Download MSc in Biochemistry Dissertation Project – 2nd Cycle Student´s

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(1O-Ser/Thr) and STn (αsialyl(26)αGalpNAc(1O-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
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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
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