Journal: bioRxiv

Article Title: N-glycans show distinct spatial distribution in mouse brain

doi: 10.1101/2023.05.30.542954

Figure Lengend Snippet: Optimized lectin fluorescence in brain slices. A) Schematic highlighting controls for determining specific binding of lectins to brain slices. The binding of lectins (gray) with a reporter such as a fluorescent tag (star) reveals the spatial distribution of glycan classes on a tissue slide. Specific binding of the lectin to the glycan structures is confirmed by determining its sensitivity to appropriate glycosidases as well as through inhibition under saturating concentrations of a competitive carbohydrate. B) The specificity of ConA binding to its substrate on the layers of the cerebellum is confirmed via removal of the N-glycans by incubation with PNGase F and inhibition of the lectin binding by saturating conditions of mannose and glucose (200 mM each). Scale bar = 200 μm.

Article Snippet: Tissues were washed again three times with TBS and incubated with 25 μg/mL of lectins (Vector Labs: ConA FL-1001, GNL B-1245, AAL FL-1391, PHA-E FL-1121, RCA-I B-1085, SNA FL-1301, MAL-I FL-1311) in TBS-T for 1 h at RT in darkness with gentle shaking.

Techniques: Fluorescence, Binding Assay, Inhibition, Incubation

Journal: bioRxiv

Article Title: N-glycans show distinct spatial distribution in mouse brain

doi: 10.1101/2023.05.30.542954

Figure Lengend Snippet: N-glycan binding lectins highlight distinct structural elements at the Purkinje cell junction of the cerebellum. In addition to diffuse binding across layers of the cerebellum, the lectins GNL and ConA showed bright punctate staining consistent with synapses (white arrows) around Purkinje cell bodies (*). Binding of AAL and PHA-E showed increased signal in the molecular layer with a few diffuse punctate structures noted. PHA-E and RCA showed bright signal in multiple luminal structures consistent with microvasculature (red arrows). Scale bar = 20 μm. A schematic with common lectin-binding sites is shown for reference.

Article Snippet: Tissues were washed again three times with TBS and incubated with 25 μg/mL of lectins (Vector Labs: ConA FL-1001, GNL B-1245, AAL FL-1391, PHA-E FL-1121, RCA-I B-1085, SNA FL-1301, MAL-I FL-1311) in TBS-T for 1 h at RT in darkness with gentle shaking.

Techniques: Binding Assay, Staining

Journal: Frontiers in Immunology

Article Title: Effects of Receptor Specificity and Conformational Stability of Influenza A Virus Hemagglutinin on Infection and Activation of Different Cell Types in Human PBMCs

doi: 10.3389/fimmu.2022.827760

Figure Lengend Snippet: Binding of biotinylated lectins and viruses to human PBMCs. The MDCK and CHO cells and PBMCs were incubated for 1 hour with (A) biotinylated Sambucus nigra lectin (SNA, final concentration 0.4 µg/ml) or biotinylated Maackia amurensis lectin 1 (MAL-1, final concentration 1 µg/ml) at room temperature or (B) with biotinylated IAVs Mem-H1N1(final concentration 200 µg/ml) or Mal-H1N1 (final concentration 10 µg/ml) at 4 °C. Lectin or virus binding was detected as mean fluorescence intensity (MFI) after staining with streptavidin–APC. Cells were then fixed and stained for subpopulation as described in Material and Methods and subjected to flow cytometric analysis. Data of one representative donor are shown.

Article Snippet: For staining with plant lectins, the cells were incubated for 1 h at room temperature with biotinylated Sambucus nigra lectin (SNA) (Vector Labs, B-1305, final concentration 0,4 µg/ml) or Maackia amurensis lectin (MAL I) (Vector Labs, M-1315, final concentration 1 µg/ml) and washed twice with the lectin buffer (0.01 mM MnCl 2 , 0.1 mM CaCl 2 , 0.1 mM MgCl 2 , 0.1 % BSA in PBSdef).

