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1. (Article ID: 4895)
 
Fontana C, Conde-Alvarez R, Stahle J, Holst O, Iriarte M, Zhao Y, Arce-Gorvel V, Hanniffy S, Gorvel JP, Moriyón I, Widmalm G
Structural Studies of Lipopolysaccharide Defective Mutants from Brucella melitensis Identify a Core Oligosaccharide Critical in Virulence
Journal of Biological Chemistry 291(14) (2016) 7727-7741
 

The structures of the lipooligosaccharides from Brucella melitensis mutants affected in the WbkD and ManBcore proteins have been fully characterized using NMR spectroscopy. The results revealed that disruption of wbkD gives rise to a rough lipopolysaccharide (R-LPS) with a complete core structure (β-D-Glcp-(1→4)-α-Kdop-(2→4)[β-D-GlcpN-(1→6)-β-D-GlcpN-(1→4) [β-D-GlcpN-(1→6)]-β-D-GlcpN-(1→3)-α-D-Manp-(1→5)]-α-Kdop-(2→6)-β-D-GlcpN3N4P-(1→6)-α-D-GlcpN3N1P), in addition to components lacking one of the terminal β-D-GlcpN and/or the β-D-Glcp residue (48 and 17%, respectively). These structures were identical to those of the R-LPS from B. melitensis EP, a strain simultaneously expressing both smooth and R-LPS, also studied herein. In contrast, disruption of manBcore gives rise to a deep rough pentasaccharide core (β-D-Glcp-(1→4)-α-Kdop-(2→4)-α-Kdop-(2→6)-β-D-GlcpN3N4P-(1→ 6)-α-D-GlcpN3N1P) as the major component (63%), as well as a minor tetrasaccharide component lacking the terminal β-D-Glcp residue (37%). These results are in agreement with the predicted functions of the WbkD (glycosyltransferase involved in the biosynthesis of the O-antigen) and ManBcore proteins (phosphomannomutase involved in the biosynthesis of a mannosyl precursor needed for the biosynthesis of the core and O-antigen). We also report that deletion of B. melitensis wadC removes the core oligosaccharide branch not linked to the O-antigen causing an increase in overall negative charge of the remaining LPS inner section. This is in agreement with the mannosyl transferase role predicted for WadC and the lack of GlcpN residues in the defective core oligosaccharide. Despite carrying the O-antigen essential in B. melitensis virulence, the core deficiency in the wadC mutant structure resulted in a more efficient detection by innate immunity and attenuation, proving the role of the [β-D-GlcpN-(1→6)-β-D-GlcpN-(1→4)[β-D-GlcpN-(1→6)]-β-D-GlcpN-(1→3)-α-D-Manp-(1→5)] structure in virulence.

mutant, Gram-negative bacteria, Brucella melitensis, lipopolysaccharide (LPS), nuclear magnetic resonance (NMR), WadC

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2. (Article ID: 6383)
 
Fontana C, Widmalm G
Primary Structure of Glycans by NMR Spectroscopy
Chemical Reviews 123(3) (2023) 1040-1102
 

Glycans, carbohydrate molecules in the realm of biology, are present as biomedically important glycoconjugates and a characteristic aspect is that their structures in many instances are branched. In determining the primary structure of a glycan, the sugar components including the absolute configuration and ring form, anomeric configuration, linkage(s), sequence, and substituents should be elucidated. Solution state NMR spectroscopy offers a unique opportunity to resolve all these aspects at atomic resolution. During the last two decades, advancement of both NMR experiments and spectrometer hardware have made it possible to unravel carbohydrate structure more efficiently. These developments applicable to glycans include, inter alia, NMR experiments that reduce spectral overlap, use selective excitations, record tilted projections of multidimensional spectra, acquire spectra by multiple receivers, utilize polarization by fast-pulsing techniques, concatenate pulse-sequence modules to acquire several spectra in a single measurement, acquire pure shift correlated spectra devoid of scalar couplings, employ stable isotope labeling to efficiently obtain homo- and/or heteronuclear correlations, as well as those that rely on dipolar cross-correlated interactions for sequential information. Refined computer programs for NMR spin simulation and chemical shift prediction aid the structural elucidation of glycans, which are notorious for their limited spectral dispersion. Hardware developments include cryogenically cold probes and dynamic nuclear polarization techniques, both resulting in enhanced sensitivity as well as ultrahigh field NMR spectrometers with a 1H NMR resonance frequency higher than 1 GHz, thus improving resolution of resonances. Taken together, the developments have made and will in the future make it possible to elucidate carbohydrate structure in great detail, thereby forming the basis for understanding of how glycans interact with other molecules.

NMR, glycan

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