Expression of lysosomal acid lipase mutants detected in three patients with cholesteryl ester storage disease

Lysosomal acid lipase (LAL) gene mutations were identified in threepatients with cholesteryl ester storage disease (CESD). Direct sequencingof genomic DNA revealed that: patient 1 was a compound heterozygote for aP181L mutation and an A to G3' splice site substitution that causesskipping of exon 7, with a loss of 49 amino acids from LAL (delta 205-253);patient 2 was a compound heterozygote for a G66V mutation and a 5' splicesite mutation (G to A) that leads to skipping of exon 8 (delta 254-277);and patient 3 was a compound heterozygote for a L273S mutation and anunidentified null allele. Furthermore, patients 2 and 3 showed a novel G-2Apolymorphism that could be detected by an Xbal restriction fragment lengthpolymorphism. All these mutants and a previously reported H274Y allele wereexpressed in vitro in HeLa cells using the vaccinia T7 expression system.The resulting recombinant proteins were inactive towards cholesteryl oleateand trioleylglycerol, demonstrating the direct involvement of thesemutations in the pathogenesis of CESD. Immunoblotting of normal LALexpressed in HeLa cells revealed four major molecular forms, at least twoof high molecular mass (54 and 50-51 kDa) and two of low molecular mass (42and 43 kDa). L273S and P181L substitutions and delta 254-277 were shown toresult in altered LAL molecular forms, some of which suggest thatpost-translational processing may interfere with the catalytic activity ofLAL.