Journal: Cell

Article Title: Small RNAs are modified with N-glycans and displayed on the surface of living cells

doi: 10.1016/j.cell.2021.04.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: All lectins were bought biotinylated from Vector labs: biotin-wheat germ agglutinin (WGA), biotin-concanavalin A (ConA), and biotin-Maackia Amurensis Lectin II (MAAII).

Techniques: Recombinant, Staining, Blocking Assay, Plasmid Preparation, High Performance Liquid Chromatography, Protein Extraction, Expressing, Software

Journal: BMC Biotechnology

Article Title: Enhanced transduction of colonic cell lines in vitro and the inflamed colon in mice by viral vectors, derived from adeno-associated virus serotype 2, using virus-microbead conjugates bearing lectin

doi: 10.1186/1472-6750-7-83

Figure Lengend Snippet: Infectivity analysis of AAV2 treated with sulfo-NHS-LC-biotin . AAV2.CMV-LacZ was treated with varying concentrations of sulfo-NHS-LC-biotin at room temperature for 45 min in the dark, followed by the addition of excess glycine to terminate the biotinylation reaction. The resulting biotinylated AAV2 preparations were applied to HeLa cells (5 × 10 6 AAV2 particles per well), which had been grown in 24-well plates at 37°C for 24 hr (initial cell number, 5 × 10 4 cells per well), and incubated at 37°C for 48 hr. Cells were fixed with glutaraldehyde, stained for β-galactosidase (LacZ) activity using X-gal as the substrate. Then, the number of infected cells, which were stained blue, in each well was counted under a light microscope (-○-). The same analysis was also performed on biotinylated AAV2 preparations, to which excess Neutralite avidin (100 μg per 5 × 10 6 AAV2 particles) had been added (-●-). Each datum shown is the average number of infected cells per well with a standard deviation (n = 26).

Article Snippet: The following lectins in biotinylated form were obtained from Vector Laboratories [the carbohydrate structure(s), to which each lectin binds, is indicated in bracket]: Con A from Jack bean ( Canavalia ensiformis ) seeds [mannose]; agglutinin from horse gram ( Dolichos biflorus ) seeds [ N -acetylgalactosamine]; agglutinin from peanuts ( Arachis hypogaea ) [galactosyl (β-1,3) N -acetylgalactosamine]; agglutinin I from castor bean ( Ricinus communis ) seeds [galactose and N -acetylglucosamine]; agglutinin from soybean ( Glycine max ) seeds [ N -acetylgalactosamine and galactose]; agglutinin I from furze gorse ( Ulex europaeus ) seeds [fucose]; and agglutinin from wheat germ ( Triticum vulgaris ) [ N -acetylglucosamine].

Techniques: Infection, Incubation, Staining, Activity Assay, Light Microscopy, Avidin-Biotin Assay, Standard Deviation

Journal: BMC Biotechnology

Article Title: Enhanced transduction of colonic cell lines in vitro and the inflamed colon in mice by viral vectors, derived from adeno-associated virus serotype 2, using virus-microbead conjugates bearing lectin

doi: 10.1186/1472-6750-7-83

Figure Lengend Snippet: Effect of the co-attachment of lectins to the microbead surfaces on the infectivity of AAV2-microbead conjugates . AAV2.CMV-LacZ was biotinylated with sulfo-NHS-LC-biotin at 50 μg/ml, followed by the removal of non-virion-associated biotinylation reagent by dialysis. AAV2-microbead conjugates were prepared by the attachment of biotinylated AAV2 particles to the surfaces of avidin-coated fluorescent microbeads (480 nm in diameter) (9.2 AAV2 particles per microbead). To these AAV2-microbead conjugates, a biotinylated form of each lectin was added in excess (0.2 μg biotinylated lectin per 10 7 avidin-coated microbeads), followed by the removal of unbound lectin molecules by centrifugation. The infectivity of these AAV2-microbead conjugates with and without lectin was analyzed on HeLa, COLO 205, and MIP-101 cell lines. Cells were cultured in 24-well plates at 37°C for 24 hr (initial cell number per well: HeLa, 5 × 10 4 ; COLO 205, 1 × 10 5 ; MIP-101, 7.5 × 10 4 ). AAV2-microbead conjugates bearing each lectin, along with free unmodified AAV2.CMV-LacZ, free biotinylated AAV2.CMV-LacZ, and AAV2-microbead conjugates without lectin, were applied to target cells (a total of 1 × 10 7 AAV2 particles per well) and incubated at 37°C for 48 hr. Cells were fixed with glutaraldehyde and stained for β-galactosidase activity using X-gal as the substrate. Then, the number of infected cells in each well was counted under a light microscope. Each datum shown is the average number of infected cells per well with a standard deviation (n = 16). A, free, unmodified AAV2.CMV-LacZ; B, free, biotinylated AAV2.CMV-LacZ; C, AAV2-microbead conjugates without lectin; D – J, AAV2-microbead conjugates bearing lectin (D, Con A; E, horse gram agglutinin; F, peanut agglutinin; G, castor bean agglutinin I; H, soybean agglutinin; I, furze gorse agglutinin I; and J, wheat germ agglutinin).

