RESUMEN
Consensus sequence 30-36 LKDRHDF of human alpha-interferons is inserted into the C- and N-terminal sequences of the artificial protein albeferon using genetic engineering methods in order to obtain artificial proteins with antiviral activity. Albeferon, obtained by Dolgikh et al. (Protein Eng., 1996, vol. 9, pp. 195-201), already contains the IFN-alpha 2 130-137 fragment and possesses antiproliferative activity comparable with that of IFN-alpha 2. According to CD spectroscopy, both proteins have regular secondary structures similar to that of the precursor protein. They exhibit antiviral activities, and the activity of one of them is comparable with that of IFN-alpha 2. At the same time, their cytotoxic properties are displayed only at relatively high concentrations, which substantially exceed the minimal antiviral concentrations.
Asunto(s)
Antivirales/farmacología , Interferón-alfa/farmacología , Péptidos/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Dicroismo Circular , Secuencia Conservada , Cisteína/química , Cisteína/genética , Evaluación Preclínica de Medicamentos/métodos , Epítopos/genética , Expresión Génica , Humanos , Interferón-alfa/química , Interferón-alfa/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/genética , Conformación Proteica , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Serina/química , Serina/genética , Relación Estructura-ActividadRESUMEN
The synthetic peptide SLTCLVKGFY, corresponding to the 364-373 amino acid sequence of the human IgG heavy chain (Immunorphin), was found to compete with [125I] beta-endorphin for binding by high-affinity receptors on T lymphocytes isolated from the blood of healthy donors (Ki 0.6 nM). The fragments 3-10, 4-10, 5-10, and 6-10 of Immunorphin also inhibited the binding (Ki 2.2, 3.4, 8.0, and 15 nM, respectively). Specificity of these receptors was studied: they turned out to be insensitive to naloxone and, therefore, are not opioid. The Kd values of the specific binding of 125I-labeled Immunorphin and its 6-10 fragment to the receptor were found to be 7.4 and 36.3 nM, respectively.
Asunto(s)
Inmunoglobulina G/metabolismo , Cadenas Pesadas de Inmunoglobulina/metabolismo , Oligopéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Unión Competitiva , Humanos , Regiones Constantes de Inmunoglobulina , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas gamma de Inmunoglobulina , Oligopéptidos/genética , Oligopéptidos/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , betaendorfina/metabolismoRESUMEN
The antiproliferative and immunosuppressive in vitro effects of immunocortin, a synthetic adrenocorticotropin-like (ACTH-like) decapeptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH, whose sequence corresponds to segment 11-20 of the variable part of the human IgG1 heavy chain, were studied. At concentrations of 10(-11)-10(-7) M, immunocortin was found to inhibit the growth of the human MT-4 T-lymphoblastoid cell line, to suppress the blast transformation of thymocytes, and to decrease the spontaneous mobility of peritoneal macrophages and their bactericidal action toward the virulent strain Salmonella typhimurium 415. By using a 125I-labeled "addressing" fragment of ACTH ¿[125I]ACTH-(13-24)¿, we showed that MT-4 cells express specific receptors for ACTH (Kd 97 pM). Immunocortin and human ACTH (but not the heavy chain of IgG1) competitively inhibited the binding of [125I]ACTH-(13-24) to these receptors with Ki1 of 0.38 and Ki2 of 0.34 nM, respectively. Specific receptors for ACTH (Kd 5.8 nM) on mouse thymocytes were detected and characterized. The unlabeled immunocortin was shown to complete with labeled ACTH-(13-24) for binding to these receptors (Ki = 1.8 nM) and this binding of immunocortin to receptors on thymocytes activates adenylate cyclase from these cells and increases the intracellular concentration of cAMP.
Asunto(s)
Hormona Adrenocorticotrópica/genética , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Oligopéptidos , Hormona Adrenocorticotrópica/metabolismo , Animales , Humanos , Activación de Macrófagos , Macrófagos/metabolismo , Ratones , Oligopéptidos/síntesis química , Oligopéptidos/química , Oligopéptidos/genética , Oligopéptidos/metabolismo , Receptores de Corticotropina/metabolismo , Salmonella typhimuriumRESUMEN
L-Glutamic acid at a concentration of 0.1 microM was found to induce differentiation of the cell line of HL-60 promyelocytic leukemia into granulocytes or neutrophils. The HL-60 cells have no specific glutamate-binding sites, but L-glutamic acid influences the reception of several cytokines by these cells. At a concentration of 0.1 microM, L-glutamic acid completely inhibits the high-affinity binding of 125I-labeled human recombinant interleukin-1 beta (Kd = 0.32 nM) to the HL-60 cells, but does not affect their low-affinity binding (Kd = 13.3 nM) and does not change the total number of the IL-1 beta-binding sites. Preincubation of the HL-60 cells with 0.1 microM of L-glutamic acid increases 2.5 times the number of receptors for 125I-labeled human recombinant tumor necrosis factor beta. These results suggest that L-glutamic acid plays an important role in the differentiation of the blood myeloid cells.
