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Glycoproteomics and glycomics investigation of membrane N ‐glycosylproteins from human colon carcinoma cells
Author(s) -
VercoutterEdouart AnneSophie,
Slomianny MarieChristine,
DekeyzerBeseme Olivia,
Haeuw JeanFrançois,
Michalski JeanClaude
Publication year - 2008
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200800151
Subject(s) - enterocyte , glycan , glycoprotein , glycosylation , glycoproteomics , glycomics , biochemistry , membrane glycoproteins , chemistry , lectin , western blot , transmembrane protein , microbiology and biotechnology , membrane protein , proteomics , biology , membrane , receptor , gene , small intestine
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 α chains integrin and dipeptidyl isomerase IV. By lectin‐blot analysis, significant changes of α‐2,3‐ and α‐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.