Fig 1.
Histology of C. rubrum and dissection protocol to obtain the three different fractions.
(A) Photo of a C. rubrum colony. (B) Transversal cryosection of a demineralized branch stained with Hemalin-Eosin and acetified Aniline blue. (C) Schematic representation of C. rubrum organization. OE: Oral epithelium; Mgl: Mesoglea; Po: Polyps; Ske: Axial skeleton; SE: Skeletogenic epithelium; GC: Gastrodermal canals; Scl: Scleroblasts/Free sclerites. (D) Dissection protocol to obtain the three fractions for qPCR and western blot.
Table 1.
Characteristics of CruCA isozymes in C. rubrum.
Fig 2.
Clustal Omega alignment of CruCA proteins.
100%, 80%, and 60% conserved amino acids are shaded in black, dark grey, and light grey, respectively (GeneDoc software, score table BLOSUM62). The N-terminus signal peptide sequences are colored in blue. The predicted GPI-anchor site is indicated by a violet rectangle in the C-terminus of CruCA5. The three zinc-binding histidines are indicated by red boxes. The proton shuttle residue is indicated by inverted green triangle. The two gatekeeper residues are indicated by inverted yellow triangles. The cysteines involved in disulfide bonds are represented as follow: the Cys57~Cys250 (CruCA2 nomenclature), common to all extracellular cnidarian α-CAs, is shown with orange triangles; the Cys138~Cys284, specific to the secreted cnidarian α-CAs cluster, is shown with inverted orange triangles. The multiple phosphorylation sites in the C-terminus tail of CruCA5 are shown by a full circle above the predicted residues.
Fig 3.
Relative gene expression of CruCA isozymes and western blot on proteins extracted from different tissue fractions of C. rubrum.
(A) Gene expression in total tissues, relative to RPLP0 expression. Letters represent statistical differences based on one-way ANOVA (p<0.05). (B) Gene expression in tissues enriched in calcifying cells and in polyps, normalized to RPLP0 and relative to the expression in total tissues. Asterisks indicate values statistically different between fractions based on Student’s t-test with p<0.05. Error bars represent standard error of the mean. (C) Western blot with anti-CruCA3 (29 kDa) (12% polyacrylamide gel). (D) Western blot with anti-CruCA4 (33 kDa) (18% polyacrylamide gel). (E) Loading control western blot with anti-actin (42 kDa) (12% polyacrylamide gel). Lane 1: Total tissues extract; lane 2: Tissues enriched in calcifying cells fraction; lane 3: Polyps fraction. MW: molecular weight of Precision Plus Protein™ All Blue Standard (Bio-Rad).
Fig 4.
Phylogenetic relationships of 71 cnidarian α-CA protein sequences.
α-CAs from human (HsCAI-XIV), two calcareous sponges: Sycon ciliatum (SciCA1-9), Leucosolenia complicata (LcoCA1-6); and from cnidarians: Corallium rubrum (CruCA1-6), Stylophora pistillata (SpiCA1-16), Acropora millepora (AmiCA1-10), Nematostella vectensis (NveCA1-7), Aiptasia pallida (ApaCA1-10), and Hydra magnipapillata (HmaCA1-15), as well as the CARPs identified in different anthozoan databases, i.e Anthopleura elegantissima (AelCARP1-2), Favia sp. (FaviaCARP), Porites australiensis (PauCARP1-2), Pocillopora damicornis (PdaCARP). Sequences were aligned with Clustal Omega and the tree was constructed using PhyML and Bayesian inference methods. The presented topology results from the PhyML method. Node support values indicate PhyML-aLRT values / MrBayes-bootstrap posterior probabilities. Only values above 50% are indicated. The bacterial CA sequences from Pectobacterium atrosepticum, Klebsiella pneumoniae and Nostoc sp. were used as outgroup. Cnidarian α-CAs could be grouped in three main clusters: i) the cytosolic and mitochondrial α-CAs with no disulfide bond, ii) the secreted and membrane bound α-CAs with the canonical disulfide bond, and iii) the secreted α-CAs with two putative disulfide bonds, the canonical and the cnidarian specific disulfide bond. Genes encoding the SpiCA8, 9, 12, the SpiCA13, 14, and the NveCA6, 7 are found closely associated on the same contig. CARP specific innovations and disulfide bonds are indicated for cnidarians. The bottom right inset presents the number of CA and CARP isozymes for each species. Sp.: species; C: calcifying organism (CaCO3 skeletons); S: symbiotic organism.
Fig 5.
Cnidarian Carbonic Anhydrase Related Proteins (CARPs).
(A) The histidine residue is encoded by the codon C.A.C/T. Single mutation resulting from transition (grey arrow head) or transversion (grey small arrow) encodes for the amino acids (one letter code) depicted in the top left panel. (B) Amino acids, and their codons, found in place of the three zinc-binding histidines within the CARP (cytosolic and secreted) identified among cnidarians. (C-D) 3D structure was calculated for each of the full length cnidarian CARP. Pictures are superimposition of the different cytosolic (C) and secreted (D) CARPs models, showing the three residues listed in the left table (B) as well as the overall structure. Black arrowheads indicate the two putative cyst-cyst bridges on the secreted CARPs structure.
Fig 6.
Predicted 3D structure of CruCA isozymes.
(A) Ribbon diagram of each CruCA 3D structure modeling. The three histidines coordinating the zinc ion at the active site are shown in bright colors. The “acatalytic” isoform, CruCA2, displays histidine, arginine and tyrosine residues in yellow. (B) Superimposition of the six CruCAs showing their high structural homology. (C) Superimposition of the three zinc-binding histidines of each CruCA, and histidine, arginine and tyrosine for CruCA2 in yellow.