• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biochemjBJ Latest papers and much more!
Biochem J. Aug 15, 1998; 334(Pt 1): 283–295.
PMCID: PMC1219690

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.

Abstract

Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-alpha-D-galactosaminide results in profound changes in mucin oligosaccharide chains. To analyse in depth the effect of this drug, we first determined the structure of mucin oligosaccharide chains synthesized by HT-29 MTX cells and the changes induced by permanent drug exposure. Mucins from untreated cells contained nine monosialylated structures (core types 1, 2, 3 and 4) and four disialylated structures (types 1, 2 and 4). Core 1 structures predominated, in particular NeuAcalpha2-3Galbeta1-3GalNAc-ol. Exposure of HT-29 MTX cells to benzyl-N-acetyl-alpha-D-galactosaminide from days 2-21 resulted in a decrease in intracellular mucins and both their sialic acid and galactose content, and an increased T (Galbeta1-3GalNAcalpha-O-Ser/Thr) and Tn (GalNAcalpha-O-Ser/Thr) antigenicity. A 3-fold increase in both Galbeta1-3GalNAc alpha2, 3-sialyltransferase activity and mRNA expression was detected. At the ultrastructural level, T-antigen was not detectable in mucin droplets in control cells, but was strongly expressed in intracytoplasmic vesicles in treated cells. In these cells, MUC1 and MUC3 transcripts were up-regulated, whereas MUC2, MUC5B and MUC5AC were down-regulated. Furthermore, constitutive and secretagogue-induced MUC5AC secretion was reduced and no mucus layer was detected. In conclusion, benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation and altered regulation of MUC5AC secretion.

