Interleukin-22 regulates B3GNT7 expression to induce fucosylation of glycoproteins in intestinal epithelial cells
2021; Elsevier BV; Volume: 298; Issue: 2 Linguagem: Inglês
10.1016/j.jbc.2021.101463
ISSN1083-351X
AutoresDaniela J. Carroll, Mary W. N. Burns, Lynda Mottram, Daniel C. Propheter, Andrew Boucher, Gabrielle M. Lessen, Ashwani Kumar, Stacy A. Malaker, Chao Xing, Lora V. Hooper, Ulf Yrlid, Jennifer J. Kohler,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoInterleukin (IL)-22 is a cytokine that plays a critical role in intestinal epithelial homeostasis. Its downstream functions are mediated through interaction with the heterodimeric IL-22 receptor and subsequent activation of signal transducer and activator of transcription 3 (STAT3). IL-22 signaling can induce transcription of genes necessary for intestinal epithelial cell proliferation, tissue regeneration, tight junction fortification, and antimicrobial production. Recent studies have also implicated IL-22 signaling in the regulation of intestinal epithelial fucosylation in mice. However, whether IL-22 regulates intestinal fucosylation in human intestinal epithelial cells and the molecular mechanisms that govern this process are unknown. Here, in experiments performed in human cell lines and human-derived enteroids, we show that IL-22 signaling regulates expression of the B3GNT7 transcript, which encodes a β1-3-N-acetylglucosaminyltransferase that can participate in the synthesis of poly-N-acetyllactosamine (polyLacNAc) chains. Additionally, we find that IL-22 signaling regulates levels of the α1-3-fucosylated Lewis X (Lex) blood group antigen, and that this glycan epitope is primarily displayed on O-glycosylated intestinal epithelial glycoproteins. Moreover, we show that increased expression of B3GNT7 alone is sufficient to promote increased display of Lex-decorated carbohydrate glycan structures primarily on O-glycosylated intestinal epithelial glycoproteins. Together, these data identify B3GNT7 as an intermediary in IL-22-dependent induction of fucosylation of glycoproteins and uncover a novel role for B3GNT7 in intestinal glycosylation. Interleukin (IL)-22 is a cytokine that plays a critical role in intestinal epithelial homeostasis. Its downstream functions are mediated through interaction with the heterodimeric IL-22 receptor and subsequent activation of signal transducer and activator of transcription 3 (STAT3). IL-22 signaling can induce transcription of genes necessary for intestinal epithelial cell proliferation, tissue regeneration, tight junction fortification, and antimicrobial production. Recent studies have also implicated IL-22 signaling in the regulation of intestinal epithelial fucosylation in mice. However, whether IL-22 regulates intestinal fucosylation in human intestinal epithelial cells and the molecular mechanisms that govern this process are unknown. Here, in experiments performed in human cell lines and human-derived enteroids, we show that IL-22 signaling regulates expression of the B3GNT7 transcript, which encodes a β1-3-N-acetylglucosaminyltransferase that can participate in the synthesis of poly-N-acetyllactosamine (polyLacNAc) chains. Additionally, we find that IL-22 signaling regulates levels of the α1-3-fucosylated Lewis X (Lex) blood group antigen, and that this glycan epitope is primarily displayed on O-glycosylated intestinal epithelial glycoproteins. Moreover, we show that increased expression of B3GNT7 alone is sufficient to promote increased display of Lex-decorated carbohydrate glycan structures primarily on O-glycosylated intestinal epithelial glycoproteins. Together, these data identify B3GNT7 as an intermediary in IL-22-dependent induction of fucosylation of glycoproteins and uncover a novel