Mastroianni Jennifer R, Ouellette André J
Department of Pathology and Laboratory Medicine, University of California, Irvine, California 92697-4800.
Department of Pathology and Laboratory Medicine, University of California, Irvine, California 92697-4800; Department of Microbiology and Molecular Genetics, University of California, Irvine, California 92697-4800.
J Biol Chem. 2009 Oct 9;284(41):27848-27856. doi: 10.1074/jbc.M109.050773. Epub 2009 Aug 17.
Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric alpha-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric alpha-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative alpha-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as alpha-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1-4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell alpha-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.
潘氏细胞是一种分泌性上皮细胞谱系，从小肠隐窝底部释放富含宿主防御肽和蛋白质的致密核心颗粒。肠道α-防御素在小鼠中被称为隐窝素（Crps），在潘氏细胞分泌物中含量极高，并且具有内在的抗蛋白水解能力。因此，我们测试了这样一个假设，即源自潘氏细胞的肠道α-防御素在小鼠大肠腔中以功能状态持续存在。为了验证这一想法，从小鼠远端结肠腔中纯化出的假定Crps进行了生化特性分析，并在体外测定了其杀菌肽活性。这些肽在酸性尿素聚丙烯酰胺凝胶电泳（acid-urea-PAGE）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）中与隐窝素对照肽迁移率相同，从而鉴定为假定的Crps。基质辅助激光解吸电离飞行时间质谱实验表明，假定的α-防御素的分子量与六种最丰富的已知Crps以及每种Crps的N端截短形式相匹配，并且这些肽含有六个半胱氨酸残基，与α-防御素的特征一致。N端测序明确揭示了肽的N端对应于Crps 1-4和6的全长、（去亮氨酸）截短和（去亮氨酸-精氨酸）截短的N端。来自小鼠大肠腔的Crps在低微摩尔范围内具有杀菌作用。因此，分泌到小肠腔中的潘氏细胞α-防御素在整个肠道中以完整且有功能的形式持续存在，这表明这些肽可能在远离其在小肠隐窝上游分泌点的结肠腔中介导肠道固有免疫。
