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 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
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Busch, R.
Right arrow Articles by Mellins, E. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Busch, R.
Right arrow Articles by Mellins, E. D.
The Journal of Immunology, 1998, 160: 734-743.
Copyright © 1998 by The American Association of Immunologists

Aberrant Intermolecular Disulfide Bonding in a Mutant HLA-DM Molecule: Implications for Assembly, Maturation, and Function1

Robert Busch2,*, Robert C. Doebele{dagger}, Emily von Scheven{ddagger}, Jimothy Fahrni* and Elizabeth D. Mellins*

* Department of Pediatrics, Stanford University Medical Center, Stanford, CA 94305; {dagger} School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and {ddagger} Department of Pediatric Rheumatology, University of California at San Francisco Medical Center, San Francisco, CA 94143.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
HLA-DM (abbreviated DM) is an MHC-encoded glycoprotein that catalyzes the selective release of peptides, including class II-associated invariant chain peptides, from MHC class II molecules. To perform its function, DM must assemble in the endoplasmic reticulum (ER), travel to endosomes, and interact productively with class II molecules. We have described previously an EBV-transformed B cell line, 7.12.6, which displays a partial Ag presentation defect and expresses a mutated DM ß-chain with Cys79 replaced by Tyr. In this study, we show that HLA-DR molecules in 7.12.6 have a defect in peptide loading and accumulate class II-associated invariant chain peptides (CLIP). Peptide loading is restored by transfection of wild-type DMB. The mutant DM molecules exit the ER slowly and are degraded rapidly, resulting in greatly reduced levels of mutant DM in post-Golgi compartments. Whereas wild-type DM forms noncovalent {alpha}ß dimers, such dimers form inefficiently in 7.12.6; many mutant DM ß-chains instead form a disulfide-bonded dimer with DM {alpha}. Homodimers of DM ß are also detected in 7.12.6 and in the {alpha}-chain defective mutant, 2.2.93. We conclude that during folding of wild-type DM, the native conformation is stabilized by a conserved disulfide bond involving Cys79ß and by noncovalent contacts with DM {alpha}. Without these interactions, DM ß can form malfolded structures containing interchain disulfide bonds; malfolding is correlated with ER retention and accelerated degradation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DM is an accessory molecule for endosomal peptide loading of MHC class II molecules (reviewed in 1 . Newly synthesized MHC class II molecules assemble in the endoplasmic reticulum (ER)3 with the invariant chain (Ii) and are transported to endocytic compartments, where Ii is proteolytically truncated to a nested set of class II-associated Ii peptides called CLIP. Class II molecules are loaded with antigenic peptides following release of CLIP from the Ag-binding groove. A role for DM in peptide loading was discovered by using EBV-B cell mutants that were unable to present soluble protein Ags and alloantigens to CD4+ T cells (2). Class II molecules in these mutants are expressed at normal levels, but lack expression of specific Ab determinants (such as that recognized by the anti-DR3 mAb, 16.23) and are unstable in SDS, suggesting inefficient loading with endosomal peptides. In the mutants, synthesis of DM {alpha}- and/or ß-chains is defective, and transfection of the nonexpressed gene(s) reconstitutes a normal class II phenotype (3, 4, 5). DM may have several interrelated functions in normal peptide loading. The first is to release Ii degradation products, including CLIP, from class II molecules newly arrived in endosomes. This function was revealed by studies showing that CLIP-class II complexes accumulate in DM-null cells (6, 7, 8, 9, 10, 11) and that purified DM catalyzes dissociation of these complexes in vitro (12, 13, 14). Secondly, DM can bind to MHC class II molecules during peptide exchange and may stabilize them against denaturation, aggregation, and/or proteolysis, thus preserving peptide binding sites (15, 16, 17, 18). Following loading with endosomal peptides, DM may facilitate additional rounds of peptide exchange, so that the final peptide repertoire is biased toward peptides that form kinetically stable complexes (12, 14, 19, 20, 21).

DM is a relatively nonpolymorphic type I transmembrane glycoprotein consisting of a 35-kDa {alpha}-chain and a 29-kDa ß-chain (5, 22, 23, 24, 25, 26) (cf Fig. 1GoA). The extracellular domains of both chains of DM are homologous to those of MHC class I and class II glycoproteins (27). This is seen most clearly in the membrane-proximal Ig superfamily-like domains, which share 20 to 37% of amino acids with classical MHC molecules. The similarity is lower (14–25% identity) in the membrane-distal domains, which correspond to the polymorphic Ag-binding groove of classical MHC molecules. There are two cysteines at positions 11 and 79 in the ß1 domain of HLA-DM, which are conserved among DM homologues from all species sequenced to date (22, 27, 28, 29, 30). The equivalent pair of cysteines in the {alpha}2 domain of class I and the ß1 domain of classical class II molecules is known from x-ray structures to form a disulfide bond (Fig. 1Go, A and B). In addition, there are five cysteines not found in classical MHC molecules, three in the ß1 domain and two in the {alpha}1 domain (Fig. 1GoA). Whether DM has a ligand-binding groove in the membrane-proximal domain similar to that of classical MHC molecules is unclear, but attempts to reveal peptide-binding activity for DM have failed (14, 18).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 1. Structure of DM and derivation of the mutant EBV-B cell line, 7.12.6. A, Schematic illustrating the distribution of cysteine residues in the extracytoplasmic domains of DM {alpha}- and ß-chains (22). The Cys79ß residue mutated in 7.12.6 is shown in bold. Horizontal lines indicate disulfide bonds inferred from homology to Ig superfamily domains; the dashed horizontal line indicates a disulfide bond inferred from homology to the MHC Ag binding domain, results shown in this work, and unpublished data (E.v.S.). B, Predicted location of the putative disulfide bond between cysteines ß11 and ß79, mapped on the crystal structure of HLA-DR1 (where the homologous bond is formed between cysteines 15 and 79 (51)). C, Derivation and simplified genomic MHC class II maps of mutant cells used in this study. All cells are derived ultimately from the DR1, DR3 heterozygous EBV-B cell line, T5-1 (35). 8.1.6 cells were obtained by random mutagenesis and selection for loss of the DR1 allele (35). They carry a hemizygous deletion spanning part of the MHC class II region between DMB and DRA (3, 52). Derivation of 2.2.93 cells from T5-1 involved selection for loss of the DR3-bearing MHC haplotype, retransfection of DR3, and selection for loss of the DM-dependent 16.23 epitope (4). 8.1.6 has a wild-type Ag presentation phenotype. The other cells are defective for Ag presentation due to different point mutations in the remaining DM allele (3, 4).

 
Wild-type DM is assembled into an {alpha}ß heterodimer in the ER and exported through the Golgi apparatus into class II-rich endocytic compartments, which in EBV-transformed B cells have characteristics of prelysosomes (5, 28, 31, 32) (E. Stang, C. Guerra, M. A. Amaya, Y. Paterson, O. Bakke, and E. D. M., submitted). Endocytic targeting of DM is directed by an intrinsic, tyrosine-based motif in the ß-chain (33, 34). In addition, association with Ii may contribute to endosomal transport, at least in the mouse (28, 34). Little is known about how DM interacts with class II molecules during peptide exchange, except that the extracytoplasmic domains of DM are sufficient for function in vitro (12) and that specific regions on the class II molecule, defined by mAb inhibition and by a mutation in the {alpha}2 domain of HLA-DR3, are involved (8, 14).

