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• W2146222746 abstract "Leishmania parasites are the causative agents of leishmaniasis, manifesting itself in a species-specific manner. The glycan epitopes on the parasite are suggested to be involved in the Leishmania pathogenesis. One of such established species-unique glycan structures is the poly-β-galactosyl epitope (Galβ1–3)n found on L. major, which can develop cutaneous infections with strong inflammatory responses. Interestingly, the polygalactosyl epitope is also suggested to be involved in the development of the parasites in its host vector, sand fly. Thus, the recognition of the galactosyl epitope by lectins expressed in host or sand fly should be implicated in the species-specific manifestations of leishmaniasis and in the parasite life cycle, respectively. We recently reported that one host β-galactoside-binding protein, galectin-3, can distinguish L. major from the other species through its binding to the poly-β-galactosyl epitope, proposing a role for galectin-3 as an immunomodulator that could influence the L. major-specific immune responses in leishmaniasis. Here we report that galectin-9 can also recognize L. major by binding to the L. major-specific polygalactosyl epitope. Frontal affinity analysis with different lengths of poly-β-galactosyllactose revealed that the galectin-9 affinity for polygalactose was enhanced in proportion to the number of Galβ1–3 units present. Even though both galectins have comparable affinities toward the polygalactosyl epitopes, only galectin-9 can promote the interaction between L. major and macrophages, suggesting distinctive roles for the galectins in the L. major-specific development of leishmaniasis in the host. Leishmania parasites are the causative agents of leishmaniasis, manifesting itself in a species-specific manner. The glycan epitopes on the parasite are suggested to be involved in the Leishmania pathogenesis. One of such established species-unique glycan structures is the poly-β-galactosyl epitope (Galβ1–3)n found on L. major, which can develop cutaneous infections with strong inflammatory responses. Interestingly, the polygalactosyl epitope is also suggested to be involved in the development of the parasites in its host vector, sand fly. Thus, the recognition of the galactosyl epitope by lectins expressed in host or sand fly should be implicated in the species-specific manifestations of leishmaniasis and in the parasite life cycle, respectively. We recently reported that one host β-galactoside-binding protein, galectin-3, can distinguish L. major from the other species through its binding to the poly-β-galactosyl epitope, proposing a role for galectin-3 as an immunomodulator that could influence the L. major-specific immune responses in leishmaniasis. Here we report that galectin-9 can also recognize L. major by binding to the L. major-specific polygalactosyl epitope. Frontal affinity analysis with different lengths of poly-β-galactosyllactose revealed that the galectin-9 affinity for polygalactose was enhanced in proportion to the number of Galβ1–3 units present. Even though both galectins have comparable affinities toward the polygalactosyl epitopes, only galectin-9 can promote the interaction between L. major and macrophages, suggesting distinctive roles for the galectins in the L. major-specific development of leishmaniasis in the host. Innate immunity plays a critical role in the protection against pathogen invasion. The recognition of invading pathogens initiates different innate immune responses, including elimination of pathogens, antigen presentation to initiate acquired immunity, and elicitation of signaling cascades, which regulate local/systemic immune responses (1Medzhitov R. Janeway Jr., C.A. Science. 