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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{ddagger}, Fausto Ulloa{ddagger}, Thécla Lesuffleur§, Alain Barbat§, Véronique Carrière§, Isabelle Kim{ddagger}, Francisco X. Real{ddagger}, 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; {ddagger} 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 sugar analogue 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 determine the effects of prolonged inhibition of sialylation, differentiated HT-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 accumulation in intracytoplasmic vesicles of brush border–associated glycoproteins like dipeptidylpeptidase-IV, the mucin-like glycoprotein MUC1, and carcinoembryonic antigen which are no longer expressed at the apical membrane. The block occurs beyond the cis-Golgi as substantiated by endoglycosidase treatment and biosynthesis analysis. In contrast, the polarized expression of the basolateral glycoprotein GP 120 is not modified. Underlying these effects we found that (a) like in mucins, NeuAc{alpha}2-3Gal-R is expressed in the terminal position of the oligosaccharide species associated with the apical, but not the basolateral glycoproteins of the cells, and (b) treatment with GalNAc-{alpha}-O-benzyl results in an impairment of their sialylation. These effects are reversible upon removal of the drug. It is suggested that {alpha}2-3 sialylation is involved in apical targeting of brush border membrane glycoproteins and 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, 16 Avenue 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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