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Functional Contributions of N- and O-Glycans to L-Selectin Ligands in Murine and Human Lymphoid Organs

2011; Elsevier BV; Volume: 178; Issue: 1 Linguagem: Inglês

10.1016/j.ajpath.2010.11.009

ISSN

1525-2191

Autores

Hanayo Arata-Kawai, Mark S. Singer, Annette Bistrup, Annemieke van Zante, Yangqing Wang, Yuki Ito, Xingfeng Bao, Stefan Hemmerich, Minoru Fukuda, Steven D. Rosen,

Tópico(s)

Proteoglycans and glycosaminoglycans research

Resumo

L-selectin initiates lymphocyte interactions with high endothelial venules (HEVs) of lymphoid organs through binding to ligands with specific glycosylation modifications. 6-Sulfo sLex, a sulfated carbohydrate determinant for L-selectin, is carried on core 2 and extended core 1 O-glycans of HEV-expressed glycoproteins. The MECA-79 monoclonal antibody recognizes sulfated extended core 1 O-glycans and partially blocks lymphocyte-HEV interactions in lymphoid organs. Recent evidence has identified the contribution of 6-sulfo sLex carried on N-glycans to lymphocyte homing in mice. Here, we characterize CL40, a novel IgG monoclonal antibody. CL40 equaled or surpassed MECA-79 as a histochemical staining reagent for HEVs and HEV-like vessels in mouse and human. Using synthetic carbohydrates, we found that CL40 bound to 6-sulfo sLex structures, on both core 2 and extended core 1 structures, with an absolute dependency on 6-O-sulfation. Using transfected CHO cells and gene-targeted mice, we observed that CL40 bound its epitope on both N-glycans and O-glycans. Consistent with its broader glycan-binding, CL40 was superior to MECA-79 in blocking lymphocyte-HEV interactions in both wild-type mice and mice deficient in forming O-glycans. This superiority was more marked in human, as CL40 completely blocked lymphocyte binding to tonsillar HEVs, whereas MECA-79 inhibited only 60%. These findings extend the evidence for the importance of N-glycans in lymphocyte homing in mouse and indicate that this dependency also applies to human lymphoid organs. L-selectin initiates lymphocyte interactions with high endothelial venules (HEVs) of lymphoid organs through binding to ligands with specific glycosylation modifications. 6-Sulfo sLex, a sulfated carbohydrate determinant for L-selectin, is carried on core 2 and extended core 1 O-glycans of HEV-expressed glycoproteins. The MECA-79 monoclonal antibody recognizes sulfated extended core 1 O-glycans and partially blocks lymphocyte-HEV interactions in lymphoid organs. Recent evidence has identified the contribution of 6-sulfo sLex carried on N-glycans to lymphocyte homing in mice. Here, we characterize CL40, a novel IgG monoclonal antibody. CL40 equaled or surpassed MECA-79 as a histochemical staining reagent for HEVs and HEV-like vessels in mouse and human. Using synthetic carbohydrates, we found that CL40 bound to 6-sulfo sLex structures, on both core 2 and extended core 1 structures, with an absolute dependency on 6-O-sulfation. Using transfected CHO cells and gene-targeted mice, we observed that CL40 bound its epitope on both N-glycans and O-glycans. Consistent with its broader glycan-binding, CL40 was superior to MECA-79 in blocking lymphocyte-HEV interactions in both wild-type mice and mice deficient in forming O-glycans. This superiority was more marked in human, as CL40 completely blocked lymphocyte binding to tonsillar HEVs, whereas MECA-79 inhibited only 60%. These findings extend the evidence for the importance of N-glycans in lymphocyte homing in mouse and indicate that this dependency also applies to human lymphoid organs. High endothelial venules (HEVs) in secondary lymphoid organs, such as lymph nodes, Peyer's patches, and tonsils, are essential for immune surveillance by supporting the recruitment, or homing, of lymphocytes from the blood.1Butcher E.C. Picker L.J. Lymphocyte homing and homeostasis.Science. 