Amand Helene L, Rydberg Hanna A, Fornander Louise H, Lincoln Per, Nordén Bengt, Esbjörner Elin K
Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
Biochim Biophys Acta. 2012 Nov;1818(11):2669-78. doi: 10.1016/j.bbamem.2012.06.006. Epub 2012 Jun 15.
Cell surface proteoglycans (PGs) appear to promote uptake of arginine-rich cell-penetrating peptides (CPPs), but their exact functions are unclear. To address if there is specificity in the interactions of arginines and PGs leading to improved internalization, we used flow cytometry to examine uptake in relation to cell surface binding for penetratin and two arginine/lysine substituted variants (PenArg and PenLys) in wildtype CHO-K1 and PG-deficient A745 cells. All peptides were more efficiently internalized into CHO-K1 than into A745, but their cell surface binding was independent of cell type. Thus, PGs promote internalization of cationic peptides, irrespective of the chemical nature of their positive charges. Uptake of each peptide was linearly dependent on its cell surface binding, and affinity is thus important for efficiency. However, the gradients of these linear dependencies varied significantly. Thus each peptide's ability to stimulate uptake once bound to the cell surface is reliant on formation of specific uptake-promoting interactions. Heparin affinity chromatography and clustering experiments showed that penetratin and PenArg binding to sulfated sugars is stabilized by hydrophobic interactions and result in clustering, whereas PenLys only interacts through electrostatic attraction. This may have implications for the molecular mechanisms behind arginine-specific uptake stimulation as penetratin and PenArg are more efficiently internalized than PenLys upon interaction with PGs. However, PenArg is also least affected by removal of PGs. This indicates that an increased arginine content not only improve PG-dependent uptake but also that PenArg is more adaptable as it can use several portals of entry into the cell.
细胞表面蛋白聚糖（PGs）似乎能促进富含精氨酸的细胞穿透肽（CPPs）的摄取，但其确切功能尚不清楚。为了研究精氨酸与PGs之间的相互作用是否存在特异性从而导致内化作用增强，我们使用流式细胞术检测了野生型CHO-K1细胞和PG缺陷型A745细胞中穿膜肽及其两种精氨酸/赖氨酸取代变体（PenArg和PenLys）的摄取与细胞表面结合的关系。所有肽在CHO-K1细胞中的内化效率均高于A745细胞，但其细胞表面结合与细胞类型无关。因此，PGs促进阳离子肽的内化，而与阳离子电荷的化学性质无关。每种肽的摄取与其细胞表面结合呈线性相关，因此亲和力对摄取效率很重要。然而，这些线性相关性的梯度差异显著。因此，每种肽一旦与细胞表面结合后刺激摄取的能力依赖于形成特定的促进摄取的相互作用。肝素亲和层析和聚类实验表明，穿膜肽和PenArg与硫酸化糖的结合通过疏水相互作用得以稳定并导致聚类，而PenLys仅通过静电吸引相互作用。这可能对精氨酸特异性摄取刺激背后的分子机制有影响，因为穿膜肽和PenArg与PGs相互作用时比PenLys更有效地内化。然而，PenArg受PGs去除的影响也最小。这表明精氨酸含量的增加不仅提高了PG依赖的摄取，而且PenArg更具适应性，因为它可以利用多种进入细胞的途径。
