P- and E-Selectins Recognize Sialyl 6-Sulfo Lewis X, the Recently Identified L-Selectin Ligand

doi:10.1006/bbrc.2000.3768

Biochemical and Biophysical Research Communications

Volume 278, Issue 1, 11 November 2000, Pages 90-96

https://doi.org/10.1006/bbrc.2000.3768 Get rights and content

Abstract

Recently we identified sialyl 6-sulfo Le^x as a major L-selectin ligand on high endothelial venules of human peripheral lymph nodes. In this study we investigated the ligand activity of sialyl 6-sulfo Le^x to E- and P-selectins and compared it with the binding activity of conventional sialyl Le^x, by using cultured human lymphoid cells expressing both carbohydrate determinants. The results of the recombinant selectin binding studies and the nonstatic monolayer cell adhesion assays indicated that both sialyl 6-sulfo Le^x and conventional sialyl Le^x served as ligand for E- and P-selectins, while L-selectin was quite specific to sialyl 6-sulfo Le^x. Anti-PSGL-1 antibodies as well as O-sialoglycoprotein endopeptidase treatment almost completely abrogated the binding of P-selectin but barely affected the binding of E-selectin, indicating that these carbohydrate determinants carried by O-glycans of PSGL-1 selectively serves as a ligand for P-selectin, while the ligand for E-selectin is not restricted to PSGL-1 nor to O-sialoglycoprotein endopeptidase-sensitive glycans. The binding of L-selectin was markedly reduced by O-sialoglycoprotein endopeptidase treatment but only minimally affected by anti-PSGL-1 antibodies, indicating that O-glycans carrying sialyl 6-sulfo Le^x were the major L-selectin ligands, while PSGL-1 was only a minor core protein for L-selectin in these cells. These results indicated that each member of the selectin family has a distinct ligand binding specificity.

