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 g...

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Autores principales:	Laleh Sheikhi Moghaddam, Ayobami Adegbite, Pumtiwitt C. McCarthy
Formato:	article
Lenguaje:	EN
Publicado:	BMC 2021
Materias:	Bioluminescence assay CMP-Glo UDP-Glo Kinetics Neisseria meningitidis Medicine R Biology (General) QH301-705.5 Science (General) Q1-390
Acceso en línea:	https://doaj.org/article/2f3e5c8a803243cb9034b87acf7cf0e0
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spelling	oai:doaj.org-article:2f3e5c8a803243cb9034b87acf7cf0e02021-11-21T12:28:01ZInvestigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase10.1186/s13104-021-05831-11756-0500https://doaj.org/article/2f3e5c8a803243cb9034b87acf7cf0e02021-11-01T00:00:00Zhttps://doi.org/10.1186/s13104-021-05831-1https://doaj.org/toc/1756-0500Abstract 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 Km of 629.2 ± 101.4 µg/mL and a Vmax of 0.8965 ± 0.05823 µM/min was obtained. Using UDP-Glo, Km and Vmax 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.Laleh Sheikhi MoghaddamAyobami AdegbitePumtiwitt C. McCarthyBMCarticleBioluminescence assayCMP-GloUDP-GloKineticsNeisseria meningitidisMedicineRBiology (General)QH301-705.5Science (General)Q1-390ENBMC Research Notes, Vol 14, Iss 1, Pp 1-7 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Bioluminescence assay CMP-Glo UDP-Glo Kinetics Neisseria meningitidis Medicine R Biology (General) QH301-705.5 Science (General) Q1-390
spellingShingle	Bioluminescence assay CMP-Glo UDP-Glo Kinetics Neisseria meningitidis Medicine R Biology (General) QH301-705.5 Science (General) Q1-390 Laleh Sheikhi Moghaddam Ayobami Adegbite Pumtiwitt C. McCarthy Investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase
description	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 Km of 629.2 ± 101.4 µg/mL and a Vmax of 0.8965 ± 0.05823 µM/min was obtained. Using UDP-Glo, Km and Vmax 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.
format	article
author	Laleh Sheikhi Moghaddam Ayobami Adegbite Pumtiwitt C. McCarthy
author_facet	Laleh Sheikhi Moghaddam Ayobami Adegbite Pumtiwitt C. McCarthy
author_sort	Laleh Sheikhi Moghaddam
title	Investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase
title_short	Investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase
title_full	Investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase
title_fullStr	Investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase
title_full_unstemmed	Investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase
title_sort	investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional neisseria meningitidis serogroup w capsule polymerase
publisher	BMC
publishDate	2021
url	https://doaj.org/article/2f3e5c8a803243cb9034b87acf7cf0e0
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Investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase

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