Structure of the O-linked carbohydrate chains of porcine zona pellucida glycoproteins

The N-linked carbohydrate chains of porcine zona pellucida glycoproteins were released by digestion with peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F and subsequently separated from the O-glycoprotein 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 mixture of O-linked oligosaccharide alditols was achieved by a combination of chromatographic techniques comprising gel-permeation chromatography on Bio-Gel P-4 and P-6, anion-exchange FPLC on Mono Q, and high-pH anion-exchange chromatography on CarboPac PA-1. The primary structures of 32 O-glycans were determined by one- and two-dimensional 1H-NMR spectroscopy. The major part of the analyzed compounds contain a combination of the structural elements Galβ1-4GlcNAcβ1-3Galβ1-3GalNAc-ol, Galβ1-4(6SO4-)GlcNAc, and α2-3-linked Neu5Gc or Neu5Ac. This series of compounds has the following structure, where n = 0 to >6: [Neu5Gc/Acα2-3]0-1[Galβ1-4(6SO4-)GlcNAcβ1-3](n)Galβ1- 4GlcNAcβ1-3Galβ1-3GalNAc-ol. In addition, smaller compounds were identified in which the Galβ1-3GalNAc-ol core is substituted by Neu5Gc/Ac α2-6-linked to GalNAc-ol and/or Neu5Gc/Ac α2-3-linked to Gal. Furthermore, oligosaccharides were obtained in which the distribution of 6-O-sulfated GlcNAc residues differs from that in the above-mentioned general structure, and a small portion of the oligosaccharides has the GlcNAcβ1-3GalNAc-ol core structure. Analysis of the endo-β-galactosidase digests of pools of N- and O-glycans indicated that the two types of oligosaccharides contain qualitatively similar poly(N-acetyllactosamine) chains. In the case of the N-linked carbohydrate chains, multiple branching of the core structures occurs, resulting in an even larger heterogeneity than observed for the O-linked carbohydrate chains.