role for B3GNT7 in intestinal glycosylation. Glycosylation is a ubiquitous posttranslational modification that produces a diverse array of cellular glycans. Glycan assembly is complex, and the process is driven by transcriptional regulation of a portfolio of cellular "glycogenes," the relative abundance of glycoprotein substrates, and availability of nucleotide sugar donor substrates. Glycans are synthesized in a sequential manner, where glycosyltransferases with distinct substrate specificities extend structures by transferring activated sugars to acceptor substrates in an α- or β-linkage, generating a glycan repertoire that is displayed on cell surfaces, secreted proteins, and within certain organelles. Glycans participate in the regulation of diverse biological processes, including proper protein folding and secretion, cellular adhesion and signaling, and immune cell trafficking (1Ohtsubo K. Marth J.D. Glycosylation in cellular mechanisms of health and disease.Cell. 2006; 126: 855-867Google Scholar, 2Moremen K.W. Tiemeyer M. Nairn A.V. Vertebrate protein glycosylation: Diversity, synthesis and function.Nat. Rev. Mol. Cell Biol. 2012; 13: 448-462Google Scholar, 3Neelamegham S. Mahal L.K. Multi-level regulation of cellular glycosylation: From genes to transcript to enzyme to structure.Curr. Opin. Struct. Biol. 2016; 40: 145-152Google Scholar). In the intestine, mucosal glycans are typically found attached to membrane-bound or secreted mucin-like glycoproteins through O-glycosidic linkages (O-glycosylation). GalNAc-type O-glycosylation begins in the Golgi apparatus with the transfer of N-acetylgalactosamine (GalNAc) to the hydroxyl group of a serine (Ser) or threonine (Thr) residue. This core structure can be elaborated into linear or branched structures. A common elaboration is the addition of poly-N-acetyllactosamine (polyLacNAc). polyLacNAc is a repeating copolymer of galactose (Gal) and N-acetylglucosamine (GlcNAc) produced by the concerted action of galactosyltransferases and GlcNAc-transferases. These extended O-glycans can be further decorated with sialic acid, fucose, sulfate groups, or ABO- or Lewis-type histo-blood group antigens, generating a wide diversity of possible structures (4Brockhausen I. Stanley P. O-GalNAc glycans.in: Varki A. Cummings R.D. Esko J.D. Stanley P. Hart G.W. Aebi M. Darvill A.G. Kinoshita T. Packer N.H. Prestegard J.H. Schnaar R.L. Seeberger P.H. Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2015: 113-123Google Scholar). For example, 6-sulfation of GlcNAc residues results in keratan sulfate II (KS II) structures (5Caterson B. Melrose J. Keratan sulfate, a complex glycosaminoglycan with unique functional capability.Glycobiology. 2018; 28: 182-206Google Scholar). Notably, mucosal O-glycans decorated with fucose have been identified as key regulators of both health and disease in the gut (6Goto Y. Uematsu S. Kiyono H. Epithelial glycosylation in gut homeostasis and inflammation.Nat. Immunol. 2016; 17: 1244-1251Google Scholar, 7Pickard J.M. Chervonsky A.V. Intestinal fucose as a mediator of host-microbe symbiosis.J. Immunol. 2015; 194: 5588-5593Google Scholar). L-fucose is a monosaccharide found in multiple classes of cell surface glycans. Incorporation of fucose into glycans is catalyzed by fucosyltransferases (FUTs). Thirteen human FUTs have been identified. FUTs catalyze the transfer of fucose from the guanosine diphosphate (GDP)—fucose donor to acceptor substrates in an α1-2-, α1-3-, α1-4- or α1-6-linkage, or α-linked to a serine or threonine side chain. Among these FUTs, ten are known to be involved in terminal fucosylation of glycan structures by decorating them with α1-2- (FUT1 and FUT2) or α1-3/4-linked fucose (FUT3–7 and FUT9–11) (8Becker D.J. Lowe J.B. Fucose: Biosynthesis and biological function in mammals.Glycobiology. 