Amino acids at the N- and C-termini of human glutamate carboxypeptidase II are required for enzymatic activity and proper folding
Language English Country England, Great Britain Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
15206943
DOI
10.1111/j.1432-1033.2004.04209.x
PII: EJB4209
Knihovny.cz E-resources
- MeSH
- Amino Acids chemistry metabolism MeSH
- Antigens, Surface chemistry metabolism MeSH
- DNA Primers MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Glutamate Carboxypeptidase II chemistry metabolism MeSH
- Catalysis MeSH
- Humans MeSH
- Protein Folding * MeSH
- Base Sequence MeSH
- Blotting, Western MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Amino Acids MeSH
- Antigens, Surface MeSH
- DNA Primers MeSH
- FOLH1 protein, human MeSH Browser
- Glutamate Carboxypeptidase II MeSH
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.
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