Specific recognition and cleavage of galectin-3 by Leishmania major through species-specific polygalactose epitope.

Lipophosphoglycan is a major surface molecule of Leishmania, protozoa parasites, which are the causative agents of leishmaniasis, a disease that annually afflicts millions of people worldwide. The oligosaccharide structures of lipophosphoglycan varies among species, and epitopes of these species-specific oligosaccharides are suggested to be implicated in the interaction of Leishmania with macrophages as well as species-specific tissue tropism observed in leishmaniasis. The recognition of the species-specific variation of oligosaccharides is likely to be mediated by host carbohydrate-binding proteins, lectins, but the identities of the lectins remain elusive. Galectin-3 is a mammalian soluble beta-galactoside-binding lectin and is expressed in macrophages, dendritic cells, and keratinocytes, as well as fibroblasts, all of which are present in the site of Leishmania infection. In this paper, we found that galectin-3 binds to lipophosphoglycan of Leishmania major but not to those of Leishmania donovani through L. major-specific polygalactose epitopes. Association of galectin-3 with L. major led to the cleavage of galectin-3, resulting in truncated galectin-3 containing the C-terminal lectin domain but lacking the N-terminal domain implicated in lectin oligomerization. This cleavage was inhibited by the galectin-3 antagonist lactose, as well as 1,10-ortho-phenanthroline, suggesting that galectin-3 is cleaved by zinc metalloproteases after its binding to lipophosphoglycans. The modulation of various innate immunity reactions by galectin-3 is affected by its oligomerization; therefore, we propose the L. major-specific truncation of galectin-3 may contribute to the species-specific immune responses induced by Leishmania.

Leishmaniasis is a tropical parasitic affliction affecting populations in Asia, Africa, South America, and southern Europe, and it is listed by the World Health Organization as one of the six most important parasitic diseases worldwide (1). Depending on the species of Leishmania, the disease manifests itself as visceral, mucosal (rarely), or cutaneous (1,2). In the initial stages of infection, extracellular, flagellated Leishmania (called promastigotes) are transmitted to humans through the bite of sand flies. The promastigotes are engulfed by macrophages, where they differentiate to non-flagellated amastigotes and multiply (1,3). Among the several Leishmania species, Leishmania donovani infection results in a visceral form of the disease, such as hepatosplenomegaly and a very limited inflammatory response at the primary site of infection (1). On the other hand, infection by Leishmania major develops in dermal tissues as cutaneous lesions (often non-lethal), with massive recruitment of leukocytes to the site of infection, suggesting that the cutaneous forms of the disease are correlated with a vigorous inflammatory response (1). It has been proposed that the difference in the induction of initial immune responses between L. major and L. donovani is accounted for partly by variation in molecules expressed on the surface of these Leishmania and by the interaction of such species-specific molecules with tissue macrophages or various cells present at the site of infection (2).
Two major molecules expressed on the surface of Leishmania are leishmanolysin (gp63) (5 ϫ 10 5 molecules/cell) and lipophosphoglycan (LPG) 1 (1ϳ5 ϫ 10 6 molecules/cell) (2). Leishmanolysin is a highly conserved glycosylphosphatidylinositolanchored membrane protein and a member of the metzincin class zinc proteinase family, which includes mammalian matrix metalloproteases-2 and 9 (4 -6). In contrast to leishmanolysin, the structure of LPG varies dramatically among Leishmania species (7)(8)(9)(10). LPG consists of four domains (Fig. 1A), a small oligosaccharide cap structure, a repeating phosphorylated (P-) saccharide region, phosphosaccharide (P-saccharide) core, and a phosphatidylinositol anchor (2,(7)(8)(9)(11)(12)(13). Structural analyses of LPG from several species of Leishmania reveal the variability of sequences in both the cap structure (Fig.  1A, see the structures of X) and the oligosaccharide substituents of the repeating P-saccharide units (Fig. 1A, see the structures of R) among species (2,12,13). The species-specific epitopes of LPGs have been suggested to be implicated in the species specificity of Leishmania (12,13). In particular, the L. major-specific epitopes, ␤-polygalactose residues (Gal␤1-3) n on the repeating P-saccharide units have been implicated both in the adhesion of L. major to the midgut of its sand fly vector (14,15) and in the interaction of L. major with macrophages (16,17). However, the receptor proteins that have affinity for the L. major-specific ␤-polygalactose epitope have not yet been well defined, as all macrophage receptors identified to date recognize common epitopes present on the surface of both L. major and L. donovani (18).
