WT, Wild type.

 
Together, these findings suggested that the CLIP accumulation in 8.39.7 cells reflected an intrinsic property of DR3 molecules carrying the {beta}R74Q mutation. This suggestion was confirmed by experiments showing that unlike wild-type DR3/CLIP complexes, which are SDS unstable (Fig. 2C; Ref. 42), SDS-stable mutant DR3 ({beta}R74Q)-CLIP complexes could be detected in 8.39.7 cells by Western blotting with the anti-CLIP mAb CerCLIP (Figs. 2C and 5A).

To quantify the effect of the {beta}R74Q mutation on CLIP release, we purified DR molecules from wild-type 8.1.6 and mutant 8.39.7 cells, loaded them with biotin-labeled CLIP, and tracked CLIP release (Fig. 2D). Release of bio-CLIP from {beta}R74Q mutant DR3 molecules was reproducibly {approx}3- to 5-fold slower than from wild-type DR3, whether dissociation was measured in NP40 (a detergent believed not to influence peptide dissociation rates; D. Zaller, unpublished data) or in OG, a detergent that accelerates peptide release (27). The increased stability of mutant DR3-CLIP complexes to dissociation was also seen in the presence of DM (data not shown). We concluded that mutation {beta}R74Q causes CLIP accumulation on DR3 molecules by slowing CLIP release.

Most DR3 mutants that confer elevated CLIP map to the P4 and P6 pockets

To explore what other mutations might confer CLIP retention phenotypes similar to those seen for the {beta}R74Q mutation in 8.39.7, we generated a second series of DR mutants, using a different approach. We introduced random mutations into DRA*0101 and DRB1*0301 cDNAs, using error-prone PCR. Mutant cDNA libraries were cloned into a retroviral vector and introduced into mutant EBV-B cell lines lacking endogenous expression of DRA (9.22.3; Fig. 3A) or DRB1 (9.4.3; Fig. 3B). Populations of transfected cells were screened for CLIP accumulation at the cell surface by FACS with CerCLIP.1. Next, we used high fidelity PCR to rescue and sequence the transduced DRA and DRB1 cDNAs from clones with consistently elevated CLIP (Table II).



View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 3. Retroviral transduction strategy for introduction of mutant DR cDNAs into chain-defective EBV-B cell lines. Libraries of randomly mutagenized DRA and DRB1*0301 cDNAs were packaged in retroviruses (hexagons). A, Viruses carrying mutant DRA cDNAs were used to transduce DRA-null 9.22.3 cells. Pairing of transduced mutant DR {alpha} chains with endogenous DRB1*0301 and DRB3*0101 restores expression of DR3 and DRw52a molecules, respectively, carrying the point mutation. B, Mutant DRB1*0301 cDNAs were introduced into DR{beta}1-null 9.4.3 cells; here, the mutant DR3 {beta}-chain pairs with the endogenous {alpha} chain to form a mutant DR3 {alpha}{beta} dimer, whereas the endogenous DRw52 molecule remains wild type (wt). C, 5.2.4 cell (DRA0, DRB0, DMB0) were first transduced with viruses carrying DRA cDNA. Selected cells were then transduced with mutant DRB3*0101 cDNAs. DR3-expressing cells were then subjected to FACS and cloned.

 

View this table:
[in this window]
[in a new window]
  		Table II. HLA-DR mutant clones with elevated CLIPa

 
For those clones harboring multiple point mutations, it was important to determine which of the mutations were associated with elevated CLIP. To this end, each individual mutation was recreated by overlap extension PCR, introduced into either DRA0 or DRB10 cells (as appropriate), and rescreened for its effect on CLIP levels (Fig. 4). For clones initially carrying single-point mutations, it was possible that the chromosomal integration site or other clonal variables, rather than the mutations, were responsible for the high-CLIP phenotypes. Thus, the single-mutation cDNAs were also reintroduced into appropriate recipient cells, generating polyclonal populations in which clonal variables should average out. A summary of CLIP levels, corrected for variation in total DR expression, for all single-point mutants is shown in Fig. 4B. All but one ({alpha}G20E) of the mutant cDNAs restored levels of total DR expression similar to those of the corresponding wild-type cDNA under control of the same retroviral promoter (Fig. 4A and data not shown). {alpha}G20E expressed DR protein at ~60% of wild-type levels at the cell surface (not shown) and in total cellular extracts (Fig. 5). Of the mutations listed in Table II, {beta}S11F, {beta}S13Y, {beta}Q70R, {beta}K71E, {beta}K71N, and {alpha}G20E all elevated CLIP. The remaining mutations had little if any effect on CLIP accumulation. Mutations {beta}S11F and {beta}K71E caused levels of CLIP accumulation approaching those seen in DM0 cell lines (e.g., 9.5.3). The phenotypes seen for {beta}S13Y, {beta}Q70R, and {beta}K71N were less pronounced. Side by side comparisons showed that the phenotypes of all these mutants were more pronounced than that of 8.39.7 cells (data not shown and Fig. 5A).