Asunto(s)
Ácido Glutámico/farmacología , Granulocitos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Receptores de Citocinas/metabolismo , Unión Competitiva , Diferenciación Celular/efectos de los fármacos , Ácido Glutámico/metabolismo , Granulocitos/citología , Células HL-60 , Humanos , Interleucina-1/metabolismo , Radioisótopos de Yodo , Linfotoxina-alfa/metabolismo , Neutrófilos/citología , Receptores de Citocinas/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
Conformational and dynamic properties of the synthetic peptides, amino acid sequence of which correspond to biologically active fragments of the following human cytokines: alpha 2, gamma interferons and interleukins 1 beta, 2, were studied in aqueous solution and dimethylsulfoxide by one-dimensional and two-dimensional 1H-NMR spectroscopy (400 MHz). The analysis of nuclear Overhauser effect data, values of vicinal J3(NH-C alpha H) coupling constants of amide protons and their temperature coefficients of chemical shifts indicate an unordered flexible conformation of the peptides in aqueous solutions. The structural and dynamic features of the peptides investigated are discussed together with their biological activity.
Asunto(s)
Interferón-alfa/química , Interferón gamma/química , Interleucina-1/química , Interleucina-2/química , Secuencia de Aminoácidos , Dimetilsulfóxido/química , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación Proteica , Protones , Agua/químicaRESUMEN
A comparative study of the conformational and dynamics properties of the ACTH-like linear peptides, sequences of which correspond to amino acid residues 11-20 of the heavy chain of human immunoglobulin G1 Eu, residues 78-85 of human pro-interleukin-1 alpha and site 10-18 of human ACTH, was performed in aqueous solution and dimethylsulfoxide by 1H-NMR spectroscopy at 400 MHz. The peptides were shown to possess an unordered unfolded flexible conformation in aqueous solution. The revealed structural and dynamic features of the peptides are discussed together with biological activity of this class of compounds.
Asunto(s)
Adyuvantes Inmunológicos/química , Hormona Adrenocorticotrópica/análogos & derivados , Hormona Adrenocorticotrópica/química , Péptidos/química , Conformación Proteica , Humanos , Espectroscopía de Resonancia MagnéticaRESUMEN
A specific interaction of [3H]Glu with T lymphocytes from the blood of healthy donors (Kd = 0.236 microM) was revealed and described. It was found that unlabeled quisqualate, a structural analogue of L-glutamic acid, and unlabeled dipeptides Ala-Glu, Glu-Ala, and Glu-Glu competitively inhibit the specific binding of [3H]Glu to T lymphocytes (with Ki 0.19, 2.4, 3.4, and 1.2 microM, respectively). Binding experiments with conjugates of labeled and unlabeled glutamic acid with dextran showed that the receptors of [3H]Glu are localized on the outer surface of the plasma membrane of T lymphocytes.
