RESUMEN
Catalytic antibodies possess a dual function that enables both antigen recognition and degradation. However, their time-consuming preparation is a significant drawback. This study developed a new method for quickly converting mice monoclonal antibodies into catalytic antibodies using site-directed mutagenesis. Three mice type monoclonal antibodies targeting hemagglutinin molecule of influenza A virus could be transformed into the catalytic antibodies by deleting Pro95 in CDR-3 of the light chain. No catalytic activity was observed for monoclonal antibodies and light chains. In contrast, the Pro95-deleted light chains exhibited a catalytic activity to cleave the antigenic peptide including the portion of conserved region of hemagglutinin molecule. The affinity of the Pro95-deleted light chains to the antigen increased approximately 100-fold compared to the wild-type light chains. In the mutants, three residues (Asp1, Ser92, and His93) come closer to the appropriate position to create the catalytic site and contributing to the enhancement of both catalytic function and immunoreactivity. Notably, the Pro95-deleted catalytic light chains could suppress influenza virus infection in vitro assay, whereas the parent antibody and the light chain did not. This strategy offers a rapid and efficient way to create catalytic antibodies from existing antibodies, accelerating the development for various applications in diagnostic and therapeutic applications.
Asunto(s)
Anticuerpos Catalíticos , Anticuerpos Monoclonales , Animales , Ratones , Anticuerpos Monoclonales/inmunología , Anticuerpos Catalíticos/metabolismo , Anticuerpos Catalíticos/inmunología , Anticuerpos Catalíticos/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Mutagénesis Sitio-Dirigida , Virus de la Influenza A/inmunología , Dominio Catalítico , Humanos , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/metabolismo , Anticuerpos Antivirales/inmunología , Ratones Endogámicos BALB CRESUMEN
AIMS: Arrhythmogenic cardiomyopathy (AC) is a severe heart disease predisposing to ventricular arrhythmias and sudden cardiac death caused by mutations affecting intercalated disc (ICD) proteins and aggravated by physical exercise. Recently, autoantibodies targeting ICD proteins, including the desmosomal cadherin desmoglein 2 (DSG2), were reported in AC patients and were considered relevant for disease development and progression, particularly in patients without underlying pathogenic mutations. However, it is unclear at present whether these autoantibodies are pathogenic and by which mechanisms show specificity for DSG2 and thus can be used as a diagnostic tool. METHODS AND RESULTS: IgG fractions were purified from 15 AC patients and 4 healthy controls. Immunostainings dissociation assays, atomic force microscopy (AFM), Western blot analysis and Triton X-100 assays were performed utilizing human heart left ventricle tissue, HL-1 cells and murine cardiac slices. Immunostainings revealed that autoantibodies against ICD proteins are prevalent in AC and most autoantibody fractions have catalytic properties and cleave the ICD adhesion molecules DSG2 and N-cadherin, thereby reducing cadherin interactions as revealed by AFM. Furthermore, most of the AC-IgG fractions causing loss of cardiomyocyte cohesion activated p38MAPK, which is known to contribute to a loss of desmosomal adhesion in different cell types, including cardiomyocytes. In addition, p38MAPK inhibition rescued the loss of cardiomyocyte cohesion induced by AC-IgGs. CONCLUSION: Our study demonstrates that catalytic autoantibodies play a pathogenic role by cleaving ICD cadherins and thereby reducing cardiomyocyte cohesion by a mechanism involving p38MAPK activation. Finally, we conclude that DSG2 cleavage by autoantibodies could be used as a diagnostic tool for AC.
