MSc in BiochemistryDissertation Project – 2nd Cycle

BACKGROUND

OBJECTIVES

1

Glycosylation is the most complex and widespread process of posttranslational modification of proteins and lipids, with an unsurpassed capacity to generate a wide array of structures (Annu. Rev. Biomed. Eng. 2007, 9, 121–167). The large polypeptide GalNAc-transferase (GalNAc-Ts) family catalyzes the transfer of N-acetylgalactosamine (GalNAc) from a sugar donor UDP-GalNAc to Ser/Thr side chains of many cell-surface proteins (Figure 1A) (Glycobiology 2012, 22, 736-756). GalNAc-Ts show distinct, although partly overlapping, kinetic properties and acceptor substrate specificities, and the available repertoire of GalNAc-Ts therefore determine which and where proteins are O-glycosylated (Trends in Cell Biology 2011, 21, 149-158). In this context, mucin-type proteins are the main templates catalysed by GalNAc-Ts. In addition, aberrant glycosylation is a universal feature of cancer (Figure 1B) and alterations in cell expression of GalNAc-Ts also occur during malignant transformation (Nat. Rev. Cancer 2015, 15, 540-555). With this project, we aim to develop a structural based discovery protocol, to unveil GalNAc-Ts family specificities and dynamics of mucin MUC1 protein upon O-glycosylation, vitally needed to understand GalNAc-Ts mechanism of action in health and to develop inhibitors for diseases like cancer.

Figure 1. A. Schematic view of GalNAc-Ts O-glycosylation process. B. Glycan structure in normal and cancer cells.

Student´s Name: Student email address: No.Supervisor ( s) : Filipa MarceloSupervisor(s) email address: filipa.marcelo@fct.unl.ptLab/Institution: Lab 302 and 106 FCT-UNLTITLE: Deciphering the secrets of GalNAc O-glycosylation.

The access to the structural knowledge of MUC1 O-glycosylation features will permit to understand GalNAc-Ts binding specificities. Specific objectives for project:

a) To determine the order and kinetics of MUC1 O-glycosylation by GalNAc-T2 and T4. b) To elucidate the effects on MUC1 conformation upon GalNAc addition during O-

glycosylation process. c) Unravel GalNAc-T2 and T4 specificities.

B.A. GalNAc-Ts

Mucin protein

MSc in BiochemistryDissertation Project – 2nd Cycle

PROJECT DESCRIPTION

TIMELINE (use fill tool for the cells)Month 1 Month 2 Month 3 Month 4 Month 5 Month 6 Month 7 Month 8 Month 9 Month 10

Task 1Task 2Task 3Thesis

2

The project focuses on the application of NMR methods to follow the glycosylation process of mucins by GalNAc-Ts, unveiling new structural, conformational and dynamic insights at atomic level of this biological event from the receptor (GalNAc-Ts) and protein acceptor (Mucin MUC1) viewpoint. MUC1 tandem repeats consisting of 20 amino acids each have 5 acceptor sites. Concerning GalNAc-Ts, we select GalNAc-T2, and T4 isoforms to probe the MUC1 O-glycosylation pathway. T2 will glycosylate 3 of the 5 potential O-glycosylation sites in MUC1 tandem repeat while T4 transferred GalNAc to the 2 remaining sites in the MUC1 sequence. The present master project is organized in 3 tasks:

Task 1: Expression of 15N and 13C isotopically labelled MUC1 containing 4 tandem repeats MUC1 domains (MUC1-4TR). GalNAc-T2 and GalNAc-T4 are currently available in the lab.

Task 2: Fast heteronuclear NMR experiments of MUC1-4TR will be carried out in presence of T2 and T4 to monitor the chemical shift displacements of MUC1 resonances until complete glycosylation, determining the order and kinetics of glycosylation process and if possible the conformational alteration of MUC1 structure upon glycosylation.

Task 3: Study of the intermolecular interactions of selected peptides substrates with GalNAc-T2 and T4 will be carried out by NMR, namely STD-NMR and trNOESY. STD-NMR experiments will be performed to monitor the interaction and identify the binding epitopes in the substrates while trNOESY experiments will be accomplished to determine the bioactive conformation.

Therefore, the student will have contact with distinct NMR techniques in the context of protein and glycoconjugates structure and recognition using well-defined methodologies implemented and currently used in the NMR group (Curr. Protein Pept. Sci. 2012, 13, 816-830; J. Am. Chem. Soc. 2013, 135, 16418 – 16428; Chem. Eur. J. 2014, 20, 16147-16155; J. Am. Chem. Soc. 2015, 137, 12438-12441). In this sense, with this project the student will be educated and trained to setup and analyse distinct NMR experiments as well as to deduce the glycosylation pathway and specificity of MUC1 O-glycosylation by specific GalNAc-Ts.