The skeletome of the red coral Corallium rubrum indicates an independent evolution of biomineralization process in octocorals

Abstract

Background: The process of calcium carbonate biomineralization has arisen multiple times during metazoan evolution. In the phylum Cnidaria, biomineralization has mostly been studied in the subclass Hexacorallia (i.e. stony corals) in comparison to the subclass Octocorallia (i.e. red corals); the two diverged approximately 600 million years ago. The precious Mediterranean red coral, Corallium rubrum, is an octocorallian species, which produces two distinct high-magnesium calcite biominerals, the axial skeleton and the sclerites. In order to gain insight into the red coral biomineralization process and cnidarian biomineralization evolution, we studied the protein repertoire forming the organic matrix (OM) of its two biominerals.

Results: We combined High-Resolution Mass Spectrometry and transcriptome analysis to study the OM composition of the axial skeleton and the sclerites. We identified a total of 102 OM proteins, 52 are found in the two red coral biominerals with scleritin being the most abundant protein in each fraction. Contrary to reef building corals, the red coral organic matrix possesses a large number of collagen-like proteins. Agrin-like glycoproteins and proteins with sugar-binding domains are also predominant. Twenty-seven and 23 proteins were uniquely assigned to the axial skeleton and the sclerites, respectively. The inferred regulatory function of these OM proteins suggests that the difference between the two biominerals is due to the modeling of the matrix network, rather than the presence of specific structural components. At least one OM component could have been horizontally transferred from prokaryotes early during Octocorallia evolution.

Conclusion: Our results suggest that calcification of the red coral axial skeleton likely represents a secondary calcification of an ancestral gorgonian horny axis. In addition, the comparison with stony coral skeletomes highlighted the low proportion of similar proteins between the biomineral OMs of hexacorallian and octocorallian corals, suggesting an independent acquisition of calcification in anthozoans.

Keywords: Axial skeleton; Biomineralization; Corallium rubrum; Evolution; Organic matrix; Proteomics; Sclerites.

Fig. 1

Phylogenetic relationship of anthozoan species used in the present study. Anthozoans are divided into two subclasses: Octocorallia and Hexacorallia. In Octocorallia, 3 species are represented: the red coral Corallium rubrum (order Alcyonacea), which is the model of the present study (red frame), and 2 other species, the soft coral Dendronephthya gigantea (order Alcyonacea) and the blue coral Heliopora coerulea (order Helioporacea). In Hexacorallia, 4 species are represented: 2 stony corals (order Scleractinia) and 2 sea anemones (order Actinaria). Phylogeny is based on www.tolweb.fr. The presence of axial skeleton and/or sclerites and the CaCO₃ polymorph are indicated by colored stars

Fig. 2

Distribution of the proteins identified in the organic matrix of the red coral axial skeleton and sclerites. a Among the total 102 proteins identified in the C. rubrum biominerals, 79 are present in the axial skeleton (left) and 75 are present in the sclerites (right), 52 being shared between the two. The Venn diagram shows the number of shared and non-shared proteins between the 4 extracted organic matrices: SOMax: soluble organic matrix of the axial skeleton, IOMax: insoluble matrix of the axial skeleton, SOMsc: soluble organic matrix of the sclerites, and IOMsc: insoluble organic matrix of the sclerites. b Protein domains identified in the proteome of the red coral biominerals and their distribution using circle representation in SOMax (dark blue), IOMax (blue), SOMsc (yellow) and IOMsc (red). ADAM10 desintegrin and metalloproteinase, AMOP adhesion-associated domain present in MUC4 and other proteins, BPI bactericidal permeability-increasing protein/lipopolysaccharide-binding protein/cholesteryl ester transfer protein N-terminal domain, CA carbonic anhydrase, EGF-like epidermal growth factor, EF-hand a calcium-binding domain, F5/8 type C coagulation factor 5/8 C-terminal domain, FAS1 fasciclin-like, HYR hyalin repeat, IG-like immunoglobulin-like, KAZAL serine protease inhibitor, LamG laminin G, LCD low complexity domain, NIDO extracellular domain of unknown function in nidogen, PAM peptidylglycine α-amidating monooxygenase, PHM peptidylglycine α-hydroxylating monooxygenase, ShK toxic stichodactyla toxin, SCP Cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily proteins, TIMP tissue inhibitor of metalloproteinase, vWA von Willebrand type A, vWD von Willebrand type D, UBA52 Ubiquitin A-52, UOMPs uncharacterized organic matrix proteins, WAP whey acidic protein ‘four-disulfide core’

