Steinert Steffen, Lee Esther, Tresset Guillaume, Zhang Dawei, Hortsch Ralf, Wetzel Richard, Hebbar Sarita, Sundram Jeyapriya Raja, Kesavapany Sashi, Boschke Elke, Kraut Rachel
Institute of Bioengineering and Nanotechnology, A*Star, Singapore, Singapore.
PLoS One. 2008 Aug 13;3(8):e2933. doi: 10.1371/journal.pone.0002933.
The uptake and intracellular trafficking of sphingolipids, which self-associate into plasma membrane microdomains, is associated with many pathological conditions, including viral and toxin infection, lipid storage disease, and neurodegenerative disease. However, the means available to label the trafficking pathways of sphingolipids in live cells are extremely limited. In order to address this problem, we have developed an exogenous, non-toxic probe consisting of a 25-amino acid sphingolipid binding domain, the SBD, derived from the amyloid peptide Abeta, and conjugated by a neutral linker with an organic fluorophore. The current work presents the characterization of the sphingolipid binding and live cell trafficking of this novel probe, the SBD peptide. SBD was the name given to a motif originally recognized by Fantini et al in a number of glycolipid-associated proteins, and was proposed to interact with sphingolipids in membrane microdomains.
METHODOLOGY/PRINCIPAL FINDINGS: In accordance with Fantini's model, optimal SBD binding to membranes depends on the presence of sphingolipids and cholesterol. In synthetic membrane binding assays, SBD interacts preferentially with raft-like lipid mixtures containing sphingomyelin, cholesterol, and complex gangliosides in a pH-dependent manner, but is less glycolipid-specific than Cholera toxin B (CtxB). Using quantitative time-course colocalization in live cells, we show that the uptake and intracellular trafficking route of SBD is unlike that of either the non-raft marker Transferrin or the raft markers CtxB and Flotillin2-GFP. However, SBD traverses an endolysosomal route that partially intersects with raft-associated pathways, with a major portion being diverted at a late time point to rab11-positive recycling endosomes. Trafficking of SBD to acidified compartments is strongly disrupted by cholesterol perturbations, consistent with the regulation of sphingolipid trafficking by cholesterol.
CONCLUSIONS/SIGNIFICANCE: The current work presents the characterization and trafficking behavior of a novel sphingolipid-binding fluorescent probe, the SBD peptide. We show that SBD binding to membranes is dependent on the presence of cholesterol, sphingomyelin, and complex glycolipids. In addition, SBD targeting through the endolysosomal pathway in neurons is highly sensitive to cholesterol perturbations, making it a potentially useful tool for the analysis of sphingolipid trafficking in disease models that involve changes in cholesterol metabolism and storage.
鞘脂类会自行聚集形成质膜微结构域，其摄取和细胞内运输与许多病理状况相关，包括病毒和毒素感染、脂质贮积病以及神经退行性疾病。然而，用于标记活细胞中鞘脂类运输途径的方法极为有限。为了解决这一问题，我们开发了一种外源性、无毒的探针，它由一个25个氨基酸的鞘脂类结合结构域（SBD）组成，该结构域源自淀粉样肽β-淀粉样蛋白，并通过一个中性连接子与一种有机荧光团相连。目前的工作展示了这种新型探针SBD肽的鞘脂类结合特性及活细胞运输情况。SBD是Fantini等人最初在一些糖脂相关蛋白中识别出的一个基序的名称，据推测它可与膜微结构域中的鞘脂类相互作用。
方法/主要发现：根据Fantini的模型，SBD与膜的最佳结合取决于鞘脂类和胆固醇的存在。在合成膜结合试验中，SBD以pH依赖的方式优先与含有鞘磷脂、胆固醇和复合神经节苷脂的类脂筏样脂质混合物相互作用，但比霍乱毒素B（CtxB）对糖脂的特异性更低。通过对活细胞进行定量时间进程共定位，我们发现SBD的摄取和细胞内运输途径不同于非类脂筏标记物转铁蛋白以及类脂筏标记物CtxB和Flotillin2-GFP的运输途径。然而，SBD穿过一条部分与类脂筏相关途径相交的内溶酶体途径，在后期大部分转向rab11阳性的再循环内体。胆固醇扰动会强烈干扰SBD向酸化区室的运输，这与胆固醇对鞘脂类运输的调节作用一致。
结论/意义：目前的工作展示了一种新型鞘脂类结合荧光探针SBD肽的特性及运输行为。我们表明SBD与膜的结合依赖于胆固醇、鞘磷脂和复合糖脂的存在。此外，SBD在神经元中通过内溶酶体途径的靶向作用对胆固醇扰动高度敏感，这使其成为分析涉及胆固醇代谢和储存变化的疾病模型中鞘脂类运输的潜在有用工具。