2003; 13: 41R-53RGoogle Scholar, 9Schneider M. Al-Shareffi E. Haltiwanger R.S. Biological functions of fucose in mammals.Glycobiology. 2017; 27: 601-618Google Scholar). Fucosylation is abundant in the mammalian gut and α1-2-fucosylation—primarily produced by Fut2—has emerged as a key regulator of commensal bacterial colonization and maintenance of bacterial symbiosis (10Pickard J.M. Maurice C.F. Kinnebrew M.A. Abt M.C. Schenten D. Golovkina T.V. Bogatyrev S.R. Ismagilov R.F. Pamer E.G. Turnbaugh P.J. Chervonsky A.V. Rapid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness.Nature. 2014; 514: 638-641Google Scholar). Recent studies have explored the molecular mechanisms that control this process and implicate the interleukin (IL)-10 family member, IL-22, as the primary regulator of intestinal epithelial fucosylation in mice (10Pickard J.M. Maurice C.F. Kinnebrew M.A. Abt M.C. Schenten D. Golovkina T.V. Bogatyrev S.R. Ismagilov R.F. Pamer E.G. Turnbaugh P.J. Chervonsky A.V. Rapid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness.Nature. 2014; 514: 638-641Google Scholar, 11Goto Y. Obata T. Kunisawa J. Sato S. Ivanov I.I. Lamichhane A. Takeyama N. Kamioka M. Sakamoto M. Matsuki T. Setoyama H. Imaoka A. Uematsu S. Akira S. Domino S.E. et al.Innate lymphoid cells regulate intestinal epithelial cell glycosylation.Science. 2014; 345: 1254009Google Scholar, 12Pham T.A. Clare S. Goulding D. Arasteh J.M. Stares M.D. Browne H.P. Keane J.A. Page A.J. Kumasaka N. Kane L. Mottram L. Harcourt K. Hale C. Arends M.J. Gaffney D.J. et al.Epithelial IL-22RA1-mediated fucosylation promotes intestinal colonization resistance to an opportunistic pathogen.Cell Host Microbe. 2014; 16: 504-516Google Scholar). In the intestine, IL-22 is produced by type 3 innate lymphoid cells (ILC3s) (13Spits H. Di Santo J.P. The expanding family of innate lymphoid cells: Regulators and effectors of immunity and tissue remodeling.Nat. Immunol. 2011; 12: 21-27Google Scholar). The downstream function of IL-22 is mediated through ligation of its heterodimeric receptor comprised of IL-22 receptor subunit alpha 1 (IL-22Rα1) and IL-10R2 (14Xie M.H. Aggarwal S. Ho W.H. Foster J. Zhang Z. Stinson J. Wood W.I. Goddard A.D. Gurney A.L. Interleukin (IL)-22, a novel human cytokine that signals through the interferon receptor-related proteins CRF2-4 and IL-22R.J. Biol. Chem. 2000; 275: 31335-31339Google Scholar, 15Kotenko S.V. Izotova L.S. Mirochnitchenko O.V. Esterova E. Dickensheets H. Donnelly R.P. Pestka S. Identification of the functional interleukin-22 (IL-22) receptor complex: The IL-10R2 chain (IL-10Rbeta ) is a common chain of both the IL-10 and IL-22 (IL-10-related T cell-derived inducible factor, IL-TIF) receptor complexes.J. Biol. Chem. 2001; 276: 2725-2732Google Scholar), which signals through activation of signal transducer and activator of transcription 3 (STAT3) (16Lejeune D. Dumoutier L. Constantinescu S. Kruijer W. Schuringa J.J. Renauld J.C. Interleukin-22 (IL-22) activates the JAK/STAT, ERK, JNK, and p38 MAP kinase pathways in a rat hepatoma cell line. Pathways that are shared with and distinct from IL-10.J. Biol. Chem. 2002; 277: 33676-33682Google Scholar). IL-22 signaling plays a critical role in maintenance of the intestinal epithelial barrier by inducing genes necessary for intestinal epithelial cell proliferation, tissue regeneration, tight junction fortification, and induction of intestinal epithelial fucosylation. Despite the growing body of evidence that demonstrates the involvement of IL-22 in mouse intestinal fucosylation, whether IL-22 promotes intestinal fucosylation in humans and the glycosyltransferases involved remain to be explored. Herein, we