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MSc in Biochemistry Dissertation Project – 2nd Cycle Student´s Name: Student email address: No. Supervisor: Filipa Marcelo Co-supervisor: Jorge Dias Supervisor(s) email address:[email protected]; [email protected] Lab/Institution: Lab 303, 106 FCT-UNL TITLE:DECODING MUC1 GLYCOSYLATION AND RECOGNITION BACKGROUND Alteration of glycome signature of the MUC1 glycoprotein is a hallmark of cancer that yield new glycan epitopes with a paramount importance in cancer cell growth and crucial for tumour metastasis (Nat Rev Cancer.2015, 15, 540). The MUC1 glycoprotein contains a variable number of tandem repeats (20 to 200) of a 20 amino acid (aa) peptide sequence, GVTSAPDTRPAPGSTAPPAH, with 5 potential sites of O-glycosylation (in bold). In a normal tissue, MUC1 is heavily decorated with branched oligosaccharides (Glycobiology 2000 10, 439–449). In tumour cells, different MUC1 epitopes, such as the Tn(αGalpNAc(1O-Ser/Thr) and STn (αsialyl(26)αGalpNAc(1O-Ser/Thr)) are now exposed to the immune system (Nat. Rev. Cancer, 2009, 9, 874). Indeed, MUC1 tumour-associated carbohydrate antigens are attractive targets for the development of cancer immunotherapies (Chem. Soc. Rev. 2013 42, 4421). Abnormal glycosylation of MUC1 results from changes in expression and/or activity of certain glycosyltransferases (GTs) like GalNAc-transferase (GalNAc-Ts) and Sialyl-transferase (Sialyl-Ts) families (Proc Natl AcadSci U S A2014, 111, E4066). In this sense, unveiling the structural features of Oglycosylation and sialylation of MUC1 will permit to understand the mechanism and binding specificities of these key enzymes. With this project we will investigate the glycosylation of MUC1 by the GalNAc-T2 and T4 and the sialyltransferase ST6GalNAc-I by using NMR spectroscopy methods. The generated glycan-derived MUC1 structures will be used for molecular recognition studies towards human macrophage galactose C-type lectin (MGL) using a protocol previously established by us (Chem. Eur. J.2014, 20, 16147). MGL is C-type lectin that is expressed in dendritic cells (DCs) and macrophages and binds both Tn- and STn motifs of tumour cells having the ability to discriminate between healthy and cancerous tissues (Cancer ImmunolImmunother. 2007, 56, 1225). Figure 1. Glycome signature of healthy cells (left panel) and cancer cells (right panel). OBJECTIVES 1 MSc in Biochemistry Dissertation Project – 2nd Cycle Specific objectives for the master project: a) To decipher the glycosylation order for the different acceptor sites of MUC1 by GalNAc-T2, GalNAc-T4 and ST6GalNAc-I. b) To elucidate the effects on MUC1 conformation upon GalNAc and Sialic addition during glycosylation process. c) To accomplish binding affinity studies of Tn and STn-MUC1 derived structures towards MGL protein. PROJECT DESCRIPTION A multidisciplinary project that will combine recombinant protein expression and purification with NMR spectroscopy has been delineated to address the goals of the project. The Project is organized in 3 tasks: Task 1: Expression and purification of recombinant of MUC1-4TR and MGL for NMR studies. Recombinant expression and purification of isotopically labelled 15N-MUC1 containing 4 tandem repeats (MUC1-4TR) will be carried out. As well expression of the non-labelled extracellular domain of MGL will be accomplished. Task 2: Glycosylation of MUC1-4TR by selected glycosyltransferases Heteronuclear NMR experiments of 15N-labeled MUC1-4TR (1H15N-HSQC) will be carried out first in presence of GalNAc-T2 and then after addition of GalNAc-T4. Chemical shift perturbation (CSP) of MUC1 resonances will be monitor until complete O-glycosylation. The final glycan-derived products of GalNAc-T2 and T4 will be further engaged into glycosylation with ST6GalNAc-I. Once again CSP analysis of MUC1 peptide resonances will be monitor until complete sialylation. The MUC1 conformation upon GalNAc and Sialic addition during glycosylation process will be investigated by NOE-based experiments Task 3: Binding affinity studies of Tn and STn-MUC1 derived structures to MGL protein CSP of MUC1 resonances in 1H15N-HSQC spectrum of the multi Tn and STn MUC1-4TR products will be monitor in presence of MGL. As well ligand-based NMR binding techniqueswill be used namelysaturation transfer difference NMR (STD-NMR) in order to define, at atomic level,the glycan and peptide epitopes. For STD-NMR mono glycosylated Tn-and STn MUC1 structures with only 20 aas will be used. These compounds were previously obtained and are currently available in the lab for NMR binding studies. 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 1 Task 2 Task 3 Thesis 2