A new antigenic epitope appears in the catalytic subunit of viscumin during intracellular transport.

The plant toxin viscumin (60 kD) consists of B- ("binding") and A- ("active") subunits joined by a disulfide bond. The B-subunit is a lectin interacting with galactose-containing glycolipids and glycoproteins of the cell surface. The A-subunit possesses N-glycosidase activity which modifies 28S ribosomal RNA. This results in irreversible inhibition of protein synthesis. After binding and receptor-mediated endocytosis viscumin-containing vesicles are transported to endoplasmic reticulum where the A- (catalytic) subunit is subsequently translocated to cytosol. It is possible that translocation of A-subunit requires its unfolding. For identification of epitopes which might appear during such unfolding, we developed hybridomas producing monoclonal antibodies against denatured viscumin A-chain. Resistance of hybridoma cells to cytotoxic action of viscumin suggests antibody-toxin interaction inside these cells. TA7 hybridoma cells against an epitope which appears only in denatured viscumin are insensitive to the toxin. This suggests that antibody-toxin interaction occurs before transmembrane translocation of the catalytic A-chain into the cytoplasm. Consequently, toxin resistance of TA7 hybridoma cells implies the appearance of a new epitope in viscumin during its intracellular transportation inside of vesicles. Sixty five octapeptides have been synthesized and epitopes have been identified for monoclonal TA7 antibody and immune mouse serum by means of ELISA. Based on the epitopic mapping the peptide A96-ETHLFTGT-T105 was chemically synthesized and binding of this peptide to the monoclonal antibody TA7 and conformation of antigenic determinant (L100-FTGT-T105) was investigated by means of (1)H-NMR spectroscopy.

[Epitope of the catalytic subunit of viscumin, exposed on its interaction with a lipid bilayer]. / Epitop kataliticheskoi sub''edinitsy viskumina, poiavliaiushchiisia pri ee vzaimodeistvii s lipidnym bisloem.

It was previously shown that the catalytic subunit of the plant toxin viscumin induces aggregation of small unilamellar liposomes and this process is inhibited by the mab_TA7 monoclonal antibody produced to the denatured catalytic subunit of viscumin (Agapov, I.I. et al., FEBS Lett., 1999, vol. 464, pp. 63-66). The interaction of the synthetic F101-T105 and A96-T105 fragments of the viscumin catalytic subunit with the mab_TA7 monoclonal antibody was studied by 1H NMR spectroscopy. The results of this study demonstrated that only the A96-T105 fragment is capable of binding to mab_TA7. A nuclear Overhauser effect observed in the antigen-antibody complex and registered on the resonances of the free peptide and exchanging between the free state and the antibody-bound state was analyzed; the mab_TA7 antigen determinant (H99-T105) was identified; and its conformation and orientation within the complex with the antibody were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

[Bacteriorhodopsin retains the conformation of Val69-Gly72 fragment during functioning]. / Sokhranenie konformatsii fragmenta Val69-Gly72 bakteriorodopsina pri ego funktsionirovanii.

Effect of the monoclonal antibody (MAb) 5B6 produced to the solubilized preparation of bacteriorhodopsin on the protein photocycle was studied to examine conformational rearrangements on the surface of functioning bacteriorhodopsin molecule. Using the methods of solid phase enzyme immunoassay, peptide phage display, and 1H NMR spectroscopy, we demonstrated that the epitope recognized by MAb 5B6 is the Val69-Pro-Phe-Gly72 fragment of the protein, with the aromatic ring of Phe71 and the methyl groups of Val69 participating in the binding. MAb 5B6 exerted no significant effect on the photocycle of bacteriorhodopsin solubilized in Triton X-100 at pH 6.2 and 7.4, which suggested that, when functioning, bacteriorhodopsin retains the conformation and position of its Val69-Pro-Phe-Gly72 fragment.

Spatial structure of the M2 transmembrane segment of the nicotinic acetylcholine receptor alpha-subunit.

A synthetic peptide corresponding to the transmembrane segment M2 (residues 236-267) of the alpha-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been studied by two dimensional 1H-NMR spectroscopy in a chloroform-methanol (1:1) mixture containing 0.1 M LiClO4. Reconstruction of the spatial structure of M2 from the NMR data resulted in an alpha-helix formed by residues 241-263. Distribution of the molecular hydrophobicity potential on the helix surface is very similar to that in five-helix bundles of proteins with a known three dimensional structure: two hydrophilic bands located on the opposite helix sides separated by strong hydrophobic zones.

Conformation of surface exposed N-terminus part of bacteriorhodopsin studied by transferred NOE technique.