Asunto(s)
Anticuerpos Catalíticos , Cardiomiopatías , Humanos , Ratones , Animales , Miocitos Cardíacos/metabolismo , Cadherinas/metabolismo , Desmogleína 2/genética , Anticuerpos Catalíticos/metabolismo , Adhesión Celular/genética , Autoanticuerpos/metabolismo , Cardiomiopatías/metabolismo , Inmunoglobulina G/metabolismo , Desmogleína 3/metabolismo , Desmosomas/metabolismoRESUMEN
One of the primary pathological mechanisms underlying Alzheimer's disease (AD) is the deposition of amyloid ß-protein (Aß42) aggregates in the brain. In this study, a catalytic anti-oligomeric Aß42 scFv antibody, HS72, was identified by screening a human antibody library, its ability to degrade Aß42 aggregates was defined, and its role in the reduction of Aß burden in the AD mouse brain was evaluated. HS72 specifically targeted Aß42 aggregates with an approximately 14-68 kDa range. Based on molecular docking simulations, HS72 likely catalyzed the hydrolytic cleavage of the His13-His14 bond of Aß42 chains in an Aß42 aggregate unit, releasing N/C-terminal fragments and Aß42 monomers. Degradation of Aß42 aggregates by HS72 triggered a considerable disassembly or breakdown of the Aß42 aggregates and greatly reduced their neurotoxicity. Aß deposit/plaque load in the hippocampus of AD mice was reduced by approximately 27% after 7 days (once daily) of intravenous HS72 administration, while brain neural cells were greatly restored and their morphology was drastically improved. The above efficacies of HS72 were all greater than those of HT7, a simple anti-oligomeric Aß42 scFv antibody. Although a catalytic anti-oligomeric Aß42 antibody may have a slightly lower affinity for Aß42 aggregates than a simple anti-oligomeric Aß42 antibody, the former may display a stronger overall efficacy (dual efficacy of induction and catalysis) than the latter (induction alone) in clearing Aß42 aggregates and improving histopathological changes in AD brain. Our findings on the catalytic antibody HS72 indicate the possibility of functional evolution of anti-oligomeric Aß42 antibodies and provide novel insights into the immunotherapy of AD.
Asunto(s)
Enfermedad de Alzheimer , Anticuerpos Catalíticos , Ratones , Humanos , Animales , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/patología , Anticuerpos Catalíticos/metabolismo , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/metabolismo , Encéfalo/metabolismo , Ratones TransgénicosRESUMEN
Histones have vital roles in chromatin functioning and gene transcription. At the same time, they are pernicious in intercellular space because they stimulate systemic inflammatory and toxic responses. Myelin basic protein (MBP) is the major protein of the axon myelin-proteolipid sheath. Antibody-abzymes with various catalytic activities are specific features of some autoimmune diseases. IgGs against five individual histones (H2B, H1, H2A, H3, and H4) and MBP were isolated from the blood of experimental autoimmune encephalomyelitis-prone C57BL/6 mice by affinity chromatography. Abzymes corresponding to various stages of EAE development, including spontaneous EAE, myelin oligodendrocyte glycoprotein (MOG)- and DNA-histone complex-accelerated onset, as well as acute and remission stages, were analyzed. IgG-abzymes against MBP and five individual histones showed unusual polyreactivity in complex formation and enzymatic cross-reactivity in the specific hydrolysis of H2B histone. All IgGs against MBP and individual histones in 3-month-old mice (zero time) demonstrated from 4 to 11 different H2B hydrolysis sites. Spontaneous development of EAE during 60 days led to a significant change in the type and number of H2B hydrolysis sites by IgGs against the five histones and MBP. Mouse treatment with MOG and DNA-histone complex changed the type and number of H2B hydrolysis sites compared to zero time. The minimum number (3) of different H2B hydrolysis sites was found for IgGs against H3 20 days after mouse immunization with DNA-histone complex, whereas the maximum number (33) for anti-H2B IgGs was found 60 days after mouse treatment with DNA-histone complex. Overall, this is the first study to demonstrate that at different stages of EAE evolution, IgG-abzymes against five individual histones and MBP could significantly differ in the specific sites and number of H2B hydrolysis sites. Possible reasons for the catalytic cross-reactivity and significant differences in the number and type of histone H2B cleavage sites were analyzed.
