Antibody mapping of the linear epitopes of CMY-2 and SHV-1 β-lactamases
Antibody mapping of the linear epitopes of CMY-2 and SHV-1 β-lactamases
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DOI：
10.1128/aac.48.10.3980-3988.2004
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发表时间：
2004-10-01
影响因子：
4.9
通讯作者：
Bonomo, RA
中科院分区：
文献类型：
--
作者：
Hujer, AM;Bethel, CR;Bonomo, RA
当抗-内酰胺酶抗体用于临床或研究时，确定识别的氨基酸对于解释这些抗体的敏感性和特异性至关重要。为此，我们利用SPOT合成方法绘制了CW-2和SHV-1 β -内酰胺酶的表位。利用抗SHV-1和抗CMY-2多克隆抗体分别鉴定出SHV-1和CMY-2中8个线性表位和7个线性表位。SHV-1的表位分别定位在abl28 ~ 38、42 ~ 54、88 ~ 100、102 ~ 114、170 ~ 182、186 ~ 194、202 ~ 210和276 ~ 288的Ambler位点。在横跨氨基酸102至114的表位中，丙氨酸和X-Scan分析表明D104， Y105， P107和S109是抗体识别的必需残基。在含有170 ~ 182氨基酸的表位中，N170、L173、P174、G175和D176具有免疫优势。在CMY-2 β -内酰胺酶中，成熟酶的4 ~ 16、70 ~ 79,211 ~ 223,274 ~ 286,289 ~ 298,322 ~ 334和343 ~ 358氨基酸定义了主要的线性表位。对位于类似于A类β -内酰胺酶（V211至V223）的omega环区域的识别位点的详细分析表明，氨基酸Q215至E219在抗体结合中起重要作用。CW-2 β -内酰胺酶与10倍摩尔过量的抗cmy -2抗体孵育60分钟，对硝基蛋白水解的抑制作用大于80%。10倍摩尔过量的抗SHV-1抗体使SHV-1的活性降低69%。对CMY-2和SHV-1结构的分析表明，这种水解活性的降低可能部分是由于抗体与omega环的直接结合，从而阻碍了底物进入活性位点。
Knowledge of the amino acids that define recognition of anti-beta-lactamase antibodies is critical to the interpretation of sensitivity and specificity of these antibodies when they are used in a clinical or research setting. To this end, we mapped the epitopes of the CW-2 and SHV-1 beta-lactamases by using the SPOT synthesis method. Eight linear epitopes in SHV-1 and seven linear epitopes in CMY-2 were identified by using anti-SHV-1 and anti-CMY-2 polyclonal antibodies, respectively. The epitopes of SHV-1 were mapped to amino acids at the Ambler positions ABL 28 to 38, 42 to 54, 88 to 100, 102 to 114, 170 to 182, 186 to 194, 202 to 210, and 276 to 288. In the epitope spanning amino acids 102 to 114, alanine and X-Scan analysis demonstrated that D104, Y105, P107, and S109 are essential residues for antibody recognition. In the epitope containing amino acids 170 to 182, N170, L173, P174, G175, and D176 were immunodominant. In CMY-2 beta-lactamase, amino acids 4 to 16,70 to 79,211 to 223,274 to 286,289 to 298,322 to 334, and 343 to 358 of the mature enzyme defined the major linear epitopes. A detailed analysis of the recognition sites that are located in an area analogous to the omega loop of class A beta-lactamases (V211 to V223) showed that the amino acids Q215 to E219 are important in antibody binding. Incubation of CW-2 beta-lactamase with a 10-fold molar excess of anti-CMY-2 antibody for 60 min resulted in greater than 80% inhibition of nitrocefin hydrolysis. A 10-fold molar excess of anti-SHV-1 antibody reduced the activity of SHV-1 by 69%. Analysis of the CMY-2 and SHV-1 structures suggest that this reduction of hydrolytic activity may be due in part to the direct binding of antibodies to the omega loop, thereby hindering access of substrate to the active site.