title: Substrate recognition of the membrane-associated sialidase NEU3 requires a hydrophobic aglycone.
abstract: The human neuraminidases (NEU) consist of a family of four isoforms (NEU1-NEU4). Members of this enzyme family are proposed to have important roles in health and disease through regulation of the composition of cellular sialosides. The NEU3 isoform is a membrane-associated enzyme that cleaves glycolipid substrates. However, few reports have examined the substrate specificity of the enzyme for non-natural substrates. We report here a series of 11 synthetic trisaccharides that feature modifications of the aglycone or the Neu5Ac residue of an octyl β-sialyllactoside. The time course of substrate cleavage by NEU3 was monitored using an electrospray ionization mass spectrometry assay to obtain relative rates (k(rel)). We observed that NEU3 substrate activity was directly dependent upon the hydrophobicity of the aglycone but had no apparent requirement for features of the ceramide headgroup. We also observed that trisaccharides with incorporated azide groups in the Neu5Ac residue at either C9 or the N5-Ac position were substrates, and in the case of the N5-azidoacetyl derivative, the activity was superior to that of GM3. However, the incorporation of larger aryl groups was tolerated only at C9, but not at N5-Ac. We propose a two-site model for enzyme recognition, requiring interaction at both the Neu5Ac residue and the hydrophobic aglycone.
authors.affiliation: Alberta Ingenuity Centre for Carbohydrate Science, Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Mahendra S
authors.initials: MS
authors.lastname: Sandbhor
authors.forename: Naoto
authors.initials: N
authors.lastname: Soya
authors.forename: Amgad
authors.initials: A
authors.lastname: Albohy
authors.forename: R Blake
authors.initials: RB
authors.lastname: Zheng
authors.forename: Jonathan
authors.initials: J
authors.lastname: Cartmell
authors.forename: David R
authors.initials: DR
authors.lastname: Bundle
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Cairo
mesh_terms: D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000201:enzymology; D005583:Fourier Analysis; D006017:Glycolipids / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
chemical_list: D006017:Glycolipids; C465436:Neu3 protein, human; D009439:Neuraminidase