report that IL-22 signaling in human intestinal epithelial cells modulates expression of the B3GNT7 transcript and induces α1-3-fucosylation of glycoproteins, including those displaying mucosal O-linked glycans. We also show that overexpression of B3GNT7 is sufficient to cause increased fucosylation of O-linked glycans, thus identifying an unexpected mechanism by which intestinal fucosylation can be regulated. To interrogate the effect of IL-22 signaling on glycosyltransferase gene expression in human intestinal epithelial cells, we used differentiated Caco-2 BBe1 cells, a subclone of the Caco-2 human colorectal adenocarcinoma cell line that displays small-intestine-like morphology and biochemical properties (17Peterson M.D. Mooseker M.S. Characterization of the enterocyte-like brush border cytoskeleton of the C2BBe clones of the human intestinal cell line, Caco-2.J. Cell Sci. 1992; 102: 581-600Google Scholar, 18Devriese S. Van den Bossche L. Van Welden S. Holvoet T. Pinheiro I. Hindryckx P. De Vos M. Laukens D. T84 monolayers are superior to Caco-2 as a model system of colonocytes.Histochem. Cell Biol. 2017; 148: 85-93Google Scholar). Differentiated Caco-2 BBe1 cells were exposed to 10 ng/ml of recombinant human (rh) IL-22 for 4 h, and their global transcriptome was evaluated by RNA sequencing. In this model system, IL-22 significantly modulated the expression of 379 genes, including recognized IL-22-responsive transcripts (TIFA, SOCS3, DUOX2, STAT3, etc.) (Table S1). Consistent with mouse epithelial expression data (10Pickard J.M. Maurice C.F. Kinnebrew M.A. Abt M.C. Schenten D. Golovkina T.V. Bogatyrev S.R. Ismagilov R.F. Pamer E.G. Turnbaugh P.J. Chervonsky A.V. Rapid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness.Nature. 2014; 514: 638-641Google Scholar, 11Goto Y. Obata T. Kunisawa J. Sato S. Ivanov I.I. Lamichhane A. Takeyama N. Kamioka M. Sakamoto M. Matsuki T. Setoyama H. Imaoka A. Uematsu S. Akira S. Domino S.E. et al.Innate lymphoid cells regulate intestinal epithelial cell glycosylation.Science. 2014; 345: 1254009Google Scholar, 12Pham T.A. Clare S. Goulding D. Arasteh J.M. Stares M.D. Browne H.P. Keane J.A. Page A.J. Kumasaka N. Kane L. Mottram L. Harcourt K. Hale C. Arends M.J. Gaffney D.J. et al.Epithelial IL-22RA1-mediated fucosylation promotes intestinal colonization resistance to an opportunistic pathogen.Cell Host Microbe. 2014; 16: 504-516Google Scholar), in vitro stimulation of human cells with IL-22 led to the induction of FUT2. Additionally, IL-22 induced significant changes in expression of eight additional genes encoding glycosyltransferases or their direct regulators (Fig. 1A). Among the glycosyltransferase genes regulated by IL-22, induction of B3GNT7 showed the highest significance. To investigate the pathway by which IL-22 induces B3GNT7 expression, we first corroborated the RNA-sequencing data by performing quantitative real-time PCR (RT-qPCR) analysis. We found that B3GNT7 gene expression is upregulated by IL-22 in differentiated Caco-2 BBe1 cells (approximately fivefold increase; Fig. 1B). Increased B3GNT7 expression was observed as soon as 2 h after IL-22 treatment (Fig. S1A), and no further increase in B3GNT7 expression was achieved when the IL-22 concentration was increased to 100 ng/ml (Fig. S1B). To determine whether B3GNT7 upregulation was dependent on activation of downstream IL-22 signaling, we first incubated differentiated Caco-2 BBe1 cells with an IL22Rα1 blocking antibody or isotype control. As shown in Figure 1B, IL-22-induced upregulation of B3GNT7 gene expression was significantly inhibited (approximately 78% inhibition) in the presence of the IL22Rα1 blocking antibody, but not the isotype control. To evaluate the generality of these observations, we also examined T84 cells, which are derived from a lung metastasis of a colon carcinoma (19Dharmsathaphorn K. McRoberts J.A. Mandel K.G. Tisdale L.D. Masui H. A human colonic tumor cell line that maintains vectorial electrolyte transport.Am. J. Physiol. 