Article Snippet: The following lectins in biotinylated form were obtained from Vector Laboratories [the carbohydrate structure(s), to which each lectin binds, is indicated in bracket]: Con A from Jack bean ( Canavalia ensiformis ) seeds [mannose]; agglutinin from horse gram ( Dolichos biflorus ) seeds [ N -acetylgalactosamine]; agglutinin from peanuts ( Arachis hypogaea ) [galactosyl (β-1,3) N -acetylgalactosamine]; agglutinin I from castor bean ( Ricinus communis ) seeds [galactose and N -acetylglucosamine]; agglutinin from soybean ( Glycine max ) seeds [ N -acetylgalactosamine and galactose]; agglutinin I from furze gorse ( Ulex europaeus ) seeds [fucose]; and agglutinin from wheat germ ( Triticum vulgaris ) [ N -acetylglucosamine].

Techniques: Infection, Avidin-Biotin Assay, Centrifugation, Cell Culture, Incubation, Staining, Activity Assay, Light Microscopy, Standard Deviation

Journal: Nature Communications

Article Title: Highly modified and immunoactive N-glycans of the canine heartworm

doi: 10.1038/s41467-018-07948-7

Figure Lengend Snippet: Glycan epitopes in D. immitis . a A summary of neutral and anionic N-glycan structures highlighting major antennal modifications; for a fuller set of defined structures refer to Supplementary Table . b The presence of HexNAc and phosphorylcholine residues in L3 larvae was probed using CGL3 (red) and TEPC 15 (green) by indirect fluorescence microscopy; also shown are the DAPI staining (for DNA) and DIC (differential interference contrast) images and the scale bar corresponds to 10 µm. c MALDI-TOF–MS of the AEAB-labelled neutral N-glycome before and after hydrofluoric acid treatment indicating loss of antennal fucose and phosphorylcholine residues. d The binding of Coprinopsis galectin (CGL3; 10 µg/ml), wheat germ agglutinin (WGA; 10 µg/ml) and human mannose binding lectin (MBL) to the Dirofilaria glycans increases after hydrofluoric acid treatment, while that to anti-phosphorylcholine (TEPC 15 IgA monoclonal) decreases. The charts indicate the uncorrected fluorescence values with the standard deviations (mean of 10 spots; analysed with an unpaired two-tailed parametric t -test with a 95% confidence level) as well as a negative control (spotting buffer only; example array scans are shown for the WGA and MBL data). Note that previous data indicate that CGL3 can recognise LacdiNAc, whereas WGA is known to bind HexNAc n motifs and MBL to a wide range of glycans including those with terminal mannose or N -acetylglucosamine residues. Refer to Supplementary Figures – and for (i) western blotting and further micrograph data (including controls) on epitopes recognised by CGL3 and TEPC 15, (ii) a summary of MS data on proteins affinity purified on CGL3- and TEPC 15-Sepharose, (iii) further MBL binding experiments (before and after endoglycosidase H treatment) to the immobilised glycome pools and fractionated immobilised glycans as well as data on other lectin interactions to the natural glycans and to defined di- and tri-saccharides

Article Snippet: The slides were incubated with either dilutions of dog sera (one control and two infected), biotinylated fungal CGL3 and CCL2 (see above) or biotinylated forms of wheat germ agglutinin, Aleuria aurantia lectin, concanavalin A or tomato lectin (Vector Laboratories; B1025, B1395, B1005 and B1175), murine TEPC 15 IgA monoclonal (Sigma-Aldrich; M1421), recombinant human mannose binding lectin (Biotechne; 9085-MB-050) or natural human C-reactive protein (MPBio; 215231505) followed by the relevant secondary and/or tertiary antibodies.

Techniques: Fluorescence, Microscopy, Staining, Binding Assay, Two Tailed Test, Negative Control, Western Blot, Affinity Purification

Journal: bioRxiv

Article Title: Dual function of the O-antigen WaaL ligase of Aggregatibacter actinomycetemcomitans

doi: 10.1101/2022.10.31.514599

Figure Lengend Snippet: Electrophoretic mobility and glycosylation of EmaA expressed in hemolytic E. coli (HEC) strains. A) Immunoblot. Wild type HEC (wild type), HEC transformed with plasmid expressing emaA ( emaA + , dashed arrows), HEC co-transformed with plasmids expressing emaA and A. actinomycetemcomitans waaL ( emaA + / waaL + , solid arrows). Equivalent amounts of outer membrane fraction were isolated and separated by electrophoresis using 4–15% gradient polyacrylamide-SDS Tris-Glycine gels. The proteins were transferred to nitrocellulose and probed with an anti-stalk EmaA monoclonal antibody. The arrows indicate the electrophoretic mobility of the expressed EmaA. Note the other well defined higher molecular weight bands that also display an electrophoretic mobility shift when co-expressed with A. actinomycetemcomitans waaL . Immunoreactive material at the top of the immunoblot corresponds to EmaA aggregates associated with the stacking gel. B). Lectin blot. The same outer membrane fractions were prepared as above and probed with biotinylated Concanavalin A to detect glycans associated with any protein bands. Image color was inverted to make the bands easier to identify. The band corresponding to the EmaA monomers co-expressed with A. actinomycetemcomitans waaL are indicated by a white arrow. Note the aggregated protein near the top of the gel, also indicated with an arrow.

Article Snippet: The membrane was blocked using a Carbo-Free Blocking Solution (Vector Laboratories, Newark, CA) and probed with biotinylated mannose-recognizing Concanavalin A (Vector Laboratories) for 1 hour.

Techniques: Western Blot, Transformation Assay, Plasmid Preparation, Expressing, Isolation, Electrophoresis, Molecular Weight, Electrophoretic Mobility Shift Assay