Role of MUC2 mucin associated FCGBP in innate host defense against Entamoeba histolytica

The mucus layer is the first line of innate host defense in the gastrointestinal tract against potential pathogens and dangerous compounds. The mucus bilayer in the colon is primarily composed of MUC2 mucin, however recent studies have described other proteins secreted with colonic mucus. One of these proteins is FCGBP which is abundantly expressed in mucus and hypothesized to stabilize the mucus layer. This study aims to characterize the biosynthesis and packaging of FCGBP with MUC2 mucin and how it is regulated in response to the colonic parasite Entamoeba histolytica ( Eh). Eh cysteine protease 5 ( Eh CP5) degrades MUC2 mucin in the C terminus to invade the colon. We hypothesize that MUC2 and FCGBP together play important roles in innate host defense. The aims of the study are, 1) to determine if FCGBP co‐localizes with MUC2 mucin and 2) to quantify a functional role for FCGBP with MUC2 against Eh . To elucidate if FCGBP is synthesized and packaged within mucin granule vesicles, granules were isolated from human goblet cells LS174T by differential centrifugation and purified through sucrose gradients. Granules were treated with the detergent CHAPS to solubilize the granule lipid bilayer and the presence of MUC2 and FCGBP determined by Western blotting. To determine if FCGBP mRNA expression was altered in response to Eh , live Eh were incubated with goblet cells for 10, 20, and 30 min and quantified by RT‐PCR. To determine if Eh could degrade FCGBP in a similar fashion as MUC2, secreted proteases from WT Eh and Eh CP5‐ were incubated with mucin granules and enzymatic cleavage of FCGBP and MUC2 determined by Western blotting using antibodies specific for the C‐terminus of MUC2 and FCGBP. FCGBP was associated with mucin granules and following treatment with the mild detergent CHAPS, increased immunoreactivity for FCGBP and MUC2 suggesting that FCGBP was localized within granules and not on the granule lipid bilayer. FCGBP partitioned in the same high density fractions as MUC2 granules purified by sucrose density gradient demonstrating that FCGBP was localized with MUC2 within mucin granules. Interestingly, both MUC2 and FCGBP mRNA expressions in goblet cells were co‐ordinately increased in a time‐dependent fashion in response to live Eh and the positive mucus secretagogues, PMA and PGE 2 . Secreted Eh cysteine protease rapidly degraded both MUC2 and FCGBP as visualized by Western blotting, with FCGBP being completely degraded within 1 min of treatment whereas MUC2 was degraded 50%. Incubation with SPs derived from Eh CP5‐ inhibited the degradation of both MUC2 and FCGBP suggesting that degradation of FCGBP occurred prior to MUC2 by destabilizing MUC2. This study demonstrates that FCGBP is localized within MUC2 mucin granules and are secreted together to form an integral part of mucosal innate host defense. Strikingly, both MUC2 and FCGBP mRNA transcripts were co‐ordinately regulated in response to Eh . Importantly, Eh cysteine proteinase rapidly degraded FCGBP prior to cleaving MUC2 in the C‐terminus. Taken together, these data suggest that FCGBP plays an integral role with MUC2 mucin in innate host defense and in conferring rheological properties of the mucus gel in the colon. Support or Funding Information Supported by CIHR