2Department of Endocrinology, Kagawa Medical University, Kagawa 761-0793, Japan; 3Plastic and Reconstructive Surgery, Kagawa Medical University, Kagawa 761-0793, Japan; 4Galpharma Co., Ltd., Kagawa 761-0703, Japan; 5Immunology and Immunopathology, Kagawa Medical University, Kagawa 761-0793, Japan; 6Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa 199-01, Japan; and 7Department of Plastic and Reconstructive Surgery, Tokyo Medical and Dental University, Tokyo 113-8510, Japan
Received on October 12, 2001; accepted on November 26, 2001.
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Abstract |
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Key words: chemoattractant/eosinophil/frontal affinity chromatography/galectin
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Introduction |
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Human galectin-9 was first identified as a putative autoantigen in patients with Hodgkins disease and was suggested to play an important role in the regulation of cellular interactions of the immune system (Tureci et al., 1997). Mouse galectin-9 has been cloned and characterized by Wada and Kanwar (1997)
. They showed that galectin-9 was capable of inducing apoptosis of thymocytes in vitro (Wada et al., 1997
). These reports, together with our previous finding that human ecalectin/galectin-9 has potent eosinophil chemoattractant (ECA) activity (Matsumoto et al., 1998
), suggest that galectin-9, in addition to other members of the galectin family (especially galectins-1 and -3), plays multiple roles in the immune system. To understand the mechanism underlying the immune-modulating effects of galectins, it is important to elucidate the function of each CRD in relation to its carbohydrate binding specificity.
In a previous study (Matsushita et al., 2000) we demonstrated that ECA activity appears to be unique to galectin-9. Among the four human galectins studied, galectins-1 and -3 had negligible ECA activity. Galectin-8 showed low ECA activity, that is, less than 10% of that of galectin-9. We also demonstrated that the carbohydrate binding activity of galectin-9 is indispensable for ECA activity. In addition, experiments involving the N- and C-terminal CRDs of galectin-9 showed that these CRDs may exist at least in part as dimers and/or multimers and exert comparable ECA activity that is substantially lower than that of full-length galectin-9. Based on these results, we postulated that galectin-9 exerts its ECA activity via cross-linking of galactoside-containing glycoconjugates on the surface of eosinophils. There are three possibilities for the interaction between the two CRDs and cell surface glycoconjugates: (1) the two domains bind to two distinct glycoconjugates; (2) the two domains bind to two identical glycoconjugates, and thus galectin-9 functions by inducing homo dimer/multimer formation of the molecules; and (3) the two domains bind to two different oligosaccharide moieties on the same glycoconjugates. In the present study we examined these possibilities by using mutant galectin-9 molecules consisting of two N- terminal CRDs (galectin-9NN) and two C-terminal CRDs (galectin-9CC) and by determining the carbohydrate binding specificities of the two CRDs. Moreover, three isoforms of galectin-9 with different linker peptides were used to examine the significance of the linker peptide structure as to ECA activity.
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Results |
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The ECA activity of galectin-9CC at 0.1 µM was similar to that of eotaxin-1 (100 ng/ml), a typical ECA. Galectin-9NN showed somewhat lower activity than that of galectin-9CC at this concentration. The overall dose-response data, however, were closely similar among the wild type (galectin-9M) and mutant galectins (Figure 3). In a separate experiment, we compared the ECA activity among the three isoforms of galectin-9 (Figure 4). The dose-response curve for galectin-9S, the isoform with the shortest linker peptide, was comparable with that for galectin-9M. As it was difficult to obtain a high concentration preparation, the ECA activity of galectin-9L at concentrations lower than 0.14 µM was measured. Galectin-9L showed similar ECA activity to that of the other isoforms over the concentration range tested.
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Discussion |
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The finding in this study that artificial tandem repeattype galectins, galectins-9NN and -9CC, showed ECA activity indistinguishable from that of wild-type galectin-9 is consistent with previous results concerning galectins-9NT and -CT. Although galectin-8 has low ECA activity, the tandem repeattype structure is not a sufficient condition for acquisition of the activity, because the two galectin-1 molecules linked by a linker peptide of galectin-8 were completely inactive in the ECA activity assay (unpublished data). These observations indicate that the ECA activity depends on the specific sugar-binding character of an individual CRD and that the two CRDs of galectin-9 interact with the same or a closely related sugar chain when it acts as an ECA.
We performed detailed analysis of the sugar-binding specificity of galectins-9NT and -9CT using a panel of 33 oligosaccharides and frontal affinity chromatography. Galectins-9NT and -9CT exhibited comparable binding affinity for most glycoprotein N-glycans, the highest affinity being for N-linked tetraantennary complex-type glycans with N-acetyllactosamine units. Fucosylation and sialylation reduced the affinity of the oligosaccharides for both CRDs. On the other hand, the binding affinity for nearly half of the glycolipid glycans tested varied by about one order of magnitude between the two CRDs. In addition, a tetraantennary glycan strongly inhibited the ECA activity of galectin-9 but not that of eotaxin. Collectively, these results suggest that galectin-9 binds to eosinophil surface glycoprotein(s) via tri- and/or tetraantennary N-linked glycans with N-acetyllactosamine units. This interaction may result in dimerization/multimerization of the cell surface glycoproteins/receptors and thereby initiate intracellular signaling pathways linked to chemotactic responses.
