The molecular defect leading to Fabry disease: structure of human alpha-galactosidase.

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 alpha-galactosidase (alpha-GAL) causes an accumulation of its substrate, which ultimately leads to Fabry disease symptoms. Here, we present the structure of the human alpha-GAL glycoprotein determined by X-ray crystallography. The structure is a homodimer with each monomer containing a (beta/alpha)8 domain with the active site and an antiparallel beta 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 alpha-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 alpha-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 alpha-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.

[1]  D. Calhoun,et al.  Purification and characterization of human alpha-galactosidase A expressed in insect cells using a baculovirus vector. , 2000, Protein expression and purification.

[2]  M Krawczak,et al.  The human gene mutation database , 1998, Nucleic Acids Res..

[3]  Z. Fujimoto,et al.  Crystal Structure of Rice α-Galactosidase Complexed with D-Galactose* , 2003, Journal of Biological Chemistry.

[4]  Ylva Gavel,et al.  Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering , 1990, Protein engineering.

[5]  S. Withers,et al.  Identification of Asp-130 as the catalytic nucleophile in the main alpha-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase. , 2000, Biochemistry.

[6]  R. Desnick.  α-Galactosidase A deficiency. Fabry disease , 2001 .

[7]  G J Kleywegt,et al.  Not your average density. , 1997, Structure.

[8]  Robert J. Desnick,et al.  Improvement in cardiac function in the cardiac variant of Fabry's disease with galactose-infusion therapy. , 2001, The New England journal of medicine.

[9]  S. Kaneko,et al.  alpha-Galactosidase from cultured rice (Oryza sativa L. var. Nipponbare) cells. , 2002, Phytochemistry.

[10]  S. Withers,et al.  Glycosidase mechanisms: anatomy of a finely tuned catalyst. , 1999, Accounts of chemical research.

[11]  S. B. Needleman,et al.  A general method applicable to the search for similarities in the amino acid sequence of two proteins. , 1970, Journal of molecular biology.

[12]  R. Schiffmann,et al.  Enzymatic and functional correction along with long-term enzyme secretion from transduced bone marrow hematopoietic stem/progenitor and stromal cells derived from patients with Fabry disease. , 1999, Experimental hematology.

[13]  K. J. Dean,et al.  Studies on human liver alpha-galactosidases. I. Purification of alpha-galactosidase A and its enzymatic properties with glycolipid and oligosaccharide substrates. , 1979, The Journal of biological chemistry.

[14]  S. Malcolm,et al.  Mutation analysis in patients with the typical form of Anderson-Fabry disease. , 1993, Human molecular genetics.

[15]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[16]  Andrea Schmidt,et al.  Structure of the xylanase from Penicillium simplicissimum , 1998, Protein science : a publication of the Protein Society.

[17]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[18]  P. Hantzopoulos,et al.  Expression of the human α-galactosidase A in Escherichia coli K-12 , 1987 .

[19]  D. Garboczi,et al.  Structural basis of Fabry disease. , 2002, Molecular genetics and metabolism.

[20]  Yingsi Chen,et al.  Expression and characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A produced in Pichia pastoris. , 2000, Protein expression and purification.

[21]  R. Desnick,et al.  Human alpha-N-acetylgalactosaminidase-molecular cloning, nucleotide sequence, and expression of a full-length cDNA. Homology with human alpha-galactosidase A suggests evolution from a common ancestral gene. , 1990, The Journal of biological chemistry.

[22]  R. Desnick,et al.  Human α-galactosidase A: characterization of the N-linked oligosaccharides on the intracellular and secreted glycoforms overexpressed by Chinese hamster ovary cells , 1998 .

[23]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[24]  A. Zhu,et al.  Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes. , 1996, Archives of biochemistry and biophysics.

[25]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[26]  A. Halpern,et al.  Weighted neighbor joining: a likelihood-based approach to distance-based phylogeny reconstruction. , 2000, Molecular biology and evolution.

[27]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[28]  B Henrissat,et al.  Structural and sequence-based classification of glycoside hydrolases. , 1997, Current opinion in structural biology.

[29]  C. Eng,et al.  Safety and efficacy of recombinant human alpha-galactosidase A replacement therapy in Fabry's disease. , 2001, The New England journal of medicine.

[30]  B. Migeon,et al.  Signal sequence and DNA-mediated expression of human lysosomal alpha-galactosidase A. , 1987, European journal of biochemistry.

[31]  J. Tager,et al.  The identity of α-galactosidase B from human liver , 1977 .

