Relationships between membrane binding, affinity and cell internalization efficacy of a cell-penetrating peptide: penetratin as a case study.
Relationships between membrane binding, affinity and cell internalization efficacy of a cell-penetrating peptide: penetratin as a case study.
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DOI：
10.1371/journal.pone.0024096
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Sagan S
中科院分区：
文献类型：
--
作者：
Alves ID;Bechara C;Walrant A;Zaltsman Y;Jiao CY;Sagan S
Penetratin是一种带正电荷的细胞穿透肽（CPP），能够结合带负电荷的膜组分，如糖胺聚糖和阴离子脂质。穿透素与这些细胞表面组分的这种主要相互作用是否意味着肽将进一步内化尚不清楚。使用质谱法，在三种不同的细胞系中定量洗涤后剩余的内化和膜结合的穿透素的量：野生型（WT）、糖胺聚糖-（GAGneg）和唾液酸缺陷型（SAneg）细胞。此外，渗透素的相互作用的亲和性和动力学的膜模型组成的纯脂质和膜片段从所提到的细胞系进行了研究，以及这种相互作用的热力学使用等离子体共振和量热法。在三种细胞系中，1 μM的渗透素内化具有相同的功效，但在SAneg>WT> GAG neg中，10 μM的渗透素内化更好。穿透素与这些细胞相互作用所释放的热量遵循内化效率的排序。Penetratin对WT细胞的亲和力为10 nM，对SAneg和GAGneg细胞以及磷脂模型膜的亲和力为µM。细胞清洗后剩余的膜结合穿透蛋白在WT和GAGneg细胞中相似，这表明这些结合位点依赖于膜磷脂。穿透素与碳水化合物的相互作用更表面和可逆，而与磷脂的相互作用更强，可能是因为肽可以插入脂肪酸链之间。这些结果表明，穿透素在细胞表面的积累和高亲和力结合并不反映肽的内化功效。总之，这些数据进一步支持易位（膜磷脂相互作用）作为低小分子浓度下穿膜蛋白使用的内化途径，而胞吞作用在较高浓度下被激活，并且需要肽在GAG和GAG簇上的积累。
Penetratin is a positively charged cell-penetrating peptide (CPP) that has the ability to bind negatively charged membrane components, such as glycosaminoglycans and anionic lipids. Whether this primary interaction of penetratin with these cell surface components implies that the peptide will be further internalized is not clear. Using mass spectrometry, the amount of internalized and membrane bound penetratin remaining after washings, were quantified in three different cell lines: wild type (WT), glycosaminoglycans- (GAGneg) and sialic acid-deficient (SAneg) cells. Additionally, the affinity and kinetics of the interaction of penetratin to membrane models composed of pure lipids and membrane fragments from the referred cell lines was investigated, as well as the thermodynamics of such interactions using plasmon resonance and calorimetry. Penetratin internalized with the same efficacy in the three cell lines at 1 µM, but was better internalized at 10 µM in SAneg>WT>GAGneg. The heat released by the interaction of penetratin with these cells followed the ranking order of internalization efficiency. Penetratin had an affinity of 10 nM for WT cells and µM for SAneg and GAGneg cells and model membrane of phospholipids. The remaining membrane-bound penetratin after cells washings was similar in WT and GAGneg cells, which suggested that these binding sites relied on membrane phospholipids. The interaction of penetratin with carbohydrates was more superficial and reversible while it was stronger with phospholipids, likely because the peptide can intercalate between the fatty acid chains. These results show that accumulation and high-affinity binding of penetratin at the cell-surface do not reflect the internalization efficacy of the peptide. Altogether, these data further support translocation (membrane phospholipids interaction) as being the internalization pathway used by penetratin at low micromolecular concentration, while endocytosis is activated at higher concentration and requires accumulation of the peptide on GAG and GAG clustering.
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影响因子：
3.4
作者：
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通讯作者：
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DOI：
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