Cysteine residues in human lysosomal acid lipase are involved in selective cholesteryl esterase activity

Pagani, Franco; Pariyarath, Rajalakshmi; Stuani, Cristiana; García, Rafael Martínez; Baralle, Francisco E.

doi:10.1042/bj3260265

Cited by 13 publications

(4 citation statements)

References 32 publications

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“…In Yeh1, the first glycine of this lipase motif is replaced by a serine and in Yeh2 it is replaced by an alanine. The functionally important cysteine residues of LIPA (Cys248 and Cys257), however, are conserved in Yeh1 only (30,40). The predicted molecular masses of these lipases range from 62 to 66 kDa, which are in good agreement with the experimentally determined 70-kDa molecular mass of the enriched yeast steryl ester hydrolase activity (45).…”

Section: Yeh1 Yeh2supporting

confidence: 73%

The Saccharomyces cerevisiae YLL012/YEH1, YLR020/YEH2, and TGL1 Genes Encode a Novel Family of Membrane-Anchored Lipases That Are Required for Steryl Ester Hydrolysis

Köffel

Tiwari

Falquet

et al. 2005

Molecular and Cellular Biology

132

115

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Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. The formation of steryl esters is well characterized, but the mechanisms that control steryl ester mobilization upon cellular demand are less well understood. We have identified a family of three lipases of Saccharomyces cerevisiae that are required for efficient steryl ester mobilization. These lipases, encoded by YLL012/ YEH1, YLR020/YEH2, and TGL1, are paralogues of the mammalian acid lipase family, which is composed of the lysosomal acid lipase, the gastric lipase, and four novel as yet uncharacterized human open reading frames. Sterols are essential lipids of eukaryotic cells. They are present in two major forms: free sterols and steryl esters. Free sterols are synthesized in the endoplasmic reticulum (ER) membrane but are greatly enriched at the plasma membrane, which harbors 90% of the free-sterol pool of a cell (27). Steryl esters, on the other hand, serve as a storage form for fatty acids and sterols that are deposited in intracellular lipid bodies or particles. The conversion of free sterols and acyl coenzymes A to steryl esters is localized to the ER (14). The formation of steryl esters is important to maintain sterol homeostasis, as the steryl ester pool conceptually serves to buffer both excess and a lack of free sterols (12). The genes required for the synthesis of steryl esters in yeast have been identified, and mutants that lack steryl esters are viable, indicating that their synthesis is not essential under standard growth conditions (52, 53). The reverse process, however, how endogenously synthesized steryl esters are mobilized from their stores and hydrolyzed to release free sterols and fatty acids, is less well understood, even though cleavage of this ester bond is generally thought to be catalyzed by a lipase.Lipases constitute a heterogeneous family of proteins with carboxyl esterase activity and are activated by a lipid-water interface (for reviews, see references 34 and 51). Crystallographic analysis revealed that lipases are remarkably similar in structure despite low overall sequence conservation. They belong to the alpha/beta hydrolase fold family of enzymes with diverse hydrolytic functions. Their catalytic domain consist of a predominantly parallel beta-sheet structure of eight beta sheets connected by helical loops of various lengths that contain the catalytic-triad residues serine, aspartic acid, and histidine (10,37,42). The nucleophilic serine of this triad is itself part of the nearly ubiquitous lipase consensus sequence motif GXSXG (17). These three amino acids, assisted by the dipolar oxyanion hole, which stabilizes the charge distribution of the transition state, catalyze the hydrolysis of the ester bond (37).Only a few gene products with lipolytic activity against neutral lipids have been characterized in Saccharomyces cerevisiae. Tgl1 has been proposed to be a triglyceride-specific lipase on the basis of its homology to lipases from humans and rats, but enzymat...

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Section: Yeh1 Yeh2supporting

confidence: 73%

The Saccharomyces cerevisiae YLL012/YEH1, YLR020/YEH2, and TGL1 Genes Encode a Novel Family of Membrane-Anchored Lipases That Are Required for Steryl Ester Hydrolysis

Köffel

Tiwari

Falquet

et al. 2005

Molecular and Cellular Biology

132

115

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“…Although it has been suggested that CESD may be less severe than WD due to a residual level of enzymatic activity (10,14), direct proof of this hypothesis is still lacking. Furthermore, as the cholesteryl esterase and triacylglycerol lipase activities of LAL can be separated (15,16), it is also conceivable that some LAL mutants could selectively retain a residual activity toward one of these two substrates. We have been able to show the direct involvement of some LAL mutants in the pathogenesis of acid lipase deficiency in general, but the accuracy of the methodology used (transient expression in mammalian cells) did not allow us to establish firmly whether some of the mutants result in low levels of activity (4) which would be responsible for the benign course of CESD.…”

