The purpose of this project is to determine the structure of proteoglycans as well as to understand the role that they play in the function of tissues and during developmental events. We have been using immunology, peptide mapping and molecular biology to characterize the core proteins of proteoglycans from cartilage and basement membrane. Basement membrane proteoglycans from glomerular and EHS tissue have been found to be immunologically similar. Peptide mapping of these proteoglycans show them to contain a immunologically similar domain of Mr=45,000 but the glomerular proteoglycan lacks a Mr-44,000 domain found in the EHS proteoglycan. Genetic clones to both the cartilage and basement membrane proteoglycan have been obtained by screening expressing vectors with antibodies to their core proteins. We have sequenced nearly 2/3 of the cartilage proteoglycans core protein and discovered a globular domain at the carboxyl terminus that has extensive homology with lectin binding proteins and a serine-glycine rich domain, for glycosaminoglycan attachment, that contains SGXXXXSGXX as a consensus spacing. Immunological evidence indicate our clones to the basement membrane are from the heparan sulfate attachment region. We are also using chondrocytes as a model system to study gene expression and determine the mechanisms by which teratogens disrupt the synthesis of proteoglycans and other matrix components during development. An immortalized chondrocyte line has been developed for this purpose by infecting fetal rat costal chondrocytes with the myc oncogene. Retinoic acid, a teratogen which produces limb and facial malformations in vivo is also known to alter chondrogenesis in vitro. We have found that retinoic acid inhibits the synthesis for type II procollagen, cartilage proteoglycan core protein, and link protein while stimulating the synthesis of type III collagen and fibronectin. Furthermore, these alterations in protein synthesis retinoic acid acts to change the phenotype of the chondrocytes, possibly by affecting transcriptional activity.
