Robert S. Haltiwanger
Professor and GRA Eminent Scholar in Biomedical Glycosciences, Complex Carbohydrate Research Center
- My laboratory identifies and characterizes O-linked glycans in a wide variety of contexts. My initial work concentrated on O-GlcNAc, in which I identified proteins bearing the modification, mapped sites of modification, and identified the enzyme that adds O-GlcNAc to proteins (OGT). In my own laboratory, I have focused on O-fucose and O-glucose glycans on Epidermal Growth Factor-like repeats (EGF repeats) and Thrombospondin Type 1 Repeats (TSRs). This research has contributed greatly to the identification and characterization of the unique enzymes required for synthesis of these glycans, including demonstrating that the Fringe family of Notch modulators are 3-N-acetylglucosaminyltransferases, molecular cloning, expression and characterization of POFUT1 and POFUT2, initial identification and characterization of the 3-glucosyltransferase modifying O-fucose on TSRs, demonstrating that the Notch-pathway gene Rumi encodes POGLUT1, and identifying the two xylosyltransferases responsible for elongating O-glucose. We recently identified two novel enzymes that add O-glucose to a novel site on EGF repeats: POGLUT2 and POGLUT2. We have all of these enzymes overexpressed and have used them to glycosylate EGF repeats and TSRs in vitro in sufficient quantities for structural studies (NMR and/or X-ray crystallography). In collaboration with others, we have recently solved the structure of three of these enzymes co-crystallized with their acceptor substrates: Rumi/POGLUT1 with an EGF repeat, XXYLT1 with and EGF-O-Glc-Xyl, and POFUT2 with a TSR. We have developed glycomic methods for analyzing O-fucose and O-glucose structures and glycoproteomic methods for site-specific O-glycan analysis on EGF repeats and TSRs. We mapped sites of glycosylation on mouse Notch1 and demonstrated which sites are necessary for Fringe enzymes to mediate their effects. We have also begun to use what we have learned about Notch glycosylation to develop inhibitors of Notch activity. In particular, we have demonstrated that fucose analogs can be used to inhibit Notch activity in a ligand-specific manner. This is the first example of a small-molecule inhibitor that functions in a ligand-specific manner.