Abstract
The role of proteoglycans in the central nervous system (CNS) is a rapidly evolving field and has major implications in the field of CNS injury. Chondroitin sulfate proteoglycans (CSPGs) increase in abundance following damage to the spinal cord and inhibit neurite outgrowth. Major advances in understanding the interaction between outgrowing neurites and CSPGs has created a need for more robust and quantitative analyses to further our understanding of this interaction. We report the use of a high-throughput assay to determine the effect of various post-translational modifications of aggrecan upon neurite outgrowth from NS-1 cells (a PC12 cell line derivative). Aggrecan contains chondroitin sulfate, keratan sulfate, and N-linked oligosaccharides (N-glycans), each susceptible to removal through different enzymatic digestions. Using a sequential digestion approach, we found that chondroitin sulfate and N-glycans, but not keratan sulfate, contribute to inhibition of neurite outgrowth by substrate-bound aggrecan. For the first time, we have shown that N-linked oligosaccharides on aggrecan contribute to its inhibition of neuritogenesis.
Highlights
Proteoglycans (PGs) are a class of complex macromolecules commonly associated with the cell surface and the extracellular matrix
NS-1 Cells Extend Neurites in a Dose-Dependent Fashion When Exposed to Varying Amounts of nerve growth factor (NGF)
We report the use of a high-throughput assay to determine the effect of various post-translational modifications of the chondroitin sulfate proteoglycans (CSPGs) aggrecan upon neurite outgrowth from NS-1 cells
Summary
Proteoglycans (PGs) are a class of complex macromolecules commonly associated with the cell surface and the extracellular matrix. PGs are comprised of a core protein that is modified post-translationally by attachment of a variety of glycosaminoglycans (GAGs) and N-linked oligosaccharides. Of particular importance is the formation of a glial scar following spinal cord injury and the increased abundance of chondroitin sulfate proteoglycans (CSPGs) in these scars. The increased CSPG content of the glial scar is a major impediment to neuronal regeneration and it is believed that by overcoming this impediment, one can promote reconnection of the disconnected portions of the spinal cord. Much research in the role of proteoglycans in CNS physiology focuses on the CSPGs and their ability to inhibit neurite outgrowth. Numerous investigations have indicated that CSPGs inhibit outgrowth of neurons [2,3,4,5,6]. CSPGs may stimulate neurite outgrowth [7,8]
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