Crystal structure of human lysosomal acid lipase and its implications in cholesteryl ester storage disease

Lysosomal acid lipase (LAL) is a serine hydrolase that hydrolyzes cholesteryl ester (CE) and TGs delivered to the lysosomes into free cholesterol and fatty acids. LAL deficiency due to mutations in the LAL gene (LIPA) results in accumulation of TGs and cholesterol esters in various tissues of the body leading to pathological conditions such as Wolman's disease and CE storage disease (CESD). Here, we present the first crystal structure of recombinant human LAL (HLAL) to 2.6 Å resolution in its closed form. The crystal structure was enabled by mutating three of the six potential glycosylation sites. The overall structure of HLAL closely resembles that of the evolutionarily related human gastric lipase (HGL). It consists of a core domain belonging to the classical α/β hydrolase-fold family with a classical catalytic triad (Ser-153, His-353, Asp-324), an oxyanion hole, and a “cap” domain, which regulates substrate entry to the catalytic site. Most significant structural differences between HLAL and HGL exist at the lid region. Deletion of the short helix, 238NLCFLLC244, at the lid region implied a possible role in regulating the highly hydrophobic substrate binding site from self-oligomerization during interfacial activation. We also performed molecular dynamic simulations of dog gastric lipase (lid-open form) and HLAL to gain insights and speculated a possible role of the human mutant, H274Y, leading to CESD. Lysosomal acid lipase (LAL) is a serine hydrolase that hydrolyzes cholesteryl ester (CE) and TGs delivered to the lysosomes into free cholesterol and fatty acids. LAL deficiency due to mutations in the LAL gene (LIPA) results in accumulation of TGs and cholesterol esters in various tissues of the body leading to pathological conditions such as Wolman's disease and CE storage disease (CESD). Here, we present the first crystal structure of recombinant human LAL (HLAL) to 2.6 Å resolution in its closed form. The crystal structure was enabled by mutating three of the six potential glycosylation sites. The overall structure of HLAL closely resembles that of the evolutionarily related human gastric lipase (HGL). It consists of a core domain belonging to the classical α/β hydrolase-fold family with a classical catalytic triad (Ser-153, His-353, Asp-324), an oxyanion hole, and a “cap” domain, which regulates substrate entry to the catalytic site. Most significant structural differences between HLAL and HGL exist at the lid region. Deletion of the short helix, 238NLCFLLC244, at the lid region implied a possible role in regulating the highly hydrophobic substrate binding site from self-oligomerization during interfacial activation. We also performed molecular dynamic simulations of dog gastric lipase (lid-open form) and HLAL to gain insights and speculated a possible role of the human mutant, H274Y, leading to CESD. Lysosomal acid lipase (LAL) is a serine hydrolase enzyme located in the lysosome, where it breaks down cholesteryl esters (CEs) and TGs into free cholesterol and fatty acids (1Goldstein J.L. Dana S.E. Faust J.R. Beaudet A.L. Brown M.S. Role of lysosomal acid lipase in the metabolism of plasma low density lipoprotein. Observations in cultured fibroblasts from a patient with cholesteryl ester storage disease.J. Biol. Chem. 1975; 250: 8487-8495Abstract Full Text PDF PubMed Google Scholar, 2Sando G.N. Rosenbaum L.M. Human lysosomal acid lipase/cholesteryl ester hydrolase. Purification and properties of the form secreted by fibroblasts in microcarrier culture.J. Biol. Chem. 1985; 260: 15186-15193Abstract Full Text PDF PubMed Google Scholar, 3Brown M.S. Kovanen P.T. Goldstein J.L. Regulation of plasma cholesterol by lipoprotein receptors.Science. 1981; 212: 628-635Crossref PubMed Scopus (592) Google Scholar). LAL belongs to a gene family of mammalian acid lipases that includes gastric and lingual lipases (4Lohse P. Lohse P. Chahrokh-Zadeh S. Seidel D. The acid lipase gene family: three enzymes, one highly conserved gene structure.J. Lipid Res. 1997; 38: 880-891Abstract Full Text PDF PubMed Google Scholar). The enzyme is synthesized by almost all cells except erythrocytes. Defective LAL activity/diminished acid lipase have been associated with two autosomal recessive traits in humans: Wolman's disease (WD) and CE storage disease (CESD) (5Anderson R.A. Bryson G.M. Parks J.S. Lysosomal acid lipase mutations that determine phenotype in Wolman and cholesterol ester storage disease.Mol. Genet. Metab. 1999; 68: 333-345Crossref PubMed Scopus (70) Google Scholar, 6Ries S. Aslanidis C. Fehringer P. Carel J.C. Gendrel D. Schmitz G. A new mutation in the gene for lysosomal acid lipase leads to Wolman disease in an African kindred.J. Lipid Res. 1996; 37: 1761-1765Abstract Full Text PDF PubMed Google Scholar). However, the clinical and biochemical phenotypes manifested by WD and CESD are significantly different. WD is an early onset disease that primarily affects infants and results in demise in the first three to twelve months of life (7Browne M. Somers G. Savoia H. Kukuruzovic R. Wolman's disease in an infant.Br. J. Haematol. 