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Class I Molecules and Heavy Chains in the Assembly of MHC Distinct Distinct Functions for the Glycans of Tapasin and Heavy Chains in the Assembly of MHC Class I Molecules This information is current as Syed Monem Rizvi, Natasha Del Cid, Lonnie Lybarger and of September 28, 2021. Malini Raghavan J Immunol 2011; 186:2309-2320; Prepublished online 24 January 2011; doi: 10.4049/jimmunol.1002959 http://www.jimmunol.org/content/186/4/2309 Downloaded from References This article cites 44 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/186/4/2309.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • 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 by guest on September 28, 2021 *average 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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Distinct Functions for the Glycans of Tapasin and Heavy Chains in the Assembly of MHC Class I Molecules Syed Monem Rizvi,* Natasha Del Cid,*,† Lonnie Lybarger,‡ and Malini Raghavan* Complexes of specific assembly factors and generic endoplasmic reticulum (ER) chaperones, collectively called the MHC class I peptide-loading complex (PLC), function in the folding and assembly of MHC class I molecules. The glycan-binding chaperone calreticulin (CRT) and partner oxidoreductase ERp57 are important in MHC class I assembly, but the sequence of assembly events and specific interactions involved remain incompletely understood. We show that the recruitments of CRT and ERp57 to the PLC are codependent and also dependent upon the ERp57 binding site and the glycan of the assembly factor tapasin. Furthermore, the ERp57 binding site and the glycan of tapasin enhance b2m and MHC class I heavy (H) chain recruitment to the PLC, with the ERp57 binding site having the dominant effect. In contrast, the conserved MHC class I H chain glycan played a minor role in CRT recruitment into the PLC, but impacted the recruitment of H chains into the PLC, and glycan-deficient H chains were impaired Downloaded from for tapasin-independent and tapasin-assisted assembly. The conserved MHC class I glycan and tapasin facilitated an early step in the assembly of H chain–b2m heterodimers, for which tapasin–ERp57 or tapasin–CRT complexes were not required. Together, these studies provide insights into how PLCs are constructed, demonstrate two distinct mechanisms by which PLCs can be stabilized, and suggest the presence of intermediate H chain-deficient PLCs. The Journal of Immunology, 2011, 186: 2309–2320. he MHC class I molecule is a heterotrimer composed of (3). An oligosaccharide structure containing three glucose resides http://www.jimmunol.org/ a heavy (H) chain, a L chain (b2-microglobulin [b2m]), (Glc3Man9GlcNAc2) is initially attached to asparagine residues T and an 8- to 9-residue peptide. MHC class I H chain on newly synthesized glycoproteins. This oligosaccharide struc- assembly with b2m and peptide is facilitated by a multiprotein ture is modified to a monoglucosylated form by glucosidases I and peptide-loading complex (PLC) composed of the TAP, an as- II, which allows for recognition by calnexin and CRT. Following sembly factor tapasin, the thiol oxidoreductase ERp57, and the correct folding, the glycoprotein is deglucosylated by glucosidase endoplasmic reticulum (ER) chaperones calreticulin (CRT) and II, resulting in release from calnexin and CRT (4). Previous calnexin (1). TAP is an ATP-binding cassette transporter involved studies have shown that MHC class I binding to CRT and TAP is in translocation of peptides from the cytosol into the ER. Tapasin impacted by blocking glucosidases I and II with the inhibitor acts as a bridge between the PLC components and TAP, and castanospermine, or by inducing MHC class I deglycosylation by by guest on September 28, 2021 facilitates loading of high-affinity peptides onto MHC class I point mutations of a highly conserved N-linked glycosylation site molecules. Following loading of high-affinity peptides, MHC at asparagine 86 (5–7). class I molecules dissociate from the PLC and are transported to CRT plays a more critical role in MHC class I assembly than the cell surface for recognition by CD8+ T cells (1). calnexin (8, 9), with CRT deficiency resulting in reduced cell The specific function of each protein in MHC class I assembly surface MHC class I (10, 11), enhanced intracellular trafficking and the sequence of assembly events are not completely unders- rates of MHC class I molecules (10, 11), and reduced steady-state tood. MHC class I folding is facilitated by the lectin chaperones levels of MHC class I H chains and tapasin (12). We recently calnexin and CRT. Calnexin and