Department of Blood Biochemistry and Molecular Biology, Beijing Institute of Transfusion Medicine, Beijing, China.
J Gene Med. 2013 May;15(5):205-14. doi: 10.1002/jgm.2711.
Previous studies have suggested that reducing the positive charge of melittin could increase endosomal release activity and improve branched polyethylenimine (BPEI)-mediated transfection. AR-23 is a melittin-related peptide from Rana tagoi, which shows 81% sequence identity with melittin but has less positively-charged residues than melittin. The present study aimed to investigate the mechanistic and functional aspects of the interaction of AR-23 with mammalian cells and thus improve BPEI-mediated gene transfection.
AR23 and two AR-23 analogs (AR-20 without positively-charged residues and AR-26 with the same positively-charged residues as melittin) were analyzed. Circular dichroism (CD) spectrometry was used to analyze the secondary structures of the peptides. Peptide-induced depolarization of cell membrane, the membrane-lytic activity of the peptides, and their potency with respect to enhancing the cellular uptake of calcein were evaluated. The physicochemical characters of complexes were measured and the effect of the peptides on BPEI-mediated transfection was determined.
The CD spectra results indicated that a positive charge in AR-23 played a crucial role in maintaining the α-helical conformation, whereas an extra positive charge could not increase α-helical formation. AR-23 displayed a similar depolarization ability to melittin. However, AR-23 showed a lower membrane lytic activity under physiological conditions and a higher lytic activity at endosomal pH than melittin and AR-26, which possess more positive charges. Compared to melittin and AR-26, AR-23, with a higher endosomal escaping activity, resulted in a higher enhancement of BPEI-mediated gene transfection, as well as the maintainance of a lower cytotoxicity.
We suggest that AR-23 may be considered as a potential enhancer for improving the transfection efficiency of cationic polymers.
先前的研究表明，降低蜂毒素的正电荷可以增加内体释放活性，从而改善支链聚乙烯亚胺（BPEI）介导的转染。AR-23 是一种来自中国林蛙的蜂毒素相关肽，与蜂毒素具有 81%的序列同一性，但带正电荷的残基比蜂毒素少。本研究旨在探讨 AR-23 与哺乳动物细胞相互作用的机制和功能，从而提高 BPEI 介导的基因转染。
分析了 AR23 及其两种 AR-23 类似物（不含正电荷残基的 AR-20 和与蜂毒素具有相同正电荷残基的 AR-26）。圆二色性（CD）光谱法用于分析肽的二级结构。评估肽诱导细胞膜去极化、肽的膜裂解活性以及它们增强 calcein 细胞摄取的效力。测量了复合物的物理化学性质，并确定了肽对 BPEI 介导转染的影响。
CD 光谱结果表明，AR-23 中的正电荷在维持 α-螺旋构象中起着关键作用，而额外的正电荷不能增加 α-螺旋形成。AR-23 显示出与蜂毒素相似的去极化能力。然而，在生理条件下，AR-23 的膜裂解活性较低，在内涵体 pH 下的裂解活性高于带更多正电荷的蜂毒素和 AR-26。与蜂毒素和 AR-26 相比，具有更高内涵体逃逸活性的 AR-23 导致 BPEI 介导的基因转染增强，同时保持较低的细胞毒性。
我们认为 AR-23 可以被认为是一种提高阳离子聚合物转染效率的潜在增强剂。
