Hoskins L C, Boulding E T, Larson G
Department of Medicine, Veterans Affairs Medical Center and Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 1997 Mar 21;272(12):7932-9. doi: 10.1074/jbc.272.12.7932.
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.
为了从红细胞上裂解血型A免疫决定簇（霍斯金斯，L.C.，拉尔森，G.，以及纳夫，G.B.（1995年）《输血》第35卷，813 - 821页），我们从人粪便细菌扭链瘤胃球菌菌株IX - 70的培养上清液中纯化并鉴定了α - N - 乙酰半乳糖胺酶（EC 3.2.1.49）活性。在疏水相互作用色谱过程中分离出了三种同工型。羟基磷灰石色谱进一步将最亲水的同工型I分离为同工型IA和IB。最疏水的同工型III与IA和IB的不同之处在于最适pH更偏酸性、耐热性更强、对烷基化剂更敏感，以及在凝胶过滤色谱过程中出现异常滞留。同工型IB在N端氨基酸序列以及对EDTA抑制的敏感性方面与IA和III不同。每种同工型都能从人血型A和AB粘蛋白糖蛋白、福斯曼半抗原以及血型A乳糖系列糖脂上裂解非还原端的α(1→3)-N - 乙酰半乳糖胺残基。IA、IB和III - PII变性同工型亚基的表观分子量（分别为158、173和201 kDa）与天然同工型的表观分子量（265、417和530 kDa）没有整数关系，但后者与天然IA的重均表观分子量（135 kDa）的比例为1.96:3.09:3.93，这表明同工型是135 kDa序列的多聚体。同工型IA和III - PII具有相同的N端氨基酸序列，该序列与另一种共生肠道细菌脆弱拟杆菌SBT3182产生的唾液酸酶的N端序列具有同源性。
