Assembly, Maturation, and Function Mutant HLA-DM Molecule

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

This information is current as Robert Busch, Robert C. Doebele, Emily von Scheven, Jimothy of September 28, 2021. Fahrni and Elizabeth D. Mellins J Immunol 1998; 160:734-743; ; http://www.jimmunol.org/content/160/2/734 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Aberrant Intermolecular Disulﬁde Bonding in a Mutant HLA-DM Molecule: Implications for Assembly, Maturation, and Function1

Robert Busch,2* Robert C. Doebele,† Emily von Scheven,‡ Jimothy Fahrni,* and Elizabeth D. Mellins*

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 ␤-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 Downloaded from 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 ␣␤ dimers, such dimers form inefficiently in 7.12.6; many mutant DM ␤-chains instead form a disulfide-bonded dimer with DM ␣. Homodimers of DM ␤ are also detected in 7.12.6 and in the ␣-chain defective mutant, 2.2.93. We conclude that during folding of wild-type DM, the native conformation is stabilized http://www.jimmunol.org/ by a conserved disulfide bond involving Cys79␤ and by noncovalent contacts with DM ␣. Without these interactions, DM ␤ can form malfolded structures containing interchain disulfide bonds; malfolding is correlated with ER retention and accelerated degradation. The Journal of Immunology, 1998, 160: 734–743.

M is an accessory molecule for endosomal peptide loading tion of the nonexpressed gene(s) reconstitutes a normal class II phe- of MHC class II molecules (reviewed in Ref. 1). Newly notype (3–5). DM may have several interrelated functions in normal synthesized MHC class II molecules assemble in the en- peptide loading. The first is to release Ii degradation products, includ- D 3 doplasmic reticulum (ER) with the invariant chain (Ii) and are trans- ing CLIP, from class II molecules newly arrived in endosomes. This ported to endocytic compartments, where Ii is proteolytically trun- function was revealed by studies showing that CLIP-class II com- by guest on September 28, 2021 cated to a nested set of class II-associated Ii peptides called CLIP. plexes accumulate in DM-null cells (6–11) and that purified DM cat- Class II molecules are loaded with antigenic peptides following re- alyzes dissociation of these complexes in vitro (12–14). Secondly, lease of CLIP from the Ag-binding groove. A role for DM in peptide DM can bind to MHC class II molecules during peptide exchange and loading was discovered by using EBV-B cell mutants that were un- may stabilize them against denaturation, aggregation, and/or proteol- ϩ able to present soluble protein Ags and alloantigens to CD4 T cells ysis, thus preserving peptide binding sites (15–18). Following loading (2). Class II molecules in these mutants are expressed at normal lev- with endosomal peptides, DM may facilitate additional rounds of pep- els, but lack expression of specific Ab determinants (such as that tide exchange, so that the final peptide repertoire is biased toward recognized by the anti-DR3 mAb, 16.23) and are unstable in SDS, peptides that form kinetically stable complexes (12, 14, 19–21). suggesting inefficient loading with endosomal peptides. In the mu- DM is a relatively nonpolymorphic type I transmembrane gly- ␣ ␤ tants, synthesis of DM - and/or -chains is defective, and transfec- coprotein consisting of a 35-kDa ␣-chain and a 29-kDa ␤-chain (5, 22–26) (cf Fig. 1A). The extracellular domains of both chains of DM are homologous to those of MHC class I and class II glyco- *Department of Pediatrics, Stanford University Medical Center, Stanford, CA proteins (27). This is seen most clearly in the membrane-proximal 94305; †School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and ‡Department of Pediatric Rheumatology, University of California at Ig superfamily-like domains, which share 20 to 37% of amino San Francisco Medical Center, San Francisco, CA 94143. acids with classical MHC molecules. The similarity is lower (14– Received for publication June 18, 1997. Accepted for publication October 25% identity) in the membrane-distal domains, which correspond 3, 1997. to the polymorphic Ag-binding groove of classical MHC mole- The costs of publication of this article were defrayed in part by the payment of cules. There are two cysteines at positions 11 and 79 in the ␤ page charges. This article must therefore be hereby marked advertisement in 1 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. domain of HLA-DM, which are conserved among DM homo- 1 Supported by grants from National Institutes of Health (AI-28809) and Arthritis logues from all species sequenced to date (22, 27–30). The equiv- ␣ ␤ Foundation to E.D.M., an Arthritis Foundation fellowship to R.B., and a Univer- alent pair of cysteines in the 2 domain of class I and the 1 sity of Pennsylvania Medical Scientist Training Program grant (T32 GM 7170) to domain of classical class II molecules is known from x-ray struc- R.C.D. tures to form a disulfide bond (Fig. 1, A and B). In addition, there 2 Address correspondence and reprint requests to Dr. R. Busch, Department of Pediatrics, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA are five cysteines not found in classical MHC molecules, three in 94305-5208. E-mail address: [email protected] ␤ ␣ the 1 domain and two in the 1 domain (Fig. 1A). Whether DM 3 Abbreviations used in this paper: ER, endoplasmic reticulum; CLIP, major his- has a ligand-binding groove in the membrane-proximal domain tocompatibility complex class II-associated invariant chain peptide; Cys, cysteine; Endo H, endoglycosaminidase H; Ii, invariant chain; Met, methionine; similar to that of classical MHC molecules is unclear, but attempts RT-PCR, reverse-transcriptase polymerase chain reaction; Tyr, tyrosine. to reveal peptide-binding activity for DM have failed (14, 18).

FIGURE 1. Structure of DM and derivation of the mutant EBV-B cell line, 7.12.6. A, Sche- matic illustrating the distribution of cysteine residues in the extracytoplasmic domains of DM ␣- and ␤-chains (22). The Cys79␤ 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 ␤11 and ␤79, mapped on the crystal structure of HLA-DR1 Downloaded from (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 heterozy- gous EBV-B cell line, T5-1 (35). 8.1.6 cells were obtained by random mutagenesis and selection http://www.jimmunol.org/ for loss of the DR1 allele (35). They carry a hem- izygous 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 pheno-

type. The other cells are defective for Ag pre- by guest on September 28, 2021 sentation due to different point mutations in the remaining DM allele (3, 4).

