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

• Published in: Chembiochem : a European journal of chemical biology 2015-04, Vol.16 (6), p.977
• Main Authors: van de Weerd, Robert, Berbis, M Alvaro, Sparrius, Marrion, Maaskant, Janneke J, Boot, Maikel, Paauw, Nanne J, de Vries, Nadine, Boon, Louis, Baba, Otto, Canada, F Javier, Geurtsen, Jeroen, Jimenez-Barbero, Jesús, Appelmelk, Ben J
• Format: Article
• Language: English
• Subjects: Bacteriology
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201402713

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 [alpha]-glucan has received relatively minor attention. Here we show that a murine monoclonal antibody (Mab) directed against glycogen cross-reacts with mycobacterial [alpha]-glucans, polymers of [alpha](1-4)-linked glucose residues with [alpha](1-6)-branch points. We identified the Mab epitope specificity by saturation transfer difference NMR and show that the [alpha](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 [alpha](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 [alpha]-glucan (amylose) is insoluble. This Mab was subsequently used to develop several techniques helpful in capsular [alpha]-glucan research. By using a capsular glucan-screening methodology based on this Mab we were able to identify several unknown genes involved in capsular [alpha]-glucan biogenesis. Additionally, we developed two methods for the detection of capsular [alpha]-glucan levels. This study therefore opens new ways to study capsular [alpha]-glucan and to identify possible targets for further research.