Analyzing the dynamic bacterial glycome with a lectin microarray approach

Abstract

Glycosylation of bacterial cell surfaces is emerging as a critical factor in symbiosis, pathogenesis, cell-cell interactions and immune evasion. The lack of high-throughput analytical tools to examine bacterial glycans has been a major obstacle to the field and has hindered closer examination of the dynamics of carbohydrate variation. We have recently developed a lectin microarray for the analysis of glycoproteins. Herein we present a rapid analytical system based on this technology for the examination of bacterial glycans. The glycosylation pattern observed distinguishes closely related Escherichia coli strains from one another, providing a facile means of fingerprinting bacteria. In addition, dynamic alterations in the carbohydrate coat of a pathogenic E. coli strain are readily observed. The fast evaluation of real-time alterations in surface-carbohydrate epitopes allows examination of the dynamic role of bacterial sugars in response to external stimuli such as the immune system.

Uncovering the Binding Specificities of Lectins with Cells for Precision Colorectal Cancer Diagnosis Based on Multimodal Imaging

“…These characteristics make lectins very useful for glycoanalysis and medicine development 16, 17, 18, 19, 20, 21. Due to their minimization of sample consumption and high throughput format for analyzing multiple targets simultaneously, lectin microarrays have been employed in the glycomics study 22, 23, 24, 25, 26. However, the interactions of lectins with glycans (dissociation constant, K d  = 10 −4 –10 −7 m ) are much weaker than the interactions of antigens with antibodies ( K d  = 10 −8 –10 −12 m ).…”

NMR analysis on the sialic acid‐binding mechanism of an R‐type lectin mutant by natural evolution‐mimicry

“…The expression vector was transformed into Escherichia coli BL21-CodonPlus (DE3)-RIL strain (Stratagene, La Jolla, CA, USA). The 15 Nlabeled or 13 C, 15 N-labeled SRC was expressed in 15 Nlabeled or 13 C, 15 N-labeled C.H.L medium, respectively (Chlorella Industry Co., Tokyo, Japan), and purified by lactose-agarose column with elution buffer (PBS buffer: 60 mM Na 2 HPO 4 Á12H 2 O, 14 mM KH 2 PO 4 , 140 mM NaCl, and 2.7 mM KCl) containing 20 mM lactose at pH 7.0. Lactose in the purified proteins was removed by PD-10 column (GE Healthcare UK Ltd., Little Chalfont, UK).…”

“…The final concentration of the protein was 0.9 mM. In addition, each mixture (1 : 80) of 13 C, 15 N-labeled or 15 N-labeled SRC and lactose (Wako Chemicals, Tokyo, Japan) or 6 0 -sialyllactose (Carbosynth Limited, Compton, UK) was also prepared for NMR chemical shift change studies or 15 N relaxation studies as sugar-bound states.…”

“…All 1 H dimensions were referenced to internal 4,4-dimethyl-4-silapentane-1-sulfate (DSS), and 13 C and 15 N were indirectly referenced to DSS [23]. 1 H, 13 C, and 15 N assignments were obtained from standard multidimensional NMR experiments described previously [24]. NMR data were processed using FELIX The zz-exchange spectroscopy and STD-[ 1 H, 13 C] HSQC spectroscopy are described in the Data S1.…”

“…1 H, 13 C, and 15 N assignments were obtained from standard multidimensional NMR experiments described previously [24]. NMR data were processed using FELIX The zz-exchange spectroscopy and STD-[ 1 H, 13 C] HSQC spectroscopy are described in the Data S1.…”