Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Doenças Reumáticas/imunologia , Anticorpos Antinucleares/imunologia , Síndrome Antifosfolipídica/imunologia , Autoanticorpos/química , Autoantígenos/química , DNA/imunologia , Humanos , Ribonucleoproteínas Nucleares Pequenas/imunologia , Proteínas Centrais de snRNPRESUMO
Anti-DNA antibodies contribute to the pathology of systemic lupus erythematosus. Their depositon in tissue lesions could result from localization of preformed immune complexes of antibodies with DNA or nucleosomes, or from cross-reaction of anti-DNA antibodies directly with tissue proteins. Structural analyses contribute to understanding their pathogenic potential. Primary structures of lupus immunoglobulin G double-stranded DNA-binding autoantibodies are determined by immunoglobulin genes with mutated variable region segments, indicative of selection by immunizing antigen. Arginine, lysine and asparagine residues in complementarity-determining region favor DNA binding. Heavy-chain variable regions make major contributions to DNA binding; affinity and specificity of binding are modulated or can be abrogated by the light-chain variable domain. Crytallographic structure is known for a few antibody-DNA complexes and several ligand-free Fab fragments. Computer modeling supplements this limited information. Structural information of lupus antibody interactions with both DNA and cross-reacting molecules will support use of ligands to inhibit tissue deposition of the antibodies and prevent lesion formation in lupus.
Assuntos
Anticorpos Antinucleares/química , Anticorpos Antinucleares/farmacologia , Animais , Anticorpos Antinucleares/genética , Complexo Antígeno-Anticorpo/metabolismo , Simulação por Computador , Cristalografia por Raios X , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Modelos Moleculares , MutaçãoRESUMO
Drs Berden and Koutouzov presented evidence that nucleosomes are antigens in lupus pathogenesis and that apoptotic cells are the source of nucleosomes. Berden's group measured persistence of circulating nucleosomes and nucleosome-antibody complexes in autoimmune mice and demonstrated nucleosomal deposition in skin of SLE patients as well as in renal lesions. Koutouzov reported that anti-nucleosomes are among the earliest autoantibodies in MRL+/+ mice, appearing several weeks before anti-DNA antibodies. Treatment of the mice with a pro-apoptotic drug, taxotere, accelerated autoantibody production and development of lesions. Herrmann proposed that persistent immunization results from reduced dead cell clearance and reduced production of immunosuppressive cytokines by defective scavenger macrophages. He also described accumulation of apoptotic cells in germinal follicles in SLE patients and attachment of nuclear antigens that are produced in apoptosis to the surface of follicular dendritic cells. Apoptosis-derived nucleosomes may be important in both the immunizing and effector arms of pathogenesis.
Assuntos
Apoptose/imunologia , Autoantígenos/imunologia , Nefrite Lúpica/imunologia , Nucleossomos/imunologia , Animais , Humanos , Nefrite Lúpica/patologiaRESUMO
It has been proposed that autoreactivity of modest affinity contributes to positive selection of a preimmunization B cell repertoire, whereas high-affinity autoreactivity leads to negative selection. This hypothesis predicts that a B cell producing a physiologically selected unmutated ssDNA-binding Ab should be a precursor of cells that respond to diverse exogenous Ags. To test this prediction, we prepared transgenic mice bearing the rearranged V(H) domain of an IgM Ab from a nonautoimmune mouse immunized with a DNA-protein complex, poly(dC)-methylated BSA. The Ab, dC1, binds both poly(dC) and ssDNA. It is encoded by V(H) and V(L) gene segments with no mutations, suggesting that the producing cell may have been selected before and activated during immunization. The dC1V(H) transgene was targeted to the IgH locus. In heterozygous mice, on a nonautoimmune C57BL/6 background, the transgene allotype was expressed on B cell surfaces and in serum Ig, but about one-third of B cells expressed the endogenous allele instead. Total serum Ig concentrations were normal and included both transgene- and endogenous gene-coded IgM and IgG. The transgene V(H) D(H)J(H) was expressed in splenic IgM cDNA with few or no mutations, and in IgG cDNA with multiple mutations. The transgene allotype was also expressed in Abs formed on immunization with thyroglobulin, pneumococcal polysaccharide, and ssDNA-methylated BSA. Consistent with the hypothesis, cells with a rearranged autoreactive V(H) domain selected for reactivity with a form of ssDNA did serve as precursors for cells producing IgM and IgG Abs to diverse Ags.