Among the DM mutants, one clone, 7.12.6, was isolated that had a less severe phenotype than mutants lacking expression of DM ß mRNA (3) (see Fig. 1GoC for derivation of this mutant and other cells used in this work). Presentation of soluble protein Ags and expression of the 16.23 determinant by 7.12.6 cells were intermediate between 8.1.6 wild-type progenitors and DM ß-null mutants, such as 9.5.3. DMA and DMB mRNA levels were normal, but sequencing revealed a missense mutation in DMB, converting codon 79 from coding for a cysteine to a tyrosine. We predicted that this mutation would destabilize the native conformation of DM by disrupting the postulated disulfide bond between Cys11ß and Cys79ß. To test this hypothesis, we have analyzed the effect of the mutation on the disulfide bond structure, assembly, and intracellular transport of DM, and further characterized its effect on DM function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
EBV-B cells

The isolation of the DM-expressing progenitor EBV-B cell line, 8.1.6 (35), its DM ß-deficient derivative, 9.5.3 (36), the related DM {alpha}-deficient cell, 2.2.93 (4), and the 7.12.6 mutant cell line (3) has been described (see Fig. 1GoC). These cells were maintained in RPMI 1640 supplemented with 25 mM HEPES, 2 mM L-glutamine, and 15% donor calf serum, and routinely screened for mycoplasma contamination and for maintenance of their 16.23 phenotype.

RT-PCR and sequencing of HLA-DM genes

The DMB mutation in 7.12.6 cells was identified initially by dideoxy sequencing of PCR-amplified DMB cDNA, using the sequencing primer, M5, as previously described (37). To search for additional mutations, mRNA was isolated from 8.1.6 and 7.12.6 cells using a Stratagene (La Jolla, CA) RNA isolation kit. DMA and DMB cDNAs were transcribed and amplified using the Superscript RT-PCR system (Life Technologies, Gaithersburg, MD), 1 µg mRNA template, and 200 ng of each primer. After reamplification using Pfu DNA polymerase (Stratagene; 30 cycles, 25 ng RT-PCR product, 200 ng of each primer), sequencing reactions were performed using the Perkin-Elmer (Foster City, CA) dye-terminator cycle-sequencing kit, and analyzed at the Stanford University (Stanford, CA) protein and nucleic acid facility. For DMB, the primers used for amplification and sequencing were DMB1 5', M7, and M13 (37); for DMA, the following primers were used: 5'-CTG TGT GGC AAG AAG GTA TGG-3', 5'-GCT GGC ATC AAA CTC TGG TCT-3', and 5'-TTG CTG ACT GGG CTC AGG AAC-3'.

Antibodies

Hybridoma cells were grown in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 25 mM HEPES, 2 x 10-5 M 2-ME, and antibiotics, and Abs were used either as tissue culture supernatant or as ascites fluid. The Abs used and their specificities are summarized in Table IGo. CerCLIP.1 and the anti-DM reagents were kind gifts of their originators, Drs. Cresswell (Yale University, New Haven, CT), Pierce (Northwestern University, Evanston, IL), Trowsdale (Imperial Cancer Research Fund, London, U.K.), and Zaller (Merck Research Laboratories, Rahway, NJ).


View this table:
[in this window]
[in a new window]
  		Table I. Reactivity of mAbs and antisera used

 
Flow-cytometric measurement of Ab binding to EBV-B cell lines

Cells (5 x 105) were incubated (45 min, 4°C) with varying concentrations of primary Ab in 50 µl RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 0.1% sodium azide, and 5% FCS, adjusted to pH 8 with NaOH, and washed twice in incubation buffer. Bound Ab was detected using saturating amounts of FITC-conjugated goat anti-mouse Ig antiserum, and cells were either counterstained with propidium iodide or fixed in 1% paraformaldehyde in PBS. Five thousand intact cells (identified by light scatter and, where applicable, by propidium iodide staining) were analyzed on Epics Elite (Coulter Corp., Miami, FL; Fig. 4Go) or FACScan (Becton Dickinson, Lincoln Park, NJ; Fig. 2Go) flow cytometers. The fluorescence profiles revealed a single population, and results are shown as median or mean fluorescence intensities.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 4. Flow-cytometric analysis of the cell surface MHC class II phenotype of 7.12.6 cells. Cells were incubated with varying concentrations of different mAbs (A, L243; B, ISCR3; C, 16.23; D, CerCLIP.1). Bound mAbs were detected by indirect immunofluorescence and quantitated by flow cytometry. The relative mAb concentration is given on the ordinate, with the highest amount of each Ab used set to 1. The abscissa shows median fluorescence intensities. All fluorescence histograms showed a single, homogeneous cell population. Similar results were obtained in two independent titrations and several additional experiments done at single mAb concentrations.

 


View larger version (76K):
[in this window]
[in a new window]
  		FIGURE 2. A point mutation in codon 79 of the DMB cDNA from 7.12.6 cells. Shown is a part of a sequencing gel of DMB cDNA from 8.1.6 and 7.12.6 cells showing the region near codon 79. The mutation is indicated by an arrow. Derived sequences are shown with codon 79 in bold type and with the mutation in 7.12.6 underlined.

 
Generation of transient transfectants

A 1.4-kbp XhoI fragment containing the full-length DMB*0101 cDNA was excised from pCDM8/DMB (22) (kindly provided by Dr. J. Trowsdale) and cloned into the episomal, EBV-based mammalian expression vector, pREP4 (Invitrogen Corp., San Diego, CA). For transient transfection, 107 cells were mixed with 30 µg pREP4 or pREP4 containing the DMB insert in a final volume of 0.5 ml PBS, electroporated (Gene Pulser equipped with capacitance extender; Bio-Rad, Hercules, CA; 330 V, 250 µF, 0.4-cm cuvette), and cultured. After recovery, cells were placed under hygromycin selection (75–100 µg/ml) and cultured for another 15 days before flow-cytometric analysis.

Biosynthetic labeling

Cultured cells were starved of methionine and cysteine in Met-/Cys- RPMI 1640 supplemented with 2 mM glutamine, 10 mM HEPES, 10% dialyzed FBS, and antibiotics for 20 to 30 min at 37°C. They were labeled with [35S]Met/Cys containing protein-labeling mix (DuPont NEN, Boston, MA) in complete Met-/Cys- medium (100 µCi [35S]Met/ml) for 30 min and chased in medium supplemented with 1 mM each of unlabeled Met and Cys for various times. Cells were harvested, washed once in PBS, and stored at -70°C until use.