Techniques: Binding Assay, Incubation, Concentration Assay, Fluorescence, Staining

Journal: Microbiology

Article Title: Characterization of Porphyromonas gingivalis sialidase and disruption of its role in host–pathogen interactions

doi: 10.1099/mic.0.000851

Figure Lengend Snippet: Reaction kinetics of 3- or 6-sialyllactose and SiaPG under different conditions. Variable concentrations of sialyllactose were exposed to SiaPG, under conditions mimicking physiological (pH 7.4 200 mM NaCl). Reactions were quenched by commencing the thiobarbituric acid assay at 1, 2 and 3 min, and the rate of Neu5Ac release determined by application of a Neu5Ac standard curve. (a) Michaelis–Menten plot, rate of 4-MU release (V0, µmol MU released min−1 mg−1 SiaPG), plotted against [3- or 6-sialyllactose] (µM) using Prism 7 (GraphPad). Error bars, sem. (b) Table summarizing Michaelis–Menten reaction kinetics and catalytic efficiency of SiaPG and MU-NANA, the table includes the k cat (4-MU release min−1), and k cat/KM (µM min−1). Data shown represent the mean (±sd) of one experiment, where each condition was repeated three times per experiment.

Article Snippet: Cells then underwent sialic acid staining with lectins from Sambucus nigra (FITC conjugated SNA, Vector Labs, specific for α2–6 Neu5Ac linkages), Maackia amuriensis (Biotinylated MAA, Vector Labs, specific for α2–3 Neu5Ac linkages).

Techniques: Acid Assay

Journal: Microbiology

Article Title: Characterization of Porphyromonas gingivalis sialidase and disruption of its role in host–pathogen interactions

doi: 10.1099/mic.0.000851

Figure Lengend Snippet: Desialylation of host-relevant glycans by SiaPG. UHPLC chromatogram showing elution of FA2G2S2 and SLeX with and without digestion by SiaPG. FA2G2S2 contains α2–6-linked Neu5Ac. Some contaminating glycans can be observed in the undigested sample, but these also appear to be desialylated. Gc=N-glycolylneuraminic acid. SLeX contains α2–3-linked Neu5Ac.

Article Snippet: Cells then underwent sialic acid staining with lectins from Sambucus nigra (FITC conjugated SNA, Vector Labs, specific for α2–6 Neu5Ac linkages), Maackia amuriensis (Biotinylated MAA, Vector Labs, specific for α2–3 Neu5Ac linkages).

Techniques:

Journal: Microbiology

Article Title: Characterization of Porphyromonas gingivalis sialidase and disruption of its role in host–pathogen interactions

doi: 10.1099/mic.0.000851

Figure Lengend Snippet: SiaPG cleaves α2-3 and α2-6 linked Neu5Ac from erythropoietin (EPO), though diacetylated sialic acid is retained. EPO was digested by SiaPG for 18 hours at 37 °C, and subjected to UHPLC-MS/MS. A deviation of MS2 ion m/z of±0.2 was permitted during spectral assignment. Sialylated structures may possess different isomers to those depicted (i.e. it is uncertain which terminal galactose residue(s) in the branched chains are sialylated). The top panel shows the UHPLC chromatogram for undigested EPO, the numbers indicate different EPO glycans, structures and further details of which are shown in Table S1. Lower panels show the UHPLC chromatogram for SiaPG digested EPO (orange), with three overlays showing MS1 ions which when fragmented produced MS2 ions with m/z corresponding to the precursor ion for Neu5Ac trisaccharide (GlcNAc-Gal-Neu5Ac, m/z=~657.25, green overlay),diactylated sialic acid trisaccharide (GlcNAc-Gal-Neu5,9Ac, m/z=~699.25, blue overlay, middle), and triacetylated sialic acid trisaccharide (GlcNAc-Gal-Neu5,8,9Ac, m/z=~741.25,brown overlay, lower).

