Assuntos
Soro Antilinfocitário/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Transplante de Coração/fisiologia , Imunossupressores/uso terapêutico , Adolescente , Adulto , Idoso , Animais , Azatioprina/uso terapêutico , Criança , Monitoramento de Medicamentos , Quimioterapia Combinada , Feminino , Rejeição de Enxerto/patologia , Transplante de Coração/imunologia , Transplante de Coração/mortalidade , Humanos , Contagem de Linfócitos , Masculino , Metilprednisolona/uso terapêutico , Pessoa de Meia-Idade , Coelhos , Estudos Retrospectivos , Taxa de SobrevidaAssuntos
Transplante de Coração/estatística & dados numéricos , Infecções/epidemiologia , Complicações Pós-Operatórias/epidemiologia , Adulto , Infecções Bacterianas/epidemiologia , Quimioterapia Combinada , Feminino , Rejeição de Enxerto/epidemiologia , Transplante de Coração/imunologia , Transplante de Coração/mortalidade , Hospitais Universitários , Humanos , Doença Iatrogênica/epidemiologia , Imunossupressores/uso terapêutico , Infecções/classificação , Masculino , Pessoa de Meia-Idade , Micoses/epidemiologia , Infecções por Protozoários/epidemiologia , Estudos Retrospectivos , Taxa de Sobrevida , Taiwan , Viroses/epidemiologiaRESUMO
Ag binding to the membrane Ig (mIg) substructure of the B cell Ag receptor leads to activation of cytoplasmic effector molecules including blk, fyn, lyn, and/or lck tyrosine kinases that are associated with receptor's dimeric Ig-alpha/Ig-beta transducer substructure. The structural basis of the apparent intermolecular transmission of this information within the receptor complex is unknown. Here we report that conservative point mutation of a sequence, S584-K597, at the cytoplasmic end of the predicted transmembrane spanning domain of the mIgM heavy chain (mu) ablates Ag-activated signal transduction, while having no detectable effect on association of mIgM with Ig-alpha/Ig-beta heterodimers. Specifically, mutation of serine584 to alanine, tyrosine587 to phenylalanine, threonine592 to valine, or lysine597 to isoleucine completely abrogated Ag-induced signal transduction leading to protein tyrosine phosphorylation and Ca2+ mobilization. Interestingly, mutants in the more peripheral of these residues, serine584 to alanine and lysine597 to isoleucine, remained responsive to a monoclonal antireceptor Ab (b-7-6) and all mutants remained responsive to polyclonal antireceptor Ab. These data implicate the polar sequence, -Y587STTVT592-, in transfer of information from ligand binding to transducer substructures within this heterooligomeric receptor complex. They further indicate that receptor activation by ligands that bind with high affinity and/or to constant region mIg epitopes is less dependent on the integrity of this motif.
Assuntos
Imunoglobulina M/fisiologia , Mutação Puntual , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais , Sequência de Aminoácidos , Sequência de Bases , Cálcio/metabolismo , Dados de Sequência Molecular , Fosforilação , Tirosina/metabolismoRESUMO
CD45 is a member of a family of membrane proteins that possess phosphotyrosine phosphatase activity, and is the source of much of the tyrosine phosphatase activity in lymphocytes. In view of its enzymatic activity and high copy number, it seems likely that CD45 functions in transmembrane signal transduction by lymphocyte receptors that are coupled to activation of tyrosine kinases. The B cell antigen receptor was found to transduce a Ca(2+)-mobilizing signal only if cells expressed CD45. Also, both membrane immunoglobulin M (mIgM) and CD45 were lost from the surface of cells treated with antibody to CD45, suggesting a physical interaction between these proteins. Finally, CD45 dephosphorylated a complex of mIg-associated proteins that appears to function in signal transduction by the antigen receptor. These data indicate that CD45 occurs as a component of a complex of proteins associated with the antigen receptor, and that CD45 may regulate signal transduction by modulating the phosphorylation state of the antigen receptor subunits.