2002; 296: 298-300Crossref PubMed Scopus (1639) Google Scholar). Some of the C-type (calcium-dependent) lectins have been demonstrated to play critical roles as pathogen recognition molecules (2Gordon S. Cell. 2002; 111: 927-930Abstract Full Text Full Text PDF PubMed Scopus (923) Google Scholar). For example, soluble collectins bind to pathogens resulting in their efficient removal (3Hoffmann J.A. Kafatos F.C. Janeway C.A. Ezekowitz R.A. Science. 1999; 284: 1313-1318Crossref PubMed Scopus (2137) Google Scholar). Another example is the membrane-associated dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). DC-SIGN binds to “self ” glycans as well as pathogen-associated glycans on Mycobacterium tuberculosis, which results in the suppression of dendritic cell functions (4Geijtenbeek T.B. Torensma R. van Vliet S.J. van Duijnhoven G.C. Adema G.J. van Kooyk Y. Figdor C.G. Cell. 2000; 100: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1443) Google Scholar, 5Geijtenbeek T.B. Kwon D.S. Torensma R. van Vliet S.J. van Duijnhoven G.C. Middel J. Cornelissen I.L. Nottet H.S. KewalRamani V.N. Littman D.R. Figdor C.G. van Kooyk Y. Cell. 2000; 100: 587-597Abstract Full Text Full Text PDF PubMed Scopus (2026) Google Scholar, 6Appelmelk B.J. van Die I. van Vliet S.J. Vandenbroucke-Grauls C.M. Geijtenbeek T.B. van Kooyk Y. J. Immunol. 2003; 170: 1635-1639Crossref PubMed Scopus (378) Google Scholar, 7Maeda N. Nigou J. Herrmann J.L. Jackson M. Amara A. Lagrange P.H. Puzo G. Gicquel B. Neyrolles O. J. Biol. Chem. 2003; 278: 5513-5516Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 8Geijtenbeek T.B. van Vliet S.J. Koppel E.A. Sanchez-Hernandez M. Vandenbroucke-Grauls C.M. Appelmelk B. van Kooyk Y. J. Exp. Med. 2003; 197: 7-17Crossref PubMed Scopus (894) Google Scholar). Thus, pathogen recognition by a lectin could induce distinct immune responses.In contrast to those C-type lectins, S-type lectins, more recently termed galectins (9Drickamer K. J. Biol. Chem. 1988; 263: 9557-9560Abstract Full Text PDF PubMed Google Scholar, 10Barondes S.H. Castronovo V. Cooper D.N. Cummings R.D. Drickamer K. Feizi T. Gitt M.A. Hirabayashi J. Hughes C. Kasai K. Cell. 1994; 76: 597-598Abstract Full Text PDF PubMed Scopus (1085) Google Scholar), have been considered as lectins that bind to self glycans because galectins bind preferentially to polylactosamine chains attached to the host cell glycoproteins/lipids (11Sato S. Hughes R.C. J. Biol. Chem. 1992; 267: 6983-6990Abstract Full Text PDF PubMed Google Scholar, 12Sparrow C.P. Leffler H. Barondes S.H. J. Biol. Chem. 1987; 262: 7383-7390Abstract Full Text PDF PubMed Google Scholar). However, a number of pathogenic entities express glycoconjugates containing β-galactosides, either similar to those of the host cells or unique to pathogens, such as the polygalactosyl epitope on protozoa Leishmania major (13Turco S.J. Descoteaux A. Annu. Rev. Microbiol. 1992; 46: 65-94Crossref PubMed Google Scholar, 14Mandrell R.E. Apicella M.A. Lindstedt R. Leffler H. Methods Enzymol. 1994; 236: 231-254Crossref PubMed Scopus (42) Google Scholar). Thus, questions such as whether some galectins can play roles as “nonself ” or pathogen-recognizing molecules have recently emerged. Indeed we and others (15Mey A. Leffler H. Hmama Z. Normier G. Revillard J.P. J. Immunol. 1996; 156: 1572-1577PubMed Google Scholar, 16Pelletier I. Sato S. J. Biol. Chem. 2002; 277: 17663-17670Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 17John C.M. Jarvis G.A. Swanson K.V. Leffler H. Cooper M.D. Huflejt M.E. Griffiss J.M. Cell Microbiol. 