Functional analysis of the α-defensin disulfide array in mouse cryptdin-4
Functional analysis of the α-defensin disulfide array in mouse cryptdin-4
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DOI：
10.1074/jbc.m406154200
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发表时间：
2004-10-15
影响因子：
4.8
通讯作者：
Ouellette, AJ
中科院分区：
文献类型：
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作者：
Maemoto, A;Qu, XQ;Ouellette, AJ
α-防御素抗微生物肽家族由独特的三二硫键阵列定义。为了测试这种不变的结构特征是否决定α-防御素杀菌活性，小鼠cryptdin-4（Crp 4）三级结构被成对的定点Ala取代Cys而破坏。在一系列Crp 4二硫化物变体中，其半胱氨酸连接性使用NMR光谱学和质谱法确认，诱变不会诱导功能丧失。相反，几种Crp 4二硫键变体的体外杀菌活性等于或大于天然Crp 4。小鼠潘氏细胞α-防御素需要通过基质金属蛋白酶-7（MMP-7）对前体进行蛋白水解活化，从而促使分析天然和突变型Crp 4和proCrp 4分子对MMP-7降解的相对敏感性。虽然天然Crp 4和proCrp 4的α-防御素部分完全抵抗蛋白水解，但所有二硫键变体都被MMP-7广泛降解。Crp 4杀菌活性被MMP-7裂解消除。因此，而不是决定α-防御素杀菌活性，Crp 4二硫键排列赋予基本的保护，免受这种关键的活化蛋白酶的降解。
The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell alpha-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and proCrp4 molecules to degradation by MMP-7. Although native Crp4 and the alpha-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining alpha-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.