A Single Polymorphic Residue Within the Peptide-Binding Cleft of MHC Class I Molecules Determines Spectrum of Tapasin Dependence This information is current as of October 2, 2021. Boyoun Park, Sungwook Lee, Euijae Kim and Kwangseog Ahn J Immunol 2003; 170:961-968; ; doi: 10.4049/jimmunol.170.2.961 http://www.jimmunol.org/content/170/2/961 Downloaded from References This article cites 30 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/170/2/961.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 October 2, 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 Errata An erratum has been published regarding this article. Please see next page or: /content/170/9/4869.2.full.pdf 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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology A Single Polymorphic Residue Within the Peptide-Binding Cleft of MHC Class I Molecules Determines Spectrum of Tapasin Dependence1 Boyoun Park, Sungwook Lee, Euijae Kim, and Kwangseog Ahn2 Different HLA class I alleles display a distinctive dependence on tapasin for surface expression and Ag presentation. In this study, we show that the tapasin dependence of HLA class I alleles correlates to the nature of the amino acid residues present at the naturally polymorphic position 114. The tapasin dependence of HLA class I alleles bearing different residues at position 114 decreases in the order of acidity, with high tapasin dependence for acidic amino acids (aspartic acid and glutamic acid), moderate dependence for neutral amino acids (asparagine and glutamine), and low dependence for basic amino acids (histidine and argi- nine). A glutamic acid to histidine substitution at position 114 allows the otherwise tapasin-dependent HLA-B4402 alleles to load high-affinity peptides independently of tapasin and to have surface expression levels comparable to the levels seen in the presence Downloaded from of tapasin. The opposite substitution, histidine to glutamic acid at position 114, is sufficient to change the HLA-B2705 allele from the tapasin-independent to the tapasin-dependent phenotype. Furthermore, analysis of point mutants at position 114 reveals that tapasin plays a principal role in transforming the peptide-binding groove into a high-affinity, peptide-receptive conformation. The natural polymorphisms in HLA class I H chains that selectively affect tapasin-dependent peptide loading provide insights into the functional interaction of tapasin with MHC class I molecules. The Journal of Immunology, 2003, 170: 961–968. http://www.jimmunol.org/ fter targeting to the endoplasmic reticulum (ER),3 nas- tapasin dependence and either cellular abundance of the ligands or cent MHC class I H chains associate with a multiprotein their binding affinities for HLA-B2705 molecule (9). Taking these complex that assists their assembly with peptide and ␤ findings and the relative lack of tapasin polymorphism into ac- A 2 ␤ microglobulin ( 2m). This complex includes calnexin, calreticulin, count (10), the MHC class I allele-dependent requirement for ta- ERp57, transporter associated with Ag processing (TAP), and ta- pasin is likely a property of the individual MHC class I alleles. pasin (1). Tapasin is indispensable for proper function of the class Recent studies indicate that the presence of serine rather than I Ag presentation pathway. In tapasin-mutant mice, the expression phenylalanine or tyrosine at position 116 in the HLA-B H chain and stability of surface MHC class I molecules are strongly re- correlates with inefficient association with the assembly complex duced (2). Even though tapasin clearly facilitates Ag presentation and correspondingly high surface expression (11, 12). Similarly, by guest on October 2, 2021 by MHC class I molecules, its precise function in this process has substitution of glutamine to alanine at position 115 of HLA-A2 not been fully elucidated. In addition to the uncertainty regarding causes a lack of association with the loading complex but the mu- the precise function of tapasin, various MHC class I molecules tant proteins are expressed on the cell surface at a level similar to differ in their dependency on tapasin for both efficient surface ex- that of wild-type HLA-A2 H chain (13). On the basis of these pression and presentation of antigenic determinants to CTL (3–8). findings, positions 115 and 116 are suggested to determine the In tapasin-negative 721.220 cells, tapasin is not required for high tapasin dependence of class I surface expression. However, among HLA-B2705 allele (B2705) surface expression