Enterocyte glycosylation is responsive to changes in extracellular conditions: implications for membrane functions

Glycobiology, 2017, vol. 27, no. 9, 847–860 doi: 10.1093/glycob/cwx041 Advance Access Publication Date: 26 June 2017 Original Article Cell Biology Enterocyte glycosylation is responsive to changes in extracellular conditions: implications for membrane functions Dayoung Park2,5, Gege Xu2,5, Mariana Barboza2,3, Ishita M Shah4, Maurice Wong2, Helen Raybould3, David A Mills4, and Carlito B Lebrilla1,2 2 Department of Chemistry, 3Department of Anatomy, Physiology and Cell Biology, and 4Department of Food Science and Technology, University of California, 1 Shields Ave, Davis, CA 95616, USA To whom correspondence should be addressed: Tel: +1-530-752-6364; Fax: +1-530-752-8995; e-mail: 1 5 These authors contributed equally to this work. Received 24 February 2017; Revised 1 May 2017; Editorial decision 1 May 2017; Accepted 5 May 2017 Abstract Epithelial cells in the lining of the intestines play critical roles in maintaining homeostasis while challenged by dynamic and sudden changes in luminal contents. Given the high density of glycosylation that encompasses their extracellular surface, environmental changes may lead to extensive reorganization of membrane-associated glycans. However, neither the molecular details nor the consequences of conditional glycan changes are well understood. Here we assessed the sensitivity of Caco-2 and HT-29 membrane N-glycosylation to variations in (i) dietary elements, (ii) microbial fermentation products and (iii) cell culture parameters relevant to intestinal epithelial cell growth and survival. Based on global LC–MS glycomic and statistical analyses, the resulting glycan expression changes were systematic, dependent upon the conditions of each controlled environment. Exposure to short chain fatty acids produced significant increases in fucosylation while further acidification promoted hypersialylation. Notably, among all conditions, increases of high mannose type glycans were identified as a major response when extracellular fructose, galactose and glutamine were independently elevated. To examine the functional consequences of this discrete shift in the displayed glycome, we applied a chemical inhibitor of the glycan processing mannosidase, globally intensifying high mannose expression. The data reveal that upregulation of high mannose glycosylation has detrimental effects on basic intestinal epithelium functions by altering permeability, host–microbe associations and membrane protein activities. Key words: cell biology, glycosylation, mass spectrometry, membrane proteins, metabolism Introduction The large surface area of the gastrointestinal tract provides abundant opportunities for direct contact with substances in the environment. Along its inner wall, at the interface of the intestinal lumen and mucosa, a single layer of epithelial cells mediate the passage of a wide composite of extracellular material, including nutrients from foods, products of microbial fermentation, as well as toxins. Proper growth and vitality of the epithelial monolayer is therefore critical for maintaining a healthy gut. A major proportion of their extracellular membrane proteins are uniquely and densely glycosylated with additions of © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: 847 848 Results Metabolism of dietary components routes glycan expression We examined whether supplementation of free monosaccharides derived from diet changes N-glycosylation outcomes on the intestinal cell surface at concentrations relevant to physiological conditions. Changes in glycosylation were assessed by globally releasing glycans from membrane proteins extracted from fully differentiated (Caco-2) and partially differentiated (HT-29) intestinal epithelial cells and analyzing the mixture by porous graphitized carbon (PGC)–LC–MS. This approach allowed us to derive comprehensive maps encompassing over 300 unique structures and monitor their expression levels individually. The glycan profile under normal growth conditions was used as a frame of reference with which to compare the glycome at varied environments. Abundant in nearly all carbohydrate-containing foods, glucose (Glc) is a soluble hexose sugar that can be efficiently metabolized by cells (Figure 1) and utilized as a primary energy source. Delivery of glucose to the proximal gut is highly regulated in healthy individuals, keeping intraluminal glucose levels fairly constant regardless of dietary load, ranging from 0.2 to 50 mM in the mammalian small intestine (Ferraris et al. 1990). After high glucose supplementation (25 mM), we observed minimal changes in the groups of glycans presented on Caco2 across all replicate samples (Table I). Within groups, individual glycan compositions showed no more than 1.7-fold changes (Figure 2A). On HT-29 cells, the abundances of nondecorated complex/hybrid glycans decreased by 20% (P < 0.05) following treatment (Table I). However, they constitute a minor component of the cell surface and sum to <4% of the total N-glycans. Overall, a high glucose environment did not have a substantial impact on cell surface N-glycosylation, indicating that absorbed glucose is ubiquitously interconverted into other activated monosaccharide forms without favoring specific biosynthetic routes (Figure 1). Similar to glucose, mannose (Man) is found in all glycoprotein-containing food products and is equally an important precursor for N-glycan synthesis. Predictably, no major changes were observed in Caco-2 cell surface glycans upon treatment with mannose, consistent with the effects observed by glucose treatment (Table I). The lack of discrete changes provides support that exogenous mannose likewise is readily utilized by multiple metabolic routes. In fact, its activated phosphorylated form (Man-6-P) can be converted into all of the monosaccharide constituents transferred onto the nascent N-glycan chain (Figure 1). In comparison, HT-29 cells grown in the presence of free mannose showed significant changes (P < 0.05) collectively in high mannose type glycans (Table I). When glycan species were evaluated individually, slight increases in Man 3, Man 7 and Man 9 were observed (Figure 2A). This data demonstrates that mannose utilization is better with exogenous mannose than via glucose interconversion, supporting earlier beliefs (Ichikawa et al. 2014). Although elementally similar to glucose, galactose (Gal), a common component of dairy and plant-based carbohydrates, is not readily metabolized and must first be converted. Unlike the effects observed by glucose treatment, significant increases (P < 0.05) were observed for all high mannose (Man 3–Man 9) (Figure 2A) and nondecorated complex/hybrid types of N-glycans (e.g. Hex5HexNAc4 and Hex5HexNAc5) in galactose-treated Caco-2 cells (Figure 2A). Correspondingly, the relative abundances of fucosylated and sialylated glycans decreased after treatment by 12–28% (P < 0.05) (Table I). This effect is show (...truncated)