Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 May 1;315(Pt 3):1055–1060. doi: 10.1042/bj3151055

Biosynthesis of the MUC2 mucin: evidence for a slow assembly of fully glycosylated units.

J K Sheehan 1, D J Thornton 1, M Howard 1, I Carlstedt 1, A P Corfield 1, C Paraskeva 1
PMCID: PMC1217261  PMID: 8645144

Abstract

The human colonic cell line PC/AA was grown to near confluency over 24 days and labelled with [14C]proline and [3H]glucose over the last 48 h in culture. The cell layer was extracted with 6 M guanidinium chloride and the mature fully glycosylated mucins were isolated at a density of 1.45 g/ml by using density-gradient centrifugation in CsCl/4 M guanidinium chloride. These mucins were identified as MUC2 with an anti-peptide antibody. The macromolecules were fragmented by reduction into two distinct populations of MUC2 subunits as assessed by agarose electrophoresis. The MUC2 mucin was polydisperse in length, ranging from 500 nm to many microns and its molecular-mass distribution, assessed by rate-zonal centrifugation, ranged from 5 x 10(6) to 40 x 10(6) Da. However, the metabolically labelled MUC2 mucins, though found throughout the whole distribution, were of much smaller average size. Since the entire distribution is not uniformly radiolabelled over 48 h, the formation of the largest species must be preceded by glycosylation and occur slowly, over several days, via the assembly of fully glycosylated units which are likely to be at least dimers [Asker, Baeckstrom, Axelsson, Carlstedt, and Hansson (1995) Biochem. J. 308, 873-880].

Full Text

The Full Text of this article is available as a PDF (747.5 KB).

