STUDY OF SAPOSINS' BIOLOGICAL FUNCTIONS

本研究的目的是阐明saposins与脂质模型膜相互作用的动力学和机理基础。我们试图确定saposins中对脂双层结合重要的氨基酸区域以及对单个saposins与特定脂/脂膜结合活性至关重要的残基。鞘脂激活蛋白是小的（80 aa）溶酶体蛋白，其具有不同的作用机制，尽管其一级序列高度相似。这些鞘脂激活蛋白或激活蛋白对于参与溶酶体中鞘糖脂/鞘脂（GSL/JSL）催化剂途径的几种水解酶的正常功能是必需的。四个（A、B、C和D）衍生自单一前体鞘脂激活蛋白原。我们假设saposins的不同功能是这些蛋白质的不同脂质/脂质膜相互作用特性的结果。拟议的研究集中在saposins B和C作为原型，因为它们与各自的酶的作用机制似乎不同。鞘脂激活蛋白B通过溶解脂质底物硫苷脂增强芳基硫酸酯酶A（阿萨）活性，鞘脂激活蛋白C在酸性pH下磷脂酰丝氨酸（pS）膜存在下诱导酸性β-葡糖苷酶构象变化.鞘脂激活蛋白C也诱导PS脂质体融合，而鞘脂激活蛋白B不诱导.这些研究旨在通过荧光和圆二色性（CD）光谱分析确定鞘脂激活蛋白-脂质和鞘脂激活蛋白-脂质-酶相互作用。蛋白质或脂质中的内在（Trp）和外在（芘）荧光探针将用作荧光共振能量转移、停流和淬灭分析的报告分子。使用生物化学和诱变技术，鞘脂激活蛋白功能的关键结构组分的位置将被表征。有效的表达系统和saposins B和C的体外检测系统的可用性为所提出的研究提供了可行性。基于鞘脂激活蛋白前体、鞘脂激活蛋白原缺陷细胞系的转基因“拯救”系统将提供鞘脂激活蛋白B和C的选定突变体的离体结构/功能相关性。拟议研究的意义涉及描述鞘脂激活蛋白的脂质结合特异性，其对鞘脂水解酶的调节具有重要作用，并影响溶酶体贮积病的表达。

The objective of the proposed studies is to elucidate kinetics and mechanistic bases of the interaction of saposins and lipid model membranes, We seek to define amino acid regions in saposins important to lipid bilayer binding and the residues critical to the binding activity of individual saposins for specific lipidl/ipid membrane. Saposins are small (80 aa) lysosomal proteins that have distinct mechanisms of action despite the high similarity of their primary sequences. These saposins or activator proteins are essential for normal function of several hydrolases involved in the glycosphingolipid/sphingolipid (GSL/JSL) catabolism pathway in lysosomes. Four (A, B, C, and D) derive from a single precursor, prosaposin. We hypothesize that the diverse functions of saposins are a result of distinct lipid/lipid membrane interaction properties of these proteins. The proposed studies focus on saposins B and C as prototypes since their mechanisms of actions with their respective enzyme appear distinct. Saposin B enhances arysulfatase A (ASA) activity by solubilizing the lipid substrate, sulfatide, and saposin C induced an acid beta- glucosidase conformational change in the presence of phosphatidylserine (pS) membranes at acidic pH. Saposin C also induces PS liposome fusion, while saposin B does not. The studies are designed to determine saposin- lipid and saposin-lipid-enzyme interactions by fluorescence and circular dichroism (CD) spectroscopic analyses. Intrinsic (Trp) and extrinsic (pyrene) fluorescent probes in proteins or lipids will be used as reporters for fluorescence resonance energy transfer, stopped-flow, and quenching analyses. Using biochemical and mutagenesis technologies, the location of key structural components for saposin functionality will be characterized. The availability of efficient expression systems and in vitro assay systems for saposins B and C provide the feasibility for the proposed studies. Transgenic "rescue" systems based on the saposin precursor, prosaposin, deficient cell lines will provide ex vivo structure/function correlation of selected mutants of saposins B and C. The significance of the proposed studies relates to the delineation of the lipid-binding specificity of saposins that have major effects on the modulation of sphingolipid hydrolases and influence on the expression of lysosomal storage diseases.