Inflammatory Stress Causes N-Glycan Processing Deficiency in Ocular Autoimmune Disease
2018; Elsevier BV; Volume: 189; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2018.10.012
ISSN1525-2191
AutoresAshley M. Woodward, Sylvain Lehoux, Flavio Mantelli, Antonio Di Zazzo, Inka Brockhausen, Stefano Bonini, Pablo Argüeso,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoHigh levels of proinflammatory cytokines have been associated with a loss of tissue function in ocular autoimmune diseases, but the basis for this relationship remains poorly understood. Here we investigate a new role for tumor necrosis factor α in promoting N-glycan–processing deficiency at the surface of the eye through inhibition of N-acetylglucosaminyltransferase expression in the Golgi. Using mass spectrometry, complex-type biantennary oligosaccharides were identified as major N-glycan structures in differentiated human corneal epithelial cells. Remarkably, significant differences were detected between the efficacies of cytokines in regulating the expression of glycogenes involved in the biosynthesis of N-glycans. Tumor necrosis factor α but not IL-1β had a profound effect in suppressing the expression of enzymes involved in the Golgi branching pathway, including N-acetylglucosaminyltransferases 1 and 2, which are required for the formation of biantennary structures. This decrease in gene expression was correlated with a reduction in enzymatic activity and impaired N-glycan branching. Moreover, patients with ocular mucous membrane pemphigoid were characterized by marginal N-acetylglucosaminyltransferase expression and decreased N-glycan branching in the conjunctiva. Together, these data indicate that proinflammatory cytokines differentially influence the expression of N-glycan–processing enzymes in the Golgi and set the stage for future studies to explore the pathophysiology of ocular autoimmune diseases. High levels of proinflammatory cytokines have been associated with a loss of tissue function in ocular autoimmune diseases, but the basis for this relationship remains poorly understood. Here we investigate a new role for tumor necrosis factor α in promoting N-glycan–processing deficiency at the surface of the eye through inhibition of N-acetylglucosaminyltransferase expression in the Golgi. Using mass spectrometry, complex-type biantennary oligosaccharides were identified as major N-glycan structures in differentiated human corneal epithelial cells. Remarkably, significant differences were detected between the efficacies of cytokines in regulating the expression of glycogenes involved in the biosynthesis of N-glycans. Tumor necrosis factor α but not IL-1β had a profound effect in suppressing the expression of enzymes involved in the Golgi branching pathway, including N-acetylglucosaminyltransferases 1 and 2, which are required for the formation of biantennary structures. This decrease in gene expression was correlated with a reduction in enzymatic activity and impaired N-glycan branching. Moreover, patients with ocular mucous membrane pemphigoid were characterized by marginal N-acetylglucosaminyltransferase expression and decreased N-glycan branching in the conjunctiva. Together, these data indicate that proinflammatory cytokines differentially influence the expression of N-glycan–processing enzymes in the Golgi and set the stage for future studies to explore the pathophysiology of ocular autoimmune diseases. Glycosylation is one of the most frequent and ubiquitous post-translational modifications in living organisms, taking place in more than half of all proteins.1Apweiler R. Hermjakob H. Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.Biochim Biophys Acta. 1999; 1473: 4-8Crossref PubMed Scopus (1492) Google Scholar It is orchestrated by the action of multiple genes encoding for glycosyltransferases and enzymes involved in the processing and turnover of glycans, such as glycosidases and sulfotransferases. Monosaccharides are the basic structural units of glycans and are unique in that they can be attached to each other in many more ways than amino acids or nucleotides, leading to enormous structural diversity. Recent estimates have indicated that there are possibly over 7000 glycan determinants in the human glycome, with a myriad of biological functions relevant to the development and physiology of different organs and tissues.2Cummings R.D. The repertoire of glycan determinants in the human glycome.Mol Biosyst. 