Enhancing effects of bile salts on the degradation of glycosphingolipids by glycosidases from bacteria of the human fecal flora.
Different concentrations of ionic and non-ionic detergents were examined for optimization of the in vitro degradations of intestinal glycosphingolipids by alpha- and beta-glycosidases from human fecal bacteria. In 5 mM Triton X-100 the enzymes hydrolyzed glycosphingolipids with lactoseries type 1 and 2 chains essentially to lactosylceramide (LacCer). In 5 mM sodium di- and trihydroxy bile salts lactosylceramide was degraded to glycosylceramide (GlcCer) in varying extent by enzymes from all five strains. The minimal bile salt concentrations for optimal 1,4-beta-galactosidase activities varied between 1 and 20 mM, i.e., close to or above the critical micellar concentrations (cmc). Dihydroxy bile salts were the most efficient in promoting conversion of LacCer to GlcCer at concentrations below 10 mM and conjugation with a taurine residue did not markedly lower the GlcCer yield. The optimal detergent concentrations for hydrolyses of the p-nitrophenyl (pnp) glycosides Gal beta 1-pnp and GalNAc alpha 1-pnp were approximately 0.05 mM for Triton X-100 and 0.5 mM for sodium taurodeoxycholate, i.e., clearly below their reported cmc values. Galabiosylceramide, globotria- and globotetraosylceramides, not degraded in the Triton X-100 micelles, were also resistant to hydrolysis using the sodium bile salts as detergents. In contrast, lactotetraosylceramide and isoglobotriaosylceramide were significantly more degraded by enzymes from a Ruminococcus gnavus strain and gangliotetraosylceramide by enzymes from a Bifidobacterium bifidum and a Bifidobacterium infantis strain using bile salt detergents. All strains but R. gnavus released terminal GalNAc from para-Forssman but not from the globotetraosylceramide or Forssman structures using 5 mM sodium deoxycholate as detergent. GM1 desialylation by two Ruminococcus torques strains and the R. gnavus and B. bifidum strains were enhanced under identical conditions. We conclude that the observed effects on glycosphingolipid hydrolyses reflects variations in the micellar presentation of the substrates. In addition, detergents seem to have a direct stimulating effect on the glycosidases, however at concentrations 10-100-times below the ones optimal for glycolipid degradations. These results with optimized bile salt concentrations, further support our previous observations that these five fecal bacterial strains produce enzymes with selected specificities towards glycosphingolipid core chains of the lactoseries type 1 and 2.
Falk
P
P
Department of Clinical Chemistry, University of Göteborg, Sahlgren's Hospital, Sweden.
Hoskins
L C
LC
Larson
G
G
D001644:Bifidobacterium / Q000201:enzymology* / Q000302:isolation & purification; D001647:Bile Acids and Salts / Q000378:metabolism / Q000494:pharmacology*; D002240:Carbohydrate Sequence; D003902:Detergents / Q000494:pharmacology; D005243:Feces / Q000382:microbiology*; D005732:Gangliosides / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D006028:Glycosphingolipids / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D017830:Octoxynol; D010459:Peptococcaceae / Q000201:enzymology* / Q000302:isolation & purification; D011092:Polyethylene Glycols / Q000494:pharmacology; D013379:Substrate Specificity
D001647:Bile Acids and Salts; D003902:Detergents; D005732:Gangliosides; D006028:Glycosphingolipids; D011092:Polyethylene Glycols; D017830:Octoxynol; D006026:Glycoside Hydrolases