Danilov Sergei M, Watermeyer Jean M, Balyasnikova Irina V, Gordon Kerry, Kugaevskaya Elena V, Elisseeva Yulia E, Albrecht Ronald F, Sturrock Edward D
Department of Anesthesiology, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Biochemistry. 2007 Aug 7;46(31):9019-31. doi: 10.1021/bi700489v. Epub 2007 Jul 14.
Angiotensin I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) is a key enzyme in cardiovascular pathophysiology. A wide spectrum of monoclonal antibodies to different epitopes on the N and C domains of human ACE has been used to study different aspects of ACE biology. In this study we characterized the monoclonal antibody (mAb) 5F1, developed against the N domain of human ACE, which recognizes both the catalytically active and the denatured forms of ACE. The epitope for mAb 5F1 was defined using species cross-reactivity, synthetic peptide (PepScan technology) and phage display library screening, Western blotting, site-directed mutagenesis, and protein modeling. The epitope for mAb 5F1 shows no overlap with the epitopes of seven other mAbs to the N domain described previously and is localized on the other side of the N domain globule. The binding of mAb 5F1 to ACE is carbohydrate-dependent and increased significantly as a result of altered glycosylation after treatment with alpha-glucosidase-1 inhibitor, N-butyldeoxynojirimycin (NB-DNJ), or neuraminidase. Out of 17 species tested, mAb 5F1 showed strict primate ACE specificity. In addition, mAb 5F1 recognized human ACE in Western blots and on paraffin-embedded sections. The sequential part of the epitope for mAb 5F1 is created by the N-terminal part of the N domain, between residues 1 and 141. A conformational region of the epitope was also identified, including the residues around the glycan attached to Asn117, which explains the sensitivity to changes in glycosylation state, and another stretch localized around the motif 454TPPSRYN460. Site-directed mutagensis and inhibition assays revealed that mAb 5F1 inhibits ACE activity at high concentrations due to binding of residues on both sides of the active site cleft, thus supporting a hinge-bending mechanism for substrate binding of ACE.
血管紧张素I转换酶（ACE，肽基二肽酶，EC 3.4.15.2）是心血管病理生理学中的关键酶。针对人ACE的N和C结构域不同表位的多种单克隆抗体已被用于研究ACE生物学的不同方面。在本研究中，我们对针对人ACE的N结构域开发的单克隆抗体（mAb）5F1进行了表征，该抗体可识别ACE的催化活性形式和变性形式。使用物种交叉反应性、合成肽（PepScan技术）和噬菌体展示文库筛选、蛋白质印迹、定点诱变和蛋白质建模来确定mAb 5F1的表位。mAb 5F1的表位与先前描述的针对N结构域的其他七种单克隆抗体的表位没有重叠，并且位于N结构域小球的另一侧。mAb 5F1与ACE的结合依赖于碳水化合物，在用α-葡糖苷酶-1抑制剂N-丁基脱氧野尻霉素（NB-DNJ）或神经氨酸酶处理后，由于糖基化改变，结合显著增加。在测试的17个物种中，mAb 5F1显示出严格的灵长类动物ACE特异性。此外，mAb 5F1在蛋白质印迹和石蜡包埋切片上识别出人ACE。mAb 5F1表位的连续部分由N结构域的N末端部分在第1和141位残基之间形成。还鉴定了表位的一个构象区域，包括与Asn117连接的聚糖周围的残基，这解释了对糖基化状态变化的敏感性，以及另一个位于基序454TPPSRYN460周围的延伸区域。定点诱变和抑制试验表明，mAb 5F1在高浓度下抑制ACE活性，这是由于活性位点裂隙两侧的残基结合，从而支持ACE底物结合的铰链弯曲机制。