Asunto(s)
Agonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/metabolismo , Ácido Quiscuálico/farmacología , Receptores de Glutamato/metabolismo , Linfocitos T/metabolismo , Unión Competitiva/efectos de los fármacos , Membrana Celular/metabolismo , Dipéptidos/farmacología , Ácido Glutámico/sangre , Humanos , Estereoisomerismo , Linfocitos T/efectos de los fármacosAsunto(s)
Lectinas Tipo C , Lectinas de Unión a Manosa , Fragmentos de Péptidos/farmacología , Fagocitosis/efectos de los fármacos , Receptores de Superficie Celular/fisiología , Receptores Fc/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Receptor de Manosa , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/químicaRESUMEN
In this work the conditions of labeling protein A with europium ions were studied and the conjugates obtained in this study were compared with traditional peroxidase conjugates currently used in immunochemistry. The conjugates of protein A with Eu3+ chelate were obtained with the use of cyclic dianhydride of diethylenetriaminepentaacetic acid (DADETPA). Conjugation methods with the use of DADETPA was shown to permit obtaining high-quality conjugates with europium chelates. Europium-labeled protein A ensured the sensitivity of the determination of adsorbed IgG at a level of 2 ng/ml and the dynamic analytical range within 3-1,000 ng/ml, which essentially exceeded similar characteristics of peroxidase conjugates with protein A. Europium-labeled protein A was used for the detection of antibodies to Francisella tularensis in the sera of humans immunized against tularemia. The sensitivity of this assay exceeded that of the enzyme immunoassay 10- to 40-fold. A conclusion was made on the possibility of using europium labelled protein A for the determination of specific antibodies to F.tularensis. This preparation may be useful in the determination of specific antibodies in low-immune sera.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Especificidad de Anticuerpos , Francisella tularensis/inmunología , Proteína Estafilocócica A , Adolescente , Adulto , Quelantes , Europio , Estudios de Evaluación como Asunto , Humanos , Técnicas para Inmunoenzimas , Inmunoglobulina M/sangre , Indicadores y Reactivos , Masculino , Ácido Pentético , Proteínas Recombinantes , Sensibilidad y EspecificidadRESUMEN
According to the type of secondary structure, cytokines are classified into three categories: alpha-spiral (IFNs-alpha, beta, omega, gamma; ILS-2, 3,4,5,6,7,9; CSFs-G, M, GM, MGF, PDGF), beta-structural (ILs-1 alpha, beta, TNFs-alpha, beta, FGF) and (alpha + beta)-structural proteins (IL-8, IFN-gamma IP-10, PF-4, bTG, GRO, 9E3). According to the type of tertiary structure, alpha-spiral proteins are grouped into IFN- and IL-2-like families and beta-structural ones into IL-1-, and TNF-like families. Two subfamilies can be identified in the IFN-like family. Theoretical and experimental evidence suggests that the genes IFNs are products of divergent or convergent evolution towards the gene of the ancient intracellular protein alpha-prothymosine, which is evolutionally in turn associated with the L7/I1 protein of two ribosomes. It is suggested that the proteins of the immunoglobulin superfamily, including cytokine receptors descended from the ancient proteins of the unicellular organisms molecular shaperons.
Asunto(s)
Evolución Biológica , Citocinas/clasificación , Secuencia de Aminoácidos , Citocinas/química , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores de Superficie Celular/química , Receptores de Superficie Celular/clasificación , Receptores Inmunológicos/química , Receptores Inmunológicos/clasificaciónRESUMEN
Recombinant human gamma-interferon is dimeric in solution at pH 7-4 as revealed by analytical gel-filtration. It was shown by circular dichroism that decreasing pH to 5.0 does not affect the secondary and tertiary structures of gamma-interferon macromolecule. It was established that heat denaturation process of gamma-interferon obeys the two-state transition model and can be described as the first-order reversible reaction. Temperature dependence of the denaturation-renaturation rate constants was shown to be consistent with the Arrhenius law. The equilibrium value of the denaturation temperature was found. Effective enthalpy of denaturation was determined both by thermodynamic and kinetic approaches. The data obtained showed that in the pH range 7-4 the dimeric IFN-gamma structure may be considered as a single cooperative thermodynamic domain. Thus, it may be concluded that gamma-interferon dimerization is necessary for the existence of the corresponding tertiary structure of the macromolecule.
Asunto(s)
Interferón gamma/química , Rastreo Diferencial de Calorimetría , Cromatografía en Gel , Dicroismo Circular , Cinética , Conformación Proteica , Desnaturalización Proteica , Proteínas Recombinantes/química , TermodinámicaRESUMEN
Human leukocyte interferon-A1 (IFN-alpha A) structure in solution was investigated by fluorescence polarization, circular dichroism and scanning microcalorimetry techniques. Using gel-filtration it was established that at neutral pH values and at concentration not exceeding 0.3 mg/ml IFN-alpha A has a dimeric configuration in solution. At pH below 5, IFN-alpha A exists as a monomer. Using circular dichroism technique the IFN-alpha A molecule was shown to preserve a native structure upon decreasing pH to 3.5. The rotational correlation time of IFN-alpha A molecule in dimeric and monomeric form was measured using fluorescence of DNS, conjugated with the protein, and fluorescence of tryptophan residues. Our data indicate that the shape of IFN-alpha A molecule may be approximated by the rigid ellipsoid of revolution with the axis ratio = 4:1. The intramolecular melting of IFN-alpha A was studied by scanning microcalorimetry and circular dichroism in the acidic pH range. Thermodynamic analysis reveals two independent cooperative transitions. These transitions can be explained by assuming that the IFN-alpha A molecule consists of two structural domains.
