PROTEOGLYCANS IN CHONDRODYSPLASIA

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The skeletal dysplasias consist of a heterogeneous group of inherited defects which are characterized by significantly altered endochondral ossification in the long bones of skull, and vertabrate of various affected species including man. With the exception of the mucopolysacharidoses, the molecular basis for the altered endochondral ossification in these affected individuals is largely unknown. One animal model which has proven useful in the study of chondrodysplasia is the Alaskan Malamute, since chondrodysplasia in these animals bears clinical and radiographic resemblance to human metaphysical chondrodysplasias. Previous biochemical studies in our laboratory of the growth plates in these animals have failed to reveal any primary defect in collagen metabolism, but ultrastructural studies of the territorial and interterritorial matrix comprising the affected growth plates reveal an abnormal organization and distribution of proteoglycan matrix granules. The objective of this proposal will be to determine whether there is a defect in proteoglycan components of the dwarf growth plates by using the techniques of biochemistry and morphology. Specifically, we will determine whether there are qualitative and quantitative differences in the physical-chemical nature of the proteoglycans present in dwarf growth plate by examining the size class of the entire proteoglycan monomers, the glycosaminoglycan constituents, and the overall size of the protein core which constitutes the back bone of the monomer. This will be accomplished by using conventional methods of extracting the proteoglycans with 4M quanidine HCl in the presence of protease inhibitors, purifying the proteoglycans by density gradient centrifugation and sizing the molecules by molecular sieve chromatography, high pressure liquid chromatography, SDS polyacrylamide electrophoresis and electron microscopy. In addition, studies will be conducted to determine whether the state of proteoglycan aggregation is altered in the dwarf growth plate. The presence of native PG aggregate, the presence and character of link proteins, and the ability of proteoglycan monomers from dwarf and normal growth plates to form aggregate (either "link" free or "link" stable) will be assessed using density gradient ultracentrifugation, molecular sieve components of the aggregate (i.e. link protein) as well as the monomer to immuno-electron microscopically examine the dwarf growth plate for evidence of altered PG aggregation. Finally, we will establish organ cultures for normal and dwarf growth plates to determine if there is in alteration in the synthesis and/or degradation of proteoglycans in the swarf utilizing incorporation of 35S-sulfate, 3H glucosamine, 3H serine into proteoglycans. Autoradiography will also be done.