RESUMEN
Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+ ions. DNA hydrolyzing activity was associated with BV 04-01 IgG, Fab, and SCA 04-01 proteins. Pronounced cleavage specificity for both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of the oligonucleotide A7C7ATATAGCGCGT7 as well as preference for cleavage within CG-rich regions of double-stranded DNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA 04-01 and two SCA 04-01 mutants (L32Phe and L27dHis) were used to model the catalytically active antibody site utilizing the previously resolved X-ray structure of (dT)3 liganded Fab 04-01. The resulting model suggested that BV 04-01 activates the target phosphodiester bond by induction of conformational strain. In addition, the antibody-DNA complex contained a potential Mg2+ ion coordination site composed of the L32Tyr and L27dHis amino acid side chains and a DNA 3'-phosphodiester group. Induction of strain and metal coordination could be constituents of a mechanism by which this antibody catalyzed DNA hydrolysis. Sequence data for BV 04-01 VH and VL genes suggested that the proposed catalytic antibody active site was germ-line encoded. This observation suggests the hypothesis that catalytic activity might represent an important but unspecified function of some antibody molecules.
Asunto(s)
Anticuerpos Catalíticos/inmunología , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/inmunología , ADN de Cadena Simple/inmunología , ADN/metabolismo , Animales , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Hidrólisis , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Cinética , Modelos Moleculares , Conformación Proteica , Células Tumorales CultivadasAsunto(s)
Anticuerpos Antinucleares/metabolismo , Anticuerpos Catalíticos/metabolismo , Enfermedades Autoinmunes/inmunología , ADN/metabolismo , Lupus Eritematoso Sistémico/inmunología , Secuencia de Bases , Desoxirribonucleasa I/metabolismo , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , PlásmidosRESUMEN
A highly effective method consisting of two affinity chromatography steps and ion-exchange and gel-filtration chromatography steps was developed for purification of autoantibodies from human sera with DNA-hydrolyzing activity. Antibody Fab fragment, which had been purified 130-fold, was shown to catalyze plasmid DNA cleavage. The flow linear dichroism technique was used for quantitative and qualitative studying of supercoiled plasmid DNA cleavage by these autoantibodies in comparison with DNase I and EcoRI restriction endonuclease. The DNA autoantibody Fab fragment was shown to hydrolyze plasmid DNA by Mg(2+)-dependent single-strand multiple nicking of the substrate. Kinetic properties of the DNA autoantibody Fab fragment were evaluated from the flow linear dichroism and agarose gel electrophoresis data and revealed a high affinity (Kobsm = 43 nM) and considerable catalytic efficiency (kappcat/Kobsm = 0.32 min-1.nM-1) of the reaction.
Asunto(s)
Autoanticuerpos/metabolismo , ADN Superhelicoidal/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Plásmidos/metabolismo , Autoanticuerpos/sangre , Autoanticuerpos/aislamiento & purificación , Celulosa/análogos & derivados , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , ADN , Humanos , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Cinética , Espectrofotometría , Factores de TiempoRESUMEN
The interaction of antibodies from blood sera of patients with autoimmune pathology, systemic lupus erythematosus with oligoribonucleotides was studied. The RNA-hydrolyzing activity was shown to be an intrinsic property of autoantibodies. Enzymic activity of antibodies in hydrolysis of poly(U) was estimated at 20-40% of that of RNase A. In contrast to known eukaryotic RNases, the autoantibodies possess a specific RNA-hydrolyzing activity for oligo r(A). The RNA-nicking activity of antibodies in hydrolysis of oligoadenylates was more higher than with hydrolysis of oligo d(A). Optimal conditions of r(pA)13 hydrolysis were selected, including the optimal of pH = 8.7.