Full Text

The Full Text of this article is available as a PDF (776K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Roussel P, Lamblin G, Lhermitte M, Houdret N, Lafitte JJ, Perini JM, Klein A, Scharfman A. The complexity of mucins. Biochimie. 1988 Nov;70(11):1471–1482. [PubMed]
  • Siddiqui J, Abe M, Hayes D, Shani E, Yunis E, Kufe D. Isolation and sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2320–2323. [PMC free article] [PubMed]
  • Gum JR, Byrd JC, Hicks JW, Toribara NW, Lamport DT, Kim YS. Molecular cloning of human intestinal mucin cDNAs. Sequence analysis and evidence for genetic polymorphism. J Biol Chem. 1989 Apr 15;264(11):6480–6487. [PubMed]
  • Gum JR, Hicks JW, Swallow DM, Lagace RL, Byrd JC, Lamport DT, Siddiki B, Kim YS. Molecular cloning of cDNAs derived from a novel human intestinal mucin gene. Biochem Biophys Res Commun. 1990 Aug 31;171(1):407–415. [PubMed]
  • Porchet N, Nguyen VC, Dufosse J, Audie JP, Guyonnet-Duperat V, Gross MS, Denis C, Degand P, Bernheim A, Aubert JP. 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. [PubMed]
  • Audie JP, Janin A, Porchet N, Copin MC, Gosselin B, Aubert JP. 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. [PubMed]
  • Guyonnet Duperat V, Audie JP, Debailleul V, Laine A, Buisine MP, Galiegue-Zouitina S, Pigny P, Degand P, Aubert JP, 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. [PMC free article] [PubMed]
  • Toribara NW, Roberton AM, Ho SB, Kuo WL, Gum E, Hicks JW, Gum JR, Jr, Byrd JC, Siddiki B, Kim YS. Human gastric mucin. Identification of a unique species by expression cloning. J Biol Chem. 1993 Mar 15;268(8):5879–5885. [PubMed]
  • De Bolós C, Garrido M, Real FX. MUC6 apomucin shows a distinct normal tissue distribution that correlates with Lewis antigen expression in the human stomach. Gastroenterology. 1995 Sep;109(3):723–734. [PubMed]
  • Bobek LA, Tsai H, Biesbrock AR, Levine MJ. 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]
  • Shankar V, Gilmore MS, Elkins RC, Sachdev GP. A novel human airway mucin cDNA encodes a protein with unique tandem-repeat organization. Biochem J. 1994 Jun 1;300(Pt 2):295–298. [PMC free article] [PubMed]
  • Lamblin G, Lhermitte M, Klein A, Houdret N, Scharfman A, Ramphal R, Roussel P. The carbohydrate diversity of human respiratory mucins: a protection of the underlying mucosa? Am Rev Respir Dis. 1991 Sep;144(3 Pt 2):S19–S24. [PubMed]
  • Kim YS, Gum J, Jr, Brockhausen I. Mucin glycoproteins in neoplasia. Glycoconj J. 1996 Oct;13(5):693–707. [PubMed]
  • Podolsky DK. Oligosaccharide structures of human colonic mucin. J Biol Chem. 1985 Jul 15;260(14):8262–8271. [PubMed]
  • Podolsky DK. Oligosaccharide structures of isolated human colonic mucin species. J Biol Chem. 1985 Dec 15;260(29):15510–15515. [PubMed]
  • Lesuffleur T, Zweibaum A, Real FX. Mucins in normal and neoplastic human gastrointestinal tissues. Crit Rev Oncol Hematol. 1994 Dec;17(3):153–180. [PubMed]
  • Lesuffleur T, Barbat A, Dussaulx E, Zweibaum A. Growth adaptation to methotrexate of HT-29 human colon carcinoma cells is associated with their ability to differentiate into columnar absorptive and mucus-secreting cells. Cancer Res. 1990 Oct 1;50(19):6334–6343. [PubMed]
  • Lesuffleur T, Porchet N, Aubert JP, Swallow D, Gum JR, Kim YS, Real FX, Zweibaum A. Differential expression of the human mucin genes MUC1 to MUC5 in relation to growth and differentiation of different mucus-secreting HT-29 cell subpopulations. J Cell Sci. 1993 Nov;106(Pt 3):771–783. [PubMed]
  • Lesuffleur T, Roche F, Hill AS, Lacasa M, Fox M, Swallow DM, Zweibaum A, Real FX. Characterization of a mucin cDNA clone isolated from HT-29 mucus-secreting cells. The 3' end of MUC5AC? J Biol Chem. 1995 Jun 9;270(23):13665–13673. [PubMed]
  • Lesuffleur T, Barbat A, Luccioni C, Beaumatin J, Clair M, Kornowski A, Dussaulx E, Dutrillaux B, Zweibaum A. Dihydrofolate reductase gene amplification-associated shift of differentiation in methotrexate-adapted HT-29 cells. J Cell Biol. 1991 Dec;115(5):1409–1418. [PMC free article] [PubMed]
  • Maoret JJ, Font J, Augeron C, Codogno P, Bauvy C, Aubery M, Laboisse CL. A mucus-secreting human colonic cancer cell line. Purification and partial characterization of the secreted mucins. Biochem J. 1989 Mar 15;258(3):793–799. [PMC free article] [PubMed]
  • Capon C, Laboisse CL, Wieruszeski JM, Maoret JJ, Augeron C, Fournet B. Oligosaccharide structures of mucins secreted by the human colonic cancer cell line CL.16E. J Biol Chem. 1992 Sep 25;267(27):19248–19257. [PubMed]
  • Kuan SF, Byrd JC, Basbaum C, Kim YS. 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]
