You Hong Xing, Qi Xiaoyang, Grabowski Gregory A, Yu Lei
Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Ohio 45267-0521, USA.
Biophys J. 2003 Mar;84(3):2043-57. doi: 10.1016/S0006-3495(03)75012-7.
Saposin C (Sap C) is a small glycoprotein required for hydrolysis of glucosylceramidase in lysosomes. The full activity of glucosylceramidase requires the presence of both Sap C and acidic phospholipids. Interaction between Sap C and acidic phospholipid-containing membranes, a crucial step for enzyme activation, is not fully understood. In this study, the dynamic process of Sap C interaction with acidic phospholipid-containing membranes was investigated in aqueous buffer using atomic force microscopy. Sap C induced two types of membrane restructuring: formation of patch-like structural domains and the occurrence of membrane destabilization. The former caused thickness increase whereas the latter caused thickness reduction in the gel-phase membrane bilayer, possibly as a result of lipid loss or an interdigitating process. Patch-like domain formation was independent of acidic phospholipids, whereas membrane destabilization is dependent on the presence and concentration of acidic phospholipids. Sap C effects on membrane restructuring were further studied using synthetic peptides. Synthetic peptides corresponding to the amphipathic alpha-helical domains 1 (designated "H1 peptide") and 2 (H2 peptide) of Sap C were used. Our results indicated that H2 contributed to domain formation but not to membrane destabilization, whereas H1 induced neither type of membrane restructuring. However, H1 was able to mimic Sap C's destabilization effect in conjunction with H2, but only when H1 was present first and H2 was added afterwards. This study provides an approach to investigate the structure-function aspects of Sap C interaction with phospholipid membranes, with insights into the mechanism(s) of Sap C-membrane interaction.
鞘脂激活蛋白C（Sap C）是一种小糖蛋白，是溶酶体中葡萄糖神经酰胺酶水解所必需的。葡萄糖神经酰胺酶的全部活性需要同时存在Sap C和酸性磷脂。Sap C与含酸性磷脂膜之间的相互作用是酶激活的关键步骤，但尚未完全了解。在本研究中，使用原子力显微镜在水性缓冲液中研究了Sap C与含酸性磷脂膜相互作用的动态过程。Sap C诱导了两种类型的膜重构：形成斑块状结构域和膜不稳定现象。前者导致厚度增加，而后者导致凝胶相膜双层厚度减小，这可能是脂质损失或叉指化过程的结果。斑块状结构域的形成与酸性磷脂无关，而膜不稳定则取决于酸性磷脂的存在和浓度。使用合成肽进一步研究了Sap C对膜重构的影响。使用了与Sap C的两亲性α-螺旋结构域1（称为“H1肽”）和2（H2肽）相对应的合成肽。我们的结果表明，H2有助于结构域的形成，但对膜不稳定没有作用，而H1既不诱导任何一种膜重构类型。然而，H1能够与H2共同模拟Sap C的去稳定作用，但前提是首先存在H1，然后添加H2。本研究提供了一种研究Sap C与磷脂膜相互作用的结构-功能方面的方法，有助于深入了解Sap C-膜相互作用的机制。