Interaction of the monoclonal antibody A5 raised against native bacteriorhodopsin (BR) with the synthetic peptide pGlu1-Ala-Gln-Ile-Thr-Gly-Arg7-NH2, corresponding to the amino acid sequence 1-7 was studied by transferred nuclear Overhauser effect (TRNOE) spectroscopy. The denaturing reagents and the specially designed pulse sequences which eliminate broad signals from the TRNOE spectra were used to favour evaluation of the TRNOE peaks. On the basis of the data obtained, the conformation of peptide bound with A5 was calculated. A model of the mutual arrangement of bacteriorhodopsin N-terminus and the first transmembrane alpha-helical segment 8-32 was proposed.

[The intensity of intratumor infiltration with polymorphonuclear neutrophils in different stages of the development of Lewis' carcinoma. The effect of the surgical removal of the primary node]. / Intensivnost' vnutriopukholevoi infil'tratsii segmentoiadernymi neitrofilami na raznykh étapakh razvitiia kartsinomy L'iuis. Vliianie operativnogo udaleniia pervichnogo uzla.

The intensity of infiltration of the primary tumour and its lung metastases with granular leucocytes and lymphocytes was studied in mice with Lewis lung carcinoma. The content of lymphocytes decreases, while that of granular leucocytes increases with the growth of the primary tumour. The relative content of lymphocytes in metastasis tumours is higher than that of granular leucocytes. The primary tumour surgery causes reliable changes in the intensity of tumour infiltration with these cells, as compared with non-operated animals.

[Conformation of fragment 66-72 of interleukin-2 complexed with a monoclonal antibody to interleukin-2]. / Konformatsiia fragmenta 66-72 interleikina-2 v komplekse s monoklonal'nym antitelom k interleikinu-2.

1H-NMR spectra of the interleukin-2 synthetic fragment Ac-Leu66-Glu-Glu-Val-Leu-Asn-Leu72-OCH3 in the presence or absence of the monoclonal antibody were analysed. The data obtained are consistent with an extended unordered conformation of the free peptide. Measurements of NOESY cross-peak intensities allowed us to determine the spatial structure of the peptide bound to the antibody. The peptide has an amphiphilic surface with hydrophobic and hydrophilic amino acid side chains clustered on the opposite sides of its alpha-helical-like structure. The hydrophobic and hydrophilic clusters are located on the opposite sides of the bound peptide's surface. The hydrophobic side chains contact the antibody surface, while the hydrophilic ones are oriented into the solvent (T. A. Balashova et al. (1991) Bioorgan. Khim. (USSR), v. 17, p. 1470-1486). Hydrolysis of the methyl ester slowly ocurs in the presence of the antibody. This process does not alter the conformation of the peptide bounded with the antibody, though decreases the peptide's affinity to the antibody.

[Study of the interaction of the monoclonal antibody to human interleukin-2 and its Fab-fragment with synthetic peptides --interleukin-2 fragments by (1)H-NMR]. / Issledovanie metodom (1)H-IaMR vzaimodeistviia monoklonal'nogo antitela k interlekina-2 cheloveka i ego Fab-fragmenta s sinteticheskimi peptidami--fragmentami interleikina-2.

Proton signals for nine synthetic peptide fragments of human interleukin-2 (region 59-78) were assigned for aqueous solutions both of pure peptides and their mixtures with LNKB-2 monoclonal antibody. The nonspecific magnetization transfer (NOE) between the antibody or its Fab-fragment and the peptides was studied upon large excess of free peptide over bound peptide. NOE spectra using modified pulse sequence, enabling to eliminate broad signals and achieve higher (peptide signal)/noise ratio were obtained. The saturation transfer experiments indicated that methyl groups of amino acid residues corresponding to Leu66,70,72, Val69 and Ala73 in interleukin-2 contact with the antibody binding site. Thus, the hydrophobic interactions are of major importance for the LNKB-2-IL-2 peptide complexes. The minimal IL-2 fragment which can still bind to LNKB-2 monoclonal antibody is -Leu70-Asn71-Leu72-.

Solution spatial structure of 'long' neurotoxin M9 from the scorpion Buthus eupeus by 1H-NMR spectroscopy.

1H-NMR spectra of Buthus eupeus neurotoxin M9 (66 amino acid residues, four disulfide bonds) reveal two slowly exchangeable conformations at acidic pH. The spatial structure of the conformer prevailing under physiologically relevant conditions has been determined from two-dimensional 1H-NMR data treated by means of a distance geometry algorithm and refined by molecular modelling. Interrelation between the structure and function of mammalian neurotoxin M9 is discussed by comparing its conformation with those of the scorpion insectotoxins which exhibit different biological specificity (insectotoxins v-2, v-3 and I5A).

[The structure of Buthus eupeus neurotoxin M9 in a solution studied by 1H-NMR spectroscopy]. / Stroenie neirotoksina M9 Buthus eupeus v rastvore po dannym spektroskopii 1H-IaMR.