Galectins belong to a ␤-galactoside-binding protein family defined by conserved peptide sequence elements involved in carbohydrate binding activity (19 -21). Up to 11 galectins (galectin-1-galectin-11) have been found in mammals, as well as many in other phyla including birds, amphibians, fish, nematodes, sponges, and fungi. Of particular interest with regard to L. major infection is galectin-3, which is expressed in and released from macrophages (22)(23)(24), dendritic cells (25), and keratinocytes (26), all of which are present in dermal tissues, the prime infection sites of Leishmania. This lectin binds ␤-galactoside-containing glycoconjugates, particularly those with a polylactosamine structure (27,28). Galectin-3 is a soluble ϳ30-kDa protein composed of two domains, a C-terminal carbohydrate recognition domain (CRD, lectin domain) and an unique N-terminal domain containing multiple repeats of a sequence rich in glycine, proline, and tyrosine (19 -21). Upon binding to glycoconjugate ligands at the cell surface, galectin-3 molecules are able to oligomerize through their N-terminal multiple repeating domains (29,30). Oligomerization of galectin-3 causes cross-linking of surface glycoproteins, leading to the stable association of galectin-3 to its receptors (19 -21). Such crosslinking is known to trigger signal transduction cascades that are involved in innate immune responses (19 -21, 31). Another consequence of galectin-3-mediated cross-linking of galectin-3 ligands is cellular adhesion; galectin-3 cross-links cells expressing its ligands, therefore leading to cell-cell adhesion (32)(33)(34)(35). More recently, it has been suggested that the persistent presence of galectin-3 on the cell surface of leukocytes through galectin-3 oligomerization results in the formation of multivalent surface galectin-3-glycoprotein lattices that have the potential to restrict the movement of certain receptors involved in immune responses (31,36,37). Together, these data suggest that galectin-3 plays several roles in immune response. However, whether galectin-3 is implicated in immune response during leishmaniasis has not been investigated.
Here, we demonstrate that galectin-3 can distinguish L. major from L. donovani by recognizing L. major-specific polygalactose epitopes. To the best of our knowledge, this is the first report suggesting that a host factor can distinguish between different species of Leishmania. The association of galectin-3 to L. major leads to the cleavage of galectin-3, and this Leishmania species-specific cleavage of cell-associated galectin-3 was also observed in L. major-infected macrophages. This truncated galectin-3 lacks the N-terminal domain crucial for galectin-3 oligomerization, which is prerequisite to the majority of galectin-3 activities. Thus, we propose that L. major-mediated formation of truncated galectin-3 may be one of the initial steps that initiate massive inflammatory responses at the early stage of L. major infection.

MATERIALS AND METHODS
Reagents-Chemicals and other reagents were obtained from Sigma unless specified otherwise. Protease inhibitors were from Roche Molecular Biochemicals.
Leishmania and Cells-L. major strain LV39, L. donovani strain 1S clone 2D, and Leishmania mexicana were kindly provided by Dr. M. Olivier (CRI-CHUL, Québec, Canada). Those Leishmania promastigotes were grown in SDM-79 medium (38) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen), antibiotics (100 g/ml penicillin, 100 g/ml streptomycin), L-glutamine (2 mM), and hemin (5 g/ml). L. major mutant Spock, which lacks the activity of ␤1,3-galactosyltransferase, and its wild type Friedlin V1 were generously provided by Dr. D. Sacks (NIAID, National Institutes of Health, Bethesda, MD) and were grown in M199 supplemented with 20% heat-inactivated fetal calf serum (Invitrogen), 20 mM HEPES, 100 M adenine, 2 mM glutamine, and antibiotics (15 was raised in our laboratory by injecting CRD, which was prepared by the method reported (30), to rabbits. Galectin-3-Recombinant human galectin-3 was purified as described previously with modification (27,35). Briefly, a clone of Escherichia coli JM109 containing an expression plasmid of human galectin-3 was used for purification. Two liters of overnight bacteria culture medium were incubated with 1 mM isopropyl-1-thio-␤-D-galactopyranoside for 3 h at 37°C to induce galectin-3 production. After sonicating bacteria in ϳ50 ml of 20 mM Tris-HCl (pH 7.5), 0.15 M NaCl, 5 mM EDTA, and 1 mM DTT (buffer A) together with a protease inhibitor mixture (Sigma), cell homogenates were centrifuged to obtain a soluble fraction. The bacterial supernatants (ϳ50 ml) were applied to 5 ml of asialofetuin-agarose (4 mg of asialofetuin/ml of gel; AminoLink Plus gel from Pierce) and after extensive washing of column (ϳ20 bed volumes) with buffer A without EDTA and DTT, galectin-3 was eluted with lactose (100 mM in buffer A without EDTA and DTT). The eluate was first dialyzed against PBS with 1 mM EDTA and then against PBS to remove lactose. Typically, 3-7 mg of galectin-3 was purified from 2-liter cultures of E. coli. The purity of galectin-3 was determined by Coomassie Brilliant Blue (CBB) and silver staining of a SDS-polyacrylamide gel.
