PATHOGENESIS OF LYSOSOMAL ACID LIPASE DEFICIENCY

Children'S Hospital Med Ctr (Cincinnati), Cincinnati OH

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Abstract

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.