The Mechanism of Fibril Formation of a Non-inhibitory Serpin Ovalbumin Revealed by the Identification of Amyloidogenic Core Regions*
The Mechanism of Fibril Formation of a Non-inhibitory Serpin Ovalbumin Revealed by the Identification of Amyloidogenic Core Regions*
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DOI：
10.1074/jbc.m110.176396
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发表时间：
2010-12
期刊：
影响因子：
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通讯作者：
N. Tanaka;Y. Morimoto;Yurika Noguchi;T. Tada;Tomonori Waku;S. Kunugi;T. Morii;Yin-Fai Lee;T. Konno;N. Takahashi
中科院分区：
文献类型：
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作者：
N. Tanaka;Y. Morimoto;Yurika Noguchi;T. Tada;Tomonori Waku;S. Kunugi;T. Morii;Yin-Fai Lee;T. Konno;N. Takahashi
卵清蛋白(OVA)是丝氨酸超家族中的一个非抑制性成员，在热诱导变性后形成纤维状聚集体。最近的研究表明，OVA原纤维的生成机制与淀粉样原纤维的形成机制相似，这与其他蛇类的聚合机制不同。在这项研究中，我们通过使用合成肽片段鉴定淀粉样蛋白核心区、定点突变和有限蛋白分解，为卵清蛋白原纤维的形成机制提供了新的见解。在蛋白质的N-末端螺旋区域，OVA在Cys73和Cys120之间具有单一的二硫键。二硫化物还原OVA的热处理导致形成长而直的纤维，这与由具有完整二硫化物的OVA形成的半弹性纤维不同。计算机预测表明，N-末端区域的螺旋B(HB)、链3A和链4-5B是高度容易β聚集的区域。与这些区域相对应的合成肽形成淀粉样纤维的事实证实了这些预测。OVA的定点突变表明，Hb中的V41a取代干扰了原纤维的形成。Hb的可溶性多肽片段与二硫键完整的全长OVA共同孵育，持续地促进长而直的纤维的形成。此外，受热诱导的卵清蛋白原纤维的N-末端螺旋区被保护，使其不发生有限的蛋白降解。这些结果表明，热诱导卵清蛋白原纤维的形成是通过一种机制发生的，该机制涉及蛋白质的N-末端螺旋区域向β-链的转变，从而形成顺序的分子间连接。
Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys73 and Cys120 in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4–5B are highly β-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to β-strands, thereby forming sequential intermolecular linkages.