␣␤ Wild-type DM is assembled into an heterodimer in the ER Table I. Reactivity of mAbs and antisera used and exported through the Golgi apparatus into class II-rich endocytic compartments, which in EBV-transformed B cells have char- Name Reactivity Ref. acteristics of prelysosomes (5, 28, 31, 32) (E. Stang, C. Guerra, M. A. Amaya, Y. Paterson, O. Bakke, and E. D. M., submitted). L243 HLA-DR ␣ 46 ISCR3 HLA-DR 47 Endocytic targeting of DM is directed by an intrinsic, tyrosine- ␤ ␤ B10.a HLA-DR 48 based motif in the -chain (33, 34). In addition, association with Ii 16.23 HLA-DR3 (DM dependent) 49 may contribute to endosomal transport, at least in the mouse (28, CerCLIP.1 CLIP N terminus 50 34). Little is known about how DM interacts with class II mole- 47G.S4 DM ␤ cytoplasmic tail 41 ␣ cules during peptide exchange, except that the extracytoplasmic 5C1 DM extracytoplasmic 15 domains domains of DM are sufficient for function in vitro (12) and that 11323 DM ␣ ϩ ␤ extracytoplasmic D. Zaller (unpublished specific regions on the class II molecule, defined by mAb in- domains data) ␣ hibition and by a mutation in the 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 ␤ mRNA (3) (see Fig. 1C for derivation of this mutant and other cells ing revealed a missense mutation in DMB, converting codon 79 used in this work). Presentation of soluble protein Ags and ex- from coding for a cysteine to a tyrosine. We predicted that this pression of the 16.23 determinant by 7.12.6 cells were intermediate mutation would destabilize the native conformation of DM by dis- between 8.1.6 wild-type progenitors and DM ␤-null mutants, such rupting the postulated disulfide bond between Cys11␤ and as 9.5.3. DMA and DMB mRNA levels were normal, but sequenc- Cys79␤. To test this hypothesis, we have analyzed the effect of the 736 ALTERED DISULFIDE BONDING IN A Cys MUTANT OF HLA-DMB mutation on the disulfide bond structure, assembly, and intracel- MA) in complete MetϪ/CysϪ medium (100 ␮Ci [35S]Met/ml) for 30 min lular transport of DM, and further characterized its effect on DM and chased in medium supplemented with 1 mM each of unlabeled Met and function. Cys for various times. Cells were harvested, washed once in PBS, and stored at Ϫ70°C until use.

Materials and Methods Immunoprecipitation EBV-B cells Cell pellets were lysed in 50 mM Tris-HCl, pH 8, 150 mM NaCl, 5 mM