2009; 5: 1087-1104Crossref PubMed Scopus (354) Google Scholar N-glycosylation constitutes a major form of glycosylation in eukaryotic cells. It starts with the transfer of a lipid-linked oligosaccharide to asparagine residues on nascent polypeptides during their translocation into the endoplasmic reticulum.3Moremen K.W. Tiemeyer M. Nairn A.V. Vertebrate protein glycosylation: diversity, synthesis and function.Nat Rev Mol Cell Biol. 2012; 13: 448-462Crossref PubMed Scopus (1099) Google Scholar Subsequent processing of this precursor ensures the efficient folding of newly synthesized glycoproteins before reaching the Golgi apparatus, where much of the diversity of glycosylation originates.4Varki A. Evolutionary forces shaping the Golgi glycosylation machinery: why cell surface glycans are universal to living cells.Cold Spring Harb Perspect Biol. 2011; 3: a005462Crossref PubMed Scopus (111) Google Scholar Localization studies have shown that glycan-processing enzymes in the Golgi have a nonuniform distribution along the cis–trans axis.5Stanley P. Golgi glycosylation.Cold Spring Harb Perspect Biol. 2011; 3: a005199Crossref PubMed Scopus (236) Google Scholar The trimming and maturation of N-glycans start in the cis-Golgi by catalytic removal of terminal mannose residues. Then, a family of mannoside N-acetylglucosaminyltransferases (MGATs) in the medial Golgi sequentially catalyzes the conversion of oligomannose structures into hybrid- and complex-type N-glycans by adding N-acetylglucosamine (GlcNAc) residues. This process appears to be essential for survival, since defects in the MGAT1 gene, which encodes for N-acetylglucosaminyltransferase 1, result in embryonic lethality.6Ioffe E. Stanley P. Mice lacking N-acetylglucosaminyltransferase I activity die at mid-gestation, revealing an essential role for complex or hybrid N-linked carbohydrates.Proc Natl Acad Sci U S A. 1994; 91: 728-732Crossref PubMed Scopus (358) Google Scholar, 7Metzler M. Gertz A. Sarkar M. Schachter H. Schrader J.W. Marth J.D. Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development.EMBO J. 1994; 13: 2056-2065Crossref PubMed Scopus (310) Google Scholar Further processing of branched structures in the trans-Golgi is crucial for the extracellular functions of N-glycans and for the formation of galectin–glycoprotein lattices, which affect a multitude of cell-adhesion and -signaling processes.8Lau K.S. Partridge E.A. Grigorian A. Silvescu C.I. Reinhold V.N. Demetriou M. Dennis J.W. Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation.Cell. 2007; 129: 123-134Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar, 9Partridge E.A. Le Roy C. Di Guglielmo G.M. Pawling J. Cheung P. Granovsky M. Nabi I.R. Wrana J.L. Dennis J.W. Regulation of cytokine receptors by Golgi N-glycan processing and endocytosis.Science. 2004; 306: 120-124Crossref PubMed Scopus (589) Google Scholar Maintenance of normal immune system homeostasis is indispensable in the prevention of dysfunction in biological systems. Autoimmune diseases are relatively frequent events that occur when the immune system turns its antimicrobial defenses against normal components of the body.10Goodnow C.C. Multistep pathogenesis of autoimmune disease.Cell. 2007; 130: 25-35Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar A diverse spectrum of autoimmune disorders has been described at the surface of eye. These can be ocular specific (eg, Mooren ulcerative keratitis), systemic (eg, Sjögren syndrome, mucous membrane pemphigoid), or secondary to other autoimmune diseases (eg, rheumatoid arthritis, systemic lupus erythematosus).11Stern M.E. Schaumburg C.S. Dana R. Calonge M. Niederkorn J.Y. Pflugfelder S.C. Autoimmunity at the ocular surface: pathogenesis and regulation.Mucosal Immunol. 