Engineering an Orthogonal O-Glycosyltransferase and Donor Sugar Pair

Despite its role in fundamental biological processes, GalNAc-type O-linked glycosylation has proven extremely difficult to study and remains one of the most poorly understood post-translational modifications. The ppGalNAcT family catalyzes the first committed step in O-glycosylation, the installation of GalNAc from the donor UDP-GalNAc onto serine and threonine (Ser/Thr) residues of acceptor polypeptides. Thus, the substrate specificity of the 20 human ppGalNAcT family members determines which proteins are O-glycosylated. Progress in the field is hampered by a limited understanding of ppGalNAcT protein substrate specificity and of the regulation underlying O-glycosylation dynamics./nThe individual ppGalNAcTs display distinct spatial and temporal expression patterns during mammalian and fly development, which may reflect their unique roles governing cell-specific glycosylation. In vitro studies have shown that ppGalNAcT family members have both unique and redundant peptide substrate specificities, although validation of these results in vivo has met with limited success. Efforts to isolate the function of individual isoforms by genetic knockdown demonstrate unpredictable functional redundancy among family members—ablation of several ppGalNAcTs in mice yields only partially penetrant phenotypes, while five ppGalNAcTs are essential for Drosophila viability. Chemically targeting one isoform with inhibitors or substrate analogs is challenging, as members of the ppGalNAcT family demonstrate a high level of structural homology and all utilize the nucleotide sugar UDP-GalNAc as a donor substrate. All current efforts to monitor the activity of individual ppGalNAcTs in vivo lack an unambiguous method that can confirm whether a target protein is naturally glycosylated by a specific ppGalNAcT in a biological context./nTowards this end, we have rationally designed an individual family member, ppGalNAcT2, with an enlarged binding pocket and a UDP-GalNAc analog with a “bump” and a chemical handle. This bump-hole pair is designed to uniquely label the protein substrates of ppGalNAcT, ultimately enabling proteomic analysis. The pair that we have developed behaves orthogonally, meaning that the ppGalNAcT2 mutant and the UDP-GalNAc analog, UDP-GalNAzMe(S), demonstrate little reactivity with the native system. UDP-GalNAzMe(S) is poorly accepted by native ppGalNAcTs in vitro and has minimal background in vivo. This pair can glycosylate and tag a known acceptor substrate of ppGalNAcT2, enabling downstream labeling with an affinity handle for enrichment and analysis./nAs described in this dissertation, our goal was to develop a ppGalNAcT and UDP-GalNAc analog pair for identification of ppGalNAcT protein substrates. Chapter 1 describes the background for this project including the biological functions of O-glycosylation, the ppGalNAcT family, and recent progress in the field. It then discusses the utility of a bump-hole strategy and significant work using bump-hole engineering in other protein families. Chapters 2 and 3 describe the enzyme and substrate panels generated for this research. Chapter 2 explores the development of a panel of ppGalNAcT mutants with enlarged binding pockets and a mammalian expression system to secrete transferase soluble domains. Chapter 3 is focused on the chemoenzymatic synthesis of a panel of UDP-GalNAc analogs with an azide chemical handle./nOngoing work is described in Chapter 4. We report the identification of a promising bump hole pair from in vitro screens with a model peptide substrate. Preliminary work demonstrating that UDP-GalNAzMe(S) is orthogonal to native ppGalNAcTs in cell lysates is also discussed. We report progress delivering UDP-GalNAzMe(S) to cells, as well as further confirmation that the analog is orthogonal to native ppGalNAcTs in cells. Ongoing work is delineated at the end of Chapter 4. We establish an ongoing collaboration with the Gerken lab at Case Western Reserve University in which a peptide library is being used to confirm that the bump-hole pair conserves ppGalNAcT substrate specificity. We also describe a strategy to measure the KM and VMAX for the candidate bump-hole pair with a model peptide substrate. Chapter 4 concludes with future directions for this research in living systems.

Advisor: Bertozzi, Carolyn R