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

Abstract

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.