Glycosylation pattern of brush border-associated glycoproteins in enterocyte-like cells: involvement of complex-type N-glycans in apical trafficking.
We have previously reported that galectin-4, a tandem repeat-type galectin, regulates the raft-dependent delivery of glycoproteins to the apical brush border membrane of enterocyte-like HT-29 cells. N-Acetyllactos-amine-containing glycans, known as galectin ligands, were found enriched in detergent-resistant membranes. Here, we analyzed the potential contribution of N- and/or O-glycans in this mechanism. Structural studies were carried out on the brush border membrane-enriched fraction using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and nano-ESI-QTOF-MS/MS. The pattern of N-glycans was very heterogeneous, with the presence of high mannose- and hybrid-type glycans as well as a multitude of complex-type glycans. In contrast, the pattern of O-glycans was very simple with the presence of two major core type 1 O-glycans, sialylated and bisialylated T-antigen structures [Neu5Acalpha2-3Galbeta1-3GalNAc-ol and Neu5Acalpha2- 3Galbeta1-3(Neu5Acalpha2-6)GalNAc-ol]. Thus, N-glycans rather than O-glycans contain the N-acetyllactosamine recognition signals for the lipid raft-based galectin-4-dependent apical delivery. In the presence of 1-deoxymannojirimycin, a drug which inhibits the generation of hybrid-type or complex type N-glycans, the extensively O-glycosylated mucin-like MUC1 glycoprotein was not delivered to the apical brush border but accumulated inside the cells. Altogether, our data demonstrate the crucial role of complex N-glycans in the galectin-4-dependent delivery of glycoproteins to the apical brush border membrane of enterocytic HT-29 cells.
UMR CNRS 8576, Unité de Glycobiologie Structurale et Fonctionnelle, IFR 147, F-59655 Villeneuve d'Ascq, France.
Willy
W
Morelle
Laurence
L
Stechly
Sabine
S
André
Isabelle
I
Van Seuningen
Nicole
N
Porchet
Hans-Joachim
HJ
Gabius
Jean-Claude
JC
Michalski
Guillemette
G
Huet
D002240:Carbohydrate Sequence; D020895:Enterocytes / Q000166:cytology*; D000939:Epitopes / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D019073:HT29 Cells; D006801:Humans; D006858:Hydrofluoric Acid / Q000378:metabolism; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D008871:Microvilli / Q000378:metabolism*; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport
D000939:Epitopes; C060192:MUC1 protein, human; D008562:Membrane Glycoproteins; D018396:Mucin-1; D011134:Polysaccharides; D006026:Glycoside Hydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D006858:Hydrofluoric Acid