Studies of Prosaposin's Physiologic Role

描述（由申请方提供）：拟定研究的目的是确定鞘脂激活蛋白原（PS）衍生鞘脂激活蛋白（鞘脂激活蛋白A、B、C和D）缺乏对鞘糖脂（GSL）代谢控制的体内影响。假设GSL催化剂是由代谢途径中这些鞘脂激活蛋白的相互作用调节的，并且这代表了基因内上位性的一个例子。此外，我们提出saposin B是控制通过该途径的流量的关键调节剂。这些研究基于以下观察结果：罕见的、不完全详细的人（B和C）和小鼠（A）孤立的鞘脂激活蛋白缺乏症与所有四种PS衍生的鞘脂激活蛋白的同时缺乏症（PS-/-）相比具有显著不同的表型（临床和生物化学）。类似地，PS-/-基因型通过掩盖孤立的B、C或A缺乏的GSL储存而导致新的表型。我们推测，这些改变的表型是由于鞘脂激活蛋白在GSL分解代谢途径的特定步骤中的相互作用和冗余造成的，这是体外/离体分析表明的。对于这些研究，将通过靶向引入特定点突变来产生具有单鞘脂激活蛋白或双鞘脂激活蛋白缺陷的小鼠，所述特定点突变基于人和小鼠模型已经产生功能完整的鞘脂激活蛋白原和“非突变的鞘脂激活蛋白”，以及分离的鞘脂激活蛋白缺陷。突变将通过在选定残基处用Phe或Ser取代Cys来破坏鞘脂激活蛋白的保守二硫键结构：离体表达分析将在ES细胞靶向之前评估这些突变对PS和鞘脂激活蛋白稳定性的总体影响。首先产生单鞘脂激活素B、C或D缺陷，并在临床、组织学和生物化学[体内和体外（例如，培养的成纤维细胞、神经元、肝细胞）]水平：我们可获得Saposin A缺陷型小鼠。将产生鞘脂激活蛋白A和B以及B和C的处置蛋白缺陷，以评价GSL催化剂中特定步骤的相互作用：例如，提出了需要鞘脂激活蛋白B和C来降解乳糖神经酰胺，以及通过包含鞘脂激活蛋白B缺陷来“拯救”鞘脂激活蛋白A缺陷表型。这些研究对GSL代谢、溶酶体贮积病表型表达和治疗有意义。

DESCRIPTION (provided by applicant): The objective of the proposed studies is to determine the in vivo effects of prosaposin (PS)-derived sphingolipid activator protein (saposins A, B, C and D) deficiencies on the control of glycosphingolipid (GSL) metabolism. The hypothesis is that GSL catabolism is modulated by the interactions of these saposins in the metabolic pathway, and that this represents an example of intragenic epistasis. Furthermore, we propose that saposin B is the key modulator that controls flow through this pathway. These studies are based on the observations that the rare, not fully detailed, human (B and C) and mouse (A) isolated saposin deficiencies have significantly different phenotypes (both clinically and biochemically), than simultaneous deficiency (PS-/-) of all four PS-derived saposins. Similarly, the PS-/- genotype leads to new phenotypes by masking the GSL storage of isolated B, C or A deficiency. We postulate that these altered phenotypes result from the interactions and redundancies of the saposins at specific steps in the GSL catabolic pathway, suggested by in vitro/ex vivo analyses. For these studies, mice with mono- or di- saposin deficiencies will be created by targeted introduction of specific point mutations that, based on human and mouse models, have produced functionally intact prosaposin and "non-mutated saposins," and isolated deficiencies of the saposins. The mutations will disrupt the conserved disulfide structure of the saposins by substitution of Phe or Ser for Cys at selected residues: ex vivo expression analyses will evaluate the overall effects of these mutations on PS and saposin stability prior to ES cell targeting. Mono-saposin B, C or D deficiencies will be created first and the resultant phenotypes characterized at the clinical, histologic and biochemical [in vivo and in vitro (e.g., cultured fibroblasts, neurons, hepatocytes)] levels: Saposin A deficient mice are available to us. Disaposin deficiencies of saposins A and B, and B and C will be created to evaluate the interactions at specific steps in GSL catabolism: e.g., the proposed need of saposins B and C for lactosylceramide degradation and the "rescue" of the saposin A deficient phenotype by inclusion of saposin B deficiency. These studies have implications for GSL metabolism, and lysosomal storage disease phenotypic expression and therapy.