Sciani JM et al. (2013), Cathepsin B/X is secreted by Echinometra lucunt...

ECB-ART-43166

J Venom Anim Toxins Incl Trop Dis 2013 Dec 16;191:33. doi: 10.1186/1678-9199-19-33.

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Cathepsin B/X is secreted by Echinometra lucunter sea urchin spines, a structure rich in granular cells and toxins.

Sciani JM , Antoniazzi MM , Neves Ada C , Pimenta DC .

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BACKGROUND: Echinometra lucunter is a common American sea urchin responsible for the majority of the marine accidents in Brazil. Although not lethal, these accidents are reported to be extremely painful. Recently, our group described the presence of toxins in its spines that contribute to the pathological reactions. Additionally, we have observed that the E. lucunter spines can regenerate when broken. In the present work we evaluated the enzymatic activities of sea urchin spine extracts in order to identify an enzyme that could contribute not only to the toxicity, but also participate in the spine growth and regeneration. RESULTS: The spine aqueous extract was tested for peptidase activity, with synthetic substrates, in the presence and absence of inhibitors and activators. For proper enzyme classification, the FRET-substrate cleavage pattern, pH-dependency activity and Western-blot analyses were performed. The spine extract was able to cleave Z-R-MCA and Abz-GIVRAK(Dnp)-OH following pre-incubation with DTT, and was inhibited by E-64. Furthermore, the double-peaked pH curve (5 and 7) and the cleavage site proportion (4:6, R↓A:A↓K) indicate the presence of both mono and dicarboxypeptidase activities. Moreover, in Western-blot analysis, the spine extract was positive for anti-cathepsin B antibody. CONCLUSIONS: E. lucunter spines extracts presented a cysteine peptidase activity that was identified as cathepsin B/X that would participate in the remodeling and growth processes of the spine, as well as in the inflammatory response to the accident.

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Genes referenced: LOC100887844 LOC100893907 LOC576823 LOC594261

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	Figure 1. Photography of the Echinometra lucunter spine tip: (A) intact spine; (B) regeneration process.
	Figure 2. Enzymatic characterization of the spine extract. Kinetic data of velocity over concentration of substrate, after the incubation of spine aqueous extract (12 μg) with (A) Z-R-MCA and (B) Abz-GIVRAK(Dnp)-OH. (C) Enzyme titration using E-64 inhibitor, performed over Abz-GIVRAK(Dnp)-OH substrate. (D) Determination of pH for optimum activity of spine aqueous extract, over Abz-GIVRAK(Dnp)-OH substrate. (E) HPLC profile, in λ = 365 nm, of the products of complete hydrolysis of the Abz-GIVRAK(Dnp)-OH substrate by spine aqueous extract. (F) Mass spectrometry analysis of the products of Abz-GIVRAK(Dnp)-OH hydrolysis by spine aqueous extract.
	Figure 3. Proteic characterization of the spine extract. (A) SDS-PAGE (10%) of aqueous extract of spine. M = molecular mass standard, SE = spine extract. Arrows indicate the molecular masses. (B) Western blotting of 5, 10 and 20 μg aqueous extract of spine, incubated with anti-cathepsin B antibody. Left lane, molecular mass standard. (C) Chromatogram obtained by gel filtration of spine aqueous extract. Dashed lines indicate the enzymatic activity.
	Figure 4. Immunohistochemical test for anti-cathepsin B antibody was performed in transversal spine sections. (A) Spine section stained with toluidin-fuchsin. (B) Spine section incubated with anti-cathepsin B. (C and D) zoomed images, corresponding to A and B, respectively. It is possible to observe the positive (brownish staining) along the decalcified matrix (B), in the same location where cells were observed in the sections stained by toluidine-fuchsin (A).

References [+] :

Brix, Cysteine cathepsins: cellular roadmap to different functions. 2008, Pubmed