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 4. Identification of single amino acid changes in DR3 that confer elevated CLIP. A, Identification of amino acid changes responsible for the high-CLIP phenotype in multiply mutated DRB clones. Transfectants carrying point mutations, recapitulating each of the multiple mutations in clones 2B4, 2D3 and 4G5, were stained with mAb L243 (anti-DR{alpha}, shaded histograms) or CerCLIP (anti-CLIP) and analyzed by FACS. Median fluorescence of secondary Ab alone, 3–5 U. DR levels in all cells were elevated 2- to 3-fold over endogenous DRw52a levels in untransduced 9.4.3 cells (data not shown). B, Hierarchy of CLIP accumulation in transfectants carrying DR{alpha} or DR{beta} point mutants in 9.22.3 and 9.4.3, respectively. Shown are relative levels of CLIP, corrected for differences in total DR expression and normalized to the levels of CLIP accumulation in the DM-null mutant, 9.5.3 (=100%). Results represent averages of three to six determinations, with SD typically <±10%. Original clones from which mutations were identified are labeled. Because of their different cellular background (cf. Fig. 3), the relative CLIP levels in DR{alpha} wild-type (wt) and {alpha}G20E mutant cells are not directly comparable with those of the DR{beta} mutants. Mutation {beta}V85M, unlike the other mutations, reproducibly results in a slightly reduced ratio of CLIP to DR. *, DR{alpha}-null 9.22.3 cells; no normalization is possible.

 
Groove mutations of DR3 stabilize complexes with CLIP

Display of informative mutants on the DR3 crystal structure (Fig. 1) revealed that they clustered in or near the Ag-binding groove, with most mutations affecting the specificity pockets that accommodate residues P4 and P6 of the bound peptide. We thus wished to determine whether the mutations affect CLIP-DR3 stability. We initially used SDS stability as a surrogate marker for stable DR-peptide binding. Lysates were prepared from mutant EBV-B cell transfectants and analyzed by SDS-PAGE without sample boiling; separated proteins were then analyzed for the presence of SDS-stable, CLIP-containing complexes by Western blotting (Fig. 5A). The anti-CLIP mAb did not detect SDS-stable CLIP complexes in cells expressing wild-type DR3. In contrast, SDS-stable CLIP-containing complexes were readily detected in cells expressing {beta}S11F and {beta}K71E DR3 mutants, consistent with the hypothesis that these mutations stabilize CLIP in the Ag-binding groove. SDS-stable CLIP complexes were also seen in mutants {beta}K71N and {alpha}G20E but were not detected in {beta}Q70R and {beta}S13Y cells, even though these mutants expressed similar levels of CLIP at the cell surface as did {beta}K71N.

We also evaluated the overall SDS stability of DR dimers, which provides some information about the fate of non-CLIP peptides in these cells. Shown are Western blots of unboiled cell extracts, probed with a mAb to DR{alpha} (Fig. 5B). The SDS-stable mutant DR3-CLIP complexes (Fig. 5A) migrated more slowly than the bulk of SDS-stable wild-type DR{alpha}{beta}-peptide complexes (Fig. 5B). Indeed, an additional, slowly migrating band appeared in DR blots of mutants {beta}S11F, {beta}K71E, {beta}K71N, and {alpha}G20E. This band represented a variable proportion of total DR molecules in the different mutants, but in all of these cells SDS-stable DR forms predominated over SDS-unstable DR dimers. In contrast, mutants {beta}S13Y and {beta}Q70R, which lacked detectable SDS-stable CLIP-DR complexes, showed a higher proportion of SDS-unstable DR dimers. To determine the contribution of DR-CLIP complexes to the distinguishable SDS stability phenotypes among DR molecules in the mutants, cell lysates were depleted of CerCLIP reactive complexes by two rounds of immunoprecipitation. Detection of residual DR molecules in cleared lysates (Fig. 5E) confirmed that the slowly migrating band represented CLIP-DR complexes. A large proportion of SDS-unstable complexes in the {beta}Q70R mutant and a smaller but substantial proportion of SDS-unstable complexes in {beta}S13Y cells also represented DR-CLIP complexes. In most of the mutants, SDS stability of the residual DR/peptide complexes (after CerCLIP clearing) resembled that of DR/peptide complexes in wild-type cells, suggesting that DM editing of these DR-peptide complexes in the mutant cells was effective once CLIP removal was accomplished. A modest but reproducible reduction in SDS stability was observed in the residual DR dimers in mutants {alpha}G20E and {beta}S13Y. The most likely explanation is a direct effect of the mutations on the SDS stability of the heterodimer, perhaps through destabilization of chain pairing. This possibility is in line with previous studies showing that factors other than peptide affinity may contribute to SDS stability (43, 44, 45). Less likely possibilities are that these molecules are occupied by short CLIP variants (aa 90–104) lacking the CerCLIP epitope or that there is reduced time for editing of DR-peptide complexes; the latter would be expected to affect mutants with the strongest CLIP retention (Fig. 4 and following text).

CLIP complexes with DR3 mutants have altered kinetic stability

For a more quantitative correlate of CLIP levels, we purified mutant DR3 molecules and tracked release of biotinylated CLIP in vitro. For DR production, we introduced DRA and wild-type or selected mutant DRB1*0301 cDNAs into DR-null 5.2.4 cells, which lack DM (Fig. 3C); thus, unlike the range of CLIP levels seen in DM+ recipient cells (Fig. 4), all mutant DR3 molecules accumulated high levels of CLIP in 5.2.4 cells (CerCLIP/ISCR3 staining ratios 0.77 to 0.87). We focused on two mutants with strong ({beta}S11F, {beta}K71E) and two with intermediate ({beta}S13Y, {beta}Q70R) phenotypes.

