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. 2013 Dec 26;8(12):e84267. doi: 10.1371/journal.pone.0084267

Comparative Study of Structural Changes Caused by Different Substitutions at the Same Residue on α-Galactosidase A

Seiji Saito 1,#, Kazuki Ohno 2,¤,#, Hitoshi Sakuraba 3,*
Editor: Eugene A Permyakov4
PMCID: PMC3873411  PMID: 24386359

Abstract

Missense mutations in the α-galactosidase A (GLA) gene comprising the majority of mutations responsible for Fabry disease result in heterogeneous phenotypes ranging from the early onset severe “classic” form to the “later-onset” milder form. To elucidate the molecular basis of Fabry disease from the viewpoint of structural biology, we comprehensively examined the effects of different substitutions at the same residue in the amino acid sequence of GLA on the structural change in the enzyme molecule and the clinical phenotype by calculating the number of atoms affected and the root-mean-square-distance value, and by coloring of the atoms influenced by the amino acid replacements. The results revealed that the severity of the structural change influences the disease progression, i.e., a small structural change tends to lead to the later-onset form and a large one to the classic form. Furthermore, the study revealed the residues important for expression of the GLA activity, i.e., residues involved in construction of the active site, a disulfide bond or a dimer. Structural study from such a viewpoint is useful for elucidating the basis of Fabry disease.

Introduction

Fabry disease (MIM 301500) is an X-linked genetic disorder resulting from a deficiency of α-galactosidase A (GLA; EC 3.2.1.22) activity [1]. GLA deficiency causes the progressive accumulation of glycolipids, predominantly globotriaosylceramide, in lysosomes of cells. The disease exhibits a wide range of clinical phenotypes, from the early-onset severe “classic” form to the “later-onset” milder one [2]. Generally, male patients with the classic form of Fabry disease, who have little or no GLA activity, develop pain in the peripheral extremities, hypohidrosis, angiokeratomas and corneal opacities in childhood or adolescence, and manifest renal, cardiac, and cerebrovascular complications in the fourth to fifth decade of life [3]. On the other hand, male patients with the later-onset form, who have residual GLA activity, develop heart and kidney disorders without the childhood symptoms [4]. Heterozygous Fabry females exhibit a wide spectrum of disease severity ranging from asymptomatic to presentation with the classic disease due to random X-chromosomal inactivation [5].

The GLA gene is localized to Xq22.1 and encodes a precursor GLA comprising a 429-amino acid polypeptide, the enzyme being glycosylated and then processed to the mature form comprising 398 amino acids, and it exists as a homodimer in lysosomes [1]. Each monomer contains a (β/α)8 barrel domain containing the active site and an anti- parallel β-sheet domain [6]. So far, more than 600 genetic mutations causing Fabry disease have been identified, and it is known that gross alterations, nonsense mutations, and most of the splicing mutations of the GLA gene lead to the classic form. However missense mutations comprising the majority of mutations result in heterogeneous phenotypes ranging from the classic form to the later-onset one.

Previously, Garman and his research group determined the GLA structure by means of X-ray crystallography and analyzed the locations of missense and nonsense mutations in the three-dimensional structure [6], [7]. Our research group studied structural changes caused by missense mutations responsible for Fabry disease by calculating the numbers of affected atoms and the root-mean-square distance (RMSD) values [8], and proposed a phenotype prediction model based on sequential and structural information [9].

In this study, we comprehensively examined different substitutions at the same residue in the amino acid sequence of GLA, focusing on their effects on the structural change in the enzyme protein and the clinical phenotype, as such investigation will provide us with information about the relationship between the enzyme structure and the disease.

Materials and Methods

GLA missense mutations

We collected GLA missense mutations and polymorphisms registered on the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/) and Fabry database (http://fabry-database.org/). From them, we selected cases in which more than two substitutions at the same residue in the amino acid sequence of GLA have been reported. Finally, we analyzed 157 amino acid substitutions at 67 residues in this study.