2010; 3: 425-442Crossref PubMed Scopus (87) Google Scholar The breakdown of self-tolerance under these conditions leads to the excessive production of cytokines that initiate and perpetuate the inflammatory cascade. Among the different cytokines found to be aberrantly expressed in autoimmune diseases, tumor necrosis factor (TNF)-α has been long recognized to be of particular importance in promoting tissue destruction.12Feldmann M. Brennan F.M. Williams R.O. Cope A.P. Gibbons D.L. Katsikis P.D. Maini R.N. Evaluation of the role of cytokines in autoimmune disease: the importance of TNF alpha in rheumatoid arthritis.Prog Growth Factor Res. 1992; 4: 247-255Abstract Full Text PDF PubMed Scopus (62) Google Scholar In the eye, and under disease conditions, TNFα is produced by a large number of stromal infiltrating cells.13Saw V.P. Dart R.J. Galatowicz G. Daniels J.T. Dart J.K. Calder V.L. Tumor necrosis factor-alpha in ocular mucous membrane pemphigoid and its effect on conjunctival fibroblasts.Invest Ophthalmol Vis Sci. 2009; 50: 5310-5317Crossref PubMed Scopus (27) Google Scholar The underlying pathologic mechanisms of TNFα are varied and multiple, and include the ability to impair the differentiation, proliferation, and viability of specific cellular targets.14Kollias G. Douni E. Kassiotis G. Kontoyiannis D. On the role of tumor necrosis factor and receptors in models of multiorgan failure, rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease.Immunol Rev. 1999; 169: 175-194Crossref PubMed Scopus (237) Google Scholar How TNFα affects the actual biology of the affected tissues remains nonetheless largely unknown. Here, we identify a novel function of TNFα in promoting N-glycan–processing deficiency at the surface of the eye through the inhibition of N-acetylglucosaminyltransferase expression in the Golgi. Moreover, we report on the finding that the alteration of N-glycan biosynthetic pathways during inflammatory stress is cytokine dependent, supporting a specific role for individual proinflammatory cytokines in the pathophysiology of tissue damage in ocular autoimmune disease. Multilayered cultures of telomerase-immortalized human corneal epithelial cells were grown as previously reported.15Argueso P. Gipson I.K. Assessing mucin expression and function in human ocular surface epithelia in vivo and in vitro.Methods Mol Biol. 2012; 842: 313-325Crossref PubMed Scopus (35) Google Scholar Briefly, cells were plated at a seeding density of 5 × 104 cells/cm2 and maintained in keratinocyte serum–free medium (Thermo Fisher Scientific, Rockville, MD) until confluence. Thereafter, cells were grown in Dulbecco's modified Eagle's medium/F12 supplemented with 10% calf serum and 10 ng/mL epidermal growth factor for 7 days to promote stratification and differentiation. Where indicated, cells were serum-starved for 1 hour and incubated with 10 or 40 ng/mL TNFα (PeproTech, Inc., Rocky Hill, NJ) or 10 ng/mL IL-1β (R&D Systems, Minneapolis, MN) in serum-free Dulbecco's modified Eagle's medium/F12. Media along with fresh cytokines were replaced every 24 hours. Conjunctival epithelium was collected by impression cytology from 17 eyes of 10 patients with ocular mucous membrane pemphigoid stage II at Fondazione GB Bietti (Rome, Italy). The mean age of the patients was 64.9 ± 9.3 years (range, 44 to 79 years). Ten specimens from 10 age-matched healthy subjects were used as a control group. The mean age of the control group was 60.3 ± 2.6 years (range, 58 to 64 years). Exclusion criteria for the control group included a history of ocular disease or eye surgery and contact lens wear. Informed consent was obtained from each recruited patient. The study protocol conformed to the ethics guidelines of the 1975 Declaration of Helsinki, and was approved by the IRB of Campus Bio Medico University of Rome (Rome, Italy; IRB number 07/06.PARComEtCBM). Human conjunctival biopsy samples, stored in paraffin, from three healthy subjects were obtained as archived material from a previously published study.16Argueso P. Tisdale A. Mandel U. Letko E. Foster C.S. Gipson I.K. The cell-layer- and cell-type-specific distribution of GalNAc-transferases in the ocular surface epithelia is altered during keratinization.Invest Ophthalmol Vis Sci. 2003; 44: 86-92Crossref PubMed Scopus (64) Google Scholar Conjunctival tissue sections from three patients with ocular mucous membrane pemphigoid were obtained from Campus Bio Medico University of Rome. For structural analyses, protein isolates (120 μg) were lyophilized, reduced in 500 μL of a 2 mg/mL dithiothreitol solution (Sigma-Aldrich, St. Louis, MO) at 