Asunto(s)
Anticuerpos Catalíticos , Encefalomielitis Autoinmune Experimental , Animales , Ratones , Histonas/metabolismo , Hidrólisis , Proteína Básica de Mielina/metabolismo , Ratones Endogámicos C57BL , ADN/metabolismo , Glicoproteína Mielina-Oligodendrócito , Anticuerpos Catalíticos/metabolismo , Inmunoglobulina GRESUMEN
Catalytic antibodies made it feasible to develop new catalysts, which had previously been the subject of research. Scientists have discovered natural antibodies that can hydrolyze substrates such as nucleic acids, proteins, and polysaccharides during decades of research, as well as several ways of producing antibodies with specialized characteristics and catalytic functions. These antibodies are widely used in chemistry, biology, and medicine. Catalytic antibodies can continue to play a role and even fully prevent the emergence of autoimmune disorders, especially in the field of infection and immunity, where the process of its occurrence and development often takes a long time. In this work, the development, design and evolution methodologies, and the expression systems and applications of catalytic antibodies, are discussed. Trial registration: not applicable.
Asunto(s)
Anticuerpos Catalíticos , Anticuerpos Catalíticos/metabolismo , Anticuerpos/genética , Proteínas , CatálisisRESUMEN
The exact mechanisms of multiple sclerosis development are still unknown. However, the development of EAE (experimental autoimmune encephalomyelitis) in Th and 2D2 mice is associated with the infringement of the differentiation profiles of bone marrow hematopoietic stem cells which are bound with the production of compounds that are harmful for human autoantibodies-abzymes that hydrolyze myelin oligodendrocyte glycoprotein, myelin basic protein, and DNA. It showed that autoimmune patients' antioxidant IgG antibodies oxidise some compounds due to their peroxidase (H2O2-dependent) and oxidoreductase (H2O2-independent) activities more effectively than those in healthy humans can. It was interesting to identify whether the redox activities of the antibodies change during the development of autoimmune diseases. Here, we analyzed the change in these redox activities of the IgGs from the blood of Th and 2D2 mice, which corresponded to different stages of the EAE development. The peroxidase activity in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) in the Th (4-fold) and 2D2 (2-fold) mice IgGs, on average, is higher than the oxidoreductase activity is. The peroxidase activity of the Th (1.9-fold) and 2D2 (3.5-fold) mice IgGs remarkably increased during the 40 days of the spontaneous development of EAE. Forty days after the immunization of the MOG peroxidase activity, the IgGs of the Th and 2D2 mice increased 5.6-6.0 times when they were compared with those that presented no increase (3 months of age). The mice IgGs were oxidized with 3,3'-diaminobenzidine (2.4-4.3-fold) and o-phenylenediamine (139-143-fold) less efficiently than they were with ABTS. However, the temper of the change in the IgG activity in the oxidation of these substrates during the spontaneous and MOG-induced development of EAE was close to that which occurred for ABTS. All of the data show that the IgG peroxidase and oxidoreductase activities of EAE mice can play an important role in their protection from toxic compounds and oxidative stress.
Asunto(s)
Anticuerpos Catalíticos , Encefalomielitis Autoinmune Experimental , Humanos , Ratones , Animales , Encefalomielitis Autoinmune Experimental/patología , Antioxidantes/farmacología , Peróxido de Hidrógeno , Ratones Endogámicos C57BL , Anticuerpos Catalíticos/metabolismo , Glicoproteína Mielina-Oligodendrócito , Peroxidasas , Oxidorreductasas , Inmunoglobulina G/metabolismoRESUMEN
The cleavage reactions of catalytic antibodies are mediated by a serine protease mechanism involving a catalytic triad composed of His, Ser, and Asp residues, which reside in the variable region. Recently, we discovered a catalytic antibody, H34 wild type (H34wt), that is capable of enzymatically cleaving an immune-check point PD-1 peptide and recombinant PD-1; however, H34wt does not contain His residues in the variable region. To clarify the reason behind the catalytic features of H34wt and the amino acid residues involved in the catalytic reaction, we performed site-directed mutagenesis focusing on the amino acid residues involved in the cleavage reaction, followed by catalytic activity tests, immunological reactivity evaluation, and molecular modeling. The results revealed that the cleavage reaction by H34wt proceeds through the action of a new catalytic site composed of Arg, Thr, and Gln. This new scheme differs from that of the serine protease mechanism of catalytic antibodies.
