PATHOGENESIS OF LYSOSOMAL ACID LIPASE DEFICIENCY

溶酶体酸性脂肪酶缺乏症的发病机制

• 批准号: 2740966
• 负责人: HONG DU
• 金额: $ 14.93万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2740966
• 关键词: Wolman's disease; cholesterol ester storage disease; disease /disorder etiology; enzyme activity; enzyme deficiency; enzyme mechanism; enzyme substrate; esterase inhibitor; gene targeting; genetic models; genetically modified animals; laboratory mouse; model design /development; phenotype; point mutation; site directed mutagenesis; sterol esterase

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.

拟议研究的目的是阐明Wolman(WD)和胆固醇酯储存(CESD)疾病的发病机制。这些疾病是由于Lal基因突变导致溶酶体酸性脂肪酶(Lal)缺乏所致。这种酶对甘油三酯和胆固醇酯底物和底物专一性的测定有活性，因此，表型还不清楚。利用我们的人和小鼠LAL基因，以及pET21A和杆状病毒表达系统，我们已经生产了足够的纯化的LAL，用于制备特异性抗体，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，并在这个基因座上创造了一只“敲除”小鼠。利用这个动物模型和我们的体外异源表达系统，我们建议：1)通过自然病史、组织学和脂类表征方法来表征LAL-/LAL-小鼠的表型。根据大鼠模型预测，我们的活体出生(至少存活21天)的LAL-/LAL-小鼠将具有WD表型。2)正常和选择的HLAL和mLAL突变形式将通过详细的动力学分析来表征，以确定对底物偏好重要的残基及其与WD和/或CESD表型的关系。这些分析将包括具有活性中心定向抑制剂的稳态和瞬时动力学，以及具有不同酰基链组成的选定底物。3)催化活性构象的基本N-糖基化占有率将通过对HLAL之间保守的共有序列进行定点突变来评估，催化活性构象将通过对HLA1、mLAL和rLAL之间保守的共有序列的定点突变来评估。4)根据体外实验结果，通过“敲入”的方法将特定的突变引入LAL-/LAL-小鼠，以确定它们的生理相关性及其与WD和CESD表型的关系。LAL基因定点突变纯合子小鼠的发展将为更全面地描述LAL的发育进行性、组织特异性参与以及作为表型发病基础的LAL差异组织表达的可能性提供必要的试剂。这些研究也将为未来WD和CESD的酶和基因治疗方法以及其他先天代谢错误的研究提供基础。