Specific Antibodies to the Fragments of Meningococcal IgA1 Protease during the Formation of Immunity to Bacterial Infections.

The features of individual fragments of IgA1 protease of Neisseria meningitidis serogroup B during the formation of immunity to bacterial infections in animals and humans were studied. The antibodies to the immunogenic regions of the studied proteins are also detected in mice infected with some bacterial pathogens and in humans with bacterial meningitis. A region of IgA1 protease was identified that is not capable of producing antibodies during immunization of animals, but that detects homologous antibodies in the blood of humans and animals recovered from bacterial infections. It has been suggested that this fragment plays a regulatory role in the process of immunogenesis.

Peculiarities of the Formation of Antimeningococcus Immunity in Mice Immunized with Fragments of N. meningitidis IgA1 Protease.

We studied immunogenicity of two recombinant proteins FR.9 and FR.11-3 created on the basis of fragments of the primary structure of N. meningitidis IgA1 protease with different molecular weights containing different sets of T and B epitopes. The proteins actively protect animals infected with live virulent culture of meningococci, serogroups A, B, and C. Analysis of CD4+, CD8+, and CD19+ lymphocyte populations in mouse blood showed predominant contribution of different cell populations to the formation of immune response to different proteins. Injection of FR.11-3 protein to animals did no affect the immunoregulatory index, hence, this protein can be used for creation of immunologically safe vaccine preparation.

Reversible Cyclic Thermal Inactivation of Oligopeptidase B from Serratia proteamaculans.

A unique property was found for oligopeptidase B from Serratia proteamaculans (PSP) as well as its mutants: they can undergo reversible thermal inactivation at 37°C, with activity being restored or even increased with respect to the initial one upon subsequent cooling. The process can be repeated several times, with the same results achieved (up to 5 cycles). This effect can be explained by a shift in the equilibrium between the inactive open form of the enzyme and the active closed one upon variation of the incubation temperature.

Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

Truncated Variants of Serratia proteamaculans Oligopeptidase B Having Different Activities.

Treatment of native psychrophilic oligopeptidase B from Serratia proteamaculans (PSP, 78 kDa) with chymotrypsin (soluble or immobilized on modified porous glass MPG-PA) in the presence of 50% glycerol leads to production of a truncated enzyme form (PSP-Chtr, ~66 kDa), which retains activity toward the low molecular weight substrate of PSP, BAPNA, but in contrast to PSP, is active toward the protein substrate azocasein. It has been shown by MALDI-TOF mass-spectrometry that PSP-Chtr lacks the N-terminal region of the molecule that envelops the catalytic domain of PSP and supposedly prevents hydrolysis of high molecular weight substrates. It has also been established that the lacking fragment corresponds to the N-terminal highest rank element of the informational structure of PSP. This finding confirms the usefulness of the method of informational structure analysis for protein engineering of enzymes. A similar treatment of PSP with immobilized trypsin also led to production of a stable truncated enzyme form (PSP-Tr, ~75 kDa) which lacked 22 C-terminal amino acid residues and completely lost enzymatic activity, presumably because of changes in the nearest environment of His652 of the catalytic triad.

[Protective properties of recombinant IgA1 protease from meningococcus].

The study of enzymatic and protective properties of recombinant IgA1 protease in active and mutant form showed that active form of IgA1 protease exhibited species - and type-specificity for mouse and human immunoglobulins. Mutant form, which did not exhibit enzymatic activity, had protective properties against meningococcal infection, induced by meningococcus serogroup A, B and C protecting the mice from lethal infection by living virulent culture of heterologous serogroups of meningococcus. Obtained results make it possible to consider IgA1 protease as a perspective preparation at the stages of development of polyvalent vaccine for protection the people from meningococcal infection of various etiology.

Oligopeptidase B from Serratia proteamaculans. III. Inhibition analysis. Specific interactions with metalloproteinase inhibitors.