Asunto(s)
Anticuerpos Catalíticos , Dominio Catalítico , Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/metabolismo , Secuencia de Aminoácidos , Receptor de Muerte Celular Programada 1 , Cadenas Ligeras de Inmunoglobulina/metabolismo , Serina Endopeptidasas/metabolismo , AminoácidosRESUMEN
Medical abzymology has made a great contribution to the development of general autoimmunity theory: it has put the autoantibodies (Ab) as the key brick of the theory to the level of physiological functionality by providing such Ab with the ability to catalyze and mediate direct and independent cytotoxic effect on cellular and molecular targets. Natural catalytic autoantibodies (abzymes) while being a pool of canonical Abs and possessing catalytic activity belong to the new group of physiologically active substances whose features and properties are evolutionary consolidated in one functionally active biomolecule. Therefore, further studies on Ab-mediated autoAg degradation and other targeted Ab-mediated proteolysis may provide biomarkers of newer generations and thus a supplementary tool for assessing the disease progression and predicting disability of the patients and persons at risks. This chapter is a summary of current knowledge and prognostic perspectives toward catalytic Abs in autoimmunity and thus some autoimmune clinical cases, their role in pathogenesis, and the exploitation of both whole molecules and their constituent parts in developing highly effective targeted drugs of the future to come, and thus the therapeutic protocols being individualized.
Asunto(s)
Anticuerpos Catalíticos , Autoinmunidad , Anticuerpos Catalíticos/metabolismo , Autoanticuerpos/metabolismo , Biomarcadores , Progresión de la Enfermedad , HumanosRESUMEN
I endeavor to share how various choices-some deliberate, some unconscious-and the unmistakable influence of many others shaped my scientific pursuits. I am fascinated by how two long-term, major streams of my research, DNA replication and purine biosynthesis, have merged with unexpected interconnections. If I have imparted to many of the talented individuals who have passed through my lab a degree of my passion for uncloaking the mysteries hidden in scientific research and an understanding of the honesty and rigor it demands and its impact on the world community, then my mentorship has been successful.
Asunto(s)
Bioquímica/historia , Replicación del ADN , Enzimas , Purinas/biosíntesis , Antiinfecciosos/química , Antiinfecciosos/farmacología , Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/metabolismo , Enzimas/química , Enzimas/metabolismo , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Masculino , Estados UnidosRESUMEN
The retro-aldolase mechanism of methodol catalysed by the catalytic antibody 33F12 is described based on the exploration of the free energy landscape obtained with QM/MM methods. The amino acids involved in the reaction have been identified, as well as their specific role played in the active site and in the flexibility of the loops. Finally, the comparison with a de novo enzyme RA95.5-8F provides a deeper understanding of catalytic differences between such different protein scaffolds.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Teoría Cuántica , Anticuerpos Catalíticos/química , Biocatálisis , Cristalografía por Rayos X , Fructosa-Bifosfato Aldolasa/química , Metanol/química , Metanol/metabolismo , Simulación de Dinámica MolecularRESUMEN
The exact mechanisms of multiple sclerosis (MS) development are still unknown, but the development of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice is associated with the violation of bone marrow hematopoietic stem cells (HSCs) differentiation profiles associated with the production of harmful for human's autoantibodies hydrolyzing myelin basic protein, myelin oligodendrocyte glycoprotein (MOG35-55), and DNA. It was shown that IgGs from the sera of healthy humans and autoimmune patients oxidize many different compounds due to their H2O2-dependent peroxidase and oxidoreductase activity in the absence of H2O2. Here we first analyzed the change in the relative redox activities of IgGs antibodies from the blood of C57BL/6 mice over time at different stages of the EAE development. It was shown that the peroxidase activity of mice IgGs in the oxidation of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is on average 6.9-fold higher than the oxidoreductase activity. The peroxidase activity of IgGs increased during the spontaneous development of EAE during 40 days, 1.4-fold. After EAE development acceleration due to mice immunization with MOG35-55 (5.3-fold), complexes of bovine DNA with methylated bovine serum albumin (DNA-metBSA; 3.5-fold), or with histones (2.6-fold), the activity was increased much faster. The increase in peroxidase activity after mice immunization with MOG35-55 and DNA-metBSA up to 40 days of experiments was relatively gradual, while for DNA-histones complex was observed its sharp increase at the acute phase of EAE (14-20 days). All data show that IgGs' redox activities can play an important role in the protection of mice from toxic compounds and oxidative stress.