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Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent
G S Cottrell1, N M Hooper, A J Turner
1Proteolysis Research Group, School of Biochemistry and Molecular Biology, The University of Leeds, Leeds, LS2 9JT United Kingdom.
Abstract:
The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.
Insights
Human cytosolic aminopeptidase P (AP-P) is a manganese-dependent enzyme crucial for degrading bradykinin. This study identifies manganese as the likely in vivo metal cofactor for cytosolic AP-P activity.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Aminopeptidase P (AP-P) is a mammalian enzyme that degrades bradykinin.
- AP-P exists in both membrane-bound and cytosolic forms, encoded by distinct genes.
- Understanding the properties of cytosolic AP-P is essential for its physiological roles.
Purpose of the Study:
- To clone, express, and characterize the human cytosolic aminopeptidase P.
- To determine the metal ion dependency and cofactor requirements of cytosolic AP-P.
- To elucidate the specific role of manganese in the enzyme's catalytic activity.
Main Methods:
- Human pancreatic cDNA library used for cloning the cytosolic AP-P gene.
- Expression of full-length cDNA in Escherichia coli and COS-1 cells.
- Enzyme activity assays, metal analysis (ICP-AES), and inhibition studies.
Main Results:
- Human cytosolic AP-P was successfully expressed and characterized.
- The enzyme hydrolyzed bradykinin and substance P, with activity dependent on metal ions.
- Inductively coupled plasma atomic emission spectroscopy revealed manganese as the primary metal cofactor.
- Enzyme activity was significantly promoted by Mn(II), and the metal-free enzyme was reactivated by Mn(II).
Conclusions:
- Human cytosolic AP-P is a single manganese-dependent enzyme.
- Manganese is the most likely in vivo metal cofactor for cytosolic AP-P.
- These findings provide insights into the catalytic mechanism and physiological function of cytosolic AP-P.