Preparation of Lipophosphoglycan and Leishmanolysin-rich Fractions-Membrane components of Leishmania were extracted as previously described by Bouvier et al. (39), with a modification. Briefly, Leishmania (ϳ3 ϫ 10 9 cells, stationary phase) were washed with PBS and resuspended in 50 ml of 10 mM Tris-HCl (pH 7.5) supplemented with 50 M 1-chloro-3-tosylamido-7-amino-2-heptanone. Cells were sonicated, and membrane-associated components were pelleted by centrifugation at 20,000 ϫ g for 20 min. The pellets were resuspended in 23 ml of ice-cold buffer containing 10 mM Tris-HCl, 140 mM NaCl, and 2% Triton X-114, and the suspension was stirred at 4°C for 1 h. After centrifugation at 8,000 ϫ g for 15 min to obtain Triton X-114-soluble material, the membrane extract was fractionated by Triton X-114 phase separation. The detergent-rich fraction was collected and diluted with 10 mM Tris-HCl (pH 7.5) supplemented with Triton X-100 (the final concentration of Triton X-100 was 1%, and the concentration of Triton X-114 was below 0.35%). This detergent-rich fraction was passed through a DEAE-Macro-Prep column (Bio-Rad). LPG-and leishmanolysin-rich fractions from the column were collected and were dialyzed against 10 mM Tris-HCl (pH 7.5).
Galectin-3 Affinity Chromatography-To prepare the galectin-3-agarose gel, purified recombinant galectin-3 (2.4 mg) was incubated with 1 ml of AminoLink Plus coupling gel (Pierce) in 0.1 M sodium phosphate and 0.15 M NaCl (pH 7.2) for 4 h at 4°C and coupled to the agarose gel according to the manufacturer's instructions (Pierce). Following equilibration in buffer B (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% Triton X-100), 1 ml of the LPG-and leishmanolysin-rich fractions was applied to the galectin-3 column (1-ml bed volume) at 4°C. The column was washed with 5 ml of buffer B, then was eluted first with 5 ml of 100 mM mannose in buffer B, followed by 100 mM lactose. The obtained fractions were fractionated by SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher & Schuell) using a Tris-glycine buffer system. After incubation with 5% skim milk in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.2% Tween 20, membranes were incubated with anti-LPG antibodies (WIC79.3 or WIC108.3), followed by anti-mouse IgG-peroxidase (Amersham Biosciences). Membrane-bound antibody complexes were visualized by exposing to Blue XB-1 film (PerkinElmer Life Sciences) after incubation with a chemiluminescence substrate for peroxidase (PerkinElmer Life Sciences). Five l of each fraction from galectin-3 affinity column chromatography were also dot-blotted onto a nitrocellulose membrane, and the presence of leishmanolysin was detected with anti-native leishmanolysin antibody (Cedarlane).
Galectin-3 Binding and Cleavage-A mouse macrophage cell line, J774A.1, was plated at 2 ϫ 10 5 cells/30-mm culture dish in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5% fetal calf serum (HyClone Laboratories, Logan, UT) and antibiotics. Leishmania promastigotes were washed with Dulbecco's modified Eagle's medium three times and added to a macrophage cell layer at the indicated macrophage: parasite ratio (1:5-1:20) in the presence or absence of lactose (50 mM). After 2 h at 37°C, the media were collected and cell/parasite-free supernatant was obtained by centrifugation at 3,000 rpm for 10 min at 4°C. The supernatants were subjected to Western blotting with anti-Mac-2 antibody to analyze the galectin-3 contents.