50°C for 90 minutes, and then alkylated with 500 μL of a 12 mg/mL iodoacetamide solution for 90 minutes at room temperature in the dark. Samples were dialyzed against 50 mmol/L ammonium bicarbonate for 24 hours at 4°C, lyophilized, and incubated with 1 mL of 50 μg/mL l-1-tosylamide-2-phenylethyl chloromethyl ketone–treated trypsin (Sigma-Aldrich) at 37°C overnight. The digested peptides were then purified using a Sep-Pak C18 (200-mg) cartridge (Waters Corp., Milford, MA), lyophilized, and incubated with 2 μL (500 units/μL) of peptide:N-glycosidase F from Flavobacterium meningosepticum (New England Biolabs, Ipswich, MA) in 200 μL of 50 mmol/L ammonium bicarbonate at 37°C for 4 hours. The mixture was further incubated with 3 μL of peptide:N-glycosidase F at 37°C overnight. The released N-glycans were purified over a Sep-Pak C18 (200-mg) cartridge. The flow-through and wash fraction containing the released N-glycans were collected, pooled, and lyophilized. The permethylation of N-glycans was performed using the NaOH:dimethyl sulfoxide slurry method. Here, lyophilized N-glycans were incubated with 1 mL of a NaOH:dimethyl sulfoxide slurry solution and 500 μL of methyl iodide (Sigma-Aldrich) for 20 to 30 minutes under vigorous shaking at room temperature. One milliliter of chloroform and 3 mL of Milli-Q water were then added, and the mixture was briefly vortexed to wash the chloroform fraction. The wash step was repeated three times. The chloroform fraction was dried, dissolved in 200 mL of 50% methanol, and loaded into a Sep-Pak C18 (200-mg) cartridge. The eluted fraction was lyophilized and dissolved in 10 μL of 75% methanol from which 1 μL was mixed with 1 μL 2,5-dihydroxybenzoic acid (Sigma-Aldrich; 5 mg/mL in 50% acetonitrile with 0.1% trifluoroacetic acid) and spotted on a matrix-assisted laser desorption/ionization polished steel target plate (Bruker Daltonics, Bremen, Germany). The profiling of permethylated N-glycans was performed at Glycomics Core, Beth Israel Deaconess Medical Center (Boston, MA). Mass spectrometry data were acquired on an UltraFlex II matrix-assisted laser desorption/ionization–time-of-flight mass spectrometer (Bruker Daltonics). Reflective positive mode was used and data recorded between m/z 500 and 6000. The mass spectrometry N-glycan profiles were acquired by the aggregation of at least 20,000 laser shots. Mass spectrometry spectra were processed using mMass software version 5.5.0 (http://www.mmass.org).17Strohalm M. Kavan D. Novak P. Volny M. Havlicek V. mMass 3: a cross-platform software environment for precise analysis of mass spectrometric data.Anal Chem. 2010; 82: 4648-4651Crossref PubMed Scopus (569) Google Scholar Mass peaks were manually annotated and assigned to a particular N-glycan composition when a match was found. Total RNA was extracted from cell cultures and impression cytology samples using an extraction reagent (TRIzol; Thermo Fisher Scientific) according to the manufacturer's protocol. Residual genomic DNA in the RNA preparation was eliminated by digestion with amplification-grade DNase I (Thermo Fisher Scientific). Five micrograms (for PCR array) or 1 μg [for real-time quantitative (qPCR)] total RNA was used for cDNA synthesis (iScript cDNA Synthesis; Bio-Rad, Hercules, CA). The analysis of 84 genes encoding for glycosylation enzymes was performed using a human glycosylation PCR array (RT2 Profiler PCR array; Qiagen, Hilden, Germany) according to the manufacturer's instructions. The array was repeated twice with independently isolated RNA. Expression values were corrected for the housekeeping genes HPRT1 and RPLP0. The 2−ΔCT and 2−ΔΔCT methods were used for relative quantitation of the number of transcripts. Gene expression levels were detected by qPCR using the SYBR Fast qPCR kit (Kapa Biosystems, Wilmington, MA) in a Mastercycler ep realplex thermal cycler (Eppendorf, Hauppauge, NY). Primer sequences for MGAT1 (forward, 5′-GGTGGAGTTGGTGGGTCATC-3′; reverse, 5′-GAGGAGAGGTCTTGCTTGCC-3′), MGAT2 (forward, 5′-CTGCTGCTGGACTCACTTC-3′; reverse, 5′-AATGCTGAAAGGAAAGAACACC-3′), and MGAT4B (forward, 5′-ACTTCATCCGCTTCCGCTTC-3′; reverse, 5′-TCCTTGTCTGACTGAGGGTTGT-3′) mRNA have been previously published.18Beheshti Zavareh R. Sukhai M.A. Hurren R. Gronda M. Wang X. Simpson C.D. Maclean N. Zih F. Ketela T. Swallow C.J. Moffat J. Rose D.R. Schachter H. Schimmer A.D. Dennis J.W. Suppression of cancer progression by MGAT1 shRNA knockdown.PLoS One. 2012; 7: e43721Crossref PubMed Scopus (32) Google Scholar, 19Lappas M. Effect of pre-existing maternal obesity, gestational diabetes and adipokines on the expression of genes involved in lipid metabolism in adipose tissue.Metabolism. 