Very Stable Two Mega Dalton High-Molecular-Mass Multiprotein Complex from Sea Cucumber Eupentacta fraudatrix

Abstract

In contrast to many human organs, only the human liver can self-regenerate, to some degree. Some marine echinoderms are convenient objects for studying the processes of regenerations of organs and tissues. For example, sea cucumbers Eupentacta fraudatrix can completely restore within several weeks, the internal organs and the whole body after their division into two or three parts. Therefore, these cucumbers are a very convenient model for studying the general mechanisms of regeneration. However, there is no literature data yet on which biomolecules of these cucumbers can stimulate the regeneration of organs and the whole-body processes. Studying the mechanisms of restoration is very important for modern biology and medicine, since it can help researchers to understand which proteins, enzymes, hormones, or possible complexes can play an essential role in regeneration. This work is the first to analyze the possible content of very stable protein complexes in sea cucumbers Eupentacta fraudatrix. It has been shown that their organisms contain a very stable multiprotein complex of about 2000 kDa. This complex contains 15 proteins with molecular masses (MMs) >10 kDa and 21 small proteins and peptides with MMs 2.0-8.6 kDa. It is effectively destroyed only in the presence of 3.0 M MgCl₂ and, to a lesser extent, 3.0 M NaCl, while the best dissociation occurs in the presence of 8.0 M urea + 0.1 M EDTA. Our data indicate that forming a very stable proteins complex occurs due to the combination of bridges formed by metal ions, electrostatic contacts, and hydrogen bonds.

Keywords: model of tissues and body regeneration; sea cucumber Eupentacta fraudatrix; very stable 2000 kDa protein complex.

Figure 1

Isolation and analysis of sea cucumber complex. FPLC gel filtration of sea cucumber homogenate proteins on a Sepharose 4B column (A). Gel filtration of the purified complex (A) on the Sepharose 4B, MM = ≈2000 kDa (B). Gel filtration of the complex after its treatment using harsh conditions: 50 mM acidic buffer (pH 2.6), 8.0 M urea, 2 mM DTT, and 1.0 M NaCl (C). In all Panels, (^__)—absorbance at 280 nm (A₂₈₀).

Figure 2

Typical examples of the time changes in the light scattering (LS) of the cucumber complex (0.005 mg/mL) in the presence of 20 mM Tris-HCl buffer (pH 7.5) containing urea, NaCl, MgCl₂, DTT, and EDTA in various concentrations and different combinations, detailed in the Figure.

Figure 3

SDS-PAGE analysis of the proteins of sea cucumber complex (25 μg) using 3.0–17.0% gradient gel. The arrows of Lane C indicate the positions of markers of molecular masses. See Materials and Methods for other details.

Figure 4

MALDI spectra of peptides and small proteins of sea cucumber complex. The fraction of peptides and small proteins was prepared by dissociation of the complex by trifluoroacetic acid in dioxane and its filtration through filters passing compounds with a MMs s <10 kDa. See Materials and Methods for other details.