The structure of the O-glycosidic oligosaccharide chains of the major Zajdela hepatoma ascites-cell-membrane glycoprotein

Abstract

Glycoprotein MI12, the major cell surface glycoprotein (molecular mass 11 0 kDa) of Zuj&lu hepatoma ascites cells, contains about 25 0-glycosidic oligosaccharide chains per molecule. They were released as oligosaccharidealditols by alkaline borohydride treatment of MU2, and purified by gel filtration on Bio-Gel P-6 followed by high-voltage paper electrophoresis. Four oligosaccharide-alditol fractions (A -D) were obtained in relative yields of 8 :6: 3 : 3.The structure of the components of fractions A-C was determined by 500-MHz 'H-NMR spectroscopy in combination with sugar composition analysis, to be as follows. Gal~(1+3)[NeuAca(2-r3)Gal~(1+4)GlcNAc~(1+6)]GalNAc-ol (C) NeuAcac(2+3)Gal/?(1+3)GalNAc-olOn the basis of sugar composition and characteristics on Bio-Gel P-6 filtration, paper electrophoresis and thin-layer chromatography, the structure of the carbohydrate component of fraction D is proposed to be as follows.

Expression and Characterization of a Lactosaminoglycan‐Carrying Glycoprotein of Zajdela Hepatoma Cell Surface

“…1). As reported previously [26], fraction IT contains a glycoprotein carrying 0-linked large glycans absent from hepatocytes. Staining of fraction 111 by silver reagent and Coomassie brilliant blue in SDS/PAGE revealed broad and diffuse bands, but discrete bands could be distinguished (Fig.…”

“…We have shown previously that poorly differentiated Zajdela ascite hepatoma cells express surface-bound specific 0-glycoproteins and N-glycoproteins carrying large glycan chains [23], which are predominant structures in tumor cells [24, 251, while the glycan structure of the 0-linked macroglycopeptide is not detected in normal hepatocytes [26].…”

“…The crude membrane fraction was obtained by differential centrifugations from labeled normal and tumor cells disrupted by nitrogen cavitation [26] and was solubilized in 10 mM Tris/HCl, pH 7.8, 0.1% Triton X-100 (Sigma). After centrifugation, the supernatant was applied to a Sepharose 6B column (Pharmacia) equilibrated in the above buffer.…”

“…Glycoproteins were treated with 1 M sodium borohydride in 100 mM NaOH aqueous solution at 37°C for 16 h in the dark [26].…”

Purification and Characterization of Endo-α-N-acetyl-galactosaminidase fromAlcaligenessp.

“…2 "'4) Since 0glycoside carbohydrates may be considerably more variable than N-glycoside carbohydrates, the O-linked oligosaccharides seem to be involved in a variety of biological phenomena such as blood group specificity, cell recognition, embryonic development and differentiation. s " '8) Recently, such oligosaccharides were found in hepatoma ascites cell membranes 9 ) and human gastric cancer cells,lO) and some relationships between O-glycoside carbohydrate chains and cancerous cells were proposed.…”