Characterization of Sialidase from an Influenza A (H3N2) Virus Strain: Kinetic Parameters and Substrate Specificity

Abstract

Neuraminidase (NA) of influenza A (H3N2) viruses was characterized after purification by gel filtration and proteolytic treatment, using the X-31 variant strain that is a reassortment between the influenza A/Victoria/3/75 (responsible for the 1975 pandemic) and the influenza A/PR/8/34 virus samples, as a model. In the purification process, NA heads, that is the spike responsible for the virus sialidase activity, were purified by filtration through a Bio-Gel polyacrylamide column. The enzyme activity was determined by periodic acid/thiobarbituric acid assay and high-performance thin-layer chromatography. The sialidase showed preference for the α-2,3-linkage over the α-2,6-linkage of sialyllactoses (K_m of 1.8 and 5.2 × 10^–4M, respectively) at pH 5.2. The enzyme acted on natural and synthetic substrates at different hydrolysis rates, as well as on human erythrocytes (A group, Rh+) and yeast (Candida albicans) cells. The active NA produced by gel filtration was characterized by different parameters of its sialidase activity, also showing to be a suitable tool for the identification of natural sialocompounds and for the screening of antisialidase drugs to treat influenza virus infections.

The binding properties of the H5N1 influenza virus neuraminidase as inferred from molecular modeling

“…All of these results imply that the N1 enzyme of the avian influenza virus binds in the binding mode that facilitates preferential cleavage of sialic acid from methyl 3"sialyllactoside over methyl 6"sialyllactoside. These results are consistent with experimental data that showed that avian influenza virus desialylates α-Neu5Ac-2,3-linked oligosaccharides at least one order faster than α-Neu5Ac-2,6-linked oligosaccharides [6] and with the data on specificity of the human influenza virus neuraminidases [4,5].…”

“…In contrast to hemagglutinin, there is only limited information on neuraminidase substrate specificity, though it has been suggested that neuraminidase preferentially recognizes the same linkage of sialic acid as the hemagglutinin of these viruses [4][5][6].…”

“…4 Based on the efficacy of zanamivir (Relenza TM ), another neuraminidase inhibitor was also developed: oseltamivir phosphate (Tamiflu TM ) [10]. Both Relenza TM , a carbohydrate-based drug, and Tamiflu TM , a carbocyclic mimetic, are potent and clinically effective anti-influenza drugs [11].…”

Influenza neuraminidase

“…Drzeniek found that early H2N2 strains cleaved 3’SiaLac about 10-fold faster than fetuin or ovomucoid, and 20 times faster than orosomucoid (α1 acid glycoprotein), Tamm-Horsfall protein from urine, or porcine submaxillary mucin 53 . A similar gradient from 3’SiaLac to fetuin to bovine mucin was found for A/Aichi/68 (H3N2) NA 54 . The extensive O-acetylation of mucin contributes to its low activity as substrate; 4-O-acetylSia is not cleaved at all, while sialic acids with O-acetyl groups at 7, 8 or 9 positions on the glycerol chain are cleaved slowly 55 .…”

Computational analysis in Influenza virus

“…The strength of the substrate binding is in the following order: 3SL > 6SL >> sialic acid. This might be a reason that an avian N1 was experimentally found to cleave the SA-α-2,3-Gal glycoconjugates better than the SA-α-2,6-Gal [27][28][29] .…”