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The Allen Institute Mouse Brain Atlas, with visualisation using the Brain Explorer software, offers a 3-dimensional view of region-specific RNA expression of thousands of mouse genes. In this Viewpoint, we focused on the region-specific expression of genes related to cellular glycosylation, and discuss their relevance towards psychoneuroimmunology. Using specific examples, we show that the Atlas validates existing observations reported by others, identifies previously unknown potential region-specific glycan features, and highlights the need to promote collaborations between glycobiology and psychoneuroimmunology researchers.
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Selectin enzymes are glycoproteins and are an important adhesion molecule in the mammalian immune system, especially in the inflammatory response and the healing process of tissues. Selectins play an important role in a variety of biological processes, including the rolling of leukocytes in endothelial cells, a process known as the adhesion cascade. It has recently been discovered and reported that the selectin mechanism plays a role in cancer and thrombosis disease. This process begins with non-covalent interactions-based selectin-ligand binding and the glycans play a role as a connector between cancer cells and the endothelium in this process. The selectin mechanism is critical for the immune system, but it is also involved in disease mechanisms, earning the selectins the nickname “Selectins-The Two Dr. Jekyll and Mr. Hyde Faces”. As a result, the drug for selectins should have a multifaceted role and be a dynamic molecule that targets the disease mechanism specifically. This chapter explores the role of selectins in the disease mechanism at the mechanism level that provides the impact for identifying the selectin inhibitors. Overall, this chapter provides the molecular level insights on selectins, their ligands, involvement in normal and disease mechanisms.
Expression of functional E-selectin ligands on the plasma membrane of carcinoma cells correlates with poor prognosis in clear cell renal cell carcinoma
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Citation Excerpt :
Among them, HECA-452 is unique antibody in recognizing a carbohydrate structure common to both sLex and sLea [4]. Moreover, HECA-452 antibody also recognizes sLex with N-acetylglucosamine (GlcNAc)-6-O-sulfation and/or galactose (Gal)-6-O-sulfation [15], and both of these sulfated forms of sLex constitute functional E-selectin ligands at least in vitro [16]. Thus, HECA-452 can detect a broad class of sialofucosylated lactosaminyl glycans that potentially function as E-selectin ligands [4].
Given the relatively high frequency of metastatic recurrence of clear cell renal cell carcinoma (ccRCC), reliable prognostic markers of ccRCC, particularly those associated with metastasis, are needed. Here, in search of those factors, we assessed the contribution of sialyl Lewis x (sLe^x) and sialyl Lewis a (sLe^a), as well as functional E-selectin ligand carbohydrates expressed on carcinoma cells, to metastasis and consequent poor prognosis in ccRCC.
Patients who underwent surgical resection (curative nephrectomy) for RCC, and whose post-operative pathological diagnosis was ccRCC (n = 117) were enrolled in this study. Expression of sLe^x/sLe^a carbohydrate antigens in ccRCC was evaluated by immunohistochemistry with an anti-sLe^x/sLe^a monoclonal antibody HECA-452. To evaluate membrane expression of sLe^x/sLe^a carbohydrate antigens quantitatively, we employed a histological scoring system used to evaluate membrane expression of human epidermal growth factor receptor 2 (HER2) in breast cancer. We also conducted an E-selectin•IgM chimera in situ binding assay to assess expression of functional E-selectin ligand carbohydrates in ccRCC. We then carried out statistical analysis to determine whether membrane expression of HECA-452-reactive sLe^x/sLe^a glycans as well as of E-selectin•IgM-binding functional E-selectin ligand carbohydrates correlates with progression-free, overall, or cancer-specific survival.
Based on HECA-452 immunochemistry, 106 of 117 ccRCC specimens expressed detectable levels of sLe^x/sLe^a glycans, primarily on the plasma membrane, and of those, 31 that showed robust membrane expression were judged as HECA-452-positive. Membrane expression of HECA-452-positive sLe^x/sLe^a glycans correlated with shortened progression-free and overall survival. Moreover, in in situ analysis, these HECA-452-positive ccRCC tissues were decorated with E-selectin•IgM chimeric proteins, calcium-dependently. Comparable analysis in normal kidney showed both HECA-452 positivity and chimera binding almost exclusively in epithelial cells that constitute proximal tubules. Membrane expression of functional E-selectin ligand carbohydrates, as detected by the E-selectin•IgM chimera, correlated more significantly with poor prognosis of patients, namely, shortened progression-free, overall and cancer-specific survival, than did HECA-452 positivity.
Expression of E-selectin•IgM-binding functional E-selectin ligand carbohydrates can serve as a reliable and potentially superior prognostic biomarker of patients with ccRCC.
A potential role for 6-sulfo sialyl Lewis X in metastasis of bladder urothelial carcinoma
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It is widely accepted that sialyl Lewis X (sLeX) and sialyl Lewis A (sLeA, also known as CA 19-9) glycans expressed on cancer cells function in E-selectin-mediated metastasis. Recently, it was reported that 6-sulfo sLeX glycans detected by the MECA-79 monoclonal antibody are expressed in roughly a quarter of gastric adenocarcinoma cases, and that these cases show a poorer prognosis than MECA-79-negative cases do. The present study was undertaken to assess expression of 6-sulfo sLeX glycans in bladder urothelial carcinoma and evaluate potential clinical implications.
We analyzed 78 specimens representing bladder urothelial carcinoma, as well as 4 bladder urothelial carcinoma cell lines, by immunostaining with a battery of anticarbohydrate antibodies. We also undertook an E-selectin·IgM chimera binding assay to assess E-selectin binding to 6-sulfo sLeX expressed on bladder urothelial carcinoma cells and performed reverse transcription polymerase chain reaction and complementary DNA transfection to determine which N-acetylglucosamine-6-O-sulfotransferases function in 6-sulfo sLeX biosynthesis in those cells. Finally, we performed double-immunofluorescence staining for MECA-79 and either CD3 or CD8 to evaluate potential association between high endothelial venule (HEV)-like vessels and tumor-infiltrating T lymphocytes.
