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

Cyril Bařinka, Petra Mlčochová, Payel Šacha, Ivan Hilgert, Pavel Majer, Barbara Slusher, Václav Hořejší, Jan Konvalinka

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)2782-2790
Number of pages9
JournalEuropean Journal of Biochemistry
Volume271
Issue number13
DOIs
StatePublished - Jul 2004
Externally publishedYes

Fingerprint

Glutamate Carboxypeptidase II
Carboxypeptidases
Amino Acids
Neurology
Prostate
Streptomyces griseus
Proteins
Metalloproteases
Enzymes
Nervous System
Drosophila
human glutamate carboxypeptidase II
Catalytic Domain
Prostatic Neoplasms
Animals
Central Nervous System
Animal Models
Clone Cells
Wounds and Injuries

Keywords

  • Metallopeptidase
  • Mutagenesis
  • NAALADase
  • Prostate cancer
  • PSMA

ASJC Scopus subject areas

  • Biochemistry

Cite this

Amino acids at the N- and C-termini of human glutamate carboxypeptidase II are required for enzymatic activity and proper folding. / Bařinka, Cyril; Mlčochová, Petra; Šacha, Payel; Hilgert, Ivan; Majer, Pavel; Slusher, Barbara; Hořejší, Václav; Konvalinka, Jan.

In: European Journal of Biochemistry, Vol. 271, No. 13, 07.2004, p. 2782-2790.

Research output: Contribution to journalArticle

Bařinka, Cyril ; Mlčochová, Petra ; Šacha, Payel ; Hilgert, Ivan ; Majer, Pavel ; Slusher, Barbara ; Hořejší, Václav ; Konvalinka, Jan. / Amino acids at the N- and C-termini of human glutamate carboxypeptidase II are required for enzymatic activity and proper folding. In: European Journal of Biochemistry. 2004 ; Vol. 271, No. 13. pp. 2782-2790.
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T1 - Amino acids at the N- and C-termini of human glutamate carboxypeptidase II are required for enzymatic activity and proper folding

AU - Bařinka, Cyril

AU - Mlčochová, Petra

AU - Šacha, Payel

AU - Hilgert, Ivan

AU - Majer, Pavel

AU - Slusher, Barbara

AU - Hořejší, Václav

AU - Konvalinka, Jan

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AB - 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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KW - Mutagenesis

KW - NAALADase

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KW - PSMA

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