Monoclonal antibodies 1G12 and 6A12 to the N-domain of human angiotensin-converting enzyme: Fine epitope mapping and antibody-based detection of ACE inhibitors in human blood
Monoclonal antibodies 1G12 and 6A12 to the N-domain of human angiotensin-converting enzyme: Fine epitope mapping and antibody-based detection of ACE inhibitors in human blood
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DOI：
10.1021/pr060658x
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发表时间：
2007-01-01
影响因子：
4.4
通讯作者：
Danilov, Sergei M.
中科院分区：
文献类型：
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作者：
Balyasnikova, Irina V.;Skirgello, Olga E.;Danilov, Sergei M.
血管紧张素转换酶(ACE)是心血管病理生理学中的一个关键酶，它由N-和C-两个同源结构域组成，每个结构域都含有一个锌依赖的活性部位。血管紧张素转换酶抑制剂是治疗高血压和心力衰竭最常用的药物之一。利用人血管紧张素转换酶N区的三维结构和21个单、双N区突变体，建立了抗人血管紧张素转换酶N区的两株单抗1G12和6A12的精细表位图谱。这两种单抗与其在血管紧张素转换酶N结构域上的表位的结合因两结构域体组织血管紧张素转换酶C结构域的存在而显著减弱，并因血液血管紧张素转换酶表面唾液酸残基的存在而进一步减弱。然而，在ACE抑制剂或EDTA的存在下，这些单抗与血液ACE的结合显著增加(5-10倍)，而这些化合物对单抗与体组织ACE结合的影响不那么明显，对于截短的N结构域的影响更小。这表明，ACE抑制剂的结合或从ACE活性中心移除锌离子会导致双域ACE分子中N-结构域和C-结构域相互排列的构象调整。结果，单抗1G12和6A12在N-结构域上的表位区域，在体细胞ACE中被C-结构域球屏蔽，在血液ACE中被定位在Asn289和Asn416上的寡糖链上的唾液酸残基屏蔽。因此，我们证明了使用这些单抗(1G12或6A12)来检测和定量人类血液中ACE抑制剂的存在的可能性。该方法可广泛应用于监测血管紧张素转换酶抑制剂的临床试验，以及开发利用这些有效药物进行个性化用药的途径。
Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N- and C-), each bearing a Zn-dependent active site. ACE inhibitors are among the most prescribed drugs in the treatment of hypertension and cardiac failure. Fine epitope mapping of two monoclonal antibodies (mAb), 1G12 and 6A12, against the N-domain of human ACE, was developed using the N-domain 3D-structure and 21 single and double N-domain mutants. The binding of both mAbs to their epitopes on the N-domain of ACE is significantly diminished by the presence of the C-domain in the two-domain somatic tissue ACE and further diminished by the presence of sialic acid residues on the surface of blood ACE. The binding of these mAbs to blood ACE, however, increased dramatically (5-10-fold) in the presence of ACE inhibitors or EDTA, whereas the effect of these compounds on the binding of the mAbs to somatic tissue ACE was less pronounced and even less for truncated N-domain. This implies that the binding of ACE inhibitors or removal of Zn2+ from ACE active centers causes conformational adjustments in the mutual arrangement of N- and C-domains in the two-domain ACE molecule. As a result, the regions of the epitopes for mAb 1G12 and 6A12 on the N-domain, shielded in somatic ACE by the C-domain globule and additionally shielded in blood ACE by sialic acid residues in the oligosaccharide chains localized on Asn289 and Asn416, become unmasked. Therefore, we demonstrated a possibility to employ these mAbs (1G12 or 6A12) for detection and quantification of the presence of ACE inhibitors in human blood. This method should find wide application in monitoring clinical trials with ACE inhibitors as well as in the development of the approach for personalized medicine by these effective drugs.