Large-scale preparation and characterization of N-linked glycopeptides from bovine fetuin.
A preparative scheme has been developed to purify asialo-glycopeptides from each of the three N-linkage sites of bovine fetuin, allowing the isolation of 100-mumols quantities of asialo-glycopeptides from 20 g of fetuin. The procedure yields seven asialo-glycopeptides which were determined to be 95% homogenous in peptide and oligosaccharide structure. The isolation scheme uses two high-capacity reverse-phase eluant systems. The primary RP-HPLC purification performed with boric acid buffered to pH 7 with triethylamine resolved sialylated tryptic glycopeptides simultaneously on the basis of glycosylation site and degree of sialylation. A second RP-HPLC purification was performed eluting isocratically with dilute phosphoric acid which resolved residual peptide and oligosaccharide heterogeneity from asialo-glycopeptides containing short peptides. Structural characterization of the products was performed utilizing 400-MHz proton NMR spectroscopy and amino acid and monosaccharide analysis. The glycopeptides contain two previously identified variant triantennary oligosaccharides which possess either Gal beta(1----4) or Gal beta(1----3) linkages to N-acetylglucosamine at one terminal branch or a biantennary oligosaccharide. These compounds should prove to be invaluable in studying carbohydrate-protein interactions, such as binding by the Gal/GalNAc lectin of mammalian hepatocytes, in the detailed three-dimensional structural analysis of complex oligosaccharides, and as purified substrates for the study of the action of glycoconjugate-modifying enzymes.
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
K G
KG
Rice
N B
NB
Rao
Y C
YC
Lee
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006020:Glycopeptides / Q000302:isolation & purification*; D009682:Magnetic Resonance Spectroscopy; D000509:alpha-Fetoproteins / Q000032:analysis*
D006020:Glycopeptides; D000509:alpha-Fetoproteins