glycan microarray screening  (Chemie GmbH)

Journal: Nature Communications

Article Title: Intelectin-2 is a broad-spectrum antimicrobial lectin

doi: 10.1038/s41467-025-67099-4

Figure Lengend Snippet: A Overlay of carbohydrate recognition domain of hItln1 (wheat, PDB ID 4WMY) and mItln2 (teal, predicted model), showing calcium (green), conserved calcium coordination residues, and the presence of alanine instead of tryptophan at position 288 in mItln2. B Binding profiles of recombinant mItln2 (25 µg/mL) to microbial (left) and mammalian (right) glycan microarrays from the National Center for Functional Glycomics (NCFG). Graphical representations of top glycan hits from the glycan arrays are included. Complete microarray data are found in Supplementary Data . Data are shown as mean ± SD ( n = 4 technical replicates). RFU, Relative Fluorescence Unit. C Glycan structures bound by mItln2 in glycan arrays. All hits contain either a terminal Gal p or 6SO 3 -Gal p , shown in red. D BLI trace of mItln2 binding to immobilized biotinylated-β-Gal p at varying concentrations, with an apparent Κ d of 1.5 µM. E Dot blot analysis of immobilized MUC2, MUC5AC, and MUC5B, probed with 0.5 μM StrepII-mItln2 in the presence of Ca 2+ or EDTA. F Images of 5 μM StrepII-mItln2 (magenta) binding to 0.01% (w/v) fluorescently labeled MUC5AC (yellow). MUC5AC without lectin treatment served as a control. Scale bars, 20 µm. G Spectroscopic assay for cross-linking of 0.01% (w/v) mucins with varying amounts of mItln2, measured by the increase in absorbance at 405 nm. Schematic of the cross-linking assay is shown on the left. Data are shown as mean ± SD ( n = 3 technical replicates). H Time-lapse images of 0.01% (w/v) fluorescently labeled MUC5AC (yellow) treated with 5 μM mItln2 (magenta). Binding of mItln2 was detected with Strep antibody. Scale bars, 20 µm. I Spectroscopic assay for cross-linking of 0.01% (w/v) lactose-functionalized glycopolymer by mItln2, measured by the increase in absorbance at 405 nm. Data are shown as mean ± SD ( n = 3 technical replicates). Structure of the lactose-functionalized trans-poly (norbornene) is shown on the top. Results in ( D ), ( E ), and ( G ) are representative of three independent experiments. Results in ( F ), ( H ), and ( I ) are representative of two independent experiments. Source data are provided as source data file.

Article Snippet: Lectin binding to mammalian glycans was assessed using the Consortium for Functional Glycomics (CFG) version 5.5 microarray and RayBiotech slides (Cat# GA-Glycan-300-1).

Techniques: Binding Assay, Recombinant, Glycoproteomics, Functional Assay, Microarray, Fluorescence, Dot Blot, Labeling, Control

Journal: Nature Communications

Article Title: Intelectin-2 is a broad-spectrum antimicrobial lectin

doi: 10.1038/s41467-025-67099-4

Figure Lengend Snippet: A Comparison of carbohydrate recognition domain of hItln1 (top, PDB ID 4WMY) and hItln2 (bottom, predicted model), showing conserved calcium coordination residues and a serine replacing tyrosine at position 309 in hItln2. ( B – D ) BLI traces of hItln2 binding to immobilized biotinylated-β-Gal f (a hItln1 ligand) ( B ), biotinylated-β-Gal p ( C ), and biotinylated-6SO 3 LacNAc ( D ). In ( B ) and ( C ), 1.5 μM protein was used. In ( D ), 10 μM protein was used. Data were normalized by background subtraction, using biotin-loaded streptavidin. E Binding of recombinant hItln2 (25 µg/mL) to a microbial glycan microarray from NCFG. Graphical representations of top glycan hits from the glycan arrays are included. Complete microarray data are found in Supplemental Data 2. Data are shown as mean ± SD ( n = 4 technical replicates). F Glycan structures bound by hItln2 in glycan arrays. All hits contain either a Gal p or ethanolamine, shown in red. G Dot blot analysis of MUC2, MUC5AC, and MUC5B, spotted on the membrane and probed with 1 μM hItln2 in the presence of Ca 2+ or EDTA. Binding of hItln2 was detected using a Strep-HRP antibody. H Images of 5 μM hItln2 (magenta) binding to 0.01% (w/v) fluorescently labeled MUC2 (yellow). Binding of hItln2 was detected with Strep antibody. Scale bars, 20 µm. Results in ( B ), ( C ), ( D ) and ( G ) are representative of three independent experiments. Result in ( H ) is representative of two independent experiments. Source data are provided as source data file.