Assuntos
Anticorpos Antinucleares/genética , DNA de Cadeia Simples/imunologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Imunoglobulina G/genética , Imunoglobulina M/genética , Região Variável de Imunoglobulina/genética , Animais , Anticorpos Antinucleares/imunologia , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Sequência de Bases , Células da Medula Óssea/imunologia , Marcação de Genes , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/biossíntese , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Poli C/imunologia , Baço/imunologiaRESUMO
Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization.
Assuntos
DNA de Cadeia Simples/imunologia , Fragmentos de Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Poli C/imunologia , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Ensaio de Imunoadsorção Enzimática/métodos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Concentração OsmolarRESUMO
This protocol describes application of single cell reverse transcription polymerase chain reaction (RT-PCR) to the study of human immunoglobulin V region usage. The procedure begins with separation of peripheral blood mononuclear cells (PBMC) from human blood. The PBMC are stained with the B cell selective marker, anti-CD19. Stained B cells are sorted by flow cytometry and deposited, consecutively, one cell into each of an array of tubes. cDNA for one or more antibody variable regions (VH and/or VL) is synthesized with a primer (or primers) complementary to sequence(s) within the constant region (Cmu, Cgamma, Ckappa and/or Clambda). The cDNA is used as template for PCR amplification with gene or gene family specific primers. A second PCR is then performed with two nested primers to increase both the specificity and quantity of V region PCR products. The purified PCR products are sequenced directly and aligned to V region germline database and the Genbank database. Single cell RT-PCR is a fast and convenient way to analyze V region gene expression. It avoids the bias that may be introduced into V region cDNA library construction by the presence of highly variable levels of mRNA in different cells. The PCR products are obtained in quantities that can be cloned into bacterial expression vectors for production of recombinant V region protein domains.
Assuntos
Linfócitos B/imunologia , Região Variável de Imunoglobulina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto , Idoso , Linfócitos B/citologia , DNA Complementar/genética , Sangue Fetal/citologia , Sangue Fetal/imunologia , Citometria de Fluxo/métodos , Expressão Gênica , Rearranjo Gênico do Linfócito B/imunologia , Genes de Imunoglobulinas/genética , Humanos , Região Variável de Imunoglobulina/sangue , Leucócitos Mononucleares/citologiaAssuntos
Anticorpos Antinucleares/imunologia , Autoanticorpos/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Animais , Anticorpos Antinucleares/genética , Autoanticorpos/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Transgênicos , Técnicas de Sonda Molecular , Ribonucleoproteínas/imunologiaAssuntos
Chaperonina 60/isolamento & purificação , Cromatografia/métodos , Fragmentos de Imunoglobulinas/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Chaperonina 60/química , Fragmentos de Imunoglobulinas/química , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Immune responses change in aging humans, but it is not known whether there is an age-associated change in the expressed B cell repertoire. We compared Ig VH cDNA libraries from circulating B cells of five elderly and three young human adults. As in young persons, nearly two-thirds of the cDNA clones from older subjects had zero to three V(H) mutations, although there was more individual variation among the elderly. V(H)4 family expression increased in older subjects, both in unmutated and in mutated cDNA clones, whereas V(H)3 family expression predominated in young adults. To test for bias toward activated cells in the cDNA libraries, we studied two older persons by both cDNA library analysis and single-cell RT-PCR. In one subject, more than 85% of VH segments were unmutated by either analysis. In the second, mutated Ig segments were much more frequent in cDNA clones than in consecutive single cells; however, V(H) family usage and high representation of particular genes were similar in both analyses. While aging humans continue to produce naive B cells with unmutated Ig genes, a shift to greater use of the V(H)4 family members and expression of particular genes may reflect changes in selection of developing B cells before affinity maturation toward reactivity with foreign antigen.
