Characterization of lysosomal acid lipase by site-directed mutagenesis and heterologous expression

Abstract

Lysosomal acid lipase (LAL) is essential for the hydrolysis of cholesterol esters and triglycerides that are delivered to the lysosomes via the low density lipoprotein receptor system. The deficiency of LAL is associated with cholesteryl ester storage disease (CESD) and Wolman's disease (WD). We cloned the human LAL cDNA and expressed the active enzyme in the baculovirus system. Two molecular forms (M(r) ~41,000 and ~40,000) with different glycosylation were found intracellularly, and ~24% of the M(r) ~46,000 form was secreted into the medium. Tunicamycin treatment produced only an inactive M(r) ~41,000 form. This result implicates glycosylation occupancy in the proper folding for active-site function. Catalytic activity was greater toward cis- than transunsaturated fatty acid esters of 4-methylumbellifcrone and toward esters with 7-carbon length acyl chains. LAL cleaved cholesterol esters and mono-, tri-, and diglycerides. Heparin had a biphasic effect on enzymatic activity with initial activation followed by inhibition. Inhibition of LAL activity by tetrahydrolipstatin and diethyl p-nitrophenyl phosphate suggested the presence of active serines in binding/catalytic domain(s) of the protein. Site-directed mutagenesis at two putative active centers, GXSXG, showed that Ser153 was important to catalytic activity, whereas Ser99 was not and neither was the catalytic nucleophile. Three reported mutations (L179P, L336P, and ΔAG302 deletion) from CESD patients were created and expressed in the Sf9 cell system. None cleaved cholesterol esters, and L179P and 1,336P cleaved only triolein at ~4% of wild-type levels. These results suggest that mechanisms, in addition to LAL defects, may operate in the selective accumulation of cholesterol esters or triglycerides in CESD and WD patients.