A. V. Bacheva

Peptide synthesis in organic media with subtilisin 72 immobilized on poly(vinyl alcohol)-cryogel carrier.

Serine proteinase subtilisin 72 was covalently attached to the beads of poly(vinyl alcohol)-cryogel, a macroporous hydrogel prepared by the freeze-thaw technique. The immobilized enzyme was examined as a catalyst in the synthesis of protected peptides Z-Ala-Ala-Xaa-Phe-pNA (Xaa = Leu, Glu, Lys) in acetonitrile/dimethylformamide mixtures. Immobilized subtilisin catalyzed with high yield the formation of peptide bonds between Phe-pNA and acyl donors including those with free carboxylic group and non-protected C-terminal basic and acidic amino acid residues.

Immunoproteasome enhances intracellular proteolysis of myelin basic protein

300 Proteasome is a multisubunit protein complex that exhibits proteolytic activity and is present in the nuclei and cytoplasm of cells. The 20S proteasome, which has a molecular weight of 700 kDa and a sedimenta tion coefficient of 20S, is present as a proteolytic core in a more complex particle, the 26S proteasome [1]. The degradation of proteins in the cell is regulated by the ubiquitinylation system, which marks the old or defective protein molecules for their recognition by the proteasome and subsequent proteolysis [2]. One of the key biological functions of the proteasome is the hydrolysis of intracellular proteins to the antigenic peptides, which are then presented on the cell surface on the major histocompatibility complex class I mole cules [3]. Recent studies indicate the existence of a molecular mechanism by which the peptides gener ated by the proteasome can also be presented on the major histocompatibility complex class II molecules [4]. The catalytic activity of a constitutive proteasome is mediated by three subunits, β1, β2, and β5, which are constitutively expressed in cells. The proteasome, which contains corresponding immunosubunits β1i, β2i, and β5i the catalytic center, is called the immuno proteasome and is significantly different from the con stitutive proteasome in its activity and substrate speci ficity. The set of antigenic peptides produced by the immunoproteasome differs from the set of peptides produced by the constitutive proteasome [5, 6]. It was recently shown that immunoproteasome not only changes the degradation spectrum of antigenic pro teins but also ensures the maintenance of protein homeostasis under conditions of oxidative stress caused by the action of interferons on the cell [7]. The amount of immunoproteasome in cells increases in various diseases (hematologic malignancies [8], rheu matoid arthritis [9], autoimmune colitis [10], Alzhe imer’s disease [11], and Huntington disease [12]). One of the most common and socially significant autoimmune diseases is multiple sclerosis (MS), which is characterized by the destruction of the myelin sheath of nerve fibers. Myelin basic protein (MBP) is a major autoantigen in multiple sclerosis. At present, the molecular mechanisms underlying the develop ment of multiple sclerosis are being actively studied. Recent studies have demonstrated an important role of both the constitutive proteasome and the immuno proteasome in the development of this disease [13]. Earlier, we studied in vitro the proteolysis of MBP by the proteasome isolated from normal mice and mice with experimental autoimmune encephalomy elitis (EAE), an experimental model of MS [14]. Dur ing further development of this research, we created a model to study the intracellular proteolysis of MBP in mammalian cells. Here we show that the intracellular hydrolysis of MBP is significantly accelerated when the proteasome–immunoproteasome balance is shifted toward the latter. It is known that the expression of the myelin basic protein in mammals is detected solely in the central and peripheral nervous systems. This protein is local ized in the membrane of specialized cells, oligoden drocytes and Schwann cells, forming the myelin sheath of axons. Unfortunately, work with primary human neuronal cultures is associated with numerous experimental difficulties (first of all, the lack of mate rial and ethical concerns). In view of this, at the first step of this work, to study the proteolysis of MBP ex vivo we created a genetic construct that made it possible to express a recombinant human MBP in mammalian cells. For this purpose, a DNA fragment 546 bp long, encoding the full length MBP, was amplificated by PCR using specific overlapping oligo nucleotides and then cloned into the pBudCE4.1/EF Immunoproteasome Enhances Intracellular Proteolysis of Myelin Basic Protein

Efficient subtilisin immobilization in chitosan, and peptide synthesis using chitosan–subtilisin biocatalytic films

The immobilization of serine protease subtilisin Carlsberg on/in chitosan, a hydrophilic biopolymer was investigated in this study. Using different techniques, biocatalysts in the form of gels, fibers and films were prepared. Subtilisin loading and biocatalytic properties of obtained samples were studied. The optimal method of enzyme incorporation was pre-mixing of chitosan acetate and enzyme solutions, then air-drying and treatment with glutaraldehyde. It was found that this form of biocomposite has the highest loading capacity (up to 20 mg protein g−1 support) and good mechanical properties. The dependence of subtilisin loading on the starting concentration of the enzyme during immobilization shows the maximum loading at [E] = 15 mg mL−1. The dependence of subtilisin loading and activity on the concentration of the glutaraldehyde was explored. The biocatalytic films were active catalysts for peptide bond formation (with 25–92% product yield) in nonaqueous media, demonstrating high activity and stability under these conditions. They are also active in aqueous media, bringing about the reverse reaction, and can therefore be used for either peptide bond formation or cleavage.

Activity and stability of native and modified subtilisins in various media.

The activity and stability of native subtilisin 72, its complex with poly(acrylic acid), and subtilisin covalently attached to poly(vinyl alcohol) cryogel were studied in aqueous and organic media by hydrolysis of specific chromogenic peptide substrates. Kinetic parameters of the hydrolysis of Glp-Ala-Ala-Leu-pNA by native subtilisin and its complex with poly(acrylic acid) were determined. Based on the comparative study of stability of native and modified subtilisins in media of various compositions, it was established that covalent immobilization of subtilisin on poly(vinyl alcohol) cryogel is the most effective approach to improve enzyme stability in water as well as in mixtures with low water content.

