Mutation of the alpha 2 domain disulfide bridge of the class I molecule HLA-A*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-A*0201. Mutation 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 recognized poorly by HLA-A*0201-restricted, peptide-specific CTLs. Cells expressing mutant HLA-A*0201 loaded with a synthetic peptide derived from the HIV-1 pol protein (476-484) were not recognized by pol IV-9-specific CTLs. Mutant C164A cells infected with influenza virus were partially recognized by influenza matrix peptide-specific CTLs, while C101S cells were not lysed. Surprisingly, endogenous peptide loading of cells expressing mutant HLA-A*0201 using a minigene coding for either the influenza A matrix peptide 58-66, or HIV-1 pol peptide 476-484, resulted in efficient CTL recognition. This suggests different structural constraints for peptide binding in the endoplasmic reticulum during biosynthesis and for binding to exported molecules on the cells surface.
Warburton
R J
RJ
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599-7290.
Matsui
M
M
Rowland-Jones
S L
SL
Gammon
M C
MC
Katzenstein
G E
GE
Wei
T
T
Edidin
M
M
Zweerink
H J
HJ
McMichael
A J
AJ
Frelinger
J A
JA
D000595:Amino Acid Sequence; D017951:Antigen Presentation / Q000276:immunology*; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D004220:Disulfides; D015684:Gene Products, pol / Q000138:chemical synthesis / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology; D015234:HLA-A Antigens / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014763:Viral Matrix Proteins / Q000138:chemical synthesis / Q000276:immunology*
D004220:Disulfides; D015684:Gene Products, pol; D015234:HLA-A Antigens; C031540:M-protein, influenza virus; D009842:Oligopeptides; D014763:Viral Matrix Proteins