Galectin-9

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Galectin-9 is one of the best-studied of the tandem-repeat galectins with glycan binding in both the N- and C-terminal domains [1]. The protein lacks a signal sequence, like most galectins, and is synthesized in the cytosol on free polyribosomes. Galectin-9 is found outside of cells and may be exported by non-classical pathways. Galectin-9 exhibits a variety of biological activities, the majority of which have focused on its immunomodulatory role toward lymphocytes, were it shows specific interactions with TIM-3, and can negatively regulate Th1 type immunity[2]. Mammalian galectin-9 exhibits affinity toward select glycan ligands, including sulfated glycans, and blood group-related glycans, and also interacts with glycans containing poly-N-acetyllactosamine (LacNAc) repeats (-3Galβ1-4GlcNAcβ1-)n through recognition of internal LacNAc repeats [3].

The crystal structure of the N-terminal carbohydrate recognition domain (CRD) been defined [3][4][5]. The GBP shows strong interactions in a metal-free manner with poly-N-acetyllactosamine sequences comprised of repeating (-3Galβ1-4GlcNAcβ1-)n by recognizing internal N-acetyllactosamine repeats[3]. Generally, it binds distinct glycan ligands from Galectin-1[6]. There are has three well-characterized linker domains between the CRDs, generated by alternative splicing[7], that may regulate cellular localization and function of the protein. Truncation of linker domain between CRDs in recombinant forms of galectin-9 stabilize the protein to proteolysis[8].


Contents

CFG Participating Investigators contributing to the understanding of this paradigm

CFG Participating Investigators (PIs) contributing to the understanding of Galectin-9 include: Linda Baum, Richard Cummings, Gabriel Rabinovich, Sachiko Sato

Progress toward understanding this GBP paradigm

This section documents what is currently known about Galectin-9, its carbohydrate ligand(s), and how they interact to mediate cell communication. Further information can be found in the GBP Molecule Page for human and mouse Galectin-9 in the CFG database.

Carbohydrate ligands

Human galectin-9 binding to glycans has been studied by a variety of techniques including glycan microarray analysis and frontal affinity chromatography.

On the CFG glycan microarray, the individual N- and C-terminal domains of recombinant dog (Canis lupus) galectin-9, generated as GST (glutathione-S-transferase) chimeras, showed similarities in glycan recognition, but also distinct differences[9]. While both domains bound well to short sulfated glycans, such as 3-O-sulfated galactose in short LacNAc structures, only the N-terminal domain bound well to many glycans expressing blood group A-related sequences and to the Forssman glycolipid-like glycans, whereas the C-terminal domain bound less well to the blood group related structures, but showed binding to a linear sialylated poly-N-acetyllactosamine pentasaccharide.

In frontal affinity chromatography, recombinant human galectin-9 was found to preferentially bind to both branched N-glycans (Kd = 0.16 μM toward tetraantennary N-glycans terminating in galactose) and glycans with poly-N-acetyllactosamine sequences (Kd = 0.09 μM toward octasaccharides with 4 repeating LacNAc groups, and this was found for both the N- and C-terminal domains. By contrast, the N-terminal, but not the C-terminal domain, showed significant binding in the low μM range to Forssman glycolipid-derived pentasaccharides and to blood group A hexasaccharide[10].

Glycan microarray analyses in microarrays with relatively short glycan species[11], showed that both the recombinant full-length human galectin-9 and the N-terminal domain displayed very similar binding patterns, and both bound to LacNAc sequences and even better to short fucosylated glycans with terminal blood group A and B trisaccharide sequences.

Cellular expression of GBP and ligands

Galectin-9 is widely expressed in various tissues (heart, lung, liver, kidney, spleen, muscle, intestine, and thymus), but weakly expressed in brain[1]. Interestingly, the rat urate transporter was reported to be 99% identical to the sequence reported for rat galectin-9 [12], suggesting that these two proteins are the same[13][14], and suggest that galectin-9 may have multiple functions, occurring as a polytopic transmembrane protein to function as the urate transporter, and as a soluble protein in its signaling and cell-binding forms.

Biosynthesis of ligands


Structure

Galectin-9 (long isoform in humans) has 355 amino acids and behaves as an ~35 kDa protein; short isoforms differ in the linker peptide length and have lower apparent sizes compared to the full-length long isoform. The crystal structure of the N-terminal carbohydrate recognition domain (CRD) been defined.[15][16][5] The GBP shows strong interactions in a metal-free manner with poly-N-acetyllactosamine sequences comprised of repeating (-3Galβ1-4GlcNAcβ1-)n by recognizing internal N-acetyllactosamine repeats [15]. Generally, it binds distinct glycan ligands from Galectin-1 [6]). There are has three well-characterized linker domains between the CRDs, generated by alternative splicing [7], that may regulate cellular localization and function of the protein. Truncation of linker domain between CRDs in recombinant forms of galectin-9 stabilize the protein to proteolysis [8].

