CMP-Neu5Ac: A Central Molecule in Bleeding Diseases and Mediator of a Novel Platelet Effector Function

项目摘要/摘要 在了解血小板数量和功能是如何调节的方面存在着根本的差距。直到我们 要更详细地了解这一点，我们将缺乏有效的治疗出血和凝血的患者 疾病。CMP-Neu5Ac是一种在血小板生物学中发挥重要作用的糖核苷酸。发布后 在血小板中，这种分子被用作细胞外唾液酸化血小板表面蛋白的底物， 从而导致体内血小板清除和活性的变化。CMP-Neu5Ac的细胞起源已公布 来自血小板和血小板表面蛋白底物的细胞外唾液酸化作用尚不清楚。 长期目标是利用分子上对血小板作为血管紧张素转换酶调节因子的作用的理解。 细胞外唾液酸化以开发血液病和癌症的基于血小板的细胞疗法。这个 本应用的总体目标是确定血小板中cMP-Neu5Ac的来源并鉴定其 生物学上相关的唾液酸化共底物。中心假说是，血小板调节细胞外 通过释放在血小板中生物合成的CMP-Neu5Ac来裂解具有重要功能的血小板蛋白 先驱物。 这将通过以下具体目标来实现：1)机械地定义功能效果 遗传性血小板减少症患者中发现的Gne酶变异体。这将通过以下方式实现 重组酶的高效表达和纯化，并进行了体外酶学鉴定。2) 确定血小板cMP-Neu5Ac的细胞来源。来自血小板、巨核细胞和 将对造血干细胞进行CMP-Neu5Ac生物合成酶的表达分析，并对 它们具有将UDP-GlcNAc酶转化为cMP-Neu5Ac的能力。3)鉴定与生物相关的血小板 胞外唾液酸化的表面蛋白底物。点击化学将被用来连接炔基改性 化学记者便于用液体鉴定新的胞外唾液酸化底物 色谱/质谱学蛋白质组学。其结果将是对细胞起源的理解 CMP-Neu5Ac在血小板谱系中的表达及细胞外血小板表面蛋白底物的鉴定 唾液酸化。这不仅对止血障碍的诊断和治疗有一定的指导意义，而且对临床治疗具有重要意义。 血栓形成，但也包括更广泛的涉及糖基改变的疾病，包括癌症和 自身免疫性疾病。 培训计划包括向Carolyn Bertozzi教授学习新的化学糖生物学技能 并在咨询专家的指导下将这些工具应用于血小板生物学。这 作为一名独立的内科-科学家调查员，培训对我来说是至关重要的 化学生物学与血液学/输血医学的接口。

PROJECT SUMMARY/ABSTRACT There is a fundamental gap in understanding how platelet number and function are regulated. Until we gain a more detailed understanding of this, we will lack effective therapies for patients with bleeding and clotting diseases. CMP-Neu5Ac is a sugar nucleotide that plays an important role in the biology of platelets. After release from platelets, this molecule is used as a substrate for extracellular sialylation of platelet surface proteins, resulting in changes in platelet in vivo clearance and activity. The cellular origin of the CMP-Neu5Ac released from platelets and the platelet surface protein substrates for extracellular sialylation remain unknown. The long-term goal is to capitalize on a molecular understanding of the role of platelets as mediators of extracellular sialylation to develop platelet-based cellular therapeutics for hematologic diseases and cancer. The overall objective of this application is to determine the origin of CMP-Neu5Ac in platelets and to identify its biologically relevant sialylation cosubstrates. The central hypothesis is that platelets regulate the extracellular sialylation of functionally important platelet proteins via release of CMP-Neu5Ac that is biosynthesized in platelet precursors. This will be accomplished via the following specific aims: 1) Mechanistically define the functional effects of GNE enzyme variants found in patients with inherited thrombocytopenia. This will be accomplished by overexpression and purification of the recombinant enzyme followed by in vitro enzymatic characterization. 2) Determine the cellular origin of platelet CMP-Neu5Ac. Lysates from platelets, megakaryocytes, and hematopoietic stem cells will be analyzed for expression of CMP-Neu5Ac biosynthetic enzymes, and tested for their ability to enzymatically transform UDP-GlcNAc to CMP-Neu5Ac. 3) Identify the biologically relevant platelet surface protein substrates for extracellular sialylation. Click chemistry will be used to attach alkyne-modified chemical reporters to facilitate identification of novel extracellular sialylation substrates by liquid chromatography/mass spectrometry proteomics. The outcome will be an understanding of the cellular origin of CMP-Neu5Ac in the platelet lineage and an identification of platelet surface protein substrates for extracellular sialylation. This will have implications not only for the diagnosis and treatment of disorders of hemostasis and thrombosis, but also for a broader range of diseases involving alterations of the glycome, including cancer and autoimmune diseases. The training plan includes learning a new chemical glycobiology skillset from Professor Carolyn Bertozzi at Stanford University, and applying these tools to platelet biology with guidance from consultant experts. This training will be critical for me to develop a unique niche as an independent physician-scientist investigator at the interface of chemical biology and hematology/transfusion medicine.