Chondromucoproteins (CMP) synthesized by chick embryo chondrocytes contain three types of carbohydrates which are O-linked to a large core protein. These include chondrointin sulfate chains (CS), keratan sulfate chains (KS), and O-linked oligosaccharides. Methods will be developed to determine the compositions and monomer sequences in each of these types of structures and the relative amount of each structure in the several species of CMP that are synthesized by the chondrocytes. For sequencing the CS chains, a series of oligosaccharides, each with a linkage region sequence at its reducing terminal and a Ga1NAc, Ga1NAc-4SO4, or Ga1NAc-6SO4 at its nonreducing terminal, will be isolated. The oligosaccharides will be analyzed to determine their nonreducing terminal residue(s) as a function of increasing distance from the linkage region. Similarly, KS will be sequenced by preparing a series of oligosaccharides, each with the YS linkage region at its reducing terminal and a G1cNAc or G1cNAc-6SO4 residue at its nonreducing terminal. The oligosaccharides will be analyzed to determine their nonreducing terminal residue(s) as a function of increasing distance from the linkage region. These methods will be used to analyze the structural differences in the different CMP's produced by the chondrocytes and the structural changes in these O-linked carbohydrates that occur when culture conditions are varied. It is a long term goal of these studies to correlate these structures with the activities of the CMP's in cartilage calcification.