The isolation of the DM-expressing progenitor EBV-B cell line, 8.1.6 (35), MgCl2, and 1% Nonidet P-40, containing protease inhibitors (50 mM io- its DM ␤-deficient derivative, 9.5.3 (36), the related DM ␣-deficient cell, doacetamide, 0.2 U/ml aprotinin, 20 ␮g/ml leupeptin, 2 ␮g/ml pepstatin 2.2.93 (4), and the 7.12.6 mutant cell line (3) has been described (see Fig. and tosyllysine chloromethyl ketone, and 1 mM fresh PMSF; the iodoac- 1C). These cells were maintained in RPMI 1640 supplemented with 25 etamide also served to suppress thiol-disulfide bond exchange), at 4°C for mM HEPES, 2 mM L-glutamine, and 15% donor calf serum, and routinely 1 h. Unextracted material was pelleted at 18,300 ϫ g (14,000 rpm in a screened for mycoplasma contamination and for maintenance of their 16.23 microfuge; 30 min, 4°C). Lysates were precleared repeatedly with preim- phenotype. mune rabbit IgG and fixed, heat-treated Staphylococcus aureus Cowan I bacteria (Calbiochem, San Diego, CA). Lysates were sequentially immu- RT-PCR and sequencing of HLA-DM genes noprecipitated with protein A-Sepharose (Pharmacia, Piscataway, NJ) The DMB mutation in 7.12.6 cells was identified initially by dideoxy se- bound to 1) preimmune rabbit IgG (as negative control), 2) antiserum quencing of PCR-amplified DMB cDNA, using the sequencing primer, 11323, and 3) L243. After extensive washing in 50 mM Tris-Hcl, pH 8, M5, as previously described (37). To search for additional mutations, 150 mM Nacl, 10 mM EDTA, and 1% Nonidet P-40, immunoprecipitates mRNA was isolated from 8.1.6 and 7.12.6 cells using a Stratagene (La 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 Jolla, CA) RNA isolation kit. DMA and DMB cDNAs were transcribed ␮ Downloaded from and amplified using the Superscript RT-PCR system (Life Technologies, acetate, pH 5.5, 1% Nonidet P-40, 1 mM PMSF, and 2 g/ml tosyllysine Gaithersburg, MD), 1 ␮g mRNA template, and 200 ng of each primer. chloromethyl ketone was added, and the mixtures were incubated overnight After reamplification using Pfu DNA polymerase (Stratagene; 30 cycles, at 37°C. Mock reactions contained water instead of enzyme. Samples were 25 ng RT-PCR product, 200 ng of each primer), sequencing reactions were resolved on Laemmli SDS-PAGE minigels containing 12% acrylamide performed using the Perkin-Elmer (Foster City, CA) dye-terminator cycle- (Bio-Rad). The gels were fixed, soaked in 4% diphenyloxazole in glacial sequencing kit, and analyzed at the Stanford University (Stanford, CA) acetic acid, rehydrated, dried, and fluorographed (Hyperfilm MP; Amer- protein and nucleic acid facility. For DMB, the primers used for amplifi- sham, Arlington Heights, IL). Ј http://www.jimmunol.org/ cation and sequencing were DMB1 5 , M7, and M13 (37); for DMA, the following primers were used: 5Ј-CTG TGT GGC AAG AAG GTA TGG- Two-dimensional gel electrophoresis Ј Ј Ј Ј 3 ,5-GCT GGC ATC AAA CTC TGG TCT-3 , and 5 -TTG CTG ACT DM was immunoprecipitated from biosynthetically labeled cells, as de- Ј GGG CTC AGG AAC-3 . scribed above, except that the ␤-chain-specific mAb, 47G.S4, was used. Immunoprecipitates were eluted in nonreducing SDS-PAGE sample buffer Antibodies and resolved on 10% SDS-PAGE tube gels (1.5 mm diameter ϫ 16 cm Hybridoma cells were grown in RPMI 1640 supplemented with 10% FCS, length). The gels were then heated in reducing sample buffer (5% 2-ME, 15 Ϫ5 2mML-glutamine, 25 mM HEPES, 2 ϫ 10 M 2-ME, and antibiotics, min, 95°C), fixed on top of 12% SDS-PAGE slab gels (1.5 mm ϫ 16 cm ϫ and Abs were used either as tissue culture supernatant or as ascites fluid. 16 cm), using 1% agarose in running buffer, and re-electrophoresed. The Abs used and their specificities are summarized in Table I. CerCLIP.1 and the anti-DM reagents were kind gifts of their originators, Drs. Cress- Western blotting of whole cell lysates by guest on September 28, 2021 well (Yale University, New Haven, CT), Pierce (Northwestern University, Cells were washed twice in PBS and lysed at 5 ϫ 107 cells/ml in Nonidet Evanston, IL), Trowsdale (Imperial Cancer Research Fund, London, U.K.), P-40 lysis buffer, debris was spun out, and the supernatant (up to 106 cell and Zaller (Merck Research Laboratories, Rahway, NJ). equivalents) was mixed with concentrated nonreducing or reducing (2.5% Flow-cytometric measurement of Ab binding to EBV-B cell v/v final concentration of 2-ME) Laemmli SDS-PAGE sample buffer. Sam- lines ples were loaded either directly or after heating (95°C, 10 min) onto 10 to 12% acrylamide SDS-PAGE gels. Separated proteins were transferred to Cells (5 ϫ 105) were incubated (45 min, 4°C) with varying concentrations polyvinylidene difluoride membranes (Immobilon P, Millipore, Bedford, of primary Ab in 50 ␮l RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, MA; 70 min, 105 V) in 3 g/L Tris, 14.4 g/L glycine, and 15% v/v methanol. 0.1% sodium azide, and 5% FCS, adjusted to pH 8 with NaOH, and washed Membranes were blocked (overnight, 4°C) in 100 mM Tris-HCl (pH 7.7), twice in incubation buffer. Bound Ab was detected using saturating 200 mM NaCl, 1% (w/v) casein (Hammerstein grade; ICN Pharmaceuti- amounts of FITC-conjugated goat anti-mouse Ig antiserum, and cells were cals, Inc., Costa Mesa, CA), 0.05% (v/v) Tween-20, and 0.05% (w/v) ␤ ␣ either counterstained with propidium iodide or fixed in 1% paraformalde- NaN3, and probed with anti-DM (47G.S4 for DM or 5C1 for DM )or hyde in PBS. Five thousand intact cells (identified by light scatter and, anti-DR (B10.a) Abs at predetermined optimal dilutions in blocking buffer where applicable, by propidium iodide staining) were analyzed on Epics (1 h, room temperature). After extensive washing in PBS, 0.1% Tween-20, Elite (Coulter Corp., Miami, FL; Fig. 4) or FACScan (Becton Dickinson, horseradish peroxidase-conjugated second-step reagents (donkey anti- Lincoln Park, NJ; Fig. 2) flow cytometers. The fluorescence profiles re- rabbit Ig, Amersham; or goat anti-mouse Ig, Life Technologies) were vealed a single population, and results are shown as median or mean flu- added in PBS-Tween containing 5% nonfat dry milk. Following further orescence intensities. washes, enhanced chemiluminescence substrate was added (Renaissance; DuPont NEN), and blots were exposed to film (Hyperfilm ECL, Generation of transient transfectants Amersham). For semiquantitative comparison of DM ␤ content between cells, a cal- A 1.4-kbp XhoI fragment containing the full-length DMB*0101 cDNA was ibration curve with graded amounts of wild-type DM was constructed by excised from pCDM8/DMB (22) (kindly provided by Dr. J. Trowsdale) mixing lysates from DM-wt 8.1.6 cells and DM ␤-null mutant 9.5.3 cells and cloned into the episomal, EBV-based mammalian expression vector, in defined proportions, keeping the total cell number constant. Nonsaturat- pREP4 (Invitrogen Corp., San Diego, CA). For transient transfection, 107 ing film exposures of 47G.S4 blots were analyzed by scanning densitom- cells were mixed with 30 ␮g pREP4 or pREP4 containing the DMB insert etry on a flat-bed scanner (ES-1200C; Epson, Torrance, CA) interfaced to in a final volume of 0.5 ml PBS, electroporated (Gene Pulser equipped with a PowerCenter 150 computer (PowerComputing, Round Rock, TX), and capacitance extender; Bio-Rad, Hercules, CA; 330 V, 250 ␮F, 0.4-cm cu- band intensities were quantified using the public domain NIH Image pro- vette), and cultured. After recovery, cells were placed under hygromycin gram (developed by U.S. National Institutes of Health and available on the selection (75–100 ␮g/ml) and cultured for another 15 days before flow- internet at http://rsb.info.nih.gov/nih-image/). cytometric analysis. Biosynthetic labeling Western blotting of glycoprotein fractions or immunoprecipitates Cultured cells were starved of methionine and cysteine in MetϪ/CysϪ RPMI 1640 supplemented with 2 mM glutamine, 10 mM HEPES, 10% This procedure was a modification of an established protocol (38). For dialyzed FBS, and antibiotics for 20 to 30 min at 37°C. They were labeled glycoprotein preparations, cells were lysed at 5 ϫ 107/ml in 50 mM Tris- 35 with [ S]Met/Cys containing protein-labeling mix (DuPont NEN, Boston, HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mM MnCl2, 1 mM CaCl2, The Journal of Immunology 737