1984; 246: G204-G208Google Scholar, 20Reid L.M. Holland J. Jones C. Wolf B. Niwayama G. Williams R. Kaplan N.O. Sato G. Some of the variables affecting the success of transplantation of human tumors into the athymic nude mouse.in: Houchens D.P. Ovejera A.A. Proceedings of the Symposium on the Use of Athymic (Nude) Mice in Cancer Research. Gustav Fischer Verlag, New York, NY1978: 107-121Google Scholar). IL-22-dependent induction of B3GNT7 expression was also observed in polarized T84 cells (appoximately 19-fold increase; Fig. S2). Polarized T84 cells were treated with an IL22Rα1 blocking antibody or isotype control, followed by IL-22 treatment. B3GNT7 gene expression was increased by IL-22 treatment but significantly inhibited (approximately 67% inhibition) in the presence of the blocking antibody, but not the isotype control (Fig. S2). Next, to determine whether B3GNT7 gene expression was dependent on STAT3 activation, we incubated differentiated Caco-2 BBe1 cells with 0.5 μM niclosamide, a pharmacological inhibitor of STAT3 signaling (21Ren X. Duan L. He Q. Zhang Z. Zhou Y. Wu D. Pan J. Pei D. Ding K. Identification of niclosamide as a new small-molecule inhibitor of the STAT3 signaling pathway.ACS Med. Chem. Lett. 2010; 1: 454-459Google Scholar), or vehicle control. We observed that the IL-22-dependent increase in B3GNT7 gene expression was also inhibited in the presence of niclosamide (approximately 57% inhibition) (Fig. 1B). The moderate reduction in B3GNT7 transcript expression was commensurate with the partial inhibition of STAT3 phosphorylation we observed for this concentration of niclosamide; unfortunately, higher niclosamide concentrations could not be examined due to toxicity. To investigate whether IL-22 signaling also regulates B3GNT7 expression in nontransformed cells, we used human enteroids, which are derived from isolated small intestinal crypts. These enteroids can be passaged indefinitely while maintaining genetic and physiological features of the individual from which they were derived (22Zachos N.C. Kovbasnjuk O. Foulke-Abel J. In J. Blutt S.E. de Jonge H.R. Estes M.K. Donowitz M. Human enteroids/colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology.J. Biol. Chem. 2016; 291: 3759-3766Google Scholar). We observed that IL-22 treatment resulted in increased B3GNT7 expression in a cultured human enteroid line (approximately 2.5-fold increase; Fig. 2A). However, this increase was not observed when the IL22Rα1 blocking antibody was included. To test the generality of this observation, we repeated the experiment using enteroid lines derived from three different individuals, one of which was the line used in Figure 2A. While there was some variability among the lines, the combined data show a statistically significant increase in B3GNT7 expression in response to IL-22 (Fig. 2B). Previously, it was observed that IL-22 regulates expression of b3gnt7 in mouse colonic organoids, and this expression was IL22Rα1-dependent (12Pham T.A. Clare S. Goulding D. Arasteh J.M. Stares M.D. Browne H.P. Keane J.A. Page A.J. Kumasaka N. Kane L. Mottram L. Harcourt K. Hale C. Arends M.J. Gaffney D.J. et al.Epithelial IL-22RA1-mediated fucosylation promotes intestinal colonization resistance to an opportunistic pathogen.Cell Host Microbe. 2014; 16: 504-516Google Scholar). Using intestinal epithelial tissue from mice lacking Stat3 expression specifically in intestinal epithelial cells (Stat3ΔIEC) (23Wang Y. Kuang Z. Yu X. Ruhn K.A. Kubo M. Hooper L.V. The intestinal microbiota regulates body composition through NFIL3 and the circadian clock.Science. 