Publications

• Purification and characterization of blood group A-degrading isoforms of alpha-N-acetylgalactosaminidase from Ruminococcus torques strain IX-70.

  Hoskins L.C., Boulding E.T., Larson G.

  To cleave blood group A immunodeterminants from erythrocytes (Hoskins, L. C., Larson, G., and Naff, G. B. (1995) Transfusion 35, 813-821), we purified and characterized alpha-N-acetylgalactosaminidase (EC 3.2.1.49) activity from culture supernatants of the human fecal bacterium Ruminococcus torque ... >> More

  To cleave blood group A immunodeterminants from erythrocytes (Hoskins, L. C., Larson, G., and Naff, G. B. (1995) Transfusion 35, 813-821), we purified and characterized alpha-N-acetylgalactosaminidase (EC 3.2.1.49) activity from culture supernatants of the human fecal bacterium Ruminococcus torques strain IX-70. Three isoforms separated during hydrophobic interaction chromatography. Hydroxyapatite chromatography further resolved the most hydrophilic, isoform I, into isoforms IA and IB. The most hydrophobic, isoform III, differed from IA and IB by a more acidic pH optimum, greater heat resistance, greater sensitivity to alkylating agents, and anomalous retardation during gel filtration chromatography. Isoform IB differed from IA and III in N-terminal amino acid sequence and in sensitivity to EDTA inhibition. Each cleaved nonreducing alpha(1-->3)-N-acetylgalactosamine residues from human blood group A and AB mucin glycoproteins, Forssman hapten, and blood group A lacto series glycolipids. The apparent molecular mass of denatured isoform subunits of IA, IB, and III-PII (158, 173, and 201 kDa, respectively) bore no integer relationship to the apparent molecular mass of the native isoforms (265, 417, and 530 kDa), but the latter bore a ratio of 1.96:3.09:3.93 to the weight-average apparent molecular mass of native IA (135 kDa), suggesting that the isoforms are multimers of a 135-kDa sequence. Isoforms IA and III-PII had an identical N-terminal amino acid sequence which showed homologies to the N-terminal sequence of sialidases produced by Bacteroides fragilis SBT3182, another commensal enteric bacterium. << Less

  J Biol Chem 272:7932-7939(1997) [PubMed] [EuropePMC]

• Degradation of blood group A glycolipid A-6-2 by normal and mutant human skin fibroblasts.

  Asfaw B., Schindler D., Ledvinova J., Cerny B., Smid F., Conzelmann E.

  The degradation of blood group glycolipid A-6-2 (GalNAc(alpha1-->3)[Fuc alpha1-->2]Gal(beta1-->4)GlcNAc(beta1-->3)Gal(beta1-->4)Glc(beta1-->1')C er, IV2-alpha-fucosyl-IV3-alpha-N-acetylgalactosaminylneolact otetraosylceramide), tritium-labeled in its ceramide moiety, was studied in situ, in skin f ... >> More

  The degradation of blood group glycolipid A-6-2 (GalNAc(alpha1-->3)[Fuc alpha1-->2]Gal(beta1-->4)GlcNAc(beta1-->3)Gal(beta1-->4)Glc(beta1-->1')C er, IV2-alpha-fucosyl-IV3-alpha-N-acetylgalactosaminylneolact otetraosylceramide), tritium-labeled in its ceramide moiety, was studied in situ, in skin fibroblast cultures from normal controls, from patients with defects of lysosomal alpha-N-acetylgalactosaminidase, and from patients with other lysosomal storage diseases. Uptake of the glycolipid with apolipoprotein E-coated liposomes was linear with time and with the amount of glycolipid added. In normal cells, the expected array of less polar products and some lipids resulting from re-using the liberated sphingosine, mainly sphingomyelin and phosphatidylcholine, were formed. In alpha-N-acetylgalactosaminidase-deficient cells, the glycolipid was virtually not degraded; product formation was less than 2% of the normal control rate, suggesting that blood group A-active glycolipids contribute as storage compounds to the pathogenesis of this disease. The expected accumulation of degradation intermediates was seen in fucosidosis, and in Sandhoff, Gaucher, and Farber disease cells, whereas normal turnover rates were found in Tay-Sachs disease cells, G(M2) activator-deficient (variant AB of G(M2) gangliosidosis) and in sulfatide activator-(sap-B-) deficient cells. In G(M1) gangliosidosis and in sap precursor-deficient cells, the lysosomal glycolipid catabolism was found to be strongly retarded; accumulation of individual products could not be seen. Skin fibroblasts from patients with alpha-N-acetylgalactosaminidase deficiency (Schindler disease) cannot degrade the major blood group A glycolipid. << Less

  J. Lipid Res. 39:1768-1780(1998) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• The 1.9 A structure of human alpha-N-acetylgalactosaminidase: the molecular basis of Schindler and Kanzaki diseases.

  Clark N.E., Garman S.C.

  alpha-N-acetylgalactosaminidase (alpha-NAGAL; E.C. 3.2.1.49) is a lysosomal exoglycosidase that cleaves terminal alpha-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, a deficiency of alpha-NAGAL activity results in the lysosomal storage disorders Schindler disease and ... >> More

  alpha-N-acetylgalactosaminidase (alpha-NAGAL; E.C. 3.2.1.49) is a lysosomal exoglycosidase that cleaves terminal alpha-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, a deficiency of alpha-NAGAL activity results in the lysosomal storage disorders Schindler disease and Kanzaki disease. To better understand the molecular defects in the diseases, we determined the crystal structure of human alpha-NAGAL after expressing wild-type and glycosylation-deficient glycoproteins in recombinant insect cell expression systems. We measured the enzymatic parameters of our purified wild-type and mutant enzymes, establishing their enzymatic equivalence. To investigate the binding specificity and catalytic mechanism of the human alpha-NAGAL enzyme, we determined three crystallographic complexes with different catalytic products bound in the active site of the enzyme. To better understand how individual defects in the alpha-NAGAL glycoprotein lead to Schindler disease, we analyzed the effect of disease-causing mutations on the three-dimensional structure. << Less

  J. Mol. Biol. 393:435-447(2009) [PubMed] [EuropePMC]