The current model for the structure of hyaline cartilage proteoglycan has been elucidated, in large part, by examining the fragments produced by proteolytic cleavage of the molecule. In this laboratory, the cyanogen bromide (CNBr)-cleavage of rat chondrosarcoma proteoglycan is being studied. CNBr-treatment of this proteoglycan is being studied. CNBr-treatment of this proteoglycan produces about six peptides from the hyaluronic acid binding region (as judged by polyacrylamide gel electrophoresis). However, the chondroitin sulfate-attachment region remains essentially intact and this large peptide can be separated from the others by gel chromatography or by cesium chloride density gradient centrifuguation. The purification of the hyaluronic acid-binding region peptides has been actively pursued over the past two years. In the proposed study, the chondroitin sulfate attachment region will be fragmented by means other than CNBr treatment such as limited proteolysis and oxidative cleavage at tryptophan residues. All the peptides will be purified to homogeneity largely by high performance liquid chromatography and analyzed for carbohydrate and amino acid content and amino acid sequence. It may be necessary to deglycosylate some of the peptides to allow complete purification and analysis. The peptides which are too large for complete sequence determination will be fragmented further by proteolysis. Overlapping sequences will be obtained by cleaving the protein by alternative enzymatic or chemical treatment. Antibodies to proteoglycan, both polyclonal and monoclonal, have been obtained. More monoclonal antibodies to proteoglycan will be produced and characterized. The reactivity of these antibodies with the various peptides will aid in identifying the antigenic sites on the proteoglycan. Not only will proposed investigations aid in the further elucidation of the structural and immunological properties of proteoglycans but they will be useful for the analysis of these macromolecules in healthy and diseased articular cartilage.
NIH ExPorter
NIH ExPorter