1996; 272: 60-66Crossref PubMed Scopus (2513) Google Scholar, 2Vestweber D. Blanks J.E. Mechanisms that regulate the function of the selectins and their ligands.Physiol Rev. 1999; 79 ([Erratum appears in Physiol Rev 2000, 80: follow i]): 181-213Crossref PubMed Scopus (825) Google Scholar, 3von Andrian U.H. Mempel T.R. Homing and cellular traffic in lymph nodes.Nat Rev Immunol. 2003; 3: 867-878Crossref PubMed Scopus (995) Google Scholar, 4Miyasaka M. Tanaka T. Lymphocyte trafficking across high endothelial venules: dogmas and enigmas.Nat Rev Immunol. 2004; 4: 360-370Crossref PubMed Scopus (356) Google Scholar, 5Rosen S.D. Ligands for L-selectin: homing, inflammation, and beyond.Annu Rev Immunol. 2004; 22: 129-156Crossref PubMed Scopus (634) Google Scholar Homing consists of multiple steps: tethering and rolling of lymphocytes on HEVs, chemokine-mediated activation of lymphocyte integrins, firm arrest of the lymphocytes, and transendothelial migration.1Butcher E.C. Picker L.J. Lymphocyte homing and homeostasis.Science. 1996; 272: 60-66Crossref PubMed Scopus (2513) Google Scholar, 6Ley K. Laudanna C. Cybulsky M.I. Nourshargh S. Getting to the site of inflammation: the leukocyte adhesion cascade updated.Nat Rev Immunol. 2007; 7: 678-689Crossref PubMed Scopus (3088) Google Scholar L-selectin is a C-type lectin present on the cell surface of lymphocytes.1Butcher E.C. Picker L.J. Lymphocyte homing and homeostasis.Science. 1996; 272: 60-66Crossref PubMed Scopus (2513) Google Scholar, 2Vestweber D. Blanks J.E. Mechanisms that regulate the function of the selectins and their ligands.Physiol Rev. 1999; 79 ([Erratum appears in Physiol Rev 2000, 80: follow i]): 181-213Crossref PubMed Scopus (825) Google Scholar, 6Ley K. Laudanna C. Cybulsky M.I. Nourshargh S. Getting to the site of inflammation: the leukocyte adhesion cascade updated.Nat Rev Immunol. 2007; 7: 678-689Crossref PubMed Scopus (3088) Google Scholar Through its interactions with specific carbohydrate-based ligands expressed on HEVs,5Rosen S.D. Ligands for L-selectin: homing, inflammation, and beyond.Annu Rev Immunol. 2004; 22: 129-156Crossref PubMed Scopus (634) Google Scholar L-selectin is essential for the tethering and rolling steps. The known HEV ligands are glycoproteins, all of which have mucin segments with a characteristic high density of O-linked glycans. As identified in mice and/or humans, these ligands include CD34, podocalyxin, endomucin, MAdCAM-1, and nepmucin.4Miyasaka M. Tanaka T. Lymphocyte trafficking across high endothelial venules: dogmas and enigmas.Nat Rev Immunol. 2004; 4: 360-370Crossref PubMed Scopus (356) Google Scholar, 5Rosen S.D. Ligands for L-selectin: homing, inflammation, and beyond.Annu Rev Immunol. 2004; 22: 129-156Crossref PubMed Scopus (634) Google Scholar, 7Uchimura K. Rosen S.D. Sulfated L-selectin ligands as a therapeutic target in chronic inflammation.Trends Immunol. 2006; 27: 559-565Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar These ligands are also defined by a monoclonal antibody (mAb), MECA-79, and the complex of reactive glycoproteins is given the name PNAd (for peripheral lymph node addressin).8Streeter P.R. Rouse B.T.N. Butcher E.C. Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes.J Cell Biol. 1988; 107: 1853-1862Crossref PubMed Scopus (537) Google Scholar, 9Berg E.L. Robinson M.K. Warnock R.A. Butcher E.C. The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor.J Cell Biol. 1991; 114: 343-349Crossref PubMed Scopus (269) Google Scholar MECA-79 stains HEVs of human, mouse, and other species; it is function-blocking, in that it inhibits in vitro adherence of lymphocytes to HEVs in lymphoid organ sections, short-term homing of lymphocytes to lymph nodes in mice, and rolling of lymphocytes along HEVs in murine lymph nodes.8Streeter P.R. Rouse B.T.N. Butcher E.C. Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes.J Cell Biol. 1988; 107: 1853-1862Crossref PubMed Scopus (537) Google Scholar, 9Berg E.L. Robinson M.K. Warnock R.A. Butcher E.C. The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor.J Cell Biol. 1991; 114: 343-349Crossref PubMed Scopus (269) Google Scholar, 10von Andrian U.H. Intravital microscopy of the peripheral lymph node microcirculation in mice.Microcirculation. 1996; 3: 287-300Crossref PubMed Scopus (175) Google Scholar The minimal L-selectin recognition determinant found on PNAd components is 6-sulfo sialyl Lewis X (6-sulfo sLex),11Hemmerich S. Leffler H. Rosen S.D. Structure of the O-glycans in GlyCAM-1, an endothelial-derived ligand for L-selectin.J Biol Chem. 1995; 270: 12035-12047Crossref PubMed Scopus (275) Google Scholar, 12Mitsuoka C. Sawada-Kasugai M. Ando-Furui K. Izawa M. Nakanishi H. Nakamura S. Ishida H. Kiso M. Kannagi R. Identification of a major carbohydrate capping group of the L-selectin ligand on high endothelial venules in human lymph nodes as 6-sulfo sialyl Lewis X.J Biol Chem. 1998; 273: 11225-11233Crossref PubMed Scopus (230) Google Scholar comprised of sialyl Lewis X, modified with a sulfate ester on the C-6 position of GlcNAc (Figure 1). Ligand O-glycans can present this structure on the terminus of either a core 2 branch or an extended core 1 branch, or on both branches (Figure 1).11Hemmerich S. Leffler H. Rosen S.D. Structure of the O-glycans in GlyCAM-1, an endothelial-derived ligand for L-selectin.J Biol Chem. 1995; 270: 12035-12047Crossref PubMed Scopus (275) Google Scholar, 13Yeh J.C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Novel sulfated lymphocyte homing receptors and their control by a Core1 extension beta 1,3-N-acetylglucosaminyltransferase.Cell. 2001; 105: 957-969Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 14Hiraoka N. Kawashima H. Petryniak B. Nakayama J. Mitoma J. Marth J.D. Lowe J.B. Fukuda M. Core 2 branching beta1,6-N-acetylglucosaminyltransferase and high endothelial venule-restricted sulfotransferase collaboratively control lymphocyte homing.J Biol Chem. 2004; 279: 3058-3067Crossref PubMed Scopus (72) Google Scholar, 15Mir G.H. Helin J. Skarp K.-P. Cummings R.D. Makitie A. Renkonen R. Leppanen A. Glycoforms of human endothelial CD34 that bind L-selectin carry sulfated sialyl Lewis x capped O- and N-glycans.Blood. 2009; 114: 733-741Crossref PubMed Scopus (33) Google Scholar Although α2–3 sialylation, α1–3 fucosylation, and 6-O-sulfation are required for optimal L-selectin interaction,5Rosen S.D. Ligands for L-selectin: homing, inflammation, and beyond.Annu Rev Immunol. 2004; 22: 129-156Crossref PubMed Scopus (634) Google Scholar 6-O-sulfation in the context of an extended core 1 O-glycan (Figure 1) is essential for the MECA-79 epitope.13Yeh J.C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Novel sulfated lymphocyte homing receptors and their control by a Core1 extension beta 1,3-N-acetylglucosaminyltransferase.Cell. 2001; 105: 957-969Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar The sulfation modifications, recognized by L-selectin and MECA-79, are generated cooperatively by two GlcNAc-6-O sulfotransferases, GlcNAc6ST-116Uchimura K. Muramatsu H. Kadomatsu K. Fan Q.W. Kurosawa N. Mitsuoka C. Kannagi R. Habuchi O. Muramatsu T. Molecular cloning and characterization of an N-acetylglucosamine-6-O- sulfotransferase.J Biol Chem. 1998; 273: 22577-22583Crossref PubMed Scopus (144) Google Scholar and GlcNAc6ST-217Bistrup A. Bhakta S. Lee J.K. Belov Y.Y. Gunn M.D. Zuo F.R. Huang C.C. Kannagi R. Rosen S.D. Hemmerich S. Sulfotransferases of two specificities function in the reconstitution of high-endothelial-cell ligands for L-selectin.J Cell Biol. 1999; 145: 899-910Crossref PubMed Scopus (246) Google Scholar, 18Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.