Hawlisch H, Frank R, Hennecke M, Baensch M, Sohns B, Arseniev L, Bautsch W, Kola A, Klos A, Köhl J
Institute of Medical Microbiology, Hannover Medical School, Germany.
J Immunol. 1998 Mar 15;160(6):2947-58.
Recent cloning of the human C3a receptor (C3aR) revealed that this receptor belongs to the large family of rhodopsin-type receptors. A unique feature of the C3aR is the large second extracellular loop comprising about 175 amino acid residues. We constructed combinatorial phage Ab libraries expressing single chain Fv Abs from BALB/c mice immunized with the affinity-purified second extracellular loop of the C3aR, fused to glutathione-S-transferase. A panel of anti-C3aR single chain Fv fragments (scFvs) was selected after four rounds of panning using the second extracellular loop of the C3aR, fused to the maltose binding protein as Ag. Sequencing of the clones obtained revealed three different groups of scFvs, the epitopes of which were mapped to two distinct regions within the loop, i.e., positions 185 to 193 and 218 to 226, representing the immunodominant domains of the loop. By flow cyotmetric analyses, the scFvs bound to RBL-2H3 cells transfected with the C3aR, but not to cells transfected with the C5aR or to nontransfected RBL-2H3 cells. In addition, the scFvs bound to the human mast cell line HMC-1. Immunofluorescence studies showed C3aR expression on polymorphonuclear granulocytes and monocytes, but not on lymphocytes. In addition, no C3aR expression was observed on human erythrocytes or platelets. Surprisingly, none of the scFvs alone or in combination inhibited C3a-induced Ca2+ mobilization from RBL-2H3 cells transfected with the C3aR. In addition, C3a did not displace binding of the scFvs to the receptor, strongly suggesting that the N-terminal part of the second extracellular loop is not involved in ligand binding.
最近对人C3a受体（C3aR）的克隆表明，该受体属于视紫红质型受体的大家族。C3aR的一个独特特征是其较大的第二个细胞外环，由约175个氨基酸残基组成。我们构建了组合噬菌体抗体文库，该文库表达来自用与谷胱甘肽-S-转移酶融合的C3aR的亲和纯化第二个细胞外环免疫的BALB/c小鼠的单链Fv抗体。在用与麦芽糖结合蛋白融合的C3aR的第二个细胞外环作为抗原进行四轮淘选后，选择了一组抗C3aR单链Fv片段（scFvs）。对获得的克隆进行测序，发现了三组不同的scFvs，其表位被定位到环内的两个不同区域，即位置185至193和218至226，代表环的免疫显性结构域。通过流式细胞术分析，scFvs与转染了C3aR的RBL-2H3细胞结合，但不与转染了C5aR的细胞或未转染的RBL-2H3细胞结合。此外，scFvs与人肥大细胞系HMC-1结合。免疫荧光研究显示C3aR在多形核粒细胞和单核细胞上表达，但在淋巴细胞上不表达。此外，在人红细胞或血小板上未观察到C3aR表达。令人惊讶的是，单独或组合使用的scFvs均未抑制C3a诱导的来自转染了C3aR的RBL-2H3细胞的Ca2+动员。此外，C3a并未取代scFvs与受体的结合，这强烈表明第二个细胞外环的N末端部分不参与配体结合。