Peptide synthesis in organic media with the use of subtilisin 72 immobilized on a poly(vinyl alcohol) cryogel

Subtilisin 72 serine protease (EC 3.4.21.14) immobilized on a poly(vinyl alcohol) cryogel was used as a catalyst in the syntheses of N-protected peptide p-nitroanilides of the general formulas Z(or Boc)-Xaa-Phe-pNA (Xaa = Leu or Ala), Z-Ala-Xaa-Yaa-pNA (Xaa = Leu or Ala; Yaa = Leu or Phe), and Z-Ala-Ala-Xaa-Yaa-pNA (Xaa = Leu, Arg, or Gly; Yaa = Phe, Leu, Gly, Asp, or Glu). The syntheses were carried out in DMF-acetonitrile mixtures. A number of protected di-, tri-, and tetrapeptides were prepared in yields up to 99%. The syntheses were found to retain stereoselectivity under the conditions studied. The activation of carboxyl group of the acylating component was shown to have a positive effect upon the coupling rate.

Functional degradation of myelin basic protein. The proteomic approach

Proteolytic degradation of autoantigens is of prime importance in current biochemistry and immunology. The fundamental issue is the functional role of peptides produced in the process of change of the hydrolysis specificity during the transition from the normal to a pathologic state. In some cases identification of specific peptide fragments can be a diagnostic and prognostic criterion of the pathology progress. The subject of this work is the comparative study of degradation peculiarities of one of the major neuroantigens, myelin basic protein, by proteases activated upon the development of a pathological demyelinating process, and by proteasomes of different origin. Comparison of the specificity of the tested biocatalysts in some cases demonstrated critical changes in the set of myelin basic protein fragments capable of being presented on the major histocompatibility complex class I upon neurodegeneration, which may promote the development of autoimmune pathological processes.

Stability and catalytic properties of subtilisin in acetonitrile/dimethylformamide mixtures with low water content

Subtilisin 72 suspension retained high activity and stability in the triple mixtures acetonitrile rDMFrwater DMF .. concentration was up to 70% vrv . The synthetic activity of subtilisin suspension was investigated using the model wx w x reaction of tetrapeptide Z-Ala-Ala-Leu-Phe-pNA formation from Z-Ala-Ala-Leu-OMe and Phe-pNA S s 30 mM, E s wx w x . 6 mM; S r E molar ratio 5000:1 . In the systems containing up to 60% DMF the 95% product yield was reached within 2 h. With DMF concentration increasing to 95%, the subtilisin catalytic efficiency notably decreased, though the product yield was still 30%. In the mixture acetonitriler80%DMFrwater, the effects of enzyme concentration, the reaction time and water content on the reaction progress were studied. q 2000 Elsevier Science B.V. All rights reserved.

Chemoenzymatic synthesis of new fluorogenous substrates for cysteine proteases of the papain family

A chemoenzymatic synthesis was developed for new highly specific fluorogenic substrates for cysteine proteases of the papain family, Abz-Phe-Ala-pNA (I) and Glp-Phe-Ala-Amc (II) (Abz, pNA, Glp, and Amc are o-aminobenzoyl, p-nitroanilide, pyroglutamyl, and 4-amino-7-methylcoumaride, respectively). Substrate (I) was obtained in an aqueous-organic medium using native chymotrypsin. Substrate (II) was synthesized in DMF-MeCN by the treatment with chymotrypsin and subtilisin Carlsberg immobilized on polyvinyl alcohol cryogel. Hydrolysis of substrate (I) with papain, ficin, and bromelain was accompanied by a 15-fold increase in fluorescence intensity, and that of substrate (II), by a change in the fluorescence spectrum. Unambiguity of enzymatic hydrolysis of the substrates after the Ala residue was shown. The specific activity of the substrate hydrolysis with papain, bromelain, and ficin and was determined. Papain showed the greatest activity for both substrates. The activity of all proteases under study was essentially higher for substrate (II), than for substrate (I). The lowest detectable papain concentrations were 2.4 × 10−10 M for (I) and 1.2 × 10−11 M for (II). A high selectivity of cysteine proteases for Glp-Phe-Ala-Amc was established.

Preparation and catalytic properties of trypsin immobilized on cryogels of polyvinyl alcohol

Commercial preparations of trypsin, varying in activity, were immobilized on a cryogel of polyvinyl alcohol, activated by dialdehydes (terephthalic, succinic, or glutaric) or divinyl sulfone. All preparations of the immobilized enzyme exhibited hydrolytic activity and retained stability for 8 months. In organic media, specimens of immobilized trypsin catalyzed the synthesis of N-carbobenzoxy-L-phenylalanyl-L-arginyl-L-leucine p-nitroanilide from N-carbobenzoxy-L-phenylalanyl-L-arginine methyl ester (or N-carbobenzoxy-L-phenylalanyl-L-arginine) and L-leucine p-nitroanilide, as well as the formation of N-carbobenzoxy-L-alanyl-L-alanyl-L-arginyl-L-phenylalanine p-nitroanilide from N-carbobenzoxy-L-alanyl-L-alanyl-L-arginine and L-phenylalanine p-nitroanilide. The presence of small amounts of water in organic solvents was prerequisite to the biocatalysts manifesting synthase activity in reactions of peptide bond formation.

Native and Modified Subtilisin 72 as a Catalyst for Peptide Synthesis in Media with a Low Water Content

The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content >80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, Lys, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70–98% in DMF–MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.