Investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase

Abstract

Objective: Neisseria meningitidis is a Gram-negative bacterium that causes meningitis. N. meningitidis serogroup W (NmW) capsule polymerase synthesizes capsular polysaccharide of this serogroup. This enzyme could be a tool for meningococcal glycoconjugate vaccine development. Our long-term goal is to control activity of the NmW capsule polymerase for production of defined carbohydrates for vaccines. The enzyme lacks a simple, high-throughput activity assay. Here, we describe the use of high-throughput bioluminescence assays (CMP-Glo and UDP-Glo by Promega) to investigate NmW capsule polymerase activity. These assays detect free nucleotides produced during transfer of sugar from UDP-Galactose and CMP-Sialic Acid to an acceptor. Kinetic studies using NmW hydrolyzed polysaccharide (PS) acceptor are described as well as preliminary work with a sialic acid trimer (DP3) acceptor.

Results: In CMP-Glo kinetic studies, with constant donor (80 µM) and varied NmW hydrolyzed polysaccharide (0-2000 µg/mL), a K_m of 629.2 ± 101.4 µg/mL and a V_max of 0.8965 ± 0.05823 µM/min was obtained. Using UDP-Glo, K_m and V_max values of 13.84 ± 9.675 µM and 0.6205 ± 0.1331 µM/min were obtained with varied CMP-NeuNAc (0-80 µM) and constant acceptor (400 µg/mL) and UDP-Gal (80 µM). This is the first report of using bioluminescence assays for NmW kinetics.

Keywords: Bioluminescence assay; CMP-Glo; Kinetics; Neisseria meningitidis; UDP-Glo.

Fig. 1

Michaelis Menten plots of kinetics data obtained using CMP-Glo with hydrolyzed serogroup W acceptor and corresponding standard curves. A Data with varying acceptor (0–2000 µg/mL) and other components held constant. K_m and V_max determined to be 629.2 ± 101.4 µg/mL and a V_max of 0.8965 ± 0.05823 µM/min. B Data with varying CMP-NeuNAc (0–80 µM) and other components held constant. K_m and V_max were determined to be 13.84 ± 9.675 µM and 0.6205 ± 0.1331 µM/min. C Standard Curve for panel A. D Standard Curve for panel B. (These are one representative example of three individual experiments. All experiments were run with two replicates. Data point indicates the mean and error bars represent standard deviation)

Fig. 2

NmW capsule polymerase activity in the absence of selected substrates. A UDP-Glo assay: maximum activity observed in presence of all components. B CMP-Glo assay: maximum activity observed in the absence of UDP-galactose. Panels A and B indicate representative examples of three individual experiments. All experiments were run with two replicates. Top of bar graph indicates the mean and error bars represent standard deviation. C HPLC chromatogram of NmW capsule polymerase elongation of hydrolyzed polysaccharide acceptor in the absence of UDP-galactose and D in the absence of result shows control and reaction with no CMP-NeuNAc. Panel C shows more peaks with later retention time compared with control indicating sialylation. Panel D indicates no retention time difference between control and reaction except for one new peak evolved at an earlier retention time indicating galactosylation. RLU (Relative Light Unit) represents the amount of light produced by luminescence and RFU is (Relative Fluorescence Unit) for fluorescence

Fig. 3

UDP-Glo assay optimization with sialic acid trimer (DP3). A Assay results with different NmW enzyme amounts with maximum activity observed with 1250 ng enzyme. B Assay results using different concentrations of DP3 acceptor indicate maximum activity with 4 mM DP3. C Results with different concentrations of CMP-NeuNAc and UDP-Gal show more activity in the presence of 80 µM of both donor sugars (Mean  = 1,01,833 RLU) vs 100 µM (98,566.7). For panel A, the data point indicates the mean and error bars represent standard deviation. For panels B and C, top of bar graph indicates the mean and error bars represent standard deviation. Each panel illustrates representative examples of three individual experiments. All experiments were run with three replicates