Amino acids at the N‐ and C‐termini of human glutamate carboxypeptidase II are required for enzymatic activity and proper folding

Abstract

Human glutamate carboxypeptidase II (GCPII) is a co-catalytic metallopeptidase and its putative catalytic domain is homologous to the aminopeptidases from Vibrio proteolyticus and Streptomyces griseus. In humans, the enzyme is expressed predominantly in the nervous system and the prostate. The prostate form, termed prostate-specific membrane antigen, is overexpressed in prostate cancer and is used as a diagnostic marker of the disease. Inhibition of the form of GCPII expressed in the central nervous system has been shown to protect against ischemic injury in experimental animal models. Human GCPII consists of 750 amino acids, and six individual domains were predicted to constitute the protein structure. Here, we report the analysis of the contribution of these putative domains to the structure/ function of recombinant human GCPII. We cloned 13 mutants of human GCPII that are truncated or extended at one or both the N-and C-termini of the GCPII sequence.The clones were used to generate stably transfected Drosophila Schneider's cells, and the expression and carboxypeptidase activities of the individual protein products were determined. The extreme C-terminal region of human GCPII was found to be critical for the hydrolytic activity of the enzyme. The deletion of as few as 15 amino acids from the C-terminus was shown to completely abolish the enzymatic activity of GCPII. Furthermore, the GCPII carboxypeptidase activity was abrogated upon removal of more than 60 amino acid residues from the N-terminus of the protein. Overall, these results clearly show that amino acid segments at the N-and C-termini of the ectodomain of GCPII are essential for its carboxypeptidase activity and/or proper folding.

Structure–Activity Relationship of 18F-Labeled Phosphoramidate Peptidomimetic Prostate-Specific Membrane Antigen (PSMA)-Targeted Inhibitor Analogues for PET Imaging of Prostate Cancer

“…29 The final protein preparation in 20 mM Tris-HCl, 150 mM NaCl, pH 8.0, was concentrated to 9 mg/mL and stored at −80 ° C until further use.…”

GCPII Variants, Paralogs and Orthologs

“…Alignment was performed using the Vector NTI Advance® v. 11.0 package. extensive deletion at the N-terminus, the PSMAL protein is likely proteolytically inactive [17]. Its mRNA has been detected in human liver and kidney [16], but there are no data showing the presence of PSMAL protein in human tissues.…”

Co-expression and impact of prostate specific membrane antigen and prostate specific antigen in prostatic pathologies

“…A transmembrane glycoprotein expressed in the human prostate parenchyma, from where it was first cloned and named prostate-specific membrane antigen (PSMA) [5] has gained increased attention in diagnosis, monitoring and treatment of PC [6]. PSMA is a metallopeptidase belonging to the peptidase family M28 [7] and has apparent molecular masses of 84-100 kDa [8] with a unique three-part structure: a short cytoplasmic amino terminus that interacts with an actin filament, a single membrane-spanning domain and a large extracellular domain [9]. Several alternative isoforms have been described, including the cytosolic variants PSMA', PSM-C, PSM-D [10] and PSMA-E.…”