Tatti M, Salvioli R, Ciaffoni F, Pucci P, Andolfo A, Amoresano A, Vaccaro A M
Laboratorio Metabolismo e Biochimica Patologica, Istituto Superiore Sanità, Roma, Italy.
Eur J Biochem. 1999 Jul;263(2):486-94. doi: 10.1046/j.1432-1327.1999.00521.x.
Saposin D is generated together with three similar proteins, saposins A, B and C, from a common precursor, called prosaposin, in acidic organelles such as late endosomes and lysosomes. Although saposin D has been reported to stimulate the enzymatic hydrolysis of sphingomyelin and ceramide, its physiological role has not yet been clearly established. In the present study we examined structural and membrane-binding properties of saposin D. At acidic pH, saposin D showed a great affinity for phospholipid membranes containing an anionic phospholipid such as phosphatidylserine or phosphatidic acid. The binding of saposin D caused destabilization of the lipid surface and, conversely, the association with the membrane markedly affected the fluorescence properties of saposin D. The presence of phosphatidylserine-containing vesicles greatly enhanced the intrinsic tyrosine fluorescence of saposin D, which contains tyrosines but not tryptophan residues. The structural properties of saposin D were investigated in detail using advanced MS analysis. It was found that the main form of saposin D consists of 80 amino acid residues and that the six cysteine residues are linked in the following order: Cys5-Cys78, Cys8-Cys72 and Cys36-Cys47. The disulfide pattern of saposin D is identical with that previously established for two other saposins, B and C, which also exhibit a strong affinity for lipids. The common disulfide structure probably has an important role in the interaction of these proteins with membranes. The analysis of the sugar moiety of saposin D revealed that the single N-glycosylation site present in the molecule is mainly modified by high-mannose-type structures varying from two to six hexose residues. Deglycosylation had no effect on the interaction of saposin D with phospholipid membranes, indicating that the glycosylation site is not related to the lipid-binding site. The association of saposin D with membranes was highly dependent on the composition of the bilayer. Neither ceramide nor sphingomyelin, sphingolipids whose hydrolysis is favoured by saposin D, promoted its binding, while the presence of an acidic phospholipid such as phosphatidylserine or phosphatidic acid greatly favoured the interaction of saposin D with vesicles at low pH. These results suggest that, in the acidic organelles where saposins are localized, anionic phospholipids may be determinants of the saposin D topology and, conversely, saposin D may affect the lipid organization of anionic phospholipid-containing membranes.
鞘脂激活蛋白D与另外三种相似的蛋白——鞘脂激活蛋白A、B和C，由一种称为前体鞘脂激活蛋白的共同前体在酸性细胞器（如晚期内体和溶酶体）中共同产生。尽管据报道鞘脂激活蛋白D能刺激鞘磷脂和神经酰胺的酶促水解，但其生理作用尚未明确确立。在本研究中，我们检测了鞘脂激活蛋白D的结构和膜结合特性。在酸性pH值下，鞘脂激活蛋白D对含有阴离子磷脂（如磷脂酰丝氨酸或磷脂酸）的磷脂膜表现出极大的亲和力。鞘脂激活蛋白D的结合导致脂质表面不稳定，相反，与膜的结合显著影响鞘脂激活蛋白D的荧光特性。含有磷脂酰丝氨酸的囊泡的存在极大地增强了鞘脂激活蛋白D的内在酪氨酸荧光，鞘脂激活蛋白D含有酪氨酸但不含色氨酸残基。使用先进的质谱分析详细研究了鞘脂激活蛋白D的结构特性。发现鞘脂激活蛋白D的主要形式由80个氨基酸残基组成，并且六个半胱氨酸残基按以下顺序连接：Cys5-Cys78、Cys8-Cys72和Cys36-Cys47。鞘脂激活蛋白D的二硫键模式与先前确定的另外两种鞘脂激活蛋白B和C相同，它们也对脂质表现出很强的亲和力。共同的二硫键结构可能在这些蛋白质与膜的相互作用中起重要作用。对鞘脂激活蛋白D糖部分的分析表明，分子中存在的单个N-糖基化位点主要被含有两到六个己糖残基的高甘露糖型结构修饰。去糖基化对鞘脂激活蛋白D与磷脂膜的相互作用没有影响，表明糖基化位点与脂质结合位点无关。鞘脂激活蛋白D与膜的结合高度依赖于双层膜的组成。神经酰胺和鞘磷脂（鞘脂激活蛋白D促进其水解的鞘脂）都不促进其结合，而酸性磷脂（如磷脂酰丝氨酸或磷脂酸）的存在极大地促进了鞘脂激活蛋白D在低pH值下与囊泡的相互作用。这些结果表明，在鞘脂激活蛋白所在的酸性细胞器中，阴离子磷脂可能是鞘脂激活蛋白D拓扑结构的决定因素，相反，鞘脂激活蛋白D可能影响含阴离子磷脂膜的脂质组织。
