Glycosylation of Thrombospondin Type 1 Repeats

描述(申请人提供)：对表皮生长因子样(EGF)重复序列的O-岩藻糖修饰对于Notch功能是必不可少的。O-岩藻糖的缺失可以改变Notch的信号传递能力，也可以干扰受体折叠。去除负责将O-岩藻糖添加到EGF重复序列(Pofut1)的酶会导致小鼠胚胎死亡，类似于Notch活性的丧失。最近的研究表明，Pofut1定位于内质网，可能参与了EGF重复序列折叠的质量控制。与EGF重复序列一样，凝血酶敏感蛋白1型重复序列(TSR)是存在于许多细胞表面/分泌蛋白中的小半胱氨酸结基序，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。通过观察到血栓反应蛋白-1的抗血管生成活性映射到含有O-岩藻糖的TSR区域，强调了这些修饰在蛋白质功能调节中的潜在意义。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。在编码Pofut2的基因中插入基因陷阱载体的BayGenology ES细胞系中，Pofut2的活性降低了50%。Pofut2基因的纯合子破坏会导致在性交后7.5天之前的胚胎死亡。综上所述，TSRs的O-岩藻糖基化和EGF重复序列之间的相似性和胚胎致死性提供了证据，表明TSRs的O-岩藻糖基化对于含TSR的蛋白质的正常功能是必不可少的，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寻常的修饰，我们将精炼TSRs上的O-岩藻糖共识序列，以便我们能够更准确地预测哪些蛋白质具有这种修饰。我们将通过确定O-岩藻糖添加到TSR中的亚细胞位置以及消除O-岩藻糖基化对Pofut2底物分泌和功能的影响来研究Pofut2在TSR折叠质量控制中的潜在作用。最后，我们将进一步分析Pofut2基因缺失导致胚胎死亡的原因。综上所述，这些数据将使我们全面了解TSR蛋白修饰如何调控重要的生物蛋白，如血栓反应蛋白-1和ADAMTS家族成员，这些蛋白在与人类疾病状态相关的一系列事件中发挥作用，包括癌症(血栓反应蛋白-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)。

DESCRIPTION (provided by applicant): O-Fucose modification 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 (Pofut1) results in embryonic lethality in mice resembling loss of Notch activity. Recent studies show that Pofut1 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).