[32]  S. Ishii,et al.  Role of Ser-65 in the activity of alpha-galactosidase A: characterization of a point mutation (S65T) detected in a patient with Fabry disease. , 2000, Archives of biochemistry and biophysics.

[33]  R. Brady,et al.  Long-term correction of globotriaosylceramide storage in Fabry mice by recombinant adeno-associated virus-mediated gene transfer , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[34]  L. Hannick,et al.  The 1.9 A structure of alpha-N-acetylgalactosaminidase: molecular basis of glycosidase deficiency diseases. , 2002, Structure.

[35]  M. Shichiri,et al.  A carboxy-terminal truncation of human alpha-galactosidase A in a heterozygous female with Fabry disease and modification of the enzymatic activity by the carboxy-terminal domain. Increased, reduced, or absent enzyme activity depending on number of amino acid residues deleted. , 1996, The Journal of clinical investigation.

[36]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[37]  Huawei Qiu,et al.  A biochemical and pharmacological comparison of enzyme replacement therapies for the glycolipid storage disorder Fabry disease. , 2003, Glycobiology.

[38]  R. Desnick,et al.  Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion , 1992, The Journal of cell biology.

[39]  L. Johnson,et al.  Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure , 1999, Protein science : a publication of the Protein Society.

[40]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[41]  J. Stroh,et al.  Characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A using a baculovirus vector. , 1994, Gene.

[42]  M. O'Meara,et al.  Analysis of splice‐site mutations of the α‐galactosidase A gene in Fabry disease , 2003 .

[43]  S. Withers,et al.  Mechanisms of enzymatic glycoside hydrolysis. , 1994, Current opinion in structural biology.

[44]  R. Desnick,et al.  Structural organization of the human alpha-galactosidase A gene: further evidence for the absence of a 3' untranslated region. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[45]  C. Scriver,et al.  The Metabolic and Molecular Bases of Inherited Disease, 8th Edition 2001 , 2001, Journal of Inherited Metabolic Disease.

[46]  S. Withers,et al.  The synthesis, testing and use of 5-fluoro-α-d-galactosyl fluoride to trap an intermediate on green coffee bean α-galactosidase and identify the catalytic nucleophile , 2000 .

[47]  R M Esnouf,et al.  An extensively modified version of MolScript that includes greatly enhanced coloring capabilities. , 1997, Journal of molecular graphics & modelling.

[48]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[49]  R. Murali.  Crystallization and Preliminary X-ray Analysis of Human a-Galactosidase A Complex , 1994 .

[50]  R. Desnick,et al.  Affinity purification of alpha-galactosidase A from human spleen, placenta, and plasma with elimination of pyrogen contamination. Properties of the purified splenic enzyme compared to other forms. , 1981, The Journal of biological chemistry.

[51]  V. McKusick.  Mendelian inheritance in man , 1971 .

[52]  R. Desnick,et al.  Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease. , 1987, The Journal of biological chemistry.

[53]  S. Kornfeld,et al.  Mannose 6-phosphate receptors: new twists in the tale , 2003, Nature Reviews Molecular Cell Biology.

[54]  D. F. Moore,et al.  Enzyme replacement therapy in Fabry disease: a randomized controlled trial. , 2001, JAMA.

[55]  C. Sweeley,et al.  Biosynthesis of α-galactosidase A in cultured chang liver cells☆ , 1983 .

[56]  K. J. Dean,et al.  The identification of α-galactosidase B from human liver as an α-acetylgalactosaminidase , 1977 .

[57]  J. Fabry.  Ein Beitrag zur Kenntniss der Purpura haemorrhagica nodularis (Purpura papulosa haemorrhagica Hebrae) , 1898, Archiv für Dermatologie und Syphilis.

[58]  D. Blom,et al.  Recombinant Enzyme Therapy for Fabry Disease: Absence of Editing of Human α-Galactosidase A mRNA , 2003 .

[59]  Daniel E. Koshland,et al.  STEREOCHEMISTRY AND THE MECHANISM OF ENZYMATIC REACTIONS , 1953 .

[60]  R. Brady,et al.  Enzymatic defect in Fabry's disease. Ceramidetrihexosidase deficiency. , 1967, The New England journal of medicine.

[61]  E. Beutler,et al.  alpha-galactosidase A from human placenta. Stability and subunit size. , 1977, Biochimica et biophysica acta.

[62]  R. Desnick,et al.  Human alpha-galactosidase A: glycosylation site 3 is essential for enzyme solubility. , 1998, The Biochemical journal.