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

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 α‐glucan has received relatively minor attention. Here we show that a murine monoclonal antibody (Mab) directed against glycogen cross‐reacts with mycobacterial α‐glucans, polymers of α(1–4)‐linked glucose residues with α(1–6)‐branch points. We identified the Mab epitope specificity by saturation transfer difference NMR and show that the α(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 α(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 α‐glucan (amylose) is insoluble. This Mab was subsequently used to develop several techniques helpful in capsular α‐glucan research. By using a capsular glucan‐screening methodology based on this Mab we were able to identify several unknown genes involved in capsular α‐glucan biogenesis. Additionally, we developed two methods for the detection of capsular α‐glucan levels. This study therefore opens new ways to study capsular α‐glucan and to identify possible targets for further research. Epitope specificity of a monoclonal antibody (Mab) to mycobacterial capsular α‐glucan: Capsular α‐glucan is a potential virulence factor of the important human pathogen Mycobacterium tuberculosis. By using advanced NMR techniques we have elucidated the epitope specificity of a monoclonal antibody directed against α‐glucan and present several Mab‐based techniques that should aid future studies of this cell‐surface structure.