6-Sulfo sLeX glycans were expressed in ~20% of bladder urothelial carcinoma cases, particularly in plasmacytoid and micropapillary variants. Positive cells were also bound by E-selectin·IgM chimeras in a calcium-dependent manner. Transcripts encoding N-acetylglucosamine-6-O-sulfotransferase-2 were detected preferentially in HT-1197 bladder urothelial carcinoma cells expressing 6-sulfo sLeX, and transfection of the enzyme complementary DNA into HT-1376 cells, which do not express 6-sulfo sLeX glycans, resulted in cell surface expression of 6-sulfo sLeX. Furthermore, 6-sulfo sLeX glycans were expressed in HEV-like vessels induced in and around lymphocyte aggregates formed near carcinoma cell nests. These HEV-like vessel-associated tumor-infiltrating lymphocytes were composed primarily of CD3⁺ T cells, with a fraction of CD8⁺ cytotoxic T cells.
Our findings indicate that 6-sulfo sLeX glycans likely play 2 roles in bladder urothelial carcinoma progression: one in lymphocyte recruitment to enhance antitumor immune responses, and the other in E-selectin-mediated tumor cell adhesion to vascular endothelial cells, which is potentially associated with metastasis.
Sialic acid cyclization of human Th homing receptor glycan associated with recurrent exacerbations of atopic dermatitis
2012, Journal of Dermatological Science
Citation Excerpt :
This glycan was first identified on high endothelial venules (HEVs) as the major ligand for Lymphocyte selectin [16]. Subsequent studies have demonstrated the binding capacity of sialyl 6-sulfo Lex for Endothelial selectin and Platelet selectin [17] and the expression of sialyl 6-sulfo Lex on some peripheral CD4+CD45RO+CCR4+CCR7+ lymphocytes, resting skin-homing memory Th cells [15]. Interestingly, the selectin-binding activity of sialyl 6-sulfo Lex is abrogated via cyclization of its sialic acid moiety [18].
The molecular pathogenesis underlying recurrent exacerbations of atopic dermatitis (AD) is unclear. Some peripheral CCR4⁺ and CCR7⁺ helper memory T cells express the specific homing receptor, sialyl 6-sulfo Lewis X (G152 glycan). This glycan loses receptor activity via cyclization of its sialic acid moiety, thus becoming cyclic sialyl 6-sulfo Lewis X (G159 glycan). These findings suggest that the disordered expression of G152 and G159 glycans may be associated with recurrent exacerbations of AD.
To assess the possible association of G152 and G159 glycans, which are expressed on peripheral helper T (Th) cells, with frequency of exacerbations.
The percentage of glycan-expressing cells among peripheral blood CD4⁺CD45RO⁺ lymphocytes was determined by flow cytometry. The association of glycans with the frequency of exacerbations determined by recurrence scores as well as with current disease activity was statistically tested.
Current disease activity was significantly associated with CCR4⁺CCR7⁻ memory Th cells expressing CSLEX-1 glycan, the conventional skin-trafficking receptor without sialic-acid-cyclization activity. In contrast, the frequency of exacerbations was positively and negatively associated with CCR4⁺CCR7⁺ memory Th cells expressing G152 and G159 glycans, respectively. Receiver operating characteristics analyses indicated that the ratio of the G152⁺/G159⁺ cell percentages discriminated patients with highly recurrent AD with the best accuracy.
Flow cytometric determination of G159 and G152 glycans on peripheral helper memory T cells may be clinically useful for identifying patients with highly recurrent AD. Disordered sialic acid cyclization of G152 glycan may underlie highly recurrent AD, which may provide a novel therapeutic approach.
Induction of 6-sulfated glycans with cell adhesion activity via T-bet and GATA-3 in human helper T cells
2012, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
Sulfated glycans play important roles in the immune system through mediating cell-to-cell interactions [1–4]. Notably, 6-sulfated N-acetyllactosamine glycans, characterized by sulfation at the C-6 position of N-acetylglucosamine (GlcNAc) residue, are involved in many cell-recognition events including selectin-mediated cell adhesion [5–10], cell–cell interaction through sialic acid-binding Ig-like lectins (siglecs) [11–15], activation of CD44 in hyaluronan-binding [16–18], and dendritic cell function [19–21]. These contributions of 6-sulfated glycans in various leukocyte-related events suggest that expression of the glycans is tightly regulated in a cell type- and cell condition-dependent manner, although the underlying mechanisms remain to be clarified.
Cell surface 6-sulfated glycans play important roles in various immunological events through cell-to-cell interactions. The 6-sulfation process is mediated by 6-sulfotransferase family isoenzymes. We previously demonstrated that GlcNAc6ST-1, one of the isoenzyme genes, is induced by GATA-3 and NF-κB in human helper T (Th) cells. However, transcriptional regulation of HEC-GlcNAc6ST, another isoenzyme important in Th cells, remains unclear.
5′-RACE analysis, chromatin immunoprecipitation, and reporter assays were performed to reveal transcriptional regulation of HEC-GlcNAc6ST. RNA-knockdown and forced expression experiments were performed to demonstrate the contribution of HEC-GlcNAc6ST to the 6-sulfated glycan expression.
We identified potential binding sites of Sp1, T-bet, and GATA-3 in the HEC-GlcNAc6ST promoter. Reporter assays indicated that transfection of Sp1 enhanced the activity, whereas mithramycin A, an Sp1-specific inhibitor, repressed it. Transfection of T-bet increased the activity, which was inhibited by introducing a mutation into the potential T-bet binding site. GATA-3 alone could not elevate the activity, although co-transfection of protein kinase A, which is known to enhance IL-5 transcription in Th2 cells through phosphorylation of GATA-3, caused elevation. RNA-knockdown and forced expression of HEC-GlcNAc6ST in Jurkat cells down- and up-regulated α2,6-sialylated 6-sulfo N-acetyllactosamine, a preferential ligand for B-cell-specific CD22 antigen, respectively.
From these results, we concluded that T-bet and GATA-3 as well as Sp1 control the expression of glycan with cell-adhesion activity by regulating HEC-GlcNAc6ST transcription in Th cells.
These results may provide a clue to biological regulation of Th-cell interaction with selectins and other carbohydrate-recognition molecules by T-bet and GATA-3.

