L1503R is a member of group I mutation and has dominant-negative effect on secretion of full-length VWF multimers: an analysis of two patients with type 2A von Willebrand disease
L1503R is a member of group I mutation and has dominant-negative effect on secretion of full-length VWF multimers: an analysis of two patients with type 2A von Willebrand disease
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DOI：
10.1111/j.1365-2516.2008.01703.x
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发表时间：
2008-05-01
期刊：
影响因子：
3.9
通讯作者：
Kojima, T.
中科院分区：
文献类型：
--
作者：
Kashiwagi, T.;Matsushita, T.;Kojima, T.
2A型血管性血友病（VWD）的特征是血管性血友病因子（VWF）的血小板依赖性功能下降;这反过来又与缺乏高分子量多聚体有关。对两个不相关的2A型VWD患者的VWF基因的序列分析显示，在核苷酸4508处存在相同的、新的、杂合的T -> G颠换，导致VWF A2结构域中L1503 R的取代。这种取代，这是没有发现在60个无关的正常人，被引入到一个全长的VWF cDNA，随后在293 T细胞中表达。只有微量的突变VWF蛋白被分泌，但大部分被保留在293 T细胞中。野生型和突变型质粒的共转染实验表明疾病发展的显性负性机制;随着更多的突变型DNA被转染，培养基中的VWF分泌受损，而更多的VWF储存在细胞裂解物中。L1503 R诱导的结构变化的分子动力学模拟表明，所有原子的均方根偏差的平均值从野生型或另一个突变L1503 Q，已被报道为一个组II突变，这是易受ADAMTS 13蛋白水解的那些移动。L1503 R的蛋白质不稳定性可能是其细胞内滞留的原因，并且可能含有更多突变VWF亚基的较大VWF多聚体可能被错误加工并保留在细胞内。
Type 2A von Willebrand disease (VWD) is characterized by decreased platelet-dependent function of von Willebrand factor (VWF); this in turn is associated with an absence of high-molecular-weight multimers. Sequence analysis of the VWF gene from two unrelated type 2A VWD patients showed an identical, novel, heterozygous T -> G transversion at nucleotide 4508, resulting in the substitution of L1503R in the VWF A2 domain. This substitution, which was not found in 60 unrelated normal individuals, was introduced into a full-length VWF cDNA and subsequently expressed in 293T cells. Only trace amount of the mutant VWF protein was secreted but most of the same was retained in 293T cells. Co-transfection experiment of both wild-type and mutant plasmids indicated the dominant-negative mechanism of disease development; as more of mutant DNA was transfected, VWF secretion was impaired in the media, whereas more of VWF was stored in the cell lysates. Molecular dynamic simulations of structural changes induced by L1503R indicated that the mean value of all-atom root-mean-squared-deviation was shifted from those with wild type or another mutation L1503Q that has been reported to be a group II mutation, which is susceptible to ADAMTS13 proteolysis. Protein instability of L1503R may be responsible for its intracellular retention and perhaps the larger VWF multimers, containing more mutant VWF subunits, are likely to be mal-processed and retained within the cell.