title: A single bottleneck in HLA-C assembly.
abstract: Poor assembly of class I major histocompatibility HLA-C heavy chains results in their intracellular accumulation in two forms: free of and associated with their light chain subunit (beta(2)-microglobulin). Both intermediates are retained in the endoplasmic reticulum by promiscuous and HLA-dedicated chaperones and are poorly associated with peptide antigens. In this study, the eight serologically defined HLA-C alleles and the interlocus recombinant HLA-B46 allele (sharing the HLA-C-specific motif KYRV at residues 66-76 of the alpha1-domain alpha-helix) were compared with a large series of HLA-B and HLA-A alleles. Pulse-labeling experiments with HLA-C transfectants and HLA homozygous cell lines demonstrated that KYRV alleles accumulate as free heavy chains because of both poor assembly and post-assembly instability. Reactivity with antibodies to mapped linear epitopes, co-immunoprecipitation experiments, and molecular dynamics simulation studies additionally showed that the KYRV motif confers association to the HLA-dedicated chaperones TAP and tapasin as well as reduced plasticity and unfolding in the peptide-binding groove. Finally, in vitro assembly experiments in cell extracts of the T2 and 721.220 mutant cell lines demonstrated that HLA-Cw1 retains the ability to form a peptide-receptive interface despite a lack of TAP and functional tapasin, respectively. In the context of the available literature, these results indicate that a single locus-specific biosynthetic bottleneck renders HLA-C peptide-selective (rather than peptide-unreceptive) and a preferential natural killer cell ligand.
authors.affiliation: Laboratory of Immunology, Regina Elena National Cancer Research Institute, Centro della Ricerca Sperimentale, Via delle Messi d'Oro 156, 00158 Rome, Italy.
authors.forename: Leonardo
authors.initials: L
authors.lastname: Sibilio
authors.affiliation: Laboratory of Immunology, Regina Elena National Cancer Research Institute, Centro della Ricerca Sperimentale, Via delle Messi d'Oro 156, 00158 Rome, Italy.
authors.forename: Aline
authors.initials: A
authors.lastname: Martayan
authors.affiliation: Laboratory of Immunology, Regina Elena National Cancer Research Institute, Centro della Ricerca Sperimentale, Via delle Messi d'Oro 156, 00158 Rome, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Setini
authors.affiliation: Laboratory of Immunology, Regina Elena National Cancer Research Institute, Centro della Ricerca Sperimentale, Via delle Messi d'Oro 156, 00158 Rome, Italy.
authors.forename: Elisa Lo
authors.initials: EL
authors.lastname: Monaco
authors.affiliation: Laboratory of Immunology, Regina Elena National Cancer Research Institute, Centro della Ricerca Sperimentale, Via delle Messi d'Oro 156, 00158 Rome, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Tremante
authors.affiliation: Cell Biology Institute, Consiglio Nazionale delle Ricerche, Via E. Ramarini 32, 00016 Monterotondo Scalo Rome, Italy.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Butler
authors.affiliation: Laboratory of Immunology, Regina Elena National Cancer Research Institute, Centro della Ricerca Sperimentale, Via delle Messi d'Oro 156, 00158 Rome, Italy. Electronic address: giacomini@ifo.it.
authors.forename: Patrizio
authors.initials: P
authors.lastname: Giacomini
mesh_terms: D000483:Alleles; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001402:B-Lymphocytes / Q000276:immunology; D002460:Cell Line; D003198:Computer Simulation; D003720:Densitometry; D015235:HLA-B Antigens; D015236:HLA-C Antigens / Q000737:chemistry*; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018583:Pliability; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D001613:beta 2-Microglobulin / Q000378:metabolism
chemical_list: D015235:HLA-B Antigens; D015236:HLA-C Antigens; C047431:HLA-Cw1 antigen; D026901:Membrane Transport Proteins; D001613:beta 2-Microglobulin; C107762:tapasin