title: HLA-E: strong association with beta2-microglobulin and surface expression in the absence of HLA class I signal sequence-derived peptides.
abstract: The nonclassical class I HLA-E molecule folds in the presence of peptide ligands donated by the signal sequences of permissive class I HLA alleles, with the aid of TAP and tapasin. To identify HLA-E-specific Abs, four monoclonals of the previously described MEM series were screened by isoelectric focusing (IEF) blot and immunoprecipitation/IEF on >30 single-allele class I transfectants and HLA-homozygous B lymphoid cells coexpressing HLA-E and HLA-A, -B, -C, -F, or -G. Despite their HLA-E-restricted reactivity patterns (MEM-E/02 in IEF blot; MEM-E/07 and MEM-E/08 in immunoprecipitation), all of the MEM Abs unexpectedly reacted with beta(2)-microglobulin (beta(2)m)-free and denatured (but not beta(2)m-associated and folded) HLA-E H chains. Remarkably, other HLA-E-restricted Abs were also reactive with free H chains. Immunodepletion, in vitro assembly, flow cytometry, and three distinct surface-labeling methods, including a modified (conformation-independent) biotin-labeling assay, revealed the coexistence of HLA-E conformers with unusual and drastically antithetic features. MEM-reactive conformers were thermally unstable and poorly surface expressed, as expected, whereas beta(2)m-associated conformers were either unstable and weakly reactive with the prototypic conformational Ab W6/32, or exceptionally stable and strongly reactive with Abs to beta(2)m even in cells lacking permissive alleles (721.221), TAP (T2), or tapasin (721.220). Noncanonical, immature (endoglycosidase H-sensitive) HLA-E glycoforms were surface expressed in these cells, whereas mature glycoforms were exclusively expressed (and at much lower levels) in cells carrying permissive alleles. Thus, HLA-E is a good, and not a poor, beta(2)m assembler, and TAP/tapasin-assisted ligand donation is only one, and possibly not even the major, pathway leading to its stabilization and surface expression.
authors.affiliation: Laboratory of Immunology, Regina Elena Cancer Institute Centro Ricerco Sperimentale, Rome, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Lo Monaco
authors.forename: Leonardo
authors.initials: L
authors.lastname: Sibilio
authors.forename: Elisa
authors.initials: E
authors.lastname: Melucci
authors.forename: Elisa
authors.initials: E
authors.lastname: Tremante
authors.forename: Miloslav
authors.initials: M
authors.lastname: Suchànek
authors.forename: Vaclav
authors.initials: V
authors.lastname: Horejsi
authors.forename: Aline
authors.initials: A
authors.lastname: Martayan
authors.forename: Patrizio
authors.initials: P
authors.lastname: Giacomini
mesh_terms: D000483:Alleles; D002460:Cell Line; D005786:Gene Expression Regulation / Q000235:genetics / Q000276:immunology*; D006680:HLA Antigens / Q000235:genetics / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008024:Ligands; D008214:Lymphocytes / Q000276:immunology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000276:immunology*; D010455:Peptides / Q000276:immunology*; D017510:Protein Folding; D001613:beta 2-Microglobulin / Q000235:genetics / Q000276:immunology*; D000097625:HLA-E Antigens
chemical_list: D006680:HLA Antigens; D015395:Histocompatibility Antigens Class I; D008024:Ligands; D026901:Membrane Transport Proteins; D010455:Peptides; D001613:beta 2-Microglobulin; D000097625:HLA-E Antigens; C107762:tapasin