Translocation of analogues of the antimicrobial peptides magainin and buforin across human cell membranes
Translocation of analogues of the antimicrobial peptides magainin and buforin across human cell membranes
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DOI：
10.1074/jbc.m208762200
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发表时间：
2003-01-10
影响因子：
4.8
通讯作者：
Matsuzaki, K
中科院分区：
文献类型：
--
作者：
Takeshima, K;Chikushi, A;Matsuzaki, K
阳离子抗菌肽在天然免疫中发挥重要作用。与对肽-细菌相互作用的广泛研究相比，对肽-人细胞相互作用知之甚少。使用人宫颈癌HeLa和成纤维细胞TM 12细胞，我们研究了两种代表性抗菌肽magainin 2和buforin 2的荧光类似物的细胞摄取，并与代表性富含Arg的细胞穿透达特-（47-57）肽（YGRKKRRQRRR）进行比较。转运的剂量、时间、温度和能量依赖性表明，三种肽通过不同的机制穿过细胞膜。爪蟾抗菌肽在几十分钟的时间尺度内内化。摄取的合作浓度依赖性表明，肽形成一个孔作为中间体类似于模型膜中的观察。此外，易位与细胞毒性相结合，这对肿瘤HeLa细胞更大。与此相反，buforin肽易位在10分钟内的温度不依赖性，浓度依赖性较低的被动机制，而没有显示任何显着的细胞毒性，在最高浓度的调查（100 μ M）。达特肽的摄取与肽浓度成比例，并且在ATP耗尽时失去浓度依赖性。该肽在较高浓度下表现出中等的细胞毒性。即使在120分钟后，时间过程也没有显示饱和。buforin肽，共价连接到28-kDa的绿色荧光蛋白，也进入细胞，这表明肽作为大分子递送到细胞中的载体的效力。然而，该机制似乎与亲本肽的机制不同。
Cationic antimicrobial peptides play important roles in innate immunity. Compared with extensive studies on peptide-bacteria interactions, little is known about peptide-human cell interactions. Using human cervical carcinoma HeLa and fibroblastic TM12 cells, we investigated the cellular uptake of fluorescent analogues of the two representative antimicrobial peptides magainin 2 and buforin 2 in comparison with the representative Arg-rich cell-penetrating Tat-(47-57) peptide (YGRKKRRQRRR). The dose, time, temperature, and energy dependence of translocation suggested that the three peptides cross cell membranes through different mechanisms. The magainin peptide was internalized within a time scale of tens of minutes. The cooperative concentration dependence of uptake suggested that the peptide forms a pore as an intermediate similar to the observations in model membranes. Furthermore, the translocation was coupled with cytotoxicity, which was larger for tumor HeLa cells. In contrast, the buforin peptide translocated within 10 min by a temperature-independent, less concentration-dependent passive mechanism without showing any significant cytotoxicity at the highest concentration investigated (100 muM). The uptake of the Tat peptide was proportional to the peptide concentration, and the concentration dependence was lost upon ATP depletion. The peptide exhibited a moderate cytotoxicity at higher concentrations. The time course did not show saturation even after 120 min. The buforin peptide, covalently attached to the 28-kDa green fluorescent protein, also entered cells, suggesting a potency of the peptide as a vector for macromolecular delivery into cells. However, the mechanism appeared to be different from that of the parent peptide.