Glycosylation of Thrombospondin Type 1 Repeats

O-岩藻糖对表皮生长因子样重复序列的调节是Notch功能的关键。损失 O-岩藻糖可以改变Notch的信号传递能力，也可以干扰受体折叠。消融术 负责将O-岩藻糖添加到EGF重复序列(Pofutl)导致小鼠胚胎死亡的酶 类似于Notch活动的丧失。最近的研究表明，Pofutl定位于内质网，并可能参与其中 在EGF重复折叠的质量控制中。与EGF重复序列一样，凝血酶反应蛋白1型重复序列(TSR)也很小 半胱氨酸结基序存在于许多细胞表面/分泌蛋白中，包括凝血酶敏感蛋白-1和 ADAMTS家族成员。最近，在TSR上发现了O-岩藻糖修饰。潜在的意义 在蛋白质功能调节方面的这些修饰中，有观察到抗-HBs 凝血酶敏感蛋白-1的血管生成活性定位于含O-岩藻糖的TSR区域。 我们最近发现了一种新的酶，Pofut2，它负责将O-岩藻糖加到TSR上。 在插入基因陷阱载体的BayGenology ES细胞系中，Pofut2活性降低了50% 编码Pofut2。Pofut2基因的纯合破坏会导致7.5天前的胚胎死亡 小圈子。TSRs和EGF结合后的胚胎致死性及其O-岩藻糖基化的相似性 重复序列提供的证据表明，TSR的O-岩藻糖基化是含TSR的正常功能所必需的 蛋白质，并可能在TSR折叠的质量控制中发挥作用。为了进一步了解这一不同寻常的现象 修改后，我们将精炼TSR上的O-岩藻糖共识序列，以便我们可以更准确地 预测哪些蛋白质带有这种修饰。我们将研究Pofut2在质量控制中的潜在作用 通过确定O-岩藻糖添加到TSR的亚细胞位置来折叠TSR以及 消除Pofut2底物分泌和功能上的O-岩藻糖基化。最后，我们将进一步分析 Pofut2基因缺失导致胚胎死亡的原因。这些数据加在一起，将提供一个 全面了解TSR蛋白修饰如何调节具有生物重要性的蛋白质 作为血栓反应蛋白-1和ADAMTS家族成员，它们在与以下各项相关的广泛事件中发挥作用 人类疾病状态，包括癌症(凝血酶敏感蛋白-1的抗血管生成作用)、Weill-Marchesani 综合征(ADAMTS10缺陷)和遗传性血小板减少性紫癜(ADAMTS13缺陷)。

O-Fucose modication of epidermal growth factor-like (EGF) repeats is essential for Notch function. Loss of O-fucose can alter the ability of Notch to signal as well as interfere with receptor folding. Ablation of the enzyme responsible for addition of O-fucose to EGF repeats (Pofutl) results in embryonic lethality in mice resembling loss of Notch activity. Recent studies show that Pofutl is localized to the ER and may participate in quality control of EGF repeat folding. Like EGF repeats, thrombospondin type 1 repeats (TSRs) are small cysteine-knot motifs present in many cell surface/secreted proteins, including thromobospondin-1 and ADAMTS family members. Recently, O-fucose modifications were found on TSRs. The potential significance of these modifications in regulation of protein function is underscored by the observation that the anti- angiogenic activity of thrombospondin-1 maps to the region of the TSR bearing O-fucose. We recently identified a novel enzyme, Pofut2, that is responsible for addition of O-fucose to TSRs. Pofut2 activity is reduced 50% in a BayGenomics ES cell line with a gene trap vector inserted into the gene encoding Pofut2. Homozygous disruption of the Pofut2 gene results in embryo lethality prior to 7.5 days post coitum. Combined, the embryo lethality and the similarities between O-fucosylation of TSRs and EGF repeats provide evidence that O-fucosylation of TSRs is essential for the proper function of TSR-containing proteins and likely plays a role in quality control of TSR folding. To further understand this unusual modification, we will refine the O-fucose consensus sequence on TSRs so that we can more accurately predict which proteins bear this modification. We will examine the potential role of Pofut2 in quality control of TSR folding by determining the subcellular location where O-fucose is added to TSRs and the effects of eliminating O-fucosylation on secretion and function of Pofut2 substrates. Finally we will further analyze the cause of the embryo lethality resulting from loss of Pofut2. Together, these data will provide a comprehensive understanding of how TSR protein modification regulates biologically important proteins such as thrombospondin-1 and ADAMTS family members, which function in a wide range of events relevant to human disease states, including cancer (anti-angiogenic effects of thrombospondin-1), Weill-Marchesani syndrome (ADAMTS10 defects), and Inherited Thrombocytopenic Purpura (ADAMTS13 defects).