Section: New Lysosomal Acid Lipase Gene Mutants Explain the Phenotype Of Wolman Disease And Cholesteryl Ester Storage Diseasementioning

confidence: 99%

New lysosomal acid lipase gene mutants explain the phenotype ofWolman disease and cholesteryl ester storage disease

Pagani

Pariyarath

García

et al. 1998

Journal of Lipid Research

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Deficiency of lysosomal acid lipase (LAL) leads to either Wolman disease (WD) or the more benign cholesteryl ester storage disease (CESD). To identify the molecular basis of the different phenotypes we have characterised the LAL gene mutations in three new patients with LAL deficiency. A patient with WD was homozygote for a null allele Y303X. The other two patients, with CESD, presented either homozygosity for T267I or compound heterozygosity consisting of Q64R and an exon 8 donor splice site substitution (G ¨ A in position Ϫ 1). The mutants T267I and Q64R and the previously reported L273S, G66V, and H274Y CESD substitutions, overexpressed in stable clones, were found to be fully glycosylated and show an enzymatic activity of 3-8% of that of normal LAL. On the other hand, the ⌬ 254-277 mutant protein derived from exon 8 skipping and the Y303X protein were totally inactive. By transient transfection of hybrid minigene constructs, the CESD G ¨ A ( Ϫ 1) substitution resulted in partial exon inclusion, thus allowing the production of a small amount of normal LAL mRNA and hence of a functional enzyme. In contrast, a G ¨ A substitution observed in WD at position ϩ 1 of the same exon 8 donor site resulted in complete exon skipping and the sole production of an inactive ⌬ 254-277 protein. In conclusion, LAL genotypes determine the level of residual enzymatic activity, thus explaining the severity of the phenotype.

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“…The predicted sequence for cLIPA indicated the presence of six cysteine residues; one of them is present in the signal peptide region, while GL has only three. It is proposed that the cysteine residues that present at positions Cys 248 , Cys 257 and Cys 265 may be involved in the cholesteryl ester hydrolase activity in LIPA which is missing in GL [ 31 , 32 ].…”

Section: Discussionmentioning

confidence: 99%

Cloning, Phylogenetic Analysis and 3D Modeling of a Putative Lysosomal Acid Lipase from the Camel, Camelus dromedarius

Ataya

2012

Molecules

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Acid lipase belongs to a family of enzymes that is mainly present in lysosomes of different organs and the stomach. It is characterized by its capacity to withstand acidic conditions while maintaining high lipolytic activity. We cloned for the first time the full coding sequence of camel’s lysosomal acid lipase, cLIPA using RT-PCR technique (Genbank accession numbers JF803951 and AEG75815, for the nucleotide and aminoacid sequences respectively). The cDNA sequencing revealed an open reading frame of 1,197 nucleotides that encodes a protein of 399 aminoacids which was similar to that from other related mammalian species. Bioinformatic analysis was used to determine the aminoacid sequence, 3D structure and phylogeny of cLIPA. Bioinformatics analysis suggested the molecular weight of the translated protein to be 45.57 kDa, which could be decreased to 43.16 kDa after the removal of a signal peptide comprising the first 21 aminoacids. The deduced cLIPA sequences exhibited high identity with Equus caballus (86%), Numascus leucogenys (85%), Homo sapiens (84%), Sus scrofa (84%), Bos taurus (82%) and Ovis aries (81%). cLIPA shows high aminoacid sequence identity with human and dog-gastric lipases (58%, and 59% respectively) which makes it relevant to build a 3D structure model for cLIPA. The comparison confirms the presence of the catalytic triad and the oxyanion hole in cLIPA. Phylogenetic analysis revealed that camel cLIPA is grouped with monkey, human, pig, cow and goat. The level of expression of cLIPA in five camel tissues was examined using Real Time-PCR. The highest level of cLIPA transcript was found in the camel testis (162%), followed by spleen (129%), liver (100%), kidney (20.5%) and lung (17.4%).

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Cysteine residues in human lysosomal acid lipase are involved in selective cholesteryl esterase activity

Cited by 13 publications

References 32 publications

The Saccharomyces cerevisiae YLL012/YEH1, YLR020/YEH2, and TGL1 Genes Encode a Novel Family of Membrane-Anchored Lipases That Are Required for Steryl Ester Hydrolysis

The Saccharomyces cerevisiae YLL012/YEH1, YLR020/YEH2, and TGL1 Genes Encode a Novel Family of Membrane-Anchored Lipases That Are Required for Steryl Ester Hydrolysis

New lysosomal acid lipase gene mutants explain the phenotype ofWolman disease and cholesteryl ester storage disease

Cloning, Phylogenetic Analysis and 3D Modeling of a Putative Lysosomal Acid Lipase from the Camel, Camelus dromedarius

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