Proteochondroitin sulfate is a major structural constituent of connective tissue, and occurs as an ubiquitous component of cell surface, where it is considered to be involved in specific cell-cell, cell-matrix and cell-receptor functions. Changes in metabolism of proteochondroitin sulfate are thought to impact upon multiple diseases and conditions involving connective tissue such as arthritis, atherosclerosis, improper wound healing etc. Knowledge of the biosynthetic regulation will assist in understanding how the metabolism and fine structure of this molecule relates to the pathology of many conditions. The polysaccharide portion of the molecule is frequently the """"""""business end"""""""", with the core protein portion involved in channeling the molecule to the appropriate cellular locations. Thus, the size of the chondroitin chains and the degree and type of sulfation provide much of the specificities in function. The control in synthesis of this fine structure is directly related to the specificity of the chondroitin sulfate initiating enzymes, membrane topography at the sites of synthesis and to the concerted interaction of all the biosynthetic enzymes. Knowledge of the structure and characteristics of the individual enzymes is essential to understand this membrane organization. Thus, the following specific aims will be pursued:
Aim I. We will purify and characterize the specific enzymes involved in the addition of the first glucuronic acid and first N- acetylgalactosamine residues to the linkage trisaccharide that is attached to the core protein. The acceptor specificities of these enzymes will be determined.
Aim II. The interaction of these enzymes with each other and with the chondroitin-polymerizing glucuronosyl transferase and N- acetylgalactosamine transferase will be investigated. In addition, the interaction of all these enzymes with nascent proteochondroitin will be examined.
Aim III. We will clone and express the cDNA for the chondroitin- polymerizin glucuronosyl transferase that we have purified. These studies will provide fundamental information for examining the regulation and organization of proteochondroitin synthesis and will enable us to obtain necessary tools for future studies.