2017; 357: 912-916Google Scholar), we found that expression of b3gnt7 was significantly impaired in the ileum, but not the colon, of these mice as compared with wild-type (WT) mice (Fig. 3). Together, these results demonstrate that regulation of B3GNT7 gene expression appears to be conserved in humans and mice and via the IL-22-IL-22Rα1-STAT3 pathway. We also attempted to evaluate changes in B3GNT7 expression at the protein level but unfortunately were unable to detect endogenous B3GNT7 using any currently available antibodies. In mice, IL-22 initiates a rapid and substantial increase in intestinal epithelial fucosylation (10Pickard J.M. Maurice C.F. Kinnebrew M.A. Abt M.C. Schenten D. Golovkina T.V. Bogatyrev S.R. Ismagilov R.F. Pamer E.G. Turnbaugh P.J. Chervonsky A.V. Rapid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness.Nature. 2014; 514: 638-641Google Scholar, 11Goto Y. Obata T. Kunisawa J. Sato S. Ivanov I.I. Lamichhane A. Takeyama N. Kamioka M. Sakamoto M. Matsuki T. Setoyama H. Imaoka A. Uematsu S. Akira S. Domino S.E. et al.Innate lymphoid cells regulate intestinal epithelial cell glycosylation.Science. 2014; 345: 1254009Google Scholar, 12Pham T.A. Clare S. Goulding D. Arasteh J.M. Stares M.D. Browne H.P. Keane J.A. Page A.J. Kumasaka N. Kane L. Mottram L. Harcourt K. Hale C. Arends M.J. Gaffney D.J. et al.Epithelial IL-22RA1-mediated fucosylation promotes intestinal colonization resistance to an opportunistic pathogen.Cell Host Microbe. 2014; 16: 504-516Google Scholar). However, whether IL-22 also promotes fucosylation in human intestinal epithelial cells has not yet been explored. Therefore, to assess the effect of IL-22 on overall fucosylation, we used fucose-recognizing lectins in lectin blots to probe the fucosylation status of glycoproteins from cell lysates. As shown in Fig. S3A, incubation of differentiated Caco-2 BBe1 cells with rhIL-22 for 48 h led to enhanced binding of Lotus tetragonolobus lectin (LTL), a lectin that specifically recognizes α1-3-linked fucose, but had no detectable effect on binding of Ulex europaeus agglutinin I (UEA I), a lectin that specifically recognizes α1-2-linked fucose. These findings were unexpected as previously published data showed that IL-22 enhances Fut2 gene expression and subsequently induces small intestinal α1-2-fucosylation in mice in an IL22Rα1-dependent manner (11Goto Y. Obata T. Kunisawa J. Sato S. Ivanov I.I. Lamichhane A. Takeyama N. Kamioka M. Sakamoto M. Matsuki T. Setoyama H. Imaoka A. Uematsu S. Akira S. Domino S.E. et al.Innate lymphoid cells regulate intestinal epithelial cell glycosylation.Science. 2014; 345: 1254009Google Scholar). Thus, we next validated our in vitro findings using epithelial tissue from Stat3ΔIEC mice. First, consistent with published data, we found that Fut2 gene expression was significantly reduced in the ileum of Stat3ΔIEC mice (Fig. S3B), suggesting that Fut2 gene expression in mice is also regulated via the IL-22-IL-22Rα1-STAT3 pathway. We also compared fucosylation patterns in WT versus Stat3ΔIEC mice using UEA I and LTL to probe ileal tissue sections. As shown in Fig. S3C, both α1-2-fucosylation (examined using UEA I binding) and α1-3-fucosylation (examined using LTL binding) are present in the mouse small intestine, and Stat3 deletion resulted in reduced binding of both lectins. Inclusion of 100 mM l-fucose in the binding buffer eliminated lectin staining, demonstrating the specificity of binding (Fig. S3D). Together, these results corroborate previous findings and demonstrate for the first time the ability for IL-22 signaling to regulate α1-3-fucosylation in both mouse and human intestinal