Figure 1.
Chemical structures of the fluorescent substrates used in this study.
Figure 2.
Substrate specificity of mammalian neuraminidases.
Vmax/KM values are plotted on a logarithmic scale. Values shown are means of three independent experiments.
Table 1.
Kinetic data from substrate studies with recombinant neuraminidases.
Table 2.
Predicted protein-ligand contacts in Neu2 and Neu3.
Figure 3.
Modeling of S1 and S5 binding to the Neu2 active site.
(a) The substrate, S1, binds to the active site with expected contacts to the arginine triad and glycerol side chain. The GlcNAc residue B face is placed on the top of the hydrophobic side chain of Q112, forming a favorable interaction. The α2,3-glycosidic linkage between Neu5Ac and Gal allows S1 to form a relatively stable complex. (b) The same position for S1 as shown in (a), but with a surface representation of the Neu2 active site. (c) The α2,6-glycosidic linkage between the Neu5Ac and Gal residues in S5 makes it difficult for the substrate to fit into the active site without adopting an unfavorable conformation. The Neu5Ac residue adopts a twist boat conformation to preserve interactions with the Arg triad. The GlcNAc A face is interacting with a hydrophobic patch in the Neu2 active site, which is likely unfavorable. (d) The same pose for S5 as shown in (c), but with a surface representation of the Neu2 active site.
Figure 4.
Modeling of S1 binding to Neu3 active site.
(a) Interactions of S1 with amino acid residues in the active site of the Neu3 homology model. (b) The same position of S1 shown in (a), with a surface representation of the active site of Neu3.
Figure 5.
Neuraminidase activity in mouse brain tissues.
The activity was measured in the homogenates of whole brain tissues of wild-type C57Bl6 mice (WT) and gene-targeted C57Bl6 mice deficient in Neu3 (neu3−/−), Neu4 (neu4−/−), Neu1 (CathAS190A-neo) and double-knockout neu3−/−; neu4−/− mice against BODIPY-labeled sialylated oligosaccharides in 10 µM concentration. Values are shown as means (±S.E). N-value for each genotype is as follows: for WT and neu3−/−; neu4−/− n = 8, for neu3−/− and neu4−/− n = 6; for CathAS190A-neo n = 4. * -significantly different from WT (P<0.05) by repeated measurements ANOVA.