Abstract
Sda antigens [GalNAcβ1-4(Neu5Acα2-3)Galβ1-O-R] are present at the nonreducing termini of O-glycans of colonic mucins of humans. Previously, we reported characterization of two glycoside hydrolase (GH) family 33 α-sialidases, SiaBb1 and SiaBb2, from a symbiotic Bifidobacterium bifidum dwelling in the human intestines. In this study, we identified a third α-sialidase, SiaBb3 from B. bifidum, that is distinguished from the aforementioned two sialidases by its possession of an additional GH123 β-N-acetylgalactosaminidase domain within the same polypeptide. The purified recombinant SiaBb3 efficiently converted GM2 ganglioside [GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-ceramide], sharing the same terminal trisaccharide structure with the Sda antigen, to lactosylceramide by releasing Neu5Ac and GalNAc in the presence of 0.1% sodium cholate. Hydrolysis of the GM2 oligosaccharide proceeds with the initial release of Neu5Ac, followed by the liberation of GalNAc, which was revealed by monitoring the reactions performed using catalytically inactive mutants for each domain of SiaBb3 and by analyzing the reactions of WT SiaBb3 on fluorescence-labeled oligosaccharides. Notably, the order of hydrolysis was reversed compared with that employed by mammalian lysosomal enzymes for GM2 degradation. Comparative O-glycomic analysis using fecal mucin as a substrate unequivocally demonstrated that SiaBb3 targets the Sda antigen of mucin O-glycans. The GH33-inactive SiaBb3 mutant retained Sda antigen-containing O-glycans intact, indicating that initial hydrolysis of Neu5Ac is essential for the subsequent removal of GalNAc. Taken together, these results indicate that SiaBb3 is a bifunctional enzyme specialized for the complete degradation of Sda antigens in host mucins.