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. 1995 Nov 1;311(Pt 3):775–785. doi: 10.1042/bj3110775

Mouse gastric mucin: cloning and chromosomal localization.

L L Shekels 1, C Lyftogt 1, M Kieliszewski 1, J D Filie 1, C A Kozak 1, S B Ho 1
PMCID: PMC1136070  PMID: 7487932

Abstract

Mucins protect gastric epithelium by maintaining a favourable pH gradient and preventing autodigestion. The purpose of this study was to clone a mouse gastric mucin which would provide a foundation for analysis of mucin gene regulation. Mucin was purified from the glandular portion of gastric specimens and deglycosylated by HF solvolysis. Antibodies against native and deglycosylated mouse gastric mucin (MGM) were raised in chickens. Screening of a mouse stomach cDNA library with the anti-(deglycosylated MGM) antibody yielded partial clones containing a 48 bp tandem repeat and 768 bp of non-repetitive sequence. The 16-amino-acid tandem repeat has a consensus sequence of QTSSPNTGKTSTISTT with 25% serine and 38% threonine. The MGM tandem repeat sequence bears no similarity to previously identified mucins. The MGM non-repetitive region shares sequence similarity with human MUC5AC and, to a lesser extent, human MUC2 and rat intestinal mucin. Northern blot analysis reveals a polydisperse message beginning at 13.5 kb in mouse stomach with no expression in oesophagus, trachea, small intestine, large intestine, caecum, lung or kidney. Immunoreactivity of antibodies against deglycosylated MGM and against a synthetic MGM tandem repeat peptide was restricted to superficial mucous cells, antral glands and Brunner's glands in the pyloric-duodenal region. DNA analysis shows that MGM recognizes mouse and rat DNA but not hamster, rabbit or human DNA. The MGM gene maps to a site on mouse chromosome 7 homologous to the location of a human secretory mucin gene cluster on human chromosome 11p15. Due to sequence similarity and predominant expression in the stomach, the MGM gene may be considered a MUC5AC homologue and named Muc5ac.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adamson M. C., Silver J., Kozak C. A. The mouse homolog of the Gibbon ape leukemia virus receptor: genetic mapping and a possible receptor function in rodents. Virology. 1991 Aug;183(2):778–781. doi: 10.1016/0042-6822(91)91010-e. [DOI] [PubMed] [Google Scholar]
  2. Audie J. P., Janin A., Porchet N., Copin M. C., Gosselin B., Aubert J. P. Expression of human mucin genes in respiratory, digestive, and reproductive tracts ascertained by in situ hybridization. J Histochem Cytochem. 1993 Oct;41(10):1479–1485. doi: 10.1177/41.10.8245407. [DOI] [PubMed] [Google Scholar]
  3. Avrameas S. Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry. 1969 Jan;6(1):43–52. doi: 10.1016/0019-2791(69)90177-3. [DOI] [PubMed] [Google Scholar]
  4. Basbaum C., Gallup M., Gum J., Kim Y., Jany B. Modification of mucin gene expression in the airways of rats exposed to sulfur dioxide. Biorheology. 1990;27(3-4):485–489. doi: 10.3233/bir-1990-273-427. [DOI] [PubMed] [Google Scholar]
  5. Bhargava A. K., Woitach J. T., Davidson E. A., Bhavanandan V. P. Cloning and cDNA sequence of a bovine submaxillary gland mucin-like protein containing two distinct domains. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6798–6802. doi: 10.1073/pnas.87.17.6798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bhaskar K. R., Garik P., Turner B. S., Bradley J. D., Bansil R., Stanley H. E., LaMont J. T. Viscous fingering of HCl through gastric mucin. Nature. 1992 Dec 3;360(6403):458–461. doi: 10.1038/360458a0. [DOI] [PubMed] [Google Scholar]