Article Snippet: Cells then underwent sialic acid staining with lectins from Sambucus nigra (FITC conjugated SNA, Vector Labs, specific for α2–6 Neu5Ac linkages), Maackia amuriensis (Biotinylated MAA, Vector Labs, specific for α2–3 Neu5Ac linkages).

Techniques: Tandem Mass Spectroscopy, Produced

Journal: Microbiology

Article Title: Characterization of Porphyromonas gingivalis sialidase and disruption of its role in host–pathogen interactions

doi: 10.1099/mic.0.000851

Figure Lengend Snippet: Zanamivir inhibits P. gingivalis growth and biofilm formation on sialoglycoproteins. Bacteria were cultured for 5 days at 37 °C anaerobically, in the presence of Neu5Ac, or on surfaces coated with the glycoproteins mucin, saliva or serum. All conditions were performed in the presence or absence of zanamivir. (a) Total growth; OD600 of the culture was used to quantify bacteria in both biofilm and planktonic states. (b) Biofilm formation, quantified by resuspension of biofilms and counting the number of bacteria under microscopy. All data shown represent the mean of three biological repeats, where conditions were tested three times per experiment. Error bars, sem. Significance determined by t-test, **P=>0.01, ***P=>0.005.

Article Snippet: Cells then underwent sialic acid staining with lectins from Sambucus nigra (FITC conjugated SNA, Vector Labs, specific for α2–6 Neu5Ac linkages), Maackia amuriensis (Biotinylated MAA, Vector Labs, specific for α2–3 Neu5Ac linkages).

Techniques: Cell Culture, Microscopy

Journal: eBioMedicine

Article Title: Sialic acid blockade inhibits the metastatic spread of prostate cancer to bone

doi: 10.1016/j.ebiom.2024.105163

Figure Lengend Snippet: Sialic acid blockade can prevent/inhibit prostate cancer bone metastasis. ( a ) Inhibition of sialylation in TRAMPC2 cells using P-SiaFNEtoc detected using pan-specific Lectenz lectin flow cytometry. Cells were treated with a range of concentrations of P-SiaFNEtoc inhibitor from 2 μM to 512 μM for 72 h. The intensities were normalised to a DMSO control. ( b ) Detection of α2-6 linked sialylated N -glycans in TRAMPC2 cells using SNA lectin flow cytometry. TRAMPC2 cells treated with 64 μM P-SiaFNEtoc for 72 h had reduced levels of SNA binding indicating a reduction in α2-6 linked sialylation in these cells (unpaired t test, p = 0.0001). ( c ) Luciferase tagged TRAMPC2 cells (control or pre-treated with 64 μM P-SiaFNEtoc for 72 h) were injected into immunocompetent C57BL/6 mice via sub-cutaneous injection and tumours were monitored using in vivo bioluminescence imaging. Pre-treatment of TRAMPC2 cells with P-SiaFNEtoc (which removed sialylated glycans) significantly reduced tumour burden over 6 weeks (n = 10, Mann–Whitney test, p = 0.0233) thus suggesting that sialic acid blockade has the potential to inhibit the growth of prostate tumours. ( d ) Inhibition of sialylation in RM1 cells using P-SiaFNEtoc detected using pan-specific Lectenz lectin flow cytometry. Cells were treated with a range of concentrations of P-SiaFNEtoc inhibitor from 2 μM to 512 μM for 72 h. The intensities were normalised to a DMSO control. ( e ) Detection of α2-6 linked sialylated N -glycans in RM1 cells using SNA lectin flow cytometry. RM1 cells treated with 256 μM P-SiaFNEtoc for 72 h had reduced levels of SNA binding indicating a reduction in α2-6 linked sialylation in these cells (unpaired t test, p < 0.0001). ( f ) Luciferase tagged RM1 cells (control or pre-treated with 256 μM P-SiaFNEtoc for 72 h) were injected into immunocompetent C57BL/6 mice via intra cardiac injection. Tumours were monitored over 15 days using in vivo bioluminescence imaging. ( g , h ) Pre-treatment of RM1 cells with P-SiaFNEtoc (to remove sialylated glycans) significantly reduced the number of skeletal tumours formed (Mann–Whitney test, p = 0.0454), the incidence of tumour in left tibias (Chi-square test, p = 0.0455), and significantly increased survival time in mice (Log-rank test, p = 0.012). ( i ) Micro-CT analysis demonstrated that P-SiaFNEtoc significantly alleviated bone destruction in the trabecular bone of tibias and increased trabecular bone volume (BV/TV, p = 0.0211) and trabecular number (Tb. N, p = 0.035) (n = 9, unpaired t test, ∗p < 0.05). Representative images are shown. Scale bar is 200 μm.

