Structure-function relationships in alpha-galactosidase A.

The degradation of macromolecules, including glycopeptides and glycolipids, occurs in the lysosome via catabolic enzymes. For example, glycosidases cleave the oligosaccharides from glycoproteins and glycolipids into smaller components used by the cell. In humans, defects in these lysosomal enzymes cause lysosomal storage diseases (LSDs). In this family of diseases, a defect in a gene leads to the loss of functional enzyme, which then leads to the accumulation of substrates in the tissues. The symptoms of the particular disease depend upon the amount, toxicity and location of the substrate that accrues. Over forty different LSDs have been identified to date, including Tay–Sachs, Gaucher, Sandhoff and Fabry diseases. Fabry disease, an X-linked inherited disorder affecting 1 in every 40000 males, is characterized by chronic pain, vascular degeneration, cardiac and renal abnormalities, and other symptoms [1]. The disease was first reported in 1898 [2], and the defective protein responsible for the disease was first identified to be α-galactosidase A (α-gal A, also known as GLA and α-GAL) (E.C. 3.2.1.22) in 1967 [3]. α-Galactosidase A is encoded by the GLA gene and catalyzes the removal of terminal α-galactose groups from substrates such as glycoproteins and glycolipids. In patients with Fabry disease the loss of functional enzyme leads to the buildup of substrates, primarily globotriaosylceramide, in the tissues [1]. Most patients with Fabry disease have a single point mutation in their GLA gene, and several hundred different missense and nonsense mutations have been identified in the patient population. The desired result of clinical research on LSDs is to develop treatments that would prevent disease symptoms from ever appearing. In Gaucher disease (caused by a defect in the lysosomal enzyme β-glucosidase), enzyme replacement therapy (ERT) in paediatric patients successfully prevents disease and is currently in use in over 3500 patients from 55 countries [4,5]. Gaucher disease was the first LSD approved for treatment with recombinant ERT in 1994 [4,5], and in 2001 Fabry disease became the second. We have recently determined the three-dimensional crystal structure (at 3.25 A resolution) of the lysosomal enzyme human α-galactosidase A, the molecule which when deficient is responsible for the development of Fabry disease (Figure 1) [6]. This structure represents one of the first structures from family 27 and clan D (including over 270 proteins from different species) in the classification of glycoside hydrolases based upon similarities in sequences, active sites and mechanisms [7]. The structure was determined both alone and in complex with its catalytic product, the α-galactose monosaccharide, indicating the atomic basis for substrate recognition. The structural analysis revealed a portion of the glycoprotein’s N-linked carbohydrate, which accounts for 5–15% of the mass of α-galalactosidase A and represents over 70 different glycoforms attached to 23 different core carbohydrate structures [8]. The carbohydrate component of the glycoprotein is required for the molecule to traffic to the lysosome via the mannose-6-phosphate receptor pathway [9]. Figure 1 The structure of α-galactosidase A. The structure of the human α-galactosidase A dimer is shown in ribbon representation. The ribbon is coloured from blue to red as the polypeptide goes from N- to C-terminus. The active site is identified ... Following the recent determination of the structure of human α-galactosidase A, we are now able to ask fundamental questions about the structure and function of α-galactosidase A and its relationship to the human disease process. The α-galactosidase A structure revealed for the first time the locations and nature of the hundreds of different atomic defects responsible for Fabry disease. We are now in position to learn the details of the atomic basis for the loss of enzyme activity in patients with Fabry disease, and to tailor therapy based upon the specific defect in α-galactosidase A in different patients. In this report, we address the defects in the α-galactosidase A enzyme that lead to Fabry disease. As we are interested in the defects in the enzyme, in this work we do not include the mutations in the α-galactosidase A gene that lead to grossly altered gene products, such as insertions and deletions leading to frameshifts and splice defects. As Fabry disease represents one of the best studied LSDs, the lessons learned from the study of α-galactosidase A will lead to greater understanding of glycosidases and LSDs generally.