Identification and functional characterization of two executioner caspases in Crassostrea gigas

Abstract

Caspase-3 and caspase-7 are two key effector caspases that play important roles in apoptotic pathways that maintain normal tissue and organ development and homeostasis. However, little is known about the sequence, structure, activity, and function of effector caspases upon apoptosis in mollusks, especially marine bivalves. In this study, we investigated the possible roles of two executioner caspases in the regulation of apoptosis in the Pacific oyster Crassostrea gigas. A full-length caspase-3-like gene named Cgcaspase-3 was cloned from C.gigas cDNA, encoding a predicted protein containing caspase family p20 and p10 domain profiles and a conserved caspase active site motif. Phylogenetic analysis demonstrated that both Cgcaspase-3 and Cgcaspase-1 may function as effector caspases clustered in the invertebrate branch. Although the sequence identities between the two caspases was low, both enzymes possessed executioner caspase activity and were capable of inducing cell death. These results suggested that Cgcaspase-3 and Cgcaspase-1 were two effector caspases in C. gigas. We also observed that nucleus-localized Cgcaspase-3, may function as a caspase-3-like protein and cytoplasm-localized Cgcaspase-1 may function as a caspase-7-like protein. Both Cgcaspase-3 and Cgcaspase-1 mRNA expression increased after larvae settled on the substratum, suggesting that both caspases acted in several tissues or organs that degenerated after oyster larvae settlement. The highest caspase expression levels were observed in the gills indicating that both effector caspases were likely involved in immune or metabolic processes in C. gigas.

Figure 1. The predicted protein sequence of Cgcaspase-3.

(A) The full-length cDNA sequence and deduced amino acid sequence of Cgcaspase-3. Nucleotides and amino acids are numbered on the left-hand side. The prodomain is underlined. The p20 and p10 domains are shaded. The conserved caspase family cysteine active site motif is bold, shaded and underlined. (B) Domains analysis of Cgcaspase-3.

Figure 2. Multiple alignment of the Crassostrea gigas caspase-3 (EKC43168) with caspase-3 s from other species.

The Cgcaspase-3 protein sequence was aligned with caspase-3 from Homo sapiens (CAC88866), Mus musculus (AAH38825), Gallus gallus (NP_990056), Xenopus laevis (NP_001081225), Danio rerio (BAB32409), Strongylocentrotus purpuratus (XP_003727973), and Drosophila melanogaster (AAF55329).

Figure 3. Phylogenic analysis of effector caspases.

Consensus neighbor-joining tree with 1000 bootstrap trials by MEGA program, based on the sequences of Cgcaspase-1 (AEB54801) and Cgcaspase-3 from Crassostrea gigas, and effector caspases from other species. The caspase-7 s protein of other species were selected from Homo sapiens (AAH15799), Mus musculus (AAH05428), Gallus gallus (XP_421764), Xenopus laevis (NP_001081408), Danio rerio (AAH95327), and Strongylocentrotus purpuratus (XP_789183).

Figure 4. Activity assay of Cgcaspase-3 and Cgcaspase-1.

(A) Recombinant expression of both enzymes. The deduced Cgcaspase-3-EGFP protein molecular weight is 74 kDa and the deduced Cgcaspase-1-EGFP protein molecular weight is 61 kDa. The asterisk indicated a non-specific band. (B) DEVDase activity assay of both enzymes. (C) Detection of cell viability with the trypan blue exclusion method. Values are displayed as the mean ± SE of triplicate independent experiments. Differences determined as statistically significant are indicated by asterisks (* P<0.05 and ** P<0.01); ns, not significant.

Figure 5. Subcellular localization of Cgcaspase-3-GFP and Cgcaspase-1-GFP in HeLa cells.

The left-hand panels depict GFP staining, the middle panels depict Hoechst staining, and the right-hand panels depict merged GFP/Hoechst staining. The upper panels depict localization of the EGFP negative control, the middle panel depicts localization of the Cgcaspase-3-EGFP protein, and the lower panel depicts localization of the Cgcaspase-1-EGFP protein. The green fluorescent signal of Cgcaspase-3-GFP was most strongly focused in condensed nuclei, while the signal of Cgcaspase-1-GFP fusion protein existed primarily in the cytoplasm.

Figure 6. Developmental stage distributions of both effector caspase transcripts.

Expression pattern of Cgcaspase-3 (A) and Cgcaspase-1 (B) transcripts at different developmental stages. RS18Q primers was used as internal control primers and a pediveliger larval sample was used as the reference stage. Dates are displayed as the mean ± SE of triplicate independent experiments. E, eggs sample; D, D-shaped larval sample; U, umbo larval sample; P, pediveliger larval sample; HAS, hours after settlement. Differences determined as statistically significant are indicated by asterisks (* P<0.05 and ** P<0.01).

Figure 7. Tissue distributions of both effector caspase transcripts.

Expression pattern of Cgcaspase-3 (A) and Cgcaspase-1 (B) transcripts in different tissues. EF gene expression was used as an internal control and a gonad sample was used as the reference sample. Dates are displayed as the mean ± SE of five independent experiments. Differences determined as statistically significant are indicated by asterisks (* P<0.05 and ** P<0.01).