MUTATION OF THE ALPHA(2) DOMAIN DISULFIDE BRIDGE OF THE CLASS-I MOLECULE HLA-A-ASTERISK-0201 - EFFECT ON MATURATION AND PEPTIDE PRESENTATION

A combination of saturation and site-directed mutagenesis was utilized to disrupt the alpha(2) domain disulfide bridge of HLA-A0201. Mutati on of cysteine 101 to a serine (C101S) or of cysteine 164 to alanine ( C164A) decreased the rate of maturation of the heavy chain, the total amount of mature heavy chain within the cell, and the level of surface expression. Cells expressing these genes and loaded with a synthetic peptide derived from the influenza A matrix protein (58-66) were recog nized poorly by HLA-A0201-restricted, peptide-specific CTLs. Cells ex pressing mutant HLA-A0201 loaded with a synthetic peptide derived fro m the HIV-1 pol protein (476-484) were not recognized by pol IV-9-spec ific CTLs. Mutant C164A cells infected with influenza virus were parti ally recognized by influenza matrix peptide-specific CTLs, while C101S cells were not lysed. Surprisingly, endogenous peptide loading of cel ls expressing mutant HLA-A0201 using a minigene coding for either the influenza A matrix peptide 58-66, or HIV-1 pol peptide 476-484, resul ted in efficient CTL recognition. This suggests different structural c onstraints for peptide binding in the endoplasmic reticulum during bio synthesis and for binding to exported molecules on the cells surface.