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. 4). Titration curves obtained for the different cells using two anti-DR mAbs, L243 and ISCR3, were similar (Fig. 4, 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. 4C). The FIGURE 2. A point mutation in codon 79 of the DMB cDNA from shapes of the titration curves were similar for the different cells, 7.12.6 cells. Shown is a part of a sequencing gel of DMB cDNA from indicating that differences in the number of 16.23-reactive Ab 8.1.6 and 7.12.6 cells showing the region near codon 79. The muta- binding sites, rather than differences in affinity, accounted for the tion is indicated by an arrow. Derived sequences are shown with reduced staining of the mutants. Thus, 16.23 appeared to recognize codon 79 in bold type and with the mutation in 7.12.6 underlined. a subset of HLA-DR3 molecules that was generated most effi- ciently in the presence of normally functioning DM. Furthermore, Downloaded from 7.12.6 cells appeared to generate this subset inefficiently. plus protease inhibitors. Unextracted material was spun out, and the su- HLA-DR3 molecules synthesized in DM-negative cells are pernatant (1.5 ϫ 107 cell equivalents) was mixed with 20 ␮l Con A-Sepha- rose (Sigma Chemical Co., St. Louis, MO). After rocking overnight at 4°C, loaded inefficiently with peptides derived from exogenous pro- Con A-Sepharose pellets were washed four times in 0.75 ml lysis buffer. teins, but instead accumulate at the cell surface as complexes with For Endo H digestions, glycoproteins were eluted in 0.6% 2-ME, 1% SDS, CLIP. To measure CLIP association of HLA-DR3 molecules at the split into two aliquots, and digested with or without Endo H, as described http://www.jimmunol.org/ above for immunoprecipitates. For nonreducing/reducing analysis, glyco- surface of 7.12.6 cells, the anti-CLIP mAb, CerCLIP.1, was used proteins were boiled in Laemmli SDS-PAGE sample buffer with or without for flow cytometry (Fig. 4D). The level of CerCLIP.1 staining was 2-ME, and one-half of the eluate (7.5 ϫ 106 cell equivalents) was used for low for 8.1.6 and high for DM ␤-null 9.5.3 cells. The 7.12.6 mu- each lane. Samples were analyzed for DM chains by Western blotting, as tant also expressed large amounts of CerCLIP.1-reactive surface described for whole cell lysates. class II molecules, indicating accumulation of CLIP. HLA-DP4 Results molecules do not detectably accumulate CLIP in the absence of DM (W. Liu, E. D. M., unpublished results), and HLA-DQ mol- Sequence analysis of HLA-DMA and HLA-DMB cDNA from ecules are expressed at lower levels than DR or DP, suggesting that mutant 7.12.6 cells

the accumulated CLIP was predominantly associated with by guest on September 28, 2021 HLA-DR3 molecules on 7.12.6 cells bind the 16.23 mAb poorly, HLA-DR molecules. When corrected for differences in total and the cells present MHC class II-restricted Ags less well than HLA-DR expression, CLIP accumulation on 7.12.6 cells was 8.1.6 progenitor cells (3). These defects are similar to, but less slightly less than that seen for 9.5.3 (Table II). Together with the pronounced than those found in mutants lacking expression of intermediate 16.23 staining, this result suggested that the mutant HLA-DMA or DMB mRNA. As levels of DMA and DMB mRNA DM molecules in 7.12.6 were capable of releasing at most a small are normal in 7.12.6 (3), we examined whether the structure of amount of CLIP. The shapes of the titration curves obtained using HLA-DM is altered by sequencing PCR-amplified DMA and the anti-CLIP reagent were similar for 9.5.3 and 7.12.6, indicating DMB cDNAs. The 7.12.6 mutant line is derived from 8.1.6 pro- that the antigenic determinants on these cells differed little in af- genitor cells, which carry two copies of the DMA*0101 gene (cf finity (Fig. 4D). Fig. 1C). Sequencing of the entire DMA-coding sequence ampli- Most normally loaded endosomal peptides stabilize DR3 mol- fied from 7.12.6 cDNA revealed no differences from the wild type ecules against dissociation by SDS at room temperature (2). In (data not shown). In contrast, the DMB cDNA contained a single nucleotide substitution changing codon 79 from TGT (Cys) to contrast, complexes with CLIP are dissociated by SDS (2, 8, 39). TAT (Tyr) (3) (Fig. 2). The remainder of the sequence was iden- To assess whether DR3 molecules in 7.12.6 cells were poorly tical to the DMB*0101 sequence found in 8.1.6 (22) (data not loaded with stabilizing peptides, DR molecules from 8.1.6, 9.5.3, shown). and 7.12.6 cells were assayed for SDS stability by Western blotting of whole cell lysates (Fig. 5). As expected, DM wild-type Restoration of 16.23 binding in 7.12.6 cells by transfection 8.1.6 cells expressed mostly SDS-stable DR3 complexes, whereas of wild-type DMB HLA-DR molecules from DM-null cells were mostly SDS unsta- To test whether the Cys793Tyr mutation in DMB of 7.12.6 cells ble. The SDS stability of HLA-DR molecules from 7.12.6 cells was the cause of the peptide-loading defect, we introduced a wild- was intermediate between 8.1.6 and 9.5.3, consistent with a partial type DMB gene into 7.12.6 cells and examined 16.23 staining as defect in peptide loading. The 7.12.6 cells contained only a small the prototype marker for DM function and normal peptide loading fraction of SDS-stable molecules, as shown by the observation that (Fig. 3). When either 7.12.6 or DM␤-null cells were transfected the intensity of the monomer band did not increase substantially with wild-type DMB cDNA, expression of 16.23-reactive DR mol- after boiling (the ability to detect a rather intense dimer band in ecules was increased to levels comparable with those found in the 7.12.6 is deceptive in this regard, as the mAb used prefers dimeric DM-wt cell, 8.1.6. In addition, staining of 7.12.6 cells with a mAb molecules; E. D. M., unpublished data). The same order of SDS specific for DR3:CLIP complexes was reduced dramatically after stability (8.1.6 ϾϾ 7.12.6 Ͼ 9.5.3) was observed by immunopre- transfection with wild-type DMB (E. v. S., E. D. M., unpublished cipitation of HLA-DR3 complexes from biosynthetically labeled data). These observations strongly suggested that the peptide-load- cells after an overnight chase (data not shown). Together with ing defect in 7.12.6 cells was due to the Tyr79 mutation in DMB. other assays of DM function, the reduced SDS stability indicated 738 ALTERED DISULFIDE BONDING IN A Cys MUTANT OF HLA-DMB Downloaded from

