Sequencing the Polysaccharide Component of Proteoglycans

Rensselaer Polytechnic Institute, Troy NY

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Abstract

DESCRIPTION (provided by applicant): Proteoglycans (PGs) play an important role in a variety of diseases affecting the excretory system, respiratory system, circulatory system, skeletal system, as well as the multisystem diseases of aging and cancer. Progress has been made in the isolation, purification, and characterization of PGs from extracellular matrix; these advances have led to the sequencing of PG core proteins. The chemistry and biology of a PG, however, is dominated by the glycosaminoglycan (GAG) components for which it is named. The characterization of GAGs is most often directed towards the identification of the class to which they belong, their molecular weight, and their charge density and disaccharide analysis. We are developing a new approach to elevate the structural characterization of the PC's GAG portion to the level currently being used on the core protein. The specific aims of this proposal are: 1. To improve microsequencing methods; 2. To use x-ray crystallography and NMR spectroscopy to elucidate the secondary structure of GAG-derived oligosaccharides having biological importance; 3. To completely sequence the GAG chains of the chondroitin sulfate PG, bikunin; and 4. To initiate the sequencing of the GAG chain(s) of the major bovine brain and human liver heparan sulfate PGs to improve our understanding of GAG structure in physiology and pathophysiology. Unlike nucleic acids and proteins, PGs are polydisperse mixtures and cannot easily be prepared as homogeneous, pure substances. Recent advances in our laboratory, particularly in liquid chromatography (LC), LC/MS and LC-fluorescence has put the possibility of purifying and sequencing PGs within reach. New efforts using high resolution Fourier transform (FT) MS with electron capture dissociation (ECD) and electron detachment dissociation (EDO) should also greatly facilitate sequencing. Our targets include bikunin, currently used as a drug for the treatment of hemorrhagic shock and acute pancreatitis, and brain and liver heparan sulfate PGs. In addition to solving the sequence or primary structure, the secondary structure of the GAG chains of these PGs will be solved by approaches using spectroscopic approaches in novel solvents.