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

通过这一奖项，化学系的生命过程化学计划支持亚利桑那大学的Matthew Cordes博士和Lewis and Clark College的Greta Binford博士之间的一个合作项目，以确定蜘蛛毒素如何靶向并摧毁细胞表面的不同分子。棕色隐居蜘蛛的毒液含有导致哺乳动物细胞和组织死亡的毒素，但也有助于蜘蛛固定和/或消化昆虫猎物。已知的影响哺乳动物的毒素可以通过从一种名为鞘脂的分子中切割出一种名为胆碱的特定“头部基团”来破坏细胞表面。其他隐居蜘蛛毒素只能切断一种不同的头部基团，称为乙醇胺，这是哺乳动物的鞘脂中没有的，因此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，所以这两种毒素对捕食可能都很重要，可能是以不同的方式。科德斯博士是结构生物学家和生物化学家，宾福德博士是有毒无脊椎动物方面的生物学家和专家。他们结合自己的专业知识，确定了这些毒素如何区分不同的头部群体，以及它们对捕食者和猎物的生物学后果。这些毒素对不同的头部群的特定作用也使它们在生物技术中用于检测或操纵不同类型的细胞表面。该项目的更广泛影响包括通过指导在两个不同机构进行综合和合作研究的本科生进行外联。蜘蛛吸引了公众的兴趣，并提供了一个机会，让公众了解有关这一主题的科学调查。一个被称为“蜘蛛节”的创新项目在波特兰的实验室和科学博览会上都在策划中。这个项目的总体目标是了解隐居蜘蛛磷脂酶D毒素中底物头部基团(乙醇胺和胆碱)偏好的原因和影响。其具体目标是绘制毒素中的界面结合位点，阐明底物头部群偏好的氨基酸序列决定因素，表征隐居蜘蛛毒素家族中底物偏好的进化，并将头部群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射线结晶学、计算结构生物学、定点突变、系统发育重建、酶分析和生物分析。这项研究的信息阐明了作用于膜表面的蛋白质对自然界中两种最常见的脂类头部基团--磷胆碱和磷乙醇胺--的不同识别。该项目还揭示了毒液中毒素的招募和专门化以及节肢动物生物化学和神经生物学的有趣方面。这些毒素也可以被开发成有价值的分析工具，以探索细胞表面结构的重要差异。这一裁决反映了NSF的法定使命，并通过使用基金会的智力优势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.