Abstract

The carbohydrate structures of beta-trace protein from human cerebrospinal fluid have been elucidated. This protein carries exclusively N-linked oligosaccharides at two sites (Asn29 and Asn56). Enzymatically released N-glycans were studied by compositional and methylation analyses, high-pH anion-exchange chromatography, and liquid secondary ion mass spectrometry. All glycans were found to be of the complex type, and most (90%) of them were biantennary with no (40%), one (40%), or two (20%) N-acetylneuraminic acid residues. The rest were triantennary chains or biantennary chains with intact or truncated lactosamine repeats. The innermost N-acetylglucosamine residues of nearly all structures were found to be alpha 1,6-fucosylated. Peripheral fucose (about 20% alpha 1,3-linked to N-acetylglucosamine) was also detected. Seventy percent of the oligosaccharides contained a bisecting N-acetylglucosamine. Especially in the neutral, but also in the monosialylated oligosaccharide fractions, many incomplete antennae consisting of N-acetylglucosamine only were present. At least 20 different N-glycans were identified. Analysis of the site-specific glycosylation patterns at Asn29 and Asn56 revealed only minor differences. According to the structural features (a high degree of fucosylation, high amounts of bisecting N-acetylglucosamine, as well as terminal N-acetylglucosamine and galactose residues, and significant amounts of N-acetylneuraminic acid in alpha 2,3 linkage), this protein can be classified as "brain-type" glycosylated.