Abstract
Epiglycanin is a mucin-type glycoprotein present at the surface of TA3Ha mouse mammary tumour cells. It is a long rod-like glycoprotein with a molecular mass of 500 kDa. Its function has not yet been established but its overexpression can affect cell-cell and cell-matrix adhesion. To understand better the biological function of epiglycanin, we have studied the biochemical structure and biosynthesis of epiglycanin in TA3Ha cells. Pulse-chase labelling experiments with [(3)H]threonine revealed an early precursor with a molecular mass of approx. 300 kDa containing approx. 5-10 kDa of N-linked glycans. The precursor was gradually converted into a high-molecular-mass mature form, owing mainly, if not entirely, to O-glycosylation. The mature molecule consists of two major glycoforms that differ in sialylation. Unlike secreted mucins, epiglycanin did not form cysteine-bound multimers, providing further evidence that epiglycanin belongs to the class of membrane-associated mucins. The mature form, but not the precursor form, is shed from the cell surface. The half-life of epiglycanin on the cell surface was found to be approx. 60 h. These results provide the first detailed analysis of the biochemical structure and biosynthesis of epiglycanin.
Full Text
The Full Text of this article is available as a PDF (211.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Balch W. E., McCaffery J. M., Plutner H., Farquhar M. G. Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell. 1994 Mar 11;76(5):841–852. doi: 10.1016/0092-8674(94)90359-x. [DOI] [PubMed] [Google Scholar]
- Byrd J. C., Dahiya R., Huang J., Kim Y. S. Inhibition of mucin synthesis by benzyl-alpha-GalNAc in KATO III gastric cancer and Caco-2 colon cancer cells. Eur J Cancer. 1995;31A(9):1498–1505. doi: 10.1016/0959-8049(95)00248-h. [DOI] [PubMed] [Google Scholar]
- Carraway K. L., Fregien N., Carraway K. L., 3rd, Carraway C. A. Tumor sialomucin complexes as tumor antigens and modulators of cellular interactions and proliferation. J Cell Sci. 1992 Oct;103(Pt 2):299–307. doi: 10.1242/jcs.103.2.299. [DOI] [PubMed] [Google Scholar]
- Codington J. F., Bhavanandan V. P., Bloch K. J., Nikrui N., Ellard J. V., Wang P. S., Jeanloz R. W. Antibody to epiglycanin and radioimmunoassay to detect epiglycanin-related glycoproteins in body fluids of cancer patients. J Natl Cancer Inst. 1984 Nov;73(5):1029–1038. [PubMed] [Google Scholar]
- Codington J. F., Cooper A. G., Brown M. C., Jeanloz R. W. Evidence that the major cell suface glycoprotein of the TA3-Ha carcinoma contains the Vicia graminea receptor sites. Biochemistry. 1975 Feb 25;14(4):855–859. doi: 10.1021/bi00675a033. [DOI] [PubMed] [Google Scholar]
- Codington J. F., Deak M. R., Frim D. M., Jeanloz R. W. Evidence for the presence of an N-acetyllactosamine-type chain in epiglycanin. Arch Biochem Biophys. 1986 Nov 15;251(1):47–54. doi: 10.1016/0003-9861(86)90049-4. [DOI] [PubMed] [Google Scholar]
- Codington J. F., Haavik S. Epiglycanin--a carcinoma-specific mucin-type glycoprotein of the mouse TA3 tumour. Glycobiology. 1992 Jun;2(3):173–180. doi: 10.1093/glycob/2.3.173. [DOI] [PubMed] [Google Scholar]
- Codington J. F., Klein G., Cooper A. G., Lee N., Brown M. C., Jeanloz R. W. Further studies on the relationship between large glycoprotein molecules and allotransplantability in the TA3 tumor of the mouse: studies on segregating TA3-HA hybrids. J Natl Cancer Inst. 1978 Apr;60(4):811–818. doi: 10.1093/jnci/60.4.811. [DOI] [PubMed] [Google Scholar]
- Codington J. F., Sanford B. H., Jeanloz R. W. Cell-surface glycoproteins of two sublines of the TA3 tumor. J Natl Cancer Inst. 1973 Aug;51(2):585–591. [PubMed] [Google Scholar]