2014; 63: 250-262Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 20Ide Y. Miyoshi E. Nakagawa T. Gu J. Tanemura M. Nishida T. Ito T. Yamamoto H. Kozutsumi Y. Taniguchi N. Aberrant expression of N-acetylglucosaminyltransferase-IVa and IVb (GnT-IVa and b) in pancreatic cancer.Biochem Biophys Res Commun. 2006; 341: 478-482Crossref PubMed Scopus (41) Google Scholar Primers for GAPDH were obtained from Bio-Rad (catalog number qHsaCED0038674). The following parameters were used: 2 minutes at 95°C, followed by 40 cycles of 5 seconds at 95°C and 30 seconds at 60°C. All samples were normalized using GAPDH housekeeping gene expression. The comparative 2−ΔΔCτ method was used for relative quantitation of the number of transcripts. No template controls were run in each assay to confirm lack of DNA contamination in the reagents used for amplification. N-acetylglucosaminyltransferase activity was measured in a plate reader using a Glycosyltransferase Activity Kit (R&D Systems) according to the manufacturer's instructions. Briefly, cell lysates in radioimmunoprecipitation assay buffer were cleared of insoluble material by centrifugation at 17,115 × g and filtered through 10 kDa–cutoff centrifugal filters to remove cellular phosphate. The filters were washed with diH2O and the protein concentration of the supernatants determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Thereafter, 25 μL (10 μg) of protein was mixed with 25 μL of a working solution of donor and acceptor substrates to give a final concentration of 0.2 mmol/L uridine diphosphate–GlcNAc and 1 mmol/L mannotriose [Manα1-3(Manα1-6)Man]. The reaction was allowed to proceed for 1 hour at 37°C. The solution was then incubated with 50 μL of 0.2 μg/mL coupling phosphatase 1 for 10 minutes at room temperature. The released phosphate was visualized with malachite green by absorbance at 620 nm and converted to specific activity (expressed as pmol/minute per microgram) using a phosphate standard curve. To control for the presence of endogenous acceptor substrates in the cell lysates, the specific activity observed in parallel reactions lacking the mannotriose acceptor was subtracted from the total values. Cell viability was assessed by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay following the manufacturer's instructions (Molecular Probes, Eugene, OR). Briefly, cultures were incubated with a 1.2 mmol/L MTT solution at 37°C for 4 hours. The absorbance values of blue formazan were determined at 540 nm. Cell viability was expressed as MTT uptake in treated cells normalized to untreated cells. Cells were lysed in radioimmunoprecipitation assay buffer supplemented with complete EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland). After homogenization with a pellet pestle, the cell extracts were centrifuged at 17,115 × g for 30 minutes at 4°C, and the protein concentration of the supernatants were determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). Proteins were separated by 1% agarose gel electrophoresis, blotted onto nitrocellulose membranes, and blocked with 1% polyvinylpyrrolidone in Tris-buffered saline–Tween overnight at 4°C. Membranes were then incubated with biotin-labeled Datura stramonium lectin (DSL; 20 μg/mL; Vector Laboratories, Burlingame, CA) or Phaseolus vulgaris leucoagglutinin (PHA-L, 5 μg/mL; Vector Laboratories) for 1.5 hours at room temperature. Membranes were developed with the Vectastain ABC kit (Vector Laboratories), and glycoproteins were visualized using chemiluminescence. Densitometry was performed using ImageJ software version 1.46r (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Paraffin-embedded sections (6 μm) of conjunctival tissue were deparaffinized, rehydrated, and blocked for 1 hour with 3% bovine serum albumin in phosphate-buffered saline. Biotin-labeled DSL or PHA-L preparations (20 μg/mL) were applied in the presence