Abstract
Gel-forming mucins are large high-molecular weight secreted O-glycoproteins responsible for the gel-properties of the mucus blanket. Five orthologous gel-forming mucins have been cloned in human and mouse. Among them, the mucin MUC6 has been less studied, particularly in rodents and no anti rodent-Muc6 antibody has been reported yet. In order to further study Muc6 in mice, our aims were to obtain a specific Muc6 antibody, to validate it and to test it in Cftr deficient mice. A polyclonal serum named CP4 was isolated from a rabbit immunized by a mouse Muc6 peptide. In Western blot experiments, the antibody detected a high-molecular weight molecule secreted by the gastric tissue. Using immunohistochemistry, we showed that the antibody reacted strongly with deep glands of duodenum and ileum and mucous neck cells of gastric body. CP4 also recognized Muc6 protein secreted at the surface of the stomach and renal collecting tubules. The centroacinar cells of pancreatic tissue also reacted with the antibody. Cftr−/− mice showed a higher expression of Muc6 at both protein and RNA levels compared with their control Cftr+/+ littermates suggesting that as in the human disease, Muc6 may contribute to the formation of materials that block pancreatic acini and ducts in mouse models of cystic fibrosis. The rabbit anti-mouse Muc6 polyclonal antibody seems highly specific to the mouse mucin and will be useful to study pancreatic pathology in cystic fibrosis.
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Abbreviations
- AB-PAS:
-
Alcian Blue–periodic acid Schiff
- AEBSF:
-
4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride
- BSA:
-
Bovine serum albumin
- BCA:
-
Bicinchoninic acid
- CFTR:
-
Cystic fibrosis transmembrane conductance regulator
- DIG:
-
Digoxigenin
- IHC:
-
Immunohistochemistry
- PBS:
-
Phosphate-buffered saline
- PFA:
-
Paraformaldehyde
- RT:
-
Room temperature
- SSC:
-
Saline sodium citrate
- TCA:
-
Trichloroacetic acid
- vWF:
-
von Willebrand factor
References
Bartman AE, Buisine MP, Aubert JP, Niehans GA, Toribara NW, Kim YS, Kelly EJ, Crabtree JE, Ho SB (1998) The MUC6 secretory mucin gene is expressed in a wide variety of epithelial tissues. J Pathol 186:398–405
Chen Y, Zhao YH, Wu R (2001) In silico cloning of mouse Muc5b gene and upregulation of its expression in mouse asthma model. Am J Respir Crit Care Med 164:1059–1066
Chen Y, Zhao YH, Kalaslavadi TB, Hamati E, Nehrke K, Le AD, Ann DK, Wu R (2004) Genome-wide search and identification of a novel gel-forming mucin MUC19/Muc19 in glandular tissues. Am J Respir Cell Mol Biol 30:155–165
Culp DJ, Latchney LR, Fallon MA, Denny PA, Denny PC, Couwenhoven RI, Chuang S (2004) The gene encoding mouse Muc19: cDNA, genomic organization and relationship to Smgc. Physiol Genomics 19:303–318
de Bolos C, Garrido M, Real FX (1995) MUC6 apomucin shows a distinct normal tissue distribution that correlates with Lewis antigen expression in the human stomach. Gastroenterology 109:723–734
De Lisle RC (1995) Increased expression of sulfated gp300 and acinar tissue pathology in pancreas of CFTR(−/−) mice. Am J Physiol 268:G717–G723
Desseyn JL (2009) Mucin CYS domains are ancient and highly conserved modules that evolved in concert. Mol Phylogenet Evol 52:284–292
Desseyn JL, Laine A (2003) Characterization of mouse Muc6 and evidence of conservation of the gel-forming mucin gene cluster between human and mouse. Genomics 81:433–436
Desseyn JL, Gouyer V, Tetaert D (2008) Architecture of the gel-forming mucins. In: Van Seuningen I (ed) The epithelial mucins: structure/function. Roles in cancer and inflammatory diseases. Research Signpost, Trivandrum, pp 1–16
Durie PR, Kent G, Phillips MJ, Ackerley CA (2004) Characteristic multiorgan pathology of cystic fibrosis in a long-living cystic fibrosis transmembrane regulator knockout murine model. Am J Pathol 164:1481–1493
Heazlewood CK, Cook MC, Eri R, Price GR, Tauro SB, Taupin D, Thornton DJ, Png CW, Crockford TL, Cornall RJ, Adams R, Kato M, Nelms KA, Hong NA, Florin TH, Goodnow CC, McGuckin MA (2008) Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis. PLoS Med 5:e54
Ho SB, Roberton AM, Shekels LL, Lyftogt CT, Niehans GA, Toribara NW (1995) Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression. Gastroenterology 109:735–747
