Fucosylated glycoproteins and fucosylated glycolipids play opposing roles in cholera intoxication.

Cholera toxin (CT) is the etiological agent of cholera. Here we report that multiple classes of fucosylated glycoconjugates function in CT binding and intoxication of intestinal epithelial cells. In Colo205 cells, knockout of B3GNT5, the enzyme required for synthesis of lacto- and neolacto-series glycosphingolipids (GSLs), reduces CT binding but sensitizes cells to intoxication. Overexpressing B3GNT5 to generate more fucosylated GSLs confers protection against intoxication, indicating that fucosylated GSLs act as decoy receptors for CT. Knockout (KO) of B3GALT5 causes increased production of fucosylated O-linked and N-linked glycoproteins, and leads to increased CT binding and intoxication. Knockout of B3GNT5 in B3GALT5 KO cells eliminates production of fucosylated GSLs but increases intoxication, identifying fucosylated glycoproteins as functional receptors for CT. These findings provide insight into molecular determinants regulating CT sensitivity of host cells.

Exo-Enzymatic Addition of Diazirine-Modified Sialic Acid to Cell Surfaces Enables Photocrosslinking of Glycoproteins.

Glycan binding often mediates extracellular macromolecular recognition events. Accurate characterization of these binding interactions can be difficult because of dissociation and scrambling that occur during purification and analysis steps. Use of photocrosslinking methods has been pursued to covalently capture glycan-dependent interactions in situ; however, use of metabolic glycan engineering methods to incorporate photocrosslinking sugar analogs is limited to certain cell types. Here, we report an exo-enzymatic labeling method to add a diazirine-modified sialic acid (SiaDAz) to cell surface glycoconjugates. The method involves the chemoenzymatic synthesis of diazirine-modified CMP-sialic acid (CMP-SiaDAz), followed by sialyltransferase-catalyzed addition of SiaDAz to desialylated cell surfaces. Cell surface SiaDAzylation is compatible with multiple cell types and is facilitated by endogenous extracellular sialyltransferase activity present in Daudi B cells. This method for extracellular addition of α2-6-linked SiaDAz enables UV-induced crosslinking of CD22, demonstrating the utility for covalent capture of glycan-mediated binding interactions.

Purification, biochemical and biophysical characterization of lysosomal ß-D-glucuronidase from an edible freshwater mussel, Lamellidens corrianus.

A lysosomal glycosidase, ß-glucuronidase, has been purified to homogeneity, from the soluble extracts of a freshwater mussel, L. corrianus, by a series of chromatography techniques involving phenyl-Sepharose, ion exchange, affinity and gel filtration chromatography. In native PAGE, ß-glucuronidase resolved into a single band and the molecular mass determined by gel filtration chromatography was found to be 250 kDa. Zymogram analysis with 4-methyl umbelliferyl ß-glucuronide substrate validated the purified enzyme as ß-glucuronidase. In SDS-PAGE, the purified enzyme was resolved into four sub-units with molecular weights around 90, 75, 65, and 50 kDa, respectively, and two of the subunits (90 and 50 kDa) cross-reacted with human ß-glucuronidase antiserum. The optimum pH and temperature of the purified glycosidase were 5.0 and 70 °C, respectively. The enzyme kinetics parameters, substrate affinity (KM) and maximum velocity (Vmax) of the purified protein estimated with p-nitrophenyl ß-D-glucuronide were 0.457 mM and 0.11867 µmol-1 min-1 mL-1, respectively. The secondary structure of ß-glucuronidase was determined in the far-UV range (190 nm to 230 nm) using CD spectroscopy. Heat denaturation plots determined by CD spectroscopy showed that the purified enzyme was stable up to 70 °C.

Comparative analysis of ß-hexosaminidase and acid phosphatase from Hydra vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1: Localization studies of acid phosphatase and ß-hexosaminidase from H. vulgaris Ind-Pune.

The present report describes a comprehensive study on comparative biochemical characterization of two lysosomal enzymes, acid phosphatase and ß-hexosaminidase in three different strains of Hydra; Hydra vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1 (self-feeder-1). Since morphology and habitat of Hydra effect lysosomal enzymes and their response to environmental pollutants, it would be interesting to identify them in different Hydra strains so as to use them as toxicity testing. Preliminary studies revealed a differential expression of acid phosphatase, ß-hexosaminidase and ß-glucuronidase in three Hydra strains. Expression of all three lysosomal enzymes in H. vulgaris Ind-Pune was low in comparison to H. vulgaris Naukuchiatal and H. magnipapillata sf-1, while their expression is comparable in H. vulgaris Naukuchiatal and H. magnipapillata sf-1. The Michaelis-Menten (KM) values for lysosomal ß-hexosaminidase using 4-nitrophenyl N-acetyl-ß-D-glucosaminide as substrate were found to be 1.3 mM, 1.1 mM and 0.8 mM, respectively for H. vulgaris Ind-Pune, H. vulgaris Naukuchiatal and H. magnipapillata sf-1. For acid phosphatase using 4-nitrophenyl-phosphate as substrate, the KM values were 0.38 mM, 1.2 mM and 0.52 mM respectively, for H. vulgaris Ind-Pune, H. vulgaris Naukuchiatal and sf-1 strains. The optimum temperature for ß-hexosaminidase was 60 °C for H. vulgaris Ind-Pune, while 50 °C was observed for H. vulgaris Naukuchiatal and sf-1 strains. The optimum pH for ß-hexosaminidase was found to be 6.0 for H. vulgaris Ind-Pune and H. vulgaris Naukuchiatal, and 5.0 for sf-1. The optimum temperature and pH of acid phosphatase was similar in all three strains, viz., 40 °C and 3.0, respectively. Preliminary localization studies using whole mount in situ hybridization revealed predominant endodermal expression of three enzymes in H. vulgaris Ind-Pune. Our results thus support the conservation of lysosomal hydrolases in Hydra.