The second bactericidal permeability increasing protein (BPI) and its revelation of the gene duplication in the Pacific oyster, Crassostrea gigas

A novel homolog of BPI was cloned from the hemocyte cDNA of Crassostrea gigas and designed as Cg-BPI2, which share the highest sequence identity with the well-known Cg-BPI (designed as Cg-BPI1). The complete cDNA of Cg-BPI2 included an open reading frame (ORF) of 1440 bp, and 3′ and 5′ untranslated regions (UTR’s) of 49 bp and 166 bp, respectively. The ORF encoded a putative protein of 479 amino acids with predicted 22-aa hydrophobic signal peptide. The phylogenetic analysis showed that one of the gene duplications could have resulted in the emergence of two homologs of BPI in oysters, which probably might have occurred after the gastropod-bivalve divergence. Furthermore, molecular modeling analysis showed that both Cg-BPIs are similar to a highly extended boomerang like shape of human BPI, consisting of an N- and C-terminal barrel and a central β-sheet. Comparison of the electrostatic surface potentials revealed that surfaces of Cg-BPI2 have more intense positive charge than that of human BPI and the Cg-BPI1. The recombinant N-terminal barrel domain showed a high affinity to LPS and can effectively kill Gram-negative bacteria. The mRNAs of two Cg-BPIs were observed in all tissues examined with the highest expression in gills. The mRNAs expression profiles in response to bacterial challenge revealed that they were inducible under infection, but with a distinct and complementary expression patterns between Cg-BPI1 and Cg-BPI2. Our findings of this second BPI gene demonstrated presence of its gene duplication for the first time in invertebrate and it appears to be one of effective LPS-binding AMPs in elimination of Gram-negative pathogens C. gigas.