Boshra Hani, Li Jun, Peters Rodney, Hansen John, Matlapudi Anjan, Sunyer J Oriol
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
J Immunol. 2004 Apr 1;172(7):4381-90. doi: 10.4049/jimmunol.172.7.4381.
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.
补体激活过程中产生的C3a、C4a和C5a过敏毒素在炎症中起关键作用。C5a是三种过敏毒素中引发生物学反应能力最强的。C5a的作用是通过其与C5a受体（C5aR，CD88）结合来介导的。迄今为止，C5aR仅在哺乳动物物种中被鉴定和克隆，其进化史仍不明确。为了深入了解C5aR的进化、保守结构域和功能，我们克隆并鉴定了虹鳟（一种硬骨鱼）中的C5aR。分离出的cDNA编码一个350个氨基酸的蛋白质，该蛋白质与其他物种的C5aR具有最高的序列相似性。基因组分析显示存在一个连续的外显子，编码整个开放阅读框。Northern印迹分析表明，虹鳟C5a受体（TC5aR）在外周血淋巴细胞（PBL）和肾脏中有显著表达。流式细胞术分析表明，针对TC5aR细胞外N端区域两个不同区域产生的两种抗体能使来自PBL的相同白细胞群体呈阳性染色。B淋巴细胞和粒细胞是抗TC5aR识别的主要细胞类型。更重要的是，这些抗体抑制了PBL向从补体激活的虹鳟血清中纯化的趋化因子组分的趋化作用。我们的数据表明，C5aR和C3aR从共同祖先分子中分化出来发生在硬骨鱼出现之前。此外，我们证明C5aR的整体结构及其在趋化作用中的作用在超过3亿年的时间里一直保持保守。
