The glycoprotein corticosteroid-binding globulin (CBG) migrates as doublet bands in PAGE 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 doublet in either PAGE or SDS-PAGE, indicating that the doublet does not arise as a result of differences in sialic acid residues. Treatment of the separated upper and lower variants of native CBG with N-glycosidase F (PNGase-F) shows a differential pattern of deglycosylation over time indicating either differences in the number, type, or location of sugars attached to each of the variants. Rate of deglycosylation is quicker and more extensive for the upper variant when compared to the lower variant. PNGase-F treatment of 1% SDS-denatured CBG does not abolish the CBG doublet seen in SDS-PAGE, indicating that there is variation in the protein moiety. Sugars could not be detected on PNGase-F treated CBG using either wheat germ aglutinin horse radish peroxidase conjugate, concavilin-A HRP conjugate, or the digoxigenin glycan detection system. While the results clearly show differences in glycosylation between the CBG variants, differences in the protein moiety may also occur to give rise to the heterogeneity seen in CBG. The latter is supported by the fact that desialated CBG migrates as two bands in IEF. Migration in IEF is based solely on charge, and since only sialic acid residues are charged in N-linked glycosylation, any heterogeneity seen for the desialated glycoprotein must reside within the protein moiety itself. The presence of O-glycosylation containing an N-acetylgalactosamine with a β 1-3 linkage to galactose could not be demonstrated using O-glycosidase. N-terminal blockage could not account for the variation, as both the upper and lower variants were able to be sequenced resulting in identical sequences for the first 13 amino acids. The data presented supports the hypothesis that the differences in the sugar as well as the protein moiety are responsible for the heterogeneity seen for CBG.
- rat CBG
- steroid binding protein