To track CLIP release, we loaded mutant DR3 molecules with biotinylated CLIP. We attached biotin to the {epsilon}-amino group of a lysine residue at the CLIP C terminus, placing the label away from the face of DR3 that interacts with DM (26, 46). Initial CLIP-bio loading was substantially less for {beta}S11F and {beta}K71E than for wild-type, {beta}S13Y, or {beta}Q70R mutant DR3 molecules (Fig. 6A). Most likely, this reflected inefficient exchange of endogenous CLIP for CLIP-bio in the former two mutants.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 6. Effects of groove mutations on CLIP release from purified DR molecules in vitro mirror the hierarchy of CLIP levels seen in cells. A, Effect of mutations on loading of DR3 molecules with C-terminally biotinylated CLIP. Mutant DR3-CLIP-bio complexes were quantified by Eu3+ fluoroimmunoassay. B and C, Spontaneous ({square}, {triangledown}, and {circ}) and DM-catalyzed ({blacksquare}, {blacktriangledown}, and •) CLIP release from wild-type (wt) and mutant DR3 molecules at pH 5.2 and 37°C. Shown are duplicate analyses of remaining complexes as a function of incubation time. Half-lives calculated by single-exponential fits to the data shown are listed in Table II. The hierarchy was reproduced in two additional experiments using acetate buffer in place of citrate-phosphate, one of which used a short CLIP variant (residues 90–104) with {epsilon}-biotinylated lysine at the C terminus (data not shown). b, {beta}.

 
To measure CLIP dissociation, release of CLIP-bio was tracked as a function of time at endosomal pH. Wild-type DR3/CLIP-bio complexes dissociated with t1/2 {approx} 8.3 h (Fig. 6, B and C, and Table III), comparable with the half-life for bio-CLIP/DR complexes from 8.1.6 progenitor cells (Fig. 2D). Dissociation was slowed substantially by mutations {beta}S13Y and {beta}Q70R and was reduced further for mutants {beta}S11F and {beta}K71E (Fig. 6, B and C and Table II). These results correlated well with the hierarchy of CLIP accumulation seen in cells.


View this table:
[in this window]
[in a new window]
  		Table III. CLIP-bio dissociation from mutant DR3 molecules

 
We also determined whether the differences in intrinsic stability were sustained in the presence of soluble HLA-DM. Under the conditions used, dissociation of wild-type DR3/CLIP-bio was substantially accelerated (t1/2 {approx} 0.72 h, a 12-fold enhancement by DM vs spontaneous dissociation), resulting in essentially complete CLIP release within a few hours (Fig. 6, B and C, and Table II). Slower rates of CLIP release were observed for all high-CLIP mutants tested (Fig. 6, B and C), and the hierarchy of half-lives was the same as in the absence of DM. Although susceptibility of unrelated peptide-DR complexes to dissociation by DM may vary over several orders of magnitude, closely related complexes tend to have similar levels of DM susceptibility (47), in line with the results shown here. In conclusion, the effects of the mutations on the kinetic stability of CLIP/DR3 complexes in vitro accounted for the hierarchy of CLIP levels seen at the surface of EBV-B cells.

To test the effects of the groove mutations on functional interaction with HLA-DM in a second assay system, we tested the ability of purified mutant DR3/CLIP complexes to associate physically with HLA-DM in vitro (Fig. 7). Wild-type DR3 molecules, as well as molecules harboring the {beta}S13Y and {beta}Q70R mutations, coprecipitated similarly with DM. In contrast, coprecipitation of {beta}S11F and {beta}K71E mutant molecules, which stabilized CLIP to a greater extent, was reduced. Coimmunoprecipitation in our assay is optimal at endosomal pH (5.0) and 37°C. These are conditions that allow peptide release during the DM-DR binding reaction. Previously reported coprecipitation experiments suggest that association with high affinity peptides impairs DM-DR coprecipitation (48) and that empty DR molecules may be the optimal substrate for DM binding (49). The effects of the {beta}S11F and {beta}K71E mutations on DM-DR coprecipitation may thus reflect inefficient generation of empty molecules due to a lack of CLIP release, rather than a direct effect of the mutations on DM affinity. Regardless of the mechanism that generates DR molecules capable of coprecipitating with DM in this assay, the result confirmed the hierarchy of effects of the mutations seen in the other assay systems.



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 7. DR3-CLIP coprecipitation with DM is reduced by mutants {beta}S11F and {beta}K71E. Purified mutant DR3-CLIP complexes were incubated with recombinant full-length DM. DM was immunoprecipitated and associated DR was detected by Western blotting with anti-DR{alpha} mAb DA6.147. A, Representative DR{alpha} blot; B, summary of results from 6–10 repeat experiments. DR{alpha} band intensities were quantified by densitometry. Binding for wild-type (WT) DR3-CLIP complexes was set to 100%.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified several mutations ({alpha}G20E, {beta}S11F, {beta}S13Y, {beta}Q70R, {beta}K71E, {beta}K71N, and {beta}R74Q) that map in or near the Ag-binding groove and increase retention of CLIP by mutant DR3 molecules at the plasma membrane. These mutants differ in several respects from mutants on the lateral face of DR that interacts with HLA-DM (26, 50). DM interaction mutants diminish the effects of soluble DM on CLIP release from DR3 molecules in vitro but do not affect CLIP release in the absence of DM. In contrast, the mutants analyzed here slow CLIP release in both the presence and the absence of soluble DM, and most increase the SDS stability of DR3-CLIP complexes. Thus, the mutations likely act by increasing the kinetic stability of CLIP-DR3 complexes.