- 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]
- Delannoy P., Kim I., Emery N., De Bolos C., Verbert A., Degand P., Huet G. Benzyl-N-acetyl-alpha-D-galactosaminide inhibits the sialylation and the secretion of mucins by a mucin secreting HT-29 cell subpopulation. Glycoconj J. 1996 Oct;13(5):717–726. doi: 10.1007/BF00702335. [DOI] [PubMed] [Google Scholar]
- Haavik S., Codington J. F., Davison P. F. Development and characterization of monoclonal antibodies against a mucin-type glycoprotein. Glycobiology. 1992 Jun;2(3):217–224. doi: 10.1093/glycob/2.3.217. [DOI] [PubMed] [Google Scholar]
- Haavik S., Nilsen M., Thingstad T., Barsett H., Renouf D. V., Hounsell E. F., Codington J. F. Specificity studies of an antibody developed against a mucin-type glycoprotein. Glycoconj J. 1999 Mar;16(3):229–236. doi: 10.1023/a:1007080405162. [DOI] [PubMed] [Google Scholar]
- Hennebicq-Reig S., Lesuffleur T., Capon C., De Bolos C., Kim I., Moreau O., Richet C., Hémon B., Recchi M. A., Maës E. Permanent exposure of mucin-secreting HT-29 cells to benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation of mucins and inhibits constitutive and stimulated MUC5AC secretion. Biochem J. 1998 Aug 15;334(Pt 1):283–295. doi: 10.1042/bj3340283. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hilkens J., Buijs F. Biosynthesis of MAM-6, an epithelial sialomucin. Evidence for involvement of a rare proteolytic cleavage step in the endoplasmic reticulum. J Biol Chem. 1988 Mar 25;263(9):4215–4222. [PubMed] [Google Scholar]
- Hong W. Protein transport from the endoplasmic reticulum to the Golgi apparatus. J Cell Sci. 1998 Oct;111(Pt 19):2831–2839. doi: 10.1242/jcs.111.19.2831. [DOI] [PubMed] [Google Scholar]
- Huet G., Hennebicq-Reig S., de Bolos C., Ulloa F., Lesuffleur T., Barbat A., Carrière V., Kim I., Real F. X., Delannoy P. GalNAc-alpha-O-benzyl inhibits NeuAcalpha2-3 glycosylation and blocks the intracellular transport of apical glycoproteins and mucus in differentiated HT-29 cells. J Cell Biol. 1998 Jun 15;141(6):1311–1322. doi: 10.1083/jcb.141.6.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huet G., Kim I., de Bolos C., Lo-Guidice J. M., Moreau O., Hemon B., Richet C., Delannoy P., Real F. X., Degand P. Characterization of mucins and proteoglycans synthesized by a mucin-secreting HT-29 cell subpopulation. J Cell Sci. 1995 Mar;108(Pt 3):1275–1285. doi: 10.1242/jcs.108.3.1275. [DOI] [PubMed] [Google Scholar]
- Jentoft N. Why are proteins O-glycosylated? Trends Biochem Sci. 1990 Aug;15(8):291–294. doi: 10.1016/0968-0004(90)90014-3. [DOI] [PubMed] [Google Scholar]
- Kemperman H., Wijnands Y., Wesseling J., Niessen C. M., Sonnenberg A., Roos E. The mucin epiglycanin on TA3/Ha carcinoma cells prevents alpha 6 beta 4-mediated adhesion to laminin and kalinin and E-cadherin-mediated cell-cell interaction. J Cell Biol. 1994 Dec;127(6 Pt 2):2071–2080. doi: 10.1083/jcb.127.6.2071. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuan S. F., Byrd J. C., Basbaum C., Kim Y. S. Inhibition of mucin glycosylation by aryl-N-acetyl-alpha-galactosaminides in human colon cancer cells. J Biol Chem. 1989 Nov 15;264(32):19271–19277. [PubMed] [Google Scholar]
- Ligtenberg M. J., Kruijshaar L., Buijs F., van Meijer M., Litvinov S. V., Hilkens J. Cell-associated episialin is a complex containing two proteins derived from a common precursor. J Biol Chem. 1992 Mar 25;267(9):6171–6177. [PubMed] [Google Scholar]
