PROTEOGLYCAN SULFATION IN CARTILAGE AND CELL-CULTURES FROM PATIENTS WITH SULFATE TRANSPORTER CHONDRODYSPLASIAS - RELATIONSHIP TO CLINICAL SEVERITY AND INDICATIONS ON THE ROLE OF INTRACELLULAR SULFATE PRODUCTION

Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gen e have been associated with a family of chondrodysplasias that include s diastrophic dysplasia (DTD), atelosteogenesis type 2 (AO2) and the l ethal condition achondrogenesis type 1B (ACG1B). There is a correlatio n between the nature of the mutations and the clinical phenotype, but our understanding of the pathophysiology of the disorder, which involv es defective sulfation of cartilage proteoglycans, is far from complet e. To evaluate the degree of proteoglycan undersulfation in vivo, we h ave extracted chondroitin sulfate proteoglycans from cartilage of twel ve patients with sulfate transporter chondrodysplasias and analyzed th eir disaccharide composition by HPLC after digestion with chondroitina se ABC. The amount of non-sulfated disaccharide was elevated in patien ts' samples (controls, 5.5% +/- 2.8 (n = 10); patients, 11% to 77%), t he highest amount being present in ACG1B patients, indicating that und ersulfation of chondroitin sulfate proteoglycans occurs in cartilage i n vivo and is correlated with the clinical severity. To investigate fu rther the biochemical mechanisms responsible for the translation of ge notype to phenotype, we have studied fibroblast cultures of patients w ith DTD, AO2 and ACG1B, and controls, by double-labelling with [S-35]s ulfate and [H-3]glucosamine. The incorporation of extracellular sulfat e, estimated by the S-35/H-3 ratio in proteoglycans, was reduced in al l patients' cells, with ACG1B cells showing the lowest values. However , disaccharide analysis of chondroitin sulfate proteoglycans showed th at these were normally sulfated or only moderately undersulfated; mark ed undersulfation was observed only after addition of the artificial g lycosaminoglycan-chain initiator, beta-D-xyloside, to the culture medi um. These results suggest that, while utilization of extracellular sul fate is impaired, fibroblasts can replenish their intracellular sulfat e pool by oxidizing sulfur-containing compounds (such as cysteine) and thus partially rescue PG sulfation under basal conditions. This rescu e pathway becomes insufficient when GAG synthesis rate is stimulated b y beta-D-syloside. These findings may explain why phenotypic consequen ces of DTDST mutations are restricted to cartilage, a tissue with high GAG synthesis rate and poor vascular supply, and imply that pharmacol ogical therapy aimed at restoring the intracellular sulfate pool might improve PG sulfation in DTD and related disorders.