Surface chemistry and linker effects on lectin–carbohydrate recognition for glycan microarrays

Abstract

Glycan microarrays are an increasingly utilised tool for analysis of protein-carbohydrate interactions and a variety of glycan-containing molecules and slide chemistries have been used to array carbohydrates on microarray surfaces. Slide surface chemistry can have significant impact on the ligand presentation, background noise, spot size and morphology and reproducibility of the arrayed molecules, which in turn impacts upon lectin-carbohydrate recognition. The linker used to attach the carbohydrate to the molecular scaffold is another variable in ligand presentation. To evaluate these effects, three different microarray surface chemistries were arrayed with the same mono-and disaccharide neoglycoconjugates and natural glycoproteins and incubated with four well-characterised plant lectins. Analogues of three monosaccharide neoglycoconjugates, with two common linkers each, were included in the test group to evaluate the linker effect on lectin recognition. Based on lowest background noise, expected lectin-ligand interaction, good spot morphology and best reproducibility, the three-dimensional hydrogel slide surface proved most suitable for lectin interrogation of carbohydrate ligands, and the more flexible phenylisothiocyanate linker afforded greater recognition of the carbohydrates by the relevant lectins.

Lectin microarray profiling and monosaccharide analysis of bovine milk immunoglobulin G oligosaccharides during the first 10 days of lactation

“…Microarray data extraction was performed as previously described (Gerlach et al., ; Kilcoyne, Gerlach, Kane, & Joshi, ). In brief, GenePix Pro v6.1.0.4 (Molecular Devices, Berkshire, UK) was used to extract raw intensity values from image files using a proprietary *.gal file which enabled the identification of 230 μm printed lectin spots using adaptive diameter (70%–130%) circular alignment.…”

“…Neutral and N ‐acetylated sugars were hydrolyzed and analyzed following a previously described method (Kilcoyne, Gerlach, Gough et al., ; Kilcoyne, Gerlach, Kane, et al., ) with minor modification. Briefly, 1 mg of each IgG sample was hydrolyzed by 2 N trifluoroacetic acid (TFA) at 100°C for 4 hr.…”

Divergent Mechanisms of Interaction of Helicobacter pylori and Campylobacter jejuni with Mucus and Mucins

“…Purified animal mucins and mucins from HT29-MTX-E12 and LS174T cells were printed onto microarray slides as described previously (22). Neoglycoconjugate (NGC) arrays were designed and printed as described previously (33). For incubations at 37°C, C. jejuni and H. pylori were cultured on agar as described above, harvested, and resuspended in Mueller-Hinton broth or in brain heart infusion (BHI) broth supplemented with 10% FBS, respectively, to an optical density at 600 nm (OD 600 ) of 0.2.…”

Glycomics Microarrays Reveal Differential In Situ Presentation of the Biofilm Polysaccharide Poly-N-acetylglucosamine on Acinetobacter baumannii and Staphylococcus aureus Cell Surfaces

“…Purified LTA from S. aureus was purchased from InvivoGen (Toulouse, France). Pure, unlabelled lectins were purchased from EY Labs (San Mateo, CA, USA) or Vector Laboratories Inc. (Burlingame, CA, USA) Neoglycoconjugates were purchased from Dextra Laboratories Ltd. (Reading, UK) and IsoSep AB (Tullinge, Sweden) or synthesised in house [65] (Tables S3 and S4). Anti-PNAG mAb (F598) was as previously generated in the Pier lab [66].…”