of 1.5% bovine serum albumin for 30 minutes at room temperature. Consecutive tissue sections incubated in parallel with 1.5% bovine serum albumin alone were used as negative controls. Endogenous peroxidase activity was subsequently blocked with 1% H2O2. Staining was performed using a horseradish peroxidase–streptavidin complex (Vectastain Elite ABC horseradish peroxidase kit; Vector Laboratories) for 30 minutes. The substrate for the peroxidase was diaminobenzidine with H2O2 (SigmaFast 3,3-diaminobenzidine with metal enhancer; Sigma-Aldrich), which yields a brown deposit. Nuclei were counterstained with hematoxylin QS (Vector Laboratories). Areas of positive staining were quantified using ImageJ software. The epithelial areas were hand-outlined and the intensity of brown pixels in the selected regions was measured using color deconvolution with the H DAB vector. Mean staining-intensity scores were normalized to area, and the intensity values from the corresponding negative controls were subtracted as background. In independent experiments, sections were stained with periodic acid-Schiff reagent for morphologic assessment. Statistical analysis was performed using Prism software version 7 (GraphPad Software, San Diego, CA). The collection of N-glycan structures expressed by any given cell or organism defines key biological process ranging from cell migration and tissue patterning to disease progression and cell death. An important prerequisite to understanding the regulation of N-glycosylation at the transcriptional level is the identification of the repertoire of oligosaccharides present on individual cell types. Matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry was used to examine the different classes of N-glycans present in differentiated human corneal epithelial cells. The most notable features of the data included the presence of complex-type biantennary (Hex3-6HexNAc4-5Fuc0-3NeuAc0-2) and high-mannose (Hex5-9HexNAc2) N-glycans (Table 1). The spectrum profile in the middle and high m/z regions revealed the presence of numerous putative N-acetyllactosamine repeats on the biantennary structures (Figure 1). Oligosaccharides with up to five or six repeating units of N-acetyllactosamine were detected, as indicated by signals at m/z 4316, 4491, 4665, 4678, 4839, 4852, 5013, 5039, and 5187. The majority of the biantennary N-glycans were fucosylated in the core (as Fuc-HexNAc at the reducing terminal) and often capped with N-acetylneuraminic acid residues on their antennae. By comparison, fewer signals consistent with tri-antennary and tetra-antennary glycans were detected in the spectrum profile. Similarly, hybrid-type N-glycans and bisecting GlcNAc structures represented a minor portion of the total N-glycan profile.Table 1Compositions and Relative Intensities of the Major Peaks in the Matrix-Assisted Laser Desorption/Ionization–Time-of-Flight Mass Spectrum of N-Glycans from Human Corneal Epithelial Cellsm/z∗All peaks were observed as [M + Na]+ ions.CompositionRelative intensity, %ObservedTheoretical1579.91579.9Hex5HexNAc249.21620.91620.9Hex4HexNAc37.51784.01784.0Hex6HexNAc290.21836.01836.1Hex3HexNAc4Fuc16.31988.11988.1Hex7HexNAc292.32029.12029.2Hex6HexNAc34.82040.22040.2Hex4HexNAc4Fuc14.32070.22070.2Hex5HexNAc411.32156.22156.2Hex4HexNAc3Fuc1NeuAc14.82192.22192.2Hex8HexNAc2100.02244.32244.3Hex5HexNAc4Fuc125.42396.32396.3Hex9HexNAc263.22418.42418.4Hex5HexNAc4Fuc213.52431.42431.4Hex5HexNAc4NeuAc117.22592.52592.5Hex5HexNAc4Fuc36.12600.52600.5Hex10HexNAc24.82605.52605.5Hex5HexNAc4Fuc1NeuAc132.22693.52693.5Hex6HexNAc5Fuc14.42779.62779.6Hex5HexNAc4Fuc2NeuAc15.22966.72966.7Hex5HexNAc4Fuc1NeuAc28.1∗ All peaks were observed as [M + Na]+ ions. Open table in a new tab The results mentioned in the previous paragraph prompted us to examine the transcriptional levels of known N-glycosylation enzymes in human corneal epithelial cells using a pathway-focused PCR array. Enzymes involved in the biosynthesis of all three major types of N-glycans, which comprise high-mannose, hybrid, and complex-type, were identified (Figure 2A). A direct comparison of the relative levels of expression revealed the presence of enzymes involved in the trimming of glucose (GANAB, PRKCSH) and mannose (MAN1A1, MAN1A2, MAN1B1), which are required