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The role of glycans in osteogenesis

Wilson, Katherine (2015) The role of glycans in osteogenesis. PhD thesis, University of York.

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Abstract

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.

Item Type: Thesis (PhD)
Keywords: Glycosylation, mesenchymal stem cells, differentiation.
Academic Units: The University of York > Biology (York)
Identification Number/EthosID: uk.bl.ethos.667700
Depositing User: Miss Katherine Wilson
Date Deposited: 12 Oct 2015 12:25
Last Modified: 24 Jul 2018 15:21
URI: http://etheses.whiterose.ac.uk/id/eprint/10403

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