title: 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.
abstract: 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.
authors.affiliation: Department of Medical Microbiology and Infection Control, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam (The Netherlands).
authors.forename: Robert
authors.initials: R
authors.lastname: van de Weerd
authors.forename: M Alvaro
authors.initials: MA
authors.lastname: Berbís
authors.forename: Marrion
authors.initials: M
authors.lastname: Sparrius
authors.forename: Janneke J
authors.initials: JJ
authors.lastname: Maaskant
authors.forename: Maikel
authors.initials: M
authors.lastname: Boot
authors.forename: Nanne J
authors.initials: NJ
authors.lastname: Paauw
authors.forename: Nadine
authors.initials: N
authors.lastname: de Vries
authors.forename: Louis
authors.initials: L
authors.lastname: Boon
authors.forename: Otto
authors.initials: O
authors.lastname: Baba
authors.forename: F Javier
authors.initials: FJ
authors.lastname: Cañada
authors.forename: Jeroen
authors.initials: J
authors.lastname: Geurtsen
authors.forename: Jesús
authors.initials: J
authors.lastname: Jiménez-Barbero
authors.forename: Ben J
authors.initials: BJ
authors.lastname: Appelmelk
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D016667:Bacterial Capsules / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics; D000939:Epitopes / Q000276:immunology*; D006003:Glycogen / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D009154:Mutation; D009161:Mycobacterium / Q000166:cytology / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D004251:DNA Transposable Elements; D000939:Epitopes; D009844:Oligosaccharides; D006003:Glycogen
keywords: STD-NMR spectroscopy; antibodies; carbohydrates; epitope specificity; glycogen monoclonal antibody; mycobacteria