Optimization of a direct spectrophotometric method to investigate the kinetics and inhibition of sialidases
- Jasvinder Kaur Hayre†1,
- Guogang Xu†2, 3,
- Luisa Borgianni1,
- Garry L Taylor4,
- Peter W Andrew5,
- Jean-Denis Docquier1 and
- Marco R Oggioni1Email author
© Hayre et al.; licensee BioMed Central Ltd. 2012
Received: 19 January 2012
Accepted: 25 September 2012
Published: 2 October 2012
Streptococcus pneumoniae expresses three distinct sialidases, NanA, NanB, and NanC, that are believed to be key virulence factors and thus, potential important drug targets. We previously reported that the three enzymes release different products from sialosides, but could share a common catalytic mechanism before the final step of product formation. 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.
In this work, we present further kinetic characterization of pneumococcal sialidases by using a direct spectrophotometric method with the chromogenic substrate p-nitrophenyl-N-acetylneuraminic acid (p- NP-Neu5Ac). Using our assay, the measured kinetic parameters of the three purified pneumococcal sialidase, NanA, NanB and NanC, were obtained and were in perfect agreement with the previously published data. The major advantage of this alternative method resides in the direct measurement of the released product, allowing to readily determine of initial reaction rates and record complete hydrolysis time courses.
We developed an accurate, fast and sensitive spectrophotometric method to investigate the kinetics of sialidase-catalyzed reactions. This fast, sensitive, inexpensive and accurate method could benefit the study of the kinetics and inhibition of sialidases in general.
KeywordsSialidase Neuraminidase Chromogenic sialic acids Kinetic assay Streptococcus pneumoniae
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 [2, 3].
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 [5–7]. 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.
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 [13–16]. The data are in good agreement with previously obtained NanA, NanB and NanC kinetic parameters, and followed the first-order reaction kinetics [5, 6]. The anti-influenza drugs Zanamivir and Oseltamivir (inhibitors of influenza virus sialidases) were also tested as inhibitors of pneumococcal sialidases, in our experimental setup.
Cloning, expression and purification of S. pneumoniae sialidases
The genes nanA (spr1536) and nanB (spr1531) were cloned from the genomic DNA of S. pneumoniae strain R6. The third sialidase gene, nanC, which is not present in R6, so was obtained from TIGR4 pneumococcal DNA (SP1326). The amplified gene segments were subsequently ligated into the commercially-available vectors, PQE30 (Qiagen), PET23b (Novagen) and PET21b (Novagen), respectively. Recombinant proteins NanA, NanB and NanC were overexpressed in Escherichia coli systems and were purified as described previously [5, 6, 17]. Protein purity was monitored by SDS-PAGE and the protein identities were confirmed by mass spectrometry.
Sialidase assays and determination of kinetic parameters
The activity of the sialidases was assayed colorimetrically and fluorometrically using the substrates p- NP-Neu5Ac (Sigma, St. Louis, Miss) and 4MU-Neu5Ac (Sigma, St- Louis, Miss), respectively. Conventional indirect assays were performed as previously described [5, 6, 17]. Briefly, the reaction mixtures containing the substrates and up to 100 nM sialidase were incubated at 37°C and stopped by the addition of 0.5 M Na2CO3 pH 9.0 for the colorimetric assay or 0.25 M glycine pH 10.0 for the fluorimetric assay. Released p-NP was detected spectrophometrically at 405 nm. The fluorescence associated with the release of 4-MU was measured at the excitation wavelength of 365 nm and an emission wavelength of 445 nm. These measurements were performed using an EnVision microplate reader (Perkin Elmer, Waltham, Mass.).
An alternative direct spectrophotometric method was optimized on the basis of the properties of the reaction substrate p- NP-Neu5Ac and its product p- NP, whose UV–vis spectra were recorded on a Cary 100 UV–vis spectrophotometer at 30°C. This method does not need to stop the reactions with the alkaline buffer and can be operated easily in multi-well flat-bottom assay plates and a microplate reader, allowing for a substantial improvement of data throughput. The variation occurring in the UV-visible spectrum upon hydrolysis of the substrate was investigated by recording, at different incubation times, spectra of 200–400 μM of p- NP-Neu5Ac in 10 mM MES buffer (pH, 6.0) in the presence of 25–700 nM of purified recombinant NanA.
The kinetic parameters of the various sialidases was determined at at 30°C in 20 mM MES buffer, pH 6.0. The initial reaction rates or complete time-course hydrolyses were recorded at a fixed wavelength of 400 nm (ΔɛM, 1,300 ± 200 M-1·cm-1) in the presence of various substrate concentrations. Enzyme concentrations were adjusted to obtain a measurable initial velocity or to record the complete reaction time-course (E0 ranged 14 to 140 nM for NanA; 160 to 9,600 nM for NanB; and 100 to 500nM for NanC). Kinetic parameters were computed using either the direct fit of the initial rates vs [S] data with the Henri-Michaelis-Menten equation of by analyzing the complete time-course reactions with the integrated form of Henri-Michaelis-Menten equation, as previously described . Each reaction was repeated at least three times. The pH and buffer dependencies of the sialidases NanA, NanB NanC were investigated in the buffers (Sodium Citrate/Disodim phosphate (100 mM, pH 4.0-5.5) and MES [2-(N-morpholino) ethanesulfonic acid] (20 mM, pH 5.5-7.0). Sialidase inhibitors used in this study, Neu5Ac2en, Zanamivir and Oseltamivir carboxylate were obtained from Sigma, GlaxoSmithKline and Roche Pharmaceutics respectively. Inhibition constants (K i s) were computed by measuring the initial reaction rates in the presence of varying concentration of inhibitors, using an competitive inhibition model, as previously described (20). Alternatively, the Vmax/K m * value, where K m * = K m (1 + [I]/K i ), was computed from the complete time-course hydrolysis in the presence of various inhibitor concentrations. The ratio (Vmax/K m )/( Vmax/K m *) (in the i. e. in the absence and presence of the inhibitor, respectively) was plotted as a function of [I], yielding a line, whose slope corresponds to 1/K i .