2003; 122: 522Crossref PubMed Scopus (4) Google Scholar). It is associated with severe liver complications, malabsorption, and growth failure (7Browne M. Somers G. Savoia H. Kukuruzovic R. Wolman's disease in an infant.Br. J. Haematol. 2003; 122: 522Crossref PubMed Scopus (4) Google Scholar). In contrast, CESD is a late onset form of the disease that occurs in both children and adults with residual (1% to ∼5%) LAL activity (8Balwani M. Breen C. Enns G.M. Deegan P.B. Honzik T. Jones S. Kane J.P. Malinova V. Sharma R. 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Lysosomal acid lipase deficiency: wolman disease and cholesteryl ester storage disease.Pril (Makedon Akad Nauk Umet Odd Med Nauki). 2014; 35: 99-106PubMed Google Scholar). The molecular mechanisms of these LAL loss-of-function mutants leading to WD and CESD have yet to be explored. Current treatment options for CESD phenotypes are limited to diets that exclude cholesterol- and TG-rich food, cholesterol-lowering drugs such as statins, and ultimately liver transplantation. Recombinant LAL replacement therapy has been shown to be effective in animal models and human clinical trials. Recently, Kanuma (sebelipase alfa) (Alexion Pharmaceuticals, Cheshire, CT) has obtained marketing authorization for the treatment of CESD in Europe and the United States with recombinant human LAL (HLAL) (13Rader D.J. Lysosomal acid lipase deficiency–a new therapy for a genetic lipid disease.N. Engl. J. Med. 2015; 373: 1071-1073Crossref PubMed Scopus (26) Google Scholar, 14Burton B.K. Balwani M. Feillet F. Baric I. Burrow T.A. Camarena Grande C. Coker M. Consuelo-Sanchez A. Deegan P. Di Rocco M. et al.A phase 3 trial of sebelipase alfa in lysosomal acid lipase deficiency.N. Engl. J. Med. 2015; 373: 1010-1020Crossref PubMed Scopus (145) Google Scholar, 15Du H. Cameron T.L. Garger S.J. Pogue G.P. Wolman ester storage disease: of human lysosomal acid lipase in Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). The gene (LIPA) that HLAL was and it consists of R.A. R. M. In of the genetic the lysosomal acid lipase/cholesteryl (LIPA) in Wolman disease to PubMed Scopus Google Scholar). It and acid with human gastric lipase and lingual lipase (4Lohse P. Lohse P. Chahrokh-Zadeh S. Seidel D. The acid lipase gene family: three enzymes, one highly conserved gene structure.J. Lipid Res. 1997; 38: 880-891Abstract Full Text PDF PubMed Google Scholar). HLAL a acid the of acids with an molecular of It six glycosylation three of which and HLAL are highly conserved of the lipase gene family O. C. D. of potential glycosylation of lysosomal acid PubMed Scopus Google Scholar). have that the and glycosylation are for LAL and O. C. D. of potential glycosylation of lysosomal acid PubMed Scopus Google Scholar, S. H. of lysosomal acid lipase by and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A of highly conserved and HLAL is present in all three and Human LAL three in the and one in the of and or has significantly hydrolase activity in a the mutants activity F. R. C. R. in human lysosomal acid lipase are in cholesteryl J. 1997; PubMed Scopus Google Scholar). The of the of HGL and dog gastric lipase have been A. S. M. L. R. C. structure of human gastric lipase and of lysosomal acid two of Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. L. M. S. P. V. R. C. structure of the form of dog gastric lipase in with a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The structure of HGL form) consists of a core and a domain, which includes a region that the site A. S. M. L. R. C. structure of human gastric lipase and of lysosomal acid two of Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). that is common to all α/β hydrolase family is the of a classical catalytic triad and an oxyanion A. S. M. L. R. C. structure of human gastric lipase and of lysosomal acid two of Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, D.L. M. B. F. M. S.J. I. J. et hydrolase PubMed Scopus Google Scholar). The of the lid region is for the substrate to the catalytic at the substrate binding site. the has the of and a binding for a its substrate A. L. M. S. P. V. R. C. structure of the form of dog gastric lipase in with a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the crystal structure of the a of HLAL was A. S. M. L. R. C. structure of human gastric lipase and of lysosomal acid two of Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In the present we have and a glycosylation and of HLAL and the crystal structure at 2.6 Å resolution in its with and a lid where a of the lid domain was performed with an to