Characterization, Recombinant Production and Structure-Function Analysis of NvCI, A Picomolar Metallocarboxypeptidase Inhibitor from the Marine Snail <i>Nerita versicolor</i>
Giovanni Covaleda-Cortés,
Martha Hernández,
Sebastián Alejandro Trejo,
Manuel Mansur,
Sergi Rodríguez-Calado,
Javier García-Pardo,
Julia Lorenzo,
Josep Vendrell,
María Ángeles Chávez,
Maday Alonso-del-Rivero,
Francesc Xavier Avilés
Affiliations
Giovanni Covaleda-Cortés
Institute of Biotechnology and Biomedicine and Departament of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
Martha Hernández
Faculty of Forestry Science, Biotechnology Center, Universidad de Concepción, Victoria 631, Barrio Universitario, 2407 Concepción, Chile
Sebastián Alejandro Trejo
Institute of Biotechnology and Biomedicine and Departament of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
Manuel Mansur
Institute of Biotechnology and Biomedicine and Departament of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
Sergi Rodríguez-Calado
Institute of Biotechnology and Biomedicine and Departament of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
Javier García-Pardo
Institute of Biotechnology and Biomedicine and Departament of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
Julia Lorenzo
Institute of Biotechnology and Biomedicine and Departament of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
Josep Vendrell
Institute of Biotechnology and Biomedicine and Departament of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
María Ángeles Chávez
Center for Protein Research, Faculty of Biology, Universidad de la Habana, 10400 La Habana, Cuba
Maday Alonso-del-Rivero
Center for Protein Research, Faculty of Biology, Universidad de la Habana, 10400 La Habana, Cuba
Francesc Xavier Avilés
Institute of Biotechnology and Biomedicine and Departament of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
A very powerful proteinaceous inhibitor of metallocarboxypeptidases has been isolated from the marine snail Nerita versicolor and characterized in depth. The most abundant of four, very similar isoforms, NvCla, was taken as reference and N-terminally sequenced to obtain a 372-nucleotide band coding for the protein cDNA. The mature protein contains 53 residues and three disulphide bonds. NvCIa and the other isoforms show an exceptionally high inhibitory capacity of around 1.8 pM for human Carboxypeptidase A1 (hCPA1) and for other A-like members of the M14 CPA subfamily, whereas a twofold decrease in inhibitory potency is observed for carboxypeptidase B-like members as hCPB and hTAFIa. A recombinant form, rNvCI, was produced in high yield and HPLC, mass spectrometry and spectroscopic analyses by CD and NMR indicated its homogeneous, compact and thermally resistant nature. Using antibodies raised with rNvCI and histochemical analyses, a preferential distribution of the inhibitor in the surface regions of the animal body was observed, particularly nearby the open entrance of the shell and gut, suggesting its involvement in biological defense mechanisms. The properties of this strong, small and stable inhibitor of metallocarboxypeptidases envisage potentialities for its direct applicability, as well as leading or minimized forms, in biotechnological/biomedical uses.