To study the binding and cleavage of purified galectin-3 by Leishmania, the parasites (1 ϫ 10 7 ) were incubated with recombinant galectin-3 (1.7 M) in 250 l of serum-free RPMI 1640 containing 25 mM Hepes: PBS (1:1 ratio) in the presence or absence of lactose (100 mM), at 4°C for 30 min for the binding assay or at 37°C with agitation (800 rpm/min) for the indicated time for the cleavage assay. After the incubation, parasite-free supernatants were obtained by spinning at 2,500 rpm for 7 min. The supernatants were fractionated by SDS-PAGE, and proteins in the gels were stained by CBB staining. Alternatively, fractionated proteins on the SDS-PAGE gels were transferred to the nitrocellulose filters and the galectin-3-related fragments were detected by anti-galectin-3 antibodies (anti-Mac-2 antibody or anti-CRD antibody). When parasites were incubated with 100 mM sugar, the concentration of NaCl in PBS was manipulated to maintain appropriate osomolarity, i.e. 317 mosmol/liter. To inhibit cleavage, various protease inhibitors were added at the concentration ranging from that recommended by the manufacture to 10 times higher. The final concentrations of ethanol or Me 2 SO (used to dissolve protease inhibitors) in the incubation mixtures were below 0.1%.

N-terminal Peptide Sequencing and Matrix-assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Analysis of Galectin-3 Fragments-
The three fragments of galectin-3 (bands A-C) were generated by incubating recombinant galectin-3 with L. major as described above and purified using asialofetuin-agarose. The fragments were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane using CAPS buffer system as previously described (40). Galectin fragments were located by staining with 0.5% Ponceau S solution. N-terminal protein sequencing of galectin-3 fragments was performed on a model 473A Applied Biosystems Sequencer by Eastern Quebec Proteomics Center (CRCHUL, Québec, Canada). MALDI-TOF was performed using a time-of-flight mass spectrometer (Voyager-DE-Pro, Per-Septive Biosystems) by Eastern Quebec Proteomic Center. Molecular masses of galectin-3 fragments were assigned based on an external mass calibration using a standard molecular marker set provided by CRCHUL.

RESULTS
Galectin-3 Specifically Binds Lipophosphoglycan of L. major but Not of L. donovani-LPG, a major surface glycoconjugate of Leishmania, contains various ␤-galactoside structures (Fig. 1A, gray boxes) (7-10). We first investigated whether galectin-3 has any affinity toward LPG from L. major and L. donovani. Membrane-integrated molecules, including glycosylphosphatidylinositol-anchored molecules such as leishmanolysin and LPG, were extracted from L. major LV39 and L. donovani and applied to galectin-3-agarose affinity columns at 4°C. After extensive washing of the column, molecules associated with the galectin-3-agarose columns were first eluted with mannose, a non-antagonist of galectin-3, followed by lactose, an antagonist of galectin-3, to elute glycoconjugates that have affinity for galectin-3. The presence of leishmanolysin and LPG-related glycoconjugates in those fractions was analyzed by Western blotting with an antibody against leishmanolysin and two monoclonal antibodies against (lipo)phosphoglycan (WIC108.3 and WIC79.3), respectively.
The majority of leishmanolysin from both L. major and L. donovani was found in the unbound (flow-through) fractions, and we could not detect any significant amount of leishmanolysin in the fractions eluted with mannose or with lactose (data not shown), indicating that leishmanolysin does not bind to galectin-3. These data are consistent with previous reports suggesting that leishmanolysin contains N-linked high mannose type oligosaccharides (41), which does not have affinity for galectin-3.
In contrast, as shown in Fig. 1B, significant proportion of L. major LPG-related glycoconjugates detected by WIC108.3 was found in the fractions eluted with lactose, an antagonist for galectin-3, but not in the fractions eluted with mannose. Thus, the data indicate that galectin-3 specifically binds LPG-related glycoconjugates of L. major. Some of LPG in the fractions eluted with lactose were also WIC79.3-positive (Fig. 1C). Unlike WIC108.3, which binds the common phosphoglycan structure of LPG of Leishmania, WIC79.3 binds to one of the L. major-specific polygalactose epitopes, (PO 4 -6(Gal␤1-3) n Gal␤1-4Man␣1-) (Fig. 1A) (17, 42). Thus, the results suggest that the fractions eluted with lactose contain one of the L. major-specific polygalactose epitopes.
