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
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
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DOI：
10.1016/s0945-053x(98)90088-9
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发表时间：
1998-10-01
期刊：
影响因子：
6.9
通讯作者：
Superti-Furga, A
中科院分区：
文献类型：
--
作者：
Rossi, A;Kaitila, I;Superti-Furga, A
遗传性发育不良硫酸盐转运蛋白(DTDST)基因突变与一系列软骨发育不良相关，包括遗传性发育不良(DTD)、2型成骨不良(AO2)和致死性软骨生成1B型(ACG1B)。突变的性质与临床表型之间存在相关性，但我们对这种疾病的病理生理学的了解还远远不完整，这涉及到软骨蛋白多糖的硫酸盐化缺陷。为了评价蛋白多糖在体内的硫酸盐化程度，我们从12例硫酸盐转运体软骨发育不良患者的软骨中提取了硫酸软骨素蛋白多糖，并用软骨素酶ABC消化后用高效液相色谱法分析了它们的双糖组成。非硫酸双糖的含量在患者样本中升高(对照组，5.5%+/-2.8(n=10)；患者，11%至77%)，最高含量出现在ACG1B患者，表明硫酸软骨素蛋白多糖在体内存在于软骨中，并与临床严重程度相关。为了进一步研究从基因型到表型转化的生化机制，我们用[S-35]硫酸盐和[H-3]氨基葡萄糖双重标记的方法，研究了DTD、AO2和ACG1B患者和对照组的成纤维细胞培养。根据S-35/H-3在蛋白多糖中的比率估计，所有患者的细胞外硫酸盐掺入都减少了，其中ACG1B细胞的参入率最低。然而，对硫酸软骨素蛋白多糖的双糖分析表明，这些蛋白多糖通常是硫酸盐化的或只有中等程度的硫酸盐化；只有在培养基中加入人工糖胺糖链引发剂β-D-木糖苷后，才能观察到明显的硫酸盐化不足。这些结果表明，虽然细胞外硫酸盐的利用受到损害，但成纤维细胞可以通过氧化含硫化合物(如半胱氨酸)来补充细胞内的硫酸盐池，从而部分挽救基础条件下的PG硫酸盐化。当β-D-syloside刺激GAG合成速率时，这一救援途径变得不足。这些发现可能解释了为什么DTDST突变的表型后果仅限于软骨，软骨是一种具有高GAG合成率和缺乏血管供应的组织，并暗示旨在恢复细胞内硫酸盐池的药物治疗可能改善DTD和相关疾病的PG硫酸盐化。
Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene have been associated with a family of chondrodysplasias that includes diastrophic dysplasia (DTD), atelosteogenesis type 2 (AO2) and the lethal condition achondrogenesis type 1B (ACG1B). There is a correlation between the nature of the mutations and the clinical phenotype, but our understanding of the pathophysiology of the disorder, which involves defective sulfation of cartilage proteoglycans, is far from complete. To evaluate the degree of proteoglycan undersulfation in vivo, we have extracted chondroitin sulfate proteoglycans from cartilage of twelve patients with sulfate transporter chondrodysplasias and analyzed their disaccharide composition by HPLC after digestion with chondroitinase ABC. The amount of non-sulfated disaccharide was elevated in patients' samples (controls, 5.5% +/- 2.8 (n = 10); patients, 11% to 77%), the highest amount being present in ACG1B patients, indicating that undersulfation of chondroitin sulfate proteoglycans occurs in cartilage in vivo and is correlated with the clinical severity. To investigate further the biochemical mechanisms responsible for the translation of genotype to phenotype, we have studied fibroblast cultures of patients with DTD, AO2 and ACG1B, and controls, by double-labelling with [S-35]sulfate and [H-3]glucosamine. The incorporation of extracellular sulfate, estimated by the S-35/H-3 ratio in proteoglycans, was reduced in all patients' cells, with ACG1B cells showing the lowest values. However, disaccharide analysis of chondroitin sulfate proteoglycans showed that these were normally sulfated or only moderately undersulfated; marked undersulfation was observed only after addition of the artificial glycosaminoglycan-chain initiator, beta-D-xyloside, to the culture medium. These results suggest that, while utilization of extracellular sulfate is impaired, fibroblasts can replenish their intracellular sulfate pool by oxidizing sulfur-containing compounds (such as cysteine) and thus partially rescue PG sulfation under basal conditions. This rescue pathway becomes insufficient when GAG synthesis rate is stimulated by beta-D-syloside. These findings may explain why phenotypic consequences of DTDST mutations are restricted to cartilage, a tissue with high GAG synthesis rate and poor vascular supply, and imply that pharmacological therapy aimed at restoring the intracellular sulfate pool might improve PG sulfation in DTD and related disorders.