Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes

Abstract

Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract. Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract. Proliferative stem cells located in the base of intestinal crypts form specialized differentiated cell types as they migrate up the villi. A continuous cell turnover occurs every four to eight days as newly differentiated cells eventually replace older cells at the tip of the villus. Self-renewing intestinal epithelial cells (IECs)1 are highly susceptible to malignant growths, which arise from imbalances in cellular proliferation, differentiation, and apoptosis. If the number of developing cells outbalances the number of mature cells undergoing apoptosis, an abnormal growth of tissue can form which, in some cases, may lead to malignant tumors. Thus, a greater understanding of the molecular details of IEC differentiation may lead to novel insight into the pathophysiology of a variety of GI diseases, including cancer. 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Comparative N-glycan profiling of colorectal cancer cell lines reveals unique bisecting GlcNAc and alpha-2,3-linked sialic acid determinants are associated with membrane proteins of the more metastatic/aggressive cell lines.J. Proteome Res. 2014; 13: 277-288Crossref PubMed Scopus (85) Google Scholar, 17.Balog C.I. Stavenhagen K. Fung W.L. Koeleman C.A. McDonnell L.A. Verhoeven A. Mesker W.E. Tollenaar R.A. Deelder A.M. Wuhrer M. N-glycosylation of colorectal cancer tissues: a liquid chromatography and mass spectrometry-based investigation.Mol. Cell. Proteomics. 2012; 11: 571-585Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Although the mechanisms that control these specific changes are not well understood, it is evident that glycosylation is critical for these cellular transformations. Determining the surface glycosylation changes of IECs in relation to their differentiation may lead to a better understanding of this malignancy. 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In differentiated cells are on the of the and are to is for the host to the types of glycans on differentiated cells as sialic acid with high A. Southwick A.M. Earle K.A. Sonnenburg J.L. A refined palate: bacterial consumption of host glycans in the gut.Glycobiology. 2013; 23: 1038-1046Crossref PubMed Scopus (125) Google Scholar). feature of the mature glycosylation is levels of the bisecting GlcNAc were also observed in the from differentiated cells, which for for the of a bisecting The bisecting GlcNAc of and has on membrane For example, the of a bisecting GlcNAc has for proteins in as which is known to be a of of in Rev. PubMed Scopus Google Scholar, M. Y. K. N. and bisecting the of the N-glycan their and Res. PubMed Scopus Google Scholar). Thus, that the of bisecting GlcNAc on differentiated cells is in the of The of expression changes are as the are in during the of which and studies be to changes to the Although not structural were changes are on the expression as and structural analyses of the surface glycans are and be a of At the of proteins on the cell surface as Caco-2 cells changes in proteins a variety of molecular changes of these proteins may as for understanding the of differentiation and has in understanding the that lead to malignant transformations. The to characterize and mass profiling is a tool for understanding results that IEC cell differentiation is by characteristic changes in the cell surface which may be specific and sensitive of differentiation Changes in on the cell membrane may be a of differentiation. Caco-2 cells are an model cell line that a characteristic on their is to that be when Caco-2 cells as changes in glycosylation expression patterns are and for their with and for with cell