Baranowska M et al. (2011), Isolation and Characterization of Adhesive Secr...

ECB-ART-42211

Evid Based Complement Alternat Med 2011 Jan 01;2011:486845. doi: 10.1155/2011/486845.

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Isolation and Characterization of Adhesive Secretion from Cuvierian Tubules of Sea Cucumber Holothuria forskåli (Echinodermata: Holothuroidea).

Baranowska M , Schloßmacher U , McKenzie JD , Müller WE , Schröder HC .

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The sea cucumber Holothuria forskåli possesses a specialized system called Cuvierian tubules. During mechanical stimulation white filaments (tubules) are expelled and become sticky upon contact with any object. We isolated a protein with adhesive properties from protein extracts of Cuvierian tubules from H. forskåli. This protein was identified by antibodies against recombinant precollagen D which is located in the byssal threads of the mussel Mytilus galloprovincialis. To find out the optimal procedure for extraction and purification, the identified protein was isolated by several methods, including electroelution, binding to glass beads, immunoprecipitation, and gel filtration. Antibodies raised against the isolated protein were used for localization of the adhesive protein in Cuvierian tubules. Immunostaining and immunogold electron microscopical studies revealed the strongest immunoreactivity in the mesothelium; this tissue layer is involved in adhesion. Adhesion of Cuvierian tubule extracts was measured on the surface of various materials. The extracted protein showed the strongest adhesion to Teflon surface. Increased adhesion was observed in the presence of potassium and EDTA, while cadmium caused a decrease in adhesion. Addition of antibodies and trypsin abolished the adhesive properties of the extract.

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Genes referenced: LOC100887844 LOC594261 prep

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	Figure 1. Scheme of equipment used for measurement of adhesion. (a) Application of the sample between two blocks; the upper block is attached to one of the beams of a laboratory balance, while the lower block is fixed. (b) Attachment of both blocks via the adhesive protein containing sample. (c) Addition of standard weights until both blocks become separated to determine the adhesive forces of the sample.
	Figure 2. Analysis and identification of adhesive protein in Cuvierian tubule extract from sea cucumber H. forskåli. Proteins were analyzed by 12% SDS PAGE and Western blotting. Gels were stained with Gel Code Blue Reagent. (A) Cuvierian tubule extract (4 M urea, 0.5 M Tris-HCl pH 7.5) purified with Ready Prep 2-D Clean up kit. (B) Western blot of Cuvierian tubule extract, incubated with antibody against precollagen D (1 : 1000 dilution) and developed with anti-rabbit IgG alkaline phosphatase. (C) Western blot detection of adhesive protein from H. forskåli, isolated by electroelution, using antibody against precollagen D of mussel M. galloprovincialis (1 : 1000 dilution) (positive control). (D) Western blot of Cuvierian tubule extract incubated with preimmune serum. (E) Western blot of Cuvierian tubule extract incubated with antibodies against isolated (electroeluted) adhesive protein from H. forskåli (dilution 1 : 1000); the serum was purified by treatment with Cuvierian tubule extract (dilution 1 : 10) at a ratio of 1 : 5. (F) Cuvierian tubule extract, separated on seminative gel. (G) Western blot of Cuvierian tubule extract, separated on seminative gel (dilution of antibody, 1 : 1000); the serum was purified by treatment with Cuvierian tubule extract (dilution 1 : 10) at a ratio of 1 : 10. M: Molecular mass markers. Arrowheads: adhesive protein (18 kDa) and dimer (36 kDa).
	Figure 3. Western blot analysis of adhesive protein from Cuvierian tubule extract, purified by immunoprecipitation. The blot was developed using antibodies against adhesive protein from H. forskåli (PoAb-Ctub) and anti-rabbit IgG alkaline phosphatase secondary antibody. (a) Adhesive protein incubated with antibody against adhesive protein from H. forskåli. (b) Protein from mussels (precollagen D), incubated with antibody against adhesive protein from Cuvierian tubules. Numbers to the left indicate molecular masses of marker proteins in kDa.

	Figure 5. Binding of adhesive protein from Cuvierian tubule extract to glass beads in the presence of various concentrations of urea. Glass beads (size, 2 mm) were incubated with adhesive protein extract in the presence of various concentrations of urea (1 M, 2 M, and 3 M; containing 0.5 M Tris-HCl, pH 7.5), as described in Materials and Methods. After washing, the glass beads were boiled in SDS sample buffer and the released protein was analyzed by 12% SDS PAGE. The gel was stained with Gel Code Blue Reagent. Relative band intensities corresponding to the adhesive protein were determined by scanning of the gel using an Odyssey Scanner and applying the Odyssey v.1.2 software to quantify the protein bands.
	Figure 6. Sections of Cuvierian tubules stained with Cason's trichrome (a, b), hematoxylin and eosin (c, d), and methylene blue and azure B (e, f). m: mesothelium; ic: inner connective tissue; gc: granular cells.
	Figure 7. Immunohistological identification of adhesive protein in sections of Cuvierian tubules. (a, b) Sections of Cuvierian tubules stained with antibody against adhesive protein (PoAb-Ctub; 1 : 100 dilution (a) and counterstained with DAPI (b)). (c, d) Sections of Cuvierian tubules stained with preimmune serum (1 : 100; (c)) and counterstained with DAPI (d). Cy3-conjugated goat anti-rabbit IgG was used as secondary antibody. m: mesothelium.
	Figure 8. Immunocytochemical localization of adhesive protein in Cuvierian tubules. (a) Sections of Cuvierian tubules incubated with antibody against adhesive protein from Cuvierian tubules (PoAb-Ctub). (b) Sections of Cuvierian tubules incubated with preimmune serum. Nanogold-labeled anti-rabbit IgG was used as secondary antibody. Arrows: nanogold particles; m: mesothelium.
	Figure 9. Effect of various concentrations of urea on adhesion to Teflon surface. Results are given in arbitrary units ± SD (n = 3).
	Figure 10. Correlation between various concentrations of protein in logarithmic scale and adhesion (standard curve). The highest concentration measured was 100 μg/mL of protein. Results are given in arbitrary units ± SD (n = 3).

	Figure 12. Neutralization of adhesion of H. forskåli extract by antibody (PoAb-Ctub). Results are given in arbitrary units ± SD (n = 3).

References [+] :

Bavington, Anti-adhesive glycoproteins in echinoderm mucus secretions. 2004, Pubmed, Echinobase