PATHOGENESIS OF LYSOSOMAL ACID LIPASE DEFICIENCY

提出的研究目的是阐明沃尔曼病（WD）和胆固醇酯沉积病（CESD）的发病机制。这些疾病是由于lal位点的突变导致溶酶体酸性脂肪酶（lal）的缺乏。该酶对甘油三酯和胆固醇酯底物和底物特异性的决定因素有活性，因此，表型尚不清楚。利用我们的人类和小鼠LAL cdna和pET 21A和杆状病毒表达系统，我们已经产生了足够的纯化LAL用于制作特异性抗体，并初步纯化和鉴定了正常和诱变的LAL。我们已经克隆了mLAL基因，并在这个位点上创建了一只“敲除”小鼠。利用该动物模型和我们的体外异源表达系统，我们提出：1)通过自然史、组织学和脂质表征方法表征lal-/lal-小鼠的表型。根据大鼠模型预测，我们的活产（存活至少21天）lal-/lal-小鼠将具有WD表型。2)将通过详细的动力学分析来确定对底物偏好重要的残基，以及它们与WD和/或CESD表型的关系，表达正常和选定的hal和mLAL突变形式。这些分析将包括稳态和瞬态动力学与活性位点定向抑制剂，并选择底物与不同的酰基链组成。3)催化活性构象的必需n -糖基化占用将通过对hLAL之间的保守一致序列进行定点突变来评估，催化活性构象将通过对hLAL、mLAL和rLAL之间的保守一致序列进行定点突变来评估。4)在体外实验结果的基础上，选择特异性突变，通过“敲入”方法导入lal-/lal-小鼠，确定其与WD和CESD表型的生理相关性和关系。lal位点的纯合突变小鼠群的发展将为更完整地描述lal的发育过程、组织特异性参与和潜在的差异组织表达提供必要的试剂，作为表型发病机制的基础。这些研究也将为未来研究WD和CESD以及其他先天性代谢错误的酶和基因治疗方法提供基础。

The objectives of the proposed studies are to elucidate the pathogenesis of Wolman (WD) and Cholesteryl Ester Storage (CESD) diseases. These diseases are due to the mutations at the lal locus that leads to a deficiency of lysosomal acid lipase (LAL). This enzyme has activity toward triglyceride and cholesteryl ester substrates and the determinates of the substrate specificity, and, therefore, the phenotypes is not understood. Using our human and mouse LAL cDNAs and pET 21A and baculovirus expression systems, we have produced sufficient purified LAL for making specific antibody, and the initial purification and characterization of normal and mutagenized LAL. We have cloned the mLAL gene and created a "knock-out" mouse at this locus. Using this animal model and our in vitro heterologous expression system, we propose to: 1) Characterize the phenotype of the lal-/lal-mouse by natural history, histologic and lipid characterization approaches. Based on the rat model anticipate that our liveborn (survival for 21 days at least) lal-/lal- mice will have a WD phenotype. 2) Normal and selected hLAL and mLAL mutant forms will be expressed characterized by detailed kinetic analyses to determine residues important for the substrate preference, and their relationship to WD and/or CESD phenotypes. These analyses will include steady-state and transient kinetics with active site directed inhibitors, and selected substrates with differing acyl chain composition. 3) The essential N-glycosylation occupancy for catalytically active conformers will be assessed by site-directed mutagenesis of the conserved consensus sequences between hLAL, catalytically active conformers will be assessed by site-directed mutagenesis of the conserved consensus sequences between hLAL, mLAL and rLAL. 4) Based on the in vitro findings, selected specific mutations will be introduced into the lal-/lal- mice by the "knock-in" approach to determined their physiologic relevance and relationship to the WD and CESD phenotypes. The development of a fleet of mice homozygous for selected point mutations at the lal locus will provide essential reagents for a more complete delineation of the developmental progressively, tissue specific involvement, and the potential for differential tissue expression of LAL as a basis for the pathogenesis of the phenotypes. These studies will also provide a basis for future studies of enzyme and gene therapeutic approaches to WD and CESD as well as other inborn errors of metabolism.