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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.
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).
Brix,
Cysteine cathepsins: cellular roadmap to different functions.
2008, Pubmed
Brix,
Cysteine cathepsins: cellular roadmap to different functions.
2008,
Pubmed
Chapman,
Emerging roles for cysteine proteases in human biology.
1997,
Pubmed
Cho,
Mosquito cathepsin B-like protease involved in embryonic degradation of vitellin is produced as a latent extraovarian precursor.
1999,
Pubmed
Cotrin,
Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides to define substrate specificity of carboxydipeptidases: assays with human cathepsin B.
2004,
Pubmed
Dubois,
Regeneration of spines and pedicellariae in echinoderms: a review.
2001,
Pubmed
,
Echinobase
Guiliano,
A gene family of cathepsin L-like proteases of filarial nematodes are associated with larval molting and cuticle and eggshell remodeling.
2004,
Pubmed
Klemencic,
Biochemical characterization of human cathepsin X revealed that the enzyme is an exopeptidase, acting as carboxymonopeptidase or carboxydipeptidase.
2000,
Pubmed
Laemmli,
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
1970,
Pubmed
Lecaille,
Biochemical properties and regulation of cathepsin K activity.
2008,
Pubmed
Lecaille,
Human and parasitic papain-like cysteine proteases: their role in physiology and pathology and recent developments in inhibitor design.
2002,
Pubmed
Magnadóttir,
Innate immunity of fish (overview).
2006,
Pubmed
Magnadóttir,
The ontogenic development of innate immune parameters of cod (Gadus morhua L.).
2004,
Pubmed
Rawlings,
MEROPS: the database of proteolytic enzymes, their substrates and inhibitors.
2012,
Pubmed
Sciani,
Pro-inflammatory effects of the aqueous extract of Echinometra lucunter sea urchin spines.
2011,
Pubmed
,
Echinobase
Sciani,
Cathepsin B/X is secreted by Echinometra lucunter sea urchin spines, a structure rich in granular cells and toxins.
2013,
Pubmed
,
Echinobase
Stachowiak,
Fluorogenic peptide substrates for carboxydipeptidase activity of cathepsin B.
2004,
Pubmed
Therrien,
Cathepsins X and B can be differentiated through their respective mono- and dipeptidyl carboxypeptidase activities.
2001,
Pubmed
Twining,
Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes.
1984,
Pubmed
Wang,
Potential role of cathepsin B in the embryonic and larval development of clam Meretrix meretrix.
2011,
Pubmed
Weiner,
Organic matrixlike macromolecules associated with the mineral phase of sea urchin skeletal plates and teeth.
1985,
Pubmed
,
Echinobase
WILKINSON,
Statistical estimations in enzyme kinetics.
1961,
Pubmed
Wilt,
Biomineralization of the spicules of sea urchin embryos.
2002,
Pubmed
,
Echinobase