When the membrane extracts of L. donovani were applied to the galectin-3-agarose column, the majority of WIC108.3-reactive molecules were found in the unbound fractions. We could not detect significant amount of L. donovani LPG in the fractions eluted with either mannose or lactose (Fig. 1D), indicating that galectin-3 does not have affinity for L. donovani LPG. Together, those results suggest that galectin-3 can distinguish L. major-derived LPG from L. donovani-derived LPG. As summarized in Fig. 1A, there are two types of ␤-galactosides, Gal␤1-4Man␣1-and (Gal␤1-3) n in the phosphoglycan domains of LPG. Although Gal␤1-4Man␣1-is found on LPG of both L. donovani and L. major (Fig. 1A, dark gray box), the polygalactose structure, (Gal␤1-3) n , is found on L. major LPG only (Fig. 1A, light gray box, R of L. major). As the above results demonstrate that galectin-3 preferentially binds to L. major LPG, it is likely that galectin-3 binds to polygalactose epitopes of L. major.
Galectin-3 Binds to L. major but Not L. donovani at 4°C-Next, we studied whether galectin-3 can also specifically bind to L. major LV39 promastigotes. Purified galectin-3 was incubated with Leishmania at 4°C for 30 min. Galectin-3 was also incubated with asialofetuin-agarose as a positive control, as galectin-3 is known to bind to asialofetuin-agarose (27). After incubation, Leishmania or asialofetuin-agarose-bound galectin-3 was separated from the unbound by centrifugation. Galectin-3 bound to the Leishmania or asialofetuin-agarose was eluted with lactose, and the amounts of galectin-3 in each fraction were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein staining with CBB. As shown in Fig. 2A, where binding occurred at 4°C, ϳ40% of applied galectin-3 was associated with L. major and ϳ80% of galectin-3 bound to asialofetuin-agarose. When galectin-3 was incubated with L. major in the presence of lactose, the binding of galectin-3 to L. major was inhibited (data not shown). In contrast, no detectable galectin-3 was bound to L. donovani ( Fig. 2A), suggesting that galectin-3 binds specifically to L. major parasites but not to L. donovani, consistent with the above data (Fig. 1, B-D).
established a L. major mutant called Spock that lacks the activity of ␤1,3-galactosyltransferase in the background of L. major Friedlin. Consequently, the phosphoglycan domain of Spock LPG is not modified with terminal polygalactosylation and exhibits structural similarity with L. donovani LPG (Fig.  1A, LPG structure of L. donovani where R is H) (15). To establish whether galectin-3 binds to L. major-specific polygalactose epitopes, the interaction of galectin-3 with L. major Friedlin or with its mutant Spock was investigated. When galectin-3 was incubated with these parasites at 4°C, a significant amount of galectin-3 bound to L. major Friedlin wild type but not to Spock mutant (Fig. 2B). Thus, the data suggest that recombinant galectin-3 binds to L. major through recognition of L. major-specific polygalactose epitopes at 4°C.
Galectin-3 Distinguishes L. major from L. donovani at 37°C-We next studied whether galectin-3 secreted from macrophages also recognizes Leishmania in a species-specific manner at 37°C. Murine macrophage cell line, J774A.1, which actively secretes galectin-3 (45), was incubated with various species of Leishmania at 37°C for 2 h. After incubation, cellfree media were obtained by centrifugation of culture media and their galectin-3 contents were determined by Western blotting with anti-Mac-2 antibody, which recognizes the N-terminal domain of galectin-3. Galectin-3 was readily detected in the cell-free media that were exposed to untreated cells or cells incubated with L. donovani or L. mexicana (Fig. 3). In contrast, we found very little intact galectin-3 in the cell-free media of cells incubated with L. major (Fig. 3). This disappearance of galectin-3 in the media was evident at cell ratios as low as 1:5 (macrophage:Leishmania ϭ 2 ϫ 10 5 :1 ϫ 10 6 ). In contrast, the reduction of galectin-3 content in the medium was inhibited in the presence of lactose (Fig. 3), whereas the presence of nonrelevant sugars, glucose or mannose did not inhibit this disappearance (data not shown). Thus, consistent with the data obtained with recombinant galectin-3 at 4°C, these results suggest that macrophage-derived galectin-3 can also distinguish L. major from L. donovani at 37°C in a manner dependent on the lectin activity of galectin-3.

