Publications

• Molecular insight into substrate recognition by human cytosolic sialidase NEU2.

  Mozzi A., Mazzacuva P., Zampella G., Forcella M.E., Fusi P.A., Monti E.

  Sialidases or neuramidases are glycoside hydrolases removing terminal sialic acid residues from sialo-glycoproteins and sialo-glycolipids. Viral neuraminidases (NAs) have been extensively characterized and represent an excellent target for antiviral therapy through the synthesis of a series of com ... >> More

  Sialidases or neuramidases are glycoside hydrolases removing terminal sialic acid residues from sialo-glycoproteins and sialo-glycolipids. Viral neuraminidases (NAs) have been extensively characterized and represent an excellent target for antiviral therapy through the synthesis of a series of competitive inhibitors that block the release of newly formed viral particles from infected cells. The human cytosolic sialidase NEU2 is the only mammalian enzyme structurally characterized and represents a valuable model to study the specificity of novel NA inhibitory drugs. Moreover, the availability of NEU2 3D structure represents a pivotal step toward the characterization of the molecular basis of natural substrates recognition by the enzyme. In this perspective, we have carried out a study of molecular docking of NEU2 active site using natural substrates of increasing complexity. Moreover, selective mutations of the residues putatively involved into substrate(s) interaction/recognition have been performed, and the resulting mutant enzymes have been preliminary tested for their catalytic activity and substrate specificity. We found that Q270 is involved in the binding of the disaccharide α(2,3) sialyl-galactose, whereas K45 and Q112 bind the distal glucose of the trisaccharide α(2,3) sialyl-lactose, corresponding to the oligosaccharide moiety of GM3 ganglioside. In addition, E218, beside D46, is proved to be a key catalytic residue, being, together with Y334, the second member of the nucleophile pair required for the catalysis. Overall, our results point out the existence of a dynamic network of interactions that are possibly involved in the recognition of the glycans bearing sialic acid. << Less

  Proteins 80:1123-1132(2012) [PubMed] [EuropePMC]

• Properties of recombinant human cytosolic sialidase HsNEU2. The enzyme hydrolyzes monomerically dispersed GM1 ganglioside molecules.

  Tringali C., Papini N., Fusi P., Croci G., Borsani G., Preti A., Tortora P., Tettamanti G., Venerando B., Monti E.

  Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to homogeneity, and its substrate specificity was studied. HsNEU2 hydrolyzed 4-methylumbelliferyl alpha-NeuAc, alpha 2-->3 sialyllactose, glycoproteins (fetuin, alpha-acid glycoprotein, transferrin, and bov ... >> More

  Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to homogeneity, and its substrate specificity was studied. HsNEU2 hydrolyzed 4-methylumbelliferyl alpha-NeuAc, alpha 2-->3 sialyllactose, glycoproteins (fetuin, alpha-acid glycoprotein, transferrin, and bovine submaxillary gland mucin), micellar gangliosides GD1a, GD1b, GT1b, and alpha 2-->3 paragloboside, and vesicular GM3. alpha 2-->6 sialyllactose, colominic acid, GM1 oligosaccharide, whereas micellar GM2 and GM1 were resistant. The optimal pH was 5.6, kinetics Michaelis-Menten type, V(max) varying from 250 IU/mg protein (GD1a) to 0.7 IU/mg protein (alpha(1)-acid glycoprotein), and K(m) in the millimolar range. HsNEU2 was activated by detergents (Triton X-100) only with gangliosidic substrates; the change of GM3 from vesicular to mixed micellar aggregation led to a 8.5-fold V(max) increase. HsNEU2 acted on gangliosides (GD1a, GM1, and GM2) at nanomolar concentrations. With these dispersions (studied in detailed on GM1), where monomers are bound to the tube wall or dilutedly associated (1:2000, mol/mol) to Triton X-100 micelles, the V(max) values were 25 and 72 microIU/mg protein, and K(m) was 10 and 15 x 10(-9) m, respectively. Remarkably, GM1 and GM2 were recognized only as monomers. HsNEU2 worked at pH 7.0 with an efficiency (compared with that at pH 5.6) ranging from 4% (on GD1a) to 64% (on alpha(1)-acid glycoprotein), from 7% (on GD1a) to 45% (on GM3) in the presence of Triton X-100, and from 30 to 40% on GM1 monomeric dispersion. These results show that HsNEU2 differentially recognizes the type of sialosyl linkage, the aglycone part of the substrate, and the supramolecular organization (monomer/micelle/vesicle) of gangliosides. The last ability might be relevant in sialidase interactions with gangliosides under physiological conditions. << Less

  J. Biol. Chem. 279:3169-3179(2004) [PubMed] [EuropePMC]

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