Cloning, Expression, Cellular Distribution, and Role in Chemotaxis of a C5a Receptor in Rainbow Trout: The First Identification of a C5a Receptor in a Nonmammalian Species

Abstract

C3a, C4a, and C5a anaphylatoxins generated during complement activation play a key role in inflammation. C5a is the most potent of the three anaphylatoxins in eliciting biological responses. The effects of C5a are mediated by its binding to C5a receptor (C5aR, CD88). To date, C5aR has only been identified and cloned in mammalian species, and its evolutionary history remains ill-defined. To gain insights into the evolution, conserved structural domains, and functions of C5aR, we have cloned and characterized a C5aR in rainbow trout, a teleost fish. The isolated cDNA encoded a 350-aa protein that showed the highest sequence similarity to C5aR from other species. Genomic analysis revealed the presence of one continuous exon encoding the entire open reading frame. Northern blot analysis showed significant expression of the trout C5a receptor (TC5aR) message in PBLs and kidney. Flow cytometric analysis showed that two Abs generated against two different areas of the extracellular N-terminal region of TC5aR positively stained the same leukocyte populations from PBLs. B lymphocytes and granulocytes comprised the majority of cells recognized by the anti-TC5aR. More importantly, these Abs inhibited chemotaxis of PBLs toward a chemoattractant fraction purified from complement-activated trout serum. Our data suggest that the split between C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. Moreover, we demonstrate that the overall structure of C5aR as well as its role in chemotaxis have remained conserved for >300 million years.

Complement C5a acts as molecular adjuvant in fish by enhancing antibody response to soluble antigen

“…The functional receptor for trout C5a (C5aR) was reported previously, which is present on B lymphocytes, granulocytes as well as macrophage-like cells [24,25]. The fact that the G-C5a fusion protein was a chemoattractant to PBLs indicates that its C5a portion was correctly folded and thus retained the ability to chemoattract trout leukocytes.…”

“…In trout the gene encoding C5aR has also been cloned and functionally characterized. Interestingly, all trout B cells expressed high levels of the C5aR [24]. Furthermore, recombinant trout C5a showed functions similar to mammalian C5a, such as respiratory burst and chemotaxis [24,25].…”

“…Interestingly, all trout B cells expressed high levels of the C5aR [24]. Furthermore, recombinant trout C5a showed functions similar to mammalian C5a, such as respiratory burst and chemotaxis [24,25]. In addition, a trout serum fraction (8-kDa particle-uptake enhancing fraction, PUEF-8) composed of trout anaphylatoxins, potentially including C5a, was found to enhance the phagocytic capacity of trout leukocytes [26].…”

“…The C5 component has been cloned and characterized in rainbow trout [23]. Activation of C5 in trout leads to the generation of the pro-inflammatory C5a, which has been shown to play important roles in the activation of trout phagocytes [24]. In trout the gene encoding C5aR has also been cloned and functionally characterized.…”

Preferential combination between the light and heavy chain isotypes of fish immunoglobulins

“…Trout leukocytes were isolated from the head kidney (HKLs) and peripheral blood (PBLs) as described previously (Boshra et al, 2004(Boshra et al, , 2005Rotllant et al, 2004). Briefly, trout blood was acquired from the caudal vein and immediately diluted (1/5) with DMEM (Invitrogen) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 25 U/ml heparin (Gibco).…”