2002; 4: 649-662Crossref PubMed Scopus (60) Google Scholar) suggest that galectin-3 can recognize such pathogens as Klebsiella pneumoniae and L. major.Galectin molecules contain one or two carbohydrate recognition domains (CRDs), 1The abbreviations used are: CRD(s), carbohydrate recognition domain(s); FAC, frontal affinity chromatography; Lac, lactose; LPG, lipophosphoglycan; PA, pyridylamine; PBS, phosphate-buffered saline; SRP, surface plasmon resonance. 1The abbreviations used are: CRD(s), carbohydrate recognition domain(s); FAC, frontal affinity chromatography; Lac, lactose; LPG, lipophosphoglycan; PA, pyridylamine; PBS, phosphate-buffered saline; SRP, surface plasmon resonance. which have a relatively similar tertiary structure with conserved peptide sequence elements, although they differ in the protein quaternary structure, i.e. the presentation of the CRDs in the molecules. The arrangement of the CRD(s) within the molecule is unique for each galectin, which are consequently classified into three types: prototype, tandem-repeat, and chimera type (18Hirabayashi J. Kasai K. Glycobiology. 1993; 3: 297-304Crossref PubMed Scopus (453) Google Scholar). Prototype galectins contain one CRD and exist as a monomer (such as galectin-7) or dimer (such as galectin-1, -2), whereas tandem-repeat galectins, such as galectin-8 and -9, contain two CRDs connected by a short linker region. Galectin-3 is chimeric type galectin, composed of one CRD and a non-CRD domain, which regulates the multivalence status of galectin-3. Galectins have different affinities for the substituted lactosamine residues (11Sato S. Hughes R.C. J. Biol. Chem. 1992; 267: 6983-6990Abstract Full Text PDF PubMed Google Scholar, 12Sparrow C.P. Leffler H. Barondes S.H. J. Biol. Chem. 1987; 262: 7383-7390Abstract Full Text PDF PubMed Google Scholar, 19Ahmed H. Allen H.J. Sharma A. Matta K.L. Biochemistry. 1990; 29: 5315-5319Crossref PubMed Scopus (69) Google Scholar) because of the structural differences in detailed carbohydrate binding pockets (20Lobsanov Y.D. Gitt M.A. Leffler H. Barondes S.H. Rini J.M. J. Biol. Chem. 1993; 268: 27034-27038Abstract Full Text PDF PubMed Google Scholar, 21Seetharaman J. Kanigsberg A. Slaaby R. Leffler H. Barondes S.H. Rini J.M. J. Biol. Chem. 1998; 273: 13047-13052Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 22Henrick K. Bawumia S. Barboni E.A. Mehul B. Hughes R.C. Glycobiology. 1998; 8: 45-57Crossref PubMed Scopus (57) Google Scholar). Hence, it is possible that some glycoconjugates, including nonself pathogenic glycans, could be recognized by various galectins, whereas others could be relatively specific to a particular galectin (23Perillo N.L. Marcus M.E. Baum L.G. J. Mol. Med. 1998; 76: 402-412Crossref PubMed Scopus (579) Google Scholar). It has also been speculated that the differences in the galectin CRD arrangements influence the cross-linking mode of the galectin ligand and contribute to their functions (24Rabinovich G.A. Rubinstein N. Toscano M.A. Biochim. Biophys. Acta. 2002; 1572: 274-284Crossref PubMed Scopus (202) Google Scholar, 25Sato S. Trends Glycosci. Glycotechnol. 2002; 14: 285-301Crossref Scopus (28) Google Scholar, 26Cooper D.N. Biochim. Biophys. Acta. 2002; 1572: 209-231Crossref PubMed Scopus (514) Google Scholar), although such a possibility remains to be investigated.Recent studies demonstrate that some galectins act as immunomodulators and cell adhesion modulators (25Sato S. Trends Glycosci. Glycotechnol. 2002; 14: 285-301Crossref Scopus (28) Google Scholar, 27Lowe J.B. Cell. 2001; 104: 809-812Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 28Liu F.T. Clin. Immunol. 2000; 97: 79-88Crossref PubMed Scopus (185) Google Scholar, 29Rabinovich G.A. Riera C.M. Landa C.A. Sotomayor C.E. Braz. J. Med. Biol. Res. 1999; 32: 383-393Crossref PubMed Scopus (28) Google Scholar, 30Perillo N.L. Pace K.E. Seilhamer J.J. Baum L.G. Nature. 