Abstract

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.

Highlights

  • We describe the identification, cDNA cloning, and biochemical characterization of a new human blood plasma glutamate carboxypeptidase (PGCP)

  • We describe a new human peptidase of the NAALADase/ prostate-specific membrane antigen (PSMA) family, a 56-kDa blood plasma glycoprotein termed PGCP, which was affinity-co-purified with the lysosomal carboxypeptidase, cathepsin A (CathA; EC 3.4.16.1)

  • We separated these proteins by FPLC ion exchange chromatography using Mono Q column (Fig. 1) and identified three of them by NH2-terminal sequencing as cathepsin D (CathD), CathA, and a so-called IP-30 [39], a 30-kDa glycoprotein induced in hematopoietic cells in response to ␥-interferon, the biological function of which is unclear

Read more

Summary

Introduction

CDNA cloning, and biochemical characterization of a new human blood plasma glutamate carboxypeptidase (PGCP). We report cloning of PGCP cDNA; characterization of its primary structure, enzymatic activity, expression, and metabolism; and its immunolocalization in human tissues and cells, which altogether suggest that PGCP is a novel glutamate carboxypeptidase secreted into the blood plasma.

Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call