title: Distinctive features of the alpha 1-domain alpha helix of HLA-C heavy chains free of beta 2-microglobulin.
abstract: Only a few monoclonal antibodies are available with a restricted specificity to HLA-C products. In the present report, we demonstrate that antibody L31, previously shown to react with beta 2m-less (free) class I MHC heavy chains, binds to an epitope (residues 66-68 of the alpha 1 domain alpha helix) present on all the HLA-C alleles corresponding to the accepted (CW1 through CW8) serologic specificities, and on a few HLA-B heavy chains sharing with HLA-C an aromatic residue at position 67. Extensive IEF blot testing of HLA homozygous, EBV-transformed B-lymphoid cells indicates that HLA-C molecules are present at significantly lower levels than HLA-B polypeptides not only at cell surface, as previously demonstrated, but also in total cellular extracts. Testing of metabolically labeled HLA-CW1, -CW5, and -CW6 transfectants and HLA homozygous lymphoid cells, particularly HLA-CW1-expressing cells, demonstrates that the L31 epitope is present on a subpopulation of naturally occurring HLA-C molecules distinct from that identified by antibody W6/32 to beta 2m-associated heavy chains. Pulse-chase experiments demonstrate that this epitope is transiently made available to antibody binding at early biosynthetic stages, but becomes hidden upon assembly with beta 2m. Thus, free HLA-C and other Y/F67+ heavy chains are characterized by distinctive antibody binding features in a region (residues 66-68) included in a previously identified HLA-C restricted motif, which has been suggested to be the primary cause of distinctive features of the antigen-binding groove, low affinity for endogenous peptide antigens and beta 2m, and preferential uptake of exogenous peptides, possibly of viral origin. We also show that HLA-CW1 heavy chains, both free and beta 2m associated, acquire sialilation. Free HLA-CW1 heavy chains are expressed at the cell surface even when unsialilated, albeit at low levels.
authors.affiliation: Center for Experimental Research, Regina Elena Institute, Rome, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Setini
authors.forename: A
authors.initials: A
authors.lastname: Beretta
authors.forename: C
authors.initials: C
authors.lastname: De Santis
authors.forename: R
authors.initials: R
authors.lastname: Meneveri
authors.forename: A
authors.initials: A
authors.lastname: Martayan
authors.forename: M C
authors.initials: MC
authors.lastname: Mazzilli
authors.forename: E
authors.initials: E
authors.lastname: Appella
authors.forename: A G
authors.initials: AG
authors.lastname: Siccardi
authors.forename: P G
authors.initials: PG
authors.lastname: Natali
authors.forename: P
authors.initials: P
authors.lastname: Giacomini
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology; D002461:Cell Line, Transformed; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000276:immunology*; D015236:HLA-C Antigens / Q000096:biosynthesis / Q000276:immunology*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D017433:Protein Structure, Secondary; D001613:beta 2-Microglobulin / Q000172:deficiency* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D015236:HLA-C Antigens; D001613:beta 2-Microglobulin