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 http://www.jimmunol.org/ 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. FIGURE 3. Transfection of wild-type DMB into 7.12.6 cells restores Similar results were obtained in two independent titrations and several 16.23 expression. 7.12.6 cells were transiently transfected with empty additional experiments done at single mAb concentrations. vector (pREP4) or with the same vector carrying a wild-type DMB insert (pREP4-DMB). After drug selection, 7.12.6 transfectants were Table II. 16.23 and CerCLIP.1 binding of EBV B cell mutants compared with similarly transfected DM-wt and DMB-null cells (8.1.6 corrected for differences in HLA-DR expression and 9.5.3, respectively) for binding of 16.23. Bound Ab was quanti- by guest on September 28, 2021 tated by incubation with goat anti-mouse Ig FITC and flow cytometry. Median fluorescence ratioa (%) Background fluorescence and L243 staining were comparable between the lines. Similar results were obtained in four experiments done 16.23 CerCLIP.1 16.23 CerCLIP.1 after two separate transfections. Cell Line L243 L243 ISCR3 ISCR3

8.1.6 45.6 6.1 46.9 6.2 9.5.3 5.3 73.3 6.8 93.0 that HLA-DR3 molecules in 7.12.6 cells have a defect in the en- 7.12.6 10.4 54.6 15.7 82.1 dosomal release of CLIP and in the acquisition of a normal com- a plement of antigenic peptides. Furthermore, DM function appears Median fluorescence intensities at saturating mAb concentrations from the experiment shown in Figure 2 were used to determine ratios, which were ex- to be greatly reduced, but not completely abolished, by the mutation. pressed as a percentage. Similar results were obtained in two independent experiments. 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 some DR alleles complexed with full-length Ii, seen early after 7.12.6 cells, HLA-DR3 synthesis, trafficking, and processing were synthesis (40). In contrast, accumulation and release of CLIP dif- analyzed by pulse-chase labeling (Fig. 6). HLA-DR molecules fered between DM wild-type and mutant cells. In 8.1.6 progenitor were immunoprecipitated and digested with Endo H to distinguish cells, small amounts of CLIP were detectable between 3 and7hof DR molecules that have traversed the medial Golgi apparatus chase, whereas in 9.5.3 and 7.12.6 mutants, CLIP accumulated to (Endo H-resistant ␤-chain, partially resistant ␣-chain) from those significantly higher levels. In both mutants, accumulation of CLIP that have not (both chains Endo H sensitive). was paralleled by the decay of class II-associated p22- and p10- All three cell lines synthesized comparable levels of HLA-DR3 processing fragments of Ii, suggesting that these fragments are molecules during the 30-min labeling period (Fig. 6, 0-h chase), precursors of CLIP. We concluded that the failure to release CLIP and the early steps of DR3 maturation and processing were similar is the earliest detectable difference in class II maturation between between the cells. DR3 molecules initially were fully sensitive to wild-type cells and the 7.12.6 mutant. 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 Abundance and trafficking of mutant DMB in 7.12.6 cells apparatus. The endosomal Ii-processing intermediates, p22Ii and We hypothesized that the DMB point mutation could diminish DM p10Ii, were undetectable until 0.5 h of chase, peaked at 3 h, and function by destabilizing the native conformation of the DM mol- decayed thereafter. At3hofchase, the amount of L243-reactive ecule, thus shortening its lifetime in the cell. To address this pos- DR3 increased above the levels seen in the pulse; this was con- sibility, steady state DM levels in the different cell lines were com- sistent with previous results showing that L243 reacts poorly with pared by Western blotting (Fig. 7A). Wild-type DM ␤-chain was The Journal of Immunology 739

implying that mutant molecules were retained in pre-Golgi compartments, most likely the ER and/or cis-Golgi reticulum. DM ␤-chains isolated from the DM ␣-null mutant, 2.2.93 (cf Fig. 1C), served as a positive control for Endo H digestion and were deglycosylated completely under these conditions. We concluded that all unpaired ␤-chains in 2.2.93, as well as a large majority of the point-mutated DM ␤-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. FIGURE 5. Intermediate SDS stability of HLA-DR3 molecules in 5 To determine whether the reduced expression of the mutant DM 7.12.6 cells. Lysates (5 ϫ 10 cell equivalents) of 8.1.6, 7.12.6, and 9.5.3 cells were resolved by nonreducing SDS-PAGE with or without molecule was due to decreased synthesis or increased turnover, boiling (lanes labeled b or nb, respectively), and DR3 ␣␤ dimers and and to compare rates of trafficking, pulse-chase analysis was per- dissociated ␤-chains were detected by Western blotting using the DR formed (Fig. 8). DM molecules were immunoprecipitated using a ␤-chain-specific mAb, B10.a. The experiment was performed twice polyclonal rabbit anti-DM antiserum, digested with Endo H, and with similar results. analyzed by SDS-PAGE. After a 30-min pulse, the antiserum pre- Downloaded from cipitated two chains of the expected m.w. from both 8.1.6 and 7.12.6 cells, with the ␤-chain band being more intense. As ex- detected as an intense, specific 29-kDa band in 8.1.6 cell, but not pected, the ␣- and ␤-chain bands were absent from the single 9.5.3 lysates. In 7.12.6, the amount of mutant DM ␤-chains was chain-deficient mutants, 2.2.93 and 9.5.3, respectively, demon- reduced by approximately fivefold compared with wild type (17 strating that the antiserum specifically recognizes both chains of and 22% of wild type in two independent quantitations). The abun- DM. In all cells, newly synthesized DM chains were sensitive to dance of DM ␣-chains was reduced by a similar amount (data not Endo H digestion, indicating that most of the DM molecules made http://www.jimmunol.org/ shown). during the 30-min labeling period had not yet reached the medial To examine the subcellular localization of the mutant DM mol- Golgi apparatus. In 8.1.6 cells, a substantial fraction of the newly ecules, DM from wild-type and mutant cells were analyzed for synthesized wild-type DM molecules became resistant to Endo H Endo H resistance, a criterion commonly used to measure export of digestion after a 45-min chase, indicating passage through the MHC molecules out of the ER past the medial Golgi apparatus (cf Golgi apparatus. Acquisition of Endo H resistance was nearly Fig. 6). Almost all of the wild-type DM molecules isolated from complete by3hofchase, although one of the two glycans on DM 8.1.6 cells were resistant to Endo H (Fig. 7B), consistent with ␣ remained sensitive throughout the chase (as expected from Ref.

accumulation in post-Golgi class II-rich prelysosomal compart- 5). The wild-type molecules persisted up until 27 h of chase. In by guest on September 28, 2021 ments (E. Stang, C. Guerra, M. A. Amaya, Y. Paterson, O. Bakke, contrast, single DM chains in 9.5.3 and 2.2.93 cells were turned and E. D. M., submitted). By contrast, only a small proportion of over completely by6hofchase and remained Endo H sensitive, the mutant DM ␤-chains in 7.12.6 cells was Endo H resistant, suggesting that they were degraded without traversing the medial

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 ϩ Endo H), or mock digested (lanes labeled Ϫ 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 CЈ are control immunoprecipitations with irrelevant Ab at0hofchase. Note that DR molecules complexed with all Ii isoforms are pre- cipitated 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. 740 ALTERED DISULFIDE BONDING IN A Cys MUTANT OF HLA-DMB

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 sub- populations were degraded by6hofchase. These ﬁndings implied that the native structure of DM was disrupted signiﬁcantly in the mutant.

