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 References This article cites 51 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/160/2/734.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 28, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 endo- plasmic 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 b-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 ab dimers, such dimers form inefficiently in 7.12.6; many mutant DM b-chains instead form a disulfide-bonded dimer with DM a. Homodimers of DM b are also detected in 7.12.6 and in the a-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 Cys79b and by noncovalent contacts with DM a. Without these interactions, DM b 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- 1 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- a b tants, synthesis of DM - and/or -chains is defective, and transfec- coprotein consisting of a 35-kDa a-chain and a 29-kDa b-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 b 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- a b 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] b a 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, cys- teine; 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). Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 735 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 res- idues in the extracytoplasmic domains of DM a- and b-chains (22). The Cys79b residue mutated in 7.12.6 is shown in bold. Horizontal lines in- dicate disulfide bonds inferred from homology to Ig superfamily domains; the dashed horizon- tal line indicates a disulfide bond inferred from homology to the MHC Ag binding domain, re- sults shown in this work, and unpublished data (E.v.S.). B, Predicted location of the putative di- sulfide bond between cysteines b11 and b79, mapped on the crystal structure of HLA-DR1 Downloaded from (where the homologous bond is formed be- tween cysteines 15 and 79 (51)). C, Derivation and simplified genomic MHC class II maps of mutant cells used in this study. All cells are de- rived 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 hap- lotype, 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). ab 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 endo- cytic compartments, which in EBV-transformed B cells have char- Name Reactivity Ref.