epithelial cells. Although LTL exhibits specificity for α1-3-fucose, it does not distinguish among multiple complex glycan epitopes that include α1-3-fucose (24Manimala J.C. Roach T.A. Li Z. Gildersleeve J.C. High-throughput carbohydrate microarray analysis of 24 lectins.Angew. Chem. Int. Ed. Engl. 2006; 45: 3607-3610Google Scholar). Lewis antigens are among the most commonly expressed α1-3-fucosylated carbohydrate epitopes on cell surfaces (25Stanley P. Cummings R.D. Structures common to different glycans.in: Varki A. Cummings R.D. Esko J.D. Stanley P. Hart G.W. Aebi M. Darvill A.G. Kinoshita T. Packer N.H. Prestegard J.H. Schnaar R.L. Seeberger P.H. Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2015: 161-178Google Scholar). Therefore, we next examined the effects of IL-22 on Lewis antigen expression in lysates from differentiated Caco-2 BBe1 cells. As shown in Figure 4A, exposure of differentiated Caco-2 BBe1 cells to rhIL-22 led to enhanced expression of the Lewis X (Lex) antigen. The increased Lex antibody binding was inhibited in the presence of 2-fluoro-peracetyl-fucose (2F-Fuc), a metabolic inhibitor of fucosylation (26Rillahan C.D. Antonopoulos A. Lefort C.T. Sonon R. Azadi P. Ley K. Dell A. Haslam S.M. Paulson J.C. Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome.Nat. Chem. Biol. 2012; 8: 661-668Google Scholar), demonstrating that increased antibody binding depends on fucosylated structures. To determine whether Lex antigen expression was also dependent on IL-22 signaling, we also cultured differentiated Caco-2 BBe1 in the presence or absence of the IL22Rα1 blocking antibody or niclosamide. We observed that the IL-22-dependent increase in Lex antigen expression was impaired following inhibition of IL-22 signaling (Fig. 4, B and C), suggesting that Lex antigen expression, like B3GNT7 expression, was also regulated via the IL-22-IL22Rα1-STAT3 pathway. To assess the effects of IL-22 on the fucosylation of nontransformed cells, we performed a limited analysis of human-derived enteroids. Enteroids derived from three individuals were treated with rhIL-22, and changes in cell surface fucosylation were examined by flow cytometry (Fig. S4). IL-22 treatment of enteroids from all three individuals resulted in increased binding of the Aleuria aurantia lectin (AAL), which recognizes fucose in multiple linkages (27Matsumura K. Higashida K. Ishida H. Hata Y. Yamamoto K. Shigeta M. Mizuno-Horikawa Y. Wang X. Miyoshi E. Gu J. Taniguchi N. Carbohydrate binding specificity of a fucose-specific lectin from Aspergillus oryzae: A novel probe for core fucose.J. Biol. Chem. 2007; 282: 15700-15708Google Scholar), and of the Lex-recognizing antibody. IL-22 did not result in increased UEA I binding to the enteroids. While the results were similar to those observed in differentiated Caco-2 BBe1 cells, sample availability was limited, and statistical analysis could not be performed. Additionally, variations in glycosylation among enteroid lines pose challenges in their analysis; therefore, subsequent experiments were performed solely in differentiated Caco-2 BBe1 cells. Fucose in the α1-3-linkage can be displayed on either N- and O-linked glycans attached to proteins (28Li J. Hsu H.C. Mountz J.D. Allen J.G. Unmasking fucosylation: From cell adhesion to immune system regulation and diseases.Cell Chem. Biol. 2018; 25: 499-512Google Scholar). To assess whether the IL-22-induced Lex antigen was present on N- or O-linked glycans, differentiated Caco-2 BBe1 cells were first incubated with IL-22. Next, PNGase F (29Norris G.E. Stillman T.J. Anderson B.F. Baker E.N. The three-dimensional structure of PNGase F, a glycosylasparaginase from Flavobacterium meningosepticum.Structure. 