The relationship among CLIP accumulation on mutant DR3 molecules in cells, CLIP release rates in vitro, and DR transit time through late endosomes, where most DM editing occurs, suggests that our in vitro assays reflect biologically meaningful levels of DM editing. For instance, the in vitro off-rates of CLIP from wild-type molecules in the presence of DM are fast (47, 51), relative to the endosomal residence time of several hours for DR in EBV-B cells), correctly predicting low levels of CLIP retention in DM+ cells. Likewise, the intermediate levels of CLIP that accumulate in the {beta}S13Y and {beta}Q70R (DM+) mutant cells correlate with in vitro CLIP off-rates on the same order of magnitude as the endosomal residence time. Whether CLIP exchange for antigenic peptides is coupled to the endosomal residence time of MHC II molecules remains unclear. However, our experiments (Fig. 5) indicate that, even in high-CLIP mutants, complexes with non-CLIP peptides acquire SDS resistance. This suggests that those mutant molecules that release CLIP then reside in endosomes long enough to undergo normal DM editing. Either egress from endosomes is delayed by DM until editing is completed or the diminished time available for editing is sufficient.

The effects of the mutations on CLIP release can be partially accounted for by molecular modeling. Several of the CLIP-stabilizing mutations ({beta}S11F, {beta}S13Y, {beta}K71E/N, {beta}R74Q) map to or near specificity pockets P4 and/or P6 of the Ag-binding groove (Fig. 1). DR3 molecules prefer acidic side chains at residue P4 of the bound peptide and long, polar, or charged side chains (K, R, E, Q, N) at P6; neither requirement is well satisfied by the P4 and P6 residues of CLIP (residues A94 and P96 of Ii, respectively). Previous studies have shown that substitution of acidic side chains at P4 of the CLIP peptide can improve CLIP binding to DR3 molecules (24, 52). We conducted molecular modeling, based on the DR3-CLIP crystal structure (4), using Swiss PDB viewer v.3.51 software (http://www.expasy.ch/spdbv/mainpage.html) and LOOK software with the CARA/MUTANT option (Molecular Applications Group, Palo Alto, CA). Modeling predicts that mutation {beta}S11F fills the lower portion of the P6 pocket, and {beta}S13Y should partially fill P4; both mutations would improve close packing with P4/P6 CLIP anchor side chains without global distortion of the Ag-binding groove. In addition, {beta}S13Y is predicted to interact with {beta}N82, which in turn hydrogen bonds with CLIP R92; these interactions may also increase the stability of CLIP binding. Mutations at {beta}K71 and {beta}R74 would be expected to diminish the preference of the P4 pocket for negatively charged residues. The {beta}Q70R mutation affects a {beta}-chain helix residue that has been implicated as a TCR contact (53) and lacks direct contact with the bound CLIP (4). Its effect on CLIP release is modest and may be due to altered interactions with adjacent DR3 residues that do contact CLIP. The mechanism by which the {alpha}G20E mutation impairs CLIP release is more enigmatic. This residue lies on a surface-exposed loop, away from the DM interaction site and adjacent to the {alpha}-chain helix that flanks the Ag-binding groove. An intriguing possibility is that this mutation slows CLIP release by impairing molecular motions of the adjacent {alpha}-chain helix that release the bound peptide.

Intriguingly, no P1 and P9 pocket mutations were isolated in these screens. A plausible hypothesis is that the methionyl residues at P1 and P9 positions of CLIP make nearly optimal interactions with their complementary specificity pockets in the DR3 allele, so it is difficult to identify mutations that improve these interactions. The P1 pocket is shaped by invariant residues of DR{alpha} and the dimorphic DR{beta} residues A85 and V86, generating a preference for midsized aliphatic side chains such as methionine (2, 54). The P9 pocket of DR3 can accommodate large aliphatic and aromatic residues and is probably a good fit for methionine as well. Different results might thus be anticipated if similar experiments were performed with DR alleles with suboptimal P1 and P9 pockets for CLIP.

MHC II molecules are highly polymorphic, with dozens to hundreds of defined alleles at the loci coding for each of the three MHC II molecules (HLA-DPA and -DPB, -DQA and -DQB, as well as DRB1 and in most haplotypes a second DRB locus (55). Selection pressures underlying the maintenance of balanced MHC II polymorphism are thought to reflect mating preferences and maximization of immune defense. Mechanisms that generate this polymorphism may include the gradual accumulation of point mutations and the introduction of short, altered sequence tracts by interlocus gene conversion (56). Interallelic gene conversion is thought to increase the number of alleles further by reshuffling polymorphic sequence tracks among pre-existing alleles in a combinatorial fashion. The result is a set of alleles representing a mosaic of polymorphic cassettes, each of which usually comprises 2–5 polymorphic residues. For the most part, this polymorphism maps to the specificity pockets lining the peptide binding groove and to residues that contact the TCR (reviewed in Ref. 1). The factors that limit MHC II polymorphism are poorly understood. Clearly, polymorphisms that impair overall folding or chain pairing would confer a selective disadvantage. However, the results reported here raise the possibility that the need to maintain efficient CLIP release also represents a significant constraint on natural MHC II polymorphism.

