title: Role of a disulfide bridge in the immune function of major histocompatibility class I antigen as studied by in vitro mutagenesis.
abstract: Polymorphic major histocompatibility class I antigens have highly conserved disulfide bridges in the second and third external domains. To study the role of a disulfide bridge, we have introduced a mutation into the mouse H-2Ld gene by oligonucleotide-directed site-specific mutagenesis, disrupting the disulfide bridge in the second domain of the protein by changing cysteine at amino acid position 101 into serine. Upon introduction of the mutant gene into L cells, the mutant transplantation antigens were synthesized, inserted into the membrane, and displayed on the cell surface, indicating that the disulfide bridge is not essential for surface expression of the H-2 antigen. Binding studies carried out with 16 monoclonal antibodies specific for the H-2Ld antigen showed that most of the allodeterminants are lost or greatly altered in the mutant antigen. Further, almost complete loss of the recognition by H-2Ld-specific alloreactive cytotoxic T cells was observed. These results indicate that polymorphic determinants are dependent on a protein folding pattern dictated by the disulfide bridge. However, two antibodies previously found to react with antigenic sites present in the first and third domains were reactive with the mutant, implying an element of domain independence with respect to the determinants recognized by these antibodies.
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics; D004220:Disulfides / Q000032:analysis; D006183:H-2 Antigens / Q000235:genetics; D006649:Histocompatibility Antigens / Q000235:genetics*; D007739:L Cells / Q000276:immunology; D008285:Major Histocompatibility Complex; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D009693:Nucleic Acid Hybridization; D011110:Polymorphism, Genetic; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014162:Transfection
chemical_list: D004220:Disulfides; D006183:H-2 Antigens; D006649:Histocompatibility Antigens