scholarly journals Glycosylation pattern of brush border-associated glycoproteins in enterocyte-like cells: involvement of complex-type N-glycans in apical trafficking

2009 ◽  
Vol 390 (7) ◽  
Author(s):  
Willy Morelle ◽  
Laurence Stechly ◽  
Sabine André ◽  
Isabelle Van Seuningen ◽  
Nicole Porchet ◽  
...  
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
T Antigen ◽  
Potential Contribution ◽  
Complex Type ◽  
Glycosylation Pattern ◽  
Hybrid Type ◽  
Flight Mass Spectrometry ◽  
Apical Trafficking ◽  
Ht 29

Abstract 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 [Neu5Acα2–3Galβ1–3GalNAc-ol and Neu5Acα2– 3Galβ1–3(Neu5Acα2–6)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.

scholarly journals GalNAc-α-O-benzyl Inhibits NeuAcα2-3 Glycosylation and Blocks the Intracellular Transport of Apical Glycoproteins and Mucus in Differentiated HT-29 Cells

1998 ◽  
Vol 141 (6) ◽  
pp. 1311-1322 ◽  
Author(s):  
Guillemette Huet ◽  
Sylviane Hennebicq-Reig ◽  
Carmen de Bolos ◽  
Fausto Ulloa ◽  
Thécla Lesuffleur ◽  
...  
Keyword(s):  
Brush Border ◽  
Intracellular Transport ◽  
Brush Border Membrane ◽  
Apical Membrane ◽  
Mucus Secretion ◽  
Membrane Glycoproteins ◽  
Terminal Position ◽  
Dipeptidylpeptidase Iv ◽  
Gp 120 ◽  
Ht 29

Exposure for 24 h of mucus-secreting HT-29 cells to the sugar analogue GalNAc-α-O-benzyl results in inhibition of Galβ1-3GalNAc:α2,3-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-α-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, NeuAcα2-3Gal-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-α-O-benzyl results in an impairment of their sialylation. These effects are reversible upon removal of the drug. It is suggested that α2-3 sialylation is involved in apical targeting of brush border membrane glycoproteins and mucus secretion in HT-29 cells.

Low-affinity transport of pyroglutamyl-histidine in renal brush-border membrane vesicles

1989 ◽  
Vol 257 (5) ◽  
pp. C971-C975 ◽  
Author(s):  
H. A. Skopicki ◽  
K. Fisher ◽  
D. Zikos ◽  
G. Flouret ◽  
D. R. Peterson
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Luminal Membrane ◽  
Renal Brush Border Membrane ◽  
Proximal Tubular ◽  
Renal Brush Border

These studies were performed to determine if a low-affinity carrier is present in the luminal membrane of proximal tubular cells for the transport of the dipeptide, pyroglutamyl-histidine (pGlu-His). We have previously described the existence of a specific, high-affinity, low-capacity [transport constant (Kt) = 9.3 X 10(-8) M, Vmax = 6.1 X 10(-12) mol.mg-1.min-1] carrier for pGlu-His in renal brush-border membrane vesicles. In the present study, we sought to demonstrate that multiple carriers exist for the transport of a single dipeptide by determining whether a low-affinity carrier also exists for the uptake of pGlu-His. Transport of pGlu-His into brush-border membrane vesicles was saturable over the concentration range of 10(-5)-10(-3) M, yielding a Kt of 6.3 X 10(-5) M and a Vmax of 2.2 X 10(-10) mol.mg-1.min-1. Uptake was inhibited by the dipeptides glycyl-proline, glycyl-sarcosine, and carnosine but not by the tripeptide pyroglutamyl-histidyl-prolinamide. We conclude that 1) pGlu-His is transported across the luminal membrane of the proximal tubule by multiple carriers and 2) the lower affinity carrier, unlike the higher affinity carrier, is nonspecific with respect to other dipeptides.

Hydrolysis and transglycosylation activities of glycosidases from small intestine brush-border membrane vesicles

2021 ◽  
Vol 139 ◽  
pp. 109940
Author(s):  
Lesbia Cristina Julio-Gonzalez ◽  
F. Javier Moreno ◽  
María Luisa Jimeno ◽  
Elisa G. Doyagüez ◽  
Agustín Olano ◽  
...  
Keyword(s):  
Small Intestine ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles
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