The Molecular Defect Leading to Fabry Disease: Structure of Human α-Galactosidase

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Abstract

Fabry disease is an X-linked lysosomal storage disease afflicting 1 in 40,000 males with chronic pain, vascular degeneration, cardiac impairment, and other symptoms. Deficiency in the lysosomal enzyme α-galactosidase (α-GAL) causes an accumulation of its substrate, which ultimately leads to Fabry disease symptoms. Here, we present the structure of the human α-GAL glycoprotein determined by X-ray crystallography. The structure is a homodimer with each monomer containing a (β/α)8 domain with the active site and an antiparallel β domain. N-linked carbohydrate appears at six sites in the glycoprotein dimer, revealing the basis for lysosomal transport via the mannose-6-phosphate receptor. To understand how the enzyme cleaves galactose from glycoproteins and glycolipids, we also determined the structure of the complex of α-GAL with its catalytic product. The catalytic mechanism of the enzyme is revealed by the location of two aspartic acid residues (D170 and D231), which act as a nucleophile and an acid/base, respectively. As a point mutation in α-GAL can lead to Fabry disease, we have catalogued and plotted the locations of 245 missense and nonsense mutations in the three-dimensional structure. The structure of human α-GAL brings Fabry disease into the realm of molecular diseases, where insights into the structural basis of the disease phenotypes might help guide the clinical treatment of patients.

Introduction

The lysosomal enzyme α-galactosidase (α-GAL or α-Gal A; EC 3.2.1.22) catalyzes the removal of galactose from oligosaccharides, glycoproteins, and glycolipids during the catabolism of macromolecules (Figure 1(a)). Deficiencies in lysosomal enzymes lead to the accumulation of substrates in the tissues, conditions known as lysosomal storage diseases. In humans, the absence of functional α-GAL leads to the accumulation of galactosylated substrates (primarily globotriaosylceramide; Figure 1(b)) in the tissues, leading to Fabry disease, an X-linked recessive disorder first described in 18981 characterized by chronic pain, ocular opacities, liver and kidney impairment, skin lesions, vascular deterioration and/or cardiac deficiencies.2., 3. Recombinant human α-GAL has the ability to restore enzyme function in patients,4., 5. and enzyme replacement therapy using α-GAL was approved in the United States in 2003 as a treatment for Fabry disease. α-GAL became the second recombinant protein approved for the treatment of a lysosomal storage disorder (after β-glucosidase, a treatment for Gaucher disease6), and α-GAL represents one of a small number of recombinant human proteins approved for the treatment of any disease. A second treatment for Fabry disease (specific for the cardiac variant of the disease) uses galactose infusion, which presumably helps stabilize the mutant α-GAL protein.7 In addition to enzyme replacement therapy and galactose infusion, gene replacement therapy using the α-GAL gene shows potential as a treatment for Fabry disease.8

There are currently two recombinant glycoprotein products, Replagal and Fabrazyme, available for enzyme replacement therapy used in the treatment of Fabry disease.4., 5. These two glycoproteins have identical amino acid sequences but are produced in different cell lines, resulting in different glycosylation at the N-linked carbohydrate attachment sites. Replagal is produced in a genetically engineered human cell line, while Fabrazyme is produced in a Chinese hamster ovary (CHO) cell line. Replagal contains a greater amount of complex carbohydrate while Fabrazyme contains a higher fraction of sialylated and phosphorylated carbohydrate.9 Because the polypeptide sequence of the two glycoproteins is identical, these differences in carbohydrate composition are solely responsible for the differences in tissue distribution and dose response of the two enzyme replacement therapies.

α-GAL has also attracted attention for its ability to convert human blood group antigens. Recombinant α-GAL has been used to convert blood of type B into blood of type O, the universal donor type,10 a process currently in clinical trials.