Immunoprecipitation

Cell pellets were lysed in 50 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM MgCl2, and 1% Nonidet P-40, containing protease inhibitors (50 mM iodoacetamide, 0.2 U/ml aprotinin, 20 µg/ml leupeptin, 2 µg/ml pepstatin and tosyllysine chloromethyl ketone, and 1 mM fresh PMSF; the iodoacetamide also served to suppress thiol-disulfide bond exchange), at 4°C for 1 h. Unextracted material was pelleted at 18,300 x g (14,000 rpm in a microfuge; 30 min, 4°C). Lysates were precleared repeatedly with preimmune rabbit IgG and fixed, heat-treated Staphylococcus aureus Cowan I bacteria (Calbiochem, San Diego, CA). Lysates were sequentially immunoprecipitated with protein A-Sepharose (Pharmacia, Piscataway, NJ) bound to 1) preimmune rabbit IgG (as negative control), 2) antiserum 11323, and 3) L243. After extensive washing in 50 mM Tris-Hcl, pH 8, 150 mM Nacl, 10 mM EDTA, and 1% Nonidet P-40, immunoprecipitates were boiled in 1 vol of 0.6% SDS, 1% 2-ME. An equal volume of 2 mU Endo H (Boehringer Mannheim Corp., Indianapolis, IN) in 50 mM sodium acetate, pH 5.5, 1% Nonidet P-40, 1 mM PMSF, and 2 µg/ml tosyllysine chloromethyl ketone was added, and the mixtures were incubated overnight at 37°C. Mock reactions contained water instead of enzyme. Samples were resolved on Laemmli SDS-PAGE minigels containing 12% acrylamide (Bio-Rad). The gels were fixed, soaked in 4% diphenyloxazole in glacial acetic acid, rehydrated, dried, and fluorographed (Hyperfilm MP; Amersham, Arlington Heights, IL).

Two-dimensional gel electrophoresis

DM was immunoprecipitated from biosynthetically labeled cells, as described above, except that the ß-chain-specific mAb, 47G.S4, was used. Immunoprecipitates were eluted in nonreducing SDS-PAGE sample buffer and resolved on 10% SDS-PAGE tube gels (1.5 mm diameter x 16 cm length). The gels were then heated in reducing sample buffer (5% 2-ME, 15 min, 95°C), fixed on top of 12% SDS-PAGE slab gels (1.5 mm x 16 cm x 16 cm), using 1% agarose in running buffer, and re-electrophoresed.

Western blotting of whole cell lysates

Cells were washed twice in PBS and lysed at 5 x 107 cells/ml in Nonidet P-40 lysis buffer, debris was spun out, and the supernatant (up to 106 cell equivalents) was mixed with concentrated nonreducing or reducing (2.5% v/v final concentration of 2-ME) Laemmli SDS-PAGE sample buffer. Samples were loaded either directly or after heating (95°C, 10 min) onto 10 to 12% acrylamide SDS-PAGE gels. Separated proteins were transferred to polyvinylidene difluoride membranes (Immobilon P, Millipore, Bedford, MA; 70 min, 105 V) in 3 g/L Tris, 14.4 g/L glycine, and 15% v/v methanol. Membranes were blocked (overnight, 4°C) in 100 mM Tris-HCl (pH 7.7), 200 mM NaCl, 1% (w/v) casein (Hammerstein grade; ICN Pharmaceuticals, Inc., Costa Mesa, CA), 0.05% (v/v) Tween-20, and 0.05% (w/v) NaN3, and probed with anti-DM (47G.S4 for DM ß or 5C1 for DM {alpha}) or anti-DR (B10.a) Abs at predetermined optimal dilutions in blocking buffer (1 h, room temperature). After extensive washing in PBS, 0.1% Tween-20, horseradish peroxidase-conjugated second-step reagents (donkey anti-rabbit Ig, Amersham; or goat anti-mouse Ig, Life Technologies) were added in PBS-Tween containing 5% nonfat dry milk. Following further washes, enhanced chemiluminescence substrate was added (Renaissance; DuPont NEN), and blots were exposed to film (Hyperfilm ECL, Amersham).

For semiquantitative comparison of DM ß content between cells, a calibration curve with graded amounts of wild-type DM was constructed by mixing lysates from DM-wt 8.1.6 cells and DM ß-null mutant 9.5.3 cells in defined proportions, keeping the total cell number constant. Nonsaturating film exposures of 47G.S4 blots were analyzed by scanning densitometry on a flat-bed scanner (ES-1200C; Epson, Torrance, CA) interfaced to a PowerCenter 150 computer (PowerComputing, Round Rock, TX), and band intensities were quantified using the public domain NIH Image program (developed by U.S. National Institutes of Health and available on the internet at http://rsb.info.nih.gov/nih-image/).

Western blotting of glycoprotein fractions or immunoprecipitates

This procedure was a modification of an established protocol (38). For glycoprotein preparations, cells were lysed at 5 x 107/ml in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mM MnCl2, 1 mM CaCl2, plus protease inhibitors. Unextracted material was spun out, and the supernatant (1.5 x 107 cell equivalents) was mixed with 20 µl Con A-Sepharose (Sigma Chemical Co., St. Louis, MO). After rocking overnight at 4°C, Con A-Sepharose pellets were washed four times in 0.75 ml lysis buffer. For Endo H digestions, glycoproteins were eluted in 0.6% 2-ME, 1% SDS, split into two aliquots, and digested with or without Endo H, as described above for immunoprecipitates. For nonreducing/reducing analysis, glycoproteins were boiled in Laemmli SDS-PAGE sample buffer with or without 2-ME, and one-half of the eluate (7.5 x 106 cell equivalents) was used for each lane. Samples were analyzed for DM chains by Western blotting, as described for whole cell lysates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sequence analysis of HLA-DMA and HLA-DMB cDNA from mutant 7.12.6 cells

HLA-DR3 molecules on 7.12.6 cells bind the 16.23 mAb poorly, and the cells present MHC class II-restricted Ags less well than 8.1.6 progenitor cells (3). These defects are similar to, but less pronounced than those found in mutants lacking expression of HLA-DMA or DMB mRNA. As levels of DMA and DMB mRNA are normal in 7.12.6 (3), we examined whether the structure of HLA-DM is altered by sequencing PCR-amplified DMA and DMB cDNAs. The 7.12.6 mutant line is derived from 8.1.6 progenitor cells, which carry two copies of the DMA*0101 gene (cf Fig. 1GoC). Sequencing of the entire DMA-coding sequence amplified from 7.12.6 cDNA revealed no differences from the wild type (data not shown). In contrast, the DMB cDNA contained a single nucleotide substitution changing codon 79 from TGT (Cys) to TAT (Tyr) (3) (Fig. 2Go). The remainder of the sequence was identical to the DMB*0101 sequence found in 8.1.6 (22) (data not shown).

Restoration of 16.23 binding in 7.12.6 cells by transfection of wild-type DMB

To test whether the Cys79->Tyr mutation in DMB of 7.12.6 cells was the cause of the peptide-loading defect, we introduced a wild-type DMB gene into 7.12.6 cells and examined 16.23 staining as the prototype marker for DM function and normal peptide loading (Fig. 3Go). When either 7.12.6 or DMß-null cells were transfected with wild-type DMB cDNA, expression of 16.23-reactive DR molecules was increased to levels comparable with those found in the DM-wt cell, 8.1.6. In addition, staining of 7.12.6 cells with a mAb specific for DR3:CLIP complexes was reduced dramatically after transfection with wild-type DMB (E. v. S., E. D. M., unpublished data). These observations strongly suggested that the peptide-loading defect in 7.12.6 cells was due to the Tyr79 mutation in DMB.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 3. Transfection of wild-type DMB into 7.12.6 cells restores 16.23 expression. 7.12.6 cells were transiently transfected with empty vector (pREP4) or with the same vector carrying a wild-type DMB insert (pREP4-DMB). After drug selection, 7.12.6 transfectants were compared with similarly transfected DM-wt and DMB-null cells (8.1.6 and 9.5.3, respectively) for binding of 16.23. Bound Ab was quantitated by incubation with goat anti-mouse Ig FITC and flow cytometry. Background fluorescence and L243 staining were comparable between the lines. Similar results were obtained in four experiments done after two separate transfections.

