PURIFICATION AND CHEMICAL CHARACTERIZATION OF BETA-TRACE PROTEIN FROMHUMAN CEREBROSPINAL-FLUID - ITS IDENTIFICATION AS PROSTAGLANDIN-D SYNTHASE

Beta-Trace protein from pooled human CSF was purified to homogeneity. An apparent molecular mass of 23-29 kDa was determined for the polypep tide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ami no-terminal sequencing of the polypeptide yielded the unique amino aci d sequence APEAQVSVQPNFQQDKFLGRWFSA24. Alignment of amino acid sequenc es obtained from tryptic peptides with the sequence previously deduced from a cDNA clone isolated by other investigators allowed the identif ication of beta-trace protein as prostaglandin D synthase [prostagland in-H-2 D-isomerase; (5Z, ha,11alpha-epidioxy-15-hydroxyprosta-5,13-die noate D-isomerase; EC 5.3.99.2]. A conservative amino acid exchange (T hr instead of Ser) was detected at amino acid position 154 of the beta -trace polypeptide chain in the corresponding tryptic peptide. The two N-glycosylation sites of the polypeptide were shown to be almost quan titatively occupied by carbohydrate. Carbohydrate compositional as wel l as methylation analysis indicated that Asn29 and Asn56 bear exclusiv ely complex-type oligosaccharide structures (partially sialylated with alpha2-3- and/or alpha2-6-linked N-acetylneuraminic acid) that are al most quantitatively alpha1-6 fucosylated at the proximal N-acetylgluco samine; approximately 70% of these molecules contain a bisecting N-ace tylglucosamine. Agalacto structures as well as those with a peripheral fucose are also present.