Because of its utility in the treatment of Fabry disease and as a reagent for converting human blood types, much effort has been put into the expression and purification of large amounts of human α-GAL. The endogenous enzyme has been purified from human placenta,11 liver cells,12 spleen cells,13 plasma,13 and fibroblasts;14 recombinant enzyme has been produced in Escherichia coli bacterial cells,15 COS monkey cells,16 CHO cells,17 baculovirus-infected Sf9 insect cells,18., 19. Pichia pastoris yeast cells,20 transduced human bone marrow cells,21 and continuously cultured genetically engineered human fibroblasts.4 Despite the ability to successfully express and purify human α-GAL since 1977, the three-dimensional structure has not been reported until now, although a crystallization report appeared in 1994.22 Structural analysis has been hindered by the heterogeneous carbohydrates on the glycoprotein, which comprise 5–15% of the mass of the secreted material and contain over 70 different species built upon 23 different core structures.23

Until 2002, there were no examples of structures of α-retaining exoglycosidases like α-GAL, from family 27 and clan D in the classification of glycoside hydrolases.24 With this report, human α-GAL becomes the third member of this family with a known structure, after chicken α-N-acetylgalactosaminidase (α-NAGAL)25 and rice α-GAL.26 After binding a substrate containing a terminal galactose with an α-glycosidic linkage, α-GAL cleaves the glycosidic linkage, leaving a free galactose in the α-anomeric configuration (Figure 1(a)). The overall retention of the α anomer in the catalytic product is achieved through a double displacement reaction mechanism, first proposed for glycosidases 50 years ago.27 Two successive nucleophilic attacks on the same anomeric carbon invert the chiral center twice, resulting in the retention of the α anomer at the completion of the reaction cycle. This mechanism requires two carboxylic acids, one acting as a nucleophile and the other acting as an acid and then a base over the course of the reaction cycle.28., 29.

We have previously determined the structure of the related enzyme chicken α-NAGAL by X-ray crystallography.25 α-NAGAL has substrate specificity and mechanism similar to α-GAL, except that α-NAGAL recognizes and cleaves a terminal galactose with an N-acetyl substituent (not a hydroxyl) on the 2 position of the galactose ring. Beginning with the α-NAGAL structure, we constructed a homology model of human α-GAL, as the two proteins share 51% identity in their amino acid sequences and there was no known structure of human α-GAL. From this model, the Fabry disease mutations partitioned into two classes: those that locally perturb the active site of the enzyme and those that adversely affect the folding of the protein.30 Here, we describe the X-ray crystal structure of human α-GAL, identical to the Replagal glycoprotein injected into patients during the course of recombinant enzyme replacement therapy for Fabry disease. To better understand the molecular basis for galactose-infusion therapy for Fabry disease, we also determined the structure of the glycoprotein bound to its catalytic product, the α-galactose monosaccharide. The structures reveal the basis for the catalytic mechanism and for substrate specificity of the enzyme, indicate conserved features of divergent members of the protein family, highlight the role of glycosylation in lysosomal trafficking and in enzyme replacement therapy, and show the atomic basis for the defects leading to Fabry disease.

Section snippets

Overall description of the structure

The structure of α-GAL was determined by X-ray crystallographic methods to a resolution limit of 3.25 Å (see Table 1 and Materials and Methods). The X-ray structure reveals α-GAL as a homodimeric glycoprotein with each monomer composed of two domains, a (β/α)8 domain containing the active site and a C-terminal domain containing eight antiparallel β strands on two sheets in a β sandwich (Figure 2(a)). After removal of the 31-residue signal sequence, the first domain extends from residues 32 to

Crystallization and X-ray data collection

α-Galactosidase (Replagal lot G302-010, TKT) was produced in the supernatant of a continuous culture of genetically modified human cells.4 α-Galactosidase was concentrated to 40 mg/ml in 20 mM Tris–HCl (pH 7.5) prior to crystallization trials. Crystals were grown in either hanging or sitting drops via vapor diffusion against a reservoir solution of 30% (w/v) polyethylene glycol (PEG) 4000 (Fluka), 100 mM Tris–HCl (pH 8.0), 200 mM ammonium sulfate. Crystals were then harvested into 35% PEG 4000,

Acknowledgements

We are very grateful to Transkaryotic Therapies Inc. for the gift of Replagal. We thank T. Allison and the other members of the Structural Biology Section for discussions. This work was supported by the Intramural Program of the National Institute of Allergy and Infectious Diseases. Data were collected at Southeast Regional Collaborative Access Team (SER-CAT) beamline at the Advanced Photon Source, Argonne National Laboratory.

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