  7. Bobek L. A., Tsai H., Biesbrock A. R., Levine M. J. Molecular cloning, sequence, and specificity of expression of the gene encoding the low molecular weight human salivary mucin (MUC7). J Biol Chem. 1993 Sep 25;268(27):20563–20569. [PubMed] [Google Scholar]
  8. Briand J. P., Andrews S. P., Jr, Cahill E., Conway N. A., Young J. D. Investigation of the requirements for O-glycosylation by bovine submaxillary gland UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosamine transferase using synthetic peptide substrates. J Biol Chem. 1981 Dec 10;256(23):12205–12207. [PubMed] [Google Scholar]
  9. Byrd J. C., Nardelli J., Siddiqui B., Kim Y. S. Isolation and characterization of colon cancer mucin from xenografts of LS174T cells. Cancer Res. 1988 Dec 1;48(23):6678–6685. [PubMed] [Google Scholar]
  10. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  11. Correa P. A human model of gastric carcinogenesis. Cancer Res. 1988 Jul 1;48(13):3554–3560. [PubMed] [Google Scholar]
  12. Danciger M., Farber D. B., Kozak C. A. Genetic mapping of three GABAA receptor-subunit genes in the mouse. Genomics. 1993 May;16(2):361–365. doi: 10.1006/geno.1993.1198. [DOI] [PubMed] [Google Scholar]
  13. Dekker J., Aelmans P. H., Strous G. J. The oligomeric structure of rat and human gastric mucins. Biochem J. 1991 Jul 15;277(Pt 2):423–427. doi: 10.1042/bj2770423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dubray G., Bezard G. A highly sensitive periodic acid-silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 15;119(2):325–329. doi: 10.1016/0003-2697(82)90593-0. [DOI] [PubMed] [Google Scholar]
  16. Eckhardt A. E., Timpte C. S., Abernethy J. L., Toumadje A., Johnson W. C., Jr, Hill R. L. Structural properties of porcine submaxillary gland apomucin. J Biol Chem. 1987 Aug 15;262(23):11339–11344. [PubMed] [Google Scholar]
  17. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  18. Gassmann M., Thömmes P., Weiser T., Hübscher U. Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J. 1990 May;4(8):2528–2532. doi: 10.1096/fasebj.4.8.1970792. [DOI] [PubMed] [Google Scholar]
  19. Gendler S. J., Lancaster C. A., Taylor-Papadimitriou J., Duhig T., Peat N., Burchell J., Pemberton L., Lalani E. N., Wilson D. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem. 1990 Sep 5;265(25):15286–15293. [PubMed] [Google Scholar]
  20. Goueli S. A., Hanten J., Davis A., Ahmed K. Polyclonal antibodies against rat liver cytosolic casein kinase II (CK-2) cross-react with CK-2 from other tissues and nuclear form (PK-N2) of the enzyme. Biochem Int. 1990;21(4):685–694. [PubMed] [Google Scholar]
  21. Griffiths B., Matthews D. J., West L., Attwood J., Povey S., Swallow D. M., Gum J. R., Kim Y. S. Assignment of the polymorphic intestinal mucin gene (MUC2) to chromosome 11p15. Ann Hum Genet. 1990 Oct;54(Pt 4):277–285. doi: 10.1111/j.1469-1809.1990.tb00383.x. [DOI] [PubMed] [Google Scholar]
  22. Gum J. R., Hicks J. W., Swallow D. M., Lagace R. L., Byrd J. C., Lamport D. T., Siddiki B., Kim Y. S. Molecular cloning of cDNAs derived from a novel human intestinal mucin gene. Biochem Biophys Res Commun. 1990 Aug 31;171(1):407–415. doi: 10.1016/0006-291x(90)91408-k. [DOI] [PubMed] [Google Scholar]
  23. Gum J. R., Jr, Hicks J. W., Lagace R. E., Byrd J. C., Toribara N. W., Siddiki B., Fearney F. J., Lamport D. T., Kim Y. S. Molecular cloning of rat intestinal mucin. Lack of conservation between mammalian species. J Biol Chem. 1991 Nov 25;266(33):22733–22738. [PubMed] [Google Scholar]
  24. Gum J. R., Jr, Hicks J. W., Toribara N. W., Rothe E. M., Lagace R. E., Kim Y. S. The human MUC2 intestinal mucin has cysteine-rich subdomains located both upstream and downstream of its central repetitive region. J Biol Chem. 1992 Oct 25;267(30):21375–21383. [PubMed] [Google Scholar]