Fig. 3

Schematic representation of the 10 collagen-like protein sequences identified in the proteome of the red coral biominerals. The collagen sequence identified in the Acropora millepora (UniProt accession number B8V7R6) organic matrix is included. a Schematic protein domain (described in the box below) representation of the different collagen proteins with their respective ID (CR_n). Colored circles indicate presence of the protein in different fractions as described in Fig. 2b. EGF-like epidermal growth factor-like, SP signal peptide, TSPN Thrombospondin N-terminal-like domain, vWA von Willebrand factor type A. Scale bars = 100 amino acids. b Genomic synteny of the CR_5, CR_39, and CR_38 genes and of the CR_9 and CR_35 genes; exons are in green boxes, introns are in blue lines, and intergenic sequences are in dotted lines. Scale bar = 1000 base pairs. c The overall proportion of collagen-like proteins and the most abundant protein, scleritin (CR_1), in each sample in percentage of weighted spectra

Fig. 4

Schematic representation of the 7 proteases and the 4 agrin-like proteins identified in the proteome of the red coral biominerals. a The peptidase S9 (CR_90) and the matrix metallopeptidase 13 (MMP13, CR_61) are specific to the axial skeleton OM. The cathepsin Z (CR_81) and one MMP12A (CR_64) are specific to the sclerites. The peptidase S8 (CR_30), the other MMP12A (CR_16) and the MMP12B (CR_20) are found in both biominerals. b CR_10, CR_60 and CR_28 are part of the same agrin-1 protein. CR_76, CR_82 and CR_62 are part of the same agrin-2 protein. Proteome ID are indicated, circle representation in the different biominerals fractions is as in Fig. 2b, and protein domains are described in the box. C345C netrin C-terminal domain, DISIN disintegrin domain, EGF-like epidermal growth factor-like, F5/8 type C coagulation factor 5/8 C-terminal domain, FOLN follistatin-N-terminal domain-like, LDLa low-density lipoprotein receptor domain class A, ShK toxin stichodactyla toxin, SP signal peptide, TM transmembrane, TY thyroglobulin type I repeats, WAP four-disulfide core domain

Fig. 5

Conservation of the C. rubrum biomineral proteomes with other anthozoan species. Venn diagram depicts the conservation of the 102 C. rubrum OM proteins within anthozoan species. Among the 102 proteins, 77 share sequence similarity with hexacorallian (Scleractinia and Actiniaria) and octocorallian proteins, 1 (CR_72) is shared between Actiniaria and Octocorallia, and 24 are only found in octocorallians. Among the latter 24, 18 are shared in octocorallians and 6 are specific to C. rubrum. The 18 octocorallian proteins are listed and the 6 C. rubrum specific proteins are detailed as in Fig. 3b (pI: isoelectric point). Sequence names in blue correspond to proteins remarkable for their acidic pI and number of negative charges at pH = 8; names in red correspond to protein remarkable for their alkaline pI and number of positive charges at pH = 8 (see Additional file 3 sheet#2)

Fig. 6

Schematic representation of eleven putatively conserved proteins identified in OM of the red coral and the scleractinian coral biominerals and their percentage of identity and similarity. Names, Proteome_ID and sequence description of the 11 C. rubrum proteins are indicated (left). Their presence in the different biomineral fractions is represented with circle as in Fig. 2b, and protein domains are described in the box. The 11 putative homologs were present in the published skeletome of 3 scleractinian species [16, 18, 21] and the OM of C. rubrum biominerals of our study (see Additional file 3 sheet#2). Percentage of identity and similarity between proteins of C. rubrum and proteins identified in the skeletome of the 3 other scleractinian species (Additional file 11) were calculated using https://www.bioinformatics.org/sms2/ident_sim.html and are indicated in the table (right; N: no putative homolog). For each scleractinian protein, GenBank accession number is indicated and published sequence name is given in brackets. Scale bars = 100 amino acids. AMOP adhesion-associated domain present in MUC4 and other proteins, Cadh cadherin repeats, Cadh_C cadherin_C, CAP cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 protein, CA carbonic anhydrase, EGF epidermal growth factor, HH_Nterm hedgehog, IG-like immunoglobulin-like, IGc2 immunoglobulin C-2 type, LamG laminin G, LCD low complexity domain, LDLa low-density lipoprotein receptor domain class A, LY low-density lipoprotein receptor YWTD domain, NIDO extracellular domain of unknown function in nidogen, SH2 Src homology 2, SP signal peptide, TM transmembrane, Tryp_SPc trypsin-like serine protease, TSP1 thrombospondin-1, vWA von Willebrand type A, vWD von Willebrand type D