Identification, characterization and analysis of expression of gene encoding carboxypeptidase A in Anopheles...

Carboxypeptidases are the digestive enzymes which cleave single amino acid residue from c-terminus of the protein. Digestive carboxypeptidase A gene regulatory elements in insects have shown their efficiency to drive midgut specific expression in transgenic mosquitoes. However no endogenous promoter has been reported for Indian malaria vector Anopheles culicifacies which is major vector in Indian subcontinent. Here we report cloning of carboxypeptidase A gene in the An. culicifacies A including its 5' upstream regions and named AcCP. In the upstream region of the gene an arthropod initiator sequence and two repeat sequences of the particular importance TTATC and GTTTT were also identified. The 1290 base pairs open reading frame encodes a protein of 48.5kDa. The coding region of the gene shares 82% and 72% similarity at nucleotide level with Anopheles gambiae and Ae. aegypti carboxypeptidase A gene, respectively. The peak expression of the gene was found to be at 3h after blood feeding and this is limited to midgut only. Based on the protein sequence, 3D structure of the AcCP was predicted and the active centre of the enzyme was predicted to consist of GLN 183, GLU 186, HIS 308 and Ser 309 amino acid residues. Comparison of the protein sequence among different genera revealed the conservation of zinc binding residues. Phylogenetically, AcCP was found most closely related to An. gambiae.