Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity.
Glutamate carboxypeptidase II (GCPII) is a membrane peptidase expressed in the prostate, central and peripheral nervous system, kidney, small intestine, and tumor-associated neovasculature. The GCPII form expressed in the central nervous system, termed NAALADase, is responsible for the cleavage of N-acetyl-L-aspartyl-L-glutamate (NAAG) yielding free glutamate in the synaptic cleft, and is implicated in various pathologic conditions associated with glutamate excitotoxicity. The prostate form of GCPII, termed prostate-specific membrane antigen (PSMA), is up-regulated in cancer and used as an effective prostate cancer marker. Little is known about the structure of this important pharmaceutical target. As a type II membrane protein, GCPII is heavily glycosylated. In this paper we show that N-glycosylation is vital for proper folding and subsequent secretion of human GCPII. Analysis of the predicted N-glycosylation sites also provides evidence that these sites are critical for GCPII carboxypeptidase activity. We confirm that all predicted N-glycosylation sites are occupied by an oligosaccharide moiety and show that glycosylation at sites distant from the putative catalytic domain is critical for the NAAG-hydrolyzing activity of GCPII calling the validity of previously described structural models of GCPII into question.
Barinka
Cyril
C
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, 166 10 Praha 6, Czech Republic.
Sácha
Pavel
P
Sklenár
Jan
J
Man
Petr
P
Bezouska
Karel
K
Slusher
Barbara S
BS
Konvalinka
Jan
J
D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D015870:Gene Expression; D043425:Glutamate Carboxypeptidase II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D007313:Insecta; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics
D000954:Antigens, Surface; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II