1995; 378: 736-739Crossref PubMed Scopus (935) Google Scholar, 31Perillo N.L. Uittenbogaart C.H. Nguyen J.T. Baum L.G. J. Exp. Med. 1997; 185: 1851-1858Crossref PubMed Scopus (266) Google Scholar, 32Sato S. Ouellet N. Pelletier I. Simard M. Rancourt A. Bergeron M.G. J. Immunol. 2002; 168: 1813-1822Crossref PubMed Scopus (208) Google Scholar, 33Matsumoto R. Matsumoto H. Seki M. Hata M. Asano Y. Kanegasaki S. Stevens R.L. Hirashima M. J. Biol. Chem. 1998; 273: 16976-16984Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 34Cooper D.N. Barondes S.H. Glycobiology. 1999; 9: 979-984Crossref PubMed Scopus (282) Google Scholar, 35Hughes R.C. Biochem. Soc. Trans. 1997; 25: 1194-1198Crossref PubMed Scopus (108) Google Scholar). We recently found that galectin-3 binds to L. major through L. major-specific poly-β-galactosyl epitope (Galβ1–3)n (16Pelletier I. Sato S. J. Biol. Chem. 2002; 277: 17663-17670Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Galectin-3 binds to L. major but not to the other species, L. donovani or L. mexicana, which do not express the polygalactosyl epitope (16Pelletier I. Sato S. J. Biol. Chem. 2002; 277: 17663-17670Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). It has been proposed that the species-specific manifestations of Leishmania in human, ranging from fatal visceral infections to mild cutaneous lesions, are accounted for in part by the species-specific glycoconjugates (see Fig. 1) (36Berman J.D. Clin. Infect. Dis. 1997; 24: 684-703Crossref PubMed Scopus (704) Google Scholar, 37Moody S.F. Handman E. McConville M.J. Bacic A. J. Biol. Chem. 1993; 268: 18457-18466Abstract Full Text PDF PubMed Google Scholar, 38Moody S.F. Acta Tropica. 1993; 53: 184-204Crossref Scopus (24) Google Scholar). Interestingly, the L. major-specific polygalactosyl epitope is implicated in two stages of the Leishmania life cycle: the host-pathogen interactions (39Handman E. Goding J.W. EMBO J. 1985; 4: 329-336Crossref PubMed Scopus (150) Google Scholar, 40Kelleher M. Bacic A. Handman E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6-10Crossref PubMed Scopus (69) Google Scholar) and the attachment of Leishmania to its sand fly vector (41Butcher B.A. Turco S.J. Hilty B.A. Pimenta P.F. Panunzio M. Sacks D.L. J. Biol. Chem. 1996; 271: 20573-20579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 42Pimenta P.F. Turco S.J. McConville M.J. Lawyer P.G. Perkins P.V. Sacks D.L. Science. 1992; 256: 1812-1815Crossref PubMed Scopus (200) Google Scholar). It is likely that such roles of the polygalactosyl epitopes are mediated by β-galactoside-binding proteins such as galectins. However, galectin-3 is hitherto the only lectin that has been shown to have an affinity for this unique epitope (16Pelletier I. Sato S. J. Biol. Chem. 2002; 277: 17663-17670Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Various galectins are expressed in dermal tissues (initial site of the Leishmania infection) which is composed of phagocytic cells, fibroblasts, and epithelial cells as well as emigrated lymphocytes. Thus, it is possible that other galectins recognize L. major. Because each galectin possesses a unique structure, binding of different galectins to the species-specific ligands could affect the different manifestations of the infections, especially the initial immune responses. However, it remains to be determined whether other galectins have any affinities to the L. major-specific glycan epitopes and whether interactions between the parasites and different galectins influence the development of immune responses in a manner different from that of galectin-3.Here we show that galectin-9 can bind to L. major in a species-specific manner through L. major-specific poly-β-galactose (Galβ1–3)1∼4. Even though that both galectin-3 and -9 recognize