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 are characterizing functional domains of tissue specific proteoglycans by isolation and characterization of peptide fragments derived from proteolytically cleaved proteoglycan. Genetic clones of proteoglycans are being isolated by screening expressing vectors with antibodies directed against the core protein of the proteoglycan. Particular attention has been directed toward studying the heparan sulfate proteoglyca1 of basement membranes which we have found to contain a basement membrane specific 400,000 MW core protein. We have isolated heparan sulfate free peptide of 200,000 MW from the proteoglycan. These observations, in combination with electron microscopic examination of the intact proteoglycans, indicate the heparan sulfate side chains are located asymetically, on only one region of the core protein. We are also using chondrocytes to determine the mechanisms by which teratogens disrupt the synthesis of proteoglycans and other matrix components during developent. Retinoic acid, a teratogen which produces limb and facial malformations in vivo is also known to disrupt chondrogenesis in vitro. We have found that retinoic acid inhibits the synthesis of type II procollagen, and cartilage proteoglycan core protein, while stimulating the synthesis of type III collagen and fibronectin. Furthermore, these alterations in protein sysnthesis are due to changes in mRNA levels of the proteins. These data suggest that retinoic acid acts to change the phenotype of the chondrocytes, possibly by affecting transcriptional activity.
