Characterization of an Alpha Type Carbonic Anhydrase from Paracentrotus lividus Sea Urchin Embryos

Abstract

Carbonic anhydrases (CA) are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the sea urchin, CA has a role in the formation of the calcitic skeleton during embryo development. Here, we report a newly identified mRNA sequence from embryos of the sea urchin Paracentrotus lividus, referred to as Pl-can. The complete coding sequence was identified with the aid of both EST databases and experimental procedures. Pl-CAN is a 447 aa-long protein, with an estimated molecular mass of 48.5 kDa and an isoelectric point of 6.83. The in silico study of functional domains showed, in addition to the alpha type CA-specific domain, the presence of an unexpected glycine-rich region at the N-terminal of the molecule. This is not found in any other species described so far, but probably it is restricted to the sea urchins. The phylogenetic analysis indicated that Pl-CAN is evolutionarily closer to human among chordates than to other species. The putative role(s) of the identified domains is discussed. The Pl-can temporal and spatial expression profiles, analyzed throughout embryo development by comparative qPCR and whole-mount in situ hybridization (WMISH), showed that Pl-can mRNA is specifically expressed in the primary mesenchyme cells (PMC) of the embryo and levels increase along with the growth of the embryonic skeleton, reaching a peak at the pluteus stage. A recombinant fusion protein was produced in E. coli and used to raise specific antibodies in mice recognized the endogenous Pl-CAN by Western blot in embryo extracts from gastrula and pluteus.

Ion Pathways in Biomineralization: Perspectives on Uptake, Transport, and Deposition of Calcium, Carbonate, and Phosphate

“…Three carbonic anhydrase enzymes specific to PMCs, have been identified. 48 It is likely that carbonic anhydrase contributes to the regulation of carbonate homeostasis, shifting the equilibrium in the direction of carbonate production, when protons are removed. Additional sources of carbonate may be carbonate transporters from the blastocoel, or synthesis from the oxygen processed during respiration in the mitochondria.…”

Comparison of Carbonic Anhydrases for CO2 Sequestration

“…The culture volume was not given, but 1 g of wet E. coli cell pellet was reported to yield 1 mg of CA [50]. Marine species, such as mussels, diatoms and sea urchin exhibit functional CAs, which were mainly designated to the α-class [51][52][53][54]. In the literature, primarily their esterase activity rather than their hydratase activity was determined.…”

Molecular Characterization of Carbonic Anhydrase II (CA II) and Its Potential Involvement in Regulating Shell Formation in the Pacific Abalone, Haliotis discus hannai

“…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.…”