datasheet1_Biotin-Labelled Clavulanic Acid to Identify Proteins Target for Haptenation in Serum: Implications in Allergy Studies.pdf

Clavulanic acid (CLV) and amoxicillin, frequently administered in combination, can be independently involved in allergic reactions. Protein haptenation with β-lactams is considered necessary to activate the immune system. The aim of this study was to assess the suitability of biotinylated analogues of CLV as probes to study protein haptenation by this β-lactam. Two synthetic approaches afforded the labeling of CLV through esterification of its carboxylic group with a biotin moiety, via either direct binding (CLV-B) or tetraethylenglycol linker (CLV-TEG-B). The second analogue offered advantages as solubility in aqueous solution and potential lower steric hindrance for both intended interactions, with the protein and with avidin. NMR reactivity studies showed that both CLV and CLV-TEG-B reacts through β-lactam ring opening by aliphatic amino nitrogen, however with different stability of resulting conjugates. Unlike CLV conjugates, that promoted the decomposition of clavulanate fragment, the conjugates obtained with the CLV-TEG-B remained linked, as a whole structure including biotin, to nucleophile and showed a better stability. This was a desired key feature to allow CLV-TEG-B conjugated protein detection at great sensitivity. We have used biotin detection and mass spectrometry (MS) to detect the haptenation of human serum albumin (HSA) and human serum proteins. MS of conjugates showed that HSA could be modified by CLV-TEG-B. Remarkably, HSA preincubation with CLV excess only reduced moderately the incorporation of CLV-TEG-B, which could be attributed to different protein interferences. The CLV-TEG-B fragment with opened β-lactam was detected bound to the 404–430HSA peptide of the treated protein. Incubation of human serum with CLV-TEG-B resulted in the haptenation of several proteins that were identified by 2D-electrophoresis and peptide mass fingerprinting as HSA, haptoglobin, and heavy and light chains of immunoglobulins. Taken together, our results show that tagged-CLV keeps some of the CLV features. Moreover, although we observe a different behavior in the conjugate stability and in the site of protein modification, the similar reactivity indicates that it could constitute a valuable tool to identify protein targets for haptenation by CLV with high sensitivity to get insights into the activation of the immune system by CLV and mechanisms involved in β-lactams allergy.

克拉维酸（Clavulanic acid, CLV）与阿莫西林（amoxicillin）常联合给药，二者均可单独引发过敏反应。β-内酰胺类（β-lactams）药物介导的蛋白质半抗原化（protein haptenation）被认为是激活免疫系统的必要前提。本研究旨在评估克拉维酸的生物素化类似物（biotinylated analogues）作为探针，用于探究该类β-内酰胺类药物引发蛋白质半抗原化的适用性。本研究通过两种合成策略，借助生物素基团（biotin moiety）对克拉维酸的羧基（carboxylic group）进行酯化（esterification）修饰，分别得到直接结合型探针CLV-B，以及通过四乙二醇连接臂（tetraethylenglycol linker）相连的探针CLV-TEG-B。相较于前者，第二种类似物在水溶液中的溶解性更优，且在与蛋白质、亲和素（avidin）的预期相互作用中空间位阻（steric hindrance）更低。核磁共振反应性研究（NMR reactivity studies）显示，克拉维酸与CLV-TEG-B均可通过脂肪族氨基氮（aliphatic amino nitrogen）引发β-内酰胺环开环（β-lactam ring opening），但二者形成的结合物（conjugates）稳定性存在差异。与克拉维酸结合物会促进克拉维酸片段（clavulanate fragment）的降解不同，CLV-TEG-B形成的结合物可保持包含生物素的完整结构与亲核试剂（nucleophile）结合，且稳定性更佳——这一关键理想特性可实现CLV-TEG-B标记蛋白质的高灵敏度检测。我们采用生物素检测与质谱（mass spectrometry, MS）技术，检测了人血清白蛋白（human serum albumin, HSA）与人血清蛋白的半抗原化情况。结合产物的质谱分析结果表明，HSA可被CLV-TEG-B修饰。值得注意的是，预先用过量克拉维酸孵育HSA仅能中等程度降低CLV-TEG-B的结合率，这一现象可归因于不同的蛋白质干扰效应。研究还检测到开环的CLV-TEG-B片段结合于经处理的HSA的404–430位肽段中。将CLV-TEG-B与人血清共同孵育后，可引发多种蛋白质的半抗原化，通过二维电泳（2D-electrophoresis）与肽质量指纹图谱（peptide mass fingerprinting）鉴定，这些蛋白包括人血清白蛋白、结合珠蛋白（haptoglobin）以及免疫球蛋白（immunoglobulins）的重链与轻链。综上，本研究结果表明，带标记的克拉维酸保留了部分克拉维酸的固有特性。尽管我们观察到二者在结合物稳定性与蛋白质修饰位点上存在差异，但相似的反应活性表明，CLV-TEG-B可作为一种极具价值的工具，用于高灵敏度鉴定克拉维酸引发半抗原化的蛋白质靶点，从而深入解析克拉维酸激活免疫系统的机制，以及β-内酰胺类药物过敏的相关致病机制。