RESUMO
Three mutants of the anaphylatoxin C5a were prepared with positions 2, 64 and 70, respectively, substituted by tryptophan. The last mutant was additionally labelled at Cys27 for fluorescence energy transfer (FET) measurements. The structural integrity and biological activity of the molecules were not affected. Fluorescence anisotropy decay (FAD) measurements showed that the rotational correlation time for tryptophan decreases in the order: [Trp2]rhC5a > [Trp64]rhC5a > [Trp70]rhC5a, indicating an increasing mobility of the side chain. Measurements of the fluorescence energy transfer from Trp70 to the 1,5-AEDANS group at Cys27 yielded a distance distribution of 2.4 +/- 0.8 nm. This value is compatible with the C-terminal chain being arranged as a slightly stretched helix pointing away from the body of the molecule.
Assuntos
Anafilatoxinas/química , Complemento C5a/química , Triptofano/química , Sequência de Aminoácidos , Anafilatoxinas/genética , Dicroísmo Circular , Complemento C5a/genética , Cisteína , Transferência de Energia , Polarização de Fluorescência , Corantes Fluorescentes , Humanos , Dados de Sequência Molecular , Mutação , Naftalenossulfonatos , Proteínas Recombinantes/química , Espectrofotometria Ultravioleta , Reagentes de Sulfidrila , Fatores de Tempo , Triptofano/genéticaRESUMO
By site-directed mutagenesis of a human complement factor C5a cDNA clone, we have designed a hybrid anaphylatoxin in which three amino acid residues in the C-terminal sequence of human C5a were exchanged to create the native C-terminal human C3a (hC3a) sequence Leu-Gly-Leu-Ala-Arg. This hybrid anaphylatoxin rC5a-(1-69)-LGLAR exhibited true C3a and C5a activity when tested in the guinea pig ileum contraction assay. Quantitative measurements of ATP release from guinea pig platelets revealed about 1% intrinsic C3a activity for this hybrid, while the C5a activity was essentially unchanged. Competitive binding assays confirmed that the rC5a-(1-69)-LGLAR mutant was able to displace radioiodinated rhC5a with a KI of approx. 40 nM and hC3a with a KI of approx. 3.7 microM from guinea pig platelets. Since the C-termini of both human C3a and C5a anaphylatoxins are known to interact with their respective receptors, we conclude that the same peptidic sequence, LGLAR, is able to bind to and activate two different receptors, the C3a receptor as well as the C5a receptor. This clone provides a novel tool for the identification of further receptor-binding residues in both anaphylatoxins, since any mutants may be tested for altered C3a and C5a activity simultaneously.
Assuntos
Anafilatoxinas/farmacologia , Complemento C3a/farmacologia , Complemento C5a/farmacologia , Mutagênese Sítio-Dirigida , Trifosfato de Adenosina/sangue , Sequência de Aminoácidos , Anafilatoxinas/genética , Animais , Ligação Competitiva , Plaquetas/metabolismo , Complemento C3a/química , Complemento C3a/genética , Complemento C5a/química , Complemento C5a/genética , Cobaias , Íleo/fisiologia , Dados de Sequência Molecular , Contração Muscular , Receptor da Anafilatoxina C5a , Receptores de Complemento/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli. A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene. The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor. After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets. The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture.