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.

Fig. 1.

Biochemical and morphological validation of Caco-2 differentiation. A, Alkaline phosphatase (ALP) activity during Caco-2 cell growth. Standard deviation is indicated by error bars (n = 3). B, SEM images of the apical side of the Caco-2 cell monolayer at different stages of growth. Microvilli were fully formed by day 21.

Fig. 2.

Global glycan profiling of all detected glycans based on the sum of their abundances as a function of growth time. Summed abundances have been grouped by glycan subtype: high mannose, neutral complex/hybrid (C/H), fucosylated C/H, and sialylated C/H. Error bars indicate standard deviation between three biological replicates.

Fig. 3.

Heatmap display of statistically significant glycan compositions from the undifferentiated state (day 5) to the post-differentiated state (day 24). Pair-wise comparisons were conducted between undifferentiated and differentiated cell samples. Log values of abundances range from low (green) to high (red). Compositions that increased in abundance are arranged from lowest to highest abundance. Glycans that showed a decrease in abundance are ordered from highest to lowest abundance. Each glycan composition is labeled as high mannose (HM), neutral (N), fucosylated (F), sialylated (S), or fucosylated and sialylated (FS) glycans.

Fig. 4.

MS/MS identification of PGC-resolved isomers. A, Extracted compound chromatograms of two isomeric forms (a and b) of Hex₆HexNAc₅Fuc₁NeuAc₂ at different times points during Caco-2 differentiation. B, MS/MS spectra of isomers a and b showing the distinct monosaccharide fragmentation patterns.

Fig. 5.

N-glycan changes of statistically significant glycans on undifferentiated versus differentiated cell surfaces. Glycans possessing the same structural features were grouped together. Values are displayed as average abundances with standard deviation bars (n = 3).

Fig. 6.

Membrane proteins identified in undifferentiated and fully differentiated cells. Venn diagrams illustrating the overlap of Caco-2 cell membrane A, proteins and B, glycoproteins. C, PANTHER molecular function analysis of unique glycoproteins.

Fig. 7.

Comparative normalized expression levels of relevant target genes involved in the glycosylation pathway. Numbered dots indicate genes that increased expression more than fourfold (threshold) as described in the tables. A, undifferentiated versus partially differentiated, B, undifferentiated versus fully differentiated, and C, partially versus fully differentiated Caco-2 cells.