Dimerization and multimerization defects of von Willebrand factor due to mutated cysteine residues
Dimerization and multimerization defects of von Willebrand factor due to mutated cysteine residues
复制标题
DOI：
10.1111/j.1538-7836.2003.00435.x
复制
发表时间：
2004-02-01
影响因子：
10.4
通讯作者：
Eikenboom, JCJ
中科院分区：
文献类型：
--
作者：
Tjernberg, P;Vos, HL;Eikenboom, JCJ
在1型和3型von Willebrand病患者中，已鉴定出导致von Willebrand因子（VWF）D3结构域（多聚化区域）和羧基末端（二聚化区域）半胱氨酸残基丢失的错义突变。我们已经研究了这些结构变化如何导致定量VWF缺陷，以及它们如何干扰二聚化和多聚化过程。在293 T细胞中的瞬时转染测定中研究了人重组VWF的多聚化区域（C1130 F，C1149 R）和二聚化区域（C2671 Y，C2739 Y，C2754 W）中的突变的影响。所有突变导致培养基中VWF分泌减少和细胞内滞留。氨基末端突变体C1130 F和C1149 R由于缺乏高分子量（HMW）多聚体而显示受损的多聚化，在与野生型（wt）VWF的共转染实验中，多聚体模式与杂合子1型患者的模式一致。羧基末端突变体C2739Y和C2754W显示VWF分泌强烈减少至几乎不存在，与3型VWD一致。C2739 Y和C2754 W的多聚体模式的特征在于不存在HMW多聚体、过量的单体和插入的奇数多聚体条带，表明二聚化缺陷。羧基末端突变体C2671Y是不同的，具有轻度减少的分泌，中间细胞内滞留和正常的多聚化模式。我们的结论是，根据定量VWF缺乏症的表型，所有半胱氨酸突变体显示受损的分泌，虽然在体外的VWF的减少似乎低于患者，这表明额外的，可能提高清除率，在体内的机制。
In patients classified with type 1 and type 3 von Willebrand disease missense mutations resulting in the loss of cysteine residues in the D3-domain (multimerization area) and in the carboxy-terminus (dimerization area) of the von Willebrand factor (VWF) have been identified. We have investigated how these structural changes result in a quantitative VWF deficiency and how they interfere with the dimerization and multimerization processes. The effect of mutations in the multimerization area (C1130F, C1149R) and in the dimerization area (C2671Y, C2739Y, C2754W) of human recombinant VWF were investigated in transient transfection assays in 293T cells. All mutations resulted in reduced secretion of VWF in the medium and in intracellular retention. The amino-terminal mutants C1130F and C1149R showed impaired multimerization by lacking high molecular weight (HMW) multimers, in cotransfection experiments with wild-type (wt) VWF, the multimeric pattern was consistent with the pattern in the heterozygous type 1 patients. The carboxy-terminal mutants C2739Y and C2754W showed strongly reduced to nearly absent secretion of VWF, consistent with type 3 VWD. The multimeric pattern of C2739Y and C2754W is characterized by the absence of HMW multimers, an excess of monomers and intervening odd-numbered multimeric bands, indicating a dimerization defect. The carboxy-terminal mutant C2671Y is different, with mildly reduced secretion, intermediate intracellular retention and a normal multimerization pattern. We conclude that, in accordance with a phenotype of quantitative VWF deficiency, all cysteine mutants show impaired secretion, although the decrease of VWF in vitro appears lower than in the patients, suggesting additional, possibly heightened clearance, mechanisms in vivo.