Lidell ME, Bara J, Hansson GC.. Mapping of the 45M1 epitope to the C-terminal cysteine-rich part of the human MUC5AC mucin. FEBS J 275: 481-489

Department of Medical Biochemistry, Göteborg University, Sweden.
FEBS Journal (Impact Factor: 4). 03/2008; 275(3):481-9. DOI: 10.1111/j.1742-4658.2007.06215.x
Source: PubMed


Mucins are large glycoproteins protecting mucosal surfaces throughout the body. Their expressions are tissue-specific, but in disease states such as cystic fibrosis, inflammation and cancer, this specificity can be disturbed. MUC5AC is normally expressed in the mucous cells of the epithelia lining the stomach and the trachea, where it constitutes a major component of the gastric and respiratory mucus. A number of mAbs have been raised against the gastric M1 antigen, an early marker for colonic carcinogenesis. Several of these mAbs recognize epitopes present on MUC5AC, suggesting that MUC5AC is the antigen. However, some of the mAbs raised against the gastric M1 antigen are widely used as antibodies against MUC5AC, despite the fact that their specificity for MUC5AC has not been clearly shown. In this study, we have tested the reactivity of the latter antibodies against a recombinantly expressed C-terminal cysteine-rich part of human MUC5AC. We demonstrate for the first time that the widely used mAb 45M1, as well as 2-12M1 and 166M1, are true antibodies against MUC5AC, with epitopes located in the C-terminal cysteine-rich part of the mucin.

