Abstract
Ld652Y cells, which are derived from the gypsy moth, Lymantria dispar, undergo apoptosis upon infection with various nucleopolyhedroviruses (NPVs). To gain insight into the mechanisms underlying NPV-induced apoptosis of Ld652Y cells, we cloned and characterized an L. dispar homologue (ld-caspase-1) of Spodoptera frugiperda effector caspase sf-caspase-1. The ld-caspase-1 gene had an open reading frame of 882bp, encoding a polypeptide of 294 amino acid residues with a predicted molecular mass of 33,304Da. Multiple alignment analysis demonstrated that Ld-caspase-1 had a high degree of amino acid sequence identity (71.4-83.6%) with those of lepidopteran caspase-1 proteins sequenced previously, and consensus sequences of a catalytic site, 174QACQG178, and three cleavage sites, 23DEGDA27, 179DKLDA183, and 190TETDG194, were completely or nearly completely conserved among these homologous caspases. When expressed in Escherichia coli, Ld-caspase-1 exhibited substantial cleavage activity for the synthetic substrate Ac-DEVD-AMC, which is preferentially cleaved by human caspase-3, and negligible activity towards Ac-IETD-AMC and Ac-LEHD-AMC, which are preferentially cleaved by human caspases-8 and -9, respectively. Ld-caspase-1 transiently expressed in Ld652Y cells executed apoptosis and stimulated caspase-3-like protease activity, which were both suppressed by the pancaspase inhibitor z-VAD-fmk. Apoptosis induction and activation of caspase-3-like protease by transiently expressed Ld-caspase-1 were also observed in the lepidopteran cell lines Sf9 and BM-N, and dipteran cell line S2. Taken together, these results indicate that the Ld-caspase-1 cloned in this study functions as an effector caspase and is responsible for apoptosis execution in insect cells destined to undergo apoptosis.
