RESUMEN
Expression of recombinant antibodies in mammalian cells is one of key problems in immunobiotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively done in yeast cells. We obtained expression strains of the methylotrophic beast Pichia pastoris producing single chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab) and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant Kd and the first order constant (k2) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (Phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and single chain antibody A.17 expressed in CHO and E. coli cells respectively.
Asunto(s)
Anticuerpos Catalíticos/biosíntesis , Anticuerpos Monoclonales/biosíntesis , Expresión Génica , Pichia , Proteínas Recombinantes/biosíntesis , Anticuerpos de Cadena Única/biosíntesis , Animales , Anticuerpos Catalíticos/genética , Anticuerpos Monoclonales/genética , Células CHO , Cricetinae , Cricetulus , Humanos , Proteínas Recombinantes/genética , Anticuerpos de Cadena Única/genéticaRESUMEN
Disseminated sclerosis is currently regarded as a CNS autoimmune disease. One of the mechanisms behind this pathology is antibody (AB) formation. In this context, recent data on AB with proteolytic activity are of importance because they participate in selective proteolysis of myelin proteins in patients with disseminated sclerosis. This paper focuses on AB-proteases associated with disseminated sclerosis and site-specificity of antibody-mediated proteolysis of myelin basic protein. Protocol of serodiagnostic algorithm to be used in clinical practice is described.
Asunto(s)
Autoanticuerpos/metabolismo , Esclerosis Múltiple/inmunología , Péptido Hidrolasas/metabolismo , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Células Presentadoras de Antígenos/inmunología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Epítopos , Humanos , Datos de Secuencia Molecular , Esclerosis Múltiple/diagnóstico , Esclerosis Múltiple/etiología , Proteína Básica de Mielina/inmunología , Proteína Básica de Mielina/metabolismo , Pruebas Serológicas , Especificidad por SustratoAsunto(s)
Antídotos/metabolismo , Antídotos/farmacología , Inmunoglobulinas/metabolismo , Inmunoglobulinas/farmacología , Compuestos Organofosforados/antagonistas & inhibidores , Secuencia de Aminoácidos , Western Blotting , Humanos , Inmunoglobulinas/biosíntesis , Datos de Secuencia Molecular , Organofosfonatos/química , Organofosfonatos/metabolismo , Compuestos Organofosforados/aislamiento & purificación , Compuestos Organofosforados/toxicidad , Biblioteca de Péptidos , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónAsunto(s)
Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Catalíticos/aislamiento & purificación , Anticuerpos Catalíticos/metabolismo , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Catalíticos/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Bovinos , Fluoresceína-5-Isotiocianato , Hidrólisis , Cinética , Albúmina Sérica Bovina/metabolismo , Especificidad por SustratoRESUMEN
A model is developed for the investigation of an enzymatic diffusion-reaction system. The aim is to analyze the dynamic behavior of three different species, the modification of their enzymatic kinetic properties and the existence of complex behaviors resulting of the catalytic activity induced by immobilization of acetylcholinesterase into artificial membrane enzymatically inactive. We report results that make possible the characterization and prediction of complex behaviors as well as a qualitative/quantitative analysis of the system stability via bifurcation diagram which allows to study: i) the effect of the initial substrate concentration in the reservoir and ii) the effect of reaction-permeation modulus of the membrane as bifurcation parameters.
