Purification and characterization of blood group A-degrading isoforms of alpha-N-acetylgalactosaminidase from Ruminococcus torques strain IX-70.
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.
Hoskins
L C
LC
Department of Medicine, Veterans Affairs Medical Center and Case Western Reserve University, Cleveland, Ohio 44106, USA.
Boulding
E T
ET
Larson
G
G
D000017:ABO Blood-Group System; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006017:Glycolipids / Q000378:metabolism; D006095:Gram-Positive Cocci / Q000201:enzymology*; D006596:Hexosaminidases / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D048809:alpha-N-Acetylgalactosaminidase
D000017:ABO Blood-Group System; D006017:Glycolipids; D007202:Indicators and Reagents; D007527:Isoenzymes; D006596:Hexosaminidases; C482722:NAGA protein, human; D048809:alpha-N-Acetylgalactosaminidase