It is noteworthy that galectin-9NT but not galectin-9CT shows exceptionally high affinity for Forssman pentasaccharide among the glycolipid glycans examined. It is postulated that Forssman antigen is involved in the pathogenesis of autoimmune diseases such as Graves disease and Hashimotos thyroiditis (Ariga et al., 1991). It is possible that recognition of Forssman antigen by the N-terminal CRD of galectin-9 is an important step for some unknown immune-modulating activity of galectin-9.
Wada and Kanwar (1997) reported the presence of an isoform (intestine isoform) of mouse galectin-9 with a 31-amino-acid insertion in the linker peptide region. When analyzing galectin-9 cDNA clones expressed in Jurkat cells, we found three isoforms of human galectin-9 (galectins-9S, -9M, and -9L): galectin-9M corresponds to authentic galectin-9, and galectins-9L and -9S have a 32-amino-acid insertion and a 12-amino-acid deletion, respectively, in the linker peptide. The expression of these isoforms was detected at the protein level in Jurkat cells (data not shown). Although the linker peptide of galectin-9L is about four times larger than that of galectin-9S, the three isoforms showed comparable ECA activity. The amino acid insertions of galectins-9L and -9M, compared to galectin-9S, are characterized by high proline contents, that is, 13 proline residues out of 44 amino acid residues (galectin-9L) and 5 out of 12 (galectin-9M). Because glycine and proline residues are common within the beta-turn, it is not likely that the linker peptides can form an extended structure. The Chou-Fasman prediction algorithm supports this premise. Hence, in spite of the large difference in linker peptide size, there may be little difference among the isoforms in configuration between N- and C-terminal CRDs. We cannot, however, exclude the possibility that the three isoforms play different roles in yet unknown function(s) of galectin-9, that is, other than ECA activity.
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Materials and methods |
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Expression and purification of recombinant proteins
E. coli BL21 cells carrying each expression plasmid were grown in 2x YT medium supplemented with 2% (w/v) glucose and 100 µg/ml ampicillin to an optical density of 0.7 at 600 nm. The expression of fusion proteins was induced by the addition of 0.1 mM isopropyl-ß-D-thiogalactopyranoside, and the cultures were continued for 2 h at 37°C (galectins-9S, -9M, -9NN and -9CC) or for 3 h at 30°C (galectin-9L). The cell pellet obtained from 500-ml culture was suspended in 90 ml of 10 mM TrisHCl (pH 7.5) containing 0.5 M NaCl, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride, and then sonicated for 10 min. The sonicate was supplemented with 10 ml of 10% (w/v) Triton X-100 and then stirred for 30 min at 4°C, followed by centrifugation at 15,000 x g for 30 min. The resulting supernatant was subjected to lactose-agarose (Seikagaku, Tokyo) affinity chromatography. Protein concentrations were determined with BCA protein assay reagent (Pierce) and bovine serum albumin as a standard.
In vitro chemotaxis
ECA activity was evaluated in vitro as described previously (Matsumoto et al., 1998; Hirashima et al., 1992
). Briefly, CD16-negative-eosinophils were enriched by applying peripheral blood leukocytes from healthy volunteers to a discontinuous density gradient of Percoll (Amersham Pharmacia Biotech), followed by immunomagnetic treatment of the cells with anti-CD16 immunoglobulin (Dako S., Glostrup, Denmark). The purity and viability of the purified eosinophils were >97% and >95%, respectively. ECA activity was evaluated using a 48-well chamber (Neuro Probe) containing a polyvinylpyrrolidone-free membrane of 5 µm pore size. Human eosinophils (0.51 x 106/ml) and various concentrations of a test chemoattractant were placed in the top and bottom chambers, respectively. Each assay was performed in triplicate. After 12-h incubation at 37°C under a humidified atmosphere of 5% CO2, the membrane separating the two chambers was removed and placed in Diff-Quick stain (Baxter Healthcare). Stained eosinophils were counted under a microscope. Human eotaxin-1 (Seikagaku) was used as a control.
Hemagglutination assays
Hemagglutination activity was assessed by the method of Nowak et al. (1976). Briefly, assay samples were prepared by serial twofold dilution of recombinant proteins in a 96-well microtiter plate. After the addition of bovine serum albumin and glutaraldehyde-fixed trypsin-treated rabbit erythrocytes to final concentrations of 0.25% (w/v) and 1% (v/v), respectively, the reaction mixture was incubated for 1 h at room temperature. The minimum concentration required for hemagglutination was visually determined.
Frontal affinity chromatography
The interactions between the N- and C-terminal CRDs of galectin-9 and synthetic oligosaccharides were studied by frontal affinity chromatography. The recombinant proteins dissolved in 0.1 M NaHCO3 (pH 8.3) containing 0.25 M NaCl and 0.1 M lactose were coupled to HiTrap NHS-activated column following the manufacturers instructions. The galectin-immobilized agarose beads were taken out from the cartridge and then packed into a stainless steel column (4 x 10 mm). Determination of the elution volume of the analyte (PA-oligosaccharides) and the calculation of Kd value were carried out as described previously (Kasai and Oda, 1986; Hirabayashi et al., 2000
; Arata et al., 2001
).
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Acknowledgment |
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Abbreviations |
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Footnotes |
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References |
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