L. major, only galectin-9 promotes L. major-macrophage interaction. Thus the data suggest the distinctive roles of galectin-9 in the L. major-specific pathogenesis of leishmaniasis.MATERIALS AND METHODSReagents—Chemicals and other reagents were obtained from Sigma unless specified otherwise. Antibodies against galectin-1, -3, and -9 were raised in our laboratories by injecting recombinant galectins to rabbits (16Pelletier I. Sato S. J. Biol. Chem. 2002; 277: 17663-17670Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 33Matsumoto R. Matsumoto H. Seki M. Hata M. Asano Y. Kanegasaki S. Stevens R.L. Hirashima M. J. Biol. Chem. 1998; 273: 16976-16984Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar).Leishmania and Cells—L. major strain LV39 and L. donovani strain 1S clone 2D were kindly provided by Dr. M. Olivier (CRI-CHUL, Qué-bec, Canada). Those Leishmania promastigotes were grown in SDM-79 medium without biopterin (43Cunningham M.L. Titus R.G. Turco S.J. Beverley S.M. Science. 2001; 292: 285-287Crossref PubMed Scopus (107) Google Scholar) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen), antibiotics (100 μg/ml penicillin, 100 μg/ml streptomycin), 2 mm l-glutamine, and 5 μg/ml hemin, as published before (16Pelletier I. Sato S. J. Biol. Chem. 2002; 277: 17663-17670Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). L. major mutant Spock, which lacks the activity of β1,3-galactosyltransferase, and its wild type Friedlin V1 (generously provided by Dr. D. Sacks (NIAID)) were grown in medium 199 (Invitrogen) supplemented with 20% heat-inactivated fetal calf serum, 20 mm HEPES, 100 μm adenine, 2 mm glutamine, and antibiotics (41Butcher B.A. Turco S.J. Hilty B.A. Pimenta P.F. Panunzio M. Sacks D.L. J. Biol. Chem. 1996; 271: 20573-20579Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Leishmania at stationary phase were used for experiments.For some experiments, the promastigote population was separated into procyclic- and metacyclic-rich fractions using Ficoll gradients as published by Spath and Beverley (44Spath G.F. Beverley S.M. Exp. Parasitol. 2001; 99: 97-103Crossref PubMed Scopus (277) Google Scholar). Briefly, stationary phase cultures of Leishmania (∼109 parasites) were centrifuged at 5,000 × g for 10 min at room temperature and resuspended in 3 ml of serum-free Dulbecco's modified Eagle's medium (Invitrogen). The cell suspensions were then loaded onto a Ficoll gradient composed, from the bottom, of 2 ml of 20%, 2 ml of 15%, 2 ml of 10%, and 2 ml of 5% Ficoll diluted in 4× serum-free medium 199. The gradient was next centrifuged at 1,300 × g for 10 min at room temperature. The metacyclic promastigotes were recovered on the top of the 10% Ficoll layer, and the procyclic ones were recovered in the 15% Ficoll layer. The parasites in those fractions were washed twice with RPMI 1640 complemented with 25 mm HEPES and used immediately for the experiments.Purification of Galectins, Preparation of Galectin Affinity Columns and Alexa 488-labeled Galectins—Recombinant human galectins were purified as described previously (16Pelletier I. Sato S. J. Biol. Chem. 2002; 277: 17663-17670Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 32Sato S. Ouellet N. Pelletier I. Simard M. Rancourt A. Bergeron M.G. J. Immunol. 2002; 168: 1813-1822Crossref PubMed Scopus (208) Google Scholar, 45Oka T. Murakami S. Arata Y. Hirabayashi J. Kasai K. Wada Y. Futai M. Arch. Biochem. Biophys. 