Lamash NE and Dolmatov IY (2013), Proteases from the regenerating gut of the holo...

ECB-ART-42795

PLoS One 2013 Jan 01;83:e58433. doi: 10.1371/journal.pone.0058433.

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Proteases from the regenerating gut of the holothurian Eupentacta fraudatrix.

Lamash NE , Dolmatov IY .

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Four proteases with molecular masses of 132, 58, 53, and 47 kDa were detected in the digestive system of the holothurian Eupentacta fraudatrix. These proteases displayed the gelatinase activity and characteristics of zinc metalloproteinases. The 58 kDa protease had similar protease inhibitor sensitivity to that of mammalian matrix metalloproteinases. Zymographic assay revealed different lytic activities of all four proteases during intestine regeneration in the holothurian. The 132 kDa protease showed the highest activity at the first stage. During morphogenesis (stages 2-4 of regeneration), the highest activity was measured for the 53 and 58 kDa proteases. Inhibition of protease activity exerts a marked effect on regeneration, which was dependent on the time when 1,10-phenanthroline injections commenced. When metalloproteinases were inhibited at the second stage of regeneration, the restoration rates were decreased. However, such an effect proved to be reversible, and when inhibition ceased, the previous rate of regeneration was recovered. When protease activity is inhibited at the first stage, regeneration is completely abolished, and the animals die, suggesting that early activation of the proteases is crucial for triggering the regenerative process in holothurians. The role of the detected proteases in the regeneration processes of holothurians is discussed.

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Species referenced: Echinodermata
Genes referenced: LOC594261 LOC752081 LOC756768

