O-glycosylation of cysteine-rich modules - The overall goal of our research is to determine the structures, modification sites, biosynthesis, and functions of sugars (O-glycans) linked to two cysteine-rich domains: Epidermal Growth Factor-like Repeats (EGFs) and Thrombospondin Type 1 Repeats (TSRs). Both EGFs and TSRs are found in numerous cell-surface and extracellular proteins. We focus on two modifications on EGFs, O-fucose (added by Protein O- fucosyltransferase 1, POFUT1) and O-glucose (added by Protein O-glucosyltransferase 1, POGLUT1). We also study O-fucose (added by POFUT2) on TSRs. All three enzymes modify hydroxyl groups of specific serines or threonines in well characterized consensus sequences within EGFs or TSRs. Knockouts of these enzymes are embryonic lethal in mice, and mutations in POFUT1 or POGLUT1 cause human genetic disorders, demonstrating the biological importance of these modifications. We propose to address several unanswered questions about the proteins modified by these O-glycans. For instance, we want to determine the O-fucose proteome. To understand O-fucose function, we need to know which proteins are modified. While database searches with the consensus sequences for POFUT1 and POFUT2 have successfully identified many target proteins, recent data has revealed a different cysteine-rich domain modified with O- fucose, an EMI domain in Multimerin1, which was not identified in our searches. Here we describe an unbiased approach to identify O-fucosylated proteins using a bioorthogonal probe, 6-alkynyl fucose (6AF), that is efficiently and preferentially incorporated into O-fucosylated proteins in cells. We expect to confirm a number of predicted substrates for POFUT1 and POFUT2, which will provide novel targets to study, but also to identify proteins that did not appear in database searches. We also want to examine the structure and function of O-fucose and O-glucose modifications on NOTCH3. In the past few years we have mapped O-fucose glycans to sites on NOTCH1 and NOTCH2 using glycoproteomic methods and determined which sites play biologically important roles. NOTCH3 is known to play important roles in vascular homeostasis, and mutations in NOTCH3 cause CADASIL, a devastating, autosomal dominant vascular disorder. The molecular mechanisms resulting in CADASIL are poorly understood. CADASIL mutations add or remove cysteines in NOTCH3 EGFs, which are predicted to disrupt glycosylation. We will map glycosylation sites on NOTCH3 isolated from vascular smooth muscle cells and evaluate how CADASIL mutations affect its glycosylation status. Finally, POFUT1 and POFUT2 are both soluble enzymes located in the lumen of the endoplasmic reticulum (ER), but their donor substrate, GDP-fucose, is synthesized in the cytosol. It is not known how GDP-fucose is transported into the ER. Here we describe a CRISPR-Cas9 screen to identify the putative ER GDP-fucose transporter. These studies will extend our understanding of the structure and function of O- glycans on cysteine-rich domains and their potential roles in diseases.
