Characterization of two pathogenic mutations in cystathionine beta-synthase: Different intracellular locations for wild-type and mutant proteins
Characterization of two pathogenic mutations in cystathionine beta-synthase: Different intracellular locations for wild-type and mutant proteins
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DOI：
10.1016/j.gene.2013.08.021
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发表时间：
2013-11-15
期刊：
影响因子：
3.5
通讯作者：
De Lucca, M.
中科院分区：
文献类型：
--
作者：
Casique, L.;Kabil, O.;De Lucca, M.
胱硫醚β-合酶（CBS）是一种吡哆醛5 '-磷酸（PLP）依赖性酶，催化同型半胱氨酸与丝氨酸缩合生成胱硫醚。同型胱氨酸尿症是一种常染色体隐性遗传疾病，通常由CBS活性缺乏引起。在此，我们描述了一种新的CBS突变（c.260 C>A（p.T87 N））和一种先前报道的变体（c.700 G>A（p.D234 N）），发现于委内瑞拉同型胱氨酸尿症患者，一种对维生素B-6无反应，一种对维生素6有反应。两种突变蛋白在原核和真核细胞中体外表达，发现与野生型CBS相比，在HEK-293细胞中的可溶性表达较低（19%T87N和23%D234N）。获得的突变蛋白的残余活性为3.5%T87N和43%D234N。凝胶排阻色谱显示T87 N突变体有聚集的趋势，而D234 N突变体的分布与野生型酶相似。使用免疫荧光显微镜，一个意想不到的差异，在细胞内的定位观察野生型和突变体蛋白质。虽然T87 N突变体表现出点状外观，但野生型蛋白质均匀分布在细胞内。有趣的是，D234 N蛋白显示两种分布。这项研究表明，致病性CBS突变产生不稳定的蛋白质，不能（T87 N）或部分不能（D234 N）组装成一个功能酶，这意味着这些突变可能是负责同型胱氨酸尿症表型。(C)2013爱思唯尔有限公司版权所有。
Cystathionine beta-synthase (CBS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the condensation of homocysteine with serine to generate cystathionine. Homocystinuria is an autosomal recessive disorder commonly caused by a deficiency of CBS activity. Here, we characterized a novel CBS mutation (c.260C>A (p.T87N)) and a previously reported variant (c.700G>A (p.D234N)) found in Venezuelan homocystinuric patients, one nonresponsive and one responsive to vitamin B-6. Both mutant proteins were expressed in vitro in prokaryotic and eukaryotic cells, finding lower soluble expression in HEK-293 cells (19% T87N and 23% D234N) compared to wild-type CBS. Residual activities obtained for the mutant proteins were 3.5% T87N and 43% D234N. Gel exclusion chromatography demonstrated a tendency of the T87N mutant to aggregate while the distribution of the D234N mutant was similar to wild-type enzyme. Using immunofluorescence microscopy, an unexpected difference in intracellular localization was observed between the wild-type and mutant proteins. While the T87N mutant exhibited a punctate appearance, the wild-type protein was homogeneously distributed inside the cell. Interestingly, the D234N protein showed both distributions. This study demonstrates that the pathogenic CBS mutations generate unstable proteins that are unable (T87N) or partially unable (D234N) to assemble into a functional enzyme, implying that these mutations might be responsible for the homocystinuria phenotype. (C) 2013 Elsevier B.V. All rights reserved.