Lrcasp9 shares similarity in structural motifs with human caspase-9 and is activated following bacterial infection and anti-viral vaccination

Abstract

Among various caspases, caspase-9 plays a crucial role in the initiation phase of apoptotic cascade. To investigate about it in a high-valued freshwater fish species rohu (), we cloned and characterized full-length caspase-9 cDNA (Lrcasp9) and analyzed its expression following bacterial infections and anti-viral vaccinations. The Lrcasp9 consisted of 1619-bp nucleotides (nt) having an ORF of 1302 nt encoding a polypeptide of 433 amino acids (aa) with a molecular mass of ∼ 48.20 kDa. Structurally, Lrcasp9 comprised of one CARD domain (1-89 aa) and one CASc domain (161-430 aa). The CASc domain consisted of one large subunit (p20) spanning from 168 to 300 aa, and a small sub unit (p10) from 343 to 430 aa. The caspase family signature histidine active motif HSAYDCCVVIILSHG, cysteine active motif KPKLFFIQACGG and pentapeptide "QACGG" active sites present in the p20 domain of Lrcasp9 was conserved across fish species, mouse and human caspase-9. Phylogenetically, it was closely related to common carp caspase-9 and exhibited significant similarity (90.1%) and identity (85.3%) in their amino acid sequence. In the uninfected fish, Lrcasp9 gene expression was highest (~ 5.3-fold) in blood and lowest in gill. In response to and infection and rhabdoviral vaccination, Lrcasp9 gene expression was significantly ( > 0.05) enhanced in gill, liver, kidney and spleen, and also in vitro during cell death, suggesting activation of the intrinsic apoptotic pathway in bacterial infections and anti-viral vaccination in .