The conserved salt bridge in human α-defensin 5 is required for its precursor processing and proteolytic stability
The conserved salt bridge in human α-defensin 5 is required for its precursor processing and proteolytic stability
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DOI：
10.1074/jbc.m801851200
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发表时间：
2008-08-01
影响因子：
4.8
通讯作者：
Lu, Wuyuan
中科院分区：
文献类型：
--
作者：
Rajabi, Mohsen;de Leeuw, Erik;Lu, Wuyuan
哺乳动物α -防御素主要在白细胞和上皮细胞中表达，在对微生物感染的先天和适应性免疫反应中发挥重要作用。六个不变的半胱氨酸残基形成了三个必不可少的二硫键和一个非典型β隆起所需的Gly残基，在所有已知的哺乳动物α -防御素的其他不同序列中完全保守。此外，一对带相反电荷的残基（Arg/Glu）在分子的突出环上形成盐桥，是高度保守的。为了研究保守的Arg(6)-Glu（14）盐桥在人α -防御素5 （HD5）中的结构和功能作用，我们化学制备了HD5及其前体proHD5以及它们相应的盐桥破坏稳定类似物E14Q-HD5和E57Q-proHD5。Glu-to-Gln突变虽然显著降低了HD5的氧化折叠效率，但对proHD5的折叠没有影响。牛胰蛋白酶在体外有效且正确地处理了proHD5，但自发降解了E57Q-proHD5。值得注意的是，HD5对胰蛋白酶有抗性，而E14Q-HD5对胰蛋白酶高度敏感。此外，胰蛋白酶对E14Q-HD5的降解是由环区Arg(13)-Gln（14）肽键的断裂引发的，这是一个灾难性的蛋白水解事件，直接导致整个防御蛋白分子被快速消化。E14Q突变没有改变HD5对金黄色葡萄球菌的杀菌活性，但显著增强了对大肠杆菌的杀灭能力。相比之下，在测试浓度下，proHD5和E57Q-proHD5对两种菌株基本无活性。我们的研究结果证实，保守盐桥在HD5中的主要功能是确保proHD5的正确加工和随后在体内成熟α -防御素的稳定。
Mammalian alpha-defensins, expressed primarily in leukocytes and epithelia, play important roles in innate and adaptive immune responses to microbial infection. Six invariant cysteine residues forming three indispensable disulfide bonds and one Gly residue required structurally for an atypical beta-bulge are totally conserved in the otherwise diverse sequences of all known mammalian alpha-defensins. In addition, a pair of oppositely charged residues (Arg/Glu), forming a salt bridge across a protruding loop in the molecule, is highly conserved. To investigate the structural and functional roles of the conserved Arg(6)-Glu(14) salt bridge in human alpha-defensin 5 (HD5), we chemically prepared HD5 and its precursor proHD5 as well as their corresponding salt bridge-destabilizing analogs E14Q-HD5 and E57Q-proHD5. The Glu-to-Gln mutation, whereas significantly reducing the oxidative folding efficiency of HD5, had no effect on the folding of proHD5. Bovine trypsin productively and correctly processed proHD5 in vitro but spontaneously degraded E57Q-proHD5. Significantly, HD5 was resistant to trypsin treatment, whereas E14Q-HD5 was highly susceptible. Further, degradation of E14Q-HD5 by trypsin was initiated by the cleavage of the Arg(13)-Gln(14) peptide bond in the loop region, a catastrophic proteolytic event resulting directly in quick digestion of the whole defensin molecule. The E14Q mutation did not alter the bactericidal activity of HD5 against Staphylococcus aureus but substantially enhanced the killing of Escherichia coli. By contrast, proHD5 and E57Q-proHD5 were largely inactive against both strains at the concentrations tested. Our results confirm that the primary function of the conserved salt bridge in HD5 is to ensure correct processing of proHD5 and subsequent stabilization of mature alpha-defensin in vivo.