Natural MHC alleles can and do accommodate a low level of residual CLIP (2), but accumulation of high levels of CLIP would be expected to impair MHC II function in Ag presentation. Our observations suggest that single point mutations can result in a degree of enhanced stabilization of DR3-CLIP complexes likely to impact presentation of non-CLIP peptides to T cells, even in the presence of DM. Strikingly, most point mutations that were found here to elevate CLIP recur in naturally occurring DRB alleles, although as part of polymorphic cassettes spanning more than one residue. Y at position {beta}13 occurs in DR7 and DRB5 molecules; R at position {beta}70 occurs in DR9, 10, 14, and subtypes of other DRB1 alleles; E at position {beta}71 occurs in subtypes of many major allele groupings, such as the DRB1*0402 variant of the DR4 family; and Q at {beta}74 occurs in DR7, subtypes of DR3, DRB3*02, -*03, and subtypes of -*01 (55). Mutation {beta}S11F is not represented among naturally occurring DRB alleles, but a large hydrophobic residue, L, is seen in DR1.

MHC II point mutations that substantially inhibit CLIP exchange may be subject to negative selection, which would either tend to eliminate these mutations or force rapid accumulation of additional point mutations that counteract the initial increase in CLIP affinity. This constraint might go some way toward explaining some specific patterns of natural MHC polymorphism. Consistent with our notion, the amino acid changes that substantially elevate CLIP in the context of the DRB1*0301 allele are not seen among natural variants of the DR3 family (the {beta}R74Q mutation, which confers a very subtle phenotype, is seen in DR3 variants, DRB1*0311 and -*0317). We also note that a three-residue polymorphic cassette, which confers resistance of DR4 (*0402) and other allele families to rheumatoid arthritis and contains the {beta}K71E substitution, has only a slight ({approx}1.6- to 3.2-fold under various conditions) stabilizing effect on DR4-CLIP complexes (57). In contrast, the phenotype associated with {beta}K71E as a single-point mutation in DR3 is much more noticeable. Thus, as a placeholder peptide, the removal of which is essential for subsequent peptide loading, CLIP may act as a powerful factor shaping patterns of MHC II polymorphism. Importantly, this concept will be readily testable by looking for compensating effects on CLIP levels of multiple amino acid changes that travel together as polymorphic cassettes in naturally occurring MHC II alleles.


   Acknowledgments
 
We thank Hyman M. Scott and Drs. Carolyn Bergman and Jeremy Caldwell for help with experiments and Drs. Garry Nolan, Susan Pierce, and Peter Cresswell for reagents.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI-28809 (to E.D.M.), the Siegelman fellowship (to R.B.), and a Cancer Research Institute Fellowship (to A.P.). Back

2 Current address: KineMed, Inc., 5980 Horton Street, Suite 470, Emeryville, CA 94608. Back

3 R.B. and E.D.M. contributed equally to this paper. Back

4 Address correspondence and reprint requests to Dr. Elizabeth D. Mellins, Department of Pediatrics, Center for Clinical Sciences Research, Room 2115, 300 Pasteur Drive, Stanford University School of Medicine, Stanford, CA 94305-5164. E-mail address: mellins{at}stanford.edu Back

5 Abbreviations used in this paper: CLIP, class II-associated invariant chain peptides; OG, n-octyl-{beta}-D-glucopyranoside. Back