Asunto(s)
Interferón-alfa/metabolismo , Leucocitos/metabolismo , Rastreo Diferencial de Calorimetría , Cromatografía en Gel , Dicroismo Circular , Polarización de Fluorescencia , Humanos , Concentración de Iones de Hidrógeno , Conformación Proteica , Proteínas Recombinantes/metabolismoRESUMEN
Data on NMR-spectroscopy studies of the structure-function interrelation in immunoglobulins G and their proteolytic fragments are reviewed. Relationship between structural and dynamic characteristics of immunoglobulins G and their functional properties is discussed.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/análisis , Fragmentos Fc de Inmunoglobulinas/análisis , Inmunoglobulina G/análisis , Proteína de Bence Jones/análisis , Humanos , Espectroscopía de Resonancia Magnética , Conformación ProteicaRESUMEN
Melting of protein A from Staphylococcus aureus has been studied in neutral medium by the methods of microcalorimetry and circular dichroism. The melting process of protein A is shown to consist of, at least, 5 independent transitions. The transition with the heat absorption maximum at 38 degrees is ascribed to the melting of the C-terminal domain of protein A. The analysis of amino acid sequences has shown the existence of structural basis for differences in heat stability of the Fc-binding domains of protein A.
Asunto(s)
Proteína Estafilocócica A , Secuencia de Aminoácidos , Calor , Conformación Proteica , Desnaturalización ProteicaRESUMEN
A high degree of correlation between the capability of subclasses of human immunoglobulins G to form aggregates due to thermal treatment, and their complement-binding activity was established. On the basis of the experimental data obtained by the methods of light scattering, circular dichroism, microcalorimetry, it was supposed that "hinge" region of immunoglobulins G participates in the initial stage of thermal aggregation and in the activation of the process of complement binding.
Asunto(s)
Activación de Complemento , Inmunoglobulina G/análisis , Sitios de Unión de Anticuerpos , Dicroismo Circular , Enzimas Activadoras de Complemento/análisis , Complemento C1/análisis , Complemento C1q , Calor , Humanos , Fragmentos Fab de Inmunoglobulinas/análisis , Fragmentos Fc de Inmunoglobulinas/análisis , Conformación ProteicaRESUMEN
Synthetic decapeptide corresponding to ACTH-like sequence of the variable part of the heavy chain of immunoglobulin G1 Eu was studied by two-dimensional 1H-NMR spectroscopy (400 MHz). A complete assignment of signals in the peptide spectrum was made. The decapeptide was shown not to have any ordered spatial structure, and was characterized by a high extent of flexibility of the oligopeptide chain, except for the peptide bond with an N-terminal residue.
Asunto(s)
Hormona Adrenocorticotrópica/análisis , Cosintropina/análisis , Inmunoglobulina G/análisis , Cadenas Pesadas de Inmunoglobulina/análisis , Región Variable de Inmunoglobulina/análisis , Fragmentos de Péptidos/análisis , Cosintropina/inmunología , Humanos , Espectroscopía de Resonancia Magnética , Fragmentos de Péptidos/inmunologíaRESUMEN
Conformational properties of the Fc- and pFc'-fragments of human myeloma immunoglobulins G of the first and third subclasses were studied by 1H-NMR method (270 and 400 MHz). It was found that the globular structures (domains) of the Fc-fragments of IgG1 and IgG3 in solution are characterized by high segmental mobility, and have no significant differences in their spatial arrangement. Comparative analysis of the spectra obtained at different temperatures (30-70 degrees C) revealed that the Fc-fragment of IgG3 has a more heat-stable conformation than the Fc of IgG1. The intramolecular mobility of the Fc-fragment increased upon lowering the pH. The partial assignment of the signals in the NMR spectra of the Fc-fragments of immunoglobulins G1 and G3 was carried out, and the pKa values for histidines of the pFc'-fragment of IgG1 were determined.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/análisis , Inmunoglobulina G/análisis , Humanos , Espectroscopía de Resonancia Magnética , Conformación ProteicaRESUMEN
A chemical modification of carboxylic groups of monoclonal human cryoglobulin M has been studied. The modification by a chromophoric carbodiimide was accompanied by complete loss of IgM cryoprecipitating properties. The number of carboxylic groups important for biological activity was estimated by the Tsou method and found to be 2. The cryoprecipitation dependence on ionic strength has been investigated and the number of ions per binding site isolated upon formation of intermolecular ion couples has been estimated. Mechanism of cryoprecipitation stipulated by intermolecular cooperative electrostatic interactions is proposed.