Cloning and characterization of the guinea pig C5a anaphylatoxin receptor: interspecies diversity among the C5a receptors.
Cloning and characterization of the guinea pig C5a anaphylatoxin receptor: interspecies diversity among the C5a receptors.
复制标题
豚鼠 C5a 过敏毒素受体的克隆和表征：C5a 受体的种间多样性。
DOI：
10.1093/intimm/10.3.275
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发表时间：
1998
影响因子：
4.4
通讯作者：
Hugli,TE
中科院分区：
文献类型：
--
作者：
Fukuoka,Y;Ember,JA;Yasui,A;Hugli,TE
过敏性毒素C5a受体(C5aR，人CD88)在介导炎症和宿主防御过程中发挥着重要作用。最近用C5aR基因敲除小鼠证明了C5aR参与宿主防御机制的直接证据。C5aR基因缺陷的小鼠不能清除肺内注入的细菌。豚鼠系统可能是独一无二的，因为它表现出与人类补体成分的交叉反应，以及对过敏毒素的高度敏感性。因此，我们从一个巨核细胞cDNA文库中克隆了豚鼠C5aR基因。豚鼠C5aR的氨基酸序列与人的同源性为67%，与狗的同源性为61.6%，与小鼠的同源性为60.2%，与大鼠的同源性为63.6%。流式细胞仪分析证实豚鼠C5aR在COS-7细胞中瞬时表达，在L细胞成纤维细胞中稳定表达。[125I]C5a的竞争性结合实验和C5a刺激的钙动员实验证明，稳定表达C5aR的L细胞表达了功能性C5aR。豚鼠C5aR的N端胞外区比其他物种的C5aR的胞外区短5~7个残基，序列同源性仅为11%。其他外膜环在五个物种中的保守性也很差(8-33%)。跨膜片段在不同物种之间高度保守(46-86%)。豚鼠C5aR与人C5a结合，因此对C5a结合至关重要的残基在这些物种之间是保守的。来自多个物种的C5aR的序列比较使得配体结合位点的保守元件得以阐明。
The anaphylatoxin C5a receptor (C5aR, CD88 in man) plays a prominent role in mediating inflammatory and host defense processes. Direct evidence of C5aR involvement in host defense mechanisms was demonstrated recently using C5aR knockout mice. Mice deficient in C5aR were unable to clear intrapulmonary-instilled bacteria. The guinea pig system is perhaps unique for exhibiting cross-reactivity with human complement components and its high sensitivity to anaphylatoxins. Therefore, we cloned the guinea pig C5aR from a megakaryocyte cDNA library. The deduced amino acid sequence of guinea pig C5aR is 67% identical to human, 61.6% to dog, 60.2% to mouse and 63.6% to rat C5aR. Transient expression of guinea pig C5aR in COS-7 cells and stable expression on L cell fibroblasts were confirmed by FACS analysis. Competitive binding studies using [125I]C5a and stimulation of calcium mobilization by C5a proved that functional C5aR was expressed on these stably transfected L cells. The N-terminal extracellular region of guinea pig C5aR was five to seven residues shorter than the same region in C5aR from other species and sequence homology was limited to 11%. Other outer membrane loops were also poorly conserved (8-33%) when compared across five species. Transmembrane segments were highly conserved between these various species (46-86%). Guinea pig C5aR binds human C5a, therefore residues critical for C5a binding have been conserved between these species. Sequence comparison of C5aR from multiple species permits conserved elements of the ligand binding sites to be elucidated.