A Murine Monoclonal Antibody to Glycogen: Characterization of Epitope-Fine Specificity by Saturation Transfer Difference (STD) NMR Spectroscopy and Its Use in Mycobacterial Capsular alpha-Glucan Research

作者：van de Weerd Robert; Alvaro Berbis M; Sparrius Marrion; Maaskant Janneke J; Boot Maikel; Paauw Nanne J; de Vries Nadine; Boon Louis; Baba Otto; Javier Canada F; Geurtsen Jeroen; Jimenez Barbero Jesus^*; Appelmelk Ben J

来源：ChemBioChem, 2015, 16(6): 977-989.

DOI：10.1002/cbic.201402713

• antibodies
• carbohydrates
• epitope specificity
• glycogen monoclonal antibody
• mycobacteria
• STD-NMR spectroscopy

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a major pathogen responsible for 1.5 million deaths annually. This bacterium is characterized by a highly unusual and impermeable cell envelope, which plays a key role in mycobacterial survival and virulence. Although many studies have focused on the composition and functioning of the mycobacterial cell envelope, the capsular -glucan has received relatively minor attention. Here we show that a murine monoclonal antibody (Mab) directed against glycogen cross-reacts with mycobacterial -glucans, polymers of (1-4)-linked glucose residues with (1-6)-branch points. We identified the Mab epitope specificity by saturation transfer difference NMR and show that the (1-4)-linked glucose residues are important in glucan-Mab interaction. The minimal epitope is formed by (linear) maltotriose. Notably, a Mycobacterium mutant lacking the branching enzyme GlgB does not react with the Mab; this suggests that the (1-6)-branches form part of the epitope. These seemingly conflicting data can be explained by the fact that in the mutant the linear form of the -glucan (amylose) is insoluble. This Mab was subsequently used to develop several techniques helpful in capsular -glucan research. By using a capsular glucan-screening methodology based on this Mab we were able to identify several unknown genes involved in capsular -glucan biogenesis. Additionally, we developed two methods for the detection of capsular -glucan levels. This study therefore opens new ways to study capsular -glucan and to identify possible targets for further research.

• 出版日期2015-4-13