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Autoanticuerpos/metabolismo , Encefalomielitis Autoinmune Experimental/enzimología , Células Madre Hematopoyéticas/inmunología , Oxidorreductasas/metabolismo , Peroxidasas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/patología , Humanos , Peróxido de Hidrógeno/farmacología , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína Básica de Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/administración & dosificación , Oxidación-Reducción , Oxidorreductasas/inmunología , Fragmentos de Péptidos/administración & dosificación , Peroxidasas/inmunologíaRESUMEN
Immunoglobulins are known to combine various effector mechanisms of the adaptive and the innate immune system. Classical immunoglobulin functions are associated with antigen recognition and the initiation of innate immune responses. However, in addition to classical functions, antibodies exhibit a variety of non-canonical functions related to the destruction of various pathogens due to catalytic activity and cofactor effects, the action of antibodies as agonists/antagonists of various receptors, the control of bacterial diversity of the intestine, etc. Canonical and non-canonical functions reflect the extreme human antibody repertoire and the variety of antibody types generated in the organism: antigen-specific, natural, polyreactive, broadly neutralizing, homophilic, bispecific and catalytic. The therapeutic effects of intravenous immunoglobulins (IVIg) are associated with both the canonical and non-canonical functions of antibodies. In this review, catalytic antibodies will be considered in more detail, since their formation is associated with inflammatory and autoimmune diseases. We will systematically summarize the diversity of catalytic antibodies in normal and pathological conditions. Translational perspectives of knowledge about natural antibodies for IVIg therapy will be also discussed.
Asunto(s)
Anticuerpos Biespecíficos/genética , Anticuerpos Catalíticos/genética , Enfermedades Autoinmunes/inmunología , Isotipos de Inmunoglobulinas/genética , Enfermedades Neurodegenerativas/inmunología , Inmunidad Adaptativa , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/metabolismo , Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/metabolismo , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/metabolismo , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/terapia , Humanos , Inmunidad Innata , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Isotipos de Inmunoglobulinas/química , Isotipos de Inmunoglobulinas/clasificación , Isotipos de Inmunoglobulinas/metabolismo , Inmunoglobulinas Intravenosas/uso terapéutico , Pruebas Inmunológicas , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/terapiaRESUMEN
For breast-fed infants, human milk is a source of various nutrients (e.g., proteins, peptides, antibodies) and bioactive components that promote neonatal growth and protect infants from viral and bacterial infection. Moreover, in terms of infant nutrition and protection the functions of many human milk components are very different from those of blood and other biological fluids of healthy adults. For example, catalytic antibodies ("abzymes") with synthetic activities (protein, oligosaccharide, and lipid kinase activities) have been found in human breast milk that are absent in the blood of healthy people. Abzymes with hydrolyzing functions have been detected not only in milk, but also in the blood of patients with autoimmune diseases. Obviously, feeding newborns human milk has a very specific role and it is a unique aspect of mammalian nutrition. Ribonuclease and DNase autoantibodies or abzymes are found in milk and blood of lactating women, but not in blood sera of healthy men and nonpregnant woman. Here, we present the first evidence that human milk secretory IgA molecules (sIgA) can effectively hydrolyze ribooligonucleotides containing 23 different bases [(pN)23 ribooligonucleotides] and 4 microRNAs: miR-9-5p, miR-219-2-3p, miR-137, and miR-219a-5p. Ribonuclease activity is an inherent property of sIgAs. We showed that 7 individual sIgAs hydrolyzed the ribooligonucleotides (pA)23, (pU)23, and (pC)23 nonspecifically and with comparable efficiency, whereas hydrolysis of the 4 microRNAs by sIgAs was site-specific. Sites of hydrolysis of 4 microRNAs by IgG from blood of patients with schizophrenia have been previously identified. The sites of hydrolysis of 4 microRNAs by sIgA-abzymes were very different from the previously identified sites of hydrolysis by IgG in patients with schizophrenia. In addition, in contrast to IgG, milk sIgAs efficiently hydrolyzed microRNAs in their loop and duplex regions.
