Faecal mucin sulphatase : identification, purification and characterisation of a novel enzyme and it relevance to ulcerative colitis
Colonic mucus is extensively sulphated and this may be critical in determining its resistance to deglycosylation by bacteria. The presence in faeces of a bacterial mucin sulphatase which might compromise mucosal resistance may have an important role in colitis. This thesis describes the purification and characterisation of a human faecal mucin sulphatase and its study in ulcerative colitis. A mucin sulphatase assay was established using as a specific substrate [35S]-labelled human colonic mucin, produced by culture of colonic biopsies for 24 hr in 95% O2, 5% CO2 in the presence of K2[35S]O4. Mucin extracted from these biopsies was purified by high performance gel filtration. Substrate purity was confirmed by caesium chloride density gradient centrifugation and SDS-PAGE. Following detection in normal faeces of a novel mucin sulphatase, it was purified 750x by sequential high performance gel filtration and ion-exchange chromatography. The purified enzyme migrated as a single band on SDS-PAGE with a relative m.w. of 15,000D, pI of 4.0 and a pH optimum of 4.5 (faecal pH ranges from 4.5 to 7.0). It has a Km of 41.9 mmol/L and a Vmax of 1.17 katal/kg for the substrate glucose-6-sulphate. Deglycosylation of mucin by a faecal glycosidase preparation was enhanced nearly 5-fold by the addition of the mucin sulphatase, demonstrating the importance of mucin sulphation in determining its resistance to glycosidase attack. Screening of 13 faecal bacterial species showed sulphatase production by Bacteroides fragilis and B. thetaiotaomicron. A mucin sulphatase prepared from B. thetaiotaomicron has a Km of 43 mmol/l and pH optimum of 5 for glucose-6-sulphatate similar, to the mucin sulphatase isolated from human faeces. Faecal sulphatase activity was assayed in bacteria-free faecal homogenates from patients with ulcerative colitis [UC] (n = 22), Crohn's disease [CD] (n = 14), and healthy subjects (n = 17). Sulphatase activity was increased in UC (median 80.2 range 6.9-1063 Units/mg protein) but not in CD (median 36.6, range 5.7-106.6 U/mg) compared to controls (median 11.3 range 3.0-53.5 U/mg) (P< 0.01, Kruskal-Wallis ANOVA by ranks). Patients with active UC had higher sulphatase activity (n = 12, median 105.5 range 22.5-1063 U/mg) than inactive UC (n = 10), median 38.4 range 6.9-249 U/mg) (P< 0.05). Bismuth subsalicylate which has been reported effective when given as enemas in UC, inhibited mucin sulphatase non-competitively by > 95% at a concentration of 4g/l. Bacterial mucin sulphatase is likely to be critical in determining the rate of deglycosylation of secreted colonic mucin. It may have an important role in the pathogenesis of ulcerative colitis. Safe inhibitors of this enzyme deserve further trial in the therapy of colitis.