Biosynthesis and antimicrobial evaluation of backbone-cyclized α-defensins.
Biosynthesis and antimicrobial evaluation of backbone-cyclized α-defensins.
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DOI：
10.1021/bi201430f
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发表时间：
2011-12-06
期刊：
影响因子：
2.9
通讯作者：
Camarero, Julio A.
中科院分区：
文献类型：
--
作者：
Garcia, Angie E.;Tai, Kenneth P.;Puttamadappa, Shadakshara S.;Shekhtman, Alexander;Ouellette, Andre J.;Camarero, Julio A.
防御素是抗菌肽，对哺乳动物的先天免疫防御很重要。在细菌抗原的刺激下，肠α-防御素被分泌到肠腔中，在那里它们具有有效的杀菌活性。 Cryptdin-4 (Crp4) 是一种在小鼠小肠潘氏细胞中表达的 α-防御素，也是已知的隐蛋白亚型中杀菌力最强的。 Crp4 的结构由三链反平行 β 折叠组成，但第四个和第五个半胱氨酸残基之间缺少三个氨基酸，这使得它们与其他 α-防御素不同。该结构还表明，折叠结构中的 α-氨基和 C 端羧基非常接近 (d ≈ 3 Å)。我们在这里展示了使用修饰的蛋白质剪接单元或内含肽来生物合成骨架环化的 Crp4。我们的数据表明，环化Crp4可以通过使用这种方法在体外和体内生物合成，尽管在细胞内产生时表达产量明显较低。所得的环状防御素保留了天然的α-防御素折叠，并且与天然Crp4相比，对多种革兰氏阳性和革兰氏阴性细菌表现出相同或更好的杀菌活性。对于 Crp4 的天然变体或环化变体，没有观察到针对人红细胞的可检测的溶血活性。此外，当与人血清一起孵育时，两种形式的Crp4也表现出高度的降解稳定性。总而言之，这些结果表明主链环化防御素在开发新型基于肽的抗菌化合物中具有潜力。
Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. Upon stimulation by bacterial antigens, enteric α-defensins are secreted into the intestinal lumen where they have potent microbicidal activities. Cryptdin-4 (Crp4) is an α-defensin expressed in Paneth cells of the mouse small intestine and the most bactericidal of the known cryptdin isoforms. The structure of Crp4 consists of a triple-stranded antiparallel β-sheet, but lacks three amino acids between the fourth and fifth cysteine residues, making them distinct from other α-defensins. The structure also reveals that the α-amino and C-terminal carboxylic groups are in proximity (d ≈ 3 Å) in the folded structure. We present here the biosynthesis of backbone-cyclized Crp4 using a modified protein splicing unit or intein. Our data show that cyclized Crp4 can be biosynthesized by using this approach both in vitro and in vivo, although the expression yield was significantly lower when produced inside the cell. The resulting cyclic defensins retained the native α-defensin fold and showed equivalent or better microbicidal activities against several Gram-positive and Gram-negative bacteria when compared to native Crp4. No detectable hemolytic activity against human red blood cells was observed for either the native or cyclized variants of Crp4. Moreover, both forms of Crp4 also showed high stability to degradation when incubated with human serum. Altogether, these results indicate the potential for backbone cyclized defensins in the development of novel peptide-based antimicrobial compounds.
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影响因子：
4.4
作者：
Foureau, David M.;Mielcarz, Daniel W.;Buzoni-Gatel, Dominique
通讯作者：
Buzoni-Gatel, Dominique
影响因子：
3.4
作者：
Arnett, Eusondia;Lehrer, Robert I.;Seveau, Stephanie
通讯作者：
Seveau, Stephanie
影响因子：
4.9
作者：
Camarero, JA;Muir, TW
通讯作者：
Muir, TW
DOI：
10.1073/pnas.96.24.13703
发表时间：
1999-11-23
影响因子：
11.1
作者：
Bessette, PH;Åslund, F;Georgiou, G
通讯作者：
Georgiou, G
DOI：
10.1073/pnas.0504613102
发表时间：
2005-09-27
影响因子：
11.1
作者：
Clark, RJ;Fischer, H;Craik, DJ
通讯作者：
Craik, DJ