Structural bases of Wolman disease and cholesteryl ester storage disease

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Abstract

To elucidate the bases of Wolman disease (WD) and cholesteryl ester storage disease (CESD) from the viewpoint of enzyme structure, we constructed a structural model of human lysosomal acid lipase (LAL) using molecular modeling software Modeller. The results revealed that the residues responsible for WD/CESD tend to be less solvent-accessible than others. Then, we examined the structural changes in the LAL protein caused by the WD/CESD mutations, using molecular modeling software TINKER. The results indicated that conformational changes of the functionally important residues and/or large conformational changes tend to cause the severe clinical phenotype (WD), whereas small conformational changes tend to cause the mild clinical phenotype (CESD), although there have been several exceptions. Further structural analysis is required to clarify the relationship between the three-dimensional structural changes and clinical phenotypes.

Highlights

► We built a structural model of human lysosomal acid lipase. ► The residues responsible for WD/CESD tend to be more interior than the others. ► Large conformational changes tend to cause the severe clinical phenotype (WD). ► Conformational changes of the functionally important residues tend to cause WD. ► Small conformational changes tend to cause the mild clinical phenotype.

Introduction

Lysosomal acid lipase (LAL, EC 3.1.1.13) is essential for the hydrolysis of triglycerides and cholesteryl esters in lysosomes. A deficiency of LAL activity causes the accumulation of triglycerides and cholesteryl esters in lysosomes, leading to two autosomal recessive genetic disorders, Wolman disease (WD; McKusick 278000) and cholesteryl ester storage disease (CESD; McKusick 21500) [1]. Patients with WD, an early-onset severe phenotype, usually succumb to hepatic and adrenal failure within the first year of life. In contrast, patients with CESD exhibit a late-onset moderate phenotype.

Both WD and CESD are caused by mutations in the LAL gene (LIPA) on chromosome 10q23.2–q23.3, and they are basically distinguished by the level of residual activity of the mutant LAL [2]. In WD, the LAL activity is almost completely absent. In contrast, the mutant LAL associated with CESD exhibits residual LAL activity [2].

So far, various kinds of gene mutations causing WD and CESD have been identified [1], [3], [4], [5], [6], [7], [8], [9]. In general, nonsense mutations, deletions, and insertions in LIPA are associated with WD, although there are some exceptions (e.g., p.G245X). On the other hand, as to missense mutations, the phenotypes are heterogeneous; some of them cause WD, and the others CESD. Previous investigation revealed that patients heterozygous for a WD mutation (E8SJM + 1) and a CESD one (p.G66V, p.P181L, p.L273S or p.H274Y) exhibited the CESD phenotype [6], suggesting that CESD mutations dominantly influence the phenotype.

Roussel et al. determined the crystal structure of human gastric lipase and built the homology model of LAL with human gastric lipase as a template. As LAL displays high sequence homology with gastric lipase without any insertion or deletion (amino acid identity 59%), the reliability of their homology model of LAL is thought to be high. Based on the homology model, they gave possible explanations for some partial deletions and missense mutations [10]. However, the details of the influence of structural changes in LAL on the pathogeneses of WD and CESD remain obscure.

In this study, we performed structural analysis of LAL from a different viewpoint to obtain further insight into the bases of WD and CESD. First, we constructed a three-dimensional model of LAL, and then determined the solvent-accessible surface area (ASA) values. Then, we built structural models of the mutant LAL proteins and examined their structural changes by calculating the numbers of atoms influenced by the amino acid replacements, and determined the root-mean-square deviation (RMSD) values. Furthermore, we examined the distributions and degrees of structural changes caused by the amino acid substitutions by coloring the influenced atoms based on the distances between the wild-type and mutant ones.

Section snippets

Mutations

The missense mutations analyzed in this study are summarized in Table 1. The clinical phenotypes and residual activity were written according to the original reports.

Structural modeling of mutant LAL proteins

Structural models of mutant LAL proteins were constructed with molecular modeling software Modeller [11] and TINKER [12], [13], [14], [15], [16] using the structure of human gastric lipase (protein data bank code: 1HLG [10]) as a template. All mutant models were energy minimized until the root-mean-square gradient value was lower

Localization of the amino acid residues of which substitutions are associated with WD/CESD in the LAL molecule

We built a three-dimensional model of human LAL using human gastric lipase as a template (Figs. 1a and b). Then, we determined the locations of amino acid substitutions that are associated with WD/CESD in the human LAL molecule (Fig. 1b). The ASA values of the amino acids, of which substitutions are responsible for WD/CESD, are shown in Table 1. The average value for the residues associated with WD/CESD is 11.9 Å2, that for the others being 40.7 Å2. The results of the F-test (p < 0.01) and the

Discussion

To elucidate the molecular bases of WD/CESD, it is very important to determine the conformational changes in human LAL associated with these diseases. Roussel et al. presented a three dimensional-model of wild type LAL and examined two partial deletions and three missense mutations [10]. But, to the best of our knowledge, there has been no comprehensive research on conformational changes in human LAL caused by amino acid substitutions leading to WD/CESD.

In this study, we investigated the

Acknowledgments

This work was supported by the Program for Research on Intractable Diseases of Health and Labor Science Research Grants (HS); the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (ID: 09-15, HS); the JAPS Asia/Africa Scientific Platform Program (HS); the Japan Society for the Promotion of Science (JSPS ID: 21390314, HS); and the High-Tech Research Center Project of the Ministry of Education, Culture, Sports, Science and

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Disclosure: All authors declare no competing interest.

1

These authors equally contributed to this work.

2

Present address is Astellas Pharma Inc.

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