PRIMARY STRUCTURE OF THE N-LINKED CARBOHYDRATE CHAINS OF CALRETICULINFROM SPINACH LEAVES

Calreticulin is a multifunctional Ca2+-binding protein of the endoplas mic reticulum of most eukaryotic cells. The 56 kDa Calreticulin glycop rotein isolated from spinach (Spinacia oleracea L.) leaves was N-degly cosylated by PNGase-F digestion. The carbohydrate moiety was isolated by gel permeation chromatography and purified by high-pa anion-exchang e chromatography. The fractions were investigated by 500 MHz H-1-NMR s pectroscopy, in combination with monosaccharide analysis and fast-atom bombardment-mass spectrometry. The following carbohydrate structure c ould be established as the major component (Man(8)GlcNAc(2)): [GRAPHIC S] Heterogeneity was demonstrated by the presence of two minor compone nts being Man(7)GlcNAc(2) lacking a terminal residue (D-1 or D-3), com pared to the major component. A. cross-reactivity with an antibody aga inst the endoplasmic reticulum retention signal HDEL was also found.