1999; 361: 195-201Crossref PubMed Scopus (49) Google Scholar, 46Matsushita N. Nishi N. Seki M. Matsumoto R. Kuwabara I. Liu F.T. Hata Y. Nakamura T. Hirashima M. J. Biol. Chem. 2000; 275: 8355-8360Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar).To prepare galectin-immobilized columns, the recombinant galectins were dissolved in 0.2 m NaHCO3, pH 8.3, containing 0.5 m NaCl and 0.1 m lactose and coupled to HiTrap NHS-activated columns (Amersham Biosciences) according to the manufacturer's instructions. After washing and deactivating excess active groups by ethanolamine, the galectin-immobilized agarose beads were removed from the cartridge. Each gel matrix was suspended in PBS containing 1 mm EDTA, and the slurry was packed into a stainless steel column (4.0 × 10 mm; bed column, 0.126 ml, GL Sciences, Inc.).Alexa 488-labeled galectins were prepared according to the manufacturer's instructions (Molecular Probes) with a slight modification. Briefly, recombinant galectin in PBS containing 5 mm lactose and 20 mm HEPES was first transferred into the manufacturer-supplied bottle containing Alexa 488 carboxylic acid, succinimidyl ester, dilithium salt (Molecular Probes), and incubated at room temperature for 1 h. After terminating the labeling reaction by adding Tris-HCl, pH 7.5 (final concentration 20 mm), the reaction mixture was first applied to a PD10 column (Amersham Biosciences) to remove lactose and excess dye, and the void fraction was collected to obtain Alexa 488-labeled galectin. Alexa 488-labeled galectin was purified further by asialofetuin-agarose as published before (32Sato S. Ouellet N. Pelletier I. Simard M. Rancourt A. Bergeron M.G. J. Immunol. 2002; 168: 1813-1822Crossref PubMed Scopus (208) Google Scholar).Galectin Binding to L. major Parasites—Leishmania parasites (1 × 107) were incubated with recombinant galectin (1 ∼ 2 μm) in 250 μl of buffer consisting of 125 μl of serum-free RPMI 1640 containing 25 mm HEPES and 125 μl of PBS in the presence or absence of 100 mm lactose at 4 °C for 30 min as described previously (16Pelletier I. Sato S. J. Biol. Chem. 2002; 277: 17663-17670Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). When the binding assays were performed in the presence of lactose, the sodium chloride concentration in the PBS was adjusted to maintain appropriate osmolarity, i.e. 317 mosmol/liter. After the incubation, parasite-free supernatants were obtained by spinning at 4,500 × g for 7 min. The supernatants were fractionated by SDS-PAGE, and proteins in the gels were stained with Coomassie Brilliant Blue. Alternatively, fractionated proteins on the SDS-polyacrylamide gels were transferred to the nitrocellulose filters, and galectin was detected by the appropriate antibody against galectin.For flow cytometric analysis, Leishmania were incubated as above with Alexa 488-labeled galectin for 15 min at 4 °C. Unbound galectin was removed by a brief wash with ice-cold PBS twice, and Leishmania were fixed in 2% formaldehyde containing PBS for 10 min at 4 °C and analyzed for the relative fluorescent intensity on a FACScalibur flow cytometer (BD Biosciences, San Jose, CA) as described previously (32Sato S. Ouellet N. Pelletier I. Simard M. Rancourt A. Bergeron M.G. J. Immunol. 2002; 168: 1813-1822Crossref PubMed Scopus (208) Google Scholar).Polygalactosyllactose Derivatives and Pyridylaminated (PA) Oligosaccharides—Polygalactosyllactose derivatives were purified from the milk of the tammar wallaby (Macropus eugenii; kindly provided by Dr. Michael Messer, Department of Biochemistry, University of Sydney, Australia) as described previously (47Messer M. Green B. Aust. J. Biol. Sci. 1979; 32: 519-531Crossref PubMed Scopus (111) Google Scholar, 48Messer M. Trifonoff E. Stern W. Collins J.G. Bradbury J.H. Carbohydr. Res. 1980; 83: 327-334Crossref PubMed Scopus (85) Google Scholar, 49Collins J.G. Bradbury J.H. Trifonoff E. Messer M. Carbohydr. Res. 1981; 92: 136-140Crossref PubMed Scopus (75) Google Scholar, 50Messer M. Urashima T. Trends Glycosci. Glycotechnol. 