CRT typically interact with mo- showed that the glycan and ERp57 binding sites of CRT are im- noglucosylated glycans on substrate glycoproteins via a carbohy- portant for its recruitment into the PLC and for the assembly- drate-binding site present within a globular domain (2). Calnexin promoting functions of CRT. These studies suggested that CRT and CRT also interact with their partner ER oxidoreductase ERp57 binding to the glycans of MHC class I or tapasin or both mole- via an elongated b-stranded hairpin structure called the P domain cules could be involved in the recruitment of CRT into the PLC (12). Within the PLC, ERp57 is recruited to tapasin via a disulfide- *Department of Microbiology and Immunology, University of Michigan Medical linked interaction between C57 of ERp57 and C95 of tapasin (13), School, Ann Arbor, MI 48109; †Graduate Program in Immunology, University of and mutation of tapasin C95 abrogates tapasin–ERp57 binding Michigan Medical School, Ann Arbor, MI 48109; and ‡Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ 85724 in human cells (13–16) and reduces the efficiency of CRT re- cruitment to the PLC (14, 16). Because CRT and ERp57 are able Received for publication September 7, 2010. Accepted for publication December 9, 2010. to interact independently of other PLC components (3), P domain– This work was supported by National Institutes of Health Grants AI044115 and dependent binding between CRT and ERp57 could serve as a point AI066131 (to M.R.), by the University of Michigan Rheumatic Diseases Core Center, for the recruitment of CRT into the PLC, in addition to a glycan and by the University of Michigan Diabetes Research and Training Center. within the PLC (MHC class I and/or tapasin). To better understand Address correspondence and reprint requests to Dr. Malini Raghavan, Department of the interactions and functions mediated by the glycans of tapasin Microbiology and Immunology, 5641 Medical Science Building II, University of Mich- igan Medical School, Ann Arbor, MI 48109-5620. E-mail address: [email protected] and MHC class I molecules, in this study we used tapasin and H Abbreviations used in this article: CRT, calreticulin; DTBP, dimethyl 3,39 dithiobis- chain glycan mutants to show that the glycan of tapasin influences propionimidate 2HCl; ER, endoplasmic reticulum; HA, hemagglutinin; LIC, ligation- the recruitment of CRT into the PLC. In contrast, the conserved independent cloning; b2m, b2-microglobulin; MFI, mean fluorescence intensity; MHC class I glycan plays a minor role in CRT recruitment into the PLC, peptide-loading complex; RT, room temperature. PLC, but is important for MHC class I H chain recruitment into Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 the PLC and for the assembly of MHC class I H chain–b2m www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002959 2310 TAPASIN’S GLYCAN FACILITATES MHC CLASS I ASSEMBLY complexes. These studies provided new insights into intermediate untagged HLA-B*3503 in the MSCV-neo vectors using previously de- complexes of the MHC class I assembly pathway and suggest that scribed procedures (16, 20). A total of 5.5 mg retroviral vectors encoding intermediate complexes can function as a platform for peptide ex- tapasin, HA–HLA-A2, HA–HLA-A2(N86Q), HLA-B*3503, or control empty vectors was mixed with 4 mg pCL-Eco DNA and 0.5 mg VSV-G change. encoding plasmid, and added to a mixture of Opti-MEM (Invitrogen) and FuGENE 6 (Roche). Following incubation for 20 min at RT, the mixtures were added to BOSC cells that had been grown to 70% confluency in a 10- Materials and Methods cm tissue culture dish. Media were changed after 24 h, and after 48 h, Cell lines supernatants containing retroviruses were harvested, filtered, and used to A human melanoma cell line M553 (17) (obtained from N. Bangia, Roswell infect M553 or 721.221 cells. Infected cells were selected by treatment Park Cancer Institute) and 721.221 (18) (obtained from R. DeMars, Uni- with 1 mg/ml puromycin (Sigma-Aldrich) or 1 mg/ml geneticin (Invi- versity of Wisconsin-Madison) were grown in RPMI 1640 (Life Tech- trogen) and maintained in 0.5 mg/ml puromycin or 0.5 mg/ml geneticin. nologies). BOSC cells (obtained from K. Collins, University of Michigan) After verifying HA–HLA-A2 and HA–HLA-A2(N86Q) expression by flow (19) were grown in DMEM (Life Technologies). All growth media were cytometric analyses and by immunoblotting analyses of cell lysates with supplemented with 10% (v/v) FBS (Life Technologies), 100 mg/ml anti-HA (Covance), M553/A2 cells were transduced with the tapasin- streptomycin, and 100 U/ml penicillin (Life Technologies).
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