Title : 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 platelet 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)Ga1 beta(1----3)[NeuAc alpha(2----3)Ga1 beta(1----4)GlcNAc beta(1----6)]GalNAc-ol.
Two minor compounds were found to be NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol and NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol.