Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a physical barrier that separates these microbes from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate the degradation of the highly complex O-glycans found in mucins. In the colon, these glycans are heavily sulfated, but the specific sulfatases that are active on colonic mucins have not been identified. Here, we show that sulfatases are essential to the utilization of colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We have characterized the activity of 12 different sulfatases encoded by this species, showing that these enzymes collectively are active on all of the known sulfate linkages in colonic O-glycans. Crystal structures of 3 enzymes provide mechanistic insight into the molecular basis of substrate-specificity.Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated Oglycans in vitro and also plays a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease (IBD).Sulfatase activity is likely to be a keystone step in bacterial mucin degradation and inhibition of these enzymes may therefore represent a viable therapeutic path for treatment of IBD and other diseases.

SummaryHumans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a physical barrier that separates these microbes from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate the degradation of the highly complex O-glycans found in mucins. In the colon, these glycans are heavily sulfated, but the specific sulfatases that are active on colonic mucins have not been identified. Here, we show that sulfatases are essential to the utilization of colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We have characterized the activity of 12 different sulfatases encoded by this species, showing that these enzymes collectively are active on all of the known sulfate linkages in colonic O-glycans. Crystal structures of 3 enzymes provide mechanistic insight into the molecular basis of substrate-specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also plays a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease (IBD). Sulfatase activity is likely to be a keystone step in bacterial mucin degradation and inhibition of these enzymes may therefore represent a viable therapeutic path for treatment of IBD and other diseases.

Functional pairing between cellular glycoconjugates and tissue lectins like galectins has wide (patho)physiological significance.T heir study is facilitated by nonhydrolysable derivatives of the natural O-glycans, such as Sand Se-glycosides.T he latter enable extensive analyses by specific 77 Se NMR spectroscopy,b ut stillr emain underexplored. By using the example of selenodigalactoside (SeDG) and the human galectin-1 and-3, we have evaluated diverse 77 Se NMR detection methods and propose selective 1 H, 77 Se heteronuclear Hartmann-Hahn transfer for efficient use in competitive NMR screening against as elenoglycoside spy ligand.B yf luorescence anisotropy,c ircular dichroism, and isothermal titration calorimetry (ITC), we show that the affinity and thermodynamics of SeDGb inding by galectins are similart ot hiodigalactoside (TDG) and N-acetyllactosamine (LacNAc), confirming that Se substitution has no major impact.I TC data in D 2 Ov ersus H 2 Oa re similar for TDG and LacNAcb inding by both galectins, but as olvent effect, indicating solvent rearrangement at the binding site, is hinted at for SeDG and clearly observed for LacNAcd imers with extendedc hain length.

The synthesis and use of a uniformly 13C labelled fucosyl donor as a general building block is demonstrated.