A novel sialylhexasaccharide from human milk: purification by affinity chromatography on immobilized wheat germ agglutinin.
A sialylhexasaccharide fraction (S-5) of human milk was obtained as described by A. Kobata and V. Ginsburg [(1972) Arch. Biochem. Biophys. 150, 273-281] and labeled by reduction with NaB[3H]4. When subjected to affinity chromatography on immobilized wheat germ agglutinin (WGA), a single component representing 60% of the S-5 fraction was retarded by the column. The asialo derivative of the WGA-retarded oligosaccharide had a higher affinity for the WGA column than the native sialyloligosaccharide. The neutral hexaose was identified as lacto-N-neohexaose by sequential exoglycosidase digestions in combination with gel filtration analyses of digestion products. Enzymatic removal of the nonsialylated branch of the intact sialyloligosaccharide by jack bean beta-galactosidase and beta-N-acetylhexosaminidase resulted in a single sialyl[3H]tetraose which was identified as sialyltetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcO[3H]) by cochromatography with authentic standard and specific antibody binding. Independent evidence for the structure of the sialylhexasaccharide was obtained by 500-MHz1H NMR spectroscopy of the WGA-purified oligosaccharide before and after neuraminidase digestion. The structural data are consistent with the following, previously undescribed, sialylhexaose in human milk: (formula; see text).
Tarrago
M T
MT
Department of Biochemistry and Nutrition, Virginia Tech, Blacksburg 24061.
Tucker
K H
KH
Van Halbeek
H
H
Smith
D F
DF
D002846:Chromatography, Affinity; D004735:Energy Transfer; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008895:Milk, Human / Q000032:analysis*; D009844:Oligosaccharides / Q000302:isolation & purification*; D014909:Wheat Germ Agglutinins
D009844:Oligosaccharides; D014909:Wheat Germ Agglutinins; C057809:sialyltetrasaccharide c