-C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. A novel, high endothelial venule-specific sulfotransferase expresses 6-sulfo sialyl Lewisx, an L-selectin ligand displayed by CD34.Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar (hereafter referred to as ST-1 and ST-2). The importance of these enzymes in lymphocyte homing has been shown in studies using single- and double-knockout mice.19Gauguet J.M. Rosen S.D. Marth J.D. von Andrian U.H. Core 2 branching beta1,6-N-acetylglucosaminyltransferase and high endothelial cell N-acetylglucosamine-6-sulfotransferase exert differential control over B- and T-lymphocyte homing to peripheral lymph nodes.Blood. 2004; 104: 4104-4112Crossref PubMed Scopus (49) Google Scholar, 20Hemmerich S. Bistrup A. Singer M.S. van Zante A. Lee J.K. Tsay D. Peters M. Carminati J.L. Brennan T.J. Carver-Moore K. Leviten M. Fuentes M.E. Ruddle N.H. Rosen S.D. Sulfation of L-selectin ligands by an HEV-restricted sulfotransferase regulates lymphocyte homing to lymph nodes.Immunity. 2001; 15: 237-247Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 21Kawashima H. Petryniak B. Hiraoka N. Mitoma J. Huckaby V. Nakayama J. Uchimura K. Kadomatsu K. Muramatsu T. Lowe J.B. Fukuda M. N-acetylglucosamine-6-O-sulfotransferases 1 and 2 cooperatively control lymphocyte homing through L-selectin ligand biosynthesis in high endothelial venules.Nat Immunol. 2005; 6: 1096-1104Crossref PubMed Scopus (154) Google Scholar, 22Uchimura K. Gauguet J.M. Singer M.S. Tsay D. Kannagi R. Muramatsu T. von Andrian U.H. Rosen S.D. A major class of L-selectin ligands is eliminated in mice deficient in two sulfotransferases expressed in high endothelial venules.Nat Immunol. 2005; 6: 1105-1113Crossref PubMed Scopus (148) Google Scholar, 23Uchimura K. Kadomatsu K. El-Fasakhany F.M. Singer M.S. Izawa M. Kannagi R. Takeda N. Rosen S.D. Muramatsu T. N-acetylglucosamine 6-O-sulfotransferase-1 regulates expression of L-selectin ligands and lymphocyte homing.J Biol Chem. 2004; 279: 35001-35008Crossref PubMed Scopus (74) Google Scholar, 24van Zante A. Gauguet J.M. Bistrup A. Tsay D. von Andrian U.H. Rosen S.D. Lymphocyte-HEV interactions in lymph nodes of a sulfotransferase-deficient mouse.J Exp Med. 2003; 198: 1289-1300Crossref PubMed Scopus (42) Google Scholar ST-1/ST-2 doubly null mice show an approximately 75% deficiency in L-selectin-dependent lymphocyte homing to lymph nodes and the complete absence of MECA-79 staining of HEVs.21Kawashima H. Petryniak B. Hiraoka N. Mitoma J. Huckaby V. Nakayama J. Uchimura K. Kadomatsu K. Muramatsu T. Lowe J.B. Fukuda M. N-acetylglucosamine-6-O-sulfotransferases 1 and 2 cooperatively control lymphocyte homing through L-selectin ligand biosynthesis in high endothelial venules.Nat Immunol. 2005; 6: 1096-1104Crossref PubMed Scopus (154) Google Scholar, 22Uchimura K. Gauguet J.M. Singer M.S. Tsay D. Kannagi R. Muramatsu T. von Andrian U.H. Rosen S.D. A major class of L-selectin ligands is eliminated in mice deficient in two sulfotransferases expressed in high endothelial venules.Nat Immunol. 