Sialidases catalyze the removal of terminal sialic acid residue from various glycoconjugates and have been implicated in pathogenesis of infectious diseases
]. In fact, sialidases differ significantly in kinetic parameters, substrate specificity and catalytic properties. For example, typical sialidases hydrolyze sialiosides to release N-acetylneuraminic acid (Neu5Ac), whereas the leech intramolecular (IT) trans
-sialidase produces 2,7-anhydro-Neu5Ac selectively from α2,3- sialosides, while trypanosomal trans
-sialidase can also transfer Neu5Ac to another sugar
The major human pathogen Streptococcus pneumoniae
encodes three distinct sialidases, NanA, NanB and NanC that could be classified into three different subtypes
]. According to a recent NMR report, NanA is a classic hydrolytic sialidase, whereas NanB could be an IT trans
-sialidase similar to the leech enzyme, and NanC can handle the dual functions of both producing 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, DANA) and hydrating this general sialidase inhibitor when substrate is depleted
]. Nonetheless, it is proposed that the three could share a common catalytic mechanism before the final product formation step from a chemistry point of view. Based on these findings, a new sialidase triad is speculated, which might coordinate the sialidase action associated with pneumococcal virulence. However, the kinetic investigations of the three sialidases have not been systematically done thus far, due to the lack of an easy and steady measurement of sialidase reaction rate.
A variety of techniques have been used for the sialidase activity assays, but all present technical challenges. The classical method is the thiobarbituric acid (TBA) assay, in which released Neu5Ac reacts with TBA reagent and shows a specific absorbance measurable at 549 nm
]. Thin layer chromatography (TLC) is another method to visualize the sialidase reaction products
]. A more sophisticated method has been described by Trignali et al., which use radiolabelled gangliosides and high performance TLC separation of the reaction products
]. Both methods are still employed for the sialidase substrate specificity determination. NMR is the best way to characterize the different sialic acids released, but requires access to expensive instrumentation
]. In recent years, two artificial substrates 2’-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4MU-Neu5Ac) and 2-O
-nitrophenyl)-N-acetylneuraminic acid (p-
), were developed for fast sialidase assays
]. The α-glycosidical linkage of these substrates can be hydrolyzed by sialidases to release a measurable fluorescence (4-methylumbelliferone, 4MU) or yellow product in alkaline conditions (p-nitrophenol, p-
The structures of 4MU-Neu5Ac andpNP-Neu5Ac. (a) 4MU-Neu5Ac, Formula: C21H24NNaO11; MW: 489.41 g/mol; (b) pNP-Neu5Ac, Formula: C17H22N2O11; MW: 430.36 g/mol.
To ease the measurement of reaction rates, and on the basis of the spectral properties of the reaction substrate (p-
NP-Neu5Ac) and product (p-
NP), a direct spectrophotometric method was designed in the current study, which allows the monitoring of the concentration of the reaction product as a function of time. In contrast to previous method, it does not need to stop the reaction by alkaline buffer before every reading. The kinetic characterization of NanA, NanB and NanC was performed, as it could provide further insights into their roles in pneumococcal virulence and metabolism
]. The data are in good agreement with previously obtained NanA, NanB and NanC kinetic parameters, and followed the first-order reaction kinetics
]. The anti-influenza drugs Zanamivir and Oseltamivir (inhibitors of influenza virus sialidases) were also tested as inhibitors of pneumococcal sialidases, in our experimental setup.