RESUMEN
We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.
Asunto(s)
Complemento C5a/química , Complemento C5a/farmacología , Alanina/química , Secuencia de Aminoácidos , Secuencia de Bases , Unión Competitiva , Calcio/metabolismo , Membrana Celular/metabolismo , Complemento C3a/química , Complemento C3a/genética , Complemento C3a/farmacología , Complemento C5a/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Neutrófilos/metabolismo , Mutación Puntual , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión , Relación Estructura-Actividad , TermodinámicaRESUMEN
The fifth component of the complement cascade, C5a, was iodinated using the Bolton-Hunter reagent. Results from the present study, using the high affinity ligand, [125I]Bolton-Hunter-labeled C5a ([125I]BH-C5a), revealed a single binding site on membranes prepared from human neutrophils, U-937 cells and human monocytes. Saturation studies demonstrated Bmax values in these cells of 11.5, 47.3 and 16.6 fmol/10(6) cells, respectively. The C5a receptor demonstrated a very high affinity for [125I]BH-C5a of approximately 4 pM in each cell type. Competition studies using analogs of C5a generated a similar order of potency in each of the cell types of C5a > or = C5a(1-74), Ser66Ala > C5a(1-73) > C5a(1-69). These studies indicate that [125I]BH-C5a labels a similar receptor in neutrophil, U-937 cell and monocyte membranes. Furthermore, C5a(1-73) produced shallow inhibition curves in competition experiments in each cell type. Computer analysis of the binding data revealed two components of binding. When 10 nM unlabeled C5a was used to initiate dissociation of [125I]BH-C5a binding in neutrophil membranes, two binding components were observed. In addition, dissociation of [125I]BH-C5a binding by 10 nM unlabeled C5a in the presence of 1 mM GppNHp decreased the percentage of binding to the slowly dissociating, high affinity binding component from 84 to 58%. These results suggest that multiple states of the C5a receptor exist.
Asunto(s)
Complemento C5a/metabolismo , Monocitos/citología , Neutrófilos/citología , Unión Competitiva , Calcio/metabolismo , Membrana Celular/metabolismo , Guanilil Imidodifosfato/farmacología , Humanos , Ligandos , Receptor de Anafilatoxina C5a , Receptores de Complemento/metabolismo , Proteínas Recombinantes/metabolismo , Succinimidas , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Stimulation of human neutrophils with platelet activating factor (PAF) resulted in a transient elevation of free cytosolic calcium. Neutrophils exhibited a two-component calcium response observed as a double peak when stimulated with greater than 5 nM PAF. In contrast, leukotriene B4 (LTB4), C5a, or formylmethionyl-leucyl-phenylalanine stimulated only a single-peak calcium response. The double-peak calcium response was not elicited in human monocytes or differentiated U937 cells, which demonstrated a single peak. Pretreatment of neutrophils with a 5-lipoxygenase inhibitor or a specific LTB4-receptor antagonist selectively blocked the second calcium peak. These results suggest that PAF-mediated activation of human neutrophils results in the activation of the 5-lipoxygenase and the subsequent generation of LTB4. This LTB4 in turn elicits a secondary rise in calcium, which contributes to the overall response of neutrophils of PAF. These results demonstrate how LTB4 participates in the cellular responses elicited by PAF in human neutrophils.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Calcio/metabolismo , Neutrófilos/metabolismo , Factor de Activación Plaquetaria/farmacología , Benzoquinonas/farmacología , Humanos , Líquido Intracelular/metabolismo , Leucotrieno B4/biosíntesis , Inhibidores de la Lipooxigenasa/farmacología , Neutrófilos/efectos de los fármacos , Estimulación QuímicaAsunto(s)
Cianuros , Neutrófilos/fisiología , Benzofuranos/metabolismo , Calcio/metabolismo , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Cianuros/metabolismo , Colorantes Fluorescentes , Fura-2 , Humanos , Isoxazoles/metabolismo , Potenciales de la Membrana , Neutrófilos/citología , Neutrófilos/ultraestructuraRESUMEN
Simultaneous measurements of the calcium rise, membrane potential change, and 90 degrees light scatter (shape change) responses exhibited by neutrophils upon activation, can be obtained with identical result as that obtained when independently performing each measurement. The putative intracellular mediator diacylglycerol depolarizes membrane potential and causes a decrease in light scatter. Leukotriene B4 causes a rise in calcium and a decrease in light scatter. The chemotactic peptide, N-formylmethionyl-leucyl-phenylalanine, causes a depolarization of membrane potential, a calcium rise, and a decrease in light scatter. The fura 2 measurements of intracellular free calcium indicate that the calcium concentration of unstimulated cells is much lower than previously thought based on quin 2 studies.