Article Snippet: Lectin binding to mammalian glycans was assessed using the Consortium for Functional Glycomics (CFG) version 5.5 microarray and RayBiotech slides (Cat# GA-Glycan-300-1).

Techniques: Comparison, Binding Assay, Recombinant, Glycoproteomics, Microarray, Dot Blot, Membrane, Labeling

Journal: Nature Communications

Article Title: A comprehensive synthetic library of poly- N -acetyl glucosamines enabled vaccine against lethal challenges of Staphylococcus aureus

doi: 10.1038/s41467-024-47457-4

Figure Lengend Snippet: A Relative fluorescence unit (RFU) of F598 mAb binding with the library of 32 PNAG pentasaccharides. The glycans are grouped according to the number of NHAc units in the molecule. Each PNAG sequence is printed five times on the glycan microarray. The error bars represent the standard deviations of five individual spots. Data are presented as mean values ± standard deviation. F598 generally prefers highly acetylated PNAG sequences. Both the location and the number of NHAc units are important determinants of F598 binding. B Quantification of the preference of F598 for acetylation at each site of the PNAG pentasaccharide. The mean values are calculated from the values of the binding intensities of all 32 PNAG sequences to F598. Each PNAG sequence is printed five times on the glycan microarray. Data are presented as mean values ± standard deviation. Source data are provided as a file.

Article Snippet: Glycan microarray slides were produced as previously described , on SuperEpoxy 2 slides (SME2; ArrayIt Corp, Sunnyvale, CA) and stored vacuum sealed at −20 °C.

Techniques: Fluorescence, Binding Assay, Sequencing, Glycoproteomics, Microarray, Standard Deviation

Journal: Nature Communications

Article Title: A comprehensive synthetic library of poly- N -acetyl glucosamines enabled vaccine against lethal challenges of Staphylococcus aureus

doi: 10.1038/s41467-024-47457-4

Figure Lengend Snippet: A IgG antibody titers to the immunizing PNAG oligosaccharide in rabbit ( n = 2 per group) sera on day 35 after prime vaccination. B IgG antibody titers in pooled rabbit sera from mQβ-conjugate or 5GlcNH 2 –TT conjugate immunized animals ( n = 2 per group) as well as titer of natural human IgG in pooled human serum against native PNAG polysaccharide purified from Acinetobacter baumannii . The numbers above symbols are the average titer numbers. Titers and 95% confidence intervals (CI) were determined by linear regression using log 10 values of the average of replicate serum dilutions to determine the X intercept and 95% CI when Y = 0.5 (OD 405 nm of ELISA plate reading). C Stacked bar graphs depicting the IgG signals at the serum dilution of 1:50,000 for each rabbit ( n = 2) immunized with mQβ–PNAG0, mQβ–PNAG10, and mQβ–PNAG26 as well as pre-immune sera, respectively, on the array. The complete microarray results are provided in the file; D Normalized binding of the comprehensive library of PNAG pentasaccharides by IgG antibodies from post-immune sera of rabbits immunized with mQβ–PNAG0, mQβ–PNAG10, and mQβ–PNAG26, respectively, as well as pre-immune sera. PNAG sequences are grouped together according to the total number of acetamides in the molecules. The color scale bar is shown on the right with 100% indicating the strongest binding to a PNAG component and 0% indicating the weakest binder. For each antigen, the two rows represent sera from two rabbits per group immunized with the specific construct. Source data are provided as a file.

Article Snippet: Glycan microarray slides were produced as previously described , on SuperEpoxy 2 slides (SME2; ArrayIt Corp, Sunnyvale, CA) and stored vacuum sealed at −20 °C.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Microarray, Binding Assay, Construct