以氫氘交換質譜法進行神經蛇毒及心臟蛇毒之抗原決定位分析

Abstract

抗原決定位分析不但可更深入瞭解抗原抗體間交互作用，更能促進新型抗體藥物之研發。至今，有許多技術被應用於抗原決定位分析，如X射線晶體學、核磁共振技術、胜肽掃描技術等，各有其優缺點和限制。本研究是首篇以氫氘交換質譜技術進行蛇毒之抗原決定位分析。研究之標的為台灣眼鏡蛇毒液中純化出的神經蛇毒 (Neurotoxin, NTX) 及心臟蛇毒 (Cardiotoxin III, CTX III) 及對該兩者皆具特異性之台灣抗蛇毒馬血清。將抗原及抗原抗體複合物分別進行氫氘交換，在不同的時間點終止反應並使蛋白質變性，最後注入LC-MS系統進行水解和分析。藉由比較目標抗原各胜肽片段在與抗體結合時與未結合時之氫氘交換速率，得以推測可能的抗原決定位位點。由實驗數據可推斷，NTX與CTX III之抗原決定位皆位於β3至β5處，這些區域在抗體結合時較難被溶劑中的氘交換，故具有較低的含氘比例。此外，我們更發現NTX與抗體結合可能導致C54位點有結構上的改變。完成方法開發後，再以越南的抗蛇毒血清驗證該方法的實用性，結果符合預期。根據先前研究顯示，不同的蛇毒抗體得交叉中和蛇毒毒性。基於研發抗體藥物成本高、耗時長，篩選現有抗體對於特定蛇毒的中和效果是另一種選擇。使用自動化的氫氘交換質譜法能迅速有效地做篩選，同時提供蛋白質結構資訊，故本論文所提出之實驗方法對於蛇毒蛋白研究極具發展潛力。Epitope mapping has been considered a powerful tool that can elucidate binding mechanism and largely facilitates the development of vaccines and drugs. To date, many techniques have been developed for epitope mapping, such as X-ray crystallization, nuclear magnetic resonance, and peptide scanning. Each of them has its own benefits and limitations. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) technique was employed for epitope mapping to probe the binding sites of the short neurotoxin 1 (NTX) and the cardiotoxin III (CTX III) toward particular polyclonal antibodies in Taiwanese bivalent antivenom by comparing the differential rate of deuterium incorporation in their free and Ab-complexed forms. The control samples and the complex samples were incubated in labeling buffer for deuterium labeling respectively. At various time intervals, samples were mixed with quench buffer to quench the H/D exchange. After denaturation, the sample was injected into the LC-MS system for online pepsin digestion and further analysis. The results indicated that the putative binding regions of NTX and CTX III, showing reduced deuterium uptake upon complex formation, were both located within the triple-stranded β sheet. In addition, a conformational change at C54 in NTX was found upon binding. We further investigated the usefulness of this platform by characterizing epitopes of the polyclonal antibodies in Vietnamese snake antivenom. This is the first report that epitopes for antibodies to snake venom toxins were mapped by HDX-MS approach. It can be expected that HDX-MS will be a rapid and efficient method for the evaluation of the cross-neutralization of NTX and CTX III by any antibodies raised by other snake venoms and provides valuable information on conformational changes induced by protein-protein interactions.