Assuntos
Envelhecimento/genética , Envelhecimento/imunologia , Regulação da Expressão Gênica/imunologia , Genes de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Adulto , Idoso , DNA Complementar/química , Frequência do Gene , Biblioteca Gênica , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Humanos , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias J de Imunoglobulina/biossíntese , Cadeias J de Imunoglobulina/genética , Região Variável de Imunoglobulina/biossíntese , Dados de Sequência Molecular , Mutação/genética , Mutação/imunologia , Estrutura Terciária de Proteína/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
mAb Z22 is a highly selective IgG anti-Z-DNA Ab from an immunized C57BL/6 mouse. Previous studies showed that heavy chain CDR3 amino acids are critical for Z-DNA binding by the single chain variable fragment (scFv) comprising both V region heavy chain (VH) and V region light chain (VL) of mAb Z22 and that the VH domain alone binds Z-DNA with an affinity similar to that of whole variable fragment (Fv). To determine whether Z-DNA binding by VH alone and by Fv involves identical complementarity determining region residues, we tested effects of single or multiple amino acid substitutions in recombinant VH, scFv, and associated VH-VL heterodimers. Each recombinant product was a fusion protein with a B domain of Staphylococcal protein A (SPA). Z22VH-SPA alone was not highly selective; it bound strongly to other polynucleotides, particularly polypyrimidines, and ssDNA as well as to Z-DNA. In contrast, scFv-SPA or associated VH-VL dimers bound only to Z-DNA. VL-SPA domains bound weakly to Z-DNA; SPA alone did not bind. Introduction of multiple substitutions revealed that the third complementarity determining region of the heavy chain (CDR3H) was critical for both VH and scFv binding to Z-DNA. However, single substitutions that eliminated or markedly reduced Z-DNA binding by scFv instead caused a modest increase or no reduction in binding by VH alone. Association of VH-SPA with Z22VL-SPA restored both the effects of single substitutions and Z-DNA selectivity seen with Fv and intact Ab. Polypyrimidine and ssDNA binding by the isolated VH domain of immunization-induced anti-Z-DNA Ab resembles the activity of natural autoantibodies and suggests that VH-dependent binding to a ligand mimicked by polypyrimidines may play a role in B cell selection before immunization with Z-DNA.
Assuntos
Anticorpos Antinucleares/metabolismo , Sítios de Ligação de Anticorpos , DNA/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/metabolismo , Fragmentos de Peptídeos/imunologia , Substituição de Aminoácidos/genética , Animais , Anticorpos Antinucleares/química , Anticorpos Antinucleares/genética , Sítios de Ligação de Anticorpos/genética , DNA/metabolismo , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Polinucleotídeos/imunologia , Polinucleotídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Previous studies showed that VH domains of several anti-DNA Abs can bind DNA in the absence of VL. In the current work, we tested the VH autoreactive potential more generally, examining VH domains that did not come from known autoantibodies. Using a bacterial expression system, we produced 11 fusion proteins, each containing a VH domain and a B domain of staphylococcal protein A. The VH domains were coded in cDNA libraries from circulating B cells of healthy young adult humans. Thus, binding properties of the Ig molecules from which they came were unknown. The B cells had not been stimulated in vitro. Seven cDNA clones combined the frequently expressed VH3-23 gene segment with varied DH and JH segments. The other clones contained unmutated VH3-7, VH3-9, VH3-53, and VH4-39 segments. We compared these bacterial expression products with single-chain Fv, VH and VL domains of IgM mAb 18/2, a VH3-23-encoded, DNA-binding autoantibody. Submicromolar concentrations of 5 of the 11 VH domains bound to ssDNA. Those and one more also bound to immobilized poly(dT), and two bound to circular plasmid dsDNA. Soluble poly(dT) was the most potent inhibitor in competitive ELISA. Seven of the VH domains also bound to immobilized nuclear ribonucleoprotein, four to histone and none to thyroglobulin. Two interacted with the matrix of a Sephacryl S-100 column. The polyreactive autoantigen-binding properties of these VH domains raise the question of whether these properties may play a role in the formation of the VH repertoire of circulating B cells.
