Partial amino acid sequence analysis of rabbit MHC class II molecules isolated from two-dimensional polyacrylamide gels

Abstract

Partial N-terminal amino acid sequence analyses were performed on rabbit MHC class II molecules eluted from 2-D electrophoretic gels. Rabbit spleen cells were biosynthetically labelled with 3H-phenylalanine, 3H-tyrosine and 35S-methionine and class II molecules were immunoprecipitated with a monoclonal antibody, 2C4. The immunoprecipitates were electrophoresed on 2-D gels and as many as 15 spots were observed. Individual spots corresponding to alpha and beta chains were eluted from unfixed gels following visualization of the spots by autoradiography of 35S-Met labelled polypeptides. Ten eluted polypeptides were subjected to amino acid sequence analysis to locate Phe and Tyr residues. Comparison of these partial sequences with sequences of human class II molecules indicated that each of six beta chains and three of four alpha chains were homologous to human DQ molecules; one of the alpha chains appeared homologous to DRα or DPα. The assignment of α or β chain to the polypeptides was confirmed by radiosequence of molecules labelled with 35S-Cys residues. Thus, by a relatively simple procedure, individual MHC class II polypeptides in spleen cell lysates have been separated from each other and partial amino acid sequences have been obtained.