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 characterization 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 column. A PGCP cDNA was obtained as an expressed sequence tag clone and completed 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-amino acid protein, with five putative Asn glycosylation sites and a 21-residue signal peptide. PGCP showed significant amino acid sequence homology to several cocatalytic metallopeptidases including a glutamate carboxypeptidase II also known as N-acetyl-aspartyl-alpha-glutamate carboxypeptidase or as prostate-specific membrane antigen and expressed glutamate carboxypeptidase activity. Expression of the PGCP cDNA in COS-1 cells, followed by Western blotting and metabolic labeling showed that PGCP is synthesized as a 62-kDa precursor, which is processed to a 56-kDa mature form containing two Asn-linked oligosaccharide chains. The mature form of PGCP was secreted into the culture medium, which is consistent with its intracellular localization in secretion granules. In humans, PGCP is found principally in blood plasma, suggesting a potential role in the metabolism of secreted peptides.
Gingras
R
R
Université de Montréal, Service de Génétique Médicale, Département de Pédiatrie, Hôpital Sainte-Justine, Montréal, Québec H3T 1C5, Canada.
Richard
C
C
El-Alfy
M
M
Morales
C R
CR
Potier
M
M
Pshezhetsky
A V
AV
D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface; D001483:Base Sequence; D019556:COS Cells; D002268:Carboxypeptidases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002478:Cells, Cultured; D002874:Chromosome Mapping; D002898:Chromosomes, Human, Pair 8; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005455:Fluorescent Antibody Technique; D043425:Glutamate Carboxypeptidase II; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D000954:Antigens, Surface; D018076:DNA, Complementary; D012333:RNA, Messenger; D002268:Carboxypeptidases; C026134:glutamate carboxypeptidase; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II