Structure-function analysis of the glucose-6-phosphate transporter deficient in glycogen storage disease type Ib
Structure-function analysis of the glucose-6-phosphate transporter deficient in glycogen storage disease type Ib
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DOI：
10.1093/hmg/11.25.3199
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发表时间：
2002-12-01
影响因子：
3.5
通讯作者：
Chou, JY
中科院分区：
文献类型：
--
作者：
Chen, LY;Pan, CJ;Chou, JY
糖原累积病Ib型（GSD-Ib）是由葡萄糖-6-磷酸转运蛋白（G6 PT）缺陷引起的，G6 PT是一种10个跨膜结构域的内质网蛋白。迄今为止，在GSD-Ib患者中已发现69个G6 PT突变，包括28个错义突变和2个密码子缺失突变。我们以前的特点是15错义和一个密码子缺失突变使用pSVL为基础的表达测定。该试验缺乏灵敏度，限制了导致功能丧失的突变和残留活性较低的突变之间的区分。我们现在报告一种改进的G6 PT检测，基于腺病毒载体介导的表达系统及其在GSD-Ib患者中发现的所有30个密码子突变的功能表征中的用途。20个自然发生的突变完全消除微粒体G6 P摄取活性，而其他10个突变，包括5个先前表征的突变，部分抑制转运蛋白。这些信息将极大地促进基因型-表型相关性。我们还报告了G6 PT的结构-功能分析。除了先前报道的3个不稳定突变之外，我们现在表明G50 R、C176 R、V235 del、G339 C和G339 D突变也损害G6 PT稳定性。G6 PT的氨基末端结构域的突变分析表明，它是最佳的G6 P摄取活性所必需的。最后，我们发现，野生型和突变G6 PT的降解是抑制一个有效的蛋白酶体抑制剂，lactacystin，表明G6 PT是蛋白酶体介导的降解底物。
Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), a 10 transmembrane domain endoplasmic reticulum protein. To date, 69 G6PT mutations, including 28 missenses and 2 codon deletions, have been identified in GSD-Ib patients. We previously characterized 15 of the missense and one codon deletion mutations using a pSVL-based expression assay. A lack of sensitivity in this assay limited the discrimination between mutations that lead to loss of function and mutations that leave a low residual activity. We now report an improved G6PT assay, based on an adenoviral vector-mediated expression system and its use in the functional characterization of all 30 codon mutations found in GSD-Ib patients. Twenty of the naturally occurring mutations completely abolish microsomal G6P uptake activity while the other 10 mutations, including 5 previously characterized ones, partially inactivate the transporter. This information should greatly facilitate genotype-phenotype correlation. We also report a structure-function analysis of G6PT. In addition to the 3 destabilizing mutations reported previously, we now show that the G50R, C176R, V235del, G339C and G339D mutations also compromise the G6PT stability. Mutation analysis of the amino-terminal domain of G6PT shows that it is required for optimal G6P uptake activity. Finally, we show that degradation of both wild-type and mutant G6PT is inhibited by a potent proteasome inhibitor, lactacystin, demonstrating that G6PT is a substrate for proteasome-mediated degradation.