A novel mucin-desulfating sulfate-6-N-acetylglucosaminidase (sulfoglycosidase) from the anaerobic colonic bacterium Prevotella strain RS2

Abstract

Sulfate removal from sulfomucin is believed to be a rate-limiting step in sulfomucin degradation by bacteria from the digestive tract. A novel sulfomucin-desulfating enzyme has been discovered in the anaerobic bacterium, Prevotella strain RS2, which can grow on colonic mucin as its sole energy source. The enzyme, located in the periplasm, was assayed by measuring p-nitrophenol removal from the model substrate sulfate-6-N-acetylglucosamine-1-p-nitrophenol, sulfate-6-N-acetylglucosamine being the other product. This activity differs from that of sulfatases which remove the sulfate ester group from sulfate-6-N-acetylglucosamine and its analogues substituted at the Cl position. The enzyme has been termed a sulfate-6-N-acetylglucosaminidase or sulfoglycosidase (SGL). The SGL was purified to a single protein band of 100 kDa as analyzed by SDS-PAGE. The purified SGL protein was trypsin-digested and peptide fragments were sequenced. PCR and inverse PCR were then used to amplify the entire sg/ gene from Prevotella strain RS2 genomic DNA. After inserting the gene into a suitable plasmid, active recombinant SGL was expressed using an Escherichia coli expression system. The SGL was characterized using a selection of model substrates, and shown to be an exo-enzyme that removes non-reducing terminal sulfate-6-N-acetylglucosamine residues by glycosidic bond cleavage. When tested against its putative physiological substrate, sulfomucin, the only small molecular size product detected corresponded to a sulfate-6-N-acetylglucosamine residue. Thus the SGL can catalyze a reaction, formerly thought to be performed in bacteria by the combined actions of a N-acetylglucosamine-6-sulfatase and a N-acetylglucosaminidase. Inhibition studies on the SGL were carried out. Inhibitors of the SGL and those of the sulfatases were used to confirm the presence or absence of SGL-like activity in other bacteria that inhabit environments containing sulfomucin. Four isolates, including Prevotella strain RS2, of the thirteen strains tested, appeared to have SGL-like activity. This research on the SGL with its novel catalytic activity, suggests a new mechanism by which sulfomucin desulfation can occur. The physiological importance of the enzyme is postulated to be (i) to provide energy in the form of sulfate-6-N-acetylglucosamine, for the bacterium, (ii) to remove sulfate-6-N-acetylglucosamine groups present on mucin chains, thus creating or removing sites for different adhesins, and (iii) removal of inhibitory sulfate-6-N-acetylglucosamine groups from mucin chains that limit degradation of the chain by exoglycosidases and neuraminidases.