Effect of carbohydrate moieties on the folate hydrolysis activity of the prostate specific membrane antigen.
BACKGROUND
Prostate specific membrane antigen or PSMA has been recognized as one of the important cellular markers for prostate cancer, the expression of which is enhanced many fold in prostate cancer and other tumor neovasculature. PSMA is a type II membrane glycoprotein with a short cytoplasmic N-terminal region, a transmembrane domain, and a 701 amino acid extracellular portion with 10 potential N-linked glycosylation sites. PSMA is a folate hydrolase, which cleaves terminal glutamates from poly- and gamma-glutamated folates; and NAALADase, which hydrolyses alpha-glutamate-linked dipeptide, N-acetyl-aspartyl-glutamate (NAAG) and is a glutamate carboxypeptidase.


METHODS
In our study we have used various enzymes or site directed mutagenesis to remove sugar molecules from PSMA protein and studied its folate hydrolase function. We have performed a biochemical characterization of N-linked glycosylation of the various mutant proteins.


RESULTS
PSMA protein expressed in different prostate cancer cell lines is differentially glycosylated. Removal of sugar residues either enzymatically or by mutagenesis abolishes the enzyme activity of PSMA protein completely.


CONCLUSIONS
N-linked carbohydrate structures are important for the folate hydrolase function of the protein. Removal of sugars partially or completely causes PSMA to be enzymatically inactive, improperly folded, resulting in increased rate of degradation.
Ghosh
Arundhati
A
George O'Brien Center for Urology Research, Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA.
Heston
Warren D W
WD
D000409:Alanine; D000954:Antigens, Surface; D001216:Asparagine; D050260:Carbohydrate Metabolism; D002268:Carboxypeptidases / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004355:Drug Stability; D005492:Folic Acid / Q000378:metabolism*; D043425:Glutamate Carboxypeptidase II; D006031:Glycosylation / Q000187:drug effects; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D018613:Microscopy, Confocal; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D000954:Antigens, Surface; D011994:Recombinant Proteins; D014415:Tunicamycin; D001216:Asparagine; D005492:Folic Acid; D006596:Hexosaminidases; D002268:Carboxypeptidases; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II; D000409:Alanine