We have initiated the development of assay techniques that will allow us to understand the sulfation code in chondroitin glycosaminoglycan (GAG) chains. These assay techniques take advantage of specific chromatography techniques (ion exchange, hydrophilicity) to separate the different disaccharides and monosaccharides that comprise the GAG chain. This is the only technique capable of doing this. We have also initiated mass specrtrophotometric analysis of these separated GAG chains to begin to determine the sequence of sulfations on the different parts of the GAG chain. We have continued experiments to modify the biological activity of GAG chains by altering the level or activity of enzymes that either synthesize or degrade sulfated chondroitin. In particular, we are developing methods to specifically alter the sulfation of GAG chains in particular positions in order to evaluate if they alter the biological activity of the GAG chain. We showed that a specific reduction in 4-sulfation abolishes the biological activity of CSPGs, thus suggesting that sulfation on the 4 position of GalNAc is essential for biological activity. We have started experiments to evaluate novel intracellular signaling mechanisms in response to GAG chains. Growth cones turn in response to bound GAG chains. We have used this property of growth cone turning to assay the activity of drugs and other compounds. In particular, we have determined that the activity of particular intracellular molecular motors and other components of the cytoskeleton are essential for this growth cone turning. Modification of their activity by drugs may ultimately prove to be a useful therapeutic approach for treatment of brain injury. Because many of these mechanisms are also found in injury to heart and blood vessels, these approaches may have a more general applicability.
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