2005; 6: 1105-1113Crossref PubMed Scopus (148) Google Scholar MECA-79 positive vessels not only are present in normal lymphoid organs but also are induced in some instances of infection and inflammation.5Rosen S.D. Ligands for L-selectin: homing, inflammation, and beyond.Annu Rev Immunol. 2004; 22: 129-156Crossref PubMed Scopus (634) Google Scholar, 7Uchimura K. Rosen S.D. Sulfated L-selectin ligands as a therapeutic target in chronic inflammation.Trends Immunol. 2006; 27: 559-565Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 25Michie S.A. Streeter P.R. Bolt P.A. Butcher E.C. Picker L.J. The human peripheral lymph node vascular addressin.Am J Pathol. 1993; 143: 1688-1698PubMed Google Scholar, 26Renkonen J. Tynninen O. Häyry P. Paavonen T. Renkonen R. Glycosylation might provide endothelial zip codes for organ-specific leukocyte traffic into inflammatory sites.Am J Pathol. 2002; 161: 543-550Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 27Bistrup A. Tsay D. Shenoy P. Singer M.S. Bangia N. Luther S.A. Cyster J.G. Ruddle N.H. Rosen S.D. Detection of a sulfotransferase (HEC-GlcNAc6ST) in high endothelial venules of lymph nodes and in HEV-like vessels within ectopic lymphoid aggregates: relationship to the MECA-79 epitope.Am J Pathol. 2004; 164: 1635-1644Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 28Drayton D.L. Liao S. Mounzer R.H. Ruddle N.H. Lymphoid organ development: from ontogeny to neogenesis.Nat Immunol. 2006; 7: 344-353Crossref PubMed Scopus (541) Google Scholar The positive vessels are frequently, but not always, high-walled and are found either in organized lymphoid aggregates (tertiary lymphoid organs) or within diffuse lymphoid aggregates.26Renkonen J. Tynninen O. Häyry P. Paavonen T. Renkonen R. Glycosylation might provide endothelial zip codes for organ-specific leukocyte traffic into inflammatory sites.Am J Pathol. 2002; 161: 543-550Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 28Drayton D.L. Liao S. Mounzer R.H. Ruddle N.H. Lymphoid organ development: from ontogeny to neogenesis.Nat Immunol. 2006; 7: 344-353Crossref PubMed Scopus (541) Google Scholar Human diseases in which MECA-79-positive vessels occur include Crohn's disease, ulcerative colitis, gastritis associated with Helicobacter pylori infection, heart and kidney allograft rejection, bronchial asthma, myocarditis, rheumatoid arthritis, Hashimoto's thyroiditis, and Graves' disease. Therapeutic effects of intravenously injected MECA-79 have been found in a sheep model of asthma.29Rosen S.D. Tsay D. Singer M.S. Hemmerich S. Abraham W.M. Therapeutic targeting of endothelial ligands for L-selectin (PNAd) in a sheep model of asthma.Am J Pathol. 2005; 166: 935-944Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar MECA-79 is only partially effective in blocking lymphocyte adherence to HEVs in mouse lymph nodes,8Streeter P.R. Rouse B.T.N. Butcher E.C. Immunohistologic and functional characterization of a vascular addressin involved in lymphocyte homing into peripheral lymph nodes.J Cell Biol. 1988; 107: 1853-1862Crossref PubMed Scopus (537) Google Scholar and more notably in human tonsils.25Michie S.A. Streeter P.R. Bolt P.A. Butcher E.C. Picker L.J. The human peripheral lymph node vascular addressin.Am J Pathol. 1993; 143: 1688-1698PubMed Google Scholar, 30Clark R.A. Fuhlbrigge R.C. Springer T.A. L-Selectin ligands that are O-glycoprotease resistant and distinct from MECA-79 antigen are sufficient for tethering and rolling of lymphocytes on human high endothelial venules.J Cell Biol. 1998; 140: 721-731Crossref PubMed Scopus (56) Google Scholar Contrary to the prevailing view that only O-glycans are essential for L-selectin determinants, N-glycans of murine CD34 can present 6-sulfo sLex to L-selectin.31Mitoma J. Bao X. Petryanik B. Schaerli P. Gauguet J.M. Yu S.Y. Kawashima H. Saito H. Ohtsubo K. Marth J.D. Khoo K.H. von Andrian U.H. Lowe J.B. Fukuda M. Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment.Nat Immunol. 2007; 8: 409-418Crossref PubMed Scopus (162) Google Scholar A structural analysis of human tonsillar PNAd is compatible with the existence of similarly modified N-glycans.15Mir G.H. Helin J. Skarp K.-P. Cummings R.D. Makitie A. Renkonen R. Leppanen A. Glycoforms of human endothelial CD34 that bind L-selectin carry sulfated sialyl Lewis x capped O- and N-glycans.Blood. 2009; 114: 733-741Crossref PubMed Scopus (33) Google Scholar The importance of these N-glycans for homing to lymph nodes was explored in mice lacking the glycosyltransferases that elaborate the relevant core 2 (Core2GlcNAcT) and extended core 1 branches (Core1-β3GlcNAcT) on O-glycans31Mitoma J. Bao X. Petryanik B. Schaerli P. Gauguet J.M. Yu S.Y. Kawashima H. Saito H. Ohtsubo K. Marth J.D. Khoo K.H. von Andrian U.H. Lowe J.B. Fukuda M. Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment.Nat Immunol. 2007; 8: 409-418Crossref PubMed Scopus (162) Google Scholar (Figure 1). MECA-79 staining was abolished on lymph node HEVs in these mice, yet approximately 50% of L-selectin-dependent homing persisted. N-glycanase treatment of HEVs eliminated the residual ligand activity, thus directly implicating N-glycans. The biological significance of the N-glycans was further indicated by the finding that tomato lectin, which binds to complex N-glycans, partially reduces lymphocyte homing in wild-type mice. To validate these findings and explore their generality in human and other species, it would be desirable to have antibodies that recognize the 6-sulfo sLex structure on both N- and O-glycans. So far, two murine IgM antibodies, known as G72 and G152, have been shown to bind this determinant.12Mitsuoka C. Sawada-Kasugai M. Ando-Furui K. Izawa M. Nakanishi H. Nakamura S. Ishida H. Kiso M. Kannagi R. Identification of a major carbohydrate capping group of the L-selectin ligand on high endothelial venules in human lymph nodes as 6-sulfo sialyl Lewis X.J Biol Chem. 1998; 273: 11225-11233Crossref PubMed Scopus (230) Google Scholar Although these antibodies block staining of human lymph node HEVs by an L-selectin-IgG chimera, their activities in lymphocyte-HEV binding assays have not been determined. Moreover, these antibodies do not react with HEVs in mouse or rat,12Mitsuoka C. Sawada-Kasugai M. Ando-Furui K. Izawa M. Nakanishi H. Nakamura S. Ishida H. Kiso M. Kannagi R. Identification of a major carbohydrate capping group of the L-selectin ligand on high endothelial venules in human lymph nodes as 6-sulfo sialyl Lewis X.J Biol Chem. 1998; 273: 11225-11233Crossref PubMed Scopus (230) Google Scholar probably because of variation in sialic acid forms between species.32Mitoma J. Miyazaki T. Sutton-Smith M. Suzuki M. Saito H. Yeh J.C. Kawano T. Hindsgaul O. Seeberger P.H. Panico M. Haslam S.M. Morris H.R. Cummings R.D. Dell A. Fukuda M. The N-glycolyl form of mouse sialyl Lewis X is recognized by selectins but not by HECA-452 and FH6 antibodies that were raised against human cells.Glycoconj J. 2009; 26: 511-523Crossref PubMed Scopus (25) Google Scholar Therefore, these antibodies are not useful in small-animal models of disease. With respect to MECA-79, its reactivity is limited to a subset of O-glycans, as explained above, and its function-blocking activity is incomplete. Furthermore, it is an IgM and has limited usefulness in small animal models of chronic inflammation (S.H., unpublished observations). In the present study, we characterized a newly generated mouse IgG mAb, designated CL40. We demonstrate the reactivity of CL40 for both O-glycan and