Collaborative Research: Head Group Preference in Recluse Spider Phospholipase D Toxins

凭借这一奖项，化学部门的生命过程化学项目支持亚利桑那大学的Matthew Cordes博士和Lewis and Clark学院的Greta Binford博士之间的合作项目，以确定蜘蛛毒液毒素如何靶向和破坏细胞表面的不同分子。褐色隐遁蜘蛛的毒液含有毒素，能导致哺乳动物的细胞和组织死亡，但也有助于蜘蛛固定和/或消化昆虫猎物。已知影响哺乳动物的毒素可以通过从鞘脂分子中切割出特定的“头部基团”，即胆碱，从而破坏细胞表面。其他隐退的蜘蛛毒素只能切断另一种叫做乙醇胺的头部群，哺乳动物的鞘脂中没有这种毒素，所以这些毒素对哺乳动物的危害可能较小。许多昆虫都有这两种头群，所以这两种毒素可能对捕食很重要，也许是以不同的方式。Cordes博士是结构生物学家和生物化学家，Binford博士是有毒无脊椎动物的生物学家和专家，他们结合他们的专业知识来确定这些毒素如何区分不同的头部群体，以及对捕食者和猎物的生物学后果。这些毒素在不同头部群上的特定作用也可以使它们在生物技术中用于检测或操纵不同种类的细胞表面。这个项目更广泛的影响包括通过指导本科生在两个不同的机构进行综合和合作研究。蜘蛛吸引了公众的兴趣，并提供了向公众通报有关该主题的科学调查的机会。一个名为“蜘蛛节”的创新项目在实验室和波特兰的一个科学博览会上同时进行。该项目的总体目标是了解底物头组（乙醇胺与胆碱）对隐遁蜘蛛磷脂酶D毒素的偏好的原因和影响。具体目标是绘制毒素的界面结合位点，阐明底物头部群偏好的氨基酸序列决定因素；隐遁蜘蛛毒素科对底物偏好的进化特征并将头部群体偏好与毒素的生物效应联系起来。使用的方法包括核磁共振、x射线晶体学、计算结构生物学、定点诱变、系统发育重建、酶分析和生物分析。这项研究的信息阐明了在膜表面作用的蛋白质对自然界中两种最常见的脂质头基团，磷脂胆碱和磷酸乙醇胺的差异识别。该项目还揭示了毒素招募和毒液专业化以及节肢动物生物化学和神经生物学的有趣方面。这些毒素也可以发展成为有价值的分析工具，以探测细胞表面结构的重要差异。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is supporting a collaborative project between Dr. Matthew Cordes at the University of Arizona and Dr. Greta Binford at Lewis and Clark College, to determine how spider venom toxins target and destroy different molecules on cell surfaces. Venoms of brown recluse spiders have toxins that cause the death of cells and tissue in mammals, but also help the spiders immobilize and/or digest insect prey. The toxins that are known to affect mammals can damage cell surfaces by cutting a specific "head group", called choline, off of a molecule called a sphingolipid. Other recluse spider toxins can only cut off a different kind of head group, called ethanolamine, that mammals do not have in their sphingolipids, so these toxins may be less harmful to mammals. Many insects have both kinds of head group, so both types of toxin are probably important for predation, perhaps in different ways. Dr. Cordes, a structural biologist and biochemist, and Dr. Binford, a biologist and expert on venomous invertebrates, combine their expertise to determine how these toxins distinguish the different head groups, and what the biological consequences are for predators and their prey. The specific action of these toxins on different head groups could also make them useful in biotechnology for detecting or manipulating different kinds of cell surfaces. The broader impacts of this project involve outreach through mentorship of undergraduates doing integrative and collaborative research at two different institutions. Spiders attract public interest and afford an opportunity to inform the public on scientific investigations on the subject. An innovative program known as "SpiderFest" is conduucted both at the laboratory and at a science expo in Portland. The overall goal of this project is to understand the causes and effects of substrate head group (ethanolamine vs. choline) preference in phospholipase D toxins from recluse spiders. The specific goals are to map interfacial binding sites in the toxins, elucidate amino-acid sequence determinants of substrate head group preference; characterize the evolution of substrate preference in the recluse spider toxin family; and correlate head group preference to biological effects of the toxins. Methods to be used include NMR, X-ray crystallography, computational structural biology, site-directed mutagenesis, phylogenetic reconstruction, enzymatic assays, and biological assays. Information from this study illuminates differential recognition, by proteins that act at membrane surfaces, of the two most common lipid head groups in nature, phosphocholine and phosphoethanolamine. The project also sheds light on toxin recruitment and specialization in venoms and interesting aspects of arthropod biochemistry and neurobiology. These toxins could also be developed into valuable analytical tools to probe important differences in cell surface structure.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.