Asunto(s)
Acetilcolinesterasa/metabolismo , Enzimas Inmovilizadas/metabolismo , Membranas Artificiales , Enfermedad de Alzheimer/enzimología , Animales , Inhibidores de la Colinesterasa/farmacología , Humanos , Hidrólisis , Cinética , Actividad Motora , Músculo Esquelético/fisiología , Neuronas/fisiologíaRESUMEN
Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F1, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)2 reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP(89-104)) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an "internal image" of protease is described. The "second order" monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Endopeptidasas/metabolismo , Animales , Anticuerpos Antiidiotipos/sangre , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Antiidiotipos/aislamiento & purificación , Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Catalíticos/sangre , Anticuerpos Catalíticos/inmunología , Anticuerpos Catalíticos/aislamiento & purificación , Enfermedades Autoinmunes/inmunología , Western Blotting , Catálisis , Endopeptidasas/sangre , Endopeptidasas/inmunología , Endopeptidasas/aislamiento & purificación , Inducción Enzimática , Ensayo de Inmunoadsorción Enzimática , Escherichia , Inmunización , Cinética , Ratones , Estructura MolecularRESUMEN
The catalytic monoclonal antibody 9A8 (MA 9A8), antiidiotypic to the antibody AE-2 (MA AE2) produced to the active site of acetyl cholinesterase from human erythrocytes, was subjected to a structure-function study. The specific binding of MA 9A8 to MA AE2 (K 2.26 x 10(9) M-1) was shown by the method of surface plasmon resonance, and the functional activity of MA 9A8 was demonstrated. Unlike acetyl cholinesterase, this antibody specifically reacted with the irreversible phosphonate inhibitors of esterases. A peptide map of MA 9A8 was analyzed by MALDI mass spectrometry. The Ser99 residue of its heavy chain was shown to be within the active site of the catalytic antibody. A computer modeling of the MA 9A8 active site suggested the existence of a catalytic dyad formed by Ser99 and His35. A comparison of the tertiary structures of the MA 9A8 and the 17E8 monoclonal antibody, which also exhibited an esterase activity and was produced to the stable analogue of the reaction transition state, indicated a practically complete coincidence of the structures of their presumed active sites.
Asunto(s)
Acetilcolinesterasa/inmunología , Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/metabolismo , Eritrocitos/inmunología , Acetilcolinesterasa/efectos de los fármacos , Acetilcolinesterasa/metabolismo , Secuencia de Aminoácidos , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Inhibidores Enzimáticos/farmacología , Eritrocitos/enzimología , Histidina , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Serina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Resonancia por Plasmón de SuperficieRESUMEN
The catalytic mechanism of an anti-idiotypic antibody, 9G4H9, displaying a beta-lactamase activity was investigated. Kinetics experiments suggest that some penicillinic derivatives behave both as substrates and inactivators. Biochemical and immunological experiments strongly indicate that ampicillin may be regarded as a suicide substrate for hydrolysis by 9G4H9. The anti-idiotypic network appears as a way to create enzyme mimics with modified catalytic activities.
Asunto(s)
Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Catalíticos/metabolismo , beta-Lactamas/metabolismo , Animales , Anticuerpos Catalíticos/química , Anticuerpos Monoclonales/metabolismo , Western Blotting , Humanos , Hidrólisis , Cinética , Ratones , Penicilinas/farmacología , Especificidad por SustratoRESUMEN
A monoclonal antibody 9G4H9 that exhibits a beta-lactamase-like activity was previously obtained in accordance with the idiotypic network theory. This abzyme presents the most catalytic efficiency in amidase activity described in literature (kcat = 0.9 min-1). Some reports have demonstrated that functionality as complex as catalysis may be mimicked in this way. Comparison of the catalytic properties of both enzyme and abzyme previously allowed us to obtain better knowledge about 9G4H9 abzymatic machinery. In attempt to characterize this abzyme, the variable regions of kappa and heavy chain were cloned. We present a 'universal' method to clone the correct Vkappa gene to bypass aberrant Vkappa (abVkappa) produced by MOPC-21-derived hybridomas. Sequences obtained are compared in the GenBank database. The VH and Vkappa genes present some important sequence homology with autoantibodies suggesting a direct relationship between catalytic anti-idiotypic antibody and autoimmunity.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Anticuerpos Monoclonales/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Línea Celular , Clonación Molecular , Electroforesis en Gel de Agar , Genes de Inmunoglobulinas , Cadenas kappa de Inmunoglobulina/metabolismo , Cinética , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , ARN/metabolismo , Alineación de SecuenciaRESUMEN
The concept of "internal image" of antiidiotypic antibodies has provided the basis for eliciting catalytic antibodies. A monoclonal IgM 9A8 that was obtained as an antiidiotype to AE-2 mAb, a known inhibitor of acetylcholinesterase, displayed esterolytic activity. Study of recombinant Fab fragments and separate light and heavy chains of 9A8 confirmed that the antibody variable domain encodes the catalytic function, whereas neither part of the primary sequence of the Fab exhibited homology with the enzyme. The specific modification of the 9A8 variable domain by an active site-directed covalent inhibitor revealed the presence of an active site Ser residue. A three-dimensional modeling suggests the existence of a functional catalytic dyad Ser-His. Comparison of active sites of 9A8 and 17E8 esterolytic abzyme raised against transition-state analog revealed structural similarity although both antibodies were elicited by two different approaches.