Asunto(s)
Anticuerpos Catalíticos/inmunología , Autoanticuerpos/inmunología , Lupus Eritematoso Sistémico/inmunología , Oligorribonucleótidos/inmunología , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Oligorribonucleótidos/metabolismoRESUMEN
Catalysis by antibodies could be a frequent phenomenon if the immune system generates a sufficiently diverse number of antibody-active sites, some of which may possess catalytic activity. A catalytic antibody can be expected to do more damage than one that simply binds antigen. The best biochemical marker of systemic lupus erythematosus (SLE) is presence of autoantibodies to DNA. In the present article, we describe the DNA-hydrolyzing activity of DNA-binding autoantibodies purified from SLE patients. The substrates employed were supercoiled plasmid, radiolabeled plasmid fragments, and oligonucleotides. Hydrolysis of DNA by the antibodies was indicated by the appearance of fragments visualized by ethidium bromide staining of agarose gels or autoradiography of polyacrylamide gels. Changes in linear dichroism values were also indicative of DNA hydrolysis. The antibody activity was purified by protein A-sepharose chromatography, high-performance liquid chromatography gel filtration, and DNA-affinity chromatography. Scrupulous control studies were done to demonstrate that DNA-hydrolyzing activity really belongs to the antibodies. Purified Fab fragments showed hydrolyzing activity, whereas the Fc fragment was inactive. The specificity of DNA cleavage was investigated, and the rate parameters of hydrolysis by antibodies and conventional nucleases were compared.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Anticuerpos/metabolismo , ADN/metabolismo , Lupus Eritematoso Sistémico/inmunología , Anticuerpos/inmunología , Anticuerpos/aislamiento & purificación , Anticuerpos Catalíticos/inmunología , Anticuerpos Catalíticos/aislamiento & purificación , Secuencia de Bases , Biomarcadores , Etidio/química , Humanos , Hidrólisis , Fragmentos de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/metabolismo , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/metabolismo , Datos de Secuencia MolecularRESUMEN
Sera of patients with different types of leukemia and acquired immune deficiency syndrome (AIDS) have been examined for the presence of the anti-DNA antibodies. DNA-hydrolyzing activity of antibodies was detected in the sera of patients with chronic lymphoid leukemia (CLL), pre-B-cell acute lymphoid leukemia (pre-B-All), acute myeloleukosis (AML), and AIDS in stages III and IV of the disease. In immunofluorescence tests, the DNA-hydrolyzing antibodies reacted preferentially with proliferating cell nuclei compared with resting cells. A 35-kDa factor was identified as the target for the DNA antibodies in the cell nuclei. The DNA-hydrolyzing antibody fraction from the serum of an AIDS patient crossreacted with HIV I virus proteins gp160, gp120, and p65.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/inmunología , Anticuerpos Catalíticos/metabolismo , Autoanticuerpos/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Leucemia/inmunología , Anticuerpos Catalíticos/inmunología , Autoanticuerpos/inmunología , División Celular , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Productos del Gen pol/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Hidrólisis , Proteínas de Neoplasias , Sialoglicoproteínas/inmunología , Células Tumorales Cultivadas , Productos del Gen pol del Virus de la Inmunodeficiencia HumanaRESUMEN
A method for purifying DNA-specific catalytic antibodies based on affinity chromatography on protein A Sepharose and on both modified and non-modified DNA-cellulose as well as HPLC has been developed. The elution conditions with high yields of DNA-hydrolyzing activity of antibodies have been optimized. The biochemical and immunological properties of catalytic antibodies have been examined. The kinetic parameters of the enzyme interaction with an oligonucleotide substrate have been determined. The influence of effectors on DNA hydrolysis by antibodies has been investigated.
Asunto(s)
Anticuerpos Antinucleares/aislamiento & purificación , Anticuerpos Catalíticos/aislamiento & purificación , Lupus Eritematoso Sistémico/inmunología , Anticuerpos Antinucleares/inmunología , Anticuerpos Catalíticos/inmunología , Secuencia de Bases , Sitios de Unión de Anticuerpos , Cromatografía de Afinidad , Humanos , Hidrólisis , Datos de Secuencia MolecularRESUMEN
Blood sera of patients with autoimmune diseases scleroderma (Scl), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) have been shown to yield a specific immune response to topoisomerase I, the product of expression of a cDNA fragment cloned into lambda gt11 and monoclonal antibodies (MAB) to the enzyme. The 'topoisomerase test' is not absolutely specific for Scl. The stable positive response of autoimmune sera to anti-topoisomerase monoclonal antibodies has a specific character and is associated with the interaction of the Fab fragment of MAB with the IgG fraction of autoimmune serum. The response observed indicates the induction of anti-idiotypic antibodies against topoisomerase. The anti-idiotype, isolated by HPLC and affinity chromatography demonstrated the following functional activities: (i) the immunological reaction against DNA; (ii) high-affinity DNA-binding with topoisomerase-specific consensus; (iii) ability to compete with the native enzyme for binding with DNA and MAB to topoisomerase; (iv) immunological reaction against MAB to topoisomerase.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , ADN/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Artritis Reumatoide/inmunología , Unión Competitiva , Cromatografía Líquida de Alta Presión , Humanos , Lupus Eritematoso Sistémico/inmunología , Datos de Secuencia Molecular , Mapeo Restrictivo , Esclerodermia Sistémica/inmunologíaRESUMEN
A new method for isolation of eukaryotic topoisomerase I from calf thymus and from Jurkat-1 cells using HPLC has been developed. The method allows quantitative purification of high molecular weight topo I and two low molecular weight fractions differing by their isoelectric points. It has been suggested that these fractions be characterized as two subforms of the enzyme possessing structural and functional differences. The differences in their specific activities, sensitivity to camptothecin and in their proteolytic digestion maps have been demonstrated for the two enzymes.