N-glycan chains that terminate with 6-sulfo sLex. CL40 stains HEVs in humans and rodents, as well as HEV-like vessels at sites of inflammation. Consistent with this broad reactivity, this mAb is superior to MECA-79 in blocking lymphocyte binding to HEVs, especially in human. The new findings expand our knowledge about the glycosylation and sulfation requirements for L-selectin ligands. CL40 (murine IgG1) was provided to us by Dyax Corporation (Cambridge, MA). The antibody was originally produced at Thios Pharmaceuticals (Emeryville, CA), by immunizing ST-1/ST-2 doubly null mice21Kawashima H. Petryniak B. Hiraoka N. Mitoma J. Huckaby V. Nakayama J. Uchimura K. Kadomatsu K. Muramatsu T. Lowe J.B. Fukuda M. N-acetylglucosamine-6-O-sulfotransferases 1 and 2 cooperatively control lymphocyte homing through L-selectin ligand biosynthesis in high endothelial venules.Nat Immunol. 2005; 6: 1096-1104Crossref PubMed Scopus (154) Google Scholar with an extended core 1 structure terminating with 6-sulfo sLex (Figure 1). Murine hybridoma supernatants were screened for binding to the glycan immunogen, and CL40 was selected. G72 was a kind gift of Dr. Reiji Kannagi. The MECA-79 hybridoma was provided by Dr. Eugene Butcher. The extended core 1 and core 2 branches were synthesized by Matt Pratt and Carolyn Bertozzi33Pratt M.R. Bertozzi C.R. Syntheses of 6-sulfo sialyl Lewis X glycans corresponding to the L-selectin ligand “sulfoadhesin”.Org Lett. 2004; 6: 2345-2348Crossref PubMed Scopus (32) Google Scholar and provided to us by Thios Pharmaceuticals. 300.19L cells34Kansas G.S. Ley K. Munro J.M. Tedder T.F. Regulation of leukocyte rolling and adhesion to high endothelial venules through the cytoplasmic domain of L-selectin.J Exp Med. 1993; 177: 833-838Crossref PubMed Scopus (185) Google Scholar (mouse pre-B cell lymphoma stably transfected with full-length human L-selectin cDNA) were provided by Dr. Geoffrey Kansas. Mice deficient in GlcNAc6ST-1 (ST-1),23Uchimura K. Kadomatsu K. El-Fasakhany F.M. Singer M.S. Izawa M. Kannagi R. Takeda N. Rosen S.D. Muramatsu T. N-acetylglucosamine 6-O-sulfotransferase-1 regulates expression of L-selectin ligands and lymphocyte homing.J Biol Chem. 2004; 279: 35001-35008Crossref PubMed Scopus (74) Google Scholar GlcNAc6ST-2 (ST-2),22Uchimura K. Gauguet J.M. Singer M.S. Tsay D. Kannagi R. Muramatsu T. von Andrian U.H. Rosen S.D. A major class of L-selectin ligands is eliminated in mice deficient in two sulfotransferases expressed in high endothelial venules.Nat Immunol. 2005; 6: 1105-1113Crossref PubMed Scopus (148) Google Scholar and both ST-1 and ST-222Uchimura K. Gauguet J.M. Singer M.S. Tsay D. Kannagi R. Muramatsu T. von Andrian U.H. Rosen S.D. A major class of L-selectin ligands is eliminated in mice deficient in two sulfotransferases expressed in high endothelial venules.Nat Immunol. 2005; 6: 1105-1113Crossref PubMed Scopus (148) Google Scholar (all on the C57BL/6 background) were used. Mice doubly null for α(1,3)fucosyltransferase-IV and -VII (FTIV and FTVII)35MalÝ P. Thall A.D. Petryniak B. Rogers C.E. Smith P.L. Marks R.M. Kelly R.J. Gersten K.M. Cheng G. Saunders T.L. Camper S.A. Camphausen R.T. Sullivan F.X. Isogai Y. Hindsgaul O. von Andrian U.H. Lowe J.B. The α(1,3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E- and P-selectin ligand biosynthesis.Cell. 1996; 86: 643-653Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar were provided by the Consortium for Functional Glycomics. Mice deficient in both core 1 extension and core 2 branching enzymes (Core1βGlcNAcT and Core2GlcNAcT)36Mitoma J. Petryniak B. Hiraoka N. Yeh J.C. Lowe J.B. Fukuda M. Extended core 1 and core 2 branched O-glycans differentially modulate