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Available from: Gunnar C Hansson, Jul 16, 2015
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    • "The current studies suggest that PAM4 is reactive with the MUC5AC mucin glycoprotein. Figure 4 presents a map of the MUC5AC mucin domains with reactive epitopes indicated for several of the anti-MUC5AC MAbs employed in our studies [22-24]. CLH2 is reactive with the peptide core of the tandem repeat domain [21], and is likely a cryptic epitope within the Capan-1 tumor-derived MUC5AC. "
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    ABSTRACT: PAM4, an antibody that has high specificity for pancreatic ductal adenocarcinoma (PDAC), compared to normal pancreas, benign lesions of the pancreas, and cancers originating from other tissues, is being investigated as a biomarker for early detection, as well as antibody-targeted imaging and therapy. Therefore, the identity of the antigen bound by this monoclonal antibody (MAb) can provide information leading to improved use of the antibody. Prior results suggested the antigen is a mucin-type glycoprotein rich in cysteine disulfide bridges that provide stable conformation for the PAM4-epitope. Indirect and sandwich enzyme immunoassays (EIA) were performed to compare and contrast the reactivity of PAM4 with several anti-mucin antibodies having known reactivity to specific mucin species (e.g., MUC1, MUC4, MUC5AC, etc.). Studies designed to block reactivity of PAM4 with its specific antigen also were performed. We demonstrate that MAbs 2-11 M1 and 45 M1, each reactive with MUC5AC, are able to provide signal in a heterologous sandwich immunoassay where PAM4 is the capture antibody. Further, we identify MAbs 21 M1, 62 M1, and 463 M1, each reactive with MUC5AC, as inhibiting the reaction of PAM4 with its specific epitope. MAbs directed to MUC1, MUC3, MUC4, MUC16 and CEACAM6 are not reactive with PAM4-captured antigen, nor are they able to block the reaction of PAM4 with its antigen. These data implicate MUC5AC as a specific mucin species to which PAM4 is reactive. Furthermore, this realization may allow for the improvement of the current PAM4 serum-based immunoassay for detection of early-stage PDAC by the application of anti-MUC5AC MAbs as probes in this sandwich EIA.
    Molecular Cancer 11/2013; 12(1):143. DOI:10.1186/1476-4598-12-143 · 4.26 Impact Factor
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    • "Cells were lysed by the M-PER Mammalian Protein Extraction Reagent (#78501, Thermo Scientific, Rockford, IL, USA) in the presence of the Halt Protease Inhibitor Cocktail (Thermo Scientific #87786). The protease inhibitors were incorporated because of prior reports of sensitivity of the anti-mucin antibodies to degradation with cell lysates [34,35]. The amounts of mucins in total cell lysates were determined by western blotting using specific antibodies against MUC5AC (Santa Cruz Biotechnology #sc-21701, dilution 1:1000) and MUC5B (Santa Cruz Biotechnology #sc-20119, dilution 1:1000) or by ELISA kits (USCN LIFE SCIENCE, Houston, TX, USA, MUC5AC #E90756Hu; MUC5B #E90684Hu). "
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    ABSTRACT: The redox-active pyocyanin (PCN) is a toxic, secondary metabolite secreted by the respiratory pathogen Pseudomonas aeruginosa (PA). Previously, we have shown that mouse lungs chronically exposed to PCN develop goblet cell hyperplasia and metaplasia (GCHM) and mucus hypersecretion, fibrosis and emphysema. These pathological features are commonly found in the airways of several chronic lung diseases, including cystic fibrosis (CF), as well as in mouse airways deficient in the forkhead box A2 (FOXA2), a transcriptional repressor of goblet GCHM and mucus biosynthesis. Furthermore, PCN inhibits FOXA2 by activating the pro-GCHM signaling pathways Stat6 and EGFR. However, it is not known whether PCN-generated reactive oxygen (ROS) and nitrogen (RNS) species posttranslationally modify and inactivate FOXA2. We examined the posttranslational modifications of FOXA2 by PCN using specific antibodies against oxidation, nitrosylation, acetylation and ubiquitination. Electrophoretic mobility shift assay (EMSA) was used to examine the ability of modified FOXA2 to bind the promoter of MUC5B mucin gene. In addition, we used quantitative real time PCR, ELISA, immunofluorescence and mouse lung infection to assess whether the loss of FOXA2 function caused GCHM and mucin overexpression. Finally, we examined the restoration of FOXA2 function by the antioxidant glutathione (GSH). We found that PCN-generated ROS/RNS caused nitrosylation, acetylation, ubiquitination and degradation of FOXA2. Modified FOXA2 had reduced ability to bind the promoter of the MUC5B gene. The antioxidant GSH alleviated the modification of FOXA2 by PCN, and inhibited the overexpression of MUC5AC and MUC5B mucins. These results suggest that PCN-mediated posttranslational modifications of FOXA2 are positively correlated with GCHM and overexpression of airway mucins. Furthermore, antioxidant treatment restores the function of FOXA2 to attenuate GCHM and mucus hypersecretion.
    Respiratory research 08/2013; 14(1):82. DOI:10.1186/1465-9921-14-82 · 3.09 Impact Factor
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    • "Interestingly, this de novo expression of Muc5ac was observed in all the resistant models (TH2-type response) but in none of the susceptible models (TH1-type response) of T muris infection. Although this mucin is predominantly found in airway and stomach mucus,42,45 studies on patients with ulcerative colitis and adenocarcinomas have shown MUC5AC expression in the intestine along with MUC2.31,46 However, this is the first time that Muc5ac expression has been implicated in response to an enteric parasitic infection. "
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    ABSTRACT: Hyperplasia of mucin-secreting intestinal goblet cells accompanies a number of enteric infections, including infections by nematode parasites. Nevertheless, the precise role of mucins in host defense in nematode infection is not known. We investigated the role of the mucin (Muc2) in worm expulsion and host immunity in a model of nematode infection. Resistant (BALB/c, C57BL/6), susceptible (AKR), and Muc2-deficient mouse strains were infected with the nematode, Trichuris muris, and worm expulsion, energy status of the whipworms, changes in mucus/mucins, and inflammatory and immune responses were investigated after infection. The increase in Muc2 production, observed exclusively in resistant mice, correlated with worm expulsion. Moreover, expulsion of the worms from the intestine was significantly delayed in the Muc2-deficient mice. Although a marked impairment in the development of periodic acid Schiff (PAS)-stained intestinal goblet cells was observed in Muc2-deficient mice, as infection progressed a significant increase in the number of PAS-positive goblet cells was observed in these mice. Surprisingly, an increase in Muc5ac, a mucin normally expressed in the airways and stomach, was observed after infection of only the resistant animals. Overall, the mucus barrier in the resistant mice was less permeable than that of susceptible mice. Furthermore, the worms isolated from the resistant mice had a lower energy status. Mucins are an important component of innate defense in enteric infection; this is the first demonstration of the important functional contribution of mucins to host protection from nematode infection.
    Gastroenterology 02/2010; 138(5):1763-71. DOI:10.1053/j.gastro.2010.01.045 · 16.72 Impact Factor
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