gain insights into the of the lid region. dynamic with the enzyme performed to the domain A molecular the of of HLAL CESD mutant, H274Y, is also The HLAL was from gene with for for site and to the of the enzyme to and The synthesized also includes and was into the and the recombinant was the the and performed the to the The was by and with and The lid was by from the of and was by cells at a density of cells with a of of in a as F. S. M. J. and characterization of human lysosomal acid lipase gene (LIPA) mutation for cholesteryl ester storage disease (CESD) 2015; PubMed Scopus Google Scholar). The that the secreted recombinant was a and as F. S. M. J. and characterization of human lysosomal acid lipase gene (LIPA) mutation for cholesteryl ester storage disease (CESD) 2015; PubMed Scopus Google Scholar). The and to a of the The was by with to a of of of HLAL at The recombinant HLAL was by a in and the with as a or at The activity was a enzyme that the free cholesterol of the to a highly cholesterol and as F. S. M. J. and characterization of human lysosomal acid lipase gene (LIPA) mutation for cholesteryl ester storage disease (CESD) 2015; PubMed Scopus Google Scholar). The a and the for A with a was to the of at as F. S. M. J. and characterization of human lysosomal acid lipase gene (LIPA) mutation for cholesteryl ester storage disease (CESD) 2015; PubMed Scopus Google Scholar). In of at was with of in a and into the The was from to in with a of and of are and The in of as a of The first of the where is for was and the was as the in the glycosylation of the and HLAL was and treatment of the performed as F. S. M. J. and characterization of human lysosomal acid lipase gene (LIPA) mutation for cholesteryl ester storage disease (CESD) 2015; PubMed Scopus Google Scholar). by a the at is acid in and is acid in from in the with the for in the with of and dynamic of 3 for performed the with of and of for of to from of the to the was obtained by of enzyme to a which of at or at was to to a of and The at for The was by and for The and by The LAL was of in and at to a of obtained from recombinant in the in in a and at the of the of the a C. C. P. A. O. T. G. and with the Biol. PubMed Scopus Google Scholar). The structure was by molecular replacement R.J. PubMed Scopus Google and the HGL structure as was the P. C. C. G. of with in Biol. PubMed Scopus Google and P. for molecular Biol. PubMed Scopus Google Scholar). are in and with are at the and where the density is the of the The overall J.S. structure for Biol. PubMed Scopus Google is and the is with The Scholar). 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LAL has also been in the of in the at T. of human lysosomal acid lipase to Full Text PDF PubMed Scopus Google Scholar, F. H. Lysosomal acid lipase in lipid metabolism and Biol. PubMed Scopus Google Scholar). for HGL form) and (lid-open form) in with have been A. S. M. L. R. C. structure of human gastric lipase and of lysosomal acid two of Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. L. M. S. P. V. R. C. structure of the form of dog gastric lipase in with a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, the molecular mechanisms leading to of of these and mutations in the In we the first crystal structure of HLAL at 2.6 Å resolution in its it with crystal of HGL and and the possible role of the highly domain in regulating the catalytic site. crystal with the and HLAL We speculated that the be the crystal are of for these of the of these to the S. A from lysosomal acid lipase is to in 1996; Google Scholar). Human LAL has six belonging to the classical of HLAL in cells the of and as the to at all six glycosylation et O. C. D. of potential glycosylation of lysosomal acid PubMed Scopus Google the of of LAL of all potential glycosylation sites. that glycosylation at and are for LAL mutations of and have significant effect LAL activity with the we have a by and into A has shown that of all glycosylation of LAL a glycosylation a activity and of the enzyme to the of the cultured cells S. H. of lysosomal acid lipase by and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, results that the of three glycosylation significantly to the and glycosylation the activity and of the at Recombinant and enabled to the first structure of HLAL has structural with the crystal structure of are significant structural between HGL and HLAL at the domain where are acid with the HGL structure A. S. M. L. R. C. structure of human gastric lipase and of lysosomal acid two of Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google the density from and density for from to as However, these in the structure of It has been that the is by with the lid the by A. L. M. S. P. V. R. C. structure of the form of dog gastric lipase in with a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is of the α/β hydrolase the which is of the catalytic is