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Abstract
Domain-specific O-fucosylation is an unusual type of glycosylation, where fucose is directly attached to the serine or threonine residues of proteins via an O-linkage. O-fucosylated proteins play critical roles in a wide variety of biological events, with the most prominent being the Notch receptors. To date, only two types of domain-specific O-fucosylation have been identified in mammals: Protein O-fucosyltransferase (POFUT) 1-mediated O-fucosylation of Epidermal Growth Factor-like (EGF) repeats and POFUT2-mediated O-fucosylation of Thrombospondin Type 1 Repeats (TSRs). Both enzymes recognize a folded domain structure with specific O-fucose consensus sequences. Recently, a novel domain-specific O-fucosylation was identified on the Elastin Microfibril Interface (EMI) domain of Multimerin-1 (MMRN1) with an undefined POFUT. In Chapter 2, we present evidence that FUT10 and FUT11 are indeed POFUTs responsible for modifying EMI domains; thus, we renamed them POFUT3 and POFUT4, respectively. Similar to POFUT1 and POFUT2, POFUT3 and POFUT4 require folded EMI for efficient modification, function in the ER, and participate in a non-canonical ER quality control pathway.Glycoproteomic analysis of intact O-fucosylated peptides remains a key challenge due to several inherent obstacles, including low abundancy and lack of oxonium ions for confident assigning. In Chapter 3, we labeled POFUT1/2 target proteins with an O-fucose-specific tag, Fuc6nyl*, which greatly improved the mass spectrometric analysis of glycopeptides by generating high-intensity, O-fucose specific diagnostic ions and increasing charge density of precursor ions (Fuc6nyl* does not label POFUT3/4 target proteins). Leveraging Fuc6nyl*, we developed a glycoproteomic workflow for profiling the O-fucose proteome of POFUT1/2, which efficiently enriched and identified fucosylated peptides, covering both predicted O-fucose sites and unexpected O-fucose sites. We envision this workflow will expand the O-fucose proteome of POFUT1/2 and broaden our target pool for research.
Together these findings advance our knowledge of O-fucosylation by unveiling two novel POFUTs and providing tools for identifying previously unknown targets for POFUT1 and 2. Our understanding of these newly identified POFUTs and protein targets is still in its infancy; the work presented here provides an entry point to this emerging field of O-fucosylation.