Galectin-3 Is Cleaved by L. major after Binding to the Parasites in a Manner Dependent of Galectin-3 Lectin Activity-To
analyze the amount of galectin-3 in the media that were exposed to either macrophages or Leishmania-infected macrophages, we used anti-Mac-2 antibody, which binds to the N- membrane components, including LPG extracted from L. major LV39 promastigotes, were applied to galectin-3-agarose affinity column. After extensive washing, the column was first eluted with mannose (Man), followed by lactose (Lac). The presence of LPG in the corresponding fractions was detected by Western blotting with anti-LPG antibody, WIC108.3, which cross-reacts with LPG from all species of Leishmania (17). Protein molecular size markers were used to show relative molecular mass as a reference. L. major-derived LPG was eluted by lactose. Or, original material. C, the presence of LPG of L. major in the eluates was detected by anti-LPG antibody, WIC79.3, which recognizes one of L. major-specific polygalactose epitopes, (PO 4 -6(Gal␤1-3) 3 Gal␤1-4Man␣1). D, membrane components extracted from L. donovani were applied to galectin-3 column as above.
terminal domain of galectin-3 (Fig. 3). Therefore, it was not clear whether the L. major-mediated reduction of galectin-3 content that was revealed by anti-Mac-2 antibody was the result of 1) the L. major-specific absorption of galectin-3, which was observed at 4°C, 2) the cleavage of galectin-3 by the parasites, or 3) the combination of both. To distinguish these possibilities, Leishmania promastigotes were incubated with purified recombinant galectin-3 for 1 h at 37°C. The supernatants of the incubation mixtures were subjected to Western blotting analysis with an anti-galectin-3 antibody that recognizes the C-terminal CRD (Fig. 4A). When galectin-3 was incubated with L. major, no full-length galectin-3 (30 kDa) was detected by anti-CRD antibody. We found that 15-kDa fragment (referred as band A) recognized by anti-CRD antibody had accumulated in the supernatant that was exposed to L. major (Fig. 4A), suggesting that galectin-3 is cleaved during the incubation with L. major. Band A was not recognized by anti-Mac-2 antibody (data not shown), indicating that band A contains the C-terminal CRD but lacks the N-terminal domain. When galectin-3 was incubated with L. major in the presence of lactose, the formation of band A by L. major was significantly inhibited (Fig. 4A, lane 2), demonstrating that the cleavage was dependent on galectin-3 binding to the L. major surface. This was corroborated by the finding that the majority of galectin-3 was not cleaved by L. donovani, which is not recognized by galectin-3 (Fig. 4A, lane 3). Similarly, when incubated with L. major mutant Spock, which galectin-3 did not bind (Fig. 2B), galectin-3 was not as efficiently cleaved as by the L. major Friedlin wild type strain (Fig. 4B). We found that the cleavage activity of the Friedlin wild type was somewhat weaker than L. major LV39 used in the experiments above (Fig. 4B). Because efficient cleavage of galectin-3 was observed only by the incubation of Leishmania, which galectin-3 binds at 4°C, those data suggest that binding of galectin-3 to Leishmania is prerequisite to the cleavage of galectin-3 by Leishmania.
The Incubation of Galectin-3 with L. major Results in the Formation of Truncated Galectin-3-The kinetics of L. majordependent cleavage of galectin-3 was analyzed next by SDS-PAGE, followed by protein staining with CBB (Fig. 5, A and B). Cleavage of galectin-3 by L. major was observed as early as after 5 min of incubation, and 50% of galectin-3 was cleaved within ϳ22 min. The cleavage product, band B, that migrated at 16 kDa was first detected after 5 min of incubation, reached the maximal level after 30 min, and then gradually disappeared. Band A, corresponding to 15 kDa, first became detectable after 5 min of incubation, gradually reached a plateau by 120 min of incubation, and remained at the same level up to 240 min of incubation, suggesting that band A was not further degraded. We also confirmed that bands A and B, detected by protein staining, were galectin-3-related proteins, as both bands were readily recognized by anti-CRD antibody (Fig. 4A and data not shown). When the same incubation mixture was analyzed by Western blotting with anti-Mac-2 antibody, which recognizes the N-terminal domain of galectin-3, a band migrating at ϳ7 kDa became detectable after 5 min of incubation and persisted to the end of incubation (Fig. 5C). The amount of this ϳ7 kDa product, which was not readily detectable by CBB staining (Fig. 5A), remained relatively unchanged for up to 4 h, suggesting that this galectin-3 N-terminal fragment was not further cleaved by L. major.
