Carbonic anhydrases (CAs) are zinc ion-containing metalloenzymes that can catalyze the essential hydration of CO₂ through the simple chemical reaction: CO₂ + H₂O ⇄ HCO₃^– + H⁺ (Lindskog and Silverman, 2000). CAs play an essential role in multiple physiological processes such as pH regulation, electrolyte balance, ionic transportation, carboxylation or decarboxylation reactions, biocalcification, and tumorigenicity (Supuran, 2008, 2011; Alterio et al., 2009). CAs are important components of the CO₂-concentrating mechanisms in different groups of algae. They could increase the rate of photosynthesis (Qu et al., 2018). In erythrocytes, CA is a superabundant enzyme that plays an indispensable role in CO₂ transport by catalyzing the dehydration of plasma HCO₃^– ions (Geers and Gros, 2000; Henry and Swenson, 2000; Perry and Gilmour, 2006). The cytosolic CA in gill may contribute to provide counter ions for maintaining pH balance and ionic regulation in fish (Henry and Swenson, 2000; Marshall, 2002). In mollusk, CA seems to be contributed to shell formation via catalyzing the hydration of CO₂ (Nielsen and Frieden, 1972). This enzyme has been shown to be an effective catalyst in the calcification mechanism of coral (Rahman and Oomori, 2010).

Carbonic anhydrase isozymes were isolated in the erythroid cells of mammals and have been subsequently identified in most organisms (Meldrum and Roughton, 1933; Rudenko et al., 2015). Eight evolutionarily distinct families of CAs, including α, β, γ, δ, ζ, η, θ, and ι, have been reported in unicellular and multicellular organisms (Zolfaghari et al., 2020). Their amino acid residues share no significant identities and seem to be evolved independently from distinct inherited genes (Krishnamurthy et al., 2008; Bertucci et al., 2009; Del Prete et al., 2015; Supuran and Capasso, 2015; Kikutani et al., 2016). Among these families, α-CA is widely distributed in animals and plants (Aspatwar et al., 2010). α-CA exhibits the highest catalytic activity in the hydration reaction than β- and η-CA. On the contrary, γ-, δ-, and ζ- CA isozymes possess the lowest enzymatic efficiencies (Capasso and Supuran, 2015; Supuran and Capasso, 2015). In mammals, 16 isoforms of α-CA isozymes have been explored, of which 13 are catalytically effective and 3 are non-catalytic due to the absence of one or more functionally active histidine amino acid residues (Sly and Hu, 1995; Tashian et al., 2000). The functions of each isozyme vary pursuant to their molecular sequences, kinetic attributes, sensitivities to inhibitors, tissue distributions, and subcellular localizations (Hewett-Emmett, 2000; Lehtonen et al., 2004).

Carbonic anhydrase II is a secreted and membrane-bound α-CA that can catalyze carboxylation and decarboxylation reactions. The typical structure of CA II contains three histidine (His) residues that can bind to Zn²⁺ ion, and a proton (H⁺) shuttling residue that is responsible for converting a Zn-bound water molecule to hydroxide ion. In addition, gate-keeping signature residues (namely, Glu-106 and Thr-199 in human CA) allow excellent orientation of Zn-bound hydroxide ion to increase the nucleophilic attack of a substrate (Christianson and Fierke, 1996; Lindskog and Silverman, 2000). CA II not only participates in the hydration reaction but also plays an important role in the osmoregulatory functions of fish (Grosell et al., 2007).

The Pacific abalone is a commercially important molluscan bioresources in China, Japan, and Korean Peninsula. Haliotis discus hannai is considered as a popular seafood item worldwide due to its contents of health beneficial bioactive molecules (Suleria et al., 2017). Previous studies have characterized cytosolic CA isozymes in vertebrates and invertebrates (Pongsomboon et al., 2009; Le Roy et al., 2012; Ali et al., 2015; Pan et al., 2016; Sumi et al., 2019). However, the characterization and expression analysis of CA isozymes in Pacific abalone have not yet been reported. In this study, the complete sequence of CA II isozyme was first cloned from the mantle of H. discus hannai, and its spatiotemporal expression was determined using the molecular assay.

Carbonic anhydrases play an important role in many physiological processes by catalyzing the hydration reaction. In mollusks, CAs have been previously identified in Tridacna squamosa (Ip et al., 2017), Mytilus galloprovincialis (Perfetto et al., 2017), and H. tuberculata (Le Roy et al., 2012). To date, the identification and biomolecular characterization of CA II isoform from the Pacific abalone have not yet been reported. For the first time, the complete sequence of CA II was cloned from the mantle tissue of H. discus hannai and the molecular properties of this protein with its expression profile were determined in this study. An 18-amino-acid NH₂-terminal signal sequence was found in the CA II isozyme followed by a cleavage site, suggesting that HdhCA II might be an extracellular secretory protein (Figure 1). The N-terminal signal sequence is a key characteristic of CA secretory protein (Aldred et al., 1991). A secretory CA has been cloned from the scleractinian coral, Stylophora pistillata, and this CA is localized in calicodermis, which is responsible for the precipitation of the skeleton (Moya et al., 2008). One CA isoform was isolated from the sea urchin embryo and described as an extracellular secreted protein (Karakostis et al., 2016). The cloned sequence of HdhCA II also possesses several key features including phosphorylation sites and N-linked glycosylation sites. These phosphorylation sites are crucial for several signal transduction cascades (Ali et al., 2016). Two potential N-linked glycosylation motifs were found in HdhCA II, suggesting that HdhCA II might be a glycoprotein. Two cysteine residues found in HdhCA II might form disulfide link that is crucial for stabilizing its protein structure and regulating biological functions of this protein (Kadokura et al., 2004; Inaba et al., 2006).