Neurotoxin M9 isolated from the venom of Central Asian scorpion Buthus eupeus (66 amino acid residues, 4 disulfide bridges) has two slowly exchangeable conformations at the acidic pH. 2D-1H-NMR spectroscopy has been used to determine the polypeptide backbone foiding in the conformer that dominates under physiological conditions. The conformer contains the right alpha-helix (residues 22-31) and the antiparallel beta-sheet, which consists of the three strands (residues 1-5, 46-52, 35-40). All five Xxx-Pro bonds are in the trans configuration. Comparison of the obtained data with the crystal structure of the homologous scorpion toxin v-3 Centruroides sculpturatus (65 residues) and the solution spatial structure of the "short" type insectotoxin I5A Buthus eupeus (35 residues) shows close similarity in the first case and similarity of the types and mutual disposition of the regular secondary structure elements in the second case.

[1H-NMR study of the Naja naja oxiana neurotoxin II and its spin-labeled derivatives. Conformation of "short" neurotoxins]. / 1H-IaMR-issledovanie neirotoksina II Naja naja oxiana i ego spin-mechenykh proizvodnykh. Konformatsiia "korotkikh" neirotoksinov.

In 1H NMR spectra of neurotoxin II N. n. oxiana the chemical shift pH-dependences in H2O and 2H2O solutions were studied, and also the deuterium exchange rates and chemical shift temperature gradients were measured for the amide protons. The spin probe method was applied to assess the degree of exposure into solvent of the amide and side chain protons. With the purpose of establishing mutual disposition of certain neurotoxin II groupings, nuclear Overhauser effect was studied in the 1H NMR spectra, along with the broadening of proton resonances induced by spin labels selectively attached to epsilon-amino groups of Lys26, Lys27, Lys45 or Lys47. The mobility of these labels was determined from the EPR spectra. The methyl resonances of Val and Leu residues were assigned to a definite position in the amino acid sequence. The following pKa were determined: alpha-NH2 Leu1 (9,2), gamma-COOH Glu2 (3,7), alpha-COOH Asn62 (1,3). The protonation of a carboxyl group(s) in neurotoxin II (alpha-COOH Asn62 seems to be involved) decreases the temperature stability of the neurotoxin II conformation. On the basis of studies on neurotoxin II and some other homologous neurotoxins, the model for the "short" neurotoxin folding in solution was proposed. Comparison of experimental data for the disposition of equivalent groups in homologous neurotoxins and in the X-ray structure of erabutoxin b Laticauda semifasciata revealed that the Val46 side chain in solution might change its orientation by 180 degrees with respect to polypeptide backbone. Binding of spin labeled neurotoxin II derivatives to the acetylcholine receptor was discussed in light of the obtained data.

[Preparation and ESR study of spin-labeled derivatives of Naja naja oxiana neurotoxin II]. / Poluchenie i EPR-issledovanie spin-mechenykh proizvodnykh neirotoksina II Naja naja oxiana.

After neurotoxin II Naja naja oxiana reaction with N-hydroxysuccinimidyl 2,2,6,6-tetramethyl-4-carboxymethylpiperidine-1-oxyl, six derivatives were isolated, each containing one spin label. Their analysis (reduction, carboxymethylation, tryptic hydrolysis, isolation and identification of the spin labeled peptide) allowed to localize the label position: the epsilon-amino groups of Lys15, Lys25, Lys26, Lys44, Lys48, and alpha-amino group of Leu1. The neurotoxin II reaction with N-hydroxysuccinimidyl 2,2,5,5-tetramethyl-3-carboxypyrrolin-1-oxyl followed by chromatography afforded 10 derivatives, each having two labeled lysine residues, wherein the position of the modified residues was determined. The reactivity and microenvironment of amino groups are discussed basing on the dependence between the reaction conditions and yields. For di-spin labeled derivatives of the pyrroline series, the inter-label distances were determined by EPR from the standard curve and used for refinement of the neurotoxin conformation in solution.

The 1H nuclear-magnetic-resonance spectra and spatial structure of the Naja mossambica mossambica neurotoxin III.

The proton NMR spectra at 300 MHz of neurotoxin III from venom of Naja mossambica mossambica are reported. By the use of double resonance techniques, pH dependence chemical shifts, isotope labeling technique, and comparison with homologous neurotoxins all proton signals in the aromatic and methyl regions as well as epsilon-CH2 proton signals of some lysine residues have been assigned to individual amino acid residues and their spatial microenvironment has been determined. The results deduced on the solution structure of neurotoxin III are in complete agreement with the crystal structure of sea snake erabutoxins as well as with the previously established backbone folding and inter-residue interactions for the Naja naja oxiana short-chain neurotoxin in solution. In addition evidence has been obtained (a) that the conformation of the beta turn in the 31-34 segment depends on the ionization state of Asp-31 and His-32 side chain groups and (b) that an intricate electrostatic interaction exists in a system of ionogenic groups of the invariant Lys-27, Lys-47, Asp-31, Arg-33, Glu-38 and His-32 residues. These aspects of dynamic conformation are related to an interaction mechanism of a neurotoxin molecule and a nicotinic acetylcholine receptor.