Ho SB, Takamura K, Anway R, Shekels LL, Toribara NW, Ota H (2004) The adherent gastric mucous layer is composed of alternating layers of MUC5AC and MUC6 mucin proteins. Dig Dis Sci 49:1598–1606
Leroy X, Copin MC, Devisme L, Buisine MP, Aubert JP, Gosselin B, Porchet N (2002) Expression of human mucin genes in normal kidney and renal cell carcinoma. Histopathology 40:450–457
Leroy X, Devisme L, Buisine MP, Copin MC, Aubert S, Gosselin B, Aubert JP, Porchet N (2003) Expression of human mucin genes during normal and abnormal renal development. Am J Clin Pathol 120:544–550
Lidell ME, Bara J, Hansson GC (2008) Mapping of the 45M1 epitope to the C-terminal cysteine-rich part of the human MUC5AC mucin. FEBS J 275:481–489
Ratcliff R, Evans MJ, Cuthbert AW, MacVinish LJ, Foster D, Anderson JR, Colledge WH (1993) Production of a severe cystic fibrosis mutation in mice by gene targeting. Nat Genet 4:35–41
Reid CJ, Harris A (1998) Developmental expression of mucin genes in the human gastrointestinal system. Gut 42:220–226
Reid CJ, Harris A (1999) Expression of the MUC6 mucin gene in development of the human kidney and male genital ducts. J Histochem Cytochem 47:817–822
Reid CJ, Hyde K, Ho SB, Harris A (1997) Cystic fibrosis of the pancreas: involvement of MUC6 mucin in obstruction of pancreatic ducts. Mol Med 3:403–411
Shekels LL, Lyftogt C, Kieliszewski M, Filie JD, Kozak CA, Ho SB (1995) Mouse gastric mucin: cloning and chromosomal localization. Biochem J 311:775–785
Snouwaert JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, Smithies O, Koller BH (1992) An animal model for cystic fibrosis made by gene targeting. Science 257:1083–1088
Tetaert D, Pierre M, Demeyer D, Husson MO, Beghin L, Galabert C, Gottrand F, Beermann C, Guery B, Desseyn JL (2007) Dietary n-3 fatty acids have suppressive effects on mucin upregulation in mice infected with Pseudomonas aeruginosa. Respir Res 8:39
Thornton DJ, Rousseau K, McGuckin MA (2008) Structure and function of the polymeric mucins in airways mucus. Annu Rev Physiol 70:459–486
Toribara NW, Roberton AM, Ho SB, Kuo WL, Gum E, Hicks JW, Gum JR Jr, Byrd JC, Siddiki B, Kim YS (1993) Human gastric mucin. Identification of a unique species by expression cloning. J Biol Chem 268:5879–5885
Toribara NW, Ho SB, Gum E, Gum JR Jr, Lau P, Kim YS (1997) The carboxyl-terminal sequence of the human secretory mucin, MUC6. Analysis of the primary amino acid sequence. J Biol Chem 272:16398–16403
Tytgat KM, Buller HA, Opdam FJ, Kim YS, Einerhand AW, Dekker J (1994) Biosynthesis of human colonic mucin: Muc2 is the prominent secretory mucin. Gastroenterology 107:1352–1363
van Klinken BJ, Einerhand AW, Duits LA, Makkink MK, Tytgat KM, Renes IB, Verburg M, Buller HA, Dekker J (1999) Gastrointestinal expression and partial cDNA cloning of murine Muc2. Am J Physiol 276:G115–G124
Velcich A, Yang W, Heyer J, Fragale A, Nicholas C, Viani S, Kucherlapati R, Lipkin M, Yang K, Augenlicht L (2002) Colorectal cancer in mice genetically deficient in the mucin Muc2. Science 295:1726–1729
Voynow JA, Gendler SJ, Rose MC (2006) Regulation of mucin genes in chronic inflammatory airway diseases. Am J Respir Cell Mol Biol 34:661–665
Acknowledgments
We thank Delphine Fontaine (Inserm U837) for her excellent technical assistance in performing IHC, the Microbiology Department of the “Centre de Biologie-Pathologie Pierre-Marie Degand” (Lille Hospital) for the access to the ABI Prism 7700, MH Gevaert and RM Siminski (Service commun–Morphologie cellulaire, Université Lille 2) for slides and J. Bara (Inserm UMR S938, Paris, France) for the 45M1 antibody. This study was supported by the French Cystic Fibrosis association “Vaincre la Mucoviscidose” (J.L.D., FC0203 and IC0806) and Children’s Memorial Research Center (S.H.L. and A.H.).
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Gouyer, V., Leir, SH., Tetaert, D. et al. The characterization of the first anti-mouse Muc6 antibody shows an increased expression of the mucin in pancreatic tissue of Cftr-knockout mice. Histochem Cell Biol 133, 517–525 (2010). https://doi.org/10.1007/s00418-010-0688-8
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DOI: https://doi.org/10.1007/s00418-010-0688-8