Asunto(s)
Inmunoglobulina A Secretora/metabolismo , MicroARNs/metabolismo , Leche Humana/metabolismo , Ribonucleótidos/metabolismo , Adulto , Animales , Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/metabolismo , Femenino , Humanos , Hidrólisis , Lactancia , Leche Humana/inmunología , Oligosacáridos/análisisRESUMEN
Over thousands of monoclonal antibodies (mAbs) have been produced so far, and it would be valuable if these mAbs could be directly converted into catalytic antibodies. We have designed a system to realize the above concept by deleting Pro95, a highly conserved residue in CDR-3 of the antibody light chain. The deletion of Pro95 is a key contributor to catalytic function of the light chain. The S35 and S38 light chains have identical amino acid sequences except for Pro95. The former, with Pro95 did not show any catalytic activity, whereas the latter, without Pro95, exhibited peptidase activity. To verify the generality of this finding, we tested another light chain, T99wt, which had Pro95 and showed little catalytic activity. In contrast, a Pro95-deleted mutant enzymatically degraded the peptide substrate and amyloid-beta molecule. These two cases demonstrate the potential for a new method of creating catalytic antibodies from the corresponding mAbs.
Asunto(s)
Algoritmos , Anticuerpos Catalíticos/química , Anticuerpos Monoclonales/química , Modelos Moleculares , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Anticuerpos Catalíticos/genética , Anticuerpos Catalíticos/metabolismo , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Catálisis , Cromatografía Líquida de Alta Presión , Hidrólisis , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Multimerización de Proteína , Proteolisis , Proteínas RecombinantesRESUMEN
Abzymes with various catalytic activities are the earliest statistically significant markers of existing and developing autoimmune diseases (AIDs). Currently, schizophrenia (SCZD) is not considered to be a typical AID. It was demonstrated recently that antibodies from SCZD patients efficiently hydrolyze DNA and myelin basic protein. Here, we showed for the first time that autoantibodies from 35 SCZD patients efficiently hydrolyze RNA (cCMP > poly(C) > poly(A) > yeast RNA) and analyzed site-specific hydrolysis of microRNAs involved in the regulation of several genes in SCZD (miR-137, miR-9-5p, miR-219-2-3p, and miR-219a-5p). All four microRNAs were cleaved by IgG preparations (n = 21) from SCZD patients in a site-specific manner. The RNase activity of the abzymes correlated with SCZD clinical parameters. The data obtained showed that SCZD patients might display signs of typical autoimmune processes associated with impaired functioning of microRNAs resulting from their hydrolysis by the abzymes.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Inmunoglobulina G/metabolismo , MicroARNs/metabolismo , ARN/sangre , ARN/metabolismo , Esquizofrenia/genética , Esquizofrenia/inmunología , Adulto , Anticuerpos Catalíticos/sangre , Femenino , Humanos , Hidrólisis , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Esquizofrenia/sangre , Adulto JovenRESUMEN
Catalytic antibody 7B9, which was elicited against p-nitrobenzyl phosphonate transition-state analogue (TSA) 1, hydrolyzes a wide range of p-nitrobenzyl monoesters and thus shows broad substrate tolerance. To reveal the molecular basis of this substrate tolerance, the 7B9 Fab fragment complexed with p-nitrobenzyl ethylphosphonate 2 was crystallized and the three-dimensional structure was determined. The crystal structure showed that the strongly antigenic p-nitrobenzyl moiety occupied a relatively shallow antigen-combining site and therefore the alkyl moiety was located outside the pocket. These results support the observed broad substrate tolerance of 7B9 and help rationalize how 7B9 can catalyze various p-nitrobenzyl ester derivatives. The crystal structure also showed that three amino acid residues (AsnH33, SerH95, and ArgL96) were placed in key positions to form hydrogen bonds with the phosphonate oxygens of the transitions-state analogue. In addition, the role of these amino acid residues was examined by site-directed mutagenesis to alanine: all mutants (AsnH33Ala, SerH95Ala, and ArgL96Ala) showed no detectable catalytic activity. Coupling the findings from our structural studies with these mutagenesis results clarified the structural basis of the observed broad substrate tolerance of antibody 7B9-catalyzed hydrolyses. Our findings provide new strategies for the generation of catalytic antibodies that accept a broad range of substrates, aiding their practical application in synthetic organic chemistry.