Functional expression and cellular distribution of diastrophic dysplasia sulfate transporter (DTDST) gene mutations in HEK cells
Functional expression and cellular distribution of diastrophic dysplasia sulfate transporter (DTDST) gene mutations in HEK cells
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DOI：
10.1093/hmg/ddh242
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发表时间：
2004-10-01
影响因子：
3.5
通讯作者：
Karniski, LP
中科院分区：
文献类型：
--
作者：
Karniski, LP
软骨细胞的硫酸盐转运缺陷会导致软骨细胞外基质蛋白多糖的硫酸盐化不足。遗传性发育不良硫酸盐转运蛋白(DTDST)基因突变与四种不同严重程度的软骨发育不良有关。为了确定DTDST致病突变的特征，我们在哺乳动物HEK-293细胞中表达了DTDST介导的硫酸盐转运，并确定野生型蛋白是糖基化的，并定位于细胞膜。A715V、C653S、Q454P和R279W四个突变刺激硫酸盐转运的速度仅为野生型DTDST的39%-62%。这四种突变均在细胞膜上表达，但与野生型DTDST相比，表达的蛋白质量有所减少。Q454P突变体是独一无二的，因为它在HEK细胞中没有正确的糖基化。转导DeltaV340和G678V突变的HEK细胞与对照HEK细胞的硫酸盐转运活性无明显差异。此外，G678V突变不沿细胞膜表达，而是被困在细胞质中。当将每个DTDST突变的硫酸盐转运能力与其已被鉴定的软骨发育不良症进行比较时，我们发现严重软骨发育不良1B表型的个体在两个DTDST等位基因上都存在零突变。无论是零突变还是部分功能突变的杂合子都会导致ATL2或DTD，而较温和的隐性多发性骨痂发育不良表型则是部分功能突变的纯合子。与以前对非洲爪哇卵母细胞的研究相比，我们发现哺乳动物细胞中表型的严重程度与残留转运功能的水平之间存在很强的相关性。
Defects in sulfate transport in chondrocytes lead to undersulfation of the cartilage extracellular matrix proteoglycans. Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene have been linked to four chondrodysplasias of varying severity. To characterize disease-causing mutations of DTDST, we expressed DTDST-mediated sulfate transport in mammalian HEK-293 cells and determined that the wild-type protein is glycosylated and localized to the cell plasma membrane. Four mutations, A715V, C653S, Q454P and R279W, stimulated sulfate transport at rates only 39-62% of wild-type DTDST. These four mutations were expressed on the plasma membrane of the cell, but the amount of expressed protein was reduced when compared with wild-type DTDST. The Q454P mutant is unique in that it is not properly glycosylated in HEK cells. There was no difference in sulfate transport activity between cells transfected with either the DeltaV340 or the G678V mutations and control HEK cells. Furthermore, the G678V mutation is not expressed along the plasma membrane, but is trapped within the cytoplasm. When comparing the sulfate transport capacity of each DTDST mutation with the chondrodysplasia in which it has been identified, we find that individuals with severe achondrogenesis 1B phenotype have null mutations on both DTDST alleles. Heterozygotes for both a null mutation and a partial-function mutation result in either atelosteogenesis type 2 or DTD, whereas the milder, recessive multiple epiphyseal dysplasia phenotype is homozygous for partial-function mutations. In contrast to previous studies in Xenopus laevis oocytes, we find a strong correlation between the severity of the phenotype and the level of residual transport function in mammalian cells.