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^☆: This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture, Japan (11680648 and on priority areas 10178104, 12036227, 12217174), grants-in-aid for the Second Term Comprehensive Ten-Year Strategy for Cancer Control from the Ministry of Health and Welfare, Japan, and a grant from the Princess Takamatsu Foundation for the Promotion of Cancer Research.

^☆☆: Abbreviations used: BSA, bovine serum albumin; Fuc-T, fucosyltransferase; GlcNAc, N-acetyl-glucosamine; PBS, phosphate-buffered saline; OSGE, O-sialoglycoprotein endopeptidase; PSGL-1, P-selectin glycoprotein ligand-1; sialyl Le^x, sialyl Lewis X, NeuAcα2 → 3Galβ1 → 4[Fucα1 → 3]GlcNAcβ1 → R; sialyl 6-sulfo Le^x, sialyl 6-sulfo Lewis X, NeuAcα2 → 3Galβ1 → 4[Fucα1 → 3][SO₃⁻-6]GlcNAcβ1 → R.

²: To whom correspondence should be addressed at Program of Experimental Pathology, Research Institute, Aichi Cancer Center, 1-1 Kanokoden, Chikusaku, Nagoya 464-8681, Japan. Fax: +81-52-723-5347. E-mail: [email protected].

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Regular ArticleP- and E-Selectins Recognize Sialyl 6-Sulfo Lewis X, the Recently Identified L-Selectin Ligand☆,☆☆

Abstract

Am. J. Pathol.

Kidney Int.

Blood

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Blood

Blood

Blood

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Regular Article
P- and E-Selectins Recognize Sialyl 6-Sulfo Lewis X, the Recently Identified L-Selectin Ligand☆,☆☆