 
Inefficient peptide loading by HLA-DR3 molecules expressed in 7.12.6 cells

To examine whether the differential 16.23 staining was due to differences in affinity or the number of sites, varying concentrations of the mAb were tested for binding to 8.1.6 progenitor cells, the DMB-null mutant, 9.5.3, and 7.12.6 cells (Fig. 4Go). Titration curves obtained for the different cells using two anti-DR mAbs, L243 and ISCR3, were similar (Fig. 4Go, A and B), indicating that the binding sites for these mAbs differed little in affinity or number. For the DM-dependent 16.23 mAb, different levels of staining were observed at saturating mAb concentrations, with 7.12.6 cells binding intermediate amounts of 16.23, as expected (Fig. 4GoC). The shapes of the titration curves were similar for the different cells, indicating that differences in the number of 16.23-reactive Ab binding sites, rather than differences in affinity, accounted for the reduced staining of the mutants. Thus, 16.23 appeared to recognize a subset of HLA-DR3 molecules that was generated most efficiently in the presence of normally functioning DM. Furthermore, 7.12.6 cells appeared to generate this subset inefficiently.

HLA-DR3 molecules synthesized in DM-negative cells are loaded inefficiently with peptides derived from exogenous proteins, but instead accumulate at the cell surface as complexes with CLIP. To measure CLIP association of HLA-DR3 molecules at the surface of 7.12.6 cells, the anti-CLIP mAb, CerCLIP.1, was used for flow cytometry (Fig. 4GoD). The level of CerCLIP.1 staining was low for 8.1.6 and high for DM ß-null 9.5.3 cells. The 7.12.6 mutant also expressed large amounts of CerCLIP.1-reactive surface class II molecules, indicating accumulation of CLIP. HLA-DP4 molecules do not detectably accumulate CLIP in the absence of DM (W. Liu, E. D. M., unpublished results), and HLA-DQ molecules are expressed at lower levels than DR or DP, suggesting that the accumulated CLIP was predominantly associated with HLA-DR molecules. When corrected for differences in total HLA-DR expression, CLIP accumulation on 7.12.6 cells was slightly less than that seen for 9.5.3 (Table IIGo). Together with the intermediate 16.23 staining, this result suggested that the mutant DM molecules in 7.12.6 were capable of releasing at most a small amount of CLIP. The shapes of the titration curves obtained using the anti-CLIP reagent were similar for 9.5.3 and 7.12.6, indicating that the antigenic determinants on these cells differed little in affinity (Fig. 4GoD).


View this table:
[in this window]
[in a new window]
  		Table II. 16.23 and CerCLIP.1 binding of EBV B cell mutants corrected for differences in HLA-DR expression

 
Most normally loaded endosomal peptides stabilize DR3 molecules against dissociation by SDS at room temperature (2). In contrast, complexes with CLIP are dissociated by SDS (2, 8, 39). To assess whether DR3 molecules in 7.12.6 cells were poorly loaded with stabilizing peptides, DR molecules from 8.1.6, 9.5.3, and 7.12.6 cells were assayed for SDS stability by Western blotting of whole cell lysates (Fig. 5Go). As expected, DM wild-type 8.1.6 cells expressed mostly SDS-stable DR3 complexes, whereas HLA-DR molecules from DM-null cells were mostly SDS unstable. The SDS stability of HLA-DR molecules from 7.12.6 cells was intermediate between 8.1.6 and 9.5.3, consistent with a partial defect in peptide loading. The 7.12.6 cells contained only a small fraction of SDS-stable molecules, as shown by the observation that the intensity of the monomer band did not increase substantially after boiling (the ability to detect a rather intense dimer band in 7.12.6 is deceptive in this regard, as the mAb used prefers dimeric molecules; E. D. M., unpublished data). The same order of SDS stability (8.1.6 >> 7.12.6 > 9.5.3) was observed by immunoprecipitation of HLA-DR3 complexes from biosynthetically labeled cells after an overnight chase (data not shown). Together with other assays of DM function, the reduced SDS stability indicated that HLA-DR3 molecules in 7.12.6 cells have a defect in the endosomal release of CLIP and in the acquisition of a normal complement of antigenic peptides. Furthermore, DM function appears to be greatly reduced, but not completely abolished, by the mutation.



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 5. Intermediate SDS stability of HLA-DR3 molecules in 7.12.6 cells. Lysates (5 x 105 cell equivalents) of 8.1.6, 7.12.6, and 9.5.3 cells were resolved by nonreducing SDS-PAGE with or without boiling (lanes labeled b or nb, respectively), and DR3 {alpha}ß dimers and dissociated ß-chains were detected by Western blotting using the DR ß-chain-specific mAb, B10.a. The experiment was performed twice with similar results.

 
HLA-DR molecules in 7.12.6 cells fail to release CLIP, whereas other steps in HLA-DR maturation are normal

To determine which steps in class II maturation were impaired in 7.12.6 cells, HLA-DR3 synthesis, trafficking, and processing were analyzed by pulse-chase labeling (Fig. 6Go). HLA-DR molecules were immunoprecipitated and digested with Endo H to distinguish DR molecules that have traversed the medial Golgi apparatus (Endo H-resistant ß-chain, partially resistant {alpha}-chain) from those that have not (both chains Endo H sensitive).



View larger version (87K):
[in this window]
[in a new window]
  		FIGURE 6. Pulse-chase analysis of HLA-DR3 maturation in 7.12.6 cells. The indicated cells (8.1.6, DM-wt; 9.5.3, DMB-null; 7.12.6, DMB-Tyr79) were metabolically labeled and chased for the times shown. HLA-DR3 molecules were immunoprecipitated with L243, boiled under reducing conditions, digested with Endo H (lanes labeled + Endo H), or mock digested (lanes labeled - Endo H), and analyzed by SDS-PAGE. Only the informative region of the gel is shown. Bands were assigned as described previously (50, 53), and the identity of the CLIP band in 9.5.3 and 7.12.6 was verified by immunoprecipitation with CerCLIP.1 (not shown). Lanes labeled C' are control immunoprecipitations with irrelevant Ab at 0 h of chase. Note that DR molecules complexed with all Ii isoforms are precipitated poorly by L243; complexes with p41/p43Ii isoforms were not detected in these experiments. Migration of prestained m.w. markers is shown on the right. The experiment was repeated four times with similar results.

 
All three cell lines synthesized comparable levels of HLA-DR3 molecules during the 30-min labeling period (Fig. 6Go, 0-h chase), and the early steps of DR3 maturation and processing were similar between the cells. DR3 molecules initially were fully sensitive to Endo H, but converted to the mature phenotype between 0.5 and 3 h of chase, indicating export from the ER past the medial Golgi apparatus. The endosomal Ii-processing intermediates, p22Ii and p10Ii, were undetectable until 0.5 h of chase, peaked at 3 h, and decayed thereafter. At 3 h of chase, the amount of L243-reactive DR3 increased above the levels seen in the pulse; this was consistent with previous results showing that L243 reacts poorly with some DR alleles complexed with full-length Ii, seen early after synthesis (40). In contrast, accumulation and release of CLIP differed between DM wild-type and mutant cells. In 8.1.6 progenitor cells, small amounts of CLIP were detectable between 3 and 7 h of chase, whereas in 9.5.3 and 7.12.6 mutants, CLIP accumulated to significantly higher levels. In both mutants, accumulation of CLIP was paralleled by the decay of class II-associated p22- and p10-processing fragments of Ii, suggesting that these fragments are precursors of CLIP. We concluded that the failure to release CLIP is the earliest detectable difference in class II maturation between wild-type cells and the 7.12.6 mutant.

