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

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 intraand 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. Molecular & Cellular Proteomics 14: 10.1074/mcp. M115.053983, 2910–2921, 2015. 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) 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. IECs are known to have highly glycosylated surfaces (1–3). The distribution of these displayed glycans across the cell surface is sensitive to the internal state as well as the external environment of the cell (4–6). In particular, variations in glycosylation patterns have been reported to occur as a function of cellular development and cancer progression (7–10). For example, global changes in cell surface sialylation have been observed during kidney endothelial, uterine epithelial, and lymphoid cell maturation (11–15). Similar variations in glycosylation have also been observed during malignant tumor progression, where the cells undergo rapid proliferation and resistance to apoptosis. In this context, recent studies in colorectal cancer tissues have demonstrated the expression of higher levels of high mannose type glycans and bisecting N-acetylglucosamine (GlcNAc) compared with normal colorectal tissues (16, 17). Although the mechanisms that control these specific changes are not well understood, it is evident From the ‡Department of Chemistry, University of California, Davis, California 95616; §Department of Dermatology, University of California, Davis School of Medicine, Sacramento, California 95816 Received July 23, 2015, and in revised form, August 21, 2015 Published, MCP Papers in Press, September 9, 2015, DOI 10.1074/ mcp.M115.053983 Author contributions: D.P., E.M., and C.B.L. designed research; D.P., K.A.B., A.M., and A.I.M. performed research; D.P., E.M., and C.B.L. wrote the paper; D.P. analyzed glycomics and proteomics data; A.M. and A.I.M. analyzed transcriptomics data. 1 The abbreviations used are: IEC, Intestinal Epithelial cell; HM, High Mannose; C, Complex; H, Hybrid; Fuc, Fucose; NeuAc, N-Acetylneuraminic Acid; Man, Mannose; GlcNAc, N-Acetylglucosamine; ALP, Alkaline Phosphatase; p-NPP, p-Nitrophenyl Phosphate; TCC, Total Compound Chromatogram; ECC, Extracted Compound Chromatogram; SEM, Scanning Electron Microscope. Research