title: Properties of recombinant human cytosolic sialidase HsNEU2. The enzyme hydrolyzes monomerically dispersed GM1 ganglioside molecules.
abstract: 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.
authors: Department of Medical Chemistry, Biochemistry, and Biotechnology, University of Milan, Medical Faculty, Laboratorio Interdisciplinare Tecnologie Avanzate (L.I.T.A.)-Segrate, Via Fratelli Cervi 93, 20090 Segrate, Milan, Italy.
authors: Cristina
authors: C
authors: Tringali
authors: Nadia
authors: N
authors: Papini
authors: Paola
authors: P
authors: Fusi
authors: Gianluigi
authors: G
authors: Croci
authors: Giuseppe
authors: G
authors: Borsani
authors: Augusto
authors: A
authors: Preti
authors: Paolo
authors: P
authors: Tortora
authors: Guido
authors: G
authors: Tettamanti
authors: Bruno
authors: B
authors: Venerando
authors: Eugenio
authors: E
authors: Monti
mesh_terms: D000818:Animals; D002417:Cattle; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000737:chemistry* / Q000378:metabolism; D005732:Gangliosides / Q000737:chemistry; D006017:Glycolipids / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008823:Micelles; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry* / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors
chemical_list: D003902:Detergents; D005732:Gangliosides; D006017:Glycolipids; D006023:Glycoproteins; D008823:Micelles; D011994:Recombinant Proteins; D005677:G(M1) Ganglioside; D017830:Octoxynol; C403285:NEU2 protein, human; D009439:Neuraminidase