title: 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.
authors.affiliation: Institute of Biomedicine and Molecular Immunology "A. Monroy", National Research Council, Via Ugo La Malfa, 153-90146, Palermo, Italy.|Institute for Biomaterials and Biomolecular Systems, University of Stuttgart, Stuttgart, Germany.|INSERM - UMR 1162, Institute de Génétique Moléculaire, Hôpital St. Louis, 27 rue Juliette Dodu, 75010, Paris, France.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Karakostis
authors.affiliation: Institute of Biomedicine and Molecular Immunology "A. Monroy", National Research Council, Via Ugo La Malfa, 153-90146, Palermo, Italy. caterina.costa@ibim.cnr.it.
authors.forename: Caterina
authors.initials: C
authors.lastname: Costa
authors.affiliation: Institute of Biomedicine and Molecular Immunology "A. Monroy", National Research Council, Via Ugo La Malfa, 153-90146, Palermo, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Zito
authors.affiliation: Institute for Biomaterials and Biomolecular Systems, University of Stuttgart, Stuttgart, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Brümmer
authors.affiliation: Institute of Biomedicine and Molecular Immunology "A. Monroy", National Research Council, Via Ugo La Malfa, 153-90146, Palermo, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Matranga
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002256:Carbonic Anhydrases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004625:Embryo, Nonmammalian; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D007526:Isoelectric Point; D008970:Molecular Weight; D016366:Open Reading Frames; D009928:Organ Specificity; D047328:Paracentrotus / Q000145:classification / Q000196:embryology / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D000072417:Protein Domains; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D002256:Carbonic Anhydrases
keywords: Biomineralization; Development; Gene expression; Recombinant proteins; Skeletogenesis