Assuntos
Antígenos de Diferenciação/fisiologia , Linfócitos B/imunologia , Antígenos de Histocompatibilidade/fisiologia , Glicoproteínas de Membrana/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais , Animais , Antígenos de Diferenciação/genética , Cálcio/fisiologia , Linhagem Celular , Membrana Celular/fisiologia , Células Cultivadas , Células Clonais , Antígenos de Histocompatibilidade/genética , Imunoglobulina M/fisiologia , Antígenos Comuns de Leucócito , Camundongos , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Plasmocitoma , Proteínas Tirosina Fosfatases , RNA Mensageiro/genética , Baço/imunologia , TransfecçãoRESUMO
To study the molecular basis for antibody diversity and the structural basis for antigen binding, we have characterized the loss of phosphocholine (P-Cho) binding both experimentally and computationally in U10, a somatic mutant of the antibody S107. Nucleotide sequencing of U10 shows a single base change in JH1, substituting Asp-101 with Ala, over 9 A distant from the P-Cho-binding pocket. Probing with antiidiotypic antibodies suggests local, not global, conformational changes. Computational results support a specific structural mechanism for the loss of P-Cho binding. The U10 mutation eliminates the charged interaction between Asp-101 and Arg-94, which allows the Arg-94 side chain to disrupt P-Cho binding sterically and electrostatically by folding into the P-Cho-binding site. These results specifically show the importance of the Arg-94 to Asp-101 side chain salt bridge in the heavy-chain CDR3 conformation and suggest that residues distant from the binding site play an important role in antibody diversity and inducible complementarity.
Assuntos
Anticorpos/genética , Complexo Antígeno-Anticorpo , Antígenos , Rearranjo Gênico , Animais , Diversidade de Anticorpos , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Simulação por Computador , Ensaio de Imunoadsorção Enzimática , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosforilcolina/imunologia , Conformação Proteica , Relação Estrutura-AtividadeAssuntos
Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Mutação , Sequência de Aminoácidos , Animais , Autoanticorpos/genética , Sequência de Bases , Fígado/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Sondas de Oligonucleotídeos , Conformação ProteicaRESUMO
The genetic mechanism responsible for the somatic diversification of two mAbs was determined. The two PC-binding hybridomas were representative of events early and late in the immune response. The P28 cell line that produces an IgM antibody and thus represents events early in the immune response, was found to have 3 bp changes in its heavy chain variable (VH) region, with some changes in antibody affinity or specificity. The RP93 cell line that produces an IgG2a antibody and thus represents later events in the immune response, was found to have 9 bp changes in its VH region resulting in decreased affinity for PC and altered specificity. Oligonucleotides specific for linked base changes in the second hypervariable regions of both of these antibodies were used to look for previously undescribed V regions or other donor sequences that could have been responsible for these base changes. Since no donor sequences were found, we have concluded that somatic point mutation rather than gene conversion, V region replacement or the expression of an unidentified germline VH region gene is truly responsible for at least some of the somatic diversification of these antibodies.
Assuntos
Anticorpos Monoclonais/genética , Colina/análogos & derivados , Genes de Imunoglobulinas , Imunoglobulina G/genética , Imunoglobulina M/genética , Fosforilcolina/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Diversidade de Anticorpos , Sequência de Bases , Sítios de Ligação de Anticorpos , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/imunologia , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos BALB C/genética , Camundongos Endogâmicos BALB C/imunologia , Dados de Sequência Molecular , MutaçãoRESUMO
The S107 myeloma cell line expresses the germ-line sequence of the T15 antiphosphocholine (P-Cho) antibody, which is the major antibody made by BALB/c mice in response to P-Cho, either on a variety of bacterial polysaccharides or when attached to a protein carrier. We have previously reported that a somatic mutant of the S107 cell line produces an antibody that has lost the ability to bind P-Cho and has acquired binding for double-stranded DNA. This antibody has a substitution of an alanine for a glutamic acid at residue 35 in the heavy chain variable region. We now show that this amino acid substitution is due to a single A-C transversion, which is the only nucleotide change in the heavy and light chain variable regions. Further, it appears that this change is due to somatic mutation rather than to gene conversion.
Assuntos
Autoanticorpos/genética , Colina/análogos & derivados , DNA de Neoplasias/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Fosforilcolina/imunologia , Plasmocitoma/imunologia , Animais , Sequência de Bases , Linhagem Celular , Variação Genética , Camundongos , Hibridização de Ácido NucleicoRESUMO
We have examined the molecular mechanism and impact of somatic diversification on the T15 heavy chain variable region gene in vivo and in vitro. Somatic point mutation appears to be responsible for the changes we have observed in both hybridomas from early and late in the immune response and in the S107 myeloma cell line in culture. By identifying S107 mutants with decreases in antigen binding, we have shown that a single point mutation can cause the loss of binding to the eliciting antigen and the acquisition of binding to another antigen. Furthermore, in this case a point mutation of the T15 heavy chain variable region gene caused the conversion of an important protective antibody to an autoantibody. While the S107 cell line frequently generates both constant and variable region mutants, hybridomas appear to have relatively stable variable region genes and unstable constant region genes which in some cases result in mutants with increased binding.