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Protein Science (2004), 13:1627-1635. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity

Cyril Barinka1, Pavel Sácha1,3, Jan Sklenár2,3, Petr Man2,3, Karel Bezouska2,3, Barbara S. Slusher4 and Jan Konvalinka1,3

1 Institute of Organic Chemistry and Biochemistry and
2 Institut of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
3 Department of Biochemistry, Faculty of Natural Science, Charles University, Prague, Czech Republic
4 Guilford Pharmaceuticals Inc., Baltimore, Maryland 21224, USA

(RECEIVED January 12, 2004; FINAL REVISION March 7, 2004; ACCEPTED March 8, 2004)



Abstract

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-acetylL-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.

Keywords: NAALADase; GCPII; PSMA; glycosylation; proteolytic activity; enzyme kinetics

Abbreviations: NAAG, N-acetyl-L-aspartyl-L-glutamate • NAALADase, N-acetylated-{alpha}-linked-acidic dipeptidase • GCPII, glutamate carboxypeptidase II • rhGCPII, recombinant human glutamate carboxypeptidase II • SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis • PSMA, prostate-specific membrane antigen • PBS, phosphate-buffered saline • FBS, fetal bovine serum • PNGase F, peptide-N4-(N-acetyl-{beta}-D-glucosaminyl) asparagine amidase F • MALDI-TOF, matrix-assisted laser desorption/ionization with time-of-flight detection • ER, endoplasmic reticulum • CD, catalytic domain • HPAEC-PAD, high-performance anion exchange chromatography with pulsed amperometric detection • GlcNAc, N-acetylglucosamine • Man, mannose • Fuc, fucose

Reprint requests to: Jan Konvalinka, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo n. 2, 166 10 Praha 6, Czech Republic; e-mail: konval{at}uochb.cas.cz; fax: 420220183578.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04622104.


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