Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes

LUFRANO DANIELA; COTABARREN JULIANA; GARCIA-PARDO JAVIER; FERNANDEZ-ALVAREZ ROBERTO; TORT OLIVIA; TANCO SEBASTIAN; AVILÉS FRANCESC XAVIER; LORENZO JULIA; OBREGÓN WALTER DAVID

PHYTOCHEMISTRY

PERGAMON-ELSEVIER SCIENCE LTD

Lugar: Amsterdam; Año: 2015

0031-9422

Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning,expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases,naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described.The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidaseinhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The maturepolypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of thefamily (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminusand another three located at the secondary binding site, a region described to contribute to the stabilizationof the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondarybinding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondarybinding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. TherimaPCI exhibited a molecular mass of 4234.8 Da by MALDI-TOF/MS. It displayed potent inhibitoryactivity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2?4-fold lowerinhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformaticanalysis, which showed that the main interaction established between the secondary binding site of rPCIand the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce theimportance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-typemetallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnologyand biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminalcleavage and inactivation by carboxypeptidases A and B was demonstrated.