While studying the sialosyl linkages in glycoconjugates, we found that commercially available sialidases prepared from Clostridium perfringens ATCC 10543 were contaminated with an unusual endo-beta-galactosidase (Endo-beta-GalAB) capable of liberating the blood group A- and B-trisaccharides from human ovarian cyst glycoproteins. We have isolated Endo-beta-GalAB in pure form from the culture supernatant of Clostridium perfringens and cloned the gene encoding this enzyme. The gene (eabC) consists of a 2400 by open reading frame, including a 35-residue signal peptide at the N-terminus. The mature protein (765 amino acids) has a predicted molecular mass of 87 kDa. Sequence comparisons of Endo-beta-GalAB to other microbial endo-beta-galactosidases revealed no significant sequence homology. Although a BLAST search did reveal a modest homology (34%) to a 'fucolectin-related protein of unknown function' from Streptococcus pneumoniae, this protein did not cleave blood group A- and B-glycoconjugates. Endo-beta-Gal AB has a very broad optimal pH (5.5--8.0) and is stable with little loss of activity upon storage at -20°C for up to 2 years. We have established the structures of the liberated trisaccharides from A+ porcine gastric mucin and B+ human ovarian cyst glycoprotein by NMR spectroscopy. Endo-beta-GalAB was found to effectively liberate the blood group A- and B-trisaccharide epitopes, GalNAc/Galalpha1 → 3(Fucalpha1 → 2) Gal, from these glycoconjugates. Also, Endo-beta-Gal AB was able to completely abolish the blood group B antigenicity of type B human red blood cells. Due to its unique specificity, Endo-beta-Gal AB should prove useful in studying the structure and function of blood group A- and B-containing glycoconjugates
