The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy.

E D Green,G Adelt,J U Baenziger,S Wilson,H Van Halbeek

doi:10.1016/s0021-9258(19)81354-6

Abstract

The structures of the entire population of sialylated asparagine-linked oligosaccharides present on bovine fetuin were elucidated. Asparagine-linked oligosaccharides were released from fetuin with N-glycanase, radiolabeled by reduction with NaB[3H]4, and fractionated by anion-exchange high performance liquid chromatography (HPLC), ion-suppression amine adsorption HPLC, and concanavalin A affinity chromatography. The 3H-labeled oligosaccharide fractions obtained were analyzed by 500-MHz 1H nuclear magnetic resonance spectroscopy, revealing the presence of 23 distinct oligosaccharide structures. These oligosaccharides differed in extent of sialylation (3% mono-, 35% di-, 54% tri-, and 8% tetrasialylated), number of peripheral branches (17% di- and 83% tribranched), linkage (alpha 2,3 versus alpha 2,6) and location of sialic acid moieties, and linkage (beta 1,4 versus beta 1,3) of galactose residues. This represents the first time that the asparagine-linked oligosaccharides of fetuin have been successfully fractionated and characterized as sialylated species. The sialylated oligosaccharides derived from fetuin were also used to further define the specificities of the lectins leukoagglutinating phytohemagglutinin and Ricinus communis agglutinin I. The behavior of these oligosaccharides during lectin affinity HPLC further establishes the structural features which predominate in the interaction of oligosaccharides with leukoagglutinating phytohemagglutinin and R. communis agglutinin I.

Highlights

The structures of the entirepopulation of sialylated ally and biologically related to thehuman plasma glycoprotein asparagine-linked oligosaccharides present on bovine a2HS
The ‘H-labeled oligosaccharide fractions obtained were analyzed by 500-MHz ‘H nuclear magnetic resonance spectroscopy, revealing the presence of 23 distinct oligosaccharide structures
Of the lectins leukoagglutinating phytohemagglutinin Bendel, 1986; Townsend et al, 1986; Takasaki & Kobata, and Ricinus communis agglutinin I. The behavior of these oligosaccharides during lectin affinitHyPLC further establishes thestructural featureswhich predominate in the interaction of oligosaccharides with leukoagglutinating phytohemagglutinin and R.communis agglutinin I. 1986; Yet et al, 1988).These studieshave demonstrated that the majority of Asn-linked oligosaccharides on fetuin bear three peripheral branches(Nilsson et al, 1979;Krusius & Finne, 1981;Carver & Grey, 1981; Van Halbeek, et al, 1985;Berman, 1986;Berman & Bendel, 1986; Townsend et al, 1986; Takasaki & Kobata, 1986; Yet et al, 1988),with the following underlying structure: Fetuin, the major glycoprotein in fetal calf serum, is an aglobulin of M, 48,000

Summary

Introduction

The structures of the entirepopulation of sialylated ally and biologically related to thehuman plasma glycoprotein asparagine-linked oligosaccharides present on bovine a2HS. The effects observed on the chemical shifts of the structural-reporter groups of Man, GlcNAc, and Gal residues due tothe presence of the NeuAc residues (compare N-2/D(D), N-B/D(F), and N-2/D(G) with Asialo/D in Table I and Fig. 3)) are inperfect agreement with previous reports (Vliegenthart et al, 1983; Joziasse et al, 1985; 1987).

Results

Conclusion