Assuntos
Autoanticorpos/biossíntese , Autoantígenos/imunologia , Expressão Gênica/imunologia , Biblioteca Gênica , Vetores Genéticos/imunologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/biossíntese , Sequência de Aminoácidos , Afinidade de Anticorpos , Autoanticorpos/genética , Autoanticorpos/metabolismo , Bacteriófago M13/genética , Sítios de Ligação de Anticorpos , Escherichia coli/genética , Escherichia coli/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Dados de Sequência Molecular , Ácidos Nucleicos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/químicaRESUMO
We have used single and multiple site-directed mutagenesis, and molecular modeling, to identify critical residues in the DNA binding site of MAb 2C10, an IgG anti-dsDNA autoantibody from an MRL/lpr lupus mouse. Simultaneous replacement of four Arg residues in the CDR3H abolished binding activity. With one exception, replacement of any one of these Arg residues reduced the activity to 20-50% of the unmutated scFv activity. Arg to Asp replacements had a slightly greater effect than Arg to Ala replacements. In the one exceptional case, replacement of Arg99 with Ala actually increased DNA binding five-fold and replacement by Asp had little effect. Mutation of Phe32 and Asn35 to A1a in CDRIH decreased DNA binding, whereas replacement of Arg31 with A1a had negligible effect. Ala substitution of any one of a cluster of Asp residues in CDR1L increased DNA binding three to six-fold, confirming previous findings that the L-chain of MAb 2C10 is not favorable for DNA binding. The L-chain does participate in shaping the selectivity of antigen binding, and mutation of CDR3L residue Asp92 or Asn93 caused a decrease in DNA binding activity. Directed mutagenesis, consistent with a molecular model, indicates that: several CDR amino acids contribute to DNA binding, without one residue dominating; both VH and VL CDR3 domains contribute to specificity of binding whereas the CDR1L hinders DNA binding. The results suggest a significant role for electrostatics in the interaction of DNA with MAb 2C10.
Assuntos
Anticorpos Antinucleares/metabolismo , Autoanticorpos/metabolismo , Sítios de Ligação de Anticorpos , DNA/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antinucleares/química , Anticorpos Antinucleares/genética , Afinidade de Anticorpos/genética , Autoanticorpos/química , Autoanticorpos/genética , Sítios de Ligação de Anticorpos/genética , DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-DirigidaRESUMO
The properties of duplex CTG.CAG and CGG.CCG, which are involved in the etiology of several hereditary neurodegenerative diseases, were investigated by a variety of methods, including circularization kinetics, apparent helical repeat determination, and polyacrylamide gel electrophoresis. The bending moduli were 1.13 x 10(-19) erg.cm for CTG and 1.27 x 10(-19) erg.cm for CGG, approximately 40% less than for random B-DNA. Also, the persistence lengths of the triplet repeat sequences were approximately 60% the value for random B-DNA. However, the torsional moduli and the helical repeats were 2.3 x 10(-19) erg.cm and 10.4 base pairs (bp)/turn for CTG and 2.4 x 10(-19) erg.cm and 10.3 bp/turn for CGG, respectively, all within the range for random B-DNA. Determination of the apparent helical repeat by the band shift assay indicated that the writhe of the repeats was different from that of random B-DNA. In addition, molecules of 224-245 bp in length (64-71 triplet repeats) were able to form topological isomers upon cyclization. The low bending moduli are consistent with predictions from crystallographic variations in slide, roll, and tilt. No unpaired bases or non-B-DNA structures could be detected by chemical and enzymatic probe analyses, two-dimensional agarose gel electrophoresis, and immunological studies. Hence, CTG and CGG are more flexible and highly writhed than random B-DNA and thus would be expected to act as sinks for the accumulation of superhelical density.
Assuntos
DNA/química , Doenças Neuromusculares/genética , Repetições de Trinucleotídeos/genética , DNA Super-Helicoidal/química , Eletroforese em Gel de Ágar , Eletroforese em Gel Bidimensional , Humanos , Cinética , Conformação de Ácido Nucleico , Plasmídeos/químicaRESUMO
OBJECTIVE: To determine whether long-term supplementation with vitamin E enhances in vivo, clinically relevant measures of cell-mediated immunity in healthy elderly subjects. DESIGN: Randomized, double-blind, placebo-controlled intervention study. SETTING AND PARTICIPANTS: A total of 88 free-living, healthy subjects at least 65 years of age. INTERVENTION: Subjects were randomly assigned to a placebo group or to groups consuming 60, 200, or 800 mg/d of vitamin E for 235 days. MAIN OUTCOME MEASURES: Delayed-type hypersensitivity skin response (DTH); antibody response to hepatitis B, tetanus and diphtheria, and pneumococcal vaccines; and autoantibodies to DNA and thyroglobulin were assessed before and after supplementation. RESULTS: Supplementation with vitamin E for 4 months improved certain clinically relevant indexes of cell-mediated immunity in healthy elderly. Subjects consuming 200 mg/d of vitamin E had a 65% increase in DTH and a 6-fold increase in antibody titer to hepatitis B compared with placebo (17% and 3-fold, respectively), 60-mg/d (41% and 3-fold, respectively), and 800-mg/d (49% and 2.5-fold, respectively) groups. The 200-mg/d group also had a significant increase in antibody titer to tetanus vaccine. Subjects in the upper tertile of serum alpha-tocopherol (vitamin E) concentration (>48.4 micromol/L [2.08 mg/dL]) after supplementation had higher antibody response to hepatitis B and DTH. Vitamin E supplementation had no effect on antibody titer to diphtheria and did not affect immunoglobulin levels or levels of T and B cells. No significant effect of vitamin E supplementation on autoantibody levels was observed. CONCLUSIONS: Our results indicate that a level of vitamin E greater than currently recommended enhances certain clinically relevant in vivo indexes of T-cell-mediated function in healthy elderly persons. No adverse effects were observed with vitamin E supplementation.
