The role of glycans in osteogenesis

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.