STRUCTURE OF THE O-LINKED CARBOHYDRATE CHAINS OF PORCINE ZONA-PELLUCIDA GLYCOPROTEINS

The N-linked carbohydrate chains of porcine zona pellucida glycoprotei ns were released by digestion with peptide-N-4-(N-acetyl-beta-glucosam inyl)asparagine amidase F and subsequently separated from the O-glycop rotein by gel-permeation chromatography on Bio-Gel P-100. The O-linked carbohydrate chains were released from the O-glycoprotein by alkaline borohydride treatment. Fractionation of the-extremely heterogeneous m ixture of O-linked oligosaccharide alditols was achieved by a combinat ion of chromatographic techniques comprising gel-permeation chromatogr aphy on Bio-Gel P-4 and P-6, anion-exchange FPLC on Mono Q, and high-p H anion-exchange chromatography on CarboPac PA-1. The primary structur es of 32 O-glycans were determined by one- and two-dimensional H-1-NMR spectroscopy. The major part of the analyzed compounds contain a comb ination of the structural elements Gal beta 1-4GlcNA beta 1-3Gal beta 1-3GalAc-ol, Gal beta 1-4(6SO(4)(-))GlcNAc, and alpha 2-3-linked Neu5G c or Neu5Ac. This series of compounds has the following structure, whe re n = 0 to > 6: [Neu5Gc/Ac alpha 2-3](0-1)[Gal beta 1-4(6SO(4)(-))Glc NAc beta 1-3](n)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-3GalNAc-ol. In a ddition, smaller compounds were identified in which the Gal beta 1-3Ga lNAc-ol core is substituted by Neu5Gc/Ac alpha 2-6-linked to GalNAc-ol and/or Neu5Gc/Ac alpha 2-3-linked to Gal. Furthermore, oligosaccharid es were obtained in which the distribution of 6-O-sulfated GlcNAc resi dues differs from that in the above-mentioned general structure, and a small portion of the oligosaccharides has the GlcNAc beta 1-3GalNAc-o l core structure. Analysis of the endo-beta-galactosidase digests of p ools of N- and O-glycans indicated that the two types of oligosacchari des contain qualitatively similar poly(N-acetyllactosamine) chains. In the case of the N-linked carbohydrate chains, multiple branching of t he core structures occurs, resulting in an even larger heterogeneity t han observed for the O-linked carbohydrate chains.