The amino acid sequences encoded by HdhCA II displayed high identities in the functional site of the CA domain (Figure 2). The molecular structure of HdhCA II isozyme contained important functional sites, such as zinc binding ligand, proton shuttling ligand, substrate associated pocket, and Thr-199 loop site, which are known to be involved in the enzymatic activity of this protein (Esbaugh and Tufts, 2006). The active site of CA contained a hydrophobic pocket (i.e., the catalytically productive site) that could interact with a non-polar CO₂ substrate (Liang and Lipscomb, 1990) and facilitate its reaction with highly nucleophilic Zn²⁺-bound OH^– (Alterio et al., 2012). The hydrophobic binding pocket residue and histidine residues that could bind with a catalytic zinc ion were conserved in CA II of H. discus hannai (Figure 2). This suggests that HdhCA II is a functionally active CA. It has been well established that the proton-shuttling residue (His-64) is responsible for the efficient proton transfer and the high catalytic rate of CO₂ hydration in CA II of human (Supuran, 2008). A site-specific mutation (His-64 replaced by alanine) results in 20- to 30-fold decrease in the catalytic activity of CA II (Tu et al., 1989). This residue is also conserved among different species of abalone CA II.

The results of phylogenetic analysis indicated that CA II gene of H. discus hannai was evolutionarily closer to H. gigantea CA II (Figure 3). Previous studies reported that the CA II of H. tuberculata (htCA2) is placed in the molluscan CA clade and more closely linked to CA II isoform of H. gigantea (Le Roy et al., 2012).

The homology modeling of HdhCA II was performed using the 3D homology structure of human CA II with a resolution of 1.07 Å as template (Figure 4). The Zn²⁺ coordinated with three conserved histidine residues comprise the zinc-binding site (Christianson and Alexander, 1989). The evaluation results also supported the structural conservation of the cloned HdhCA II gene, with amino acids in favorable positions.

The expression of HdhCA II mRNA was detected in all tested tissues with mantle as the site of highest expression (Figure 5) which is in agreement with the previous report (Le Roy et al., 2012; Ip et al., 2017). The expression analysis suggests that HdhCA II could involve in mantle function such as shell formation. HdhCA II might also involve in acid–base regulation, ion transport, and modulation of ionic concentration (Miyashita et al., 2012).

The temporal expression profile of HdhCA II during ontogenesis revealed that HdhCA II mRNA was expressed throughout the early developmental stages, with shell formation stage having the highest level (Figure 6). This result of analysis suggests that HdhCA II plays an important role during larval shell formation. Previous studies have reported that the functional inhibition of CA in Paracentrotus lividus and Heliocidaris tuberculata can prevent the deposition of calcium carbonate in the larval skeleton formation (Zito et al., 2015).

The expression of HdhCA II mRNA in the mantle tissue was examined with ISH using an antisense CA II mRNA as a probe. The gene distributed in the mantle can speculate the participation of these genes in the biomineralization process during shell formation (Suzuki and Nagasawa, 2013). The expression of genes at the mantle edge and mantle pallial have participated in the synthesis of prismatic and nacreous layers, respectively (Takeuchi and Endo, 2006; Inoue et al., 2010). The expression patterns of HdhCA II transcript were detected in the epithelium layer of the mantle and mantle pallial (Figure 5). The mantle can secrete biomineralization protein in outer epithelial cells to modulate shell formation (Jablonski, 1990; Werner et al., 2013). Based on the in situ results of the present study, we speculated that HdhCA II might be involved in the shell formation by catalyzing the hydration of CO₂.

This is the first study of molecular characterization and expression of HdhCA II mRNA in different tissues and developmental stages of the Pacific abalone. HdhCA II was highly expressed in the mantle tissue implying that it might be participated in the shell formation process. The expression patterns of HdhCA II during larval developmental stages imply that this enzyme is involved in the shell germination of abalone. The ISH results demonstrated that the signals were found in the mantle epithelial cells, indicating that this gene might be essential for the shell formation by controlling the regular deposition of CaCO₃. The findings of our current research could help us to understand the functional role of CA in the shell formation of abalone or be useful for the development of aquaculture methods in this abalone species.

The original contributions presented in the study are publicly available. This data can be found here: NCBI GenBank, accession number: MT876410, https://www.ncbi.nlm.nih.gov/nuccore/MT876410.1.

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of CNU (approval number: CNU IACUC-YS-2020-5).

KK conceptualized and designed the experiments and prepared the manuscript. MS designed and conducted the experiment, analyzed the data, and wrote the manuscript. ZS conducted the ISH, analyzed the data, and prepared graphs. KS analyzed the data. SC and KC helped to plan the experiment, revised the manuscript, and gave intellectual input to improve it. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.