Inhibition of the novel oligopeptidase B from Serratia proteamaculans (PSP) by basic pancreatic trypsin inhibitor, Zn2+ ions, and o- and m-phenanthroline was investigated. A pronounced effect of calcium ions on the interaction of PSP with inhibitors was demonstrated. Inversion voltamperometry and atomic absorption spectrometry revealed no zinc ions in the PSP molecule. Hydrophobic nature of the enzyme inhibition by o- and m-phenanthroline was established.

Oligopeptidase B from Serratia proteamaculans. II. Enzymatic characteristics: substrate analysis, influence of calcium ions, pH and temperature dependences.

Enzymatic properties of a novel oligopeptidase B from psychrotolerant gram-negative microorganism Serratia proteamaculans (PSP) were studied. The substrate specificity of PSP was analyzed using p-nitroanilide substrates, and the influence of calcium ions on the enzyme activity was studied. Hydrolysis of oligopeptides by PSP was studied using melittin as the substrate. Optimal conditions for the PSP activity (pH and temperature) have been established. It was found that PSP shares some properties with oligopeptidases B from other sources containing two Asp/Glu residues in the S2 site, but it differs significantly in some characteristics. The S2 site of PSP contains only one Asp460 residue. The secondary specificity of PSP has a number of specific features: an unusual substrate inhibition by peptides with hydrophobic residues at the P2 position, as well as the drastic influence of calcium ions on substrate characteristics of the enzyme. It is assumed that the PSP molecule contains a large hydrophobic substrate-binding site, and significant conformational rearrangements of the enzyme active site are induced by Ca(2+) binding and by the formation of the enzyme-substrate complex. The temperature characteristics of PSP (high activity at low temperature as well as low apparent temperature optimum (25°C)) confirm that PSP is a psychrophilic enzyme.

Effect of enteropeptidase on survival of cultured hippocampal neurons under conditions of glutamate toxicity.

The effects of full-size bovine enteropeptidase (BEK) and of human recombinant light chain enteropeptidase (L-HEP) on survival of cultured hippocampal neurons were studied under conditions of glutamate excitotoxicity. Low concentrations of L-HEP or BEK (0.1-1 and 0.1-0.5 nM, respectively) protected hippocampal neurons against the death caused by 100 µM glutamate. Using the PAR1 (proteinase-activated receptor) antagonist SCH 79797, we revealed a PAR1-dependent mechanism of neuroprotective action of low concentrations of enteropeptidase. The protective effect of full-size enteropeptidase was not observed at the concentrations of 1 and 10 nM; moreover, 10 nM of BEK caused death of 88.9% of the neurons, which significantly exceeded the cell death caused by glutamate (31.9%). Under conditions of glutamate cytotoxicity the survival of neurons was 26.8% higher even in the presence of 10 nM of L-HEP than in the presence of 10 nM BEK. Pretreatment of cells with 10 nM of either form of enteropeptidase abolished the protective effect of 10 nM thrombin under glutamate cytotoxicity. High concentrations of BEK and L-HEP caused the death of neurons mainly through necrosis.

[Isolation and determination of activity of IgA1 protease from Neisseria meningitidis].

A method of the isolation and purification of IgAl protease from a culture of Neisseria meningitidis serogroup A has been developed. Three inactivated intermediates of the production of the meningococcal vaccine, a culture liquid, as well as a supernatant and sediment obtained by the precipitation of bacterial cells by cetavlon, served as a starting material. The purity of IgA1 protease was determined by SDS-PAGE. An immunoenzyme assay for determining the IgA1 protease activity has been devised. The yield of the enzyme with a specific activity of 0.5 to 4 million units/mg from 103 g of the cetavlon precipitate (40 l of culture liquid) was about 600 mug. It was shown that IgAl protease isolated from serogroup A meningococcus is capable of protecting experimental animals (mice) infected with meningococcus of serogroup B.

[Biodegradable polymer microparticles with entraped herbal extracts: preparation with supercritical carbon dioxide and use for tissue repair].

