GalNAc-{alpha}-O-benzyl Inhibits NeuAc{alpha}2-3 Glycosylation and Blocks the Intracellular Transport of Apical Glycoproteins and Mucus in Differentiated HT-29 Cells

doi:10.1083/jcb.141.6.1311

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© The Rockefeller University Press, 0021-9525/1998//1311 $5.00
The Journal of Cell Biology, Volume 141, Number 6, , 1998 1311-1322

Articles

GalNAc- ${alpha}$ -O-benzyl Inhibits NeuAc ${alpha}$ 2-3 Glycosylation and Blocks the Intracellular Transport of Apical Glycoproteins and Mucus in Differentiated HT-29 Cells

Guillemette Huet^*, Sylviane Hennebicq-Reig^*, Carmen de Bolos, Fausto Ulloa, Thécla Lesuffleur, Alain Barbat, Véronique Carrière, Isabelle Kim, Francisco X. Real, Philippe Delannoy^||, and Alain Zweibaum

^* Unité de Recherches sur la Biologie et la Physiopathologie des Cellules Mucipares, Institut National de la Sante et de la Recherche Medicale (INSERM) U377, 59045 Lille Cedex, France; Unitat de Biologia Cellular i Molecular, Institut Municipal d'Investigació Mèdica, Universitat Autónoma de Barcelona, E-08003, Barcelona, Spain; Unité de Recherches sur la Différenciation Cellulaire Intestinale, INSERM U178, 94807 Villejuif Cedex, France; and ^|| Laboratoire de Chimie Biologique, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR)-111, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq Cedex, France

Exposure for 24 h of mucus-secreting HT-29 cells to the sugaranalogue GalNAc- ${alpha}$ -O-benzyl results in inhibition of Galβ1-3GalNAc: ${alpha}$ 2,3-sialyltransferase, reduced mucin sialylation, and inhibition of their secretion(Huet, G., I. Kim, C. de Bolos, J.M. Loguidice, O. Moreau,B. Hémon, C. Richet, P. Delannoy, F.X. Real., and P.Degand. 1995. J. Cell Sci. 108:1275–1285). To determinethe effects of prolonged inhibition of sialylation, differentiatedHT-29 populations were grown under permanent exposure to GalNAc- ${alpha}$ -O-benzyl.This results in not only inhibition of mucus secretion, but also in a dramatic swelling of the cells and the accumulationin intracytoplasmic vesicles of brush border–associatedglycoproteins like dipeptidylpeptidase-IV, the mucin-like glycoproteinMUC1, and carcinoembryonic antigen which are no longer expressedat the apical membrane. The block occurs beyond the cis-Golgias substantiated by endoglycosidase treatment and biosynthesisanalysis. In contrast, the polarized expression of the basolateralglycoprotein GP 120 is not modified. Underlying these effectswe found that (a) like in mucins, NeuAc ${alpha}$ 2-3Gal-R is expressedin the terminal position of the oligosaccharide species associatedwith the apical, but not the basolateral glycoproteins of thecells, and (b) treatment with GalNAc- ${alpha}$ -O-benzyl results in an impairment of their sialylation. These effects are reversibleupon removal of the drug. It is suggested that ${alpha}$ 2-3 sialylationis involved in apical targeting of brush border membrane glycoproteinsand mucus secretion in HT-29 cells.

Abbreviations used in this paper: CEA, carcinoembryonic antigen; DPP-IV, dipeptidylpeptidase-IV; GalNAc- ${alpha}$ -O-benzyl, benzyl-2-acetamido-2-deoxy- ${alpha}$ -D-galactopyranoside; MAA, Maackia amurensis agglutinin; MTX, methotrexate; PNA, Arachis hypogaea agglutinin; SNA, Sambucus nigra agglutinin. The abbreviations for sialyltransferases are according to the new systematic nomenclature proposed by Tsuji et al. (1996).

Address all correspondence to Alain Zweibaum, INSERM U178, 16Avenue Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France.Tel.: (33) 1-45-59-50-41. Fax: (33) 1-46-77-02-33. E-mail: zweibaum{at}infobiogen.fr

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