Undersulfation of Proteoglycans Synthesized by Chondrocytes from a Patient with Achondrogenesis Type 1B Homozygous for an L483P Substitution in the Diastrophic Dysplasia Sulfate Transporter

Abstract

). To ascertain the consequences of the sulfate transport defect on proteoglycan synthesis, we studied the structure and sulfation of proteoglycans in cartilage tissue and in fibroblast and chondrocyte cultures from a fetus with achondrogenesis 1B. Proteoglycans extracted from epiphyseal cartilage and separated on agarose gels migrated more slowly than controls and stained poorly with alcian blue. The patient's cultured cells showed reduced incorporation of [ S]sulfate relative to [3 H]glucosamine, impaired uptake of sulfate, and higher resistance to chromate toxicity compared to control cells. Epiphyseal chondrocytes cultured in alginate beads synthesized proteoglycans of normal molecular size as judged by gel filtration chromatography, but undersulfated as judged by ion exchange chromatography and by the amount of nonsulfated disaccharide. High performance liquid chromatography analysis of chondroitinase-digested proteoglycans showed that sulfated disaccharides were present, although in reduced amounts, indicating that at least in vitro, other sources of sulfate can partially compensate for sulfate deficiency. A t1475c transition causing a L483P substitution in the eleventh transmembrane domain of the sulfate/chloride antiporter was present on both alleles in the patient who was the product of a consanguineous marriage. The results indicate that the defect of sulfate transport is expressed in both chondrocytes and fibroblasts and results in the synthesis of proteoglycans bearing glycosaminoglycan chains which are poorly sulfated but of normal length.Achondrogenesis type 1B (ACG-1B) 1 (1) is an autosomal recessive, lethal chondrodysplasia with severe underdevelopment of the skeleton, extreme micromelia, and death before or immediately after birth because of thoracic hypoplasia (1-8). Both radiological and histological features differentiate ACG-1B from achondrogenesis type 1A and achondrogenesis type 2 (3). In ACG-1B, cartilage matrix contains abnormally coarse collagen fibers and stains poorly with cationic dyes which have affinity for sulfated proteoglycans (toluidine blue or alcian blue) (4,8). Evidence of undersulfation of proteoglycans (PGs) in cartilage on the basis of histological findings and in fibroblast cultures, together with the demonstration of insufficient formation of activated sulfate metabolites, was obtained recently in one patient with ACG-1B (8). Further studies have revealed that ACG-1B is caused not by a defect in the metabolic activation of sulfate, as originally concluded (8), but by a sulfate uptake defect caused by mutations in the sulfate transporter gene (9) originally isolated as the locus responsible for the non-lethal disorder, diastrophic dysplasia (10). The pathogenesis of the severe developmental defect of the skeleton seen in ACG-1B is believed to involve undersulfation of cartilage PGs, as suggested by both the staining properties of cartilage and its markedly reduced sulfate content (9), but direct demonstration of undersulfation of individual cartilage PGs is lacking. We h...

Maintenance of chondroitin sulfation balance by chondroitin-4-sulfotransferase 1 is required for chondrocyte development and growth factor signaling during cartilage morphogenesis

“…In branchial arches (white arrows) and AER of limb buds (black arrowheads) in C4st1 mutant (gt/gt) embryos, a C4st1 exon I-specific signal (E) is present, but an exon II/III-specific signal (F) is absent. (Hronowski and Anastassiades, 1988;Ippolito et al, 1983), and it has also been reported that undersulfation of proteoglycans leads to reduced Alcian Blue staining (Rossi et al, 1996); thus, the reduction in Alcian Blue staining probably reflects a reduction in GAG content and C4S levels in the cartilage growth plate. Closer inspection of mutant skeletons revealed reduced bone length, but increased width in long bones as well as the iliac bone ( Fig.…”

Anion translocation through an Slc26 transporter mediates lumen expansion during tubulogenesis

“…Once significant lumen has formed, Ci-Slc26aα may then shuttle sulfate into the cytoplasm, providing the substrate for the sulfation of luminal glycoaminoglycans that will facilitate further lumen expansion. It has been reported that the proteoglycans synthesized by chondrocytes from a patient with achondrogenesis type 1B homozygous for an L483P substitution in SLC26A2 were severly undersulfated (26). Ci-Slc26aα L485P, which is unable to transport sulfate, fails to support lumen expansion, validating the importance of the structural element at and around this residue in maintaining the protein function in vivo.…”

“…4B). Ci-Slc26aα L485P, which corresponds to the L483P mutation in SLC26A2 from patients with achondrogenesis type 1B (26), also failed to rescue the lumen formation defect (Fig. 4D).…”

Functional Analysis of Diastrophic Dysplasia Sulfate Transporter

“…Taken together, these results suggested that chondrocytes evidently possess the DTDST sulfate/chloride antiport system. deficient Culture-Undersulfation of proteoglycans is a major consequence of impairment of the DTDST transport system in chondrocytes (7). Thus, we studied the growth factor response of chondrocytes associated with undersulfated proteoglycans.…”