located at an the between a and a is located a between and and is in with and His-353, which to the to with the of the of at the between the core and in HLAL the a of the catalytic is by and two and a which with the of the site is by hydrophobic and two with to the catalytic The domain is a of to and It a lid that a closed in at the between and core The lid region is by an and two short that the site. and the lid as with the and of the lid A. L. M. S. P. V. R. C. structure of the form of dog gastric lipase in with a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It is from the structure that the catalytic is and substrate in the site. The of be to to the The closed lid of HLAL is of three short and the form of the lid is of two A. L. M. S. P. V. R. C. structure of the form of dog gastric lipase in with a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the overall structural between the HGL and of the of the lid region significant structural an of Å in human with the domain of an and the catalytic activity and of lipase and 1997; PubMed Scopus Google Scholar, B. C. lipase and and of and J. 37: PubMed Scopus Google Scholar). However, such a for HLAL has been to The structure also density for three of the and the three potential and to The of HLAL six in the with three in and form a the are as in the of and are in the lid region and are the catalytic is Å from catalytic of the lid region from the structure a hydrophobic the a for lipid substrate was by the structure of the A. L. M. S. P. V. R. C. structure of the form of dog gastric lipase in with a Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). mutations and of and in the domain have been associated with CESD F. R. R. C. B. G. of lysosomal acid lipase mutants in three patients with cholesteryl ester storage Genet. 1996; PubMed Scopus Google Scholar, Y. the serine 1999; PubMed Scopus Google Scholar). mutants residual activity F. R. R. C. B. G. of lysosomal acid lipase mutants in three patients with cholesteryl ester storage Genet. 1996; PubMed Scopus Google Scholar). In we have a by a acid 238NLCFLLC244, that is the and its role in regulating the catalytic site. We speculated that the of the lid to the substrate and to catalytic the catalytic activity of was significantly with the in enzyme The profile that the of and the the to its molecular that the of activity of is to the or of the its highly hydrophobic site. we an to the site of in with and in enzyme site with of such as the serine and Y. the serine 1999; PubMed Scopus Google Scholar). between and enzymes, by a with a which the catalytic site to the of an We have which to the in the site of the enzyme in an with activity and highly to the at at of these at that the hydrophobic site is by the lid region and its leads to an form in the of lipid It is possible that the lid and substrate binding in to the enzyme from and simulations have been to gain insights into domain and of H. G. Role of in the of from molecular PubMed Scopus Google Scholar). the HLAL we performed of HLAL and at both and in the closed a between and is leads to a of to is located at a and that the lid and is in to the core domain We at is and a with and the lid in a closed form. at is in with and leads to the of the lid into an to the of the we a of the form at the a at in the a significant is between and that leads to of the lid region HLAL is from that of an HGL and A. C. S. G. simulations of human and dog gastric insights into domain PubMed Scopus Google Scholar). simulations of HGL an and and in the lid of the conditions and or A. C. S. G. simulations of human and dog gastric insights into domain PubMed Scopus Google Scholar). the from the core domain also in HGL A. C. S. G. simulations of human and dog gastric insights into domain PubMed Scopus Google Scholar). However, in we the We have reported that mutating to the common loss-of-function in CESD in significant of lipase activity in to and F. S. M. J. and characterization of human lysosomal acid lipase gene (LIPA) mutation for cholesteryl ester storage disease (CESD) 2015; PubMed Scopus Google Scholar). We speculated that the between and core and the The of the lid domain the hydrophobic substrate binding site in the closed form is for the enzyme in an and in that the In HLAL in the is in a with and is by two and and the between the and a region we a of the at and the to that of the The results that of with significantly the to of the lid region to We that potential between the lid and core and the lid from the leading to by hydrophobic such as and However, to structural of HLAL with to that or or the activity of a to an effective for WD and CESD are the The and structure have been in the with with cholesteryl ester cholesteryl ester storage disease dog gastric lipase human gastric lipase human lysosomal acid lipase lysosomal acid lipase lid molecular glycosylation Wolman's disease