2002; 14: 153-176Crossref Scopus (49) Google Scholar). Briefly, 4 volumes of chloroform/methanol mixture (2:1, v/v) were added to the wallaby milk. After being thoroughly mixed and separated by centrifugation at 4 °C and 3,500 × g for 30 min, the methanol/water upper fraction containing oligosaccharides was obtained, and solvents were removed by evaporation followed by freeze-drying. The oligosaccharides (∼100 mg) were fractionated by gel filtration on a Sephadex G-25 column (100 × 2.5 cm, Amersham Biosciences), and polygalactosyllactose derivatives were purified further by gel filtration on a Bio-Gel P-4 (100 × 2.5 cm, Bio-Rad), as published before. The purified oligosaccharides were labeled with PA following the manufacturer's instructions (Takara Shuzo, Tokyo, Japan). The purity of PA-labeled oligosaccharides was confirmed as described previously (51Arata Y. Hirabayashi J. Kasai K. J. Biol. Chem. 2001; 276: 3068-3077Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).Frontal Affinity Chromatography (FAC) Analysis—FAC analysis was carried out as described previously (51Arata Y. Hirabayashi J. Kasai K. J. Biol. Chem. 2001; 276: 3068-3077Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 52Hirabayashi J. Arata Y. Kasai K. J. Chromatogr. 2000; 890: 261-271Crossref PubMed Scopus (64) Google Scholar, 53Hirabayashi J. Hashidate T. Arata Y. Nishi N. Nakamura T. Hirashima M. Urashima T. Oka T. Futai M. Muller W. Yagi F. Kasai K. Biochim. Biophys. Acta. 2002; 1572: 232-254Crossref PubMed Scopus (813) Google Scholar). Briefly, PA-oligosaccharide was dissolved in PBS containing 1 mm EDTA and applied to the column through a 2-ml sample loop connected to the Rheodyne 7725 injector. The sample loop and the column were immersed in a 20 °C water bath. The flow rate was controlled by a Shimadzu LC-10Advp pump at 0.25 ml/min. Elution of PA-oligosaccharide from the column was monitored by a Shimadzu PR10AxL fluorescence detector at 400 nm (excitation at 320 nm). The elution volume of PA-oligosaccharide of interest (Vf) was determined as described previously. The elution volume of PA-oligosaccharide, which has no affinity to galectins (V0), was determined by using PA-rhamnose.The total amount of immobilized galectin in the column, Bt is first determined by using PA-lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4GlcOPA) using the equation described previously, [A]0·(Vf−V0)=BtKd·(Vf−V0)(Eq. 1) where Kd is the dissociation constant between interacting biomolecules, Bt is the total amount of immobilized ligand, [A]0 is the initial concentration of the PA-oligosaccharide of interest (A), Vf is the elution volume of A, and V0 is the elution volume of PA-rhamnose, which has no affinity toward galectins. As in the assays employed in this study, [A]0, the initial concentration of the PA-oligosaccharides, was 10 nm, which was negligibly small compared with Kd. Thus, Vf approached the maximum value, Vf, which is independent of [A]0, and we used the following equation to obtain the values of Kd of each galectin toward a polygalactosyllactose derivative. Vf-V0=Bt/Kd(Eq. 2) Infection of Macrophages with L. major Parasites—Stationary phase L. major promastigotes at a cell concentration of 107 cells/ml were labeled with cell tracker green (4.65 μg/ml, Molecular Probes) following the manufacturer's instruction. J774A.1 macrophages (2 × 105 cells) were incubated in 0.5 ml of RPMI 1640 and 25 mm HEPES with the labeled parasites (5 × 106 cells) in the absence or presence (1.6 μm) of galectins and incubated for 30 min at 37 °C. Following the incubation, parasites that were not associated with macrophages were removed by washing three times with RPMI 1640 and 25 mm HEPES, and cells were then lysed in PBS containing 1% Triton X-100. Fluorescenceassociated macrophages, which corresponds to the number