Received for publication December 13, 2002. Accepted for publication February 19, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. McFarland, B. J., C. Beeson. 2002. Binding interactions between peptides and proteins of the class II major histocompatibility complex. Med. Res. Rev. 22:168.[Medline]
  2. Rammensee, H. G., T. Friede, S. Stevanoviic. 1995. MHC ligands and peptide motifs: first listing. Immunogenetics 41:178.[Medline]
  3. Cresswell, P.. 1994. Assembly, transport, and function of MHC class II molecules. Annu. Rev. Immunol. 12:259.[Medline]
  4. Ghosh, P., M. Amaya, E. Mellins, D. C. Wiley. 1995. The structure of an intermediate in class II MHC maturation: CLIP bound to HLA-DR3. Nature 378:457.[Medline]
  5. Eastman, S., M. Deftos, P. C. DeRoos, D. H. Hsu, L. Teyton, N. S. Braunstein, C. J. Hackett, A. Rudensky. 1996. A study of complexes of class II invariant chain peptide: major histocompatibility complex class II molecules using a new complex- specific monoclonal antibody. Eur. J. Immunol. 26:385.[Medline]
  6. Jasanoff, A., S. Song, A. R. Dinner, G. Wagner, D. C. Wiley. 1999. One of two unstructured domains of Ii becomes ordered in complexes with MHC class II molecules. Immunity 10:761.[Medline]
  7. Nakagawa, T. Y., A. Y. Rudensky. 1999. The role of lysosomal proteinases in MHC class II-mediated antigen processing and presentation. Immunol. Rev. 172:121.[Medline]
  8. Villadangos, J. A., H. L. Ploegh. 2000. Proteolysis in MHC class II antigen presentation: who’s in charge?. Immunity 12:233.[Medline]
  9. Rabinowitz, J. D., M. Vrljic, P. M. Kasson, M. N. Liang, R. Busch, J. J. Boniface, M. M. Davis, H. M. McConnell. 1998. Formation of a highly peptide-receptive state of class II MHC. Immunity 9:699.[Medline]
  10. Sette, A., S. Southwood, J. Miller, E. Appella. 1995. Binding of major histocompatibility complex class II to the invariant chain-derived peptide, CLIP, is regulated by allelic polymorphism in class II. J. Exp. Med. 181:677.[Abstract/Free Full Text]
  11. Rudensky, A. Y., P. Preston-Hurlburt, S. C. Hong, A. Barlow, C. A. Janeway, Jr.. 1991. Sequence analysis of peptides bound to MHC class II molecules. Nature 353:622.[Medline]
  12. Chicz, R. M., R. G. Urban, W. S. Lane, J. C. Gorga, L. J. Stern, D. A. Vignali, J. L. Strominger. 1992. Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size. Nature 358:764.[Medline]
  13. Hunt, D. F., H. Michel, T. A. Dickinson, J. Shabanowitz, A. L. Cox, K. Sakaguchi, E. Appella, H. M. Grey, A. Sette. 1992. Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-Ad. Science 256:1817.[Abstract/Free Full Text]
  14. Busch, R., R. C. Doebele, N. S. Patil, A. Pashine, E. D. Mellins. 2000. Accessory molecules for MHC class II peptide loading. Curr. Opin. Immunol. 12:99.[Medline]
  15. Mellins, E., P. Cameron, M. Amaya, S. Goodman, D. Pious, L. Smith, B. Arp. 1994. A mutant human histocompatibility leukocyte antigen DR molecule associated with invariant chain peptides. J. Exp. Med. 179:541.[Abstract/Free Full Text]
  16. Riberdy, J. M., P. Cresswell. 1992. The antigen-processing mutant T2 suggests a role for MHC-linked genes in class II antigen presentation. J. Immunol. 148:2586.[Abstract]
  17. Fung-Leung, W. P., C. D. Surh, M. Liljedahl, J. Pang, D. Leturcq, P. A. Peterson, S. R. Webb, L. Karlsson. 1996. Antigen presentation and T cell development in H2-M-deficient mice. Science 271:1278.[Abstract]
  18. Martin, W. D., G. G. Hicks, S. K. Mendiratta, H. I. Leva, H. E. Ruley, L. Van Kaer. 1996. H2-M mutant mice are defective in the peptide loading of class II molecules, antigen presentation, and T cell repertoire selection. Cell 84:543.[Medline]
  19. Miyazaki, T., P. Wolf, S. Tourne, C. Waltzinger, A. Dierich, N. Barois, H. Ploegh, C. Benoist, D. Mathis. 1996. Mice lacking H2-M complexes, enigmatic elements of the MHC class II peptide-loading pathway. Cell 84:531.[Medline]
  20. Bikoff, E. K., G. Wutz, G. A. Kenty, C. H. Koonce, E. J. Robertson. 2001. Relaxed DM requirements during class II peptide loading and CD4+ T cell maturation in BALB/c mice. J. Immunol. 166:5087.[Abstract/Free Full Text]
  21. Gautam, A. M., M. Yang, P. J. Milburn, R. Baker, A. Bhatnagar, J. McCluskey, T. Boston. 1997. Identification of residues in the class II-associated Ii peptide (CLIP) region of invariant chain that affect efficiency of MHC class II- mediated antigen presentation in an allele-dependent manner. J. Immunol. 159:2782.[Abstract]
  22. Liang, M. N., C. Lee, Y. Xia, H. M. McConnell. 1996. Molecular modeling and design of invariant chain peptides with altered dissociation kinetics from class II MHC. Biochemistry 35:14734.[Medline]
