Characterization, Recombinant Production and Structure-Function Analysis of NvCI, A Picomolar Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita versicolor.

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.

Sequence and comparison of mitochondrial genomes in the genus Nerita (Gastropoda: Neritimorpha: Neritidae) and phylogenetic considerations among gastropods.

In the present study, we determined the mitochondrial DNA (mtDNA) sequence of three Neritas, Nerita versicolor, Nerita tessellata, and Nerita fulgurans. We present an analysis of the features of their gene content and genome organization and compare these within the genus Nerita, and among the main gastropod groups. The new sequences were used in a phylogenetic analysis including all available gastropod mitochondrial genomes. Genomic lengths were quite conserved, being 15,866bp for N. versicolor, 15,741bp for N. tessellata and 15,343bp for N. fulgurans. Intergenic regions were generally short; genes are transcribed from both strands and have a nucleotide composition high in A and T. The high similarity in nucleotide content of the different sequences, gene composition, as well as an identical genomic organization among the Nerita species compared in this study, indicates a high degree of conservation within this diverse genus. Values ​​of Ka/Ks of the 13 protein coding genes (PCGs) of Nerita species ranged from 0 to 0.18, and suggested different selection pressures in gene sequences. Bayesian phylogenetic analyses using concatenated DNA sequences of the 13 PCGs and the two rRNAs, and of amino acid sequences strongly supported Neritimorpha and Vetigastropoda as sister groups.