Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 50/70, Basel, Switzerland.
Biochemistry. 2011 May 31;50(21):4650-64. doi: 10.1021/bi1019429. Epub 2011 May 3.
Many cell-penetrating peptides (CPPs) bind to glycosaminoglycans (GAG) located on the extracellular side of biological tissues. CPP binding to the cell surface is intimately associated with clustering of surface molecules and is usually followed by uptake into the cell interior. We have investigated the uptake mechanism by comparing CPPs which bind, but cannot induce, GAG clustering with those which do induce GAG clustering. We have synthesized the tryptophan-labeled CPP nona-l-arginine (WR(9)) and its monodispersely PEGylated derivate (PEG(27)-WR(9)) and have compared them with respect to glycan binding, glycan clustering, and their uptake into living cells. Both CPPs bind to the GAG heparin with high affinity (K(D) ∼ 100 nM), but the PEGylation prevents the GAG clustering. Thus, it is possible to uncouple and analyze the contributions of GAG binding and GAG clustering to the biological CPP uptake. The uptake of PEG-WR(9) into CH-K1 cells is confined to intracellular vesicles, where colocalization with transferrin attests to an endocytic uptake. Transfection experiments with plasmid DNA for GFP revealed poor GFP expression, suggesting that endocytic uptake of PEG-WR(9) is compromised by insufficient release from endocytic vesicles. In contrast, WR(9) shows two uptake routes. At low concentration (<5 μM), WR(9) uptake occurs mainly through endocytosis. At higher concentration, WR(9) uptake is greatly enhanced, showing a diffuse spreading over the entire cytoplasm and nucleus-a phenomenon termed "transduction". Transduction of WR(9) leads to a higher GFP expression as compared to PEG-WR(9) endocytosis but also damages the plasma membrane as evidenced by SYTOX Green staining. The results suggest that GAG binding without and with GAG clustering induce two different pathways of CPP uptake.
许多细胞穿透肽（CPPs）与位于生物组织细胞外表面的糖胺聚糖（GAG）结合。CPP 与细胞表面的结合与表面分子的聚集密切相关，通常随后被摄取到细胞内部。我们通过比较结合但不能诱导 GAG 聚集的 CPP 与诱导 GAG 聚集的 CPP 来研究摄取机制。我们合成了色氨酸标记的 CPP 壬基精氨酸（WR（9））及其单分散聚乙二醇化衍生物（PEG（27）-WR（9）），并比较了它们在糖结合、糖聚集及其进入活细胞方面的情况。两种 CPP 都与 GAG 肝素具有高亲和力（K（D）∼100 nM），但 PEG 化阻止了 GAG 聚集。因此，有可能将 GAG 结合和 GAG 聚集对生物 CPP 摄取的贡献解耦并进行分析。PEG-WR（9）进入 CH-K1 细胞的摄取仅限于细胞内囊泡，其中与转铁蛋白的共定位证明了内吞作用的摄取。用 GFP 的质粒 DNA 进行转染实验显示 GFP 表达较差，表明 PEG-WR（9）的内吞作用因从内吞体中释放不足而受到损害。相比之下，WR（9）显示出两种摄取途径。在低浓度（<5 μM）下，WR（9）的摄取主要通过内吞作用发生。在较高浓度下，WR（9）的摄取大大增强，在整个细胞质和核中呈现弥漫性扩散——这种现象称为“转导”。与 PEG-WR（9）的内吞作用相比，WR（9）的转导导致 GFP 表达更高，但也如 SYTOX Green 染色所示损害了质膜。结果表明，没有 GAG 聚集和有 GAG 聚集的 GAG 结合诱导了 CPP 摄取的两种不同途径。
