Purification and chemical characterization of beta-trace protein from human 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 polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequencing of the polypeptide yielded the unique amino acid sequence APEAQVSVQPNFQQDKFLGRWFSA. Alignment of amino acid sequences obtained from tryptic peptides with the sequence previously deduced from a cDNA clone isolated by other investigators allowed the identification of beta-trace protein as prostaglandin D synthase [prostaglandin-H2 D-isomerase; (5Z, 13E)-(15S)-9 alpha, 11 alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase; EC 5.3.99.2]. A conservative amino acid exchange (Thr 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 quantitatively occupied by carbohydrate. Carbohydrate compositional as well as methylation analysis indicated that Asn29 and Asn56 bear exclusively complex-type oligosaccharide structures (partially sialylated with alpha 2-3- and/or alpha 2-6-linked N-acetylneuraminic acid) that are almost quantitatively alpha 1-6 fucosylated at the proximal N-acetylglucosamine; approximately 70% of these molecules contain a bisecting N-acetylglucosamine. Agalacto structures as well as those with a peripheral fucose are also present.
Hoffmann
A
A
CSF-Laboratory, Department of Neurology, Medizinische Hochschule Hannover, Hannover, F.R.G.
Conradt
H S
HS
Gross
G
G
Nimtz
M
M
Lottspeich
F
F
Wurster
U
U
D000595:Amino Acid Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000134:cerebrospinal fluid* / Q000737:chemistry; D054834:Lipocalins; D008745:Methylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D014357:Trypsin / Q000378:metabolism
D054834:Lipocalins; D010446:Peptide Fragments; D014357:Trypsin; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase