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

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.

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

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.

Systemic lupus erythematosus: molecular cloning of fourteen recombinant DNase monoclonal kappa light chains with different catalytic properties.

BACKGROUND: DNase antibodies can play an important role in the pathogenesis of different autoimmune pathologies. METHODS: An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with systemic lupus erythematosus (SLE) was used. The small pools of phage particles displaying DNA binding light chains with different for DNA were isolated by affinity chromatography on DNA-cellulose and the fraction eluted with 0.5M NaCl was used for preparation of individual monoclonal light chains (MLChs, 28kDa). Forty-five of 451 individual colonies were randomly chosen for a study of MLChs with DNase activity. The clones were expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography followed by gel filtration, and studied in detail. RESULTS: Fifteen of 45 MLChs efficiently hydrolyzed DNA, and fourteen of them demonstrated various optimal concentrations of KCl or NaCl in a 1-100mM range and showed one or two pH optima in a 4.8-9.1 range. All MLChs were dependent on divalent metal cations: the ratio of relative DNase activity in the presence of Mn(2+), Ca(2+), Mg(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each MLCh preparation. Fourteen MLChs demonstrated a comparable affinity for DNA (260-320nM), but different kcat values (0.02-0.7min(-1)). CONCLUSIONS: These observations suggest an extreme diversity of DNase abzymes from SLE patients. GENERAL SIGNIFICANCE: SLE light chain repertoire can serve as a source of new types of DNases.

Systemic lupus erythematosus: molecular cloning and analysis of recombinant DNase monoclonal κ light chain NGK-1.

Because DNase antibodies are cytotoxic, enter the nucleus and cause DNA fragmentation inducing cell death by apoptosis, they can play an important role in the pathogenesis of different autoimmune pathologies and especially systemic lupus erythematosus (SLE). The interesting goal of catalytic antibodies research is not only to study a possible biological role of such antibodies, but also to develop in future new human and animal therapies that use the advantages offered by abzymes. An immunoglobulin κ light chain library from SLE patients was cloned into a phagemid vector. Phage particles displaying recombinant monoclonal antibody light chains (MLChs) capable of binding DNA were isolated by affinity chromatography on DNA-cellulose. Sixteen of the 46 MLChs efficiently hydrolyzed DNA; one MLCh (approximately 27-28kDa) was expressed in Escherichia coli and purified by metal chelating and gel filtration. MLCh NGK-1 was electrophoretically homogeneous and demonstrated a positive answer with mouse IgGs against light chains of human antibodies after western blotting. SDS-PAGE in a gel containing DNA demonstrated that the MLCh hydrolyzes DNA and is not contaminated by canonical DNases. The DNase MLCh was activated by several metal ions. The protein sequence of the DNase MLCh has homology with mammalian DNases I and shares with them several identical or similar (with the same side chain functionality) important amino acid residues, which are necessary for DNA hydrolysis and binding of Mg(2+) and Ca(2+) ions. The affinity of DNA for this first example of a MLCh (K(M) = 0.3 microM) was 150- to 200-fold higher than for human DNase I.

Systemic lupus erythematosus: molecular cloning of several recombinant DNase monoclonal kappa light chains with different catalytic properties.

An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of three patients with systemic lupus erythematosus was used. Phage particles displaying DNA binding light chains were isolated by affinity chromatography on DNA-cellulose, and the fraction eluted by an acidic buffer (pH 2.6) was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Thirty three of 687 individual colonies obtained were randomly chosen for study of MLCh DNase activity. Nineteen of 33 clones contained MLChs with DNase activity. Four preparations of MLChs were expressed in Escherichia coli in soluble form, purified by metal chelating chromatography followed by gel filtration, and studied in detail. Detection of DNase activity after SDS-PAGE in a gel containing DNA demonstrated that the four MLChs are not contaminated by canonical DNases. The MLChs demonstrated one or two pH optima. They were inactive after the dialysis against ethylenediaminetetraacetic acid but could be activated by several externally added metal ions; the ratio of relative activity in the presence of Mg(2+) , Mn(2+) , Ni(2+) , Ca(2+) , Zn(2+) , and Co(2+) was individual for each MLCh preparation. K(+) and Na(+) inhibited the DNase activity of various MLChs at different concentrations. Hydrolysis of DNA by all four MLCh was saturable and consistent with Michaelis-Menten kinetics. These clones are the first examples of recombinant MLChs possessing high affinity for DNA (Km = 3-9 nM) and demonstrating high kcat values (3.4-6.9 min(-1) ). These observations suggest that the systemic lupus erythematosus light chain repertoire can serve as a source of new types of DNases.

