Studies of Prosaposin's Physiologic Role

描述（由申请人提供）：拟议研究的目的是确定丙皂苷（PS）衍生鞘脂激活蛋白（皂苷A、B、C和D）缺乏对鞘脂糖（GSL）代谢控制的体内影响。假设GSL分解代谢是由代谢途径中这些皂苷的相互作用调节的，这代表了基因内上位的一个例子。此外，我们提出皂苷B是通过这一途径控制流动的关键调节剂。这些研究是基于这样的观察，即罕见的，不完全详细的，人类（B和C）和小鼠(A)分离的皂苷缺乏具有显著不同的表型（临床和生化），而同时缺乏（PS-/-）所有四种PS衍生的皂苷。同样，PS-/-基因型通过掩盖分离的B、C或A缺陷的GSL储存而导致新的表型。我们假设这些改变的表型是由皂苷在GSL分解代谢途径中特定步骤的相互作用和冗余引起的，这是通过体外/离体分析得出的结论。在这些研究中，将通过有针对性地引入特定的点突变来产生单皂苷或二皂苷缺乏的小鼠，这些突变基于人类和小鼠模型，已经产生了功能完整的皂苷和“非突变的皂苷”，并分离出皂苷缺乏。突变将通过在选定的残基上用Phe或Ser取代Cys来破坏皂苷的保守二硫结构：体外表达分析将在ES细胞靶向之前评估这些突变对PS和皂苷稳定性的总体影响。首先会产生单皂苷B、C或D缺陷，并在临床、组织学和生化[体内和体外（如培养成纤维细胞、神经元、肝细胞）]水平上表现出相应的表型特征：皂苷A缺陷小鼠可供我们使用。将创建皂苷A和B以及B和C的二磷酸腺苷缺乏症，以评估GSL分解代谢中特定步骤的相互作用：例如，提出的乳糖神经酰胺降解需要皂苷B和C，以及通过包含皂苷B缺乏症来“拯救”皂苷A缺陷表型。这些研究对GSL代谢和溶酶体贮积病的表型表达和治疗具有启示意义。

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.