A Putative Mucin-degrading Operon and a 6-Sulfo-N-Acetyl-β-D-Glucosaminidase in Prevotella Strain RS2

Abstract

A protective layer of mucus lines the surface of the gastrointestinal tract. Agents or processes that interrupt the continuity of this layer can potentially compromise the integrity of the mucosa, causing disease. The anaerobic bacterium Prevotella strain RS2 utilises the major structural component of the mucus layer, the mucous glycoprotein or mucin, as an energy source. Prevotella strain RS2 possesses a novel mucin-desulfating sulfatase (MdsA) which removes the protective sulfate ester group from N-acetyl-β-D-glucosamine-6-sulfate sugar residues of mucin oligosaccharide side chains. The desulfation of sulfated sugar residues in mucin is thought to be the rate-limiting step in mucin degradation. The gene sequence for MdsA has previously been determined. The 4650 bp of genomic DNA downstream of the gene encoding MdsA was amplified by inverse PCRs and sequenced. Two complete open reading frames, mdsC and mdsD, were identified and are predicted to encode putative proteins of 362 and 1073 amino acids respectively. The deduced MdsC protein exhibited homology to the catalytic domain of aminoglycoside phosphotransferases and may perform a similar function. The deduced MdsD protein is predicted to be transported to the periplasm and/or outer membrane, and based on homology, may have a multiprotein proteolytic function similar to the tricorn protease. Northern and Western blot analyses suggest mdsA, mdsC and mdsD are coexpressed and their expression is increased when Prevotella strain RS2 is grown on mucin. This work is the first mucin-degrading operon of genes to be described. Also during the course of this research, a novel desulfating enzyme, 6-sulfo-N-acetyl-β-D-glucosaminidase(6-SNG) was discovered in Prevotella strain RS2. The enzyme can remove intact N-acetyl-β-D-glucosamine-6-sulfate residues from sulfated sugar substrates and is different to MdsA which removes the sulfate ester group. A procedure for the partial purification of active 6-SNG has been determined. The 6-SNG is predicted to corresponded to a 67.0 kDa SDS-PAGE protein band though several isoenzymes or fragmentation products were detected during the purification. The 6-SNG is located in the periplasm of Prevotella strain RS2 and is expressed at increased levels when grown on mucin. Inhibition studies for the partially purified enzyme have identified a number of potential enzyme inhibitors. This work represents the first 6-SNG to be described that may play a role in mucin-desulfation.