Article Snippet: Slides were incubated for 3 h at room temperature with FITC-conjugated SNA lectin (Vector labs, FL-1301-2) at 1:500 or FITC-conjugated MAL I Lectin (Vector labs, FL-1311-2) at 1:500.

Techniques: Inhibition, Flow Cytometry, Control, Binding Assay, Luciferase, Injection, In Vivo, Imaging, MANN-WHITNEY, Micro-CT

Journal: Nature Communications

Article Title: Human-type sialic acid receptors contribute to avian influenza A virus binding and entry by hetero-multivalent interactions

doi: 10.1038/s41467-022-31840-0

Figure Lengend Snippet: a Graphical summary of methods that were employed to establish density gradients on HEK ΔSia cells and LAMP I. The Sia repertoire exposed on HEK ΔSia cells was specifically tuned by transfection with different concentrations of ST6Gal1 and/or ST3Gal4. Co-transfected LAMP I will be secreted in a C-terminally biotinylated form that can be attached to streptavidin BLI sensors for comparing entry into HEK ΔSia cells with binding to LAMP I with a similar sialylation pattern in a BLI assay. b , c Comparison of virus binding rates to avian-type and human-type receptors. Absolute binding rates (nm/300 s), normalized to a particle number of 10 9 particles of each virus strain, were determined. b Binding to sensors fully loaded with LAMP I produced in HEK ΔSia cells co-transfected with 25 ng ST6Gal1 or 25 ng ST3Gal4 yielding high levels of LacNAc termini carrying 2-6Sia (magenta bars) or 2-3Sia (black bars). c Binding to sensors fully loaded with 2-6S(LN)2 (magenta bars) or 2-3S(LN)2 (black bars). Relative binding rates were plotted as a function of relative receptor density for human H3N2 strains BK79 ( d ) and WU95 ( e ), avian H5N1 strain HU02 ( f ) and human H1N1 strain PR8 ( g ). Black lines show binding to LAMP I proteins obtained by co-expression with a concentration range of sialyltransferases ST6Gal1 ( d , e ) or ST3Gal4 ( f , g ). Magenta lines show binding to LAMP I proteins obtained by co-expression of a concentration range of ST6Gal1 in combination with 25 ng ST3Gal4 ( d , e ) or a concentration range of ST3Gal4 in combination with 25 ng ST6Gal1 ( f , g ). 2-6Sia density or 2-3Sia density on LAMP I was quantified by SNA or MAL I binding respectively and plotted on the x-axis as relative SNA ( d , e ) or MAL I ( f , g ) signal reflecting the relative receptor density. Data from biological replicates were fitted into single curves of which R-sq values are indicated. ( b n = 7, 3, 8, 8, 3, 3, 4, 4, 3, 3, 9 and 4 for each bar from left to right; c n = 6, 6, 4, 8, 3, 3, 4, 7, 3, 3, 5 and 4 for each bar from left to right). Data are presented as mean values +/−S.D. Panel a was Created with BioRender.com. Source data are provided as a Source Data file.

Article Snippet: Cells firstly were incubated with biotinylated-lectins (MAL I, Vector Labs; SNA, Vector labs) for 1 hr and then complexed with Streptavidin for 1 hr, (Alexa Fluor ™ 568 conjugate (1 mg/mL),Thermo Fisher).

Techniques: Transfection, Binding Assay, Produced, Expressing, Concentration Assay