Cooperative, Heparan Sulfate-Dependent Cellular Uptake of Dimeric Guanidinoglycosides
Cooperative, Heparan Sulfate-Dependent Cellular Uptake of Dimeric Guanidinoglycosides
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DOI：
10.1002/cbic.201000399
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发表时间：
2010-11-02
期刊：
影响因子：
3.2
通讯作者：
Tor, Yitzhak
中科院分区：
文献类型：
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作者：
Dix, Andrew V.;Fischer, Lucile;Tor, Yitzhak
寡聚精氨酸和富含胍的分子转运蛋白已被证明有助于多种生物学相关货物的细胞内递送。几种这样的转运蛋白已被认为与细胞表面硫酸乙酰肝素蛋白聚糖相互作用，作为其细胞进入途径的一部分。与其他富含胍的转运蛋白不同，细胞摄取纳摩尔浓度的胍基糖苷完全依赖于硫酸乙酰肝素。由于不同的细胞在其表达水平和/或细胞表面硫酸乙酰肝素蛋白聚糖的组成上不同，因此人们可能能够利用这种差异来选择性地靶向某些细胞类型。为了系统地研究它们与细胞表面相互作用的性质，通过使用新霉素、巴龙霉素和妥布霉素作为支架来合成单体和二聚胍基糖苷。这些转运蛋白在胍基的数量和3D排列上不同。在产生相应的荧光链霉亲和素-藻红蛋白-Cy 5缀合物后，通过流式细胞术在野生型和突变型中国仓鼠卵巢细胞中测量其细胞摄取。所有衍生物在缺乏硫酸乙酰肝素的突变体细胞中的摄取可忽略不计。减少胍基团的数量会减少摄取，但这些基团的三维排列对细胞递送不太重要。尽管用单体载体制备的缀合物在表达硫酸乙酰肝素链的突变细胞中显示出显著降低的摄取，其中硫酸化模式改变，但用二聚胍基糖苷制备的缀合物可以克服这种缺陷并在这种缺陷细胞中保持高水平的摄取。这一发现表明，细胞摄取依赖于转运蛋白的化合价和细胞表面受体上硫酸基团的含量和排列。化学修饰或羧基还原肝素衍生物的竞争研究证实了这些观察结果。总之，这些发现表明，增加转运蛋白的化合价保留了硫酸乙酰肝素特异性，并提供了可以根据细胞表面硫酸乙酰肝素蛋白聚糖的特定组成区分不同细胞类型的试剂。
Oligoarginine and guanidinium-rich molecular transporters have been shown to facilitate the intracellular delivery of a diverse range of biologically relevant cargos. Several such transporters have been suggested to interact with cell-surface heparan sulfate proteoglycans as part of their cell-entry pathway. Unlike for other guanidinium-rich transporters, the cellular uptake of guanidinoglycosides at nanomolar concentrations is exclusively heparan sulfate dependent. As distinct cells differ in their expression levels and/or the composition of cell-surface heparan sulfate proteoglycans, one might be able to exploit such differences to selectively target certain cell types. To systematically investigate the nature of their cell-surface interactions, monomeric and dimeric guanidinoglycosides were synthesized by using neomycin, paromomycin, and tobramycin as scaffolds. These transporters differ in the number and 3D arrangement of their guanidinium groups. Their cellular uptake was measured by flow cytometry in wild-type and mutant Chinese hamster ovary cells after the corresponding fluorescent streptavidin-phycoerythrin-Cy5 conjugates had been generated. All derivatives showed negligible uptake in mutant cells lacking heparan sulfate. Decreasing the number of guanidinium groups diminished uptake, but the three dimensional arrangement of these groups was less important for cellular delivery. Whereas conjugates prepared with the monomeric carriers showed significantly reduced uptake in mutant cells expressing heparan sulfate chains with altered patterns of sulfation, conjugates prepared with the dimeric guanidinoglycosides could overcome this deficiency and maintain high levels of uptake in such deficient cells. This finding suggests that cellular uptake depends on the valency of the transporter and both the content and arrangement of the sulfate groups on the cell-surface receptors. Competition studies with chemically desulfated or carboxy-reduced heparin derivatives corroborated these observations. Taken together, these findings show that increasing the valency of the transporters retains heparan sulfate specificity and provides reagents that could distinguish different cell types based on the specific composition of their cell-surface heparan sulfate proteoglycans.