Asunto(s)
Anticuerpos Monoclonales/química , Imitación Molecular , Secuencia de Aminoácidos , Animales , Anticuerpos Catalíticos/metabolismo , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Cartilla de ADN , Humanos , Ratones , Modelos Químicos , Datos de Secuencia MolecularRESUMEN
A catalytic IgG (Ab2) displaying a beta-lactamase-like activity was previously obtained by using the antiidiotypic pathway: the particularity of this antibody is that it is a true antiidiotype of the beta-lactamase active site. We have previously demonstrated that this IgG has retained some of the structural information displayed by the beta-lactamase active site, evident from data that polyclonal anti-Ab2 antibodies (Ab3) recognize beta-lactamase. In this article, we investigated the catalytic mechanism of the abzyme compared to that of the enzyme. The experimental data allow us to hypothesize the catalytic residues required for catalysis.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , beta-Lactamasas/inmunología , beta-Lactamasas/metabolismo , Animales , Anticuerpos Antiidiotipos/metabolismo , Bacillus cereus/enzimología , Dominio Catalítico , Inmunoglobulina G/metabolismo , Cinética , RatonesRESUMEN
Peptide T has a sequence (Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr) belonging to HIV envelope that is involved in the interaction with CD(4) receptor of T lymphocytes. Research of protease activities towards this peptide is very relevant for AIDS therapy. Characterization of specificity of subtilisin Carlsberg towards this very hydrophilic peptide is proposed by using high-performance liquid chromatography and mass spectrometry. Peptide T was totally hydrolysed by the protease after 24 h. Separation of hydrophilic fragments was perfected with an hydrophilic stationary phase and a reversed acetonitrile gradient. Peptide masses were determined by ion spray mass spectrometry. Four primary and one secondary hydrolysis products were found, corresponding to cleavage at the carboxylic side of threonine. Specifities of subtilisin Carlsberg towards the Segments 19 to 26 of bovine pancreatic ribonuclease A, an homologous fragment of peptide T, and peptide T were compared.
RESUMEN
Antigen mimicry by anti-idiotypic antibodies is investigated as a reliable strategy to achieve molecular imprinting of an enzymatic activity. A monoclonal anti-idiotypic antibody (Ab2-9G4H9) was elicited by using a monoclonal antibody (Ab1-7AF9) specific for the beta-lactamase active site. Catalytic features of Ab2 were characterized with beta-lactamase substrates. The antibody combining site appeared to have retained a part of the catalytic specificity. The relevance of the idiotypic mimicry concept for the generation of catalytic antibodies was further demonstrated by eliciting a third generation antibody (Ab3), which was shown to recognize beta-lactamase: the complete internal image properties of Ab2 9G4H9, including binding and catalytic properties, were thus checked.
Asunto(s)
Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Monoclonales/metabolismo , Imitación Molecular , beta-Lactamasas/inmunología , beta-Lactamas/metabolismo , Animales , Catálisis , Ratones , Estructura MolecularRESUMEN
In accord with the original approach that we proposed, catalytic antibodies may be produced by using the anti-idiotypic pathway according to antigen/antibody complementarity rules. The generation and screening of the idiotypic Ab1, the central point on which are anchored the interactions with both the antigen (enzyme) and the anti-idiotypic abzyme, represent a crucial step for the success of this approach. We herein propose to describe a strategy for which we have developed a number of assays, aiming at selecting the proper Ab1, with desired features, likely to elicit an anti-idiotypic catalytic antibody. beta-Lactamase from Bacillus cereus was chosen as the example illustrating our arguments.
Asunto(s)
Anticuerpos Antiidiotipos/biosíntesis , Anticuerpos Catalíticos/biosíntesis , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Catalíticos/inmunología , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/farmacología , Bacillus cereus/enzimología , Sitios de Unión , Ensayo de Inmunoadsorción Enzimática/métodos , Cinética , Penicilina G/farmacología , Ingeniería de Proteínas , beta-Lactamasas/inmunología , beta-Lactamasas/metabolismoRESUMEN
Approaches aiming at eliciting antibodies (Abs) that catalyze specific chemical transformations are numerous. Most of the developed methods are based on the chemical steps of the reaction catalyzed rather than on the structure of known enzyme active sites. The authors have developed an approach that rests on the mimicry properties of the idiotypic network of immune regulation. Recent results, together with the existence of natural catalytic Abs in autoimmune diseases, indicate the need to better understand the regulation properties of immune response, in order to improve the efficiency of tailor-made catalytic Abs.