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Title: The role of glycans in osteogenesis
Author: Wilson, Katherine
ISNI:       0000 0001 2423 8097
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2015
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Glycosylation is an abundant post translational modification of proteins, which occurs in the endoplasmic reticulum and Golgi. Nearly every secreted and plasma membrane localised protein is glycosylated. Mesenchymal stromal/stem cells (MSCs) are adult stem cells that can self-renew and differentiate into multiple cell types including: osteoblasts, adipocytes and chondrocytes. Due to their tri-lineage capacity and their immunoregulatory functions, MSCs are attractive tools for the treatment of multiple diseases and conditions. Key to their use in regenerative medicine is knowledge about what influences a MSC to differentiate into a particular cell type. Despite previous studies describing distinct glycan profiles of cells at different stages of development, whether glycans play a functional role in directing MSC differentiation is currently unknown. Here, utilizing an immortalized primary human MSC line (hTERT-MSCs), the N-glycans from MSCs and osteoblasts were harvested using the Filter aided N-glycan Separation (FANGS) method. Following permethylation, N-glycans were analysed by mass spectrometry (MALDI-TOF/TOF). This method allowed the reliable, quantitative, relative abundance of different glycan structures to be compared between the two cell types for the first time. Complex N-glycans were significantly more abundant in osteoblasts compared to MSCs. The N-glycan profiles of different hTERT-MSC lines were not significantly different, despite variations in differentiation potential. The glycosylation pathway was genetically disrupted by targeting a subunit of the Conserved Oligomeric Complex (COG) with a shRNA. As predicted, since COG is responsible for the tethering of vesicles carrying Golgi resident enzymes, Cog4 knock-down hTERT-MSCs had disrupted N- and O-glycan synthesis. Interestingly, Cog4 knock-down hTERT-MSCs showed reduced osteogenic capacity with reduced levels of mineralised extracellular matrix (ECM). Surprisingly, the chemical inhibition of complex N-glycan synthesis increased ECM mineralisation, whilst inhibition of O-glycan synthesis or proteoglycan sulfation mimicked the Cog4 knock-down cells with reduced mineralisation. These results showed a novel role of both N-and O-glycans in osteogenesis.
Supervisor: Ungar, Daniel ; Genever, Paul Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available