Structural studies on the O-linked carbohydrate chains of human platelet glycocalicin

Glycocalicin (140 kDa), constituting the main part of glycoprotein Ib (160 kDa), was released from the human platelet membrane by the action of a Ca2+-dependent protease, present in the platelet cytoplasm and liberated during sonication of the platelet suspension. After activation of the protease by Ca2+, the sonicated plateled suspension was subjected to differential centrifugation. The supernatant was applied to a column of wheat germ agglutinin linked to Sepharose 4B; glycocalicin was eluted from the column with 2.5% (w/v) N-acetylglucosamine. Glycocalicin was found to contain 40% carbohydrate by weight, representing N- as well as O-glycosidically linked carbohydrate chains. The O-glycosidic chains were split off by alkaline cleavage in the presence of 3H-labelled NaBH4. The liberated 3H-labelled oligosaccharide-alditols were fractionated on a DEAE-Sephadex A-25 column. The structures of the oligosaccharide-alditols were investigated by 500-MHz 1H-NMR spectroscopy. The major compound was identified as NeuAc alpha(2->3)Galbeta(1->3)[NeuAcalpha(2->3)Galbeta(1->4)GlcNAcbeta(1->6)]GalNAc-ol. Two minor compounds were found to be NeuAcalpha(2->3)Galbeta(1->3)[NeuAcalpha(2->6)]GalNAc-ol and NeuAcalpha(2->3)Galbeta(1->3)GalNAc-ol

Identification of a tetrasialylated monofucosylated tetraantennary N-linked carbohydrate chain in human platelet glycocalicin

Glycocalicin (140 kDa), the main constituent of the glycoprotein Ib alpha-chain (150 kDa) of the human platelet membrane, contains 4 putative N-glycosylation sites. For the structural analysis of the N-glycosidic carbohydrate chains of glycocalicin, the glycoprotein has been subjected to the hydrazinolysis procedure. The acidic carbohydrate chains obtained were fractionated by ion-exchange chromatography on DEAE-Sephadex A-25, subsequently analysed by sugar analysis, anion-exchange chromatography on Mono Q HR 5/5 500 MHz 1H-NMR spectroscopy. A novel tetrasialylated monofucosylated tetraantennary chain was identified in the glycoprotein. It could also be deduced that in all structures the alpha2¨6-linked NeuAc is attached exclusively at the Gal beta 1¨4GlcNAc beta 1¨2Man alpha 1¨3 antenna, whereas the other antennae can be terminated with alpha2¨3-linked NeuAc. As minor constituents sialylated N-linked carbohydrate chains with a terminal Fuc alpha1¨2Gal beta1¨. sequence were detected