Biodegradable microparticles based on poly-D,L-lactide with entrapped mixture of herbal water-soluble extracts of Plantago major and Calendula officinalis were prepared. For preparation of these microparticles the previously developed method based on the usage of supercritical carbon dioxide (SC-CO2) was proposed. Microparticles were obtained by two techniques: 1) by preparing porous polymer monolith containing entrapped mixture of herbal extracts, which was then reduced to fine microparticles (ca. 0.1 mm) by dry ice grinding (called here as "monolithisation technique") and 2) by spraying of this polymer/extracts mixture through a jet (spray technique). In vitro release kinetic profile of herbal extract mixture was found to depend on the microparticle preparation technique, on the microparticle structure as well as on the initial ratio polymer/extracts (w/w). The microparticles were used for gastric ulcer treatment in a rat model. The extracts released from microparticles were found to accelerate tissue repair.

Oligopeptidase B from Serratia proteamaculans. I. Determination of primary structure, isolation, and purification of wild-type and recombinant enzyme variants.

A novel trypsin-like protease (PSP) from the psychrotolerant gram-negative microorganism Serratia proteamaculans was purified by ion-exchange chromatography on Q-Sepharose and affinity chromatography on immobilized basic pancreatic trypsin inhibitor (BPTI-Sepharose). PSP formed a tight complex with GroEL chaperonin. A method for dissociating the GroEL-PSP complex was developed. Electrophoretically homogeneous PSP had molecular mass of 78 kDa; the N-terminal amino acid sequence 1-10 was determined, and mass-spectral analysis of PSP tryptic peptides was carried out. The enzyme was found to be the previously unknown oligopeptidase B (OpdB). The S. proteamaculans 94 OpdB gene was sequenced and the producer strain Escherichia coli BL-21(DE3) pOpdB No. 22 was constructed. The yield of expressed His(6)-PSP was 1.5 mg/g biomass.

[Keratinase of an anaerobic thermophilic bacterium Thermoanaerobacter sp. strain 1004-09 isolated from a hot spring in the Baikal Rift zone].

A thermophilic anaerobic bacterial strain 1004-09 belonging to the genus Thermoanaerobacter and capable of growth on protein substrates such as albumin, gelatin, casein, and alpha and beta-keratins was isolated from the Urinskii hot spring (Barguzin river valley, Republic of Buryatia, Russia). A 150-kDa serine proteinase was revealed in the strain supernatant; it exhibited optimal activity at 60 degrees C and pH 9.3 and was capable of keratin hydrolysis. A number of characteristics for the strain 1004-09 keratinase were established including activation by SDS and NaCl and residual activity (15% to the activity of the intact protein) in the presence of 10% ethanol and acetone.

[Selective inhibitors of plasmepsin II from Plasmodium falciparum based on pepstatin].

A number of new inhibitors of plasmepsin II (PlmII) from Plasmodium falciparum, one of the key factors of malarial parasite survival, were synthesized. The inhibitors are analogues of pepstatin with various variants of Ala residue substitutions. Effects of the inhibitors on human PlmII and cathepsin D were studied. Inhibition of PlmII by the substrate was found, which required the use of the modified Henderson method for the determination of inhibition constants. Two synthesized inhibitors were shown to exhibit a pronounced selectivity to PlmII (K(i) = 5.5 and 5 nM) in comparison with cathepsin D (K(i) = 230 and 3000 nM, respectively).

[VI Symposium on "Chemistry of Proteolytic Enzymes"].

The VI Symposium on the Chemistry of Proteolytic Enzymes took place in Moscow on April 23-25, 2007. It was dedicated to the memory of Corresponding Member of the Russian Academy of Sciences Vladimir Konstantinovich Antonov. At the symposium, 40 reports were delivered and 103 posters were presented in the following sections: (1) expression of genes, isolation and general characterization of proteases; (2) structure-function studies of proteases; (3) regulation of the activity of proteolytic enzymes; (4) regulatory functions of proteolytic enzymes; (5) proteases in biotechnology, protein engineering, and peptide synthesis; and (6) proteolysis and medicine. In addition to Russian scientists, researchers from the United States, the Netherlands, France, Ukraine, Belarus, Azerbaijan and Uzbekistan took part in the work of the symposium. Note that, in the five years since the V Symposium, the geography of Russian scientific centers working in the area of proteolysis has been considerably extended. Participating in the forum were researchers from, Novosibirsk, Tomsk, Penza, and Stavropol in addition to scientists from Moscow, Saint Petersburg, Petrozavodsk, Kazan, Nizhni Novgorod, and Krasnodar.