for the conversion of oligomannose structures into branched N-glycans. The expression of MGAT1, involved in initiating the synthesis of hybrid and complex-type N-glycans; MAN2A1, required for the synthesis of complex-type N-glycans; as well as transferases carrying out core fucosylation (FUT8) and the poly-N-acetyllactosamine extension (B3GN2, B4GALT1, B4GALT2, B4GALT3), was also observed. The relatively low levels of MGAT3 were consistent with the low amount of bisecting GlcNAc structures found in human corneal epithelial cells. Although some discrepancies were identified between gene expression and glycan content, such as the relative high levels of MGAT4B involved in the synthesis of tri-antennary structures, the transcriptional data on the major part echoed the N-glycosylation profile obtained by mass spectrometry. To assess the effects of proinflammatory conditions on the N-glycosylation pathway, cultures of human corneal epithelial cells were treated with IL-1β and TNFα, two key mediators of the inflammatory response known to exacerbate damage during chronic disease. Figure 2B depicts the changes in the relative expression of genes involved in N-glycan processing as scatterplots using a twofold cutoff (Supplemental Table S1). The effect of IL-1β was limited to the down-regulation of MGAT3 and MAN1C1 at 24 and 48 hours, respectively. MGAT4C, a putative and poorly characterized N-acetylglucosaminyltransferase, was the only up-regulated gene in these experiments. The effect of TNFα, on the other hand, was much greater than that of IL-1β. At 24 hours, TNFα promoted down-regulation of four genes involved in the trimming of glucose and mannose (GANAB, MAN1B1, MAN2A2, MAN1C1), whereas 13 genes were down-regulated at 48 hours and included those necessary for the trimming (GANAB, PRKCHS, MOGS, MAN1B1, MAN2A2, MAN1C1) and elongation (MGAT1, MGAT2, MGAT3, MGAT4B, MGAT5B, B4GALT3) of the N-glycan chains. Observation of the response to IL-1β and TNFα evidenced major differences between the efficacies of both cytokines in regulating the transcription of MGAT genes that create complex-type N-glycan structures (Figure 2C). The MGAT family of genes is involved in encoding enzymes that transfer GlcNAc to glycoproteins transiting the medial Golgi, forming mono-, bi-, tri-, and tetra-antennary branched N-glycans (Figure 3A). In an effort to validate the data obtained by PCR array showing reduced MGAT gene expression after TNFα treatment, qPCR experiments were performed using primers against three of the most highly expressed branching enzymes in corneal epithelial cells, MGAT1, MGAT2, and MGAT4B, which contribute to the formation of mono-, bi-, and tri-antennary structures. The measured levels of expression in these experiments confirmed that TNFα had a significant effect in lowering the number of MGAT transcripts at 48 hours, with little or no effect at 24 hours (Figure 3B). Since TNFα can directly activate a spectrum of genomic targets within the first few hours of treatment,21Tian B. Nowak D.E. Jamaluddin M. Wang S. Brasier A.R. Identification of direct genomic targets downstream of the nuclear factor-kappaB transcription factor mediating tumor necrosis factor signaling.J Biol Chem. 2005; 280: 17435-17448Crossref PubMed Scopus (196) Google Scholar our results suggest an indirect mechanism of control by which the gene network downstream of TNF signaling regulates MGAT expression. To further determine whether a reduction in the number of MGAT transcripts is correlated with impaired enzymatic activity, a glycosyltransferase-activity assay was performed in whole-cell lysates isolated from human corneal epithelial cells. Here, the ability of endogenous N-acetylglucosaminyltransferases to transfer uridine diphosphate–GlcNAc to mannotriose was determined by measuring free phosphate released from uridine diphosphate. As expected, the differences in N-acetylglucosaminyltransferase activity were evident after 48 hours of TNFα stimulation (Figure 3C), when the levels of MGAT transcripts were significantly reduced. In control experiments, treatment with TNFα did not have a significant effect on cell viability (Figure 3D). Next, lectins were used to measure the levels of branched oligosacc
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