Paris Sébastien, Burlacu Alina, Durocher Yves
Animal Cell Technology Group, Bioprocess Sector, Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, Canada.
J Biol Chem. 2008 Mar 21;283(12):7697-704. doi: 10.1074/jbc.M705424200. Epub 2008 Jan 23.
Polyethyleneimines (PEIs) are efficient non-viral vectors for gene transfer. Heparan sulfate proteoglycans have been proposed to be the cell-surface receptors for PEI.DNA complexes (polyplexes). Here, we investigated if syndecan-1 (SDC1) and syndecan-2 (SDC2) are involved in PEI-mediated transfection. Following addition of polyplexes to HEK293 cells, green fluorescent protein-tagged SDCs rapidly formed clusters with PEI that were dependent of lipid raft integrity. However, although SDC1 overexpression slightly enhanced PEI-mediated gene expression, SDC2 dramatically inhibited it. Confocal microscopy analysis showed that SDC1.polyplex endocytosis occurred within minutes after addition of polyplexes, whereas SDC2.polyplex endocytosis took hours. Expression of SDC1 cytoplasmic deletion mutants revealed that the SDC1 cytoplasmic tail is required for gene expression, but not for clustering or endocytosis, whereas overexpression of SDC1/SDC2 chimeras showed that the SDC2 ectodomain is responsible for the inhibitory effect on gene transfer. This study provides evidence that SDCs may have opposing effects on PEI-mediated transfection.
聚乙烯亚胺（PEIs）是用于基因转移的高效非病毒载体。硫酸乙酰肝素蛋白聚糖被认为是PEI-DNA复合物（多聚体）的细胞表面受体。在此，我们研究了syndecan-1（SDC1）和syndecan-2（SDC2）是否参与PEI介导的转染。将多聚体添加到HEK293细胞后，绿色荧光蛋白标记的SDCs迅速与PEI形成簇，这依赖于脂筏的完整性。然而，虽然SDC1过表达略微增强了PEI介导的基因表达，但SDC2却显著抑制了它。共聚焦显微镜分析表明，SDC1-多聚体的内吞作用在添加多聚体后几分钟内发生，而SDC2-多聚体的内吞作用则需要数小时。SDC1细胞质缺失突变体的表达表明，SDC1细胞质尾巴对于基因表达是必需的，但对于簇的形成或内吞作用并非必需，而SDC1/SDC2嵌合体的过表达表明，SDC2胞外域对基因转移具有抑制作用。这项研究提供了证据表明SDCs可能对PEI介导的转染具有相反的作用。