  • Huang J, Byrd JC, Yoon WH, Kim YS. Effect of benzyl-alpha-GalNAc, an inhibitor of mucin glycosylation, on cancer-associated antigens in human colon cancer cells. Oncol Res. 1992;4(11-12):507–515. [PubMed]
  • Huet G, Kim I, de Bolos C, Lo-Guidice JM, Moreau O, Hemon B, Richet C, Delannoy P, Real FX, 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. [PubMed]
  • 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. [PubMed]
  • Duk M, Steuden I, Duś D, Radzikowski C, Lisowska E. Application of chemically desialylated and degalactosylated human glycophorin for induction and characterization of anti-Tn monoclonal antibodies. Glycoconj J. 1992 Jun;9(3):148–153. [PubMed]
  • Hovenberg HW, Davies JR, Carlstedt I. Different mucins are produced by the surface epithelium and the submucosa in human trachea: identification of MUC5AC as a major mucin from the goblet cells. Biochem J. 1996 Aug 15;318(Pt 1):319–324. [PMC free article] [PubMed]
  • Aminoff D, Gathmann WD, McLean CM, Yadomae T. Quantitation of oligosaccharides released by the beta-elimination reaction. Anal Biochem. 1980 Jan 1;101(1):44–53. [PubMed]
  • Baenziger JU, Natowicz M. Rapid separation of anionic oligosaccharide species by high performance liquid chromatography. Anal Biochem. 1981 Apr;112(2):357–361. [PubMed]
  • Lo-Guidice JM, Wieruszeski JM, Lemoine J, Verbert A, Roussel P, Lamblin G. Sialylation and sulfation of the carbohydrate chains in respiratory mucins from a patient with cystic fibrosis. J Biol Chem. 1994 Jul 22;269(29):18794–18813. [PubMed]
  • Papac DI, Wong A, Jones AJ. Analysis of acidic oligosaccharides and glycopeptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Chem. 1996 Sep 15;68(18):3215–3223. [PubMed]
  • Lamblin G, Boersma A, Klein A, Roussel P, van Halbeek H, Vliegenthart JF. Primary structure determination of five sialylated oligosaccharides derived from bronchial mucus glycoproteins of patients suffering from cystic fibrosis. The occurrence of the NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)] GlcNAc beta(1----.) structural element revealed by 500-MHz 1H NMR spectroscopy. J Biol Chem. 1984 Jul 25;259(14):9051–9058. [PubMed]
  • Recchi MA, Harduin-Lepers A, Boilly-Marer Y, Verbert A, Delannoy P. Multiplex RT-PCR method for the analysis of the expression of human sialyltransferases: application to breast cancer cells. Glycoconj J. 1998 Jan;15(1):19–27. [PubMed]
  • Yonezawa S, Byrd JC, Dahiya R, Ho JJ, Gum JR, Griffiths B, Swallow DM, Kim YS. Differential mucin gene expression in human pancreatic and colon cancer cells. Biochem J. 1991 Jun 15;276(Pt 3):599–605. [PMC free article] [PubMed]
  • Crepin M, Porchet N, Aubert JP, Degand P. Diversity of the peptide moiety of human airway mucins. Biorheology. 1990;27(3-4):471–484. [PubMed]
  • Dufosse J, Porchet N, Audie JP, Guyonnet Duperat V, Laine A, Van-Seuningen I, Marrakchi S, Degand P, Aubert JP. Degenerate 87-base-pair tandem repeats create hydrophilic/hydrophobic alternating domains in human mucin peptides mapped to 11p15. Biochem J. 1993 Jul 15;293(Pt 2):329–337. [PMC free article] [PubMed]
  • Pringault E, Arpin M, Garcia A, Finidori J, Louvard D. A human villin cDNA clone to investigate the differentiation of intestinal and kidney cells in vivo and in culture. EMBO J. 1986 Dec 1;5(12):3119–3124. [PMC free article] [PubMed]
  • Hennebicq-Reig S, Tetaert D, Soudan B, Kim I, Huet G, Briand G, Richet C, Demeyer D, Degand P. O-Glycosylation and cellular differentiation in a subpopulation of mucin-secreting HT-29 cell line. Exp Cell Res. 1997 Aug 25;235(1):100–107. [PubMed]
  • Peterson GL. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. [PubMed]
  • Gendler SJ, Lancaster CA, Taylor-Papadimitriou J, Duhig T, Peat N, Burchell J, Pemberton L, Lalani EN, Wilson D. Molecular cloning and expression of human tumor-associated polymorphic epithelial mucin. J Biol Chem. 1990 Sep 5;265(25):15286–15293. [PubMed]
  • Debailleul V, Laine A, Huet G, Mathon P, d'Hooghe MC, Aubert JP, Porchet N. Human mucin genes MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6 express stable and extremely large mRNAs and exhibit a variable length polymorphism. An improved method to analyze large mRNAs. J Biol Chem. 1998 Jan 9;273(2):881–890. [PubMed]
  • Dall'Olio F, Malagolini N, Guerrini S, Serafini-Cessi F. Resistance to methotrexate is associated with selective changes of alpha 2,6- and alpha 2,3-sialyltransferase activities toward N-acetyllactosaminic sequences in human colon cancer cell line HT-29. Biochem Biophys Res Commun. 1993 Oct 29;196(2):714–720. [PubMed]
  • 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]
  • Specian RD, Oliver MG. Functional biology of intestinal goblet cells. Am J Physiol. 1991 Feb;260(2 Pt 1):C183–C193. [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...