Selected References

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

  1. Asker N., Baeckström D., Axelsson M. A., Carlstedt I., Hansson G. C. The human MUC2 mucin apoprotein appears to dimerize before O-glycosylation and shares epitopes with the 'insoluble' mucin of rat small intestine. Biochem J. 1995 Jun 15;308(Pt 3):873–880. doi: 10.1042/bj3080873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carlstedt I., Herrmann A., Hovenberg H., Lindell G., Nordman H., Wickström C., Davies J. R. 'Soluble' and 'insoluble' mucins--identification of distinct populations. Biochem Soc Trans. 1995 Nov;23(4):845–851. doi: 10.1042/bst0230845. [DOI] [PubMed] [Google Scholar]
  3. Carlstedt I., Herrmann A., Karlsson H., Sheehan J., Fransson L. A., Hansson G. C. Characterization of two different glycosylated domains from the insoluble mucin complex of rat small intestine. J Biol Chem. 1993 Sep 5;268(25):18771–18781. [PubMed] [Google Scholar]
  4. Carlstedt I., Lindgren H., Sheehan J. K., Ulmsten U., Wingerup L. Isolation and characterization of human cervical-mucus glycoproteins. Biochem J. 1983 Apr 1;211(1):13–22. doi: 10.1042/bj2110013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlstedt I., Sheehan J. K., Corfield A. P., Gallagher J. T. Mucous glycoproteins: a gel of a problem. Essays Biochem. 1985;20:40–76. [PubMed] [Google Scholar]
  6. Carlstedt I., Sheehan J. K. Macromolecular properties and polymeric structure of mucus glycoproteins. Ciba Found Symp. 1984;109:157–172. doi: 10.1002/9780470720905.ch11. [DOI] [PubMed] [Google Scholar]
  7. Carrato C., Balague C., de Bolos C., Gonzalez E., Gambus G., Planas J., Perini J. M., Andreu D., Real F. X. Differential apomucin expression in normal and neoplastic human gastrointestinal tissues. Gastroenterology. 1994 Jul;107(1):160–172. doi: 10.1016/0016-5085(94)90073-6. [DOI] [PubMed] [Google Scholar]
  8. Chang S. K., Dohrman A. F., Basbaum C. B., Ho S. B., Tsuda T., Toribara N. W., Gum J. R., Kim Y. S. Localization of mucin (MUC2 and MUC3) messenger RNA and peptide expression in human normal intestine and colon cancer. Gastroenterology. 1994 Jul;107(1):28–36. doi: 10.1016/0016-5085(94)90057-4. [DOI] [PubMed] [Google Scholar]
  9. Corfield A. P., Clamp J. R., Casey A. D., Paraskeva C. Characterization of a sialic-acid-rich mucus glycoprotein secreted by a premalignant human colorectal adenoma cell line. Int J Cancer. 1990 Dec 15;46(6):1059–1065. doi: 10.1002/ijc.2910460619. [DOI] [PubMed] [Google Scholar]
  10. Corfield A. P., Wagner S. A., Paraskeva C., Clamp J. R., Durdey P., Reuter G., Schauer R. Loss of sialic acid O-acetylation in human colorectal cancer cells. Biochem Soc Trans. 1992 May;20(2):94S–94S. doi: 10.1042/bst020094s. [DOI] [PubMed] [Google Scholar]
  11. Dekker J., Strous G. J. Covalent oligomerization of rat gastric mucin occurs in the rough endoplasmic reticulum, is N-glycosylation-dependent, and precedes initial O-glycosylation. J Biol Chem. 1990 Oct 25;265(30):18116–18122. [PubMed] [Google Scholar]
  12. Devine P. L., Layton G. T., Clark B. A., Birrell G. W., Ward B. G., Xing P. X., McKenzie I. F. Production of MUC1 and MUC2 mucins by human tumor cell lines. Biochem Biophys Res Commun. 1991 Jul 31;178(2):593–599. doi: 10.1016/0006-291x(91)90149-2. [DOI] [PubMed] [Google Scholar]
  13. Gendler S. J., Spicer A. P. Epithelial mucin genes. Annu Rev Physiol. 1995;57:607–634. doi: 10.1146/annurev.ph.57.030195.003135. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Hanisch F. G., Hanski C., Hasegawa A. Sialyl Lewis(x) antigen as defined by monoclonal antibody AM-3 is a marker of dysplasia in the colonic adenoma-carcinoma sequence. Cancer Res. 1992 Jun 1;52(11):3138–3144. [PubMed] [Google Scholar]
  16. Klomp L. W., van Rens L., Strous G. J. Identification of a human gastric mucin precursor: N-linked glycosylation and oligomerization. Biochem J. 1994 Dec 15;304(Pt 3):693–698. doi: 10.1042/bj3040693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Layton G. T., Devine P. L., Warren J. A., Birrell G., Xing P. X., Ward B. G., McKenzie I. F. Monoclonal antibodies reactive with the breast carcinoma-associated mucin core protein repeat sequence peptide also recognise the ovarian carcinoma-associated sebaceous gland antigen. Tumour Biol. 1990;11(5):274–286. doi: 10.1159/000217661. [DOI] [PubMed] [Google Scholar]
  18. Ogata S., Uehara H., Chen A., Itzkowitz S. H. Mucin gene expression in colonic tissues and cell lines. Cancer Res. 1992 Nov 1;52(21):5971–5978. [PubMed] [Google Scholar]
  19. Paraskeva C., Buckle B. G., Sheer D., Wigley C. B. The isolation and characterization of colorectal epithelial cell lines at different stages in malignant transformation from familial polyposis coli patients. Int J Cancer. 1984 Jul 15;34(1):49–56. doi: 10.1002/ijc.2910340109. [DOI] [PubMed] [Google Scholar]
  20. Sheehan J. K., Carlstedt I. Size heterogeneity of human cervical mucus glycoproteins. Studies performed with rate-zonal centrifugation and laser light-scattering. Biochem J. 1987 Aug 1;245(3):757–762. doi: 10.1042/bj2450757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sheehan J. K., Oates K., Carlstedt I. Electron microscopy of cervical, gastric and bronchial mucus glycoproteins. Biochem J. 1986 Oct 1;239(1):147–153. doi: 10.1042/bj2390147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Thornton D. J., Carlstedt I., Sheehan J. K. Identification of glycoproteins on nitrocellulose membranes and gels. Methods Mol Biol. 1994;32:119–128. doi: 10.1385/0-89603-268-X:119. [DOI] [PubMed] [Google Scholar]
  24. Thornton D. J., Davies J. R., Kraayenbrink M., Richardson P. S., Sheehan J. K., Carlstedt I. Mucus glycoproteins from 'normal' human tracheobronchial secretion. Biochem J. 1990 Jan 1;265(1):179–186. doi: 10.1042/bj2650179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Thornton D. J., Devine P. L., Hanski C., Howard M., Sheehan J. K. Identification of two major populations of mucins in respiratory secretions. Am J Respir Crit Care Med. 1994 Sep;150(3):823–832. doi: 10.1164/ajrccm.150.3.8087358. [DOI] [PubMed] [Google Scholar]
  26. Thornton D. J., Holmes D. F., Sheehan J. K., Carlstedt I. Quantitation of mucus glycoproteins blotted onto nitrocellulose membranes. Anal Biochem. 1989 Oct;182(1):160–164. doi: 10.1016/0003-2697(89)90735-5. [DOI] [PubMed] [Google Scholar]
  27. Thornton D. J., Howard M., Devine P. L., Sheehan J. K. Methods for separation and deglycosylation of mucin subunits. Anal Biochem. 1995 May 1;227(1):162–167. doi: 10.1006/abio.1995.1266. [DOI] [PubMed] [Google Scholar]
  28. Thornton D. J., Sheehan J. K., Lindgren H., Carlstedt I. Mucus glycoproteins from cystic fibrotic sputum. Macromolecular properties and structural 'architecture'. Biochem J. 1991 Jun 15;276(Pt 3):667–675. doi: 10.1042/bj2760667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tytgat K. M., Büller H. A., Opdam F. J., Kim Y. S., Einerhand A. W., Dekker J. Biosynthesis of human colonic mucin: Muc2 is the prominent secretory mucin. Gastroenterology. 1994 Nov;107(5):1352–1363. doi: 10.1016/0016-5085(94)90537-1. [DOI] [PubMed] [Google Scholar]
  30. Vavasseur F., Dole K., Yang J., Matta K. L., Myerscough N., Corfield A., Paraskeva C., Brockhausen I. O-glycan biosynthesis in human colorectal adenoma cells during progression to cancer. Eur J Biochem. 1994 Jun 1;222(2):415–424. doi: 10.1111/j.1432-1033.1994.tb18880.x. [DOI] [PubMed] [Google Scholar]
  31. Voorberg J., Fontijn R., Calafat J., Janssen H., van Mourik J. A., Pannekoek H. Assembly and routing of von Willebrand factor variants: the requirements for disulfide-linked dimerization reside within the carboxy-terminal 151 amino acids. J Cell Biol. 1991 Apr;113(1):195–205. doi: 10.1083/jcb.113.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES