V. A. Nesmeyanov

Immunoregulatory properties of hexapeptide isolated from porcine bone marrow cell culture

Myelopeptide 1 (MP-1) is hexapeptide originally isolated from porcine bone marrow cell culture. It was synthesized and its immunoregulatory properties were studied. MP-1 caused a 1.5-2-fold dose-dependent increase of antibody production in the culture of mouse immune lymph node cells. It abolished Con A induction of T suppressors in the suspension of mouse spleen cells and counteracted the inhibitory effect of T suppressors on antibody production. The inoculation of MP-1 (1 x 10(-9) g/mouse) to mice two weeks after their gamma-irradiation (2 Gy) resulted in an increase of antibody production up to 80.2 +/- 15.5% as compared to that in the irradiated control 37.6 +/- 12.0%. Immunofluorescent analysis revealed the specific binding of MP-1 with receptors on the target cells in the suspension of mouse spleen cells. It is supposed that MP-1 participates in the immunoregulatory processes in the living organism.

Muramyl peptide-binding sites are located inside target cells

Using flow cytometry and fluorescence polarization analysis, specific muramyl peptide‐binding sites were shown to be located inside T‐lymphocytes, macrophages and neuroblastoma cells, but not inside B‐cells. No binding sites were found on the cell surface. The number of binding sites for each cell type was determined. Two types of binding sites were observed for myelomonocytic WEH1‐3 cells with K fd values of 21 and 540 nM. Inhibition analysis demonstrated that for effective binding, an intact glycopeptide molecule and D‐configuration of isoglutamine residue are important.

Crystal structure of an anti‐interleukin‐2 monoclonal antibody Fab complexed with an antigenic nonapeptide

The three‐dimensional structure of the Fab fragment of a monoclonal antibody (LNKB‐2) to human interleukin‐2 (IL‐2) complexed with a synthetic antigenic nonapeptide, Ac‐Lys‐Pro‐Leu‐Glu‐Glu‐Val‐Leu‐Asn‐Leu‐OMe, has been determined at 3.0 Å resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen–antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR‐L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly α‐helical conformation similar to that in the epitope fragment 64–72 of the IL‐2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL‐2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody–antigen complexation involves a significant rearrangement of the epitope‐containing region of the IL‐2 with retention of the α‐helical character of the epitope fragment.

Biochemical characterization of glucosaminylmuramyldipeptide binding sites of murine macrophages.

By using radioligand analysis, murine peritoneal macrophages were shown to express several hundred high‐affinity cell surface GMDP‐binding sites (K a 350 pM). Photoaffinity labeling followed by SDS‐PAGE enabled us to identify 32–34 and 38 kDa proteins inside these cells that bound GMDP specifically.

Specific binding of glucosaminylmuramyl peptides to histones

Intracellular N‐acetylglucosaminylmuramyl peptide‐binding proteins of murine macrophages and myelomonocytic WEHI‐3 cells were characterized. SDS‐PAGE and Western blotting revealed proteins with molecular masses of 18, 32 and 34 kDa retaining the ability to specifically bind glucosaminylmuramyl dipeptide. The inhibition analysis demonstrated that only biologically active muramyl peptides but not inactive analogs or fragments of glucosaminylmuramyl dipeptide could inhibit glucosaminylmuramyl dipeptide‐binding to these proteins. Purification of these proteins and sequencing of peptides obtained after in‐gel trypsin digestion enabled us to identify the above mentioned proteins as histones H1 and H3. These findings suggest that nuclear histones might be target molecules for muramyl peptides.

N-acetylglucosamine-containing muramyl peptides directly affect macrophages

In this study flow cytometry was used to show that macrophages were the major population of murine peritoneal exudate cells (MPEC), increasing Ia expression upon treatment with N-acetylglucosaminyl-beta 1-4-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP). Modulation of Ia expression resulted from direct action of GMDP on macrophages, rather than from effect of cytokines released by T-cells. The effect of GMDP on two populations of macrophages, namely, slow and rapid responding, was studied in detail. Rapid responding cells were represented by Ia-positive macrophages: GMDP augmented their Ia expression. In contrast, slow responding subpopulation was represented by initially Ia-negative macrophages, in which GMDP induced de novo synthesis of Ia-antigens. The ability to induce Ia expression was also characteristic for other adjuvant-active N-acetylglucosamine-containing muramyl peptides (GMPs). Macrophages were shown to engulf GMPs by endocytosis. Activation of macrophages by GMDP resulted in an increase in their phagocytic activity.

Isolation and characterization of the ALP1 protease from Aspergillus fumigatus and its protein inhibitor from Physarium polycephalum

It is known that Aspergillus fumigatus secretes a serine protease ALP1 of the subtilisin family in the presence of extracellular protein substrates. We found conditions of A. fumigatus culturing that provide a high ALP1 activity inside cells without induction by extracellular proteins. The identity of the properties of the secreted and intracellular enzymes was shown. A thermostable protein inhibitor of the ALP1 protease was isolated from the plasmodium of myxomycete Physarum polycephalum. Its molecular mass is 32–33 kDa. It inhibits the ALP1 protease activity with IC50 of 0.14 μM and was also shown to be a less efficient inhibitor of the activity of HIV-1 protease (IC50 2.5 μM).

Monoclonal antibodies to botulinum neurotoxins of types A, B, E, and F

Mouse monoclonal antibodies to health-threatening botulinum neurotoxins (BoNTs) of types A, B, E, and F have been produced and characterized. The antibodies are capable of interacting with a toxin inside the respective natural toxic complex. A sandwich ELISA for the quantitative detection of botulotoxins has been developed on based on the antibodies. The detection limits of the test systems for BoNTs A, B, E, and F is 0.4, 0.5, 0.1, and 2.4 ng/ml, respectively. The assay quantitatively detects BoNTs in canned meat and vegetables. Two antibodies, BNTA-4.1 and BNTA-9.1, both separately and in combination, are capable of neutralizing the natural botulinum toxic complex of type A in vivo; a combination of antibodies neutralizes a higher dose of the toxin. It has been shown that the antibody BNTA-4.1 binds specifically to the light (catalytic) chain of the toxin, and the antibody BNTA-9.1 interacts with the heavy chain. We believe that monoclonal antibodies BNTA-4.1 and BNTA-9.1 hold promise for developing therapeutic antibodies to treat BoNT/A-caused botulism in an emergency.

Production of interleukin 2 in serum-free medium

The production of interleukin 2 by rat splenocytes, stimulated with Con A in serum-free medium, was studied. Under optimal conditions the stimulation in serum-containing and serum-free media resulted in identical interleukin 2 titers. The addition of phorbol myristate acetate prior or simultaneously with Con A did not augment interleukin 2 production. In contrast, preincubation of lymphocytes in serum-free medium for 24-36 hrs brought about a 8-10-fold increase of interleukin 2 titer. The interleukin 2 titer 4 hrs after the induction of preincubated lymphocytes with Con A was about 20% of maximal activity. The possible mechanism of early interleukin 2 production is under discussion.

The three-dimensional structure of the antigen-binding fragment of a monoclonal antibody to human interleukin-2 in two crystal forms at 2.2 and 2.9 Å resolution

The three-dimensional structure of the antigen-binding fragment of a monoclonal antibody to human interleukin-2 was determined in two crystal forms by the X-ray method of molecular replacement at 2.2 and 2.9 Å resolutions. The spatial structure of the protein and the stereochemistry of its antigen-binding site were analyzed.