Purification and Characterization of Blood Group A-degrading Isoforms of α-N-Acetylgalactosaminidase from Ruminococcus torques Strain IX-70

Abstract

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 ␣-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 ␣(133)-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.

Purification and Characterization of a‐N‐Acetylgalactosaminidase from Clostridium perfringens

“…The problem with the chicken enzyme is the high concentration and low pH required for deantigenation. Several prokaryotic sources have been studied, but only one of these enzymes has been puri ed to homogeneity (27) and no deantigenation studies with it were performed on the A 2 epitope. The clostridial enzyme shows particular promise because it is active at pH 6.2 to 7.2, the pH of erythrocyte units, and is active against the type A 2 epitope.…”

Distribution of α-N-acetylgalactosaminidases among marine bacteria of the phylum Bacteroidetes, epiphytes of marine algae of the Seas of Okhotsk and Japan

“…The closest related enzyme of this family is a single, comprehensively characterized α N acetylgalac tosaminidase from the anaerobic pathogenic bacte rium Elisabethkingia meningoseptica of the phylum Bacteroidetes, which has already found application in biotechnology [12]. The α N acetylgalactosamini dases of the family GH109 realize the mechanism of hydrolysis of the O glycoside bond with the involve ment of NAD + [12,13] in contrast to the enzymes of the bacteria Clostridium perfringens [8] and Rumino coccus torques [10] of the phylum Firmicutes, which are included in the family GH36 and appear to be the classical glycoside hydrolases (http://www.cazy.org/ GH36_bacteria.html).…”

“…The practical solution of the problem is hampered by the absence of economically advanta geous sources of the enzymes, which act effectively under conditions where erythrocytes retain viability (neutral pH, temperature of 20-25°C, and low ionic strength). Therefore, the search for new sources of specific, stable, and efficient enzymes for the large scale modification of A erythrocytes is topical.To date, α N acetylgalactosaminidases with the required properties were revealed in the anaerobic bacteria of terrestrial origin, Clostridium perfringens [6-9] and Ruminococcus torques [10,11] of the phy lum Firmicutes and Elisabethkingia meningoseptica of the phylum Bacteroidetes [12][13][14]. The evidence of α N acetylgalactosaminidases from marine bacteria is scarce.…”

“…To date, α N acetylgalactosaminidases with the required properties were revealed in the anaerobic bacteria of terrestrial origin, Clostridium perfringens [6][7][8][9] and Ruminococcus torques [10,11] of the phy lum Firmicutes and Elisabethkingia meningoseptica of the phylum Bacteroidetes [12][13][14]. The evidence of α N acetylgalactosaminidases from marine bacteria is scarce.…”

Modifying the red cell surface: towards an ABO‐universal blood supply

“…Nevertheless, the group did report conversion of 'strong' A (assumingly A 1 ) blood group RBCs. Another yet uncloned enzyme with apparent neutral pH was identified in Ruminococcus torgues and shown to partially remove A antigens of A RBCs (Hoskins et al, 1997), although complete removal was not achieved as evidenced by serology (Hoskins & Boulding, 2001). One common feature of all these enzymes used in the past is that they all function efficiently with simple aGal(NAc) monosaccharide substrates and have broad specificities for a-linked Gal and GalNAc terminal sugars including a1-3 and a1-4 linked.…”