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

S. Sheriff, Hong Du, G. A. Grabowski

Research output: Contribution to journalArticle

77 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)27766-27772
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number46
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

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Sterol Esterase
Cholesterol Ester Storage Disease
Mutagenesis
Cholesterol Esters
Site-Directed Mutagenesis
Wolman Disease
Triglycerides
Glycosylation
Catalytic Domain
Esters
Triolein
Paraoxon
Sf9 Cells
Tunicamycin
LDL Receptors
Baculoviridae
Diglycerides
Lysosomes
Catalyst activity
Serine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of lysosomal acid lipase by site-directed mutagenesis and heterologous expression. / Sheriff, S.; Du, Hong; Grabowski, G. A.

In: Journal of Biological Chemistry, Vol. 270, No. 46, 01.01.1995, p. 27766-27772.

Research output: Contribution to journalArticle

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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.",
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N2 - 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.

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