Biological roles of GBP-ligand interaction

It has been shown that galectin-9 binds to a unique glycoprotein ligand Tim-3 expressed in Th1 and Th17 cells[17][18][19][20]. In addition, galectin-9 can interact with protein disulfide isomerase (PDI) at the cell surface, increasing retention of PDI on the surface and altering surface redox potential[21]. Galectin-9 null-mice have interesting phenotypes related to immune regulation. Galectin-9 null-mice develop acute and memory responses to Herpes Simplex Virus (HSV) that are of greater magnitude and better quality than those that occur in wild-type infected animals[22]; they exhibit increased resistance to influenza A virus compared to wild-type mice [23]; and they exhibit susceptibility to experimentally-induced autoimmune disease [17]. Galectin-9 expression is elevated in peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus[24]. Galectin-9 is the only tandem-repeat galectin that has been administered in animal models of disease to assess therapeutic potential [17][25][26]. Galectin-9 exhibits the ability induce apoptosis in some lymphocytes [2][21] and this can be inhibited by inclusion of lactose or inhibitors. Galectin-9 has eosinophil chemoattractant activity (26), and the term Ecalectin was given to a variant of T lymphocyte-derived galectin-9 that was found to be an eosinophil chemoattractant [27].

CFG resources used in investigations

The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for Galectin-9.

Glycan profiling


Glycogene microarray

Probes for human galectin-9 have been included in all versions of the CFG glycogene chip, and probes for mouse galectin-9 are included on versions 2, 3, and 4.

Knockout mouse lines

CFG-generated Galectin-9 knockout mice have been used to study the biological functions of this paradigm GBP. (CFG PI data)

Glycan array

Investigators have used CFG carbohydrate compounds and glycan array screening to study ligand binding specificity of Galectin-9 (for example, click here). To see all glycan array results for Galectin-9, click here.

Related GBPs

Galectin-4 (CFG data), galectin-6, galectin-8 (CFG data), and galectin-12 (CFG data).

