A70 FECAL-ADHERENT MUCUS IS A NON-INVASIVE SOURCE OF PRIMARY HUMAN MUC2 FOR STRUCTURAL AND FUNCTIONAL CHARACTERIZATION IN HEALTH AND DISEASE

• Published in: Journal of the Canadian Association of Gastroenterology 2023-03, Vol.6 (Supplement_1), p.38-39
• Main Authors: Fancy, N, Melvin, M, N, N, Kazemian, N, D’Aloisio, L, Davidson-Hunt, S, Chadee, K, Pakpour, S, Ghosh, S, Gibson, D, Zandberg, W, Bergstrom, K
• Format: Article
• Language: English
• Subjects: Poster Presentations
• ISSN: 2515-2084; 2515-2092
• DOI: 10.1093/jcag/gwac036.070

Abstract Background The gel-forming O-glycoprotein Mucin-2 (MUC2) is a key mediator of host-microbe homeostasis in part by forming a barrier to segregate inflammatory microbes from distal colon tissues. While animal models have provided insights into how Muc2 functions, relatively few studies have explored human MUC2. This is due to difficulty in accessing primary MUC2 which has been traditionally seen as firmly adherent to tissues and thus attainable mainly through invasive approaches (e.g. surgery, biopsies, etc), or via transformed cell lines (e.g. LS174T). This highlights a need to find alternative sources of MUC2. Purpose The purpose of this study is to develop a non-invasive method to analyze human MUC2 from healthy persons and determine if this can be applied to disease states. Recent studies have shown a significant portion of MUC2 is bound to feces (Bergstrom and Shan et al, 2020). We therefore reasoned this fecal MUC2 was accessible for both purification and structural and functional characterization. Method We purified MUC2 from feces via established extraction methods used for tissues. The mucins were resolved by composite urea agarose polyacrylamide gel electrophoresis (UreaAgPAGE) and analyzed by in-gel staining with Alcian Blue (AB), or Western blot for lectins and MUC2. Mucins were subjected to both proteomics to confirm enrichment of MUC2; and O-glycomics via non-reductive ammonia-catalyzed β-elimination followed by capillary electrophoresis (CE) and mass spectrometry in parallel with Type III porcine gastric mucin (PGM) O-glycans for comparison. Purified O-glycans were tested functionally via microbial growth assays with Bacteroides spp, and the ability to be metabolized into short-chain fatty acids (SCFA). Mucus barrier function was also visualized directly on Carnoy's-fixed paraffin-embedded (CFPE) fecal sections followed by dual staining for bacteria by FISH, and MUC2 and/or lectins. Result(s) Confocal imaging of CFPE fecal sections revealed a microbiota-encapsulating barrier layer of varying thickness among various healthy human subjects. UreaAgPAGE showed high molecular weight bands (~1 – 2 MDa) by AB staining. Proteomics and western analysis confirmed MUC2 enrichment in fecal mucin preparations. Western analysis via a lectin panel showed human MUC2 bound several lectins but was notably lacking in signal for Sambucus nigra lectin (SNA; α2,6-linked sialic acid) or Ulex europaeus agglutinin I (UEA1; α1,2-linked fucose). O-glycomics rev