基于生物发光的脑膜炎奈瑟菌 W 群荚膜聚合酶双功能测定动力学参数的检测方法研究。

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

机构信息

Bioenvironmental Sciences Program, Morgan State University, 1700 East Cold Spring Lane, Baltimore, MD, 21251, USA.

Department of Chemistry, Morgan State University, 1700 East Cold Spring Lane, Baltimore, MD, 21251, USA.

出版信息

BMC Res Notes. 2021 Nov 18;14(1):417. doi: 10.1186/s13104-021-05831-1.

DOI:10.1186/s13104-021-05831-1

PMID:34794506

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8600345/

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 of 629.2 ± 101.4 µg/mL and a V of 0.8965 ± 0.05823 µM/min was obtained. Using UDP-Glo, K and V 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.

摘要

目的

脑膜炎奈瑟菌是一种革兰氏阴性细菌，可引起脑膜炎。脑膜炎奈瑟菌血清群 W（NmW）荚膜聚合酶合成该血清群的荚膜多糖。这种酶可能是开发脑膜炎球菌糖缀合物疫苗的工具。我们的长期目标是控制 NmW 荚膜聚合酶的活性，以生产用于疫苗的特定碳水化合物。该酶缺乏简单、高通量的活性测定法。在这里，我们描述了使用高通量生物发光测定法（Promega 的 CMP-Glo 和 UDP-Glo）来研究 NmW 荚膜聚合酶的活性。这些测定法检测在将糖从 UDP-半乳糖和 CMP-唾液酸转移到受体时产生的游离核苷酸。还描述了使用 NmW 水解多糖（PS）受体的动力学研究，以及与唾液酸三聚体（DP3）受体的初步工作。

结果

在 CMP-Glo 动力学研究中，使用恒定的供体（80 μM）和变化的 NmW 水解多糖（0-2000 μg/mL），获得 K 值为 629.2 ± 101.4 μg/mL 和 V 值为 0.8965 ± 0.05823 μM/min。使用 UDP-Glo，当变化 CMP-NeuNAc（0-80 μM）和恒定受体（400 μg/mL）和 UDP-Gal（80 μM）时，获得 K 和 V 值分别为 13.84 ± 9.675 μM 和 0.6205 ± 0.1331 μM/min。这是首次报道使用生物发光测定法研究 NmW 动力学。