1994; 2: 1049-1059Google Scholar) was used to release N-linked glycans in cell lysates, while StcE (30Malaker S.A. Pedram K. Ferracane M.J. Bensing B.A. Krishnan V. Pett C. Yu J. Woods E.C. Kramer J.R. Westerlind U. Dorigo O. Bertozzi C.R. The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 7278-7287Google Scholar) was used on live cells to cleave peptides heavily modified with GalNAc-type O-glycans. We confirmed the effectiveness of the PNGase F treatment by monitoring the change in apparent molecular weight of LAMP1, a 40 kDa polypeptide that is normally modified with up to 18 N-linked glycans (Fig. S5). While PNGase F treatment reduced Lex levels detected by immunoblot, this reduction was modest and not significant (Fig. 5, A and B). In contrast, StcE treatment led to a robust and statistically significant reduction in Lex antigen levels (Fig. 5, C and D). Together, these results, combined with the observation that IL-22 induces expression of known O-glycosylated glycoproteins (e.g., DMBT1, MUC13, ICAM1, etc.) (Table S1), implicate the involvement of IL-22 in the transcriptional regulation and downstream fucosylation of heavily O-glycosylated glycoproteins in human intestinal epithelial cells. Because PNGase F may not have equal accessibility to all N-linked glycans, our results do not exclude the possibility that IL-22 also induces α1-3 fucosylation of N-glycoproteins. We next considered mechanisms by which IL-22 might induce increased α1-3-fucosylation. While the previously described IL-22-induced increase in α1-2-fucosylation in mice was attributed to increased Fut2 expression (11Goto Y. Obata T. Kunisawa J. Sato S. Ivanov I.I. Lamichhane A. Takeyama N. Kamioka M. Sakamoto M. Matsuki T. Setoyama H. Imaoka A. Uematsu S. Akira S. Domino S.E. et al.Innate lymphoid cells regulate intestinal epithelial cell glycosylation.Science. 2014; 345: 1254009Google Scholar), we did not detect IL-22-dependent changes in expression of FUTs capable of adding α1-3-fucose. Specifically, RT-qPCR analysis of FUT4 and FUT9, genes encoding FUTs primarily responsible for Lex synthesis (31Cailleau-Thomas A. Coullin P. Candelier J.J. Balanzino L. Mennesson B. Oriol R. Mollicone R. FUT4 and FUT9 genes are expressed early in human embryogenesis.Glycobiology. 2000; 10: 789-802Google Scholar), revealed no IL-22-dependent change in gene expression (Fig. S6). Additionally, inspection of RNA-sequencing data and subsequent validation using RT-qPCR revealed modest or no IL-22 dependent changes in expression of other FUT-encoding genes nor of genes encoding enzymes involved in GDP-fucose synthesis (Table S2). Thus, we considered other possible mechanisms to account for the increase in α1-3-fucosylation. Specifically, we hypothesized that the observed increase in α1-3-fucosylation could be due to increased production of glycan structures that are substrates for α1-3-fucosylation. B3GNT7, the glycosyltransferase gene that was most significantly upregulated by IL-22 treatment, encodes an N-acetylglucosaminyltransferase involved in the biosynthesis of polyLacNAc repeats of keratan sulfate (32Seko A. Yamashita K. beta1,3-N-Acetylglucosaminyltransferase-7 (beta3Gn-T7) acts efficiently on keratan sulfate-related glycans.FEBS Lett. 2004; 556: 216-220Google Scholar, 33Kitayama K. Hayashida Y. Nishida K. Akama T.O. Enzymes responsible for synthesis of corneal keratan sulfate glycosaminoglycans.J. Biol. Chem. 2007; 282: 30085-30096Google Scholar), which could potentially be fucosylated to produce the Lex antigen. Indeed, we observed that Caco-2 BBe1 cells treated with IL-22 showed increased staining with Lycopersicon esculentum lectin (LEL), a lectin that recognizes GlcNAc residues in both chitin and polyLacNAc chains (F
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