  25. Gum J. R., Jr, Hicks J. W., Toribara N. W., Siddiki B., Kim Y. S. Molecular cloning of human intestinal mucin (MUC2) cDNA. Identification of the amino terminus and overall sequence similarity to prepro-von Willebrand factor. J Biol Chem. 1994 Jan 28;269(4):2440–2446. [PubMed] [Google Scholar]
  26. Gum J. R., Jr Mucin genes and the proteins they encode: structure, diversity, and regulation. Am J Respir Cell Mol Biol. 1992 Dec;7(6):557–564. doi: 10.1165/ajrcmb/7.6.557. [DOI] [PubMed] [Google Scholar]
  27. Guyonnet Duperat V., Audie J. P., Debailleul V., Laine A., Buisine M. P., Galiegue-Zouitina S., Pigny P., Degand P., Aubert J. P., Porchet N. Characterization of the human mucin gene MUC5AC: a consensus cysteine-rich domain for 11p15 mucin genes? Biochem J. 1995 Jan 1;305(Pt 1):211–219. doi: 10.1042/bj3050211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hansson G. C., Baeckström D., Carlstedt I., Klinga-Levan K. Molecular cloning of a cDNA coding for a region of an apoprotein from the 'insoluble' mucin complex of rat small intestine. Biochem Biophys Res Commun. 1994 Jan 14;198(1):181–190. doi: 10.1006/bbrc.1994.1026. [DOI] [PubMed] [Google Scholar]
  29. Ho S. B., Niehans G. A., Lyftogt C., Yan P. S., Cherwitz D. L., Gum E. T., Dahiya R., Kim Y. S. Heterogeneity of mucin gene expression in normal and neoplastic tissues. Cancer Res. 1993 Feb 1;53(3):641–651. [PubMed] [Google Scholar]
  30. Ho S. B., Roberton A. M., Shekels L. L., Lyftogt C. T., Niehans G. A., Toribara N. W. Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression. Gastroenterology. 1995 Sep;109(3):735–747. doi: 10.1016/0016-5085(95)90380-1. [DOI] [PubMed] [Google Scholar]
  31. Kieliszewski M. J., Kamyab A., Leykam J. F., Lamport D. T. A Histidine-Rich Extensin from Zea mays Is an Arabinogalactan Protein. Plant Physiol. 1992 Jun;99(2):538–547. doi: 10.1104/pp.99.2.538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kozak C. A., Peyser M., Krall M., Mariano T. M., Kumar C. S., Pestka S., Mock B. A. Molecular genetic markers spanning mouse chromosome 10. Genomics. 1990 Nov;8(3):519–524. doi: 10.1016/0888-7543(90)90039-w. [DOI] [PubMed] [Google Scholar]
  33. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  34. Lunsford R. D., Jenkins N. A., Kozak C. A., Liang L. F., Silan C. M., Copeland N. G., Dean J. Genomic mapping of murine Zp-2 and Zp-3, two oocyte-specific loci encoding zona pellucida proteins. Genomics. 1990 Jan;6(1):184–187. doi: 10.1016/0888-7543(90)90465-7. [DOI] [PubMed] [Google Scholar]
  35. Majors J. E., Varmus H. E. Nucleotide sequencing of an apparent proviral copy of env mRNA defines determinants of expression of the mouse mammary tumor virus env gene. J Virol. 1983 Sep;47(3):495–504. doi: 10.1128/jvi.47.3.495-504.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Meezaman D., Charles P., Daskal E., Polymeropoulos M. H., Martin B. M., Rose M. C. Cloning and analysis of cDNA encoding a major airway glycoprotein, human tracheobronchial mucin (MUC5). J Biol Chem. 1994 Apr 29;269(17):12932–12939. [PubMed] [Google Scholar]
  37. Nguyen V. C., Aubert J. P., Gross M. S., Porchet N., Degand P., Frézal J. Assignment of human tracheobronchial mucin gene(s) to 11p15 and a tracheobronchial mucin-related sequence to chromosome 13. Hum Genet. 1990 Dec;86(2):167–172. doi: 10.1007/BF00197699. [DOI] [PubMed] [Google Scholar]
  38. Polson A., von Wechmar M. B., van Regenmortel M. H. Isolation of viral IgY antibodies from yolks of immunized hens. Immunol Commun. 1980;9(5):475–493. doi: 10.3109/08820138009066010. [DOI] [PubMed] [Google Scholar]