Diversity of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL/MpJ-lpr mice.

It has been shown for the first time that polyclonal IgG abzymes (Abzs) with DNase activity from the sera of autoimmune-prone MRL/MpJ-lpr mice can be separated by isoelectric focusing into many subfractions having the isoelectric points (pI) from 4.5 to 9, with the maximal activity for Abzs with pI = 6.5-9.0. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions demonstrating a range of affinities for DNA and different levels of the relative DNase activities (RDA) due to intrinsically bound metals and after addition of external Mg(2+) , Mn(2+) , Ca(2+), and Mg(2+) +Ca(2+). Some fractions significantly increase RDAs in the presence of external ions (Mg(2+) + Ca(2+) > Mg(2+) > Mn(2+) > Ca(2+)), while each of this cofactor can also inhibit or have no influence on the RDAs of another fractions. It is known that complexes of DNA with histones and other proteins of apoptotic cells are the primary immunogens in systemic lupus erythematosus (SLE). Bovine serum albumin (BSA) and methylated BSA (mBSA) increase the RDAs of only some fractions, while have no effect or inhibit other IgG fractions. The ratio of the RDAs in the presence of all metal ions, BSA, and mBSA was individual for every abzyme fraction. Mn(2+) and Ca(2+) stimulated accumulation of only relaxed form of supercoiled DNA (scDNA) in the case of all subfractions, while in the presence of Mg(2+) antibodies (Abs) of some subfractions (and in the presence of Mn(2+) +Ca(2+) all subfractions) produced relaxed DNA (rDNA) and linear DNA (linDNA) in a variable extent. The data obtained show that the polyclonal Abzs of mice may be a cocktail of Abs directly to DNA, RNA, and their complexes with proteins and anti-idiotypic Abs to active centers of different nucleases. The diversity of the physicochemical and kinetic characteristics of the Abzs seems to be significantly widened when pre-diseased mice spontaneously develop the disease.

Extreme Diversity of IgGs Against Histones, DNA, and Myelin Basic Protein in the Cerebrospinal Fluid and Blood of Patients with Multiple Sclerosis.

It was recently shown that IgGs from sera of multiple sclerosis (MS) patients are active in the hydrolysis of DNA and myelin basic protein (MBP). We first analyzed the relative concentration of antibodies against five histones (H1, H2a, H2b, H3, and H4) in the cerebrospinal fluid (CSF) and serum of patients with MS. The relative concentrations of blood and CSF IgGs against histones and their activity in the hydrolysis of five histones varied greatly from patient to patient. However, all 28 IgG preparations were hydrolyzed from one to five histones. Relative activities and correlation coefficients among the activities of IgGs from serum and CSF in the hydrolysis of five histones (H1, H2a, H2b, H3, and H4), DNA, and MBP were calculated. It was shown that auto-IgGs from CSF and sera of MS patients are extremely heterogeneous in their affinity to histones, MBP, and DNA. The heterogeneity of IgG-abzymes hydrolyzing DNA, MBP, and histones from CSF and sera was also demonstrated using their isoelectrofocusing. The isofocusing profiles DNase, MBP-, and histone-hydrolyzing activities of IgGs may be very different for various individuals, but the total IgG subfractions with all their activities are distributed from pH 3 to 10.