- McCool D. J., Forstner J. F., Forstner G. G. Synthesis and secretion of mucin by the human colonic tumour cell line LS180. Biochem J. 1994 Aug 15;302(Pt 1):111–118. doi: 10.1042/bj3020111. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miller S. C., Codington J. F., Klein G. Further studies on the relationship between allotransplantability and the presence of the cell surface glycoprotein epiglycanin in the TA3-MM mouse mammary carcinoma ascites cell. J Natl Cancer Inst. 1982 Jun;68(6):981–988. [PubMed] [Google Scholar]
- Mizuno M., Singer S. J. A soluble secretory protein is first concentrated in the endoplasmic reticulum before transfer to the Golgi apparatus. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5732–5736. doi: 10.1073/pnas.90.12.5732. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Moniaux N., Nollet S., Porchet N., Degand P., Laine A., Aubert J. P. Complete sequence of the human mucin MUC4: a putative cell membrane-associated mucin. Biochem J. 1999 Mar 1;338(Pt 2):325–333. [PMC free article] [PubMed] [Google Scholar]
- Shekels L. L., Hunninghake D. A., Tisdale A. S., Gipson I. K., Kieliszewski M., Kozak C. A., Ho S. B. Cloning and characterization of mouse intestinal MUC3 mucin: 3' sequence contains epidermal-growth-factor-like domains. Biochem J. 1998 Mar 15;330(Pt 3):1301–1308. doi: 10.1042/bj3301301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sheng Z. Q., Hull S. R., Carraway K. L. Biosynthesis of the cell surface sialomucin complex of ascites 13762 rat mammary adenocarcinoma cells from a high molecular weight precursor. J Biol Chem. 1990 May 25;265(15):8505–8510. [PubMed] [Google Scholar]
- 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]
- 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]
- Thingstad T., Haavik S., Hansen K., Sletten K., Codington J. F., Barsett H. Human carcinoma-associated antigen (HCA), isolated from the endometrial carcinoma cell line KLE-1 and ascitic fluid of a patient with ovarian carcinoma; comparison with epiglycanin. Eur J Pharm Sci. 1998 Apr;6(2):121–129. doi: 10.1016/s0928-0987(97)00076-6. [DOI] [PubMed] [Google Scholar]
- Tytgat K. M., Bovelander F. J., Opdam F. J., Einerhand A. W., Büller H. A., Dekker J. Biosynthesis of rat MUC2 in colon and its analogy with human MUC2. Biochem J. 1995 Jul 1;309(Pt 1):221–229. doi: 10.1042/bj3090221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tytgat K. M., Opdam F. J., Einerhand A. W., Büller H. A., Dekker J. MUC2 is the prominent colonic mucin expressed in ulcerative colitis. Gut. 1996 Apr;38(4):554–563. doi: 10.1136/gut.38.4.554. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Van den Eijnden D. H., Evans N. A., Codington J. F., Reinhold V., Silber C., Jeanloz R. W. Chemical structure of epiglycanin, the major glycoprotein of the TA3-Ha ascites cell. The carbohydrate chains. J Biol Chem. 1979 Dec 10;254(23):12153–12159. [PubMed] [Google Scholar]
- Vos H. L., de Vries Y., Hilkens J. The mouse episialin (Muc1) gene and its promoter: rapid evolution of the repetitive domain in the protein. Biochem Biophys Res Commun. 1991 Nov 27;181(1):121–130. doi: 10.1016/s0006-291x(05)81390-7. [DOI] [PubMed] [Google Scholar]
- Watkins S. C., Slayter H. S., Codington J. F. Intracellular pathway of a mucin-type membrane glycoprotein in mouse mammary tumor cells. Carbohydr Res. 1991 Jun 25;213:185–200. doi: 10.1016/s0008-6215(00)90608-6. [DOI] [PubMed] [Google Scholar]
- Williams S. J., McGuckin M. A., Gotley D. C., Eyre H. J., Sutherland G. R., Antalis T. M. Two novel mucin genes down-regulated in colorectal cancer identified by differential display. Cancer Res. 1999 Aug 15;59(16):4083–4089. [PubMed] [Google Scholar]
- Williams S. J., Munster D. J., Quin R. J., Gotley D. C., McGuckin M. A. The MUC3 gene encodes a transmembrane mucin and is alternatively spliced. Biochem Biophys Res Commun. 1999 Jul 22;261(1):83–89. doi: 10.1006/bbrc.1999.1001. [DOI] [PubMed] [Google Scholar]