Structural modeling of mutant GLAs

Structural models of mutant GLA monomers were built by means of homology modeling using molecular modeling software, TINKER (http://dasher.wustl.edu/tinker/) [10]–[14]. The crystal structure of human GLA (PDB: 1R46) [6] was used as a template, and energy minimization was performed. The root-mean-square gradient value was set at 0.05 kcal/mol·Å.

Calculation of the number of atoms influenced by an amino acid substitution and the RMSD values between the wild type GLA and mutant GLAs

Each mutant model was superimposed on the wild type GLA structure based on the Cα atoms by the least-square-mean fitting algorithm, in which the optimal rotations and translations are found by minimizing the sum of the squared distances among all structures in the superposition [15]–[19]. We defined that the atom was affected by an amino acid substitution when the position of the atom in a mutant differed from that in the wild type structure by more than 0.15 Å. We calculated the numbers of atoms affected in the main chain and in the side chain of the enzyme, and in the active site (E170 and E231). Then, we calculated the RMSD values between the wild type GLA and mutant GLAs [15]–[19].

Determination of the solvent-accessible surface area (ASA) value

The ASA value of an amino acid residue in the wild type GLA was calculated using Stride (http://webclu.bio.wzw.tum.de/stride/) to evaluate the location of the residue in the GLA molecule.

Coloring of the atoms influenced by an amino acid substitution

To determine the influence of the amino acid substitutions geographically and semi- quantitatively, coloring of the influenced atoms in the three-dimensional structure of the enzyme molecule was performed for 12 mutants (M72I, M72R, M72V, E66G, E66K, E66Q, C56G, C56F, C56Y, W236C, W236L, and W236R) as to four positions (M72, E66, C56, and W236) in the GLA structure. The colors of affected atoms were shown on the basis of the distance between the wild type and mutant one.

Statistical analysis

To determine the differences in the number of the affected atoms and the RMSD value between the classic Fabry group and later-onset one, statistical analysis was performed using Excel 2013 (Microsoft, Redmond, WA) by means of one side Welch's t test, it being taken that there was a significant difference if p<0.05. Then, power analysis (http://www.statmethods.net/stats/power.html) was performed using G*POWER3 to evaluate statistical power for this Welch's t test [20]. In power analysis calculation, sample sizes of two groups and significant level were set to 134, 11, and 0.05, respectively.

Results

Different substitutions at the same residue in the amino acid sequence of GLA

We examined the numbers of affected atoms for the whole enzyme protein and for the active site, and the RMSD and ASA values. The results are shown in Table 1. The numbers of atoms affected in the main chain and in the side chain, and the RMSD values in the classic Fabry group were 107±129 (134), 131±152 (134), and 0.089±0.074 Å (134), respectively. The values are expressed as average ± standard deviation (number of cases). On the other hand, in the later-onset Fabry group, they were 23±36 (11), 28±50 (11), and 0.033±0.038 Å (11), respectively. The statistical analysis showed significant differences between the classic Fabry group and the later-onset Fabry group in numbers of affected atoms in the main chain (P<0.001, Welch's t test) and in the side chain (P<0.001, Welch's t test), and RMSD (P<0.001, Welch's t test). The results of the power analysis revealed that the estimated values of power were 0.70, 0.72, and 0.80 for numbers of affected atoms in the main chain and in the side chain, and RMSD, respectively. This suggests that the structural change resulting from the amino acid substitutions leading to the classic phenotype is essentially greater than that in the later-onset one, although there are some exceptional cases, i.e., in R112H and R301Q, the numbers of affected atoms and the RMSD values are apparently large, although the patients with these mutations exhibited the later-onset phenotype (Table 1). Furthermore, the results revealed that there were no later-onset Fabry cases in which the structure of the active site was affected, although there were 57 affected cases among the 134 classic Fabry ones. This suggests that a defect of the active site tends to lead to the classic phenotype.

Table 1. Different substitutions at the same residue of the amino acid sequence of α-galactosidase A.

Genotype Phenotype Number of affected atoms RMSD ASA Reference
Main chain Side chain Active site (Å) (Å2)
N34K Classic 165 153 0 0.196 25.7 Hum Genomics 2006, 2: 297–309
N34S Classic 6 1 0 0.029 25.7 Am J Hum Genet 1993, 53: 1186–97
P40L Classic 80 83 0 0.111 0.8 J Invest Med 2000, 48: 227–35
P40S Classic 15 12 0 0.032 0.8 FEBS Lett 1990, 259: 353–6
M42T Classic 9 9 0 0.023 3.8 Mol Genet Metab 2002, 76: 23–30
M42V Classic 12 13 0 0.028 3.8 Eur J Hum Genet 1996, 4: 219–24
G43R Classic 413 471 12 0.177 0.0 Mol Med 2002, 8: 306–12
G43V Classic 219 250 1 0.126 0.0 Mol Genet Metab 2002, 76: 23–30
H46R Classic 25 27 0 0.043 0.0 Mol Med 1997, 3: 174–82
H46Y Hetero 83 119 0 0.088 0.0 Hum Mutat 2001, 18: 459
R49C Classic 125 136 0 0.109 55.3 Pharmacogenet Genomics 2008, 18: 773–80
R49G Classic 182 197 0 0.169 55.3 Mol Med 2002, 8: 306–12
R49L Classic 240 259 4 0.201 55.3 Hum Mol Genet 1994, 3: 667–9
R49P Classic 174 209 2 0.125 55.3 Hum Mutat 2001, 18: 459
R49S Classic 361 365 2 0.251 55.3 Eur J Hum Genet 1996, 4: 219–24
C52R Classic 195 242 15 0.136 49.0 Hum Mutat 1996, 8: 38–43
C52S Classic 2 1 0 0.022 49.0 Hum Mol Genet 1994, 3: 1795–9
C52Y Classic 56 75 0 0.073 49.0 Biochim Biophys Acta 2010, 1802: 247–52
C56F Classic 67 78 0 0.128 38.4 Hum Mol Genet 1994, 3: 1795–9
C56G Classic 51 60 0 0.131 38.4 Am J Hum Genet 1993, 53: 1186–97
C56Y Classic 58 65 0 0.132 38.4 Eur J Hum Genet 1996, 4: 219–24
E66G Classic 45 74 0 0.062 29.2 Am J Hum Genet 2006, 79: 31–40
E66K Classic 422 503 7 0.361 29.2 Hum Mutat 2005, 25: 412
E66Q Polymorphism 23 32 0 0.048 29.2 Hum Genet 1992, 89: 29–32.
M72I Classic 38 46 0 0.054 0.0 Mol Med 2002, 8: 306–12
M72R Classic 145 198 1 0.119 0.0 Ned Tijdschr Geneeskd 200, 144: 2412–5
M72V Later-onset 7 6 0 0.026 0.0 Hum Mutat 1998, Suppl 1: S213–16
L89P Classic 6 12 0 0.023 0.0 Mol Med 1997, 3: 174–82
L89R Classic 286 337 4 0.164 0.0 Hum Mol Genet 1994, 3: 1795–9
D92H Classic 324 482 14 0.140 0.2 Eur J Hum Genet 1996, 4: 219–24
D92Y Classic 408 580 16 0.182 0.2 Mol Med 1997, 3: 174–82
D93G Classic 216 264 14 0.133 0.4 Eur J Hum Genet 1996, 4: 219–24
D93N Classic 60 131 8 0.073 0.4 J Mol Med 2005, 83: 647–54
D93V Classic 200 284 12 0.128 0.4 Hum Genomics 2006, 2: 297–309
C94S Classic 20 19 0 0.031 0.6 Hum Mutat 2001, 18: 459
C94Y Classic 180 210 0 0.142 0.6 Mol Med 1997, 3: 174–82
A97P Classic 33 31 0 0.054 9.3 Br J Dermatol 2002, 147: 545–8
A97V Later-onset 10 12 0 0.025 9.3 Mol Med 1997, 3: 174–82
R100K Classic 37 22 0 0.043 28.8 Hum Mol Genet 1994, 3: 1795–9
R100T Classic 205 232 5 0.137 28.8 Mol Med 1997, 3: 174–82
R112C Classic 26 40 0 0.037 26.1 J Invest Med 2000, 48: 227–35
R112H Later-onset 70 70 0 0.082 26.1 Hum Mol Genet 1994, 3: 1795–9
R112S Classic 25 35 0 0.034 26.1 Hum Mutat 2005, 25: 299–305
F113L Later-onset 3 2 0 0.014 4.4 Mol Med 1997, 3: 174–82
F113S Hetero 0 0 0 0.005 4.4 Hum Mutat 2001, 18: 459
G138E Classic 167 203 1 0.121 0.0 Mol Med 2002, 8: 306–12
G138R Classic 209 259 4 0.277 0.0 Mol Med 1997, 3: 174–82
C142R Classic 50 89 13 0.065 37.2 Mol Med 1999, 5: 806–11
C142Y Classic 18 25 9 0.046 37.2 Hum Genet 1995, 95: 557–61
S148N Classic 21 25 0 0.039 0.0 J Invest Med 2000, 48: 227–35
S148R Classic 128 177 9 0.104 0.0 Mol Med 1997, 3: 174–82
W162C Classic 35 35 0 0.045 26.1 Hum Genet 1996, 98: 719–26
W162R Classic 28 44 0 0.043 26.1 Am J Hum Genet 1993, 53: 1186–97
L166G Classic 91 101 1 0.081 0.6 Hum Genomics 2006, 2: 297–309
L166V Classic 14 14 0 0.031 0.6 Hum Genet 1995, 95: 557–61
D170H Classic 236 302 8 0.120 0.0 Hum Mutat 2003 Sup, 22: 258
D170V Classic 88 134 4 0.072 0.0 Mol Med 1997, 3: 174–82
G171C Classic 69 85 7 0.065 4.5 J Dermatol Sci 2008, 52: 61–4
G171D Classic 88 103 8 0.095 4.5 Hum Mutat 2005, 25: 299–305
G171R Classic 315 347 12 0.284 4.5 J Mol Med 2005, 83: 647–54
C172F Classic 12 25 2 0.039 33.5 J Hum Genet 2001, 46: 192–6
C172G Classic 10 7 2 0.028 33.5 Hum Mutat 2003, 22: 486–92
C172R Classic 18 45 3 0.049 33.5 J Invest Med 2000, 48: 227–35
C172Y Classic 12 26 2 0.041 33.5 Hum Mol Genet 1994, 3: 1795–9
G183A Hetero 35 44 0 0.063 2.1 Biochim Biophys Acta 2010, 1802: 247–52
G183D Classic 262 296 8 0.204 2.1 Mol Med 1999, 5: 806–811
G183S Classic 62 103 0 0.088 2.1 Mol Genet Metab 2002, 76: 23–30
M187T Classic 0 0 0 0.006 0.0 Hum Genomics 2006, 2: 297–309
M187V Classic 8 13 0 0.030 0.0 J Invest Med 2000, 48: 227–35
S201F Classic 0 2 0 0.007 6.6 Hum Mutat 2005, 25: 299–305
S201Y Classic 0 2 0 0.011 6.6 Hum Genomics 2006, 2: 297–309
C202Y Classic 382 434 12 0.209 0.0 Mol Med 1997, 3: 174–82
C202W Hetero 276 313 7 0.175 0.0 Hum Mol Genet 1994, 3: 503–5
P205R Classic 455 571 9 0.257 0.2 Mol Genet Metab 2002, 76: 23–30
P205T Classic 6 16 0 0.022 0.2 Eur J Hum Genet 1996, 4: 219–24
Y207C Classic 3 4 3 0.017 50.9 J Inherit Metab Dis 2009, 32: 424–40
Y207S Classic 4 5 3 0.019 50.9 Mol Genet Metab 2002, 76: 23–30
P210L Later-onset 0 0 0 0.003 93.3 New case
P210S Later-onset 0 0 0 0.003 93.3 New case
Y216C Classic 1 1 0 0.005 7.5 Biochim Biophys Acta 2010, 1802: 247–52
Y216D Classic 187 246 6 0.145 7.5 Mol Med 1997, 3: 174–82
C223R Classic 518 589 15 0.255 0.0 Mol Genet Metab 2002, 76: 23–30
C223Y Classic 451 546 16 0.220 0.0 Mol Genet Metab 2002, 76: 23–30
N224D Classic 65 67 0 0.057 0.0 Hum Mutat 1998, Suppl 1; S288–90
N224S Classic 42 47 0 0.054 0.0 J Invest Med 2000, 48: 227–35
D231G Classic 20 58 3 0.045 54.9 Exp Mol Med 2009, 31: 1–7
D231V Classic 22 64 3 0.041 54.9 Hum Mutat 2008, 29: 331
D234E Classic 20 27 1 0.043 40.9 Hum Mutat 2005, 25: 299–305
D234Y Classic 352 456 13 0.270 40.9 Mol Genet Metab 2002, 76: 23–30
S235C Classic 0 0 0 0.004 53.9 Mol Med 1999, 5: 806–811
S235F Classic 0 1 0 0.004 53.9 Hum Mutat 2008, 29: 331
W236C Classic 2 7 0 0.012 40.6 Eur J Hum Genet 1996, 4: 219–24
W236L Classic 0 2 0 0.005 40.6 Mol Med 1999, 5: 806–811
W236R Classic 6 23 0 0.025 40.6 Hum Genomics 2006, 2: 297–309
D244H Hetero 226 287 5 0.122 74.8 Mol Med 1999, 5: 806–811
D244N Classic 20 42 0 0.037 74.8 Hum Mol Genet 1994, 3: 1795–9
G258R Hetero 97 87 0 0.101 0.8 Hum Mutat 2001, 18: 459
G258V Classic 67 67 0 0.087 0.8 Hum Mutat 2008, 29: 331
D264V Classic 132 194 9 0.097 11.2 Am J Hum Genet 1993, 53: 1186–97
D264Y Classic 103 132 6 0.087 11.2 Hum Mutat 2005, 25: 299–305
D266E Classic 40 69 8 0.061 5.9 Mol Med 2002, 8: 306–12
D266H Hetero 445 596 16 0.200 5.9 J Investig Med 2000, 48: 227–35
D266N Classic 48 75 10 0.059 5.9 Clin Genet 2000, 58: 228–33
D266V Classic 26 43 7 0.039 5.9 Am J Hum Genet 1993, 53: 1186–97
D266Y Classic 39 79 10 0.047 5.9 Mol Genet Metab 2008, 95: 224–8
M267I Classic 75 105 16 0.095 4.2 Mol Med 1999, 5: 806–811
M267R Classic 132 192 15 0.097 4.2 Hum Genomics 2006, 2: 297–309
V269A Classic 10 18 2 0.029 0.0 Hum Mol Genet 1993, 2: 1051–3
V269M Classic 113 129 8 0.104 0.0 Hum Genomics 2006, 2: 297–309
G271C Classic 58 57 0 0.066 0.2 Mol Genet Metab 2002, 76: 23–30
G271S Classic 45 65 0 0.057 0.2 Hum Genomics 2006, 2: 297–309
G271V Classic 195 239 7 0.149 0.2 Hum Genomics 2006, 2: 297–309
N272K Classic 60 100 0 0.065 2.8 Hum Mol Genet 1994, 3: 1795–9
N272S Classic 2 3 0 0.013 2.8 Eur J Hum Genet 2004, 12: 678–81
Q279E Later-onset 34 26 0 0.035 20.4 Hum Genet 1992, 89: 29–32
Q279H Classic 102 123 4 0.094 20.4 Hum Mutat 2001, 18: 459
Q279R Classic 55 56 0 0.061 20.4 Hum Mutat 2003 Sup, 22: 258
Q280H Hetero 87 104 0 0.079 0.0 Hum Mutat 2001, 18: 459
Q280K Classic 30 41 0 0.040 0.0 J Mol Med 2005, 83: 647–54
T282A Classic 3 0 0 0.008 1.2 Hum Mutat 2008, 29: 331
T282N Classic 6 9 0 0.023 1.2 J Hum Genet 2001, 46: 192–6
A285D Classic 32 29 0 0.041 0.0 Hum Genomics 2006, 2: 297–309
A285P Classic 21 29 0 0.043 0.0 Hum Mutat 2005, 25: 299–305
W287C Classic 16 14 0 0.028 1.6 Mol Med 1997, 3: 174–82
W287G Classic 11 12 0 0.026 1.6 Eur J Hum Genet 1996, 4: 219–24
A288D Classic 86 96 0 0.106 0.0 Hum Mol Genet 1994, 3: 1795–9
A288P Classic 50 60 0 0.068 0.0 Mol Genet Metab 2002, 76: 23–30
P293A Classic 75 58 0 0.069 2.6 Mol Genet Metab 2002, 76: 23–30
P293T Classic 13 18 0 0.032 2.6 Hum Genomics 2006, 2: 297–309
M296I Later-onset 5 12 0 0.018 0.0 New Eng J Med 1995, 333: 288–93
M296V Later-onset 7 13 0 0.023 0.0 New Eng J Med 1991, 324: 395–399
S297C Classic 0 1 0 0.011 0.0 Mol Med 2002, 8: 306–12
S297F Classic 215 268 8 0.145 0.0 Am J Hum Genet 1993, 53: 1186–97
N298H Hetero 140 181 0 0.089 0.0 Eur J Hum Genet 1996, 4: 219–24
N298K Classic 106 183 0 0.092 0.0 Hum Mutat 1996, 8: 38–43
N298S Classic 18 14 0 0.029 0.0 Mol Med 1997, 3: 174–82
R301Q Later-onset 112 166 0 0.126 40.5 Am J Hum Genet 1990, 47: 784–9
R301P Classic 73 94 0 0.074 40.5 J Hum Genet 2001, 46: 192–6
N320K Classic 150 154 0 0.105 0.0 Biochem Biophys Res Commun 1995, 214: 1219–24
N320Y Classic 239 286 0 0.183 0.0 J Invest Med 2000, 48: 227–35
Q321E Classic 124 144 0 0.088 42.6 Mol Med 1999, 5: 806–811
Q321R Classic 22 24 0 0.034 42.6 Hum Genomics 2006, 2: 297–309
G328A Classic 145 139 0 0.100 0.0 Am J Hum Genet 1993, 53: 1186–97
G328R Classic 560 584 4 0.283 0.0 Hum Genet 1992, 89: 29–32
G328V Classic 222 239 0 0.160 0.0 Hum Mutat 2005, 25: 299–305
E358A Classic 102 175 0 0.091 90.8 Hum Mutat 2005, 25: 299–305
E358G Classic 77 136 0 0.083 90.8 Mol Med 2002, 8: 306–12
E358K Classic 571 661 9 0.293 90.8 Hum Mutat 1998, Suppl 1: S139–40
G373D Classic 103 116 0 0.116 1.0 Hum Mutat 2001, 17: 353
G373S Classic 1 0 0 0.004 1.0 Biochem Biophys Res Commun 1995, 214: 1219–24
C382W Hetero 271 265 0 0.205 0.2 Intern Med J 2002, 32: 575–84
C382Y Classic 241 245 0 0.203 0.2 Hum Mutat 2003 Sup, 22: 258
P409A Classic 13 11 0 0.049 37.2 Hum Mutat 2001, 18: 459
P409S Classic 10 4 0 0.024 37.2 Mol Med 2002, 8: 306–12
P409T Hetero 21 18 0 0.078 37.2 Hum Mutat 2001, 18: 459
T410A Later-onset 0 0 0 0.005 18.6 Clin Genet 2003, 63: 205–9
T410P Classic 77 93 0 0.089 18.6 Hum Mutat 2008, 29: 331
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Classic, the classic form of Fabry disease; Later-onset, the later-onset form; Hetero, heterozygote of Fabry disease; and Polymorphism, GLA polymorphism.

