Proteomic analysis of skeletal organic matrix from the stony coral Stylophora pistillata

Abstract

It has long been recognized that a suite of proteins exists in coral skeletons that is critical for the oriented precipitation of calcium carbonate crystals, yet these proteins remain poorly characterized. Using liquid chromatography-tandem mass spectrometry analysis of proteins extracted from the cell-free skeleton of the hermatypic coral, Stylophora pistillata, combined with a draft genome assembly from the cnidarian host cells of the same species, we identified 36 coral skeletal organic matrix proteins. The proteome of the coral skeleton contains an assemblage of adhesion and structural proteins as well as two highly acidic proteins that may constitute a unique coral skeletal organic matrix protein subfamily. We compared the 36 skeletal organic matrix protein sequences to genome and transcriptome data from three other corals, three additional invertebrates, one vertebrate, and three single-celled organisms. This work represents a unique extensive proteomic analysis of biomineralization-related proteins in corals from which we identify a biomineralization "toolkit," an organic scaffold upon which aragonite crystals can be deposited in specific orientations to form a phenotypically identifiable structure.

Fig. 1.

Predicted structure of P12, a potential BMP inhibitor. The amino acid sequence (A) and predicted secondary structure (B) of a unique SOM protein after cleavage of the export signal peptide. For the sequence, peptides sequenced by LC-MS/MS are colored orange; translated sequence confirmed from PCR amplification of S. pistillata cDNA is underlined; stop codon is marked with an asterisk; the predicted glycosylation site, Asn79 is indicated by a “+.” In the structure, the N-terminal region corresponding to a potential binding site with a bone morphogenic protein is shown in blue, and Cys potentially involved in a cystine knot fold disulfide bonds between Cys107 and Cys144, and Cys-137 and Cys181 are colored red.

Fig. 2.

CARP subfamily general pattern. (A) Conservation of residues in the CARP subfamily as predicted by ConSurf, with CARP4 as the query. Warmer (more red) and cooler (more blue) colors represent conserved and variable amino acid positions, respectively. Residues are predicted to be exposed (e), buried (b), functional (i.e., highly conserved and exposed; f), or structural (i.e., highly conserved and buried, s). Numbers indicate residue number of CARP4. (B) Schematic of the CARP subfamily of SOM proteins. An N-terminal variable region is followed by a repeat of a highly acidic region plus a highly conserved nonacidic region; the C terminus is variable.