The Cys3Tyr 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 ␤ cytoplasmic tail-specific mAb and resolved by nonreducing/reducing two-dimensional SDS-PAGE (Fig. 9A). DM precipitates from 8.1.6 cells contained two specific ␣ ␤ spots with the expected m.w. of DM - and -chains (35 and 29 FIGURE 7. Reduced steady state levels and altered subcellular lo- kDa, respectively) under reducing conditions. Both chains mi- calization of mutant DM in 7.12.6 cells. A, Comparison of wild-type Downloaded from grated to the right of the diagonal, i.e., slightly faster under non- and mutant DM levels. Nonidet P-40 lysates of 106 8.1.6, 9.5.3, and reducing than under reducing conditions, indicating that both 7.12.6 cells, or mixtures of 8.1.6 and 9.5.3 lysates containing the in- chains contained intramolecular disulfide bonds and that chain as- dicated cell equivalents of 8.1.6 while keeping the total cell number sociation was noncovalent. In 7.12.6 precipitates, monomeric DM constant, were boiled under reducing conditions and analyzed for DM ␤-chain was also seen, but monomeric ␣-chain was not coprecipi- ␤-chains by Western blotting. Mutant DM levels were about 20% of tated detectably under these conditions. This result indicated that wild type, as determined by densitometric quantitation of band inten- noncovalent heterodimers were destabilized or formed in smaller sities. Similar results were obtained in two independent experiments. http://www.jimmunol.org/ B, ER retention of mutant DM. Glycoprotein preparations from the amounts in the 7.12.6 mutant. To the left of the diagonal, vertically indicated cell lines were digested with Endo H or mock digested as aligned 29- and 35-kDa spots were observed at a nonreducing shown, and DM ␤-chains were detected by Western blotting. The po- m.w. of 56 kDa, showing covalent heterodimerization (the nonre- sitions of undigested and deglycosylated DM ␤-chains are shown (␤ duced m.w. are somewhat less than the sum of the reduced subunit and ␤s, respectively). The film was overexposed to reveal minor sub- m.w., probably due to deviations from an ideal rod shape in the populations in the mutant cells. The experiment was performed three disulfide-bonded dimer). Furthermore, a 29-kDa spot without other times with similar results. by guest on September 28, 2021

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 ␤-chain position in 9.5.3 immunoprecipitates, but unlike DM or class II ␤-chains, it was resistant to Endo H. It was much weaker than the DM ␤ band in the other cells and did not interfere with DM ␤ identiﬁcation. Similar results were obtained in four separate experiments, but the amount of Endo H-resistant mutant DM in 7.12.6 cells at3hofchase was variable. The Journal of Immunology 741

erodimer with DM ␣. We conclude that both correct pairing of DM ␤ with DM ␣ and the Cys79␤ residue are important for maintain- ing the wild-type disulﬁde bond arrangement of DM ␤.

Discussion 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 mu- Downloaded from tagenized 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 ␤. http://www.jimmunol.org/ FIGURE 9. Chain pairing and disulfide bonding of mutant DM in The reduced peptide exchange in 7.12.6 cells can be attributed 7.12.6 cells. A, Nonreducing/reducing two-dimensional SDS-PAGE chiefly to the decreased abundance of mutant DM in post-Golgi analysis of biosynthetically labeled DM ␤-chain immunoprecipitates compartments. Steady state levels of mutant DM are about one- ␣ from 8.1.6 and 7.12.6 cells. Positions of DM monomeric - and fifth of wild type due to increased turnover, and export from the ␤-chains are indicated by arrowheads, the covalent ␣-␤ dimer is in- ER is delayed. Together, this results in at least a 20-fold decrease dicated by arrows, and the ␤-␤ homodimer with an open arrowhead. The diagonal, as well as the vertical set of spots to the right of the dimer of DM levels in post-Golgi compartments, such as the prelysoso- region and the diffuse background above it, were also found in immu- mal compartments to which wild-type DM travels in 8.1.6 progen- noprecipitates from 9.5.3 cells (not shown). Note that even though the itor cells (E. Stang, C. Guerra, M. A. Amaya, Y. Paterson, O.

