In Vivo Analysis of the Role of O-Glycosylations of Von Willebrand Factor
In Vivo Analysis of the Role of O-Glycosylations of Von Willebrand Factor
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DOI：
10.1371/journal.pone.0037508
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发表时间：
2012-05-17
期刊：
影响因子：
3.7
通讯作者：
Denis, Cecile V.
中科院分区：
文献类型：
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作者：
Badirou, Idinath;Kurdi, Mohamad;Denis, Cecile V.
本项目旨在研究o -糖基化在血管性血友病因子（VWF）生命周期中的作用。总共产生了14种不同的小鼠Vwf cdna在一个或多个o -糖基化位点突变：9个单个突变体，2个双链，2个簇和1个突变体，所有9个小鼠糖基化位点突变（Del-O-Gly）。我们通过水动力注射在VWF缺陷小鼠中表达每个突变的cDNA。用花生凝集素（PNA）免疫吸附试验验证o -糖基化状态。水动力注射后肝细胞表达的野生型（WT） VWF与PNA结合的亲和力略高于内皮源性VWF。相比之下，Del-O-Gly VWF突变体不结合PNA，表明去除了o链聚糖。所有的突变体都显示出正常的多聚体模式。两个突变体Del-OGly和T1255A/T1256A的表达量比WT - VWF诱导的低50%，其体内半衰期明显缩短。在检测各突变体纠正vwf缺陷小鼠出血时间的能力时，我们发现S1486A、T1255A、T1256A和双链T1255A/T1256A不能纠正vwf缺陷小鼠出血时间。总之，我们已经证明了o -糖基化对于正常的VWF多聚和生物合成是必不可少的。此外，一些o -糖基化位点，特别是T1255和T1256残基，参与VWF血浆水平的维持，对正常止血至关重要。体外灌注实验表明，S1486残基似乎对血小板结合很重要。
The objective of this project was to study the function of O-glycosylations in von Willebrand factor (VWF) life cycle. In total, 14 different murine Vwf cDNAs mutated on one or several O-glycosylations sites were generated: 9 individual mutants, 2 doublets, 2 clusters and 1 mutant with all 9 murine glycosylation sites mutated (Del-O-Gly). We expressed each mutated cDNA in VWF deficient-mice by hydrodynamic injection. An immunosorbent assay with Peanut Agglutinin (PNA) was used to verify the O-glycosylation status. Wild-type (WT) VWF expressed by hepatocytes after hydrodynamic injection was able to bind PNA with slightly higher affinity than endothelial-derived VWF. In contrast, the Del-O-Gly VWF mutant did not bind PNA, demonstrating removal of O-linked glycans. All mutants displayed a normal multimeric pattern. Two mutants, Del-OGly and T1255A/T1256A, led to expression levels 50% lower than those induced by WT VWF and their half-life in vivo was significantly reduced. When testing the capacity of each mutant to correct the bleeding time of VWF-deficient mice, we found that S1486A, T1255A, T1256A and the doublet T1255A/T1256A were unable to do so. In conclusion we have shown that O-glycosylations are dispensable for normal VWF multimerization and biosynthesis. It also appears that some O-glycosylation sites, particularly the T1255 and T1256 residues, are involved in the maintenance of VWF plasma levels and are essential for normal haemostasis. As for the S1486 residue, it seems to be important for platelet binding as demonstrated in vitro using perfusion experiments.