Abundance and trafficking of mutant DMB in 7.12.6 cells

We hypothesized that the DMB point mutation could diminish DM function by destabilizing the native conformation of the DM molecule, thus shortening its lifetime in the cell. To address this possibility, steady state DM levels in the different cell lines were compared by Western blotting (Fig. 7GoA). Wild-type DM ß-chain was detected as an intense, specific 29-kDa band in 8.1.6 cell, but not 9.5.3 lysates. In 7.12.6, the amount of mutant DM ß-chains was reduced by approximately fivefold compared with wild type (17 and 22% of wild type in two independent quantitations). The abundance of DM {alpha}-chains was reduced by a similar amount (data not shown).



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 7. Reduced steady state levels and altered subcellular localization of mutant DM in 7.12.6 cells. A, Comparison of wild-type and mutant DM levels. Nonidet P-40 lysates of 106 8.1.6, 9.5.3, and 7.12.6 cells, or mixtures of 8.1.6 and 9.5.3 lysates containing the indicated cell equivalents of 8.1.6 while keeping the total cell number constant, were boiled under reducing conditions and analyzed for DM ß-chains by Western blotting. Mutant DM levels were about 20% of wild type, as determined by densitometric quantitation of band intensities. Similar results were obtained in two independent experiments. B, ER retention of mutant DM. Glycoprotein preparations from the indicated cell lines were digested with Endo H or mock digested as shown, and DM ß-chains were detected by Western blotting. The positions of undigested and deglycosylated DM ß-chains are shown (ß and ßs, respectively). The film was overexposed to reveal minor subpopulations in the mutant cells. The experiment was performed three times with similar results.

 
To examine the subcellular localization of the mutant DM molecules, DM from wild-type and mutant cells were analyzed for Endo H resistance, a criterion commonly used to measure export of MHC molecules out of the ER past the medial Golgi apparatus (cf Fig. 6Go). Almost all of the wild-type DM molecules isolated from 8.1.6 cells were resistant to Endo H (Fig. 7GoB), consistent with accumulation in post-Golgi class II-rich prelysosomal compartments (E. Stang, C. Guerra, M. A. Amaya, Y. Paterson, O. Bakke, and E. D. M., submitted). By contrast, only a small proportion of the mutant DM ß-chains in 7.12.6 cells was Endo H resistant, implying that mutant molecules were retained in pre-Golgi compartments, most likely the ER and/or cis-Golgi reticulum. DM ß-chains isolated from the DM {alpha}-null mutant, 2.2.93 (cf Fig. 1GoC), served as a positive control for Endo H digestion and were deglycosylated completely under these conditions. We concluded that all unpaired ß-chains in 2.2.93, as well as a large majority of the point-mutated DM ß-chains in 7.12.6, were retained in pre-Golgi compartments. Endo H resistance of a small subpopulation of mutant DM molecules implied that this subset was able to escape from ER retention and to mediate the small amount of endosomal peptide exchange revealed by phenotypic analysis of class II molecules in 7.12.6 cells.

To determine whether the reduced expression of the mutant DM molecule was due to decreased synthesis or increased turnover, and to compare rates of trafficking, pulse-chase analysis was performed (Fig. 8Go). DM molecules were immunoprecipitated using a polyclonal rabbit anti-DM antiserum, digested with Endo H, and analyzed by SDS-PAGE. After a 30-min pulse, the antiserum precipitated two chains of the expected m.w. from both 8.1.6 and 7.12.6 cells, with the ß-chain band being more intense. As expected, the {alpha}- and ß-chain bands were absent from the single chain-deficient mutants, 2.2.93 and 9.5.3, respectively, demonstrating that the antiserum specifically recognizes both chains of DM. In all cells, newly synthesized DM chains were sensitive to Endo H digestion, indicating that most of the DM molecules made during the 30-min labeling period had not yet reached the medial Golgi apparatus. In 8.1.6 cells, a substantial fraction of the newly synthesized wild-type DM molecules became resistant to Endo H digestion after a 45-min chase, indicating passage through the Golgi apparatus. Acquisition of Endo H resistance was nearly complete by 3 h of chase, although one of the two glycans on DM {alpha} remained sensitive throughout the chase (as expected from 5 . The wild-type molecules persisted up until 27 h of chase. In contrast, single DM chains in 9.5.3 and 2.2.93 cells were turned over completely by 6 h of chase and remained Endo H sensitive, suggesting that they were degraded without traversing the medial Golgi apparatus. Export from the ER was also delayed in 7.12.6 cells, as Endo H-resistant forms were not detected until 3 h after labeling. Both the Endo H-resistant and Endo H-sensitive subpopulations were degraded by 6 h of chase. These findings implied that the native structure of DM was disrupted significantly in the mutant.



View larger version (72K):
[in this window]
[in a new window]
  		FIGURE 8. Increased turnover and decreased rates of ER export of mutant DM. The indicated cells were labeled biosynthetically and chased for the times shown. Cell lysates were immunoprecipitated using antiserum 11323, and the precipitates were digested with Endo H or mock digested, as shown. Only the informative region of the gel is shown. There was a faint band (labeled *) close to the ß-chain position in 9.5.3 immunoprecipitates, but unlike DM or class II ß-chains, it was resistant to Endo H. It was much weaker than the DM ß band in the other cells and did not interfere with DM ß identification. Similar results were obtained in four separate experiments, but the amount of Endo H-resistant mutant DM in 7.12.6 cells at 3 h of chase was variable.

 
The Cys->Tyr mutation at position 79 in DMB of 7.12.6 cells does not prevent heterodimerization, but causes aberrant disulfide bonding

To examine disulfide bonding and assembly of wild-type and mutant DM heterodimers and single chains, DM was immunoprecipitated from Nonidet P-40 extracts of biosynthetically labeled 8.1.6 and 7.12.6 cells using a DM ß cytoplasmic tail-specific mAb and resolved by nonreducing/reducing two-dimensional SDS-PAGE (Fig. 9GoA). DM precipitates from 8.1.6 cells contained two specific spots with the expected m.w. of DM {alpha}- and ß-chains (35 and 29 kDa, respectively) under reducing conditions. Both chains migrated to the right of the diagonal, i.e., slightly faster under nonreducing than under reducing conditions, indicating that both chains contained intramolecular disulfide bonds and that chain association was noncovalent. In 7.12.6 precipitates, monomeric DM ß-chain was also seen, but monomeric {alpha}-chain was not coprecipitated detectably under these conditions. This result indicated that noncovalent heterodimers were destabilized or formed in smaller amounts in the 7.12.6 mutant. To the left of the diagonal, vertically aligned 29- and 35-kDa spots were observed at a nonreducing m.w. of 56 kDa, showing covalent heterodimerization (the nonreduced m.w. are somewhat less than the sum of the reduced subunit m.w., probably due to deviations from an ideal rod shape in the disulfide-bonded dimer). Furthermore, a 29-kDa spot without other vertically aligned spots was seen at a nonreducing m.w. of 47 kDa, consistent with homodimerization of DM ß.