of L. major bound to or internalized by macrophages, were measured using a fluorometric plate reader (PerSeptive Biosystems).RESULTSAnalysis of the Binding Properties of Galectin-1 and -3 toward Polygalactosyllactose Derivatives by FAC—We have recently developed an assay system to assess the binding affinity of various galectins for oligosaccharides by employing FAC analysis with fluorescence-tagged oligosaccharides (53Hirabayashi J. Hashidate T. Arata Y. Nishi N. Nakamura T. Hirashima M. Urashima T. Oka T. Futai M. Muller W. Yagi F. Kasai K. Biochim. Biophys. Acta. 2002; 1572: 232-254Crossref PubMed Scopus (813) Google Scholar). Thus, we next established a system to search for lectins that can bind to and distinguish L. major from other Leishmania species through recognition of the poly-β-galactosyl epitope. To achieve this goal, galectin-1, which is ubiquitously expressed in various tissues, and galectin-3 were first used to assess the affinities for the epitope. For the FAC analysis, we used purified poly-β-galactosyllactose instead of L. major-derived lipophosphoglycans (LPGs) because the LPGs prepared from L. major are a mixture of various lengths of the polygalactosyl epitope (Galβ1–3)n, ranging from one to three repeats (54McConville M.J. Thomas-Oates J.E. Ferguson M.A. Homans S.W. J. Biol. Chem. 1990; 265: 19611-19623Abstract Full Text PDF PubMed Google Scholar) (R in Fig. 1). Various lengths of poly-β-galactosyllactose derivatives (Galβ1–3)nGalβ1–4Glc, were purified from the milk of the tammar wallaby (48Messer M. Trifonoff E. Stern W. Collins J.G. Bradbury J.H. Carbohydr. Res. 1980; 83: 327-334Crossref PubMed Scopus (85) Google Scholar, 49Collins J.G. Bradbury J.H. Trifonoff E. Messer M. Carbohydr. Res. 1981; 92: 136-140Crossref PubMed Scopus (75) Google Scholar), and those derivatives were labeled with PA residues. One of the PA-polygalactosyllactose derivatives dissolved in PBS-EDTA at a concentration of 10 nm was applied continuously to a column containing either galectin-1 or -3 agarose. Elution profiles of PA-labeled sugars from galectin-1 and -3 are shown collectively in Fig. 2. As control, PA-rhamnose, which does not show any affinity for galectins, was also applied to the column.Fig. 2Elution profiles of various PA-labeled poly-β-galactosyllactose derivatives after application to immobilized galectin-1 and -3 columns. PA-labeled poly-β-galactosyllactose derivatives were dissolved in EDTA-PBS at a concentration of 10 nm, and 2 ml of each solution was applied to the column (10 × 4.0 mm, 0.126 ml) through a 2-ml loop (inner diameter, 0.75 mm) at a flow rate of 0.25 ml/min at 20 °C. Each elution pattern of PA-labeled oligosaccharide was superimposed on that of PA-rhamnose so that the retardation could be seen. The structures of PA-oligosaccharide are shown above the elution patterns. To obtain the Kd and Bt values, the total amount of immobilized galectin in the column was calculated by analyzing the concentration dependence of retardation of PA-lacto-N-fucopentaose I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4GlcOPA) with Equation 1, as described previously (77Arata A. Sekiguchi M. Hirabayashi J. Kasai K. Biol. Pharm. Bull. 2001; 24: 14-18Crossref PubMed Scopus (5) Google Scholar). The Bt values of galectin-1 and -3 were 2.3 and 0.14 nmol, respe" @default.
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• W2146222746 date "2003-06-01" @default.
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• W2146222746 title "Specific Recognition of Leishmania major Poly-β-galactosyl Epitopes by Galectin-9" @default.
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• W2146222746 doi "https://doi.org/10.1074/jbc.m302693200" @default.
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