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Ésteres/metabolismo , Nitrobencenos/metabolismo , Biocatálisis , Ésteres/química , Hidrólisis , Modelos Moleculares , Estructura Molecular , Nitrobencenos/química , Relación Estructura-ActividadRESUMEN
Histones and their posttranslational modified forms play pivotal roles in chromatin functioning and gene transcription. Also, histones are harmful when they enter the intercellular space; their administration to animals results in systemic inflammatory and toxic responses. Autoantibodies having enzymatic activities (abzymes) are the specific feature of several autoimmune and viral diseases. Electrophoretically homogeneous IgGs containing no canonical proteases were purified from sera of HIV-infected patients by using several affinity chromatographies. In contrast to known canonical proteases, Abs from HIV-infected patients hydrolyzed exclusively only histones but no other control globular proteins. The H3 and H4 histone cleavage sites by antihistone IgGs were determined by matrix-assisted laser desorption/ionization mass spectrometry for the first time. Two clusters of H3 hydrolysis contain major (â) and minor (*) cleavage sites: 18-K*Q*LAâTK*AâAR*KSâA*P-30 and 34-G*VK*KPHR*YRPGTVA*L*R-50. H4 histone has only 1 cluster of cleavage sites containing additionally moderate (↓) cleavage sites: 15-AâKRâHRâKVLR↓D*NIQ↓GIT*K-31. Sites of these histones cleavage correspond mainly to their known epitopes. It was surprising that most of the cleavage sites of histones are involved in the interaction with DNA of nucleosome core. Because histones act as damage-associated molecules, abzymes against H3 and H4 can play important role in pathogenesis of AIDs and probably other viral and immune diseases.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Autoanticuerpos/metabolismo , Infecciones por VIH/inmunología , Histonas/química , Inmunoglobulina G/metabolismo , Adolescente , Adulto , Secuencia de Aminoácidos , Anticuerpos Catalíticos/biosíntesis , Anticuerpos Catalíticos/aislamiento & purificación , Autoanticuerpos/biosíntesis , Autoanticuerpos/aislamiento & purificación , Sitios de Unión , Biocatálisis , ADN/química , ADN/metabolismo , Epítopos/química , Femenino , VIH/inmunología , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/patología , Histonas/inmunología , Histonas/metabolismo , Humanos , Hidrólisis , Sueros Inmunes/química , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/aislamiento & purificación , Masculino , Modelos Moleculares , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Proteolisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Catalytic antibodies are a promising model for creating highly specific biocatalysts with predetermined activity. However, in order to realize the directed change or improve their properties, it is necessary to understand the basics of catalysis and the specificity of interactions with substrates. In the present work, a structural and functional study of the Fab fragment of antibody A5 and a comparative analysis of its properties with antibody A17 have been carried out. These antibodies were previously selected for their ability to interact with organophosphorus compounds via covalent catalysis. It has been established that antibody A5 has exceptional specificity for phosphonate X with bimolecular reaction rate constants of 510 ± 20 and 390 ± 20 min^(-1)Ð^(-1) for kappa and lambda variants, respectively. 