Glycosylation of Thrombospondin Type 1 Repeats

血小板反应蛋白 1 型重复序列的糖基化

• 批准号: 7266505
• 负责人: Robert S. Haltiwanger
• 金额: $ 35.16万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7266505
• 关键词: ADAMTS; Ablation; Adult; Affect; Alleles; Biological Process; CD36 Antigens; CD36 gene; Cell Surface Proteins; Cell surface; Cells; Characteristics; Consensus Sequence; Cysteine; Data; Databases; Defect; Development; Disruption; Drosophila genus; ES Cell Line; Embryo; Embryonic Development; Endopeptidases; Enzymes; Epidermal Growth Factor; Event; Family; Family member; Fucose; Fucosyltransferase 1; Future; Genes; Golgi Apparatus; Heterozygote; Homozygote; In Vitro; Inherited; Lead; Localized; Location; Malignant Neoplasms; Maps; Modification; Mus; Mutagenesis; Mutation; Pathway interactions; Peptide Hydrolases; Physiological; Play; Polysaccharides; Post-Translational Protein Processing; Process; Proteins; Quality Control; Range; Regulation; Research Personnel; Role; Signal Transduction; Site; Specificity; Staging; Syndrome; Thrombocytopenic Purpura; Thrombospondin 1; Time; Tissues; Ursidae Family; Weaning; concept; day; design; glycosylation; human disease; in vivo; member; mutant; notch protein; novel; programs; protein folding; protein function; receptor; vector

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).

描述（由申请人提供）：表皮生长因子样（EGF）重复序列的O-岩藻糖修饰对Notch功能至关重要。O-岩藻糖的损失可以改变Notch的信号能力以及干扰受体折叠。负责将O-岩藻糖添加到EGF重复序列（Pofut 1）的酶的消融导致小鼠胚胎死亡，类似于Notch活性的丧失。最近的研究表明，Pofut 1定位于ER，并可能参与EGF重复折叠的质量控制。与EGF重复序列一样，血小板反应蛋白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，包括血小板反应蛋白-1和ADAMTS家族成员。最近，在TSR上发现了O-岩藻糖修饰。这些修饰在调节蛋白质功能中的潜在意义通过以下观察而得到强调：血小板反应蛋白-1的抗血管生成活性映射到含有O-岩藻糖的TSR区域。我们最近发现了一种新的酶，Pofut 2，负责添加O-岩藻糖的TSR。在BayGenomics ES细胞系中，Pofut 2活性降低50%，其中基因捕获载体插入编码Pofut 2的基因中。Pofut 2基因的纯合破坏导致胚胎在交配后7.5天之前死亡。结合，胚胎致死率和TSRs和EGF重复序列的O-岩藻糖基化之间的相似性提供了证据，证明TSRs的O-岩藻糖基化对于含TSRs的蛋白质的正常功能是必不可少的，并且可能在TSR折叠的质量控制中起作用。为了进一步了解这种不寻常的修饰，我们将改进TSR上的O-岩藻糖共有序列，以便我们可以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的亚细胞位置以及消除O-岩藻糖基化对Pofut 2底物分泌和功能的影响来研究Pofut 2在TSR折叠质量控制中的潜在作用。最后，我们将进一步分析Pofut 2缺失导致胚胎死亡的原因。总而言之，这些数据将全面了解TSR蛋白修饰如何调节生物学上重要的蛋白质，例如血小板反应蛋白-1和ADAMTS家族成员，这些蛋白质在与人类疾病状态相关的广泛事件中发挥作用，包括癌症（血小板反应蛋白-1的抗血管生成作用）、韦尔-马切萨尼综合征（ADAMTS 10缺陷）和遗传性血小板减少性紫癜（ADAMTS 13缺陷）。