Slagboom Julien, Otvos Reka A, Cardoso Fernanda C, Iyer Janaki, Visser Jeroen C, van Doodewaerd Bjorn R, McCleary Ryan J R, Niessen Wilfried M A, Somsen Govert W, Lewis Richard J, Kini R Manjunatha, Smit August B, Casewell Nicholas R, Kool Jeroen
AIMMS Division of BioMolecular Analysis, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands.
AIMMS Division of BioMolecular Analysis, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands; Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, Vrije Universiteit, Amsterdam, The Netherlands.
Toxicon. 2018 Jun 15;148:213-222. doi: 10.1016/j.toxicon.2018.04.022. Epub 2018 May 4.
Venoms from snakes are rich sources of highly active proteins with potent affinity towards a variety of enzymes and receptors. Of the many distinct toxicities caused by envenomation, neurotoxicity plays an important role in the paralysis of prey by snakes as well as by venomous sea snails and insects. In order to improve the analytical discovery component of venom toxicity profiling, this paper describes the implementation of microfluidic high-resolution screening (HRS) to obtain neurotoxicity fingerprints from venoms that facilitates identification of the neurotoxic components of envenomation. To demonstrate this workflow, 47 snake venoms were profiled using the acetylcholine binding protein (AChBP) to mimic the target of neurotoxic proteins, in particular nicotinic acetylcholine receptors (nAChRs). In the microfluidic HRS system, nanoliquid chromatographic (nanoLC) separations were on-line connected to both AChBP profiling and parallel mass spectrometry (MS). For virtually all neurotoxic elapid snake venoms tested, we obtained bioactivity fingerprints showing major and minor bioactive zones containing masses consistent with three-finger toxins (3FTxs), whereas, viperid and colubrid venoms showed little or no detectable bioactivity. Our findings demonstrate that venom interactions with AChBP correlate with the severity of neurotoxicity observed following human envenoming by different snake species. We further, as proof of principle, characterized bioactive venom peptides from a viperid (Daboia russelli) and an elapid (Aspidelaps scutatus scutatus) snake by nanoLC-MS/MS, revealing that different toxin classes interact with the AChBP, and that this binding correlates with the inhibition of α7-nAChR in calcium-flux cell-based assays. The on-line post-column binding assay and subsequent toxin characterization methodologies described here provide a new in vitro analytic platform for rapidly investigating neurotoxic snake venom proteins.
蛇毒是高活性蛋白质的丰富来源，对多种酶和受体具有强大的亲和力。在蛇毒中毒引起的众多不同毒性中，神经毒性在蛇以及有毒海螺和昆虫致猎物麻痹过程中起着重要作用。为了改进毒液毒性分析中的分析发现环节，本文描述了微流控高分辨率筛选（HRS）的实施，以从毒液中获取神经毒性指纹图谱，这有助于识别蛇毒中毒的神经毒性成分。为了展示此工作流程，使用乙酰胆碱结合蛋白（AChBP）对47种蛇毒进行了分析，以模拟神经毒性蛋白的靶标，特别是烟碱型乙酰胆碱受体（nAChRs）。在微流控HRS系统中，纳升液相色谱（nanoLC）分离与AChBP分析和并行质谱（MS）在线连接。对于几乎所有测试的具有神经毒性的眼镜蛇科蛇毒，我们获得了生物活性指纹图谱，显示出主要和次要生物活性区，其中包含与三指毒素（3FTxs）一致的质量数，而蝰蛇科和游蛇科毒液显示出很少或没有可检测到的生物活性。我们的研究结果表明，毒液与AChBP的相互作用与不同蛇种致人中毒后观察到的神经毒性严重程度相关。作为原理证明，我们进一步通过nanoLC-MS/MS对蝰蛇科（罗素蝰蛇）和眼镜蛇科（南非盾鼻蛇）蛇的生物活性毒液肽进行了表征，揭示了不同毒素类别与AChBP相互作用，并且这种结合与基于钙流细胞的测定中α7-nAChR的抑制相关。本文所述的在线柱后结合测定和随后的毒素表征方法为快速研究具有神经毒性的蛇毒蛋白提供了一个新的体外分析平台。
