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HLA-E: Strong Association with β₂-Microglobulin and Surface Expression in the Absence of HLA Class I Signal Sequence-Derived Peptides¹

Elisa Lo Monaco^*, Leonardo Sibilio^*, Elisa Melucci^*, Elisa Tremante^*, Miloslav Suchànek, Vaclav Horejsi, Aline Martayan^* and Patrizio Giacomini^2,*

^* Laboratory of Immunology, Regina Elena Cancer Institute Centro Ricerco Sperimentale, Rome, Italy; EXBIO Praha, Vestec, Czech Republic; and Institute of Molecular Genetics, Prague, Czech Republic

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 β₂-microglobulin (β₂m)-free and denatured (but not β₂m-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 β₂m-associated conformers were either unstable and weakly reactive with the prototypic conformational Ab W6/32, or exceptionally stable and strongly reactive with Abs to β₂m 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, β₂m assembler, and TAP/tapasin-assisted ligand donation is only one, and possibly not even the major, pathway leading to its stabilization and surface expression.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Italian Ministry of Health Agreement 91 (to P.G.) and Project AV0Z50520514 (to V.H.).

² Address correspondence and reprint requests to Dr. Patrizio Giacomini, Laboratory of Immunology, Regina Elena Cancer Institute Centro Ricerco Sperimentale, Via delle Messi d’Oro 156, 00158 Rome, Italy. E-mail address: giacomini{at}ifo.it

³ Abbreviations used in this paper: β₂m, β₂-microglobulin; Endo H, endoglycosidase H; IEF, isoelectric focusing.

⁴ The online version of this article contains supplemental material.