Cleavage of Galectin-3 by L. major Is Inhibited by 1,10ortho-Phenanthroline-Specific cleavage of galectin-3 by L. major was not evident after heat treatment of Leishmania (Fig. 6, lane 8), suggesting that proteases of Leishmania are involved in the cleavage. Although Leishmania contains various proteases, the major surface protease is leishmanolysin (46 -49). To investigate which proteases are involved in the cleavage of galectin-3, we used various protease inhibitors: FIG. 2. Binding of galectin-3 to L. major through L. majorspecific polygalactose epitope at 4°C. A, recombinant galectin-3 was incubated with L. major LV39 (LM), L. donovani (LD), or asialofetuin-agarose (asF) at 4°C for 30 min. After incubation, the supernatants (Unbound) were separated from the parasites by centrifugation. After a brief wash with PBS, the parasite-associated galectin-3 (Bound) was eluted with lactose. Galectin-3 in the fractions was detected by staining SDS-PAGE gels with CBB. B, galectin-3 was incubated with L. major (Friedlin) mutant Spock or its wild type (WT) at 4°C for 30 min as above. metalloprotease inhibitors (1,10-ortho-phenanthroline (OPA), EDTA, phosphoramidon), serine protease inhibitors (Pefabloc ® SC, aprotinin), a cysteine protease inhibitor (E-64), a cysteine and serine protease inhibitor (leupeptin), an aminopeptidase inhibitor (bestatin), an aspartate protease inhibitor (pepstatin), a papain and trypsin inhibitor (antipain-dihydrochloride), and a chymotrypsin inhibitor (chymostatin). None of the inhibitors for cysteine protease or aminopeptidase exhibited significant inhibition of galectin-3 cleavage (data not shown). Phosphoramidon (data not shown) and EDTA (Fig. 6, lanes 9 and 10), two metalloprotease inhibitors, also failed to inhibit cleavage. Antipaindihydrochloride and chymostatin showed slight inhibition at concentrations (500 g/ml) 10-fold higher than the recommended concentration (data not shown). In contrast, OPA, a zinc chelator and a potent leishmanolysin inhibitor (4, 46), efficiently inhibited L. major-mediated cleavage of galectin-3 (Fig. 6, lanes 4 -7). The inhibition by OPA became evident as low as 0.5 mM (Fig. 6, lane 4). Therefore, these data suggest that zinc-dependent metalloproteases are involved in the cleavage of galectin-3 by L. major. A ϳ22-kDa fragment (referred to here as band C) was observed in the presence of OPA, although the mechanism of the formation of this fragment is not clear (Fig. 6). Although two soluble cytosolic metalloexopeptidases are reported to exhibit sensitivity to both OPA and bestatin (47), the cleavage of galectin-3 by L. major was inhibited only by OPA (Fig. 6) but not bestatin (data not shown), suggesting that these exopeptidases are not involved in the cleavage. Together, the data suggest that galectin-3 is cleaved by zinc-dependent metalloproteases, likely surface proteases, such as leishmanolysin, a major membrane component, after galectin-3 binds to the surface through L. major-specific polygalactose epitopes.
Truncated Galectin-3 Fragments Contain the Carbohydrate Recognition Domain-The N-terminal sequences of the three galectin-3 cleavage products (bands A, B, and C), were next analyzed. As shown in Fig. 7, the N-terminal sequences of bands C, B, and A revealed that galectin-3 was cleaved at amino acids 66, 91, and 103/109, respectively. MALDI-TOF analysis of molecular mass of the band B-rich fraction shows 17,625 and 16,525 Da, which closely corresponded to the calculated molecular mass of band B (17,616 Da) and the minor component of band A (16,512 Da) (data not shown), suggesting that those fragments contain the full-length CRD.