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	Figure 1. The holothurian Eupentacta fraudatrix. (A) General view of a holothurian with exposed aquapharyngeal complex and tentacles. (B) An animal with eviscerated aquapharyngeal complex, gut and gonad tubules. Abbreviations: ac, aquapharyngeal complex; g, gut; gn, gonad tubules; t, tentacles.
	Figure 2. Scheme of the consecutive stages of internal organ regeneration in the holothurian Eupentacta fraudatrix. An undamaged animal. It contains aquapharyngeal complex with tentacles, gut with mesentery, gonads (not shown) and organs of respiration (respiratory trees). Stage 0 (just after evisceration). The animal after evisceration contains only cloaca, respiratory trees and intestinal mesentery. On the anterior end of the animal the body wall is ruptured. Stage 1 The rupture on the anterior end is closed by thrombus and connective tissue. Stage 2 A connective tissue is thickening; the rudiment of AC is formed at the anterior end of the holothurian. Stage 3 A rod-like thickening, the anterior gut rudiment, begins growing backward from AC rudiment, along the torn edge of mesentery. Stage 4 The anterior gut rudiment elongates. Development of AC is continued. On this stage nerve ring and water-vascular ring canal are formed. Stage 5 The posterior rudiment becomes noticeable. Growth of AC and the anterior gut rudiment is continued. Stage 6 The main structures of AC develop, and the AC increases in size. The anterior gut rudiment continues growing backward, along the mesenteric edge, and reaches the middle of the body. Stage 7 The anterior and posterior rudiments fuse to each other to restore the entire gut. Abbreviations: ac, aquapharyngeal complex; ap, anterior primordium; c, cloaca; g, gut; m, mesentery; pac, primordium of the aquapharyngeal complex; pp, posterior primordium; rt, respiratory trees; t, tentacles.
	Figure 3. Gelatinolytic activity in the gut primordium.(A) Inverted image of zymography of the homogenates: lane 1, 1% Triton X-100 homogenate; lane 2, 1% Na-SDS homogenate. Thirty micrograms of the homogenate protein was resolved by 10% SDS-PAGE in the presence of 1 mg/ml gelatin. (B) Densitometric analysis of horizontally scanned lanes 1 and 2. Results represent the meanÂ±SE of 9 different zymograms corresponding to 3 zymograms from each of the 3 different homogenates per experimental condition (*, high correlation score between gelatinase activity and used detergent, significant with pâ‰¤0.001).
	Figure 4. The effect of storage conditions on the composition of the gut primordium lytic bands.Inverted image of zymography of the homogenates: lane 1: homogenate isolated from a frozen tissue (sample 1); lane 2: frozen homogenate after 20 days of storage (sample 2). White arrows indicate lytic zones and molecular masses of the proteinases (kDa) from the freshly isolated homogenate of the gut primordium. Black arrows indicate new lytic zones and molecular masses of the proteinases (kDa) after thawing the homogenate. Images of gelatin zymograms (10% SDS-PAGE with 0.1% gelatin).
	Figure 5. The effect of substrates on the protease activities of the gut primordium homogenate.(A) Inverted image of zymograms of the same homogenate with gelatin (1) and collagen I (2). Molecular masses (expressed in kDa) are indicated. (B) Densitometric analysis of the scanned bands. Adjusted volume (a.v.): proteinase activity represented the integrated intensity of all the pixels in the band, excluding the background. Results represent the meanÂ±SE of 6 different zymograms corresponding to 3 zymograms from each of the 2 different homogenates per experimental condition (â€“, reliable differences between the compared values, pâ‰¤0.05).
	Figure 6. Inverted image of gelatin zymograms (10% SDS-PAGE) of the same homogenate from the gut primordium.(1) Gel incubated without agent; (2) with 2.5 mM of DTT; (3) with 1% Triton X-100 and 0.1 mM PMSF; (4) with 1% Triton X-100 and the protease inhibitor cocktail; (5) with 2 mM 1,10-phenanthroline. Thirty micrograms of total tissue protein was loaded in each lane. Molecular masses (expressed in kDa) are indicated.
	Figure 7. Gelatinolytic activity in the gut primordium during the process of regeneration.(A) Inverted image of zymograms of gut primordium homogenates from noneviscerated (day 0) and eviscerated (regeneration stages 1, 2, 4, and 6) animals. Molecular masses (expressed in kDa) are indicated. (B) Densitometric analysis of horizontally scanned bands. The percentage (%) represents the integrated intensity of all the pixels in the lytic band excluding the background as a percentage of the total value of horizontally scanned bands. Results represent the meanÂ±SE of 6 different zymograms corresponding to 3 zymograms from each of the 2 different homogenates per experimental condition (*, pâ‰¤0.01 as compared with day 0, nâ€Š=â€Š6).
	Figure 8. Morphology of internal organs in regenerating Eupentacta fraudatrix after inhibition of proteases with 1,10-phenanthroline for 14 days.(A) The general view of internal organs in a control animal. There are well developed anterior gut primordium and primordium of AC. (B) A paraffin section of AC primordium of a control animal. The primordium is composed of loose connective tissue containing numerous cells. There is primordium of pharynx comprising groups of cells with microcavities. (C) Part of AC primordium of a control animal at greater magnification. (D) A paraffin section of gut primordium of a control animal. The digestive epithelium already began developing (arrowheads). (E) The general view of internal organs in an experimental animal. There is only short thickening along the edge of mesentery. The asterisk indicates the end of the thickening. (F) A paraffin section of AC primordium of an experimental animal. The primordium is composed of dense connective tissue. The primordium of pharynx is absent yet. (G) Part of AC primordium of an experimental animal at greater magnification. (H) A paraffin section of gut primordium of an experimental animal. There is only small connective tissue thickening at the edge of mesentery. Abbreviations: dc, dense connective tissue; g, gut primordium; m, mesentery; n, radial nerve cord; p, primordium of the pharynx; pac, primordium of the AC; th, connective tissue thickening; w, radial water-vascular canal.
	Figure 9. Morphology of internal organs in regenerating Eupentacta fraudatrix in 14 days after cancellation of 1,10-phenanthroline treatment.(A) The general view of internal organs in a control animal. There are well developed gut and AC. (B) A paraffin section of AC of a control animal. The AC is composed of loose connective tissue containing numerous cells. There is well-developed pharynx. (C) Part of AC of a control animal at greater magnification. (D) A paraffin section of gut of a control animal. The digestive epithelium develops high folds. (E) The general view of internal organs in an experimental animal. There are well developed gut and AC. (F) A paraffin section of AC of an experimental animal. The AC contains pharynx encircled by a layer of dense connective tissue. (G) Part of AC of an experimental animal at greater magnification. (H) A paraffin section of the gut of an experimental animal. The digestive epithelium forms small folds. Abbreviations: ac, aquapharyngeal complex; dc, dense connective tissue; f, fold of the digestive epithelium; g, gut; m, mesentery; n, radial nerve cord; p, pharynx; w, radial water-vascular canal.

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