View larger version (59K):
[in this window]
[in a new window]
  		FIGURE 9. Chain pairing and disulfide bonding of mutant DM in 7.12.6 cells. A, Nonreducing/reducing two-dimensional SDS-PAGE analysis of biosynthetically labeled DM ß-chain immunoprecipitates from 8.1.6 and 7.12.6 cells. Positions of DM monomeric {alpha}- and ß-chains are indicated by arrowheads, the covalent {alpha}-ß dimer is indicated by arrows, and the ß-ß homodimer with an open arrowhead. The diagonal, as well as the vertical set of spots to the right of the dimer region and the diffuse background above it, were also found in immunoprecipitates from 9.5.3 cells (not shown). Note that even though the ß-chain spots in the monomer and heterodimer populations of 7.12.6 were of similar intensity, an {alpha}-chain spot could only be detected in the covalent heterodimer, indicating that the efficiency of noncovalent association was poor. B, Western blotting analysis of altered disulfide bonding and trafficking of DM ß in mutants. Glycoprotein preparations from the indicated cells were boiled under nonreducing or reducing conditions, as shown, and analyzed for the migration of DM ß-chains by Western blotting. Shown are the positions of dissociated ß-chains, covalent ß-ß homodimers, and covalent {alpha}-ß heterodimers. The experiment was performed three times with similar results. C, The covalent dimer in 7.12.6 cells contains DM {alpha}. Glycoprotein preparations from 7.12.6 and from the DM {alpha}-null mutant 2.2.93 were analyzed by nonreduced SDS-PAGE, and DM {alpha}-chains were detected by Western blotting. *Indicates an unknown protein that was detected nonspecifically by 5C1 in the negative control cells.

 
The chain composition of the different disulfide-bonded forms was confirmed by Western blotting of nonreducing gels using chain-specific Abs. The anti-DM ß mAb detected the monomeric DM ß-chain in glycoprotein preparations from all cells except the DM ß-null cell, 9.5.3 (Fig. 9GoB, lanes labeled - Endo H). In addition, it detected a 47-kDa band (probably the ß-ß homodimer) in 7.12.6 and 2.2.93 cells, and a 56-kDa band in 7.12.6. The latter band was also detected by probing blots of anti-DM ß immunoprecipitates with a mAb against DM {alpha} (Fig. 9GoC), confirming that this band represented a covalent {alpha}-ß heterodimer. Together, these results indicate that wild-type DM exists as a noncovalent dimer with intramolecular disulfide bonds, that a significant proportion of DM ß-chain can homodimerize in the absence of DM {alpha}, and that the mutant DM ß-chain in 7.12.6 cells can form a covalent heterodimer with DM {alpha}. We conclude that both correct pairing of DM ß with DM {alpha} and the Cys79ß residue are important for maintaining the wild-type disulfide bond arrangement of DM ß.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our experiments on the EBV-B cell, 7.12.6, have identified a mechanism for its partial Ag presentation defect. The phenotype of 7.12.6 is consistent with inefficient function of the mutated DM molecules. In 7.12.6 cells, HLA-DR molecules mature normally, but fail to release CLIP, so that CLIP:DR3 complexes accumulate at the cell surface. Inefficient CLIP release is associated with poor endosomal peptide loading, as shown by decreased expression of the 16.23 epitope, decreased SDS stability, and decreased Ag presentation to MHC class II-restricted T cells. Furthermore, the point mutation in DM is likely to be responsible for the peptide-loading defect, as 16.23 staining is restored following transfection of wild-type DMB into 7.12.6 cells. As our study used randomly mutagenized EBV-B cell clones, additional, adventitious mutations or clonal variation may add to the severity and quality of the phenotype of 7.12.6. Nevertheless, the data strongly support the view that the major aspects of the 7.12.6 phenotype are due to the point mutation in DM ß.

The reduced peptide exchange in 7.12.6 cells can be attributed chiefly to the decreased abundance of mutant DM in post-Golgi compartments. Steady state levels of mutant DM are about one-fifth of wild type due to increased turnover, and export from the ER is delayed. Together, this results in at least a 20-fold decrease of DM levels in post-Golgi compartments, such as the prelysosomal compartments to which wild-type DM travels in 8.1.6 progenitor cells (E. Stang, C. Guerra, M. A. Amaya, Y. Paterson, O. Bakke, and E. D. M., submitted) and in other EBV-B cells (31). It will be interesting to elucidate the mechanisms that control turnover of wild-type DM, single DM chains, and mutated DM in the ER and in endosomes. Even for wild-type cells, the endosomal concentration of DM is on the order of five- to tenfold less than that of DR molecules (41) (W. Liu, E. D. M., unpublished data). Our observations showing that an even smaller amount of mutant DM in endosomes of 7.12.6 mediates detectable peptide loading are consistent with the view that DM can function catalytically in the cell. However, an additional chaperone function for DM is not ruled out by our data.

ER retention and accelerated turnover are hallmarks of recognition by the ER quality control apparatus (42) and suggest malfolding of the mutant molecules in 7.12.6. The observation that disulfide bonding of DM ß is strikingly and specifically changed in the mutants provides direct evidence for such conformational aberrations. Wild-type DM predominantly forms a noncovalent dimer, with both DM {alpha}- and ß-chains containing intramolecular disulfide bonds, as shown by off-diagonal migration in nonreducing/reducing two-dimensional PAGE analysis. In the absence of {alpha}-chain expression, about one-half of the ß-chains are monomeric under nonreducing conditions, while the other half forms a covalent ß-ß monomer. In the ER of 7.12.6 cells, monomeric and homodimerized mutant DM ß-chains are also detected, but the most abundant species is a covalent {alpha}-ß heterodimer. The efficiency of noncovalent pairing with DM {alpha} is reduced by the mutation. We have found no evidence for formation of higher order oligomers of DM chains, or of nonspecific disulfide-bonded aggregates with other proteins, suggesting that covalent chain pairing is specific in both 7.12.6 and 2.2.93 cells, despite the presence of substantial amounts of other nascent proteins in the ER (42).

Even the fraction of mutant DM molecules in 7.12.6 cells that can escape ER retention is degraded rapidly in post-Golgi compartments. This behavior differs strikingly from that of the wild-type molecule, which persists for at least 27 h in highly degradative late endocytic compartments. Thus, the conformation of the post-Golgi fraction of mutant DM also appears to be destabilized or altered compared with the wild type, allowing better targeting for post-Golgi degradation or increased exposure of proteolytic sites. While the escaping subpopulation of mutant DM may be more native-like than the population that is retained, this result implies that ER quality control is inefficient in that it does not perfectly discriminate between functionally normal and altered DM molecules.

