Purification, cDNA cloning, and expression of a new human blood plasma glutamate carboxypeptidase homologous to N-acetyl-aspartyl-alpha-glutamate carboxypeptidase/prostate-specific membrane antigen

We describe the identification, cDNA cloning, and biochemical characterizat ion of a new human blood plasma glutamate carboxypeptidase (PGCP), PGCP was co-purified from human placenta with lysosomal carboxypeptidase, cathepsin A, lysosomal endopeptidase, cathepsin D, and a gamma-interferon-inducible protein, IP-30, using an affinity chromatography on a Phe-Leu-agarose colum n. A PC:CP cDNA was obtained as an expressed sequence tag clone and complet ed at 5'-end by rapid amplification of cDNA ends polymerase chain reaction. The cDNA contained a 1623-base pair open reading frame predicting a 541-am ino acid protein, with five putative Asn glycosylation sites and a al-resid ue signal peptide. PGCP showed significant amino acid sequence homology to several cocatalytic metallopeptidases including a glutamate carboxypeptidas e II also known as N-acetyl-aspartyl-alpha-glutamate carboxypeptidase or as prostate-specific membrane antigen and expressed glutamate carboxypeptidas e activity. Expression of the PGCP cDNA in COS-l cells, followed by Western blotting and metabolic labeling showed that PGCP is synthesized as a 62-kD a precursor, which is processed to a 56-kDa mature form containing two Asn- linked oligosaccharide chains. The mature form of PGCP was secreted into th e culture medium, which is consistent with its intracellular localization i n secretion granules. In humans, PGCP is found principally in blood plasma, suggesting a potential role in the metabolism of secreted peptides.