  39. Porchet N., Nguyen V. C., Dufosse J., Audie J. P., Guyonnet-Duperat V., Gross M. S., Denis C., Degand P., Bernheim A., Aubert J. P. Molecular cloning and chromosomal localization of a novel human tracheo-bronchial mucin cDNA containing tandemly repeated sequences of 48 base pairs. Biochem Biophys Res Commun. 1991 Mar 15;175(2):414–422. doi: 10.1016/0006-291x(91)91580-6. [DOI] [PubMed] [Google Scholar]
  40. Ramesh V., Cheng S. V., Kozak C. A., Herron B. J., Shih V. E., Taylor B. A., Gusella J. F. Mapping of ornithine aminotransferase gene sequences to mouse chromosomes 7, X, and 3. Mamm Genome. 1992;3(1):17–22. doi: 10.1007/BF00355836. [DOI] [PubMed] [Google Scholar]
  41. Rose M. C., Kaufman B., Martin B. M. Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin. J Biol Chem. 1989 May 15;264(14):8193–8199. [PubMed] [Google Scholar]
  42. Rose M. C. Mucins: structure, function, and role in pulmonary diseases. Am J Physiol. 1992 Oct;263(4 Pt 1):L413–L429. doi: 10.1152/ajplung.1992.263.4.L413. [DOI] [PubMed] [Google Scholar]
  43. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Slomiany B. L., Sarosiek J., Slomiany A. Gastric mucus and the mucosal barrier. Dig Dis. 1987;5(3):125–145. doi: 10.1159/000171168. [DOI] [PubMed] [Google Scholar]
  45. Sorimachi H., Emori Y., Kawasaki H., Kitajima K., Inoue S., Suzuki K., Inoue Y. Molecular cloning and characterization of cDNAs coding for apo-polysialoglycoprotein of rainbow trout eggs. Multiple mRNA species transcribed from multiple genes contain diverged numbers of exact 39-base (13-amino acid) repeats. J Biol Chem. 1988 Nov 25;263(33):17678–17684. [PubMed] [Google Scholar]
  46. Spicer A. P., Parry G., Patton S., Gendler S. J. Molecular cloning and analysis of the mouse homologue of the tumor-associated mucin, MUC1, reveals conservation of potential O-glycosylation sites, transmembrane, and cytoplasmic domains and a loss of minisatellite-like polymorphism. J Biol Chem. 1991 Aug 15;266(23):15099–15109. [PubMed] [Google Scholar]
  47. Strous G. J., Dekker J. Mucin-type glycoproteins. Crit Rev Biochem Mol Biol. 1992;27(1-2):57–92. doi: 10.3109/10409239209082559. [DOI] [PubMed] [Google Scholar]
  48. Toribara N. W., Gum J. R., Jr, Culhane P. J., Lagace R. E., Hicks J. W., Petersen G. M., Kim Y. S. MUC-2 human small intestinal mucin gene structure. Repeated arrays and polymorphism. J Clin Invest. 1991 Sep;88(3):1005–1013. doi: 10.1172/JCI115360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Toribara N. W., Roberton A. M., Ho S. B., Kuo W. L., Gum E., Hicks J. W., Gum J. R., Jr, Byrd J. C., Siddiki B., Kim Y. S. Human gastric mucin. Identification of a unique species by expression cloning. J Biol Chem. 1993 Mar 15;268(8):5879–5885. [PubMed] [Google Scholar]
  50. Tso J. Y., Sun X. H., Kao T. H., Reece K. S., Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res. 1985 Apr 11;13(7):2485–2502. doi: 10.1093/nar/13.7.2485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Turner B. S., Bhaskar K. R., Hadzopoulou-Cladaras M., Specian R. D., LaMont J. T. Isolation and characterization of cDNA clones encoding pig gastric mucin. Biochem J. 1995 May 15;308(Pt 1):89–96. doi: 10.1042/bj3080089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Verma M., Davidson E. A. Molecular cloning and sequencing of a canine tracheobronchial mucin cDNA containing a cysteine-rich domain. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7144–7148. doi: 10.1073/pnas.90.15.7144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Wright H. T. Nonenzymatic deamidation of asparaginyl and glutaminyl residues in proteins. Crit Rev Biochem Mol Biol. 1991;26(1):1–52. doi: 10.3109/10409239109081719. [DOI] [PubMed] [Google Scholar]

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