Construction of a Natural Mucin Microarray and Interrogation for Biologically Relevant Glyco-Epitopes

Abstract

Mucins are the principal components of mucus and mucin glycosylation has important roles in defence, microbial adhesion, immunomodulation, inflammation and cancer. Mucin expression and glycosylation are dynamic, responding to changes in local environment and disease. Potentially hundreds of heterogenous glycans can substitute one mucin molecule and it is difficult to identify biologically accessible glyco-epitopes. Thirty-seven mucins, from the reproductive and gastrointestinal (GI) tracts of six species (bovine, ovine, equine, porcine, chicken and deer) and from two human-derived cell lines, were purified. Following optimisation of mucin printing to construct a novel mucin microarray, the glycoprofiles of the whole mucins were compared using a panel of lectins and one antibody. Accessible glycomotifs of GI mucins varied according to species and localisation of mucin origin, with terminal fucose, the sialyl T-antigen and N-linked oligosaccharides identified as potentially important. The occurrence of T-and sialyl T-antigen varied in bovine and ovine reproductive tract mucins, and terminal N-acetylgalactosamine (GalNAc) and sulfated carbohydrates were detected. This study introduces natural mucin microarrays as an effective tool for profiling mucin glyco-epitopes and highlights their potential for discovery of biologically important motifs in bacterial-host interactions and fertility.

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

“…Mucins were isolated from the collected mucus and were purified as described previously (16,22). In brief, mucus was solubilized with guanidine hydrochloride (final concentration, 4 M).…”

“…Samples were reduced with dithiothreitol (DTT) (Sigma-Aldrich) at a final concentration of 0.01 M for 5 h at 37°C and were alkylated with iodoacetamide (0.025 M) (SigmaAldrich). Mucin was purified using CsCl density gradient separation followed by size exclusion chromatography (16,22). Non-mucin-containing fractions from the CsCl gradient were retained for subsequent analysis.…”

“…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).…”

“…These include cell lines that produce adherent mucus layers (20,21) and novel mucin microarrays that contain natural mucins from different animal species (22). This study aimed to examine the interactions of both H. pylori and C. jejuni with mucus and mucins by harnessing these novel techniques.…”

Interaction of microbes with mucus and mucins

“…We overcame some of these hurdles through the use of a novel high throughput mucin microarray platform containing mucins from different animal species. 38 This array enabled high throughput and quantitative analysis of the interaction of fluorescently labeled bacteria with the mucins. Other advantages include efficient use of limited mucin quantities enabling optimization of the number of binding experiments that can be done and optimal presentation of the glycans, thereby maximizing the access of the bacteria to potential glycan receptors.…”