  23. van Ham, S. M., U. Gruneberg, G. Malcherek, I. Broker, A. Melms, J. Trowsdale. 1996. Human histocompatibility leukocyte antigen (HLA)-DM edits peptides presented by HLA-DR according to their ligand binding motifs. J. Exp. Med. 184:2019.[Abstract/Free Full Text]
  24. Weber, D. A., B. D. Evavold, P. E. Jensen. 1996. Enhanced dissociation of HLA-DR-bound peptides in the presence of HLA-DM. Science 274:618.[Abstract/Free Full Text]
  25. Kinsella, T. M., G. P. Nolan. 1996. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum. Gene Ther. 7:1405.[Medline]
  26. Doebele, R. C., R. Busch, H. M. Scott, A. Pashine, E. D. Mellins. 2000. Determination of the HLA-DM interaction site on HLA-DR molecules. Immunity 13:517.[Medline]
  27. Avva, R. R., P. Cresswell. 1994. In vivo and in vitro formation and dissociation of HLA-DR complexes with invariant chain-derived peptides. Immunity 1:763.[Medline]
  28. Lampson, L. A., R. Levy. 1980. Two populations of Ia-like molecules on a human B cell line. J. Immunol. 125:293.[Abstract]
  29. Watanabe, M., T. Suzuki, M. Taniguchi, N. Shinohara. 1983. Monoclonal anti-Ia murine alloantibodies crossreactive with the Ia-homologues of other mammalian species including humans. Transplantation 36:712.[Medline]
  30. Busch, R., R. C. Doebele, E. von Scheven, J. Fahrni, E. D. Mellins. 1998. Aberrant intermolecular disulfide bonding in a mutant HLA-DM molecule: implications for assembly, maturation, and function. J. Immunol. 160:734.[Abstract/Free Full Text]
  31. Guy, K., V. Van Heyningen, B. B. Cohen, D. L. Deane, C. M. Steel. 1982. Differential expression and serologically distinct subpopulations of human Ia antigens detected with monoclonal antibodies to Ia {alpha} and {beta} chains. Eur. J. Immunol. 12:942.[Medline]
  32. Clark, E. A., R. Yakoshi. 1984. Leukocyte Typing Springer-Verlag, Berlin.
  33. Stern, L. J., D. C. Wiley. 1992. The human class II MHC protein HLA-DR1 assembles as empty {alpha}{beta} heterodimers in the absence of antigenic peptide. Cell 68:465.[Medline]
  34. Sloan, V. S., P. Cameron, G. Porter, M. Gammon, M. Amaya, E. Mellins, D. M. Zaller. 1995. Mediation by HLA-DM of dissociation of peptides from HLA-DR. Nature 375:802.[Medline]
  35. Busch, R., Z. Reich, D. M. Zaller, V. Sloan, E. D. Mellins. 1998. Secondary structure composition and pH-dependent conformational changes of soluble recombinant HLA-DM. J. Biol. Chem. 273:27557.[Abstract/Free Full Text]
  36. Gorga, J. C., P. J. Knudsen, J. A. Foran, J. L. Strominger, S. J. Burakoff. 1986. Immunochemically purified DR antigens in liposomes stimulate xenogeneic cytolytic T cells in secondary in vitro cultures. Cell. Immunol. 103:160.[Medline]
  37. Schafer, P. H., J. M. Green, S. Malapati, L. Gu, S. K. Pierce. 1996. HLA-DM is present in one-fifth the amount of HLA-DR in the class II peptide-loading compartment where it associates with leupeptin-induced peptide (LIP)-HLA-DR complexes. J. Immunol. 157:5487.[Abstract]
  38. Sanderson, F., M. J. Kleijmeer, A. Kelly, D. Verwoerd, A. Tulp, J. J. Neefjes, H. J. Geuze, J. Trowsdale. 1994. Accumulation of HLA-DM, a regulator of antigen presentation, in MHC class II compartments. Science 266:1566.[Abstract/Free Full Text]
  39. Mellins, E., B. Arp, D. Singh, B. Carreno, L. Smith, A. H. Johnson, D. Pious. 1990. Point mutations define positions in HLA-DR3 molecules that affect antigen presentation. Proc. Natl. Acad. Sci. USA 87:4785.[Abstract/Free Full Text]
  40. Fling, S. P., B. Arp, D. Pious. 1994. HLA-DMA and -DMB genes are both required for MHC class II/peptide complex formation in antigen-presenting cells. Nature 368:554.[Medline]
  41. Morris, P., J. Shaman, M. Attaya, M. Amaya, S. Goodman, C. Bergman, J. J. Monaco, E. Mellins. 1994. An essential role for HLA-DM in antigen presentation by class II major histocompatibility molecules. Nature 368:551.[Medline]
  42. Mellins, E., L. Smith, B. Arp, T. Cotner, E. Celis, D. Pious. 1990. Defective processing and presentation of exogenous antigens in mutants with normal HLA class II genes. Nature 343:71.[Medline]
  43. Verreck, F. A., C. Vermeulen, A. V. Poel, P. Jorritsma, R. Amons, J. E. Coligan, J. W. Drijfhout, F. Koning. 1996. The generation of SDS-stable HLA DR dimers is independent of efficient peptide binding. Int. Immunol. 8:397.[Abstract/Free Full Text]
  44. Natarajan, S. K., L. J. Stern, S. Sadegh-Nasseri. 1999. Sodium dodecyl sulfate stability of HLA-DR1 complexes correlates with burial of hydrophobic residues in pocket 1. J. Immunol. 162:3463.[Abstract/Free Full Text]
