Proteoglycans (PGs) play an important role in a variety of diseases affecting the excretory system, respiratory system, circulatory system, skeletal system, also the multisystem diseases of aging and cancer. Progress in the isolation, purification, and characterization of proteoglycans from extracellular matrix has led to the sequencing of proteoglycan core-proteins. The polysaccharide or glycosaminoglycan (GAG) components for which a proteoglycan is named dominates its chemistry and biology. Until now, the characterization of these GAG components has focused primarily on the identification of their family (i.e., chondroitin, dermatan, heparin, etc.), molecular weight, charge density and disaccharide analysis. We are developing a new approach to elevate the structural characterization of the GAG portion of the PG to the same level used on the core protein. Because of the polydispersity and structural similarity between GAGs within a given class, scientists originally viewed GAGs as molecules of undetermined structure (or sequence) and thus, GAGs were not considered informational molecules. There are structural differences between the core proteins of various PGs including members of a particular class. Thus, GAGs are not a continuum of molecules but rather are structurally distinct. The polysaccharide portion of PGs also has structurally distinctive domains. For example, defined tetrasaccharide sequences are present at the poly- saccharide's reducing end, where it attaches to its core protein. Specific residues terminate newly formed chondroitin sulfate chains. An unusual GlcN2S3S6S residue found within heparin, also marks the center of its binding site for antithrombin III. Two unusual residues, GlcN3S6S and GlcA2S, in the heparan sulfate (HS) receptor responsible for herpes simplex virus type 1 infection. The presence of such uniquely modified sugars within 10-50 kDa polysaccharide chains suggests that these molecules contain information. Biosynthetic studies have resulted in the identification of most of the enzymes involved in GAG synthesis but have not yet lead to clear understanding of what controls the placement of sequence information within a GAG.
The specific aims of this focused proposal (07/09-06/14) are to: 1. Improve MS-based GAG microsequencing, including the development of new state-of-the-art and routine analytical methods widely available to biologists and chemists;2. Elucidate secondary structure of GAGs and their complexes with GAG-binding proteins using NMR spectroscopy and X-ray crystallography;3. Develop MS-based quantitative profiling of GAG-derived oligosaccharides using isotopically-labeled standards;4. Sequence decorin using methods developed for bikunin;and 5. Sequence an animal tissue- derived HS using isotope-labeled bioengineered model HS and HS-oligosaccharide models.
The proposed effort impacts human health by developing methods to analyze and sequence the glycosaminoglycan chains of proteoglycans. Currently it is difficult to analyze and not possible to sequence these complex polysaccharides. This has prevented a firm understanding of the structure- activity relationship of this important class of biomacromolecules.
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