Proteoglycan is a basic structural element of cartilage, critical in its physiological function and centrally involved in pathological processes causing cartilage destruction in various forms of arthritis. This application requests continued funding for studies of the primary structure and immunological properties of the proteoglycan of bovine nasal cartilage. Methods have been developed for the identification and isolation of the tryptic fragments of bovine nasal cartilage proteoglycan and several major fragments have been obtained in fractions sufficiently homogeneous to allow their biochemical and immunological characterization as well as N-terminal amino acid sequence determination. Under the present proposal, additional proteoglycan fragments will be isolated by gel chromatography or HPLC following the removal of their carbohydrate constituents by chemical or enzymatic means. Individual tryptic fragments will be enzymatically or chemically cleaved, the subfragments isolated and characterized in terms of their biochemical and immunological properties, and their amino acid sequences determined. Analysis of these isolated tryptic subfragments of proteoglycan, and of selected overlapping sequences from proteoglycan fragments produced by alternate means, should allow reconstruction of the primary structure of all or most of the intact molecule. In addition, it is proposed to utilize hybridoma technology and a solid-phase immunoassay to develop a series of monoclonal antibodies directed against specific structurally defined antigenic determinants localized to individual isolated proteoglycan subfragments. The appropriate monoclonal antibodies can be used to elucidate the structure of proteoglycans in other tissues and species, and to approach the study of the molecular biology and genetics of proteoglycans. Better knowledge of the structure, immunology and molecular biology of cartilage proteoglycan can lead to a better understanding of what happens to cartilage on a molecular level in the course of arthritis and hopefully point to more effective therapeutic modalities.
