The mammalian neuraminidase (NEU) enzymes are found in diverse cellular compartments. Members of the family, such as NEU2 and NEU1, are cytosolic or lysosomal, while NEU3 and NEU4 are membrane-associated. NEU enzymes that act on substrates in the plasma membrane could modulate cellular signaling, cell surface glycoforms and the composition of plasma membrane glycolipids. Therefore, their substrates and mechanism of action are of interest for discerning their physiological roles. We have studied the structure of the human NEU3 using molecular modeling to predict residues involved in the recognition and hydrolysis of glycolipid substrates. To test the model, we have used site-directed mutagenesis of the recombinant protein. Enzymatic studies of the relative activity of these mutants, as well as their pH profiles and inhibition by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, are reported. Using nuclear magnetic resonance spectroscopy, we confirmed that the enzyme is a retaining exo-sialidase, and we propose that the key catalytic residues of the enzyme consist of the general acid-base D50 and the nucleophilic Y370-E225 pair. Mutations of residues expected to interact directly with the sialic acid N5-acetyl (A160, M87, I105) and C7-C9 glycerol side-chain (E113, Y179, Y181) reduced enzymatic activity. We identified several active mutants of the enzyme which contain modifications at the periphery of the active site. Truncations at the N- or C-terminus of more than 10 residues abolished enzyme activity. We propose a catalytic mechanism consistent with the data and identify residues that contribute to glycolipid recognition.

The removal of sialic acid (Sia) residues from glycoconjugates in vertebrates is mediated by a family of neuraminidases (sialidases) consisting of Neu1, Neu2, Neu3 and Neu4 enzymes. The enzymes play distinct physiological roles, but their ability to discriminate between the types of linkages connecting Sia and adjacent residues and between the identity and arrangement of the underlying sugars has never been systematically studied. Here we analyzed the specificity of neuraminidases by studying the kinetics of hydrolysis of BODIPY-labeled substrates containing common mammalian sialylated oligosaccharides: 3′Sia-LacNAc, 3′SiaLac, SiaLex, SiaLea, SiaLec, 6′SiaLac, and 6′SiaLacNAc. We found significant differences in substrate specificity of the enzymes towards the substrates containing α2,6-linked Sia, which were readily cleaved by Neu3 and Neu1 but not by Neu4 and Neu2. The presence of a branching 2-Fuc inhibited Neu2 and Neu4, but had almost no effect on Neu1 or Neu3. The nature of the sugar residue at the reducing end, either glucose (Glc) or N-acetyl-D-glucosamine (GlcNAc) had only a minor effect on all neuraminidases, whereas core structure (1,3 or 1,4 bond between D-galactose (Gal) and GlcNAc) was found to be important for Neu4 strongly preferring β3 (core 1) to β4 (core 2) isomer. Neu3 and Neu4 were in general more active than Neu1 and Neu2, likely due to their preference for hydrophobic substrates. Neu2 and Neu3 were examined by molecular dynamics to identify favorable substrate orientations in the binding sites and interpret the differences in their specificities. Finally, using knockout mouse models, we confirmed that the substrate specificities observed in vitro were recapitulated in enzymes found in mouse brain tissues. Our data for the first time provide evidence for the characteristic substrate preferences of neuraminidases and their ability to discriminate between distinct sialoside targets.

In the past two decades, human neuraminidases (human sialidases, hNEUs) have been found to be involved in numerous pathways in biology. The development of selective and potent inhibitors of these enzymes will provide critical tools for glycobiology, help to avoid undesired side effects of antivirals, and may reveal new small-molecule therapeutic targets for human cancers. However, because of the high active site homology of the hNEU isoenzymes, little progress in the design and synthesis of selective inhibitors has been realized. Guided by our previous studies of human NEU3 inhibitors, we designed a series of C4,C7-modified analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) and tested them against the full panel of hNEU isoenzymes (NEU1, NEU2, NEU3, NEU4). We identified inhibitors with up to 38-fold selectivity for NEU3 and 12-fold selectivity for NEU2 over all other isoenzymes. We also identified compounds that targeted NEU2 and NEU3 with similar potency.