[A rate-limiting stage of enteropeptidase hydrolysis].

It has been shown for the first time that deacylation is the rate-limiting stage in the enteropeptidase-catalyzed hydrolysis of highly efficient oligopeptide substrates containing four Asp residues in positions P2-P5. On the other hand, the rate-limiting stage in the hydrolysis of low-efficiency peptide substrates containing less than four Asp or Glu residues in positions P2-P5 is acylation, as has previously been suggested for all amide and peptide substrates of serine proteases on the basis of the classic studies by Bender et al. The method of introduction of an additional nucleophile or another effector that selectively affects the deacylation stage was used to determine the rate-limiting stage in the enteropeptidase hydrolysis of Nalpha-benzyloxycarbonyl-L-lysine thiobenzyl ester, the highly efficient amide substrate GlyAsp4-Lys beta-naphthyl amide, and the low-efficiency peptide substrate VLSAADK-GNVKAAWG (where a hyphen denotes the hydrolysis site). The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

[Various effects of serine proteinases, activated protein C and duodenase, on mast cells].

Some serine proteinases of haemostasis can regulate blood clotting and inflammation acting at proteinase-activated receptors (PARs). It is known that the anticoagulant proteinase, activated protein C (APC), exhibits anti-inflammatory effects on endothelial cells and macrophages and this involves endothelial protein C receptor--EPCR and proteinase-activated receptor--PAR1. We have studied the effect of wide range of APC concentrations on functional activity of rat peritoneal mast cells (PMC), which secrete the proinflammatory mediators, under normal conditions and during acute inflammation in rats. APC was able to reduce beta-hexosaminidase release from PMC. APC at very low concentrations (0.2-2 nM) modulated the mediator secretion from PMC under normal conditions and also during acute inflammation in rats. APC abolished the proinflammatory activity of duodenase (80 nM), the proteinase from gastrointestinal tract and mast cells. Mast cells pretreated with cathepsin G (PAR1 antagonist) or duodenase abolished protective antiinflammatory effect of low concentrations of APC on PMC degranulation. Our data indicated that blockade of the mast cells proinflammatory mediator secretion by APC involved PAR1 activation.

[Polyelectrolyte microcapsules as systems for delivery of biologically active substances].

Novel biodegradable microcapsules for delivery of biologically active substances (BAS) were prepared by layer-by-layer (LbL) adsorption of oppositely charged polyelectrolytes, namely sodium alginate (Alg) and poly-L-lysine (PLL). To immobilize these BAS, porous spherical CaCO3 microparticles were used as templates. The templates (cores) were coated with several layers of oppositely charged polyelectrolytes forming shell on a core surface. The core-shell microparticles were converted into hollow microcapsules by a core dissolution after an EDTA treatment. Mild conditions for microcapsule fabrication allow to perform an entrapment of various biomolecules while keeping their bioactivity. Biocompatibility and biodegradable capability of the polyelectrolytes give a possibility to use the microcapsules as the target delivery systems. Chymotrypsin (Chym) entrapped into the microcapsules was used as a model enzyme. The immobilized enzyme was found to keep about 86% of the activity compared to a native Chym. The obtained microcapsules were stable at an acidic medium while they could be easily decomposed by trypsin treatment at an slightly alkaline medium. Chym was shown to be active after being released from the microcapsules decomposed by trypsin treatment. Thus, the microcapsules prepared by the LbL - technique can be used for the development of new type of BAS delivery systems in humans and animals.