RESUMEN
Virtually nothing is known about the structure, function, and evolutionary origins of the C3aR in nonmammalian species. Because C3aR and C5aR are thought to have arisen from the same common ancestor, the recent characterization of a C5aR in teleost fish implied the presence of a C3aR in this animal group. In this study we report the cloning of a trout cDNA encoding a 364-aa molecule (TC3aR) that shows a high degree of sequence homology and a strong phylogenetic relationship with mammalian C3aRs. Northern blotting demonstrated that TC3aR was expressed primarily in blood leukocytes. Flow cytometric analysis and immunofluorescence microscopy showed that Abs raised against TC3aR stained to a high degree all blood B lymphocytes and, to a lesser extent, all granulocytes. More importantly, these Abs inhibited trout C3a-mediated intracellular calcium mobilization in trout leukocytes. A fascinating structural feature of TC3aR is the lack of a significant portion of the second extracellular loop (ECL2). In all C3aR molecules characterized to date, the ECL2 is exceptionally large when compared with the same region of C5aR. However, the exact function of the extra portion of ECL2 is unknown. The lack of this segment in TC3aR suggests that the extra piece of ECL2 was not necessary for the interaction of the ancestral C3aR with its ligand. Our findings represent the first C3aR characterized in nonmammalian species and support the hypothesis that if C3aR and C5aR diverged from a common ancestor, this event occurred before the emergence of teleost fish.
Asunto(s)
Proteínas de la Membrana/aislamiento & purificación , Oncorhynchus mykiss , Receptores de Complemento/aislamiento & purificación , Xenopus , Secuencia de Aminoácidos , Animales , Anticuerpos Bloqueadores/química , Sitios de Unión de Anticuerpos , Northern Blotting , Southern Blotting , Calcio/antagonistas & inhibidores , Calcio/metabolismo , Complemento C3a/antagonistas & inhibidores , Complemento C3a/fisiología , Proteínas Inactivadoras de Complemento/fisiología , ADN Complementario/aislamiento & purificación , Técnica del Anticuerpo Fluorescente Indirecta , Cobayas , Humanos , Líquido Intracelular/inmunología , Líquido Intracelular/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Ratas , Receptores de Complemento/antagonistas & inhibidores , Receptores de Complemento/inmunología , Receptores de Complemento/metabolismo , Análisis de Secuencia de ADNRESUMEN
PL37 (RAARISLGPRCIKAFTE) is an antisense homology box peptide composed of aa 37-53 of C5a-anaphylatoxin and is considered to be the region essential for C5a function. Using a computer program, we designed the complementary peptides ASGAPAPGPAGPLRPMF (Pep-A) and ASTAPARAGLPRLPKFF (Pep-B). Pep-A bound to PL37 and to C5a with very slow dissociation as determined by analysis using surface plasmon resonance, whereas Pep-B failed to bind at all. C5a was inactivated by concentrations of 7 nM or more of Pep-A, and this concentration of Pep-A inhibited induction of intracellular Ca(2+) influx in neutrophils. Patch clamp electrophysiology experiments also showed the effectiveness of Pep-A in C5aR-expressing neuroblastoma cells. Furthermore, Pep-A administration prevented rats from C5a-mediated rapid lethal shock induced by an Ab to a membrane inhibitor of complement after LPS sensitization.