STUDY OF SAPOSINS' BIOLOGICAL FUNCTIONS

本研究的目的是阐明皂苷与脂质模型膜相互作用的动力学和机制基础，我们试图确定皂苷中对脂质双分子层结合重要的氨基酸区域，以及对单个皂苷与特定脂质/脂质膜结合活性至关重要的残基。皂苷是一种小的（80aa）溶酶体蛋白，尽管它们的初级序列高度相似，但具有不同的作用机制。这些皂苷或激活蛋白对于参与溶酶体中鞘糖脂/鞘脂（GSL/JSL）分解代谢途径的几种水解酶的正常功能是必需的。四种（A、B、C和D）来源于单一的前体，即丙素。我们假设皂苷的不同功能是这些蛋白质不同的脂质/脂质膜相互作用特性的结果。由于皂苷B和C与各自酶的作用机制不同，因此提出的研究重点是皂苷B和C作为原型。皂苷B通过溶解脂质底物、硫脂来增强arysulfatase A （ASA）的活性，而皂苷C在酸性ph下诱导了磷脂酰丝氨酸（pS）膜存在时的酸性β -葡萄糖苷酶构象变化。皂苷C还诱导了pS脂质体融合，而皂苷B则没有。本研究旨在通过荧光和圆二色性（CD）光谱分析测定皂苷-脂质和皂苷-脂质-酶的相互作用。蛋白质或脂质中的内在（色氨酸）和外在（芘）荧光探针将用作荧光共振能量转移，停止流动和淬火分析的报告。利用生物化学和诱变技术，对皂苷功能的关键结构成分的位置进行表征。皂苷B和C的高效表达系统和体外检测系统的可用性为提出的研究提供了可行性。基于皂素前体、皂素缺陷细胞系的转基因“拯救”系统将为皂素B和c的选择突变体提供离体结构/功能相关性。所提出的研究的意义在于描述皂素的脂质结合特异性，这种特异性对鞘脂水解酶的调节和溶酶体贮积病的表达有重要影响。

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.