New insights into the apoptotic process in mollusks: characterization of caspase genes in Mytilus galloprovincialis

Abstract

Apoptosis is an essential biological process in the development and maintenance of immune system homeostasis. Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis. Although the genes encoding caspase proteins have been described in vertebrates and in almost all invertebrate phyla, there are few reports describing the initiator and executioner caspases or the modulation of their expression by different stimuli in different apoptotic pathways in bivalves. In the present work, we characterized two initiator and four executioner caspases in the mussel Mytilus galloprovincialis. Both initiators and executioners showed structural features that make them different from other caspase proteins already described. Evaluation of the genes' tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells. Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens. To understand the mechanisms of caspase gene regulation, hemocytes were treated with UV-light, environmental pollutants and pathogen-associated molecular patterns (PAMPs) and apoptosis was evaluated by microscopy, flow cytometry and qPCR techniques. Our results suggest that the apoptotic process could be tightly regulated in bivalve mollusks by overexpression/suppression of caspase genes; additionally, there is evidence of caspase-specific responses to pathogens and pollutants. The apoptotic process in mollusks has a similar complexity to that of vertebrates, but presents unique features that may be related to recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature.

Figure 1. Modular architecture of initiator and executioner caspase proteins.

Initiator and executioner labeled as A and B, respectively. ScanProsite was used to predict the domain features of all caspase proteins. DED domain in mussel caspase-8 was predicted by the SMART software.

Figure 2. Positions of critical residues for protein structure, substrate binding and catalysis.

Green letters represent the CARD and DED domains. The caspase-8 DED domain is highlighted in green. Catalytic subunits P20 and P10 are represented in orange and blue letters, respectively. Asp (D) residues at potential cleavage site between the large and small subunits were highlighted in pink. S1 site (Arg179, Gln283) is highlighted in yellow, S2 site (Ser339) and S3 site (Arg341) are highlighted in blue and S4 site (Trp348) is highlighted in red. The catalytic cysteine conserved site (QACxG) and the catalytic dyad are marked in dark green. Numbering is based on caspase-1 residue positions.

Figure 3. Phylogenetic analysis of initiator caspases.

Neighbor-joining (NJ) phylogenetic tree for initiator caspase proteins. Numbers on branches are bootstrap percentages.

Figure 4. Phylogenetic analysis of executioner caspases.

Neighbor-joining (NJ) phylogenetic tree for executioner caspase proteins. Numbers on branches are bootstrap percentages.

Figure 5. Expression of the six caspase genes in different tissues.

Results represent the mean ± SD of 3 different samples. Fold change units were calculated by dividing the normalized expression values in the different tissues by the normalized expression values obtained in the muscle. Data were analyzed using the Student’s t-test. NS: no significant differences regarding to the expression levels detected in the muscle.

Figure 6. Histological changes related to the apoptotic process observed in hemocytes treated with UV light.

A and D: Control granulocytes and small hyalinocytes not treated with UV-light. B: Slight chromatin condensation in granulocytes observed after 3 h pt. C and F: Appearance of intracellular bodies, positive stained for DNA, inside the cytoplasm of granulocytes and hyalinocytes, respectively after 24 h pt. E: Chromatin condensation in small hyalinocytes after 6 h pt.

Figure 7. Evaluation of apoptosis induced in hemocytes treated with UV light by flow cytometry.

A: Hemocytes were divided into two populations according to their FSC and SSC characteristics. B and C: Changes of apoptotic levels during the time course in R1 and R2 populations, respectively. Results represent the mean ± SD of data from 9 hemocyte populations. Data were analyzed using the Student’s t-test. * Indicates significant differences (p<0.05).

Figure 8. Kinetics of caspase gene expression in hemocytes treated with UV light.

Results represent the mean ± SD of 3 pools (3 hemocyte populations per pool). Data were analyzed using the Student’s t-test. A: Evolution of the expression levels of initiator caspase-2 and caspase-8. a and b indicate significant differences in caspase-2 and -8, respectively. B: Evolution of the expression levels of executioner caspase-3/7-1, -3/7-2, -3/7-3 and -3/7-4. c, d, e and f indicate significant differences (p<0.05) in caspase-3/7-1, -2, -3 and -4, respectively.

Figure 9. Induction of caspase genes in hemocytes treated with different PAMPs at 1, 3 and 6 h pt.

Results represent the mean ± SD of 4 experimental hemocyte pools. Data were analyzed using the Student’s t-test. a, b, c, d, e and f indicate significant differences (p<0.05) of caspase-2, -8, 3/7-1, -3/7-2, -3/7-3 and -3/7-4, respectively.

Figure 10. Modulation of caspase gene expression in hemocytes treated with bezopyrene, phenanthrene and PCBs.

Results represent the mean ± SD of 4 experimental hemocyte pools. Data were analyzed using the Student’s t-test. a, b, c, d, e and f indicate significant differences (p<0.05) of caspase-2, -8, 3/7-1, -3/7-2, -3/7-3 and -3/7-4, respectively.