INTRAMOLECULAR CHARGE HETEROGENEITY IN PURIFIED MAJOR HISTOCOMPATIBILITY CLASS-II ALPHA-POLYPEPTIDE AND BETA-POLYPEPTIDE CHAINS

Major histocompatibility (MHC) class II antigens are heterodimeric cel l surface glycoproteins consisting of an alpha and beta chain. Althoug h one-dimensional SDS-polyacrylamide gel electrophoresis analysis of p urified MHC class II antigens shows a single diffuse band for each cha in, multiple spots of identical molecular size were observed for each chain when analyzed by two-dimensional electrophoresis. The basis of t his heterogeneity has not been clearly defined and has been predicted partially to be due to glycosylation and/or phosphorylation of the mat ure protein. To investigate the role of the three N-linked oligosaccha rides of the alpha and beta chains in determining the isoelectric poin t of each chain, affinity-purified MHC class II antigens from human an d rat sources were deglycosylated using asparagine amidase. The comple te enzymatic removal of all three N-linked oligosaccharides was confir med by SDS-polyacrylamide gel electrophoresis as well as by four diffe rent lectin-linked Western blot analyses. Two-dimensional gel analysis of the deglycosylated molecules shows no significant difference from the fully glycosylated chains. We have expressed truncated forms of th e HLA DR2 chains which lack the transmembrane and cytoplasmically expo sed regions in Escherichia coli. Two-dimensional electrophoresis of th ese single chains also reveal multiple banding patterns. The two-dimen sional banding patterns described are unaffected by exposure to acidic or basic conditions, increased gel running time in the first dimensio n, treatment of the proteins with alkaline phosphatase to remove any p otential phosphorylation, or preincubation in the presence of iodoacet amide. Multiple forms of recombinant alpha and beta chains were also o bserved in Tris-glycine-urea gels which merged into a single band in t he presence of SDS. In addition, partially fractionated bands from pre parative isoelectric focusing gels, when refocused, showed an identica l number of multiple spots spanning the same range of isoelectric poin ts. These results together suggest that each polypeptide chain of MHC class II antigens may exist in multiconformational forms, and the obse rved charge heterogeneity is independent of glycosylation and phosphor ylation of the proteins.