Abstract
In previous work we reported that long term treatment of polarized HT-29 cells by 1-benzyl-2-acetamido-2-deoxy-alpha-d-galactopyranoside (GalNAcalpha-O-bn) induced undersialylation and intracellular distribution of apical glycoproteins such as dipeptidyl peptidase IV (DPP-IV), and we suggested therefore that sialylation could act as an apical targeting signal. In this work, the apical direct biosynthetic route was studied after transfection of polarized enterocyte-like HT-29 5M12 cloned cells with a murine cDNA coding for a soluble form of DPP-IV, which was secreted into the apical medium. A 24-h treatment of transfected cells by GalNAcalpha-O-bn markedly inhibited the apical secretion and the sialylation of this soluble murine DPP-IV, which became blocked inside the cell. A similar short GalNAcalpha-O-bn treatment also induced an intracellular distribution of both endogenous transmembrane DPP-IV and proteins involved in the regulation of the apical trafficking such as the apical t-SNARE syntaxin-3 and the raft-associated protein annexin XIIIb, whereas the basolateral t-SNARE syntaxin-4 kept its normal localization. These apical membrane proteins moved efficiently from trans-Golgi network to apical carrier vesicles but failed to be transported from carrier vesicles to the apical plasma membrane. Isolation of membrane microdomains showed that GalNAcalpha-O-bn induced the formation of abnormal lipid-rich microdomains in comparison to normal rafts, as shown by their lower buoyant density and their depletion in annexin XIIIb. In conclusion, GalNAcalpha-O-bn blocks the anterograde traffic to the apical surface of polarized HT-29 cells at the transport level or docking/fusion level of carrier vesicles.
Highlights
In recent years, data of the literature have shown the role of glycosylation in the apical biosynthetic route in polarized epithelial cells
In previous work we reported that long term treatment of polarized HT-29 cells by 1-benzyl-2-acetamido2-deoxy-␣-D-galactopyranoside (GalNAc␣-O-bn) induced undersialylation and intracellular distribution of apical glycoproteins such as dipeptidyl peptidase IV (DPP-IV), and we suggested that sialylation could act as an apical targeting signal
The sialylation of N- and/or O-glycans was found inhibited on the endogenous glycoproteins DPP-IV, MUC1, and GPI-anchored carcinoembryonic antigen, and we suggested that undersialylation of glycoproteins may induce a defect in the direct apical targeting of glycoproteins (19 –21)
Summary
Data of the literature have shown the role of glycosylation in the apical biosynthetic route in polarized epithelial cells. Basolateral glycoproteins such as gp120 and gp525 kept a normal localization under GalNAc␣-O-bn treatment [19, 21] These data led us to analyze more deeply the interferences of exogenous GalNAc␣-O-bn with the intracellular processes of glycosylation in HT-29 cells. Thereafter, we reported that GalNAc␣-O-bn was extensively metabolized beyond Gal1–3GalNAc␣-O-bn, showing that the glycosylation of endogenous substrates by several glycosyltransferases could be inhibited, and in particular the sialylation of N-glycans by ␣2,3-sialyltransferase ST3Gal IV [21, 23] In this way, the sialylation of N- and/or O-glycans was found inhibited on the endogenous glycoproteins DPP-IV, MUC1, and GPI-anchored carcinoembryonic antigen, and we suggested that undersialylation of glycoproteins may induce a defect in the direct apical targeting of glycoproteins (19 –21). We and others found that GalNAc␣-O-bn treatment of different cell lines in culture did not result in the identical alterations, regarding glycosylation and intracellular trafficking [21, 23,24,25,26,27], suggesting that the cell type specificity in the cellular responses to GalNAc␣-O-bn was connected to the cell type specificity in the modification of the glycosylation pattern
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