␤-chain spots in the monomer and heterodimer populations of 7.12.6 Bakke, and E. D. M., submitted) and in other EBV-B cells (31). It by guest on September 28, 2021 were of similar intensity, an ␣-chain spot could only be detected in the will be interesting to elucidate the mechanisms that control turn- covalent heterodimer, indicating that the efficiency of noncovalent as- over of wild-type DM, single DM chains, and mutated DM in the sociation was poor. B, Western blotting analysis of altered disulfide ER and in endosomes. Even for wild-type cells, the endosomal bonding and trafficking of DM ␤ in mutants. Glycoprotein preparations concentration of DM is on the order of five- to tenfold less than from the indicated cells were boiled under nonreducing or reducing conditions, as shown, and analyzed for the migration of DM ␤-chains that of DR molecules (41) (W. Liu, E. D. M., unpublished data). by Western blotting. Shown are the positions of dissociated ␤-chains, Our observations showing that an even smaller amount of mutant covalent ␤-␤ homodimers, and covalent ␣-␤ heterodimers. The exper- DM in endosomes of 7.12.6 mediates detectable peptide loading iment was performed three times with similar results. C, The covalent are consistent with the view that DM can function catalytically in dimer in 7.12.6 cells contains DM ␣. Glycoprotein preparations from the cell. However, an additional chaperone function for DM is not 7.12.6 and from the DM ␣-null mutant 2.2.93 were analyzed by non- ruled out by our data. ␣ reduced SDS-PAGE, and DM -chains were detected by Western blot- ER retention and accelerated turnover are hallmarks of recog- ting. *Indicates an unknown protein that was detected nonspecifically nition by the ER quality control apparatus (42) and suggest mal- by 5C1 in the negative control cells. folding of the mutant molecules in 7.12.6. The observation that disulfide bonding of DM ␤ is strikingly and specifically changed in vertically aligned spots was seen at a nonreducing m.w. of 47 kDa, the mutants provides direct evidence for such conformational ab- consistent with homodimerization of DM ␤. errations. Wild-type DM predominantly forms a noncovalent ␣ ␤ The chain composition of the different disulfide-bonded forms dimer, with both DM - and -chains containing intramolecular was confirmed by Western blotting of nonreducing gels using disulfide bonds, as shown by off-diagonal migration in nonreduc- chain-specific Abs. The anti-DM ␤ mAb detected the monomeric ing/reducing two-dimensional PAGE analysis. In the absence of ␣ ␤ DM ␤-chain in glycoprotein preparations from all cells except the -chain expression, about one-half of the -chains are monomeric DM ␤-null cell, 9.5.3 (Fig. 9B, lanes labeled Ϫ Endo H). In ad- under nonreducing conditions, while the other half forms a cova- dition, it detected a 47-kDa band (probably the ␤-␤ homodimer) in lent ␤-␤ monomer. In the ER of 7.12.6 cells, monomeric and ho- 7.12.6 and 2.2.93 cells, and a 56-kDa band in 7.12.6. The latter modimerized mutant DM ␤-chains are also detected, but the most band was also detected by probing blots of anti-DM ␤ immuno- abundant species is a covalent ␣-␤ heterodimer. The efficiency of precipitates with a mAb against DM ␣ (Fig. 9C), confirming that noncovalent pairing with DM ␣ is reduced by the mutation. We this band represented a covalent ␣-␤ heterodimer. Together, these have found no evidence for formation of higher order oligomers of results indicate that wild-type DM exists as a noncovalent dimer DM chains, or of nonspecific disulfide-bonded aggregates with with intramolecular disulfide bonds, that a significant proportion of other proteins, suggesting that covalent chain pairing is specific in DM ␤-chain can homodimerize in the absence of DM ␣, and that both 7.12.6 and 2.2.93 cells, despite the presence of substantial the mutant DM ␤-chain in 7.12.6 cells can form a covalent het- amounts of other nascent proteins in the ER (42). 742 ALTERED DISULFIDE BONDING IN A Cys MUTANT OF HLA-DMB

Even the fraction of mutant DM molecules in 7.12.6 cells that they may represent aberrant products that can accumulate only can escape ER retention is degraded rapidly in post-Golgi com- when normal folding requirements are not met. partments. This behavior differs strikingly from that of the wild- type molecule, which persists for at least 27 h in highly degrada- Acknowledgments tive late endocytic compartments. Thus, the conformation of the We thank Miguel Amaya and Michael Riley for initial flow-cytometric post-Golgi fraction of mutant DM also appears to be destabilized experiments using 7.12.6; Drs. P. Cresswell, S. Pierce, D. Zaller, and J. or altered compared with the wild type, allowing better targeting Trowsdale for their kind gifts of Abs; Drs. S. Fling and D. Pious for the gift for post-Golgi degradation or increased exposure of proteolytic of 2.2.93 cells; and Drs. R. Kopito and C. Clayberger for critical review of sites. While the escaping subpopulation of mutant DM may be the manuscript. more native-like than the population that is retained, this result implies that ER quality control is inefficient in that it does not References perfectly discriminate between functionally normal and altered 1. Busch, R., and E. D. Mellins. 1996. Developing and shedding inhibitions: how DM molecules. MHC class II molecules reach maturity. Curr. Opin. Immunol. 8:51. In addition to the weak sequence homology, several lines of 2. Mellins, E., L. Smith, B. Arp, T. Cotner, E. Celis, and D. Pious. 1990. Defective processing and presentation of exogenous antigens in mutants with normal HLA evidence are consistent with a structure for DM that resembles that class II genes. Nature 343:71. of Ag-presenting MHC class I and class II proteins. Wild-type 3. Morris, P., J. Shaman, M. Attaya, M. Amaya, S. Goodman, C. Bergman, DM, like conventional class II molecules, only contains intrachain J. J. Monaco, and E. Mellins. 1994. An essential role for HLA-DM in antigen presentation by class II major histocompatibility molecules. Nature 368:551. disulfide bonds, despite the presence of five additional cysteines

4. Fling, S. P., B. Arp, and D. Pious. 1994. HLA-DMA and -DMB genes are both Downloaded from unique to DM. Furthermore, analyses of cysteine mutants strongly required for MHC class II/peptide complex formation in antigen-presenting cells. suggest that the ␤ -domain cysteines, residues 11 and 79, which Nature 368:554. 1 5. Denzin, L. K., N. F. Robbins, C. Carboy-Newcomb, and P. Cresswell. 1994. are conserved among MHC molecules, are paired in wild-type Assembly and intracellular transport of HLA-DM and correction of the class II DM. Mutating Cys79␤ of DM delays its export from the ER and antigen-processing defect in T2 cells. Immunity 1:595. 6. Riberdy, J. M., J. R. Newcomb, M. J. Surman, J. A. Barbosa, and P. Cresswell. diminishes its stability and function, implying that this cysteine is 1992. HLA-DR molecules from an antigen-processing mutant cell line are asso- important for structural integrity. The formation of aberrant disul- ciated with invariant chain peptides. Nature 360:474. ﬁde bond arrangements upon mutating Cys79␤ provides more di- 7. Sette, A., S. Ceman, R. T. Kubo, K. Sakaguchi, E. Appella, D. F. Hunt, http://www.jimmunol.org/ T. A. Davis, H. Michel, J. Shabanowitz, R. Rudersdorf, H. M. Grey, and rect evidence that this residue is normally involved in a disulﬁde R. DeMars. 1992. Invariant chain peptides in most HLA-DR molecules of an bond. The idea that residue 11␤ is the partner cysteine for Cys79␤ antigen-processing mutant. Science 258:1801. is supported by the observation that mutating residue 11 to Tyr 8. Mellins, E., P. Cameron, M. Amaya, S. Goodman, D. Pious, L. Smith, and B. Arp. 1994. A mutant human histocompatibility leukocyte antigen DR mole- results in a similar partial peptide-loading defect (E. v. S., unpub- cule associated with invariant chain peptides. J. Exp. Med. 179:541. lished). The 7.12.6 phenotype can be partially corrected by cul- 9. Miyazaki, T., P. Wolf, S. Tourne, C. Waltzinger, A. Dierich, N. Barois, H. Ploegh, C. Benoist, and D. Mathis. 1996. Mice lacking H-2 M complexes, turing the cells at reduced temperature (M. Riley, M. Amaya, E. v. enigmatic elements of the MHC class II peptide-loading pathway. Cell 84:531. S., E. D. M., unpublished). This suggests that folding and transport 10. Fung-Leung, W.-P., C. D. Surh, M. Liljedahl, P. J. D. Leturcq, P. A. Peterson, S. R. Webb, and L. Karlsson. 1996. Antigen presentation and T cell development