Structural analysis of representative amino acid substitutions

We examined different amino acid substitutions at M72, E66, C56, and W236, because they are expected to provide us with useful information for elucidating the mechanism by which structural changes caused by them influence the severity of the disease and for identifying residues essential for the maintenance of proper folding. The localization of these residues in the dimer is shown in Fig. 1. The residues are widely distributed over the GLA molecule and are distant from the catalytic residues (D170 and D231).

Figure 1. Structure of the GLA dimer and positions of the amino acid residues involved in the substitutions.

Figure 1

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The backbone is shown as a line. Subunit A and subunit B comprising the dimer are shown in light blue and green, respectively. The amino acids involved in the substitutions (C56, E66, M72 and W236) and the catalytic residues (D170 and D231) are indicated as a CPK model. Front view (left) and back view (right).

M72 (M72I, M72R, and M72V)

M72 is located on the α-helix (66–84) of the (β/α)8 barrel domain. The ASA value of this residue is 0 Å2, suggesting that it is fully buried. The numbers of atoms influenced by M72I in the main chain, side chain and active site are 38, 46 and 0, respectively, the RMSD value being 0.054 Å. The numbers of atoms influenced by M72R in the main chain, side chain and active site are 145, 198, and 1, respectively, the RMSD value being 0.119 Å. Considering the results, the structural changes in GLA caused by these amino acid substitutions are thought to be large. The patients with these mutations exhibited the classic form of Fabry disease. On the other hand, as to M72V, the numbers of atoms influenced in the main chain, side chain and active site are 7, 6 and 0, respectively, the RMSD value being 0.026 Å. This suggests that the structural change caused by M72V is small, and that it does not affect the active site. The patients with M72V exhibited the later-onset Fabry disease. Coloring of the influenced atoms allowed clear visualization of the differences in the structural changes between these cases (Fig. 2a).

Figure 2. Coloring of the atoms in the three-dimensional structure of GLA influenced by amino acid substitutions at M72 (a), E66 (b), C56 (c), and W236 (d).

Figure 2

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The backbone of GLA is shown as a line. The atoms of the substituted residues are indicated as small black spheres and the influenced atoms as large spheres. The colors of the influenced atoms show the distances between the wild type and mutant ones as follows: 0.15 Å≤cyan <0.30 Å, 0.30 Å≤green <0.45 Å, 0.45 Å≤yellow <0.60 Å, 0.60 Å≤orange <0.75 Å, and red ≥0.75 Å.

E66 (E66G, E66K and E66Q)

E66 is located on the α-helix (66–84) of the (β/α)8 barrel domain. The ASA value is 29.2 Å2, suggesting that the residue is half-exposed to the solvent. For the E66G substitution, the numbers of atoms influenced in the main chain, side chain and active site are 45, 74, and 0, respectively, the RMSD value being 0.062 Å. For the E66K substitution, the numbers of atoms affected in the main chain, side chain and active site are 422, 503, and 7, respectively, the RMSD value being 0.361 Å. The patients with such large structural changes exhibited the classic form of Fabry disease. On the other hand, as to the E66Q substitution, which has been reported to be a functional polymorphism [21], the numbers of atoms affected in the main chain, side chain, and active site are 23, 32, and 0, respectively, the RMSD value being 0.048 Å. These results suggest that the structural change is moderate and that it does not affect the active site. Fig. 2b clearly shows that the structural change caused by E66Q is restricted to a small region on the molecular surface, although those caused by E66G and E66K extend over a broad area around the substituted residue.

C56 (C56G, C56F, and C56Y)

C56 is located between two α-helices (47–50 and 66–84). The ASA value of the residue is 38.4 Å2, suggesting that it is exposed to the solvent. The C56 residue forms a disulfide bond with C63 (Fig. 3), and it plays an important role in conformation of the enzyme molecule. Fig. 2c shows the structural changes caused by the C56G, C56F, and C56Y amino acid substitutions. These amino acid substitutions at the C56 position are predicted to disturb the formation of disulfide bond between C56 and C63, and thus the mutant proteins would be excessively degraded before they are transported to the lysosomes. All of the patients with these mutations presented the classic form of Fabry disease.

