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理基础，我们试图确定皂苷中对脂质双层结合重要的氨基酸区域，以及对单个皂苷与特定的Lipoidl/IPID膜结合活性至关重要的残基。Saposins是一种小的(80aa)溶酶体蛋白，尽管它们的初级序列高度相似，但它们有不同的作用机制。这些皂苷或激活蛋白是参与溶酶体内神经鞘糖脂/鞘糖脂(GSL/JSL)分解代谢途径的几种水解酶正常功能所必需的。四种(A、B、C和D)来自单一前体--丙皂苷。我们推测，皂苷的不同功能是这些蛋白质不同的脂/脂膜相互作用性质的结果。拟议的研究集中在皂苷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。在酸性pH条件下，在磷脂酰丝氨酸(PS)膜的存在下，皂苷B通过溶解脂肪底物硫脂和皂苷C引起酸性β-葡萄糖苷酶的构象变化，从而提高芳硫酸酯A(ASA)的活性。皂苷C也能诱导PS脂质体融合，而皂苷B不能。本研究旨在通过荧光光谱分析和圆二色谱(CD)分析来确定皂苷-脂和皂苷-脂-酶的相互作用。蛋白质或脂类中的本征(Trp)和外源(Py)荧光探针将被用作荧光共振能量转移、停流和猝灭分析的报告。利用生化和诱变技术，将确定皂苷功能的关键结构成分的位置。皂苷B和C的高效表达系统和体外检测系统的可用性为拟议的研究提供了可行性。基于皂苷前体细胞系的转基因“拯救”系统将提供皂苷B和C突变体的体外结构/功能关联。本研究的意义在于揭示皂苷的脂结合专一性，它对鞘磷脂水解酶的调节和溶酶体贮藏疾病的表达具有重要影响。

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.