Protease and DNase Activities of a Very Stable High-Molecular-Mass Multiprotein Complex from Sea Cucumber Eupentacta fraudatrix

Abstract

Only some human organs, including the liver, are capable of very weak self-regeneration. Some marine echinoderms are very useful for studying the self-regeneration processes of organs and tissues. For example, sea cucumbers Eupentacta fraudatrix (holothurians) demonstrate complete restoration of all organs and the body within several weeks after their division into two parts. Therefore, these cucumbers are a prospective model for studying the general mechanisms of self-regeneration. However, there is no data available yet concerning biomolecules of holothurians, which can stimulate the processes of organ and whole-body regeneration. Investigation of these restoration mechanisms is very important for modern medicine and biology because it can help to understand which hormones, nucleic acids, proteins, enzymes, or complexes play an essential role in self-regeneration. It is possible that stable, polyfunctional, high-molecular-weight protein complexes play an essential role in these processes. It has recently been shown that sea cucumbers Eupentacta fraudatrix contain a very stable multiprotein complex of about 2000 kDa. The first analysis of possible enzymatic activities of a stable protein complex was carried out in this work, revealing that the complex possesses several protease and DNase activities. The complex metalloprotease is activated by several metal ions (Zn2+ > Mn2+ > Mg2+). The relative contribution of metalloproteases (~63.4%), serine-like protease (~30.5%), and thiol protease (~6.1%) to the total protease activity of the complex was estimated. Metal-independent proteases of the complex hydrolyze proteins at trypsin-specific sites (after Lys and Arg). The complex contains both metal-dependent and metal-independent DNases. Mg2+, Mn2+, and Co2+ ions were found to strongly increase the DNase activity of the complex.

Keywords: protease and DNase catalytic activities of the complex; sea cucumber Eupentacta fraudatrix; very stable 2000 kDa protein complex.

Figure 1

Dependence of the efficiency of azocasein (3.3 mg/mL) hydrolysis (A₄₃₆) by a stable complex (0.025 mg/mL or ~1.25 × 10⁻⁸ M) on the pH of the reaction medium (A). Effect of preincubation of a stable complex with specific inhibitors of thiol (iodoacetamide), serine (ABSF), and metalloproteases (EDTA), as well as ZnCl₂ and MnCl₂, on its activity in the hydrolysis of azocasein (B). Dependence of the efficiency of azocasein hydrolysis on concentrations of different metal ions (C).

Figure 2

MALDI spectra correspond to 0.8 mg/mL H1 (A–C) and H2A histone (D,E) in the presence of sea cucumber stable complex (0.15 mg/mL or 7.5 × 10⁻⁸ M). Below the spectra, the protein sequences of histones H1 and H2A and the sites of their hydrolyses by the stable complex are shown. Major cleavage sites are indicated by large stars (★), moderate sites by arrows (↓), and minor sites by colons (:) (A–D). Aromatic residues of the protein sequence are marked in grey, after which no histone hydrolysis is observed. RU: relative values.

Figure 3

MALDI spectra correspond to 0.8 mg/mL H2B (A,B) and H3 histone (C,D) in the presence of sea cucumber stable complex (0.15 mg/mL or 7.5 × 10⁻⁸ M). Below the spectra, the protein sequences of histones H2B and H3 and the sites of their hydrolyses by the stable complex are shown. Major cleavage sites are indicated by large stars (★), moderate sites by arrows (↓), and minor sites by colons (:) (A–D). Aromatic residues of the protein sequence are marked in grey, after which no histone hydrolysis is observed. RU: relative values.

Figure 4

MALDI spectra correspond to H4 histone (0.8 mg/mL) over time hydrolysis in the presence of sea cucumber stable complex (0.15 mg/mL or 7.5 × 10⁻⁸ M). Major cleavage sites are indicated by large stars (★), moderate sites by arrows (↓), and minor sites by colons (:) (A–D). Below the spectra, the protein sequence of histone H4 and the sites of its hydrolysis by the stable complex are shown. Aromatic residues of the protein sequence are marked in grey, after which no histone hydrolysis is observed. RU: relative values.

Figure 5

Dependence of the DNA efficiency of hydrolysis by a stable complex (0.015 mg/mL) on the pH of the reaction mixture (A). Effect of the complex preincubation with EDTA, addition of EDTA in reaction mixture, and different metal ions (2.0 mM) on the activity of a complex dialyzed against EDTA (B). Dependence of the efficiency of DNA hydrolysis by the complex dialyzed against EDTA on concentrations of different metal ions (C). Dependence of the DNase activity of the complex on the concentration of CaCl₂ at a constant optimal concentration of MgCl₂ (2.5 mM), as well as the concentration of chlorides of different metals (Ca, Cu, and Mg) at a fixed concentration (5.0 mM) of MnCl₂ (D).

Figure 6

In situ assay of DNase activity of proteins of a stable complex (15 µg) using reducing conditions. DNase activity was revealed as dark bands on the fluorescent background (lane in situ) by ethidium bromide staining. A part of the gel corresponding to the destructed complex was stained with Coomassie R250 to show the position of complex proteins (lane C-blue). Lane C corresponds to proteins with known MWs. See Materials and Methods for further details.