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 protein of proteoglycans from basement membrane. Basement membrane proteoglycans from EHS tissue consists of a MW = 400,000 core protein with 3 heparan sulfate side chains attached to one end. The core protein is divided into 2 major domains: a MW = 200,000 trypsin sensitive region containing the side chains and a MW = 200,000 trypin resistent region containing MW = 46,000 and 44,000 V-8 protease resistant sub domains. The glomerular basement membrane proteoglycan is synthesized from the same MW = 400,000 precursor protein as the EHS proteoglycan but is proteolytically processed to a MW = 250,000 core protein containing the MW = 46,000 subdomain but not the MW = 44,000 subdomain. Genetic clones to both the trypsin sensitive and trypsin resistant domains have been obtained using antibodies. The deduced sequence from these clones show the core protein to consist of a unique repeat structure with no homology to any other connective tissue component. We are also using chondrocytes as a model system to study gene expression and determine the mechanisms by which teratogens disrupt the synthesis of proteolycans and other matrix components during development. 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 of type II procollagen, cartilage proteoglycan core protein, and link protein while stimulating the synthesis of type III collagen and fibronectin. Furthermore, retinoic acid acts to change the phenotype of the chondrocytes by affecting transcriptional activity.
