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 polysaccharide or glycosaminoglycan (GAG) components for which it is named. The characterization of these GAG components has primarily been 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 on the core protein. We report success in sequencing GAG chains 10-15 saccharide residues from peptide core.
The specific aims of this proposal are to: 1. Focus reading frame sequencing of GAG chains on regions more remote from the protein core particularly sites of biological importance; 2. Utilize well developed chemical derivatization methods in solid state sequencing; 3. Apply computer simulation to the study of the kinetics of final and transient product formation; 4. Begin to elucidate the secondary structure and conformation of GAG chains; and 5. Initiate the synthesis of neo-PGs and PG model compounds for future studies aimed at understanding the tertiary structure of PGs. 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 homogeneous GAG chain for sequencing. A single tetradecasaccharide of molecular weight 4,655, has been recently prepared from heparin and 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 peptidoglycan heparin and decorin. The purification of a single, homogeneous, biologically active chain for sequencing represents a major objective of this project. However, this goal raises a concern. Will the arbitrary choice of a single GAG chain truly reflect the structure and the multiple biological functions of the polysaccharide mixture? Thus, it will also be necessary to use additional approaches that will provide sequence information on polydisperse GAG mixtures. Three approaches will be used to sequence GAGs. These are: 1. sequencing by simulation, using computer and mathematical models; 2. sequencing of large, homogeneous GAG-derived oligosaccharides; and 3. defined reading frame sequencing using a microanalytical (1-10 micrograms of GAG. 10-100 micrograms of PG) approach. The long term objective of this proposed research is to better understand the physiological role of a major component of the extracellular matrix, proteoglycans by obtaining an intimate and detailed knowledge of their structure.
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