STUDIES ON THE ROLE OF GLYCOSYLATION IN THE ORIGIN OF THE ELECTROPHORETIC VARIANTS FOR RAT CORTICOSTEROID-BINDING GLOBULIN

The glycoprotein corticosteroid-binding globulin (CBG) migrates as dou blet bands in PACE and SDS-PAGE, and as numerous bands in isoelectric focusing (IEF). This study deals with the origin of this heterogeneity . Desialation of rat CBG with neuraminidase does not abolish the doubl er in either PAGE or SDS-PAGE, indicating that the doubler does not ar ise as a result of differences in sialic acid residues. Treatment of t he separated upper and lower variants of native CBG with N-glycosidase F (PNGase-F) shows a differential pattern of deglycosylation over tim e indicating either differences in the number, type, or location of su gars attached to each of the variants. Rate of deglycosylation is quic ker and more extensive for the upper variant when compared to the lowe r variant. PNGase-F treatment of 1% SDS-denatured CBG does not abolish the CBG doubler seen in SDS-PAGE, indicating that there is variation in the protein moiety. Sugars could not be detected on PNGase-F treate d CBG using either wheat germ aglutinin horse radish peroxidase conjug ate, concavilin-A HRP conjugate, or the digoxigenin glycan detection s ystem. While the results clearly show differences in glycosylation bet ween the CBG variants, differences in the protein moiety may also occu r to give rise to the heterogeneity seen in CBG. The latter is support ed by the fact that desialated CBG migrates as two bands in IEF. Migra tion in IEF is based solely on charge, and since only sialic acid resi dues are charged in N-linked glycosylation, any heterogeneity seen for the desialated glycoprotein must reside within the protein moiety its elf The presence of O-glycosylation containing an N-acetylgalactosamin e with a beta 1-3 linkage to galactose could not be demonstrated using O-glycosidase. N-terminal blockage could not account for the variatio n, as both the upper and lower variants were able to be sequenced resu lting in identical sequences for the first 13 amino acids. The data pr esented supports the hypothesis that the differences in the sugar as w ell as the protein moiety are responsible for the heterogeneity seen f or CBG.