Isolation of a human milk sialyloligosaccharide by affinity chromatography with wheat germ agglutinin (WGA)

Abstract

Lectin affinity chromatography has been applied to the separation of the sialyloligosaccharides of human milk. A human milk sialyloligosaccharide fraction was tritium labeled and applied to a highly substituted WGA-agarose column (20 mg/ml). Only a single component from the complete sialyloligosaccharide fraction was retarded in the WGA-agarose column. The WGA-bound fraction when applied to paper chromatography migrated with identical mobility as the sialylhexasaccharide fraction (S-5) of human milk, previously isolated and described by Kobata and Ginsburg in 1972 [Arch. Biochem. Biophys., 150:273-281]. A purified sialylhexasaccharide fraction (S-5), isolated according to the method of Kobata and Ginsburg, was radiolabeled and applied to the WGA-agarose column. The WGA-bound (60%) and WGA-unbound (40%) sialylhexasaccharide fractions were isolated. The WGA-bound sialylhexasaccharide fraction was subjected to neuraminidase digestion to remove sialic acid, and the resulting neutral oligosaccharide had more affinity for the WGA-agarose column. Sequential exoglycosidase digestion of the asialo derivative of the WGA-bound fraction with jack bean β -galactosidase and β -hexosaminidase demonstrated the presence of a lacto-N-neohexaose core. The position of sialic acid in the sialyllacto-N-neohexaose was determined by simultaneous digestion of the sialylhexaose with jack bean β -galactosidase and β -hexosaminidase, which removed the non-sialylated branch from the sialylhexaose and produced a sialyltetraose. The sialyltetraose was found to be sialyltetrasaccharide c as demonstrated by its elution time on HPLC and direct binding to monospecific anti-sialyltetrasccharide c serum. The structural data indicated that the WGA-bound sialylhexaose is a sialyl derivative of lacto-N-neohexaose with sialic acid linked to the 3 branch of this core structure which represents a previously undescribed sialyloligosaccharide in human milk. The structure of the WGA-bound sialylhexaose is,