Asunto(s)
ADN-Topoisomerasas de Tipo I/aislamiento & purificación , Animales , Bovinos , Línea Celular , Cromatografía Líquida de Alta Presión , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Bacteriano/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Plásmidos , Relación Estructura-ActividadRESUMEN
A DNA-nicking activity was detected in the sera of patients with various autoimmune pathologies and was shown to be a property of autoantibodies. The DNA hydrolyzing activity, which was purified by affinity and high-performance liquid chromatography, corresponded in size to immunoglobulin M (IgM) and IgG and had a positive response to antibodies to human IgG. The DNA hydrolyzing autoantibodies were stable to acid shock and yielded a DNA degradation pattern that was different from that of deoxyribonuclease (DNase) I and blood DNase.
Asunto(s)
Autoanticuerpos/metabolismo , Enfermedades Autoinmunes/metabolismo , ADN/metabolismo , Acetatos/farmacología , Ácido Acético , Autoanticuerpos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , ADN Polimerasa I/metabolismo , Desoxirribonucleasa I/metabolismo , Electroforesis en Gel de Agar , Humanos , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulina G/metabolismo , Inmunoglobulina M/aislamiento & purificación , Inmunoglobulina M/metabolismo , Cinética , Lupus Eritematoso Sistémico/inmunología , PlásmidosRESUMEN
The principal existence of natural catalytic antibodies in the autoimmune sera is discussed. In the course of the autoimmune process, the induction of antiidiotypic antibodies against topoisomerase I has been shown in the sera of patients with scleroderma, systemic lupus erythematosus, and rheumatoid arthritis. The above antibodies were obtained in preparative amounts. Proceeding from the concept of the idiotypic network, the antibodies were suggested to be natural enzymes and their properties were studied. They appeared to be anti-DNA antibodies, competing with the native topoisomerase I for binding to anti-topoisomerase monoclonal antibodies and possessing highly specific DNA-binding activity (Kd is about 0.1 nM). The antiidiotypic antibodies specifically inhibit the topoisomerase-catalysed relaxation reaction and affect the formation of covalent DNA-protein complex. Possible involvement of antiidiotypic antibodies against topoisomerase in the catalysis of reactions of DNA transformation is analysed. Catalytic antibodies that are natural enzymes possessing DNA-nicking activity have been isolated from the blood sera of patients with different autoimmune pathologies.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/metabolismo , Catálisis , ADN/metabolismo , ADN-Topoisomerasas de Tipo I/inmunología , ADN-Topoisomerasas de Tipo I/metabolismo , Humanos , Sueros InmunesRESUMEN
The interaction of sera from 34 patients with different autoimmune diseases with the expressed fusion protein cloned in lambda gt11 vector (topoisomerase I--beta galactosidase) and monoclonal antibodies against enzyme was studied. It was demonstrated that 100% of Scl cases possessed positive activity against fusion protein. It was shown that this test is not absolutely specific for Scl, i. e. 57.1% of Sle and 84.6% of RA demonstrated positive activity against "topoisomerase test". Autoimmune sera had the positive activity against monoclonal antibodies for topoisomerase I. This activity was shown to be due to the presence of antiidiotypic antibodies against topoisomerase in the autoimmune sera.
Asunto(s)
Anticuerpos Antiidiotipos/análisis , Anticuerpos Monoclonales , Enfermedades Autoinmunes/inmunología , ADN-Topoisomerasas de Tipo I/inmunología , ADN Circular/genética , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/inmunología , Enfermedades Autoinmunes/genética , Regulación de la Expresión Génica , Humanos , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/inmunología , Pronóstico , Esclerodermia Sistémica/diagnóstico , Esclerodermia Sistémica/inmunologíaRESUMEN
Optimal conditions for the preparation of avidin-peroxidase conjugates by the periodate method were studied. A method based on hydrophobic interaction chromatography was developed for the isolation of active oligomers. The probable structure of oligomers with highest sensitivity is discussed.