Glycosylation of Thrombospondin Type 1 Repeats

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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).