Cloning of a Mucin-Desulfating Sulfatase Gene from Prevotella Strain RS2 and Its Expression Using a Bacteroides Recombinant System

Abstract

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl-and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.Mucins are high-molecular-weight glycoproteins which form the structural component of the protective mucus gel layer at the surfaces of the gastrointestinal, respiratory, and female genital tracts. Colonic mucin, particularly in the proximal region, contains significant levels of sulfate covalently bound to the mucin oligosaccharide chains, and levels of 2.0 to 6.5 g of sulfate per 100 g of mucin have been found (9, 17). Heavily sulfated mucins (sulfomucins) have many of the general lubricating and barrier functions of mucins with lower sulfate levels. There is accumulating evidence that sulfomucins may, in addition, rate-limit mucin degradation by mucin-degrading bacterial enzymes (4,7,13,15,18,24,26,27), and this role is thought to be particularly important in the colon, where approximately 10 14 bacterial cells are located (10). There have been reports of mucin-desulfating sulfatases that partially remove the sulfate from sulfomucin in a number of bacteria from the mouth, stomach, and colon and in feces. The effect of such sulfatases is to increase the susceptibility of the mucin to degradation by other mucin-degrading enzymes (4, 27). Elevated levels of bacterial mucin-desulfating sulfatases are found in feces of patients with ulcerative colitis (26). The sulfated sugar specificity of these fecal sulfatases, the number of different types present, the bacterial origin of the elevated levels, and the conditions which regulate bacterial production of such enzymes are unknown. The types of mucin-desulfating sulfatases that might be predicted include sulfatases specific for galactose-3-sulfate, g...

Anaerobic Sulfatase-maturating Enzymes, First Dual Substrate Radical S-Adenosylmethionine Enzymes

“…10C, panel 1), consistent with previous reports on Ser-type sulfatases (3,25). This result confirms that the unknown E. coli sulfatase maturation system is restricted to the Cys-type sulfatases (24).…”

A Novel Mechanism for Desulfation of Mucin: Identification and Cloning of a Mucin-Desulfating Glycosidase (Sulfoglycosidase) from Prevotella Strain RS2

“…The larger SGL peak eluting from the hydroxyapatite column was subjected to SDS-PAGE, and the major band (100 kDa) was excised. N-terminal sequencing of selected tryptic peptides was performed as previously described (28).…”

“…However, some bacteria possess mucin-desulfating sulfatases which desulfate sulfomucin, allowing glycosidases to access and act on the mucins. The mucin-desulfating sulfatases that have been described include sulfatases specific for the ␤-D-galactopyranosyl 3-sulfate, ␤-Dgalactopyranosyl 6-sulfate, and 2-acetamido-2-deoxy-␤-D-glucopyranosyl 6-sulfate (6-SO 3 -␤-GlcNAc) building blocks of the oligosaccharide chains (26,28,29).…”

Desulfurization of mucin by Pseudomonas aeruginosa: influence of sulfate in the lungs of cystic fibrosis patients

“…Mucin sulfatase activity has been identified in microbes associated with most mucosal layers, including the enteric bacteria of the genera Bacteroides and Prevotella (Tsai et al, 1991;Wright et al, 2000), oral streptococci (Jansen et al, 1997;Smalley et al, 1994), vaginal bacteria and the respiratory pathogens Pseudomonas aeruginosa and Burkholderia cepacia (Jansen et al, 1999;Tralau et al, 2007). Desulfation of the terminal sugars of mucin constitutes the first step in mucin degradation to provide carbon for growth but organisms like P. aeruginosa can also utilize human mucin as a source of sulfur (Tralau et al, 2007).…”