3D-Modeling of antibody A5 structure made it possible to establish that the reaction residue L-Y33 is located on the surface of the active site, in contrast to the A17 antibody, in which the reaction residue L-Y37 is located at the bottom of a deep hydrophobic pocket. To investigate a detailed mechanism of the reaction, A5 antibody mutants with replacements L-R51W and H-F100W were created, which made it possible to perform stopped-flow kinetics. Tryptophan mutants were obtained as Fab fragments in the expression system of the methylotrophic yeast species Pichia pastoris. It has been established that the effectiveness of their interaction with phosphonate X is comparable to the wild-type antibody. Using the data of the stopped-flow kinetics method, significant conformational changes were established in the phosphonate modification process. The reaction was found to proceed using the induced-fit mechanism; the kinetic parameters of the elementary stages of the process have been calculated. The results present the prospects for the further improvement of antibody-based biocatalysts.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Compuestos Organofosforados/metabolismo , Secuencia de Aminoácidos , Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/genética , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Biocatálisis , Dominio Catalítico , Clonación Molecular , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Cinética , Modelos Moleculares , Compuestos Organofosforados/antagonistas & inhibidores , Compuestos Organofosforados/química , Compuestos Organofosforados/inmunología , Pichia/genética , Pichia/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The antigen-binding properties of single chain Fv antibodies (scFvs) can vary depending on the position and type of fusion tag used, as well as the host cells used for expression. The issue is even more complicated with a catalytic scFv antibody that binds and hydrolyses a specific antigen. Herein, we investigated the antigen-binding and -hydrolysing activities of the catalytic anti-nucleic acid antibody 3D8 scFv expressed in Escherichia coli or HEK293f cells with or without additional amino acid residues at the N- and C-termini. DNA-binding activity was retained in all recombinant forms. However, the DNA-hydrolysing activity varied drastically between forms. The DNA-hydrolysing activity of E. coli-derived 3D8 scFvs was not affected by the presence of a C-terminal human influenza haemagglutinin (HA) or His tag. By contrast, the activity of HEK293f-derived 3D8 scFvs was completely lost when additional residues were included at the N-terminus and/or when a His tag was incorporated at the C-terminus, whereas a HA tag at the C-terminus did not diminish activity. Thus, we demonstrate that the antigen-binding and catalytic activities of a catalytic antibody can be separately affected by the presence of additional residues at the N- and C-termini, and by the host cell type.
Asunto(s)
Anticuerpos Catalíticos/metabolismo , ADN/metabolismo , Hemaglutininas/metabolismo , Histidina/metabolismo , Oligopéptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/metabolismo , Anticuerpos Catalíticos/genética , Clonación Molecular/métodos , ADN/química , División del ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Células HEK293 , Hemaglutininas/genética , Histidina/genética , Humanos , Cinética , Oligopéptidos/genética , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Análisis de Secuencia de Proteína , Anticuerpos de Cadena Única/genéticaRESUMEN
A catalytic antibody A17 and its mutants highly efficiently interact with organophosphorus pesticide paraoxon. In this work, we studied the protective properties of antibody A17-K47 in paraoxon poisoning using a mouse model. The optimal paraoxon dose simulating the acute toxic effect of organophosphorus compounds was 550 µg/kg. The pharmacokinetic parameters of A17-K47 antibody were t1/2distr =7.2±1.4 min, t1/2el =330±20 min. The antibody did not cause toxic effects when administered at a ten-fold calculated therapeutic dose (610 mg/kg). The drug did not reduce mortality from acute paraoxon poisoning; however, the absence of drug toxicity opens up prospects for its use in symptomatic treatment of chronic paraoxon poisoning.