sialyl Lewis x-type L-selectin ligand activity.J Biol Chem. 2003; 278: 9953-9961Crossref PubMed Scopus (70) Google Scholar were crossed onto the BALB/c background. FFPE blocks containing synovia from patients with rheumatoid arthritis (RA) or osteoarthritis and colons from patients with ulcerative colitis (UC) or noninflammatory conditions were obtained from surgical specimens archived in the Department of Pathology at the University of California, San Francisco. The tissue samples were obtained under Committee on Human Research approval (CHR H1060-28724). Specimens from patients with a diagnosis of RA were evaluated, and samples that demonstrated the classic features of rheumatoid arthritis including papillary hyperplasia and lymphoplasmacytic infiltration of the synovium were selected. Similarly, total colectomy specimens from patients with a diagnosis of UC were evaluated, and samples that demonstrated chronic colitis with acute inflammation and mucosal ulceration were selected. Extended core 1 and core 2 O-glycans were chemically synthesized with or without 6-O- sulfation and conjugated with biotin at their reducing termini.33Pratt M.R. Bertozzi C.R. Syntheses of 6-sulfo sialyl Lewis X glycans corresponding to the L-selectin ligand “sulfoadhesin”.Org Lett. 2004; 6: 2345-2348Crossref PubMed Scopus (32) Google Scholar The enzyme-linked immunosorbent assay (ELISA) plates (Thermo Scientific, Waltham, MA) were coated overnight with 10 μg/ml streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS. The plates were washed and incubated with twofold serial dilutions of 50 nmol/L synthetic sugars diluted in PBST (PBS containing 0.1% Tween-20). Antibodies were applied at the following final concentrations: CL40 at 5 μg/ml and MECA-79 (rat IgM), G72 supernatant12Mitsuoka C. Sawada-Kasugai M. Ando-Furui K. Izawa M. Nakanishi H. Nakamura S. Ishida H. Kiso M. Kannagi R. Identification of a major carbohydrate capping group of the L-selectin ligand on high endothelial venules in human lymph nodes as 6-sulfo sialyl Lewis X.J Biol Chem. 1998; 273: 11225-11233Crossref PubMed Scopus (230) Google Scholar (mouse IgM), and HECA-452 (rat IgM; BD Pharmingen, San Jose, CA) at 1 μg/ml. Bound antibodies were detected with alkaline phosphatase (ALP)-conjugated secondary Abs (Jackson ImmunoResearch) with PNPP as substrate (Thermo Scientific). CHO cells were transiently transfected with various combinations of cDNAs encoding glycosyltransferases and sulfotransferases as previously described.13Yeh J.C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Novel sulfated lymphocyte homing receptors and their control by a Core1 extension beta 1,3-N-acetylglucosaminyltransferase.Cell. 2001; 105: 957-969Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 31Mitoma J. Bao X. Petryanik B. Schaerli P. Gauguet J.M. Yu S.Y. Kawashima H. Saito H. Ohtsubo K. Marth J.D. Khoo K.H. von Andrian U.H. Lowe J.B. Fukuda M. Critical functions of N-glycans in L-selectin-mediated lymphocyte homing and recruitment.Nat Immunol. 2007; 8: 409-418Crossref PubMed Scopus (162) Google Scholar, 37Bierhuizen M.F. Fukuda M. Expression cloning of a cDNA encoding UDP-GlcNAc:Gal beta 1-3-GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen.Proc Natl Acad Sci USA. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar Parental CHO cells lack Core1βGlcNAcT, Core2GlcNAcT, FTIV, and FTVII.13Yeh J.C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Novel sulfated lymphocyte homing receptors and their control by a Core1 extension beta 1,3-N-acetylglucosaminyltransferase.Cell. 2001; 105: 957-969Abstract Full Text Full Text PDF PubMed Scopus (294)

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