or for presentation HLA alleles, glutamine at position 115 is highly conserved, and of viral determinants to CTL (3). In contrast, for the HLA-B4402 position 116 alone cannot explain the phenotypic differences in allele (B4402), functional Ag presentation and surface expression tapasin dependence between B4402 and B2705 or A0301 alleles are highly dependent on tapasin (3). The factors that determine the because they are identical at position 116 with aspartic acid. More- differences in the relative tapasin dependence of various MHC over, the presence of aspartic acid at 116 resulted in either in- class I molecules remain elusive. Analysis of the amino acid se- creased (in A68) or decreased (in B7) binding of tapasin to MHC quence of some ligands that are loaded into B2705 in the presence class I molecules (12, 14). Thus, other positions in the HLA H and the absence of tapasin shows no clear correlation between chain should act as a major determinant of the tapasin dependence of MHC class I molecules. In this study, we have determined the requirements of MHC class I alleles for tapasin dependence. Graduate School of Biotechnology, Korea University, Seoul, Korea Received for publication September 9, 2002. Accepted for publication November 8, 2002. Materials and Methods The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance DNA constructs with 18 U.S.C. Section 1734 solely to indicate this fact. The cDNA encoding human tapasin was subcloned into the pcDNA3.1/ 1 This work was supported by a grant from the 21C Frontier for Functional Genome hygromycin vector (Invitrogen, Carlsbad, CA). Site-directed mutants of Analysis of Human Genome. B2705 with H to D at position 114 (B27H114D), H to N at position 114 2 Address correspondence and reprint requests to Dr. Kwangseog Ahn, Graduate (B27H114N), H to R at position 114 (B27H114R), D to Y at position 116 School of Biotechnology, Korea University, 1, 5-Ga, Anam-Dong, Sungbuk-Gu, (B27D116Y), and the other substitution mutants of B4402 were made by Seoul 136-701, Korea. E-mail address: [email protected] changing the codons by PCR with Pfu DNA polymerase (Stratagene, La 3 ␤ ␤ Abbreviations used in this paper: ER, endoplasmic reticulum; 2m, 2 microglobu- Jolla, CA). All HLA cDNAs and their mutagenized derivatives were in- lin; TAP, transporter associated with Ag processing; endo H, endoglycosidase H. serted into the mammalian expression vector pcDNA3.1 (Invitrogen). Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 962 DETERMINANT FOR TAPASIN DEPENDENCE OF MHC CLASS I MOLECULES Stable cell lines and Abs cross-linked proteins were immunoprecipitated with mAb HLA-B Ab-1. Precipitates were separated by 12% SDS-PAGE and transferred to an im- Tapasin expression was restored in 721.220 cells by transfection with the mobilon-P membrane (Millipore, Bedford, MA). The membrane was in- cDNA-encoding tapasin, and transfectants were selected with 0.2 mg/ml cubated with HRP-conjugated streptavidin for1h4°C, and biotinylated hygromycin, giving rise to the 721.220.Tpn cell line. The HLA-G-specific proteins were visualized by using ECL Western blotting reagent (Pierce). mAb G233 was a gift from Dr. Y. Loke (University of Cambridge, Cam- Peptide translocation was determined after incubating microsomes with bridge, U.K.). Polyclonal rabbit K455 Ab reacts with MHC class I H chains biotin-conjugated reporter peptides in the absence of competitor for 30 min ␤ and 2m in both assembled and nonassembled forms (15). The mouse mAb at 26°C with or without 1 mM ATP. Microsomal membranes were recov- HLA-B Ab-1 (NeoMarkers, Fremont, CA) recognizes only HLA-B alleles ered by centrifugation at 75,000 ϫ g for 10 min through a 0.5 M sucrose ␤ and mAb W6/32 recognizes only the complex of H chain and 2m. The cushion in cold RM buffer. After washing with cold RM buffer twice, the anti-tapasin Ab (R.gp48N) was a gift from Dr. P. Cresswell (Yale Univer- membrane pellet was directly dissolved in sample buffer. The samples were sity, New Haven, CT). FITC-conjugated goat anti-mouse IgG Abs and analyzed on tricine/SDS-PAGE, appropriate for resolution of low mass HRP-conjugated streptavidin were purchased from Jackson ImmunoRe- polypeptides as described (18), and probed with HRP-conjugated strepta- search Laboratories (West Grove, PA) and Pierce (Rockford, IL), vidin. The relative densities of the peptide bands were determined by use respectively. of an imaging densitometer (GS-700; Bio-Rad, Richmond, CA) and Multi- Pulse-chase and immunoprecipitation Analyst densitometer software (Bio-Rad).