Abstract：
Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. We previously demonstrated that two representative bacterial polycationic isopeptides, ε-poly-l-α-lysine consisting of 25-35 l-α-lysine residues (ε-PαL25-35) and ε-poly-l-β-lysine consisting of l-β-lysine residues (ε-PβL4-13), were internalized into mammalian cells by both energy-independent direct penetration and energy-dependent endocytosis/macropinocytosis, and then diffused throughout the cytosol. In this study, we investigated the cell-penetrating activity of an ε-PαL short-chain derivative consisting of 5-14 l-α-lysine residues (ε-PαL5-14) to gain insight into the relationship between the isopeptide-chain length and the manner of cellular internalization. We prepared a conjugate of ε-PαL5-14 and a fluorescent dye (FAM) by click chemistry, and incubated the resulting polymer, ε-PαL5-14-FAM, with HeLa cells. Unlike ε-PαL25-35-FAM, ε-PαL5-14-FAM was internalized into cells only by energy-dependent endocytosis/macropinocytosis. Furthermore, a high concentration (>50 μM) was required for the internalization events. ε-PαL5-14 has a chain length almost equal to that of the membrane permeable ε-PβL4-13, which can enter cells at low concentrations. Considering that the basicity of the β-amino group is higher than that of α-amino acid at physiological pH, ε-PβL is expected to have a greater cell-penetrating capacity than ε-PαL, provided their isopeptide-chain lengths are similar, suggesting that a more extended chain derivative of ε-PβL would be more advantageous for cellular internalization of cargo proteins than ε-PαL25-35.
摘要：
细菌产生聚阳离子均聚（氨基酸），其特征在于异肽主链。我们先前证明了两种代表性的细菌聚阳离子异肽，由25-35个l-α-赖氨酸残基（ε-PαL25-35）和由l-β-赖氨酸残基（ε-PβL4-13）组成的ε-聚-l-β-赖氨酸，通过能量非依赖性直接穿透和能量依赖性内吞作用/巨细胞作用内化到哺乳动物细胞中，然后扩散到整个细胞质中。在这项研究中,我们研究了由5-14l-α-赖氨酸残基（ε-PαL5-14）组成的ε-PαL短链衍生物的细胞穿透活性，以深入了解异肽链长度与细胞内化方式之间的关系。我们通过点击化学制备了ε-PαL5-14和荧光染料（FAM）的缀合物，并培养得到的聚合物，ε-PαL5-14-FAM,HeLa细胞与ε-PαL25-35-FAM不同，ε-PαL5-14-FAM仅通过能量依赖性内吞作用/巨噬细胞作用内化到细胞中。此外，内化事件需要高浓度(>50μM)。ε-PαL5-14的链长几乎等于膜可渗透的ε-PβL4-13的链长，其可以低浓度进入细胞。考虑到生理pH下β-氨基的碱性高于α-氨基酸的碱性,预计ε-PβL比ε-PαL具有更大的细胞穿透能力，前提是它们的异肽链长度相似，这表明ε-PβL的更延伸的链衍生物将比ε-PαL25-35更有利于货物蛋白的细胞内化。
