Effects of Lipid Composition on the Entry of Cell-Penetrating Peptide Oligoarginine into Single Vesicles

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• 作者：Sabrina Sharmin ; Md. Zahidul Islam ; Mohammad Abu Sayem Karal ; Sayed Ul Alam Shibly ; Hideo Dohra ; Masahito Yamazaki
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：August 2, 2016
• 年：2016
• 卷：55
• 期：30
• 页码：4154-4165
• 全文大小：572K
• 年卷期：0
• ISSN：1520-4995

文摘

The cell-penetrating peptide R₉, an oligoarginine comprising nine arginines, has been used to transport biological cargos into cells. However, the mechanisms underlying its translocation across membranes remain unclear. In this report, we investigated the entry of carboxyfluorescein (CF)-labeled R₉ (CF-R₉) into single giant unilamellar vesicles (GUVs) of various lipid compositions and the CF-R₉-induced leakage of a fluorescent probe, Alexa Fluor 647 hydrazide (AF647), using a method developed recently by us. First, we investigated the interaction of CF-R₉ with dioleoylphosphatidylglycerol (DOPG)/dioleoylphosphatidylcholine (DOPC) GUVs containing AF647 and small DOPG/DOPC vesicles. The fluorescence intensity of the GUV membrane due to CF-R₉ (i.e., the rim intensity) increased with time to a steady-state value, and then the fluorescence intensity of the membranes of the small vesicles in the GUV lumen increased without leakage of AF647. This result indicates that CF-R₉ entered the GUV lumen from the outside by translocating across the lipid membrane without forming pores through which AF647 could leak. The fraction of entry of CF-R₉ at 6 min in the absence of pore formation, P_entry (6 min), increased with an increase in CF-R₉ concentration, but the CF-R₉ concentration in the lumen was low. We obtained similar results for dilauroyl-PG (DLPG)/ditridecanoyl-PC (DTPC) (2/8) GUVs. The values of P_entry (6 min) of CF-R₉ for DLPG/DTPC (2/8) GUVs were larger than those obtained with DOPG/DOPC (2/8) GUVs at the same CF-R₉ concentrations. In contrast, a high concentration of CF-R₉ induced pores in DLPG/DTPC (4/6) GUVs through which CF-R₉ entered the GUV lumen, so the CF-R₉ concentration in the lumen was higher. However, CF-R₉ could not enter DOPG/DOPC/cholesterol (2/6/4) GUVs. Analysis of the rim intensity showed that CF-R₉ was located only in the outer monolayer of the DOPG/DOPC/cholesterol (2/6/4) GUVs. On the basis of analyses of these results, we discuss the elementary processes by which CF-R₉ enters GUVs of various lipid compositions.