Journal: Microbiology Spectrum

Article Title: Mapping Molecular Recognition of β1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus

doi: 10.1128/Spectrum.01826-21

Figure Lengend Snippet: Carbohydrate binding by BoSGBP MLG -A using a structurally diverse saccharide microarray. Heatmap comparing the binding patterns of BoSGBP MLG -A and selected control proteins. The microarray comprised soluble saccharides of different origins (fungal, bacterial, plant, and microalgal polysaccharides or glycoproteins) (Table S1); the major backbone sequences are depicted at the bottom. The heatmap represents the relative binding intensities calculated as the percentage of the fluorescence signal intensity at 150 pg (0.5 mg/ml)/spot given by the saccharide probe most strongly bound by each protein (normalized as 100%). Results are detailed in Table S2. S. cerevisiae, Saccharomyces cerevisiae ; N. oculata , Nanochloropsis oculata ; P. palmata , Palmaria palmata ; C. albicans , Candida albicans ; M. tuberculosis , Mycobacterium tuberculosis ; h Malectin, human malectin; Tm CBM41, CBM41 of Thermotoga maritima ; m Dectin-1, murine dectin-1; Ct CBM11, CBM11 of Clostridium thermocellum ; ConA, concanavalin A; AAL, Aleuria aurantia lectin.

Article Snippet: For carbohydrate microarray binding assays, the high-throughput expression and purification of BoSGBP MLG -A was done using the established protocols of NZYTech Ltd. (Lisbon, Portugal) ( ).

Techniques: Binding Assay, Microarray, Control, Fluorescence

Journal: Microbiology Spectrum

Article Title: Mapping Molecular Recognition of β1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus

doi: 10.1128/Spectrum.01826-21

Figure Lengend Snippet: Determination of BoSGBP MLG -A oligosaccharide binding specificity and chain length requirements using sequence-defined oligosaccharides. (A) “Glucome” microarray comprising 153 sequence-defined gluco-oligosaccharides prepared as neoglycolipid (NGL) probes. Ct CBM11 (CBM11 of Clostridium thermocellum ) and Cm CBM6-2 (CBM6-2 of Cellvibrio mixtus ) were used as control proteins. The degree of polymerization (DP) and glucose linkages are indicated at the top of the panels. Some relevant carbohydrate probe sequences for binding to Ct CBM11 are depicted in panel B. G, glucose; AO, NGLs were prepared from reducing oligosaccharides by oxime ligation with an aminooxy (AO)-functionalized lipid . The binding signals are depicted as mean values of fluorescence intensities of duplicate spots for each probe arrayed at 5 fmol/spot (with error bars) and are representative of at least two independent experiments (details are in Table S4). (B) Microscale thermophoresis analysis of the interaction of BoSGBP MLG -A with sequence-defined gluco-oligosaccharides. Dose-response curves were fitted to a one-site binding model to obtain K a values. Error bars indicate the standard deviations from triplicate experiments ( n = 3). Quality of the fitting is given by the standard error of regression and the reduced chi-square (Red χ 2 ) parameters. S/N, signal-to-noise ratio. (C) Sequences of the gluco-oligosaccharides depicted from the nonreducing to the reducing end. Monosaccharide symbol representations follow the symbol nomenclature for glycans (SNFG) . The 3-linkages are underscored.

Article Snippet: For carbohydrate microarray binding assays, the high-throughput expression and purification of BoSGBP MLG -A was done using the established protocols of NZYTech Ltd. (Lisbon, Portugal) ( ).

Techniques: Binding Assay, Sequencing, Microarray, Control, Ligation, Fluorescence, Microscale Thermophoresis