Assuntos
Imunidade Celular/efeitos dos fármacos , Vitamina E/farmacologia , Idoso , Análise de Variância , Autoanticorpos/análise , Citotoxicidade Imunológica , Método Duplo-Cego , Feminino , Alimentos Fortificados , Nível de Saúde , Humanos , Hipersensibilidade Tardia/imunologia , Imunoglobulinas/análise , Masculino , Vacinação , Vitamina E/administração & dosagemAssuntos
Anticorpos Antinucleares/química , DNA/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antinucleares/genética , Anticorpos Antinucleares/imunologia , Diversidade de Anticorpos , Reações Antígeno-Anticorpo , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Sítios de Ligação de Anticorpos/imunologia , Clonagem Molecular , Cristalografia por Raios X , Genes de Imunoglobulinas , Humanos , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Conformação Proteica , TransgenesRESUMO
Bacterial production of recombinant Fab or Fv domains of antibodies is an important tool for analyzing structural correlates of antigen binding or idiotype expression. Bacterial products may be Fab or Fv molecules that assemble from separate chains in the periplasm or a single-chain Fv protein. This article describes properties and applications of a plasmid vector used for production of single-chain Fv (scFv). The expression cassette, initially designed for production of the Fv domain of anti-Z-DNA mAb Z22, has a bacterial secretion signal that permits secretion of soluble scFv into growth medium. A single B domain of staphylococcal protein A facilitates affinity purification and generic assay of products independent of antigen-binding activity. Gene segment swapping and directed mutagenesis identified structural features important for Z-DNA binding and revealed the structural similarity of autoantibody and immunization-induced antibody. The H and L regions of mAb Z22 are readily replaced by those of any cloned Ig, a library of V regions, or other proteins. Modified forms of the vector code for production of separate H or L chain V regions, which can associate with each other to form functional Fv complexes. Although there is large variation in the yield with different V regions, most clones provide enough soluble product for antigen-binding assays. Current developments are aimed at increasing the yield consistency to allow production of enough material for three-dimensional structural analysis.
Assuntos
Anticorpos Antinucleares/biossíntese , Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Proteínas Recombinantes/imunologia , Anticorpos Antinucleares/química , Anticorpos Antinucleares/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Western Blotting , Clonagem Molecular , DNA/imunologia , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fragmentos Fab das Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/biossíntese , Mutagênese Sítio-Dirigida , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes/genéticaRESUMO
To investigate the autoantibody repertoire associated with SLE, we have created phage display IgG Fab libraries from two clinically active SLE patients and from the healthy identical twin of one of these patients. The libraries from the lupus discordant twins were found to both include unusually large representations of the V(H)5 gene family. By panning with DNA, the SLE libraries each yielded IgG anti-double-stranded (ds) DNA autoantibodies, which are characteristic of lupus disease. These included a V(H)5 autoantibody from the affected twin, that has a targeted cluster of mutations that potentially improves binding affinity. The recovered IgG anti-dsDNA autoantibodies expressed the same idiotypes associated with the in vivo IgG anti-dsDNA response of the respective SLE donor. Heavy-light chain shuffling experiments demonstrated a case in which the in vitro creation of anti-dsDNA binding activity required restrictive pairing of a heavy chain with Vlambda light chains similar to those in circulating anti-dsDNA autoantibodies. By contrast, IgG anti-ds autoantibodies could not be recovered from the library from the healthy twin, or from shuffled libraries with heavy chains from the healthy twin. These repertoire analyses illustrate how inheritance and somatic processes interplay to produce lupus-associated IgG autoantibodies.