Structural and functional studies of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts)

National Institute Of Dental & Craniofacial Research

Investigators

Linked publications & trials

Abstract

The long-term goal of my work is to characterize carbohydrate synthesizing enzymes and use that knowledge to design molecules that modulate the glycosylation pathways that underly pathological states. Glycans are essential for life, and both eukaryotic and prokaryotic cells are protected by celluar or extracelluar coats consisting of carbohydrates, which may be conjugated to proteins (glycoproteins) and lipids (glycolipids). These glycans play a broad range of critical roles in modulating cellular processes. Our approach is to illuminate the mechanisms of the enzymes that are responsible for synthesizing glycan using combinations of structural, biochemical, and biophysical approaches. Glycan biosynthesis is regulated by a diverse group of enzymes, including glycosyltransferases (GTs), hydrolases, and other glycan modifying enzymes. This project focuses on characterizing the enzymes that initiate mucin-type O-glycosylation called GalNAc-Ts and therefore regulate the biological properties of proteins that become densely modified with mucin-type O-glycans as they pass through the secretory pathway. This includes mucins, the primary components of the mucus coat that envelopes all the mucosal layers of the gastrointestinal (GI), respiratory, and urogenital tracts. This coat forms the outermost face of the innate immune system, which modulates the microbiome, and forms the first barrier against physical, chemical, and infectious agents thereby protecting underlying tissues from damage. Inflammatory changes and other cellular stresses are signaled via membrane bound mucin receptors such as Mucin 1, which is abnormally expressed and glycosylated in many cancers. GalNAc-Ts (20 human isoenzymes) initiate mucin-type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) from UDP-GalNAc to a serine or threonine on a substrate to form Tn-antigen. These enzymes contain two domains that are important for activity: a catalytic domain and a lectin (sugar binding) domain. We have been characterizing the substrate specificities of individual GalNAc-Ts using structural and biochemical tools, and complementing these studies with experiments in cellulo (Ji, Samara et al. Nat Comm, 2018, Kumar et al. Nat Comm 2024). There is extensive diversity among the GalNAc-Ts within and across species, and our group and others have shown the consequence of this diversity is that each isoenzyme follows a unique set of guidelines for glycosylation. In a long-standing collaboration with Dr. Ben Schumann (Francis Crick Institute), we are studying how specific GalNac-T12 mutations found in patients with colorectal cancer alter selection of substrates in the GI tract, building on our earlier work on the structural characterization of GalNAc-T12 (Fernandez et al. PNAS, 2019). We are also currently using our structures and data to find ways to modulate GalNAc-Ts with either small molecule inhibitors or activators. For example, We are working with Drs. Samy Cecioni (University of Montreal) and Dr. Yong Sok Lee (NIAID, NIH) to design specific inhibitors of the Toxoplasma gondii enzyme TxgGalNAc-T3, which is involved in cyst wall glycosylation and influences bradyzoite cyst wall rigidity. We reason that by inhibiting T3, we can weaken the parasite and make it less resistant to drugs targeting cysts and bradyzoites.