Figure 3. GLA structure and residues involving a disulfide bond.

Figure 3

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The backbone of GLA is shown as a line. The atoms involved in the formation of a disulfide bond (C52-C94, C56-C63, C142-C172, C202-C223, and C378-C382) and the catalytic residues (D170 and D231) are shown as a CPK model. Front view (left) and back view (right).

W236 (W236C, W236L, and W236R)

W236 is located on the α-helix (236–247) of the (β/α)8 barrel domain, the ASA value being 40.6 Å2, suggesting that the residue is exposed to the solvent. As Fig. 2d shows, the structural changes caused by W236C, W236L, and W236R are small (The numbers of atom in the main chain affected by W236C, W236L, and W236R are 2, 0, and 6, respectively, and those in the side chain are 7, 2, and 23, respectively. The RMSD values for them are 0.012 Å, 0.005 Å, and 0.025 Å, respectively). None of them affects the active site. However, as W236 is located on the dimer interface of GLA (Fig. 1), and the side chain of W236 forms a hydrogen bond with E358 (Fig. 4), the amino acid substitution is thought to affect the conformation of the GLA molecule.

Figure 4. The hydrogen bond between W236 and E358.

Figure 4

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The side chain of W236 forms a hydrogen bond with E358. The backbone of GLA is shown as a ribbon model, and W236 and E358 are indicated as a stick. The hydrogen bond is shown as a yellow dotted line. Front view (left) and back view (right).

Discussion

Recently, the results of newborn screening revealed a high incidence (1 in ∼1, 250–9,000) of Fabry disease [22]–[24]. As Fabry disease can be treated with recombinant human GLAs [25]–[27], it is very important to understand the basis of the disease and to predict the outcome for patients found on screening. For this purpose, a structural study will provide us with valuable information. Garman and Garboczi reported that there are at least two classes of mutations in GLA that lead to disease progression: those near the active site and those of buried residues distant from the active site that adversely affect the folded state of the molecule, and a mild phenotype tends to be more solvent-accessible than a severe one [6]. Our research group obtained essentially the same results as those of Garman and Garboczi. Our previous study revealed that structural changes in the classic Fabry group were generally large and tended to be localized to the core region or located in the functionally important region including the active site, and that those in the later-onset group were small and localized on the surface of the molecule [8].

As further structural study, we focused on different substitutions at the same residue in the amino acid sequence of GLA, because such specific cases are useful for examining the influence of the severity of the structural changes on the disease progression and for identifying the residues important for the expression of GLA activity.

In this study, we could select 157 amino acid substitutions at 67 residues from two databases, and examined the correlation between the structural changes in GLA and the clinical phenotype. The results revealed that the structural changes leading to the later-onset Fabry disease tend to be smaller than those for the classic Fabry disease, i.e., M72 is buried and E66 is exposed to the solvent, and at both residues, amino acid substitutions causing a small structural change (M72V and E66Q) lead to later-onset Fabry disease or a functional polymorphism, and ones causing a large structural change (M72I, M72R, E66G, and E66K) result in classic Fabry disease. This study also revealed residues important for expression of the GLA activity. A structural change affecting the active site tends to lead to the classic form. C56 and W236 are thought to be involved in the formation of a disulfide bond and the dimer, respectively. Substitutions at these residues should affect proper folding and lead to classic Fabry disease, even if the structural change is small.

In conclusion, we investigated the effects of different substitutions at the same residue in the amino acid sequence of GLA on structural changes in the enzyme molecule and the clinical phenotype. The results revealed that structural changes influence the disease progression. Structural study from such a unique viewpoint is useful for elucidation of the basis of Fabry disease.

Acknowledgments

We wish to thank Dr. J. Ponder (Department of Biochemistry and Molecular Biophysics, Washington University) for providing us with the TINKER software.

Funding Statement

This work was supported by the Program for Research on Intractable Diseases of Health and Labor Science Research (to HS); the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (ID: 09-15, to HS); and the Japan Society for the Promotion of Science (JSPS ID: 23659527, to HS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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