References

  1. 1.0 1.1 Wada J, Kanwar YS. Identification and characterization of galectin-9, a novel beta-galactoside-binding mammalian lectin. J Biol Chem. 1997;272(9):6078-86
  2. 2.0 2.1 Zhu C, Anderson AC, Schubart A, Xiong H, Imitola J, Khoury SJ, Zheng XX, Strom TB, Kuchroo VK. The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity. Nat Immunol. 2005;6(12):1245-52.
  3. 3.0 3.1 3.2 Nagae M, Nishi N, Nakamura-Tsuruta S, Hirabayashi J, Wakatsuki S, Kato R. Structural analysis of the human galectin-9 N-terminal carbohydrate recognition domain reveals unexpected properties that differ from the mouse orthologue. J Mol Biol. 2008;375(1):119-35
  4. Yoshida H, Teraoka M, Nishi N, Nakakita S, Nakamura T, Hirashima M, Kamitori S. X-ray structures of human galectin-9 C-terminal domain in complexes with a biantennary oligosaccharide and sialyllactose. J Biol Chem. 2010;285(47):36969-76.
  5. 5.0 5.1 Nagae M, Nishi N, Murata T, Usui T, Nakamura T, Wakatsuki S, Kato R. Crystal structure of the galectin-9 N-terminal carbohydrate recognition domain from Mus musculus reveals the basic mechanism of carbohydrate recognition. J Biol Chem. 2006;281(47):35884-93.
  6. 6.0 6.1 Bi S, Earl LA, Jacobs L, Baum LG. Structural features of galectin-9 and galectin-1 that determine distinct T cell death pathways. J Biol Chem. 2008;283(18):12248-58
  7. 7.0 7.1 Nishi N, Itoh A, Shoji H, Miyanaka H, Nakamura T. Galectin-8 and galectin-9 are novel substrates for thrombin. Glycobiology. 2006;16(11):15C-20C.
  8. 8.0 8.1 Nishi N, Itoh A, Fujiyama A, Yoshida N, Araya S, Hirashima M, Shoji H, Nakamura T. Development of highly stable galectins: truncation of the linker peptide confers protease-resistance on tandem-repeat type galectins. FEBS Lett. 2005;579(10):2058-64
  9. Poland PA, Rondanino C, Kinlough CL, Heimburg-Molinaro J, Arthur CM, Stowell SR, Smith DF, Hughey RP. Identification and characterization of endogenous galectins expressed in Madin Darby canine kidney cells. J Biol Chem. 2011;286(8):6780-90
  10. Hirabayashi J, Hashidate T, Arata Y, Nishi N, Nakamura T, Hirashima M, Urashima T, Oka T, Futai M, Muller WE, Yagi F, Kasai K. Oligosaccharide specificity of galectins: a search by frontal affinity chromatography. Biochim Biophys Acta. 2002;1572(2-3):232-54
  11. Horlacher T, Oberli MA, Werz DB, Krock L, Bufali S, Mishra R, Sobek J, Simons K, Hirashima M, Niki T, Seeberger PH. Determination of carbohydrate-binding preferences of human galectins with carbohydrate microarrays. Chembiochem. 2010;11(11):1563-73
  12. Leal-Pinto E, Tao W, Rappaport J, Richardson M, Knorr BA, Abramson RG. Molecular cloning and functional reconstitution of a urate transporter/channel. J Biol Chem. 1997;272(1):617-25
  13. Lipkowitz MS, Leal-Pinto E, Rappoport JZ, Najfeld V, Abramson RG. Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter. J Clin Invest. 2001;107(9):1103-15.
  14. Lipkowitz MS, Leal-Pinto E, Cohen BE, Abramson RG. Galectin 9 is the sugar-regulated urate transporter/channel UAT. Glycoconj J. 2004;19(7-9):491-8
  15. 15.0 15.1 Nagae M, Nishi N, Nakamura-Tsuruta S, Hirabayashi J, Wakatsuki S, Kato R. Structural analysis of the human galectin-9 N-terminal carbohydrate recognition domain reveals unexpected properties that differ from the mouse orthologue. J Mol Biol. 2008;375(1):119-35.
  16. Yoshida H, Teraoka M, Nishi N, Nakakita S, Nakamura T, Hirashima M, Kamitori S. X-ray structures of human galectin-9 C-terminal domain in complexes with a biantennary oligosaccharide and sialyllactose. J Biol Chem. 2010;285(47):36969-76.
  17. 17.0 17.1 17.2 Seki M, Oomizu S, Sakata KM, Sakata A, Arikawa T, Watanabe K, Ito K, Takeshita K, Niki T, Saita N, Nishi N, Yamauchi A, Katoh S, Matsukawa A, Kuchroo V, Hirashima M. Galectin-9 suppresses the generation of Th17, promotes the induction of regulatory T cells, and regulates experimental autoimmune arthritis. Clin Immunol. 2008;127(1):78-88.
  18. Niwa H, Satoh T, Matsushima Y, Hosoya K, Saeki K, Niki T, Hirashima M, Yokozeki H. Stable form of galectin-9, a Tim-3 ligand, inhibits contact hypersensitivity and psoriatic reactions: a potent therapeutic tool for Th1- and/or Th17-mediated skin inflammation. Clin Immunol. 2009;132(2):184-94.
  19. Naka EL, Ponciano VC, Cenedeze MA, Pacheco-Silva A, Camara NO. Detection of the Tim-3 ligand, galectin-9, inside the allograft during a rejection episode. Int Immunopharmacol. 2009;9(6):658-62.
  20. Anderson DE. TIM-3 as a therapeutic target in human inflammatory diseases. Expert Opin Ther Targets. 2007;11(8):1005-9.
  21. 21.0 21.1 Bi S, Hong PW, Lee B, Baum LG. Galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance T-cell migration and HIV entry. Proc Natl Acad Sci U S A. 2011;108(26):10650-5
  22. Sehrawat S, Reddy PB, Rajasagi N, Suryawanshi A, Hirashima M, Rouse BT. Galectin-9/TIM-3 interaction regulates virus-specific primary and memory CD8 T cell response. PLoS Pathog. 2010;6(5):e1000882.
  23. Sharma S, Sundararajan A, Suryawanshi A, Kumar N, Veiga-Parga T, Kuchroo VK, Thomas PG, Sangster MY, Rouse BT. T cell immunoglobulin and mucin protein-3 (Tim-3)/Galectin-9 interaction regulates influenza A virus-specific humoral and CD8 T-cell responses. Proc Natl Acad Sci U S A. 2011;108(47):19001-6
  24. Wang Y, Meng J, Wang X, Liu S, Shu Q, Gao L, Ju Y, Zhang L, Sun W, Ma C. Expression of human TIM-1 and TIM-3 on lymphocytes from systemic lupus erythematosus patients. Scand J Immunol. 2008;67(1):63-70
  25. Tsuchiyama Y, Wada J, Zhang H, Morita Y, Hiragushi K, Hida K, Shikata K, Yamamura M, Kanwar YS, Makino H. Efficacy of galectins in the amelioration of nephrotoxic serum nephritis in Wistar Kyoto rats. Kidney Int. 2000;58(5):1941-52.
  26. Baba M, Wada J, Eguchi J, Hashimoto I, Okada T, Yasuhara A, Shikata K, Kanwar YS, Makino H. Galectin-9 inhibits glomerular hypertrophy in db/db diabetic mice via cell-cycle-dependent mechanisms. J Am Soc Nephrol. 2005;16(11):3222-34.
  27. Matsumoto R, Matsumoto H, Seki M, Hata M, Asano Y, Kanegasaki S, Stevens RL, Hirashima M. Human ecalectin, a variant of human galectin-9, is a novel eosinophil chemoattractant produced by T lymphocytes. J Biol Chem. 1998;273(27):16976-84

Acknowledgements

The CFG is grateful to the following PIs for their contributions to this wiki page: Linda Baum, Richard Cummings

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