Molecular Characterization of a Dual Domain Carbonic Anhydrase From the Ctenidium of the Giant Clam, Tridacna squamosa, and Its Expression Levels After Light Exposure, Cellular Localization, and Possible Role in the Uptake of Exogenous Inorganic Carbon

Abstract

A Dual-Domain Carbonic Anhydrase (DDCA) had been sequenced and characterized from the ctenidia (gills) of the giant clam, Tridacna squamosa, which lives in symbiosis with zooxanthellae. DDCA was expressed predominantly in the ctenidium. The complete cDNA coding sequence of DDCA from T. squamosa comprised 1,803 bp, encoding a protein of 601 amino acids and 66.7 kDa. The deduced DDCA sequence contained two distinct α-CA domains, each with a specific catalytic site. It had a high sequence similarity with tgCA from Tridacna gigas. In T. squamosa, the DDCA was localized apically in certain epithelial cells near the base of the ctenidial filament and the epithelial cells surrounding the tertiary water channels. Due to the presence of two transmembrane regions in the DDCA, one of the Zn²⁺-containing active sites could be located externally and the other one inside the cell. These results denote that the ctenidial DDCA was positioned to dehydrate [Formula: see text] to CO₂ in seawater, and to hydrate the CO₂ that had permeated the apical membrane back to [Formula: see text] in the cytoplasm. During insolation, the host clam needs to increase the uptake of inorganic carbon from the ambient seawater to benefit the symbiotic zooxanthellae; only then, can the symbionts conduct photosynthesis and share the photosynthates with the host. Indeed, the transcript and protein levels of DDCA/DDCA in the ctenidium of T. squamosa increased significantly after 6 and 12 h of exposure to light, respectively, denoting that DDCA could participate in the light-enhanced uptake and assimilation of exogenous inorganic carbon.

Keywords: Symbiodinium; bicarbonate; calcification; carbon dioxide; symbiosis; tridacnid.

Figure 1

Molecular characterization of the Dual Domain Carbonic Anhydrase (DDCA) of Tridacna squamosa. (A) An amino acid sequence alignment of the DDCA of T. squamosa and tgCA of T. gigas. (B) A comparison of the two CA domains of the DDCA of T. squamosa. The shaded residues indicate identical or highly similar amino acids. The signal sequence is labeled and indicated by a black box. The residues that are single underlined make up the first CA domain, while the second CA domain is double underlined. The open star marks the putative residue which attaches a GPI anchor. The asterisks indicate the gatekeeper residues that allow efficient proton-transfer. The residues that coordinate the catalytic Zn²⁺ ion are marked by hash signs. The open triangles indicate the residues that form the CO₂ binding sites. The hydrophilic residues that make up the binding sites for ${\text{HCO}}_3^{-}$ are marked by arrows. The transmembrane domains (TMs) are indicated by a red box. The lack of one hydrophilic residue that is involved in ${\text{HCO}}_3^{-}$ binding in the first CA domain of DDCA is marked by a blue box.

Figure 2

A phenogramic analysis of the Dual Domain Carbonic Anhydrase (DDCA) of Tridacna squamosa. A phenogram illustrating the relationship of the DDCA of T. squamosa with all known CAs of Homo sapiens, Tridacna gigas CA (tgCA) and CAs of several species of algae. The Ca of the bacterium Nostoc sp. (PCC7120) was used as the outgroup. The number located at each branch point represents the bootstrap value (max = 1,000).

Figure 3

Gene expression of the Dual Domain Carbonic Anhydrase (DDCA) in tissues/organs of Tridacna squamosa. The mRNA expression of the DDCA in the outer mantle (OM), inner mantle (IM), ctenidium (Cten), foot muscle (FM), byssal muscle (BM), heart, hepatopancreas (HP), and kidney (Kid) of T. squamosa kept in darkness for 12 h (control). A negative control (NTC) was included in the first lane.

Figure 4

Effects of light on the mRNA expression level of Dual Domain Carbonic Anhydrase (DDCA) in the ctenidium of Tridacna squamosa. Absolute quantification (× 10⁴ copies of transcript per ng of total RNA) of DDCA transcripts in the ctenidium of T. squamosa kept in darkness for 12 h (control), or exposed to light for 3, 6 or 12 h, or kept in darkness for another 12 h (a total of 24 h in darkness). Results represent means + S.E.M. (N = 4). Means not sharing the same letters are significantly different from each other (P < 0.05).

Figure 5

Effects of light on the protein abundance of Dual Domain Carbonic Anhydrase (DDCA) in the ctenidium of Tridacna squamosa. The protein abundance of DDCA in the ctenidium of T. squamosa kept in darkness for 12 h (control), or exposed to light for 3, 6 or 12 h, or kept in darkness for another 12 h (a total of 24 h in darkness). (A) Examples of an immunoblot of DDCA and tubulin (reference protein), and an immunoblot of DDCA using the anti-DDCA antibody pretreated with peptide competition (PC). (B) The optical density of the DDCA band for a 25 μg protein load was normalized with respect to that of tubulin. Results represent means + S.E.M. (N = 4). Means not sharing the same letter are significantly different from each other (P < 0.05).

Figure 6

Immunofluorescence localization of the Dual Domain Carbonic Anhydrase (DDCA) in the tertiary water channels (WCs) of the ctenidium of Tridacna squamosa. Immunofluorescence localization of the DDCA in the WCs of the ctenidium of T. squamosa exposed to 12 h of light (A–D) or 12 h of darkness (control; E–H). Differential interference contrast (DIC) images show the lattice formation of WCs (A,E). Anti-DDCA immunofluorescence is shown in green (B,F) with nuclei counterstained with DAPI in blue (C,G). Green and blue channels are merged and overlaid with the respective DIC images (D,H). Arrowheads indicate DDCA-immunostaining at the apical membrane of the epithelial cells surrounding the WCs. HL, hemolymph. Scale bar: 20 μm. Reproducible results were obtained from four individual clams for each experimental condition.

Figure 7

Immunofluorescence localization of the Dual Domain Carbonic Anhydrase (DDCA) in the ctenidial filaments (CFs) of the ctenidium of Tridacna squamosa. Immunofluorescence localization of the DDCA in the CFs of T. squamosa exposed to 12 h of light (A–D) or 12 h of darkness (control; E–H). Differential interference contrast (DIC) images show the structure of CFs (A,E). Anti-DDCA immunofluorescence is shown in green (B,F) with nuclei counterstained with DAPI in blue (C,G). Green and blue channels are merged and overlaid with the respective DIC images (D,H). Arrowheads indicate DDCA-immunostaining at the apical membrane of the epithelial cells located at the base of CFs and those surrounding the tertiary water channels (WCs). Scale bar: 20 μm. Reproducible results were obtained from four individual clams for each experimental condition.

Figure 8

A validation of the immunostaining of the Dual Domain Carbonic Anhydrase (DDCA) using a peptide competition assay (PCA). The immunofluorescence localization of DDCA in the ctenidial filaments (CFs) and tertiary water channels (WCs) of a ctenidium of Tridacna squamosa exposed to 12 h of light using an anti-DDCA antibody (A,C), or the same anti-DDCA antibody pre-incubated with the immunizing peptide in PCA (B,D). The anti-DDCA immunofluorescence is shown in green, overlaid with DAPI nuclei staining and differential interference contrast images. Arrowheads in (A,C) indicate DDCA-immunostaining at the apical membrane of some epithelial cells at the base of CFs and WCs. By contrast, there is a lack of anti-DDCA antibody staining in the control with PCA (B,D) in both CFs and WCs. HL, hemolymph. Scale bar: 20 μm.