Glycoproteomics and glycomics investigation of membrane N-glycosylproteins from human colon carcinoma cells.
Aberrant glycosylation of proteins is known to profoundly affect cellular adhesion or motility of tumoral cells. In this study, we used HT-29 human colon epithelial cancer cells as a cellular model of cancer progression, as they can either proliferate or differentiate into enterocyte phenotype. A glycoproteomic approach based on Con A lectin-affinity chromatography, SDS-PAGE and MS analysis, allowed the identification of membrane N-glycoproteins from Triton X-100-solubilized proteins from membrane preparation. Among them, 65% were membrane proteins, and 45% were known to be N-glycosylated, such as alpha chains integrin and dipeptidyl isomerase IV. By lectin-blot analysis, significant changes of alpha-2,3- and alpha-2,6-sialylation of membrane glycoproteins were observed between proliferating and differentiated HT-29 cells. From these results, nano-LC-MS/MS analysis of the tryptic digests of the corresponding bands was performed and led to the identification of several transmembrane glycoproteins, like members of the solute carrier family and adhesion proteins. Finally, we compared N-glycans profiles and monosaccharide composition of proliferating and enterocyte-like HT-29 cells using MALDI-MS and GC-MS analyses of permethylated derivatives. This glycomic approach allowed to underscore significant changes in N-glycans structure, in particular the expression of atypical N-acetylglucosamine (GlcNAc)-ended N-glycans in enterocyte-like HT-29 cells.
Vercoutter-Edouart
Anne-Sophie
AS
Unité de Glycobiologie Structurale et Fonctionnelle, UMR USTL/CNRS no. 8576, IFR 147, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France. anne-sophie.vercoutter@univ-lille1.fr
Slomianny
Marie-Christine
MC
Dekeyzer-Beseme
Olivia
O
Haeuw
Jean-François
JF
Michalski
Jean-Claude
JC
D002454:Cell Differentiation; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D003110:Colonic Neoplasms / Q000378:metabolism / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel; D054794:Glycomics / Q000379:methods*; D006031:Glycosylation; D019073:HT29 Cells; D006801:Humans; D037102:Lectins / Q000737:chemistry; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000378:metabolism; D008958:Models, Molecular; D011134:Polysaccharides / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
D037102:Lectins; D008562:Membrane Glycoproteins; D011134:Polysaccharides