Undersulfated chondroitin sulfate in cartilage from a miniature poodle with spondyloepiphyseal dysplasia.

In order to determine if either the proteoglycans or collagen in the cartilagenous epiphyses of a Miniature Poodle with spondyloepiphyseal dysplasia were abnormal, the cartilage was dissociatively extracted in 4 M guanidine HCl in the presence of protease inhibitors and subjected to isopycnic cesium chloride dissociative density gradient ultracentrifugation. Dissociative extraction solubilized 97% of the uronic acid and 88% of the protein. Uronic acid distributed anomalously in the density gradient in that about 1/3 was recovered in each of the D1 (1.58 g/ml), D2 (1.49 g/ml) and D3 (1.44 g/ml) fractions. Proteoglycans in the D1, D2 and D3 fractions also eluted from Sepharose CL-2B columns in a manner indicative of monomers of a smaller apparent hydrodynamic size than those from normal canine growth plate or articular cartilage. D1, D2 and D3 monomers subjected to the sodium borohydride reaction followed by chromatography on a Sepharose CL-6B column yielded glycosaminoglycan chain molecular weights of 10,200 (D1), 7600 (D2) and 6200 (D3). High pressure liquid chromatography on a Whatman Partisil 10PAC column of the chondroitinase AC II digests of D1, D2 and D3 fractions revealed that 60% of the D1, 81% of the D2 and 88% of the D3 unsaturated disaccharides eluted in the delta DiOS-delta DiHA position. Subsequent HPLC of the unsaturated disaccharides on the Hypersil APS column resulted in the recovery of 97% of the nonsulfated unsaturated disaccharides in the delta DiOS position. Associative extraction in 0.5 M guanidine followed by associative gradient ultracentrifugation resulted in the recovery of 27% of the uronic acid in the aA1 and 47% in the aA2 fractions. Two dimensional SDS gel electrophoresis of the CNBr peptides of the collagen isolated by pepsin digestion and 0.9 M NaCl precipitation revealed type II collagen. This study has demonstrated that spondyloepiphyseal dysplasia in a Miniature Poodle is characterized by cartilage containing undersulfated chondroitin sulfate proteoglycan.