Molecular cloning and characterization of first organic matrix protein from sclerites of red coral, Corallium rubrum

Abstract

We report here for the first time the isolation and characterization of a protein from the organic matrix (OM) of the sclerites of the alcyonarian, Corallium rubrum. This protein named scleritin is one of the predominant proteins extracted from the EDTA-soluble fraction of the OM. The entire open reading frame (ORF) was obtained by comparing amino acid sequences from de novo mass spectrometry and Edman degradation with an expressed sequence tag library dataset of C. rubrum. Scleritin is a secreted basic phosphorylated protein which exhibits a short amino acid sequence of 135 amino acids and a signal peptide of 20 amino acids. From specific antibodies raised against peptide sequences of scleritin, we obtained immunolabeling of scleroblasts and OM of the sclerites which provides information on the biomineralization pathway in C. rubrum.

FIGURE 1.

Silver staining of electrophoresis gels and Western blots on organic matrix extracted from sclerites of C. rubrum. a, one-dimensional SDS-PAGE (10 μg protein/lane; BisTris 12% polyacrylamide gel, silver stain molecular mass marker (M6539; Sigma) as protein marker). b, one-dimensional SDS-PAGE (10 μg protein/lane, Tris-Tricine 16.5% polyacrylamide gel, kaleidoscope polypeptide standards (161-0325; Bio-Rad) as protein marker); c, two-dimensional electrophoresis gel (Tris-Tricine 16.5% polyacrylamide gel, kaleidoscope polypeptide standards (161-0325) as protein marker) of the major protein band (18*) excised from one dimensional SDS-PAGE. d, Western blot (5 μg of proteins/lane; BisTris 12% polyacrylamide gel, Precision Plus Protein WesternC^TM standard (161-0376; Bio-Rad) as protein marker) with antibodies against phosphorylated serine. e, Western blot with antibodies against the phosphorylated threonine (5 μg of proteins/lane; BisTris 12% polyacrylamide gel, Precision Plus Protein WesternC^TM standard (161-0376) as protein marker). f, Western blot with antibodies against phosphorylated tyrosine (5 μg of proteins/lane; BisTris 12% polyacrylamide gel, Precision Plus Protein WesternC^TM standard (161-0376) as protein marker). g, Western blot with anti-scleritin antibodies (Tris-Tricine 16.5% polyacrylamide gel, Precision Plus Protein WesternC^TM standard (161-0376) as protein marker). MW, molecular mass; M, protein marker; and triangles show apparent molecular mass of proteins (in kDa).

FIGURE 2.

Nucleotide gene and amino acid sequences of scleritin. Nucleotide sequence above (GenBank accession number JQ652458) and deduced translation of the ORF (nucleotides 40–507) below. The putative signal peptide is underlined, the peptide sequence obtained after Edman degradation is highlighted in gray, the two consecutive peptides obtained by MS/MS are indicated in bold letters, the potential N-glycosylation site is highlighted in black (Asp-152), the five potential O-glycosylation sites (Thr-24, Ser-56, Ser-57, Thr-150, and Ser-154) are indicated by asterisks, and the seven potential phosphorylation sites are boxed (Thr-24, Ser-65, Ser-72, Ser-73, Thr-113, Tyr-129, and Tyr-147).

FIGURE 3.

Histology and immunohistochemistry of a longitudinal section of a demineralized branch of C. rubrum. a, overview of a section stained with hemalun/eosin/aniline blue which, respectively, stain nuclei and cytoplamic and connective regions observed with a bright field light microscope. b–h, labeling with antibodies against scleritin (orange to yellow) is merged with blue labeling of cell nuclei with DAPI: b, immunolabeling of apical region of the colony; c, immunolabeling of tissues close to a polyp; d, immunolabeling of oral epithelium; e, immunolabeling at the interface between aboral epithelium and organic matrix of axial skeleton; f, immunolabeling at the base of a branch; g, magnification of e showing labeling of organic matrix of sclerites inside the organic matrix of axial skeleton. h, magnification of f showing labeling of organic matrix of a sclerite inside a scleroblast. Observations from b to h were performed with a confocal microscope. SW, sea water; po, polyp; oe, oral epithelium; sc, scleroblast; gc, gastrodermic canal; ae, aboral epithelium; ax om, organic matrix of axial skeleton; scl om, organic matrix of sclerite. Each image was obtained by Z-stack reconstruction of 10 tissue sections, with 1-μm step.

FIGURE 4.

Diagram of sclerite incorporation in medullar part of axial skeleton at apex of a branch of C. rubrum. From the inside to the outside: medullar part of the axial skeleton (med); annular part of the axial skeleton (ann); aboral epithelium (ae); mesoglea (mes); scleroblast (scl); and oral epithelium (oe). Scleritin from the organic matrix of sclerites is indicated by yellow asterisks.