Thermal stability assay of sialidase NanA
The thermal shift assay of sialidase NanA against different buffers was performed in an iCycler iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA). In brief, to each well of a 96-well PCR plate was added 1 μl of 50 μM NanA, 0.25 μl of 500 × Sypro orange protein dye (originally 5000 × in Dimethyl sulfoxide) and 48.75 μl of buffer. The final volume was 50 μl. After sealing with optical film on top, the plate was put into the Real-Time PCR machine. The plate was then heated from 25°C to 89°C at the heating rate of 0.5°C per min. The fluorescence intensity of each well was measured at 490 nm excitation and 530 nm emission wavelength every min. The thermal shift curves were generated by the iCycler system automatically.
Optimization of a direct spectrophotometric assay to investigate the kinetics of sialidase-catalyzed reactions
Kinetic parameters for the hydrolysis of p-NP-Neu5Ac by the pneumococcal sialidases computed using the direct and the indirect spectrophotometric assays (see Materials and Methods for details)
p- NP-Neu5Ac direct method
p-NP-Neu5Ac indirect method
k cat (s -1)
K m (μM)
kcat/K m (M -1·s -1)
k cat (s -1)
K m (μM)
kcat/K m (M -1·s -1)
(3.5 ± 0.3) × 105
(8.4 ± 0.9) × 104
(2.7 ± 0.3) × 102
(1.1 ± 0.1) × 102
(3.4 ± 0.3) × 104
(1.2 ± 0.1) × 104
The effects of buffer and pH on the activity of pneumococcal sialidases
Inhibition of pneumococcal sialidases by three common neuraminidase inhibitors
Inhibition assay of pneumococcal sialidases
K i (μM)
Comparisons of existing sialidase assay methods
Resorcinol or diphenylamine
HPTLC and radiochromatoscanner
HPLC and fluorimeter
Time to run
Hazardous in case of skin contact
Cost per run
Expensive and substrate unstable
Inaccurate; Time consuming
Costly; Time consuming
Costly; Time consuming
Costly; Time consuming
Here, with both 4MU-Neu5Ac and p- NP-Neu5Ac sialidase assays and our direct spectrophotometric method, we observed that NanA, NanB and NanC show different pH optima and the activity could be affected by the buffer systems. It is intriguing that phosphate buffer at neutral pH has some negative effects on the NanA and NanC activity, but not NanB. By contrast, the three sialidases shower higher enzymatic activity in MES buffer, pH 5.5. Preliminary thermal shift assays confirmed that MES could increase the thermal stability of the sialidase.
In the present study, we also investigated the typical sialidase inhibitors Neu5Ac2en, Oseltamivir carboxylate and Zanamivir. Unlike the influenza virus sialidases, NanA is the only enzyme among the three pneumococcal sialidases that could be inhibited by Neu5Ac2en and Oseltamivir carboxylate with inhibition constants in the micromolar range . Very limited inhibitory effects of NanA were seen with Zanamivir. None of the tested inhibitors significantly inhibited NanB and NanC (K i > > 7.5 mM). Previous structural studies had indicated that the guanidinium group of Zanamivir does not add to substrate-NanA interactions, which is different from the binding pattern of influenza virus neuraminidase and could be the reason why Zanamivir is a weak inhibitor to NanA . Similar results were also observed in animal studies . Furthermore, comparison of NanA complexes with NanB structures and NanC model shows that both Zanamivir and Oseltamivir cannot interact tightly with the other pneumococcal sialidases due to the different architecture around active sites .
In this work, we developed an accurate, fast and sensitive spectrophotometric method to investigate the kinetics of sialidase-catalyzed reactions. Using our assay, the measured kinetic parameters of the three purified pneumococcal sialidase, NanA, NanB and NanC, were obtained and were in perfect agreement with the previously published data [5–7]. In view of the fact that both the substrates used in this paper, as also the relative inhibitors work well, and were in fact originally developed, for assays of viral sialidases, the method developed should be suitable for the assays of most sialidases, including most if not all viral and bacterial sialidases [21, 22]. This method, prone to automation and high-throughput screening, could ease and accelerate the screening of potential large libraries of chemical compounds to identify new inhibitors, which represent interesting and relevant anti-infective drug targets.
- p- NP-Neu5Ac p:
2-deoxy-2,3-didehydro-N-acetyl- neuraminic acid
The work was supported in part by the European Commission grants xFP7-HEALTH-222983 (PNEUMOPATH) and by Ricerca Regionale Toscana in Materia di Salute 2009–201. GX is the recipient of a European Respiratory Society Fellowship (ERS LTRF-n°93-2011) and a general program grant from National Natural Science Foundation of China (No.81170007).
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