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. We are developing a new approach to elevate the structural characterization of the PG's, GAG portion to the level currently being used on the core protein.
The specific aims of this proposal are: 1. To improve methods for microsequencing GAGs using enzymatic, chemical, electrophoretic and mass spectrometric techniques; 2. To apply computer simulation analysis to improve our understanding of GAG sequence; 3. To sequence the GAG chain(s) of the major bovine brain PG to improve our understanding of prion- based diseases; 4. To use NMR spectroscopy to elucidate the secondary structure of GAG-derived oligosaccharides having biological importance; 5. To understand the underlying structural variability in pharmaceutical heparin to improve its application as an anticoagulant/antithrombotic and to enhance its potential application to other disease states. Unlike nucleic acids and proteins, polysaccharides are polydisperse mixtures and cannot easily be prepared as homogeneous, pure substances. Advances in the fractionation of acidic oligosaccharides suggest it may now be possible to prepare a single GAG chain for sequencing. An oligosaccharide of molecular weight 7,980, has been recently prepared from heparin and purified to homogeneity. Its structure determined by nuclear magnetic resonance spectroscopy, GAG chains, of similar size, containing Xyl at their reducing end have also been prepared during the sequencing of the first 13 residues to peptidoglycan heparin. The purification of a single, homogeneous, biologically active chain for sequencing represents a major objective of this project. A challenging target will be sequenced and success in the endeavor might lead to a better understanding of BSE and Alzheimer's disease. A cocrystal structure of heparin oligosaccharides with bFGF and a solution structure with PF4 have been solved with the support of this grant.
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