Activation of Paneth cell alpha-defensins in mouse small intestine.
Activation of Paneth cell alpha-defensins in mouse small intestine.
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发表时间：
2002
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通讯作者：
T. Ayabe;D. Satchell;P. Pesendorfer;H. Tanabe;Carole L Wilson;S. Hagen;A. Ouellette
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作者：
T. Ayabe;D. Satchell;P. Pesendorfer;H. Tanabe;Carole L Wilson;S. Hagen;A. Ouellette
小肠隐窝中的潘氏细胞分泌杀微生物的α-防御素（称为Cryptdin），作为肠道先天免疫的成分。穴状蛋白的杀菌活性需要基质金属蛋白酶-7（MMP-7;基质溶解素）对前体的蛋白水解活化（Wilson，C. L.，Ouellette，A. J.，Saturday，D. P.，Ayabe，T.，洛佩斯-博阿多美国，斯特拉特曼，J.L.，Hultgren，S. J.，马特里西安湖M.，Parks，W. C.（1999）Science 286，113-117）。在这里，我们报告在小鼠潘氏细胞的cryptdin前体的细胞内加工。对纯化的天然原密码子的MMP-7酶的肽测序鉴定了在Ser（43）和瓦尔（44）之间的前区中以及在Ser（58）和Leu（59）之间的密码子肽N末端的保守切割位点。免疫染色共定位的前体prosegments和成熟的cryptdin肽潘氏细胞颗粒，提供证据的分泌。广泛的MMP-7依赖性procryptdin处理发生在潘氏细胞，如肠隐窝蛋白和蛋白质的蛋白质印迹分析所示，从颗粒富集的亚细胞组分。在体外抗菌肽测定中加入可溶性前片段抑制了反式cryptdin-3和-4的杀菌活性，表明前片段在分泌前可能具有细胞保护作用。在无菌小鼠小肠和皮下生长的胎鼠小肠无菌植入物中，激活的cryptdin水平正常。因此，MMP-7对原密码素加工的起始不需要直接暴露于细菌，并且无菌潘氏细胞的基础MMP-7含量足以加工和激活α-防御素前体。MMP-7-依赖性的procryptdin在体内的激活提供了小鼠潘氏细胞的功能性肽顶端分泌到小肠腔。
Paneth cells in small intestine crypts secrete microbicidal alpha-defensins, termed cryptdins, as components of enteric innate immunity. The bactericidal activity of cryptdins requires proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7; matrilysin) (Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113-117). Here, we report on the intracellular processing of cryptdin proforms in mouse Paneth cells. Peptide sequencing of MMP-7 digests of purified natural procryptdins identified conserved cleavage sites in the proregion between Ser(43) and Val(44) as well as at the cryptdin peptide N terminus between Ser(58) and Leu(59). Immunostaining co-localized precursor prosegments and mature cryptdin peptides to Paneth cell granules, providing evidence of their secretion. Extensive MMP-7-dependent procryptdin processing occurs in Paneth cells, as shown by Western blot analyses of intestinal crypt proteins and proteins from granule-enriched subcellular fractions. The addition of soluble prosegments to in vitro antimicrobial peptide assays inhibited the bactericidal activities of cryptdin-3 and -4 in trans, suggesting possible cytoprotective effects by prosegments prior to secretion. Levels of activated cryptdins were normal in small bowel of germ-free mice and in sterile implants of fetal mouse small intestine grown subcutaneously. Thus, the initiation of procryptdin processing by MMP-7 does not require direct bacterial exposure, and the basal MMP-7 content of germ-free Paneth cells is sufficient to process and activate alpha-defensin precursors. MMP-7-dependent procryptdin activation in vivo provides mouse Paneth cells with functional peptides for apical secretion into the small intestine lumen.