Phosphorylation at the 2-position of xylose of chondroitin sulfate was initially discovered in aggrecan from the Swarm rat chondrosarcoma. Following this discovery, phosphorylation of other glycosaminoglycan (GAG) chains including chondroitin sulfate, dermatan sulfate and heparan sulfate were found. The exact function of this modification still remains unknown. The role of phosphorylation in the intracellular sorting and control of synthesis of proteoglycans (PGs) will be investigated using tissue culture, radioisotopic labelling, and chemical analysis. The detailed structure of the glycosaminoglycan modification of the linkage region in the Swarm rat chondrosarcoma chondroitin sulfate PGs and the EHS heparan sulfate PGs will continue to be investigated. We will develop an assay for xylose phosphorylating enzyme(s), purify it and determine its chemical and physical properties, its donor and substrate specificities and subcellular distribution. We will determine the structure of the initial modification, identify the phosphorylating sites along the protein core, and obtain partial amino acid sequences for such sites. In developing the method for assaying the phosphorylating enzyme, we will use a silk fibroin or small peptides as substrates and partially purified xylosyl transferase from the Swarm rat chondrosarcoma or EHS sarcoma to prepare potential substrates. Additionally, a new mercurial method will be used to remove nonreducing unsaturated glucuronic acid residue from chondroitinase AC-treated PGs and enzymes will be used to degrade the molecule into the linkage region. This will provide high molecular weight substrates that will then be used to probe subcellular fractionations for the enzyme. Structural techniques will be used to define the properties of the phosphorylated products. The degraded linkage region preparation will be used to produce monoclonal antibodies to the linkage region. The results of the study will be used to propose a role of phosphorylation in PG synthesis.