  45. Ettinger, R. A., A. W. Liu, G. T. Nepom, W. W. Kwok. 2000. {beta}57-Asp plays an essential role in the unique SDS stability of HLA-DQA1*0102/DQB1*0602 {alpha}{beta} protein dimer, the class II MHC allele associated with protection from insulin-dependent diabetes mellitus. J. Immunol. 165:3232.[Abstract/Free Full Text]
  46. Stratikos, E., L. Mosyak, D. M. Zaller, D. C. Wiley. 2002. Identification of the lateral interaction surfaces of human histocompatibility leukocyte antigen (HLA)-DM with HLA-DR1 by formation of tethered complexes that present enhanced HLA-DM catalysis. J. Exp. Med. 196:173.[Abstract/Free Full Text]
  47. Belmares, M. P., R. Busch, K. W. Wucherpfennig, H. M. McConnell, E. D. Mellins. 2002. Structural factors contributing to DM susceptibility of MHC class II/peptide complexes. J. Immunol. 169:5109.[Abstract/Free Full Text]
  48. Kropshofer, H., S. O. Arndt, G. Moldenhauer, G. J. Hämmerling, A. B. Vogt. 1997. HLA-DM acts as a molecular chaperone and rescues empty HLA-DR molecules at lysosomal pH. Immunity 6:293.[Medline]
  49. Denzin, L. K., C. Hammond, P. Cresswell. 1996. HLA-DM interactions with intermediates in HLA-DR maturation and a role for HLA-DM in stabilizing empty HLA-DR molecules. J. Exp. Med. 184:2153.[Abstract/Free Full Text]
  50. Guerra, C. B., R. Busch, R. C. Doebele, W. Liu, T. Sawada, W. W. Kwok, M. D. Chang, E. D. Mellins. 1998. Novel glycosylation of HLA-DR{alpha} disrupts antigen presentation without altering endosomal localization. J. Immunol. 160:4289.[Abstract/Free Full Text]
  51. Zarutskie, J. A., R. Busch, Z. Zavala-Ruiz, M. Rushe, E. D. Mellins, L. J. Stern. 2001. The kinetic basis of peptide exchange catalysis by HLA-DM. Proc. Natl. Acad. Sci. USA 98:12450.[Abstract/Free Full Text]
  52. Bischof, F., W. Wienhold, C. Wirblich, G. Malcherek, O. Zevering, A. M. Kruisbeek, A. Melms. 2001. Specific treatment of autoimmunity with recombinant invariant chains in which CLIP is replaced by self-epitopes. Proc. Natl. Acad. Sci. USA 98:12168.[Abstract/Free Full Text]
  53. Lombardi, G., L. Barber, S. Sidhu, J. R. Batchelor, R. I. Lechler. 1991. The specificity of alloreactive T cells is determined by MHC polymorphisms which contact the T cell receptor and which influence peptide binding. Int. Immunol. 3:769.[Abstract/Free Full Text]
  54. Geluk, A., K. E. van Meijgaarden, A. A. Janson, J. W. Drijfhout, R. H. Meloen, R. R. de Vries, T. H. Ottenhoff. 1992. Functional analysis of DR17(DR3)-restricted mycobacterial T cell epitopes reveals DR17-binding motif and enables the design of allele-specific competitor peptides. J. Immunol. 149:2864.[Abstract]
  55. Robinson, J., M. J. Waller, P. Parham, J. G. Bodmer, S. G. Marsh. 2001. IMGT/HLA Database: a sequence database for the human major histocompatibility complex. Nucleic Acids Res. 29:210.[Abstract/Free Full Text]
  56. Martinsohn, J. T., A. B. Sousa, L. A. Guethlein, J. C. Howard. 1999. The gene conversion hypothesis of MHC evolution: a review. Immunogenetics 50:168.[Medline]
  57. Patil, N. S., A. Pashine, M. P. Belmares, W. Liu, B. Kaneshiro, J. Rabinowitz, H. McConnell, E. D. Mellins. 2001. Rheumatoid arthritis (RA)-associated HLA-DR alleles form less stable complexes with class II-associated invariant chain peptide than non-RA-associated HLA-DR alleles. J. Immunol. 167:7157.[Abstract/Free Full Text]
  58. Pious, D., L. Dixon, F. Levine, T. Cotner, R. Johnson. 1985. HLA class II regulation and structure. Analysis with HLA-DR3 and HLA-DP point mutants. J. Exp. Med. 162:1193.[Abstract/Free Full Text]
  59. Cotner, T., H. Charbonneau, E. Mellins, D. Pious. 1989. mRNA abundance, rather than differences in subunit assembly, determine differential expression of HLA-DR{beta}1 and -DR{beta}3 molecules. J. Biol. Chem. 264:11107.[Abstract/Free Full Text]
  60. Mellins, E., S. Kempin, L. Smith, T. Monji, D. Pious. 1991. A gene required for class II-restricted antigen presentation maps to the major histocompatibility complex. J. Exp. Med. 174:1607.[Abstract/Free Full Text]
  61. Levine, F., H. Erlich, B. Mach, R. Leach, R. White, D. Pious. 1985. Deletion mapping of HLA and chromosome 6p genes. Proc. Natl. Acad. Sci. USA 82:3741.[Abstract/Free Full Text]

Related articles in The JI:

IN THIS ISSUE

The JI 2003 170: 4423-4424. [Full Text]  



This article has been cited by other articles:


Home page
 J. Immunol.Home page
L.-E. Fallang, S. Roh, A. Holm, E. Bergseng, T. Yoon, B. Fleckenstein, A. Bandyopadhyay, E. D. Mellins, and L. M. Sollid
Complexes of Two Cohorts of CLIP Peptides and HLA-DQ2 of the Autoimmune DR3-DQ2 Haplotype Are Poor Substrates for HLA-DM
J. Immunol., October 15, 2008; 181(8): 5451 - 5461.
[Abstract] [Full Text] [PDF]