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

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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.

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Acknowledgments

This work has been fully supported by the EU-ITN BIOMINTEC Project, contract number 215507 to V. Matranga. The first author, K. Karakostis, has been the recipient of a Marie Curie ITN program fellowship in the frame of the above-mentioned project. We thank Dr. B. Diehl-Seifert (ed.enilno-t@trefieS.lebreab-tumleh), NanotecMARIN GmbH, Mainz, Germany, for the immunization of mice and the preparation of the anti-Pl-CAN antibodies. Authors acknowledge the technical assistance of M. Biondo for sea urchins maintenance and breeding.

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  1. Konstantinos Karakostis

    Present address: INSERM - UMR 1162, Institute de Génétique Moléculaire, Hôpital St. Louis, 27 rue Juliette Dodu, 75010, Paris, France

Authors and Affiliations

  1. Institute of Biomedicine and Molecular Immunology “A. Monroy”, National Research Council, Via Ugo La Malfa, 153-90146, Palermo, Italy

    Konstantinos Karakostis, Caterina Costa, Francesca Zito & Valeria Matranga

  2. Institute for Biomaterials and Biomolecular Systems, University of Stuttgart, Stuttgart, Germany

    Konstantinos Karakostis & Franz Brümmer

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  1. Konstantinos Karakostis
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Correspondence to Caterina Costa.

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This paper is dedicated to the memory of Valeria Matranga who did not live to see her work published.

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Supplementary Fig. S1

In vitro esterase activity assay of Pl-CAN by hydrolysis of p-nitrophenyl acetate, measured by the absorbance at 405 nm after 30 min. Each sample was tested in triplicate and the standard deviation (SD) was calculated. The CA activity of Pl-CAN was inhibited by the addition of the CA inhibitor acetazolamide (0.1 mM, Pl-CAN + inhibitor). Human CA was used as a positive control. (TIF 670 kb)

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Karakostis, K., Costa, C., Zito, F. et al. Characterization of an Alpha Type Carbonic Anhydrase from Paracentrotus lividus Sea Urchin Embryos. Mar Biotechnol 18, 384–395 (2016). https://doi.org/10.1007/s10126-016-9701-0

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