In addition to the weak sequence homology, several lines of evidence are consistent with a structure for DM that resembles that of Ag-presenting MHC class I and class II proteins. Wild-type DM, like conventional class II molecules, only contains intrachain disulfide bonds, despite the presence of five additional cysteines unique to DM. Furthermore, analyses of cysteine mutants strongly suggest that the ß1-domain cysteines, residues 11 and 79, which are conserved among MHC molecules, are paired in wild-type DM. Mutating Cys79ß of DM delays its export from the ER and diminishes its stability and function, implying that this cysteine is important for structural integrity. The formation of aberrant disulfide bond arrangements upon mutating Cys79ß provides more direct evidence that this residue is normally involved in a disulfide bond. The idea that residue 11ß is the partner cysteine for Cys79ß is supported by the observation that mutating residue 11 to Tyr results in a similar partial peptide-loading defect (E. v. S., unpublished). The 7.12.6 phenotype can be partially corrected by culturing the cells at reduced temperature (M. Riley, M. Amaya, E. v. S., E. D. M., unpublished). This suggests that folding and transport of mutant DM are temperature sensitive, as is observed for intracellular transport of viral glycoprotein mutants with disrupted disulfide bonds (43). Interestingly, mutating a cysteine in the homologous {alpha}2-domain disulfide bond of the classical class I molecule, HLA-A*0201, also results in a partial functional defect and delayed ER-to-Golgi transport (44).

The altered disulfide-bonding patterns in 2.2.93 and 7.12.6 cells show that both DM {alpha} and Cys79 of the ß-chain contribute to proper folding, assembly, and disulfide bonding of DM. The observation that the {alpha}-chain is associated with mutated DM ß in 7.12.6 (albeit mostly covalently) shows that Cys79ß is not absolutely required for the chains to associate specifically with one another. Rather, its role seems to be to maintain a stable, proteolytically resistant and transport-competent conformation of the heterodimer and to suppress interchain disulfide bonding, possibly by competing with {alpha}-chain cysteines for pairing with Cys11ß. Independently, DM {alpha} contributes to the suppression of aberrant disulfide bonds in DM ß. This is shown by the observation that wild-type DM ß can form covalent homodimers in the absence of DM {alpha} in 2.2.93 cells. This observation may be surprising in view of the fact that chain pairing of conventional class II molecules generally results in heterodimers and is isotype and even haplotype specific; however, homodimerization is not unusual for Ig superfamily proteins in general, and self-association of soluble DM ß-chains expressed in insect cells has been reported (12). As MHC class II-like heterodimers may have evolved from a homodimeric ancestor (45), and as DM diverged early from Ag-presenting MHC class I and class II molecules (27), the specific covalent dimerization of DMß in 2.2.93 might be a vestigial phenomenon. The relevance of the unusual disulfide-bonded {alpha}-ß and ß-ß dimers for normal folding of wild-type DM remains to be elucidated. Covalent dimers may occur as transient intermediates during normal folding, or they may represent aberrant products that can accumulate only when normal folding requirements are not met.


   Acknowledgments
 
We thank Miguel Amaya and Michael Riley for initial flow-cytometric experiments using 7.12.6; Drs. P. Cresswell, S. Pierce, D. Zaller, and J. Trowsdale for their kind gifts of Abs; Drs. S. Fling and D. Pious for the gift of 2.2.93 cells; and Drs. R. Kopito and C. Clayberger for critical review of the manuscript.


   Footnotes
 
1 Supported by grants from National Institutes of Health (AI-28809) and Arthritis Foundation to E.D.M., an Arthritis Foundation fellowship to R.B., and a University of Pennsylvania Medical Scientist Training Program grant (T32 GM 7170) to R.C.D. Back

2 Address correspondence and reprint requests to Dr. R. Busch, Department of Pediatrics, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5208. E-mail address: Back

3 Abbreviations used in this paper: ER, endoplasmic reticulum; CLIP, major histocompatibility complex class II-associated invariant chain peptide; Cys, cysteine; Endo H, endoglycosaminidase H; Ii, invariant chain; Met, methionine; RT-PCR, reverse-transcriptase polymerase chain reaction; Tyr, tyrosine. Back

