The Regulation Mechanism Of Caspases In Apoptosis In Pacific Oyster Crassostrea Gigas

Apoptosis is a form of programmed cell death process controlled by a family of cysteine proteases called caspases, which plays crucial roles in homeostasis, development of tissues and organs and immune defense. The apoptosis and the detailed regulation mechanism have been well studied in vertebrate, but the information in invertebrate, especiallymollusc, is still very limited. In the present study, two new members of caspase family including Cg Caspase-3 and Cg Caspase-8 were cloned and identified from Pacific oyster Crassostrea gigas. Their function and the regulation mechanism of caspase-mediated apoptosis were conducted by using the method of molecular biology, cell biology and bioinformatics.Cg Caspase-3 encodes a polypeptide of 307 amino acids with an ORF of 924 bp. Cg Caspase-8 encodes a polypeptide of 484 amino acids with an ORF of 1455 bp. Cg Caspase-3 and Cg Caspase-8 are both zymogens containing characteristic CASc domains in the C terminals and conserved caspase active site motifs. In addition, Cg Caspase-3 and Cg Caspase-8 m RNA can be examined in all tested tissues including gill, gonad(female), gonad(male), hemocytes, hepatopancrease, mantle and muscle, and the highest expression levels are observed in the mantle and hemocytes. In different development stages(from two cell embryos to umbo larvae), Cg Caspase-3 and Cg Caspase-8 m RNA can be examined with a different expression patterns. The immunofluorescence analysis indicated that Cg Caspase-3 mainly locates in the cytoplasm of hemocytes, while a small amount of Cg Caspase-3 appears in the nucleus. Cg Caspase-8 mainly locates in the cytoplasm of hemocytes.In vitro, r Cg Caspase-3 prefers an Asp-Xaa-Xaa-Asp(DXXD)-like substrate, which was similar to that of human Caspase-3. Both pan-caspase inhibitor Z-VADFMK and caspase-3 inhibitor Ac-DEVD-CHO exhibit significant inhibition on the proteolytic activity of r Cg Caspase-3. In vivo, the proliferation of HEK293 T cells transfected with Cg Caspase-3 decreases significantly, while the apoptosis ratio increases significantly. In addition, Cg Caspase-3 functions as a pattern recognition receptor(PRR). LPS pull-down, enzyme-linked immunosorbent assay and confocal microscopic analysis collectively reveal that Cg Caspase-3 binds LPS directly and surface plasmon resonance analysis further demonstrate its high binding specificity and moderate binding affinity(KD = 1.08×10-6 M) to LPS. However, r Cg Cas-3(ΔN58) displays very weak LPS-binding activity, while r Cg Cas-3(NT) did not exhibit LPSbinding capability. The binding of Cg Caspase-3 to LPS significantly inhibits its proteolytic activity toward AC-DEVD-p NA in a dose dependent manner in vitro. Compared with the control and vector transfection groups, the cell proliferation of HEK293 T cells with Cg Caspase-3 significantly decreases, while the transfection of LPS rescues the cells from apoptosis. Intracellular LPS inhibits the proteolytic activity of Cg Caspase-3 to PARP and apoptosis induced by Cg Caspase-3.Recombinant Cg Caspase-8 shows very weak activity to substrate Ac-IETD p NA. After concentration for 12 h, the proteolytic activity of Cg Caspase-8 increases. r Cg Caspase-3 can be cleaved into two submits p20 and p11 after incubation with r Cg Caspase-8. The proliferation of HEK293 T cells transfected with Cg Caspase-8 decreases significantly, while the apoptosis ratio increases significantly. The Caspase-8 inhibitor Ac-IETD-FMK exhibits significant inhibition on the proteolytic activity of r Cg Caspase-8. In addition, Cg Caspase-8 is observed to be completely colocalized with Cg Caspase-3, indicating that they may interact with each other in vivo. Cg Caspase-8 can respond to the stimulation of LPS, PGN and Vibrio splendidus. The expression levels of Cg Caspase-8 m RNA and protein show an obvious increase at 3 h after the treatment of LPS and then decrease to the normal level. In contrast, the expression of Cg Caspase-8 m RNA increases at 3 h after the treatment of V. splendidus and then decreases to the normal level. However, the protein expression of Cg Caspase-8 declines from 0 h after the treatment. Cg Caspase-8 shows a weak response to the PGN treatment, with a minor increase of m RNA expression at 6 h.In summary, Cg Caspase-3 and Cg Caspase-8 are highly conserved in evolution. They exhibitproteolytic activity with typical CASc domains and induce cell apoptosis. Meanwhile, they can respondto the treatment of PAMPs and pathogens. In addition, C. gigas has a special regulation mechanism of apoptosis, LPS can inhibit apoptosis mediated by Cg Caspase-3, which has never been reported in other vertebrates or invertebrates and probably plays a vital role in the immune defense of C. gigas. The results provide further information for the knowledge of caspase in C. gigas and laid a solid foundation for unveiling the cellular and molecular mechanisms of apoptosis in invertebrates as well.