Proteochondroitin sulfate is a major constituent of the cartilage matrix and all other connective tissue. It plays an important role in bone and connective tissue metabolism, wound healing, and in cell interactions. Defective synthesis of these molecules could impact upon diseases and conditions such as arthritis, atherosclerosis, improper wound healing, etc. Better knowledge of the biosynthesis and its regulation may assist in providing rational therapies for these conditions. Proteochondroitin sulfates consist of a protein backbone to which many glycosaminoglycan chains of varying chain length and sulfate content are covalently attached. The chondroitin chains are linked to a wide range of core protein families. The functions of these proteoglycans are not clearly defined, but may be as varied as their structures. It is unclear how the protein core and the highly anionic sulfated glycosaminoglycan structures direct these functions, but it is likely that the core protein would be involved in channeling the transport and presentation of the attached glycosaminoglycans to specific intracellular granule, cell surface, or matrix localities. Whether or not this is a main function of the protein core, it does seem apparent that the fine structure of the highly anionic glycosaminoglycans may be of paramount importance in function at each location where the proteoglycans are presented. It is not clear at present as to what key features control the biosynthesis of the fine structure of the chondroitin sulfate chains. Our goal is to examine the assembly of the polysaccharide portion of proteochondroitin sulfate. Although the main outlines of the biosynthetic pathway are now clear, several areas remain to be explored. These include the organization, sub-cellular localization, movement and interaction of nascent proteoglycan and enzyme for synthesis of the polysaccharide portion of proteochondroitin sulfate. We will use a chick embryo epiphyseal cartilage system that synthesizes at least two populations of proteochondroitins that are predominantly 6-sulfated and a mast cell system that synthesizes an intracellular proteochondroitin 4-sulfate. In order to examine the organization of the enzymes involved in synthesis, we will sub-fractionate the Golgi followed by functional characterization of the fractions. These fractions will be assayed for xylosyl transferase, two galactosyl transferases, N-acetylgalactosaminyl transferase(s), glucuronosyl transferase(s), 4-sulfotransferase, 6- sulfotransferase and specific marker enzymes. In addition, N- acetylgalactosaminyl transferase, glucuronosyl transferase and sulfotransferases will be purified to homogeneity, employing conventional protein purification techniques as well as affinity chromatographic techniques. The fundamental characteristics and specificities of these enzymes will be determined. The interactions of the enzymes with each other to form multi-enzyme complexes will be examined. This will provide the basis for the long-term goal which is to reconstitute proteochondroitin sulfate synthesis with the purified enzymes, raise antibodies to the purified enzymes and to clone them.