Received for publication June 18, 1997. Accepted for publication October 3, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Busch, R., E. D. Mellins. 1996. Developing and shedding inhibitions: how MHC class II molecules reach maturity. Curr. Opin. Immunol. 8:51.[Medline]
  2. 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]
  3. 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]
  4. 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]
  5. Denzin, L. K., N. F. Robbins, C. Carboy-Newcomb, P. Cresswell. 1994. Assembly and intracellular transport of HLA-DM and correction of the class II antigen-processing defect in T2 cells. Immunity 1:595.[Medline]
  6. Riberdy, J. M., J. R. Newcomb, M. J. Surman, J. A. Barbosa, P. Cresswell. 1992. HLA-DR molecules from an antigen-processing mutant cell line are associated with invariant chain peptides. Nature 360:474.[Medline]
  7. Sette, A., S. Ceman, R. T. Kubo, K. Sakaguchi, E. Appella, D. F. Hunt, T. A. Davis, H. Michel, J. Shabanowitz, R. Rudersdorf, H. M. Grey, R. DeMars. 1992. Invariant chain peptides in most HLA-DR molecules of an antigen-processing mutant. Science 258:1801.[Abstract/Free Full Text]
  8. 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]
  9. Miyazaki, T., P. Wolf, S. Tourne, C. Waltzinger, A. Dierich, N. Barois, H. Ploegh, C. Benoist, D. Mathis. 1996. Mice lacking H-2 M complexes, enigmatic elements of the MHC class II peptide-loading pathway. Cell 84:531.[Medline]
  10. Fung-Leung, W.-P., C. D. Surh, M. Liljedahl, P. J. 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]
  11. Martin, W. D., G. G. Hicks, S. K. Mendiratta, H. I. Leva, H. E. Ruley, L. V. 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]
  12. 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]
  13. Sherman, M. A., D. A. Weber, P. E. Jensen. 1995. DM enhances peptide binding to class II MHC by release of invariant chain-derived peptide. Immunity 3:197.[Medline]
  14. Denzin, L. K., P. Cresswell. 1995. HLA-DM induces CLIP dissociation from MHC class II {alpha}ß dimers and facilitates peptide loading. Cell 82:155.[Medline]
  15. Sanderson, F., C. Thomas, J. Neefjes, J. Trowsdale. 1996. Association between HLA-DM and HLA-DR in vivo. Immunity 4:87.[Medline]
  16. 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]
  17. 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 6:1.[Medline]
  18. Kropshofer, H., G. J. Hämmerling, A. B. Vogt. 1997. How HLA-DM edits the MHC class II peptide repertoire: survival of the fittest?. Immunol. Today 18:77.[Medline]
  19. Kropshofer, H., A. B. Vogt, G. Moldenhauer, J. Hammer, J. S. Blum, G. J. Hämmerling. 1996. Editing of the HLA-DR-peptide repertoire by HLA-DM. EMBO J. 15:6144.[Medline]
  20. 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]
  21. Van Ham, S. M., U. Grüneberg, G. Malcherek, I. Bröker, 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]
  22. Kelly, A. P., J. J. Monaco, S. Cho, J. Trowsdale. 1991. A new human HLA class II-related locus, DM. Nature 353:571.[Medline]
  23. Sanderson, F., S. H. Powis, A. P. Kelly, J. Trowsdale. 1994. Limited polymorphism in HLA-DM does not involve the peptide binding groove. Immunogenetics 39:56.[Medline]
  24. Hermel, E., J. Yuan, J. J. Monaco. 1995. Characterization of polymorphism within the H2-M MHC class II loci. Immunogenetics 42:136.[Medline]
  25. Carrington, M., A. Harding. 1994. Sequence analysis of two novel HLA-DMA alleles. Immunogenetics 40:165.[Medline]
  26. Carrington, M., M. Yeager, D. Mann. 1993. Characterization of HLA-DMB polymorphism. Immunogenetics 38:446.[Medline]
  27. Cho, S., M. Attaya, J. J. Monaco. 1991. New class II-like genes in the murine MHC. Nature 353:573.[Medline]
  28. Karlsson, L., A. Péléraux, R. Lindstedt, M. Liljedahl, P. A. Peterson. 1994. Reconstitution of an operational MHC class II compartment in nonantigen-presenting cells. Science 266:1569.[Abstract/Free Full Text]
  29. Hermel, E., J. J. Monaco. 1995. RT1. DMa and RT1. DMb: the rat homologues of H2-DMa and H2-DMb. Immunogenetics 42:446.[Medline]
  30. Niimi, M., Y. Nakai, Y. Aida. 1995. Nucleotide sequences and the molecular evolution of the DMA and DMB genes of the bovine major histocompatibility complex. Biochem. Biophys. Res. Commun. 217:522.[Medline]
  31. Sanderson, F., M. J. Klejmeer, 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]
  32. Green, J. M., R. DeMars, X. Xu, S. K. Pierce. 1995. The intracellular transport of MHC class II molecules in the absence of HLA-DM. J. Immunol. 155:3759.[Abstract]
  33. Marks, M. S., P. A. Roche, E. van Donselaar, L. Woodruff, P. J. Peters, J. S. Bonifacino. 1995. A lysosomal targeting signal in the cytoplasmic tail of the ß chain directs HLA-DM to the MHC class II compartments. J. Cell Biol. 131:(2):351.[Abstract/Free Full Text]
  34. Lindstedt, R., M. Liljedahl, P. A. Peterson, L. Karlsson. 1995. The MHC class II molecule H2-M is targeted to an endosomal compartment by a tyrosine-based targeting motif. Immunity 3:(5):561.[Medline]
  35. Gladstone, P., D. Pious. 1978. Stable variants affecting B cell alloantigens in human lymphoid cells. Nature 271:459.[Medline]
  36. 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]
  37. Shaman, J., E. von Scheven, P. Morris, M. Y. Chang, E. Mellins. 1995. Analysis of HLA-DMB mutants and -DMB genomic structure. Immunogenetics 41:117.[Medline]
  38. Freeze, H. H.. 1987. Unit 17.1. Special considerations for glycoproteins and their purification. F. M. Ausubel, and R. Brent, and R. E. Kingston, and D. D. Moore, and J. G. Seidman, and J. A. Smith, and K. Struhl, eds. Current Protocols in Molecular Biology, Vol. 3 17.1.5. John Wiley and Sons, New York.
  39. Monji, T., A. L. McCormack, J. R. Yates III, D. Pious. 1994. Invariant-cognate peptide exchange restores class II dimer stability in HLA-DM mutants. J. Immunol. 153:4468.[Abstract]
  40. Newcomb, J. R., P. Cresswell. 1993. Structural analysis of proteolytic products of MHC class II-invariant chain complexes generated in vivo. J. Immunol. 151:4153.[Abstract]
  41. 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]
  42. Hurtley, S. M., A. Helenius. 1989. Protein oligomerization in the endoplasmic reticulum. Annu. Rev. Cell Biol. 5:277.
  43. Long, D., G. H. Cohen, M. I. Muggeridge, R. J. Eisenberg. 1990. Cysteine mutants of herpes simplex virus type 1 glycoprotein D exhibit temperature-sensitive properties in structure and function. J. Virol. 4:5542.
  44. Warburton, R. J., M. Matsui, S. L. Rowland-Jones, M. C. Gammon, G. E. Katzenstein, T. Wei, M. Edidin, H. J. Zweerink, A. J. McMichael, J. A. Frelinger. 1994. Mutation of the {alpha}2 domain disulfide bridge of the class I molecule HLA-A*0201: effect on maturation and peptide presentation. Hum. Immunol. 39:261.[Medline]
  45. Kaufman, J., K. Skjoedt, J. Salomonsen. 1990. The MHC molecules of nonmammalian vertebrates. Immunol. Rev. 113:83.[Medline]
  46. Lampson, L., R. Levy. 1980. Two populations of Ia-like molecules on a human cell line. J. Immunol. 125:293.[Abstract]
  47. 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]
  48. Clark, E. A., R. Yakoshi. 1984. Human B cell and B cell blast-associated surface molecules defined with monoclonal antibodies. A. Bernard, and L. Baunsell, and J. Dausset, and S. Schlossman, eds. Leukocyte Typing 339. Springer-Verlag, Berlin.
  49. Johnson, J., T. Meo, G. Riethmüller, D. Schendel, R. Wank. 1982. Direct demonstration of an HLA-DR allotypic determinant on the low molecular weight (beta) subunit using a mouse monoclonal antibody specific for DR3. J. Exp. Med. 156:104.[Abstract/Free Full Text]
  50. 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]
  51. Stern, L. J., J. H. Brown, T. S. Jardetzky, J. C. Gorga, R. G. Urban, J. L. Strominger, D. C. Wiley. 1994. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 368:215.[Medline]
  52. 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]
  53. Nguyen, Q. V., W. Knapp, R. E. Humphreys. 1989. Inhibition by leupeptin and antipain of the intracellular proteolysis of Ii. Hum. Immunol. 24:153.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
M. van Lith and A. M. Benham
The DM{alpha} and DMbeta Chain Cooperate in the Oxidation and Folding of HLA-DM
J. Immunol., October 15, 2006; 177(8): 5430 - 5439.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. W. Lee, L. Hertel, R. K. Louie, T. Burster, V. Lacaille, A. Pashine, D. A. Abate, E. S. Mocarski, and E. D. Mellins
Human Cytomegalovirus Alters Localization of MHC Class II and Dendrite Morphology in Mature Langerhans Cells
J. Immunol., September 15, 2006; 177(6): 3960 - 3971.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. C. Doebele, A. Pashine, W. Liu, D. M. Zaller, M. Belmares, R. Busch, and E. D. Mellins
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 Polymorphism
J. Immunol., May 1, 2003; 170(9): 4683 - 4692.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. P. Belmares, R. Busch, K. W. Wucherpfennig, H. M. McConnell, and E. D. Mellins
Structural Factors Contributing to DM Susceptibility of MHC Class II/Peptide Complexes
J. Immunol., November 1, 2002; 169(9): 5109 - 5117.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. S. Patil, A. Pashine, M. P. Belmares, W. Liu, B. Kaneshiro, J. Rabinowitz, H. McConnell, and E. D. Mellins
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., December 15, 2001; 167(12): 7157 - 7168.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Busch, Z. Reich, D. M. Zaller, V. Sloan, and E. D. Mellins
Secondary Structure Composition and pH-dependent Conformational Changes of Soluble Recombinant HLA-DM
J. Biol. Chem., October 16, 1998; 273(42): 27557 - 27564.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. B. Guerra, R. Busch, R. C. Doebele, W. Liu, T. Sawada, W. W. Kwok, M.-d. Y. Chang, and E. D. Mellins
Novel Glycosylation of HLA-DR{alpha} Disrupts Antigen Presentation Without Altering Endosomal Localization
J. Immunol., May 1, 1998; 160(9): 4289 - 4297.
[Abstract] [Full Text] [PDF]

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 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
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Busch, R.
Right arrow Articles by Mellins, E. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Busch, R.
Right arrow Articles by Mellins, E. D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS