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

Abstract

The N-linked carbohydrate chains of procine zona pellucida glycoproteins were released by digestion with peptide-N4-(N-acetyl-beta-glucosaminlyl)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 relesed from the O-glycoprotein by alakaline borohydride treatment. Fractitionation 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 Galbeta1-4GlcNAcbeta1-3Galbeta1-3GalNAc-ol, Galbeta1-4(6SO4-)GlcNAc, and alpha2-3-linked Neu5Gc or Neu5Ac. This series of compounds has the following structure, where n = 0 to > 6 : [Neu5Gc/Acalpha2-3]0-1[Galbeta1-4(6SO4-)GlcNAcbeta1-3]nGalbeta1-4GlcNAcbeta1-3Galbeta1-3GalNAc-ol.

In addition, smaller compounds were identified in which the Galbeta1-3GalNAc-ol core is substituted by Neu5Gc/Ac alpha2-6-linked to GalNAc-ol and/or Neu5Gc/Ac alpha2-3-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 GlcNAcbeta1-3GalNAc-ol core structure. Analysis of the endo-beta-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.