of mutant DM are temperature sensitive, as is observed for intra- by guest on September 28, 2021 in H2-M-deficient mice. Science 271:1278. cellular transport of viral glycoprotein mutants with disrupted di- 11. Martin, W. D., G. G. Hicks, S. K. Mendiratta, H. I. Leva, H. E. Ruley, and sulfide bonds (43). Interestingly, mutating a cysteine in the homol- L. V. Kaer. 1996. H2-M mutant mice are defective in the peptide loading of class ogous ␣ -domain disulfide bond of the classical class I molecule, II molecules, antigen presentation, and T cell repertoire selection. Cell 84:543. 2 12. Sloan, V. S., P. Cameron, G. Porter, M. Gammon, M. Amaya, E. Mellins, and HLA-A*0201, also results in a partial functional defect and de- D. M. Zaller. 1995. Mediation by HLA-DM of dissociation of peptides from layed ER-to-Golgi transport (44). HLA-DR. Nature 375:802. 13. Sherman, M. A., D. A. Weber, and P. E. Jensen. 1995. DM enhances peptide The altered disulfide-bonding patterns in 2.2.93 and 7.12.6 cells binding to class II MHC by release of invariant chain-derived peptide. Immunity show that both DM ␣ and Cys79 of the ␤-chain contribute to 3:197. 14. Denzin, L. K., and P. Cresswell. 1995. HLA-DM induces CLIP dissociation from proper folding, assembly, and disulfide bonding of DM. The ob- ␣␤ ␣ ␤ MHC class II dimers and facilitates peptide loading. Cell 82:155. servation that the -chain is associated with mutated DM in 15. Sanderson, F., C. Thomas, J. Neefjes, and J. Trowsdale. 1996. Association be- 7.12.6 (albeit mostly covalently) shows that Cys79␤ is not abso- tween HLA-DM and HLA-DR in vivo. Immunity 4:87. lutely required for the chains to associate specifically with one 16. Denzin, L. K., C. Hammond, and P. Cresswell. 1996. HLA-DM interactions with intermediates in HLA-DR maturation and a role for HLA-DM in stabilizing another. Rather, its role seems to be to maintain a stable, proteo- empty HLA-DR molecules. J. Exp. Med. 184:2153. lytically resistant and transport-competent conformation of the het- 17. Kropshofer, H., S. O. Arndt, G. Moldenhauer, G. J. Ha¨mmerling, and A. B. Vogt. 1997. HLA-DM acts as a molecular chaperone and rescues empty HLA-DR mol- erodimer and to suppress interchain disulfide bonding, possibly by ecules at lysosomal pH. Immunity 6:1. competing with ␣-chain cysteines for pairing with Cys11␤. Inde- 18. Kropshofer, H., G. J. Ha¨mmerling, and A. B. Vogt. 1997. How HLA-DM edits pendently, DM ␣ contributes to the suppression of aberrant disul- the MHC class II peptide repertoire: survival of the fittest? Immunol. Today ␤ 18:77. fide bonds in DM . This is shown by the observation that wild- 19. Kropshofer, H., A. B. Vogt, G. Moldenhauer, J. Hammer, J. S. Blum, and G. J. type DM ␤ can form covalent homodimers in the absence of DM Ha¨mmerling. 1996. Editing of the HLA-DR-peptide repertoire by HLA-DM. ␣ in 2.2.93 cells. This observation may be surprising in view of the EMBO J. 15:6144. 20. Weber, D. A., B. D. Evavold, and P. E. Jensen. 1996. Enhanced dissociation of fact that chain pairing of conventional class II molecules generally HLA-DR-bound peptides in the presence of HLA-DM. Science 274:618. results in heterodimers and is isotype and even haplotype specific; 21. Van Ham, S. M., U. Gru¨neberg, G. Malcherek, I. Bro¨ker, A. Melms, and J. Trowsdale. 1996. Human histocompatibility leukocyte antigen (HLA)-DM ed- however, homodimerization is not unusual for Ig superfamily pro- its peptides presented by HLA-DR according to their ligand binding motifs. teins in general, and self-association of soluble DM ␤-chains ex- J. Exp. Med. 184:2019. pressed in insect cells has been reported (12). As MHC class II-like 22. Kelly, A. P., J. J. Monaco, S. Cho, and J. Trowsdale. 1991. A new human HLA class II-related locus, DM. Nature 353:571. heterodimers may have evolved from a homodimeric ancestor 23. Sanderson, F., S. H. Powis, A. P. Kelly, and J. Trowsdale. 1994. Limited poly- (45), and as DM diverged early from Ag-presenting MHC class I morphism in HLA-DM does not involve the peptide binding groove. Immuno- and class II molecules (27), the specific covalent dimerization of genetics 39:56. 24. Hermel, E., J. Yuan, and J. J. Monaco. 1995. Characterization of polymorphism DM␤ in 2.2.93 might be a vestigial phenomenon. The relevance of within the H2-M MHC class II loci. Immunogenetics 42:136. the unusual disulfide-bonded ␣-␤ and ␤-␤ dimers for normal fold- 25. Carrington, M., and A. Harding. 1994. Sequence analysis of two novel HLA- DMA alleles. Immunogenetics 40:165. ing of wild-type DM remains to be elucidated. Covalent dimers 26. Carrington, M., M. Yeager, and D. Mann. 1993. Characterization of HLA-DMB may occur as transient intermediates during normal folding, or polymorphism. Immunogenetics 38:446. The Journal of Immunology 743

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