title: Expression and functional characterization of human lysosomal acid lipase gene (LIPA) mutation responsible for cholesteryl ester storage disease (CESD) phenotype.
abstract: Lysosomal acid lipase (LAL) is a serine hydrolase which hydrolyzes cholesteryl ester and triglycerides delivered to the lysosomes into free cholesterol and free fatty acids. Mutations in the LAL gene (LIPA) result in accumulation of triglycerides and cholesterol esters in various tissues of the body, leading to pathological conditions such as Wolman's disease (WD) and cholesteryl ester storage disease (CESD). CESD patients homozygous for His295Tyr (H295Y) mutation have less than 5% of normal LAL activity. To shed light on the molecular basis for this loss-of-function phenotype, we have generated the recombinant H295Y enzyme and studied its biophysical and biochemical properties. No significant differences were observed in the expression levels or glycosylation patterns between the mutant and the wild type LAL. However, the H295Y mutant displayed only residual enzymatic activity (<5%) compared to the wild type. While wild type LAL is mostly a monomer at pH 5.0, the vast majority H295Y exists as a high molecular soluble aggregate. Besides, the H295Y mutant has a 20°C lower melting temperature compared to the wild type. Transient expression studies in WD fibroblasts showed that mutation of His295 to other amino acids resulted in a significant loss of enzymatic activity. A homology model of LAL revealed that His295 is located on an α-helix of the cap domain and could be important for tethering it to its core domain. The observed loss-of-function phenotype in CESD patients might arise from a combination of protein destabilization and the shift to a non-functional soluble aggregate.
authors.affiliation: Pfizer Worldwide Research, Groton, CT 06340, USA. Electronic address: francis.rajamohan@pfizer.com.
authors.forename: Francis
authors.initials: F
authors.lastname: Rajamohan
authors.affiliation: Pfizer Worldwide Research, Cambridge, MA 02139, USA.
authors.forename: Allan R
authors.initials: AR
authors.lastname: Reyes
authors.affiliation: Pfizer Worldwide Research, Cambridge, MA 02139, USA.
authors.forename: Wanida
authors.initials: W
authors.lastname: Ruangsiriluk
authors.affiliation: Pfizer Worldwide Research, Groton, CT 06340, USA.
authors.forename: Lise R
authors.initials: LR
authors.lastname: Hoth
authors.affiliation: Pfizer Worldwide Research, Groton, CT 06340, USA.
authors.forename: Seungil
authors.initials: S
authors.lastname: Han
authors.affiliation: Pfizer Worldwide Research, Groton, CT 06340, USA.
authors.forename: Nicole
authors.initials: N
authors.lastname: Caspers
authors.affiliation: Pfizer Worldwide Research, Cambridge, MA 02139, USA.
authors.forename: Meihua
authors.initials: M
authors.lastname: Tu
authors.affiliation: Pfizer Worldwide Research, Cambridge, MA 02139, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Ward
authors.affiliation: Pfizer Worldwide Research, Groton, CT 06340, USA.
authors.forename: Ravi G
authors.initials: RG
authors.lastname: Kurumbail
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002788:Cholesterol Esters / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D050356:Lipid Metabolism; D008247:Lysosomes / Q000201:enzymology* / Q000473:pathology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D002787:Sterol Esterase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015223:Wolman Disease / Q000201:enzymology* / Q000235:genetics / Q000473:pathology
chemical_list: D002788:Cholesterol Esters; D066329:Protein Aggregates; D011993:Recombinant Fusion Proteins; C503451:LIPA protein, human; D002787:Sterol Esterase
keywords: Cholesteryl ester storage disease (CESD); LAL-deficiency; Lipid metabolism; Lysosomal acid lipase (LAL); Protein aggregation; Wolman’s disease (WD)