Sialosides Containing 7- N-Acetyl Sialic Acid Are Selective Substrates for Neuraminidases from Influenza A Viruses

Abstract

Sialidases or neuraminidases are sialic-acid-cleaving enzymes that are expressed by a broad spectrum of organisms, including pathogens. In nature, sialic acids are monosaccharides with diverse structural variations, but the lack of novel probes has made it difficult to determine how sialic acid modifications impact the recognition by sialidases. Here, we used a chemoenzymatic synthon strategy to generate a set of α2-3- and α2-6-linked sialoside probes that contain 7-N-acetyl or 7,9-di-N-acetyl sialic acid as structure mimics for those containing the less stable naturally occurring 7-O-acetyl- or 7,9-di-O-acetyl modifications. These probes were used to compare the substrate specificity of several sialidases from different origins. Our results show that 7-N-acetyl sialic acid was readily cleaved by neuraminidases from H1N1 and H3N2 influenza A viruses, but not by sialidases of human or bacterial origin, thereby indicating that the influenza enzymes possess a distinctive and more promiscuous substrate binding pocket.

Keywords: N-acetyl analogue; O-acetyl sialic acid; carbohydrate; chemoenzymatic synthesis; influenza A virus neuraminidase; sialidase.

Figure 1

Structures of (a) N-acetylneuraminic acid (Neu5Ac), (b) 7-O-acetyl Neu5Ac (Neu5,7Ac₂), and (c) 7,9-di-O-acetyl Neu5Ac (Neu5,7,9Ac₃).

Scheme 1. One-Pot Three-Enzyme (OP3E) Synthesis of α2–3 (6–7)- and α2–6 (8–9)-Linked Sialosides from Man2,4diN₃ (1) or Man2,4,6triN₃ (2) Followed by Chemical Conversion of Azido Groups to N-Acetyl Groups

Azido groups in sialosides containing Neu5,7diN₃ (6 and 8) or Neu5,7,9triN₃ (7 and 9) were chemically converted to N-acetyl moieties to generate sialosides containing Neu5Ac7NAc (10 and 12) or Neu5Ac7,9diNAc (11 and 13).

Figure 2

Human and bacterial sialidase substrate specificity studies using sialyl GalβpNPs with low (a) and high (b) enzyme concentrations. Sialidase amounts for the low-concentration assays were standardized using the substrate Neu5Acα3GalβpNP. Sialidase amounts for the high-concentration enzyme assays were 4–500 fold (hNEU2, 8-fold; BiNanH2, 10-fold; Au Sialidase, 5-fold; CpNanI, 17-fold; Vc Sialidase, 4-fold; SpNanA, 100-fold; SpNanB, 100-fold; SpNanC, 500-fold; PmST1, 20-fold) higher than those used in the low-concentration enzyme assays depending on the availability of the enzymes. Abbreviations: h, human; Bi, Bifidobacterium infantis; Au, Arthrobacter ureafaciens; Cp, Clostridium perfringens; Vc, Vibrio cholerae; Sp, Streptococcus pneumoniae; PmST1, the sialidase activity of Pasteurella multocida α2–3-sialyltransfearse 1 in the presence of CMP (0.4 mM).

Figure 3

Substrate specificity analysis of IAV NAs using Siaα3/6GalβpNP and derivatives with low (a) and 10-fold higher (b) enzyme concentrations, respectively.