Two-dimensional poly(acrylamide) electrophoresis of fluoresceinated glycopeptides. Resolution and structural characterization of ovalbumin glycans.

The microheterogeneous mixture of fluoresceinated glycopeptides (FGPs) obtained from the single site of glycosylation of chicken ovalbumin was resolved by a combination of discontinuous electrophoresis in a high-density poly(acrylamide) gel (PAGE) for sizing, in conjunction with borate-PAGE. Two FGPs of similar size but with different mobilities in borate-PAGE were purified and characterized by sequential exoglycosidase digestion and sizing on the discontinuous PAGE system, as well as by methylation analysis. The two FGPs of identical size are distinct and have structures beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->3)-[beta-D-Glc pNAc-(1-->4)]-[beta-D-Glc pNAc-(1-->2)-alpha-D- Man p-(1-->6)]-beta-D-Man-p-(1-->4)-beta-D-Glc pNAc-(1-->4)-beta-D-Glc pNAc-1-->R and alpha-D-Man p-(1-->2)-alpha-D-Man p-(1-->3 or 6)-[alpha-D-Man p-(1-->3)-[alpha-D-Man p-(1-->6)]-alpha-D-Man p-(1-->6 or 3)]-beta-D-Man p-(1-->4)-beta-D-Glc pNAc-(1-->4)-beta-D-Glc pNAc-1-->R (R = Asn-(amino acids)-fluorescein).

The (1----3)-linked alpha-L-fucosyl group of the N-glycans of the Wistaria floribunda lectins is recognized by a rabbit anti-serum.

An increasing number of plant glycoproteins have been shown to possess a characteristic N-glycan component containing a beta-(1----2)-linked D-xylose unit on the core beta-D-mannose unit, and an alpha-(1----3)-linked L-fucose unit on the asparagine-linked 2-acetamido-2-deoxy-D-glucose unit. Wistaria floribunda seeds have two distinct lectins; the erythroagglutinin, WFA, and the lymphocyte mitogen, WFM. Earlier studies indicated that both lectins belong to such a class of glycoproteins.

The polypeptide of immunoglobulin G influences its galactosylation in vivo.

To examine the nature of the factors influencing the galactosylation pattern of the heavy chain of murine immunoglobulin G (IgG), cell fusion was performed between a myeloma (P3x63Ag8) and a hybridoma (Sp2HL/Bu) cell line which secrete different IgGs possessing structurally distinct CH2-linked oligosaccharide moieties. The glycosylation patterns of the IgGs of the parental and fused cells were studied. Pronase digestion of the purified heavy chains and subsequent end labeling with fluorescein isothiocyanate produced fluoresceinated glycopeptides which were detected and purified by polyacrylamide gel electrophoresis.