A chemical biology approach to characterise the substrates of the glycosyltransferase GalNAc-T4 in breast cancer
File(s)
Author(s)
Calle, Beatriz
Type
Thesis
Abstract
O-GalNAc glycosylation is one of the most abundant post-translational modifications. Aberrant O-GalNAc glycosylation is a hallmark of breast cancer. The glycosyltransferase GalNAc-T4 is thought to be heavily implicated in breast cancer, however its exact function in breast cancer and its protein substrates remain unclear. The aim of this project was therefore to characterise the protein substrates and glycosylation sites of GalNAc-T4 in the context of breast cancer and gain insight into the role of GalNAc-T4-mediated glycosylation in breast cancer. We first developed a bioorthogonal reporter system for GalNAc-T4 using the "bump-and-hole" (BH) approach. We demonstrated that BH GalNAc-T4 recapitulates the in vitro activity of the wild-type enzyme whilst displaying a switch from the native substrate to the bumped analogue. We then equipped breast cancer cells with the ability to tag the substrates of GalNAc-T4 with a chemical handle. Analysing these chemically-tagged glycoproteins by mass spectrometry (MS) allowed us to determine the substrates of GalNAc-T4 in a panel of breast cancer cell lines. We also characterised the GalNAc-T4-mediated glycosylation sites in one of the newly identified substrates by MS-glycoproteomics and postulated a possible role of GalNAc-T4mediated glycosylation on the function of the protein. To investigate the role of GalNAc-T4 and its substrates in a biologically-relevant model system, preliminary work was also performed to extend the BH approach to an in vivo setting. Deciphering the identity and the function of the breast cancer relevant-GalNAc-T4 substrates and glycosylation sites may help yield new diagnostic and therapeutic options towards breast cancer.
Date Issued
2024-09-13
Date Awarded
2025-03-01
Copyright Statement
Attribution-NonCommercial 4.0 International Licence (CC BY-NC)
License URL
Advisor
Schumann, Benjamin
Malanchi, Ilaria
Sponsor
Imperial College London
Francis Crick Institute
Grant Number
CHTP G00445
Publisher Department
Department of Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)
Rights Embargo Date
2026-02-28