Orthogonal approaches for studying glycosaminoglycan biopolymer
Grande-Allen, K. Jane
Doctor of Philosophy
The evolution of glycosaminoglycans (GAGs) in eukaryotic species has been one of great biological mysteries. This thesis investigates the composition, rate of degradation and solution characteristics of aggrecan proteoglycan derived GAGs through a variety of biochemical and biophysical means, thereby providing a window to understanding these complex biopolymers. Aim 1 covers the development and application a of novel tandem enzymatic strategy (lyase + hydrolase) to characterize the various sulfated products of GAG breakdown. Aim 2 applies strategies of analytical ultracentrifugation, crowding kinetics and a variety of biophysical methods to access GAG fine structure and molecular composition and to understand how this degradation may be modulated in vivo. Aim 3 follows directly from Aim 2 in assessing how bulk behavior of these biological polymers varies in a concentration dependent manner using rheology along with other crowders to understand scaling relationships. Overall, these aims provide a new viewpoint to understand, length, size, shape and compositional complexity of glycosaminoglycans as well as their specific degradation characteristics. This research will facilitate the development and application of biotherapeutic GAG derived polymers where changes in backbone adducts and stereochemistry could activate the colossal amount of potentially useful chemical compounds that are generally inert, dormant or only transiently active in the extracellular space.