1-benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside blocks the apical biosynthetic pathway in polarized HT-29 cells.
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.
Delacour
Delphine
D
Unité INSERM 560, Place de Verdun, 59045 Lille cedex, France.
Gouyer
Valérie
V
Leteurtre
Emmanuelle
E
Ait-Slimane
Tounsia
T
Drobecq
Hervé
H
Lenoir
Christelle
C
Moreau-Hannedouche
Odile
O
Trugnan
Germain
G
Huet
Guillemette
G
D000818:Animals; D017302:Annexins / Q000378:metabolism; D016764:Cell Polarity; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism; D005690:Galactose / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006031:Glycosylation; D019073:HT29 Cells; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D021381:Protein Transport; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins
C411946:1-benzyl-2-acetamido-2-galactopyranoside; D017302:Annexins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D018819:Dipeptidyl Peptidase 4; D005690:Galactose