IDENTIFICATION OF A CLEAVAGE SITE DIRECTING THE IMMUNOCHEMICAL DETECTION OF MOLECULAR ABNORMALITIES IN TYPE-IIA VONWILLEBRAND-FACTOR
IDENTIFICATION OF A CLEAVAGE SITE DIRECTING THE IMMUNOCHEMICAL DETECTION OF MOLECULAR ABNORMALITIES IN TYPE-IIA VONWILLEBRAND-FACTOR
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DOI：
10.1073/pnas.87.16.6306
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发表时间：
1990-08-01
影响因子：
11.1
通讯作者：
RUGGERI, ZM
中科院分区：
文献类型：
--
作者：
DENT, JA;BERKOWITZ, SD;RUGGERI, ZM
冯维勒布兰德因子亚基的蛋白水解切割对于分子的加工和/或功能可能很重要，并且在冯维勒布兰德病的某些亚型中发生改变。它导致从 225 kDa 亚基生成两个表观分子量分别为 140 kDa 和 176 kDa 的主要片段。我们现已获得化学证据来定位残基 Tyr-842 和 Met-843 之间的蛋白酶敏感键，该位点似乎反映了钙依赖性中性蛋白酶（钙蛋白酶）的特异性。针对四种合成肽产生了抗体，这些合成肽代表紧接在切割位点之前或之后或包括切割位点的序列。一种抗体（针对从 Ala-837 到 Asp-851 的片段）仅与完整亚基反应，其表位包括切割位点。所有其他片段均与 140-kDa 或 176-kDa 片段发生特异性反应，证明它们源自单次切割。在来自 5 名 IIA 型冯维勒布兰德病患者中的 4 名的纯化冯维勒布兰德因子样本中，抗肽抗体显示出与 140-kDa 或 176-kDa 片段的反应性显着降低，表明在切割位点周围存在聚集的不同分子异常。因此，在大多数 IIA 型患者中，一种常见的发病机制可能会导致较大多聚体的消失，这是由于结构变化可能会暴露出对特定蛋白酶作用的敏感键。这些研究证明了使用针对相关结构域的抗肽抗体来进行正常分子和突变分子的免疫化学区分。
Proteolytic cleavage of the von Willebrand factor subunit may be important for processing and/or function of the molecule and is altered in certain subtypes of von Willebrand disease. It results in the generation of two main fragments with apparent molecular masses of 140 kDa and 176 kDa from the 225-kDa subunit. We have now obtained chemical evidence to locate the protease-sensitive bond between residues Tyr-842 and Met-843, a site that appears to reflect the specificity of calcium-dependent neutral proteases (calpains). Antibodies were raised against four synthetic peptides that represented sequences immediately preceding or following or including the cleavage site. One antibody (against the fragment from Ala-837 through Asp-851) reacted only with the intact subunit, and its epitope included the cleavage site. All others reacted specifically with either the 140-kDa or the 176-kDa fragment, demonstrating their origin from a single cleavage. In samples of purified von Willebrand factor from four of five patients with type IIA von Willebrand disease, the anti-peptide antibodies showed markedly decreased reactivity with either the 140-kDa or the 176-kDa fragment, suggesting the existence of distinct molecular abnormalities clustered around the cleavage site. Thus, in the majority of type IIA patients, a common pathogenetic mechanism may lead to the disappearance of the larger multimers as a consequence of structural changes that may expose a sensitive bond to the action of specific proteases. These studies demonstrate the use of anti-peptide antibodies directed at a relevant structural domain for the immunochemical differentiation of normal and mutant molecules.