Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.
Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.
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鉴定形成低密度脂蛋白受体相关蛋白结合表位的凝血因子 VIII A2 结构域残基。
DOI：
10.1021/bi0520380
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发表时间：
2006
期刊：
影响因子：
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通讯作者：
Saenko,EvgueniL
中科院分区：
文献类型：
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作者：
Sarafanov,AndreyG;Makogonenko,EvgenyM;Pechik,IgorV;Radtke,Klaus-Peter;Khrenov,AlexeyV;Ananyeva,NatalyaM;Strickland,DudleyK;Saenko,EvgueniL
血液循环中凝血因子VIII （fVIII）水平的调节涉及肝受体低密度脂蛋白受体相关蛋白（LRP）。fVIII中两个主要的LRP结合位点之一位于A2结构域（A2）内，可能暴露于fVIII与血管性血友病因子的复合物内，并通过LRP参与fVIII的调控。这项工作旨在鉴定形成lrp结合位点的A2残基，先前显示涉及残基484−509。分离A2进行丙氨酸扫描诱变，随后在杆状病毒系统中表达一组突变体。在竞争和表面等离子体共振实验中，发现A2突变体K466A、R471A、R484A、S488A、R489A、R490A、H497A和K499A对LRP的亲和力降低了2 - 4倍。这与细胞培养中lrp介导的突变体内化程度降低1.3 - 1.5倍相关。将这些突变成对组合导致累积效应，即LRP的亲和力降低7 - 13倍，LRP介导的细胞内化程度降低1.6 - 2.2倍。我们得出结论，上述残基在LRP A2结合表位的形成中起关键作用。小鼠实验显示，与fVIII相比，A2在循环中的半衰期缩短了约4.5倍。A2突变体R471A/R484A或与受体相关蛋白（LRP的经典配体）共注射的A2的半衰期分别比A2延长了~ 1.9倍和~ 3.5倍。这进一步证实了突变残基对A2与LRP相互作用的重要性，并表明存在LRP依赖机制，作为活化fVIII从循环中解离的产物去除A2。
Regulation of the coagulation factor VIII (fVIII) level in circulation involves a hepatic receptor low-density lipoprotein receptor-related protein (LRP). One of two major LRP binding sites in fVIII is located within the A2 domain (A2), likely exposed within the fVIII complex with von Willebrand factor and contributing to regulation of fVIII via LRP. This work aimed to identify A2 residues forming its LRP-binding site, previously shown to involve residues 484−509. Isolated A2 was subjected to alanine-scanning mutagenesis followed by expression of a set of mutants in a baculovirus system. In competition and surface plasmon resonance assays, affinities of A2 mutants K466A, R471A, R484A, S488A, R489A, R490A, H497A, and K499A for LRP were found to be decreased by 2−4-fold. This correlated with 1.3−1.5-fold decreases in the degree of LRP-mediated internalization of the mutants in cell culture. Combining these mutations into pairs led to cumulative effects, i.e., 7−13-fold decrease in affinity for LRP and 1.6−2.2-fold decrease in the degree of LRP-mediated internalization in cell culture. We conclude that the residues mentioned above play a key role in formation of the A2 binding epitope for LRP. Experiments in mice revealed an ∼4.5 times shorter half-life for A2 in the circulation in comparison with that of fVIII. The half-lives of A2 mutant R471A/R484A or A2 co-injected with receptor-associated protein, a classical ligand of LRP, were prolonged by ∼1.9 and ∼3.5 times, respectively, compared to that of A2. This further confirms the importance of the mutated residues for interaction of A2 with LRP and suggests the existence of an LRP-dependent mechanism for removing A2 as a product of dissociation of activated fVIII from the circulation.