Infection of Inflammatory Macrophages with L. major Results in the Cleavage of Galectin-3 Associated with the Cell Surface-Finally, we next investigates whether infection with L. major leads to the cleavage of galectin-3, which is associated with the surface of macrophage. Thioglycollate-induced peritoneal macrophages were incubated with galectin-3. After unbound galectin-3 was removed, macrophages were infected with L. major LV39 or L. donovani for 2 h. As shown in Fig. 8, when macrophages were infected with L. major, the majority of surface galectin-3 on macrophages was cleaved to form band A. In contrast, incubation of macrophages with L. donovani did not lead to any significant cleavage of galectin-3. Together, the data suggest that L. major infection results in cleavage of cell surface-bound galectin-3.

DISCUSSION
Although the importance of the interaction between the species-specific molecules of Leishmania and host factors of mononuclear phagocytes present at the site of infection has been suggested (2), the identities of host molecules involved in such species-specific recognition of Leishmania remain elusive. In particular, L. major-specific polygalactose epitope has been proposed to be implicated in the species-specific recognition of the parasite by macrophages (16,17). This study provides evidence suggesting that the macrophage lectin, galectin-3, can recognize L. major through its binding to the L. major speciesspecific polygalactose (Gal␤1-3) n epitopes on the phosphoglycans. Thus, galectin-3 does not bind to L. donovani and a L. major mutant, Spock, which do not contain polygalactose epitopes on LPG (15). To the best of our knowledge, this report provides the first evidence that an animal lectin, galectin-3, is potentially responsible for the recognition of this L. major polygalactose epitope. The association of galectin-3 with L. major results in the formation of truncated galectin-3, which contains the CRD but lacks the N-terminal domain. Furthermore, we found that infection of inflammatory macrophages with L. major results in the depletion of full-length galectin-3 molecules, which are associated with the surface of macrophages. Truncated galectin-3 lacking the N-terminal domain is known to be incapable of oligomerizing (29,30). Because the oligomerization of galectin-3 is prerequisite to the majority of galectin-3's activities, including its immunomodulative activities (19 -21), these data suggest that the species-specific interaction of galectin-3 with L. major leads to the formation of a "dominant-negative" form of galectin-3.
Our data demonstrate that the association of galectin-3 with Leishmania is prerequisite to the cleavage of galectin-3 by the parasites. Among protease inhibitors to the previously reported Leishmania proteases, we found that only OPA, which inhibits zinc-dependent proteases, including a major Leishmania surface leishmanolysin, efficiently inhibits the cleavage of galec-tin-3. Furthermore, we also observed that incubation of L. major with galectin-3 does not induce the secretion of Leishmania proteases with the ability to cleave galectin-3 to the incubation media. 2 Together, our data suggest that the cleavage of galectin-3 is mediated mainly by the surface proteases rather than intracellular proteases. The major surface zinc protease of Leishmania is leishmanolysin, a member of metzincin class zinc protease (50,51). Interestingly, other members of metzincin, the mammalian zinc metalloproteases matrix metalloprotease 2 and 9, are also known to be able to cleave galectin-3 (52,53), implying that leishmanolysin may cleave galectin-3. However, our results (Fig. 7) suggest that galectin-3 is cleaved at sites that do not contain typical substrate peptide sequences reported for leishmanolysin (39). Therefore, further investigations are necessary to elucidate the enzymes responsible for the cleavage of galectin-3.
Galectin-3 belongs to a family of galectins (␤-galactosidebinding lectins), which are widely expressed in various phyla. In human, 11 galectins (galectin-1-galectin-11) have been found and 10 candidate galectin genes are identified in human genome (21). Thus, our results do not eliminate the possibility that other galectins and other potential galectinlike molecules may be able to recognize the L. major-specific polygalactose epitope. It is known that each galectin differs significantly in the recognition of galactosyl residues within oligosaccharides (27,28,54). For example, x-ray crystal structure analyses and studies of carbohydrate binding specificity of galectin-1 and 3 reveal that galectin-3 has more extended carbohydrate-recognition subsites than galectin-1, so that the CRD of galectin-3 is able to accommodate longer/ modified ␤-galactosides (55). In the case of the binding of Leishmania, we observed that galectin-1 did not show significant affinity for L. major, 3 suggesting that the polygalactose epitope binds to the galectin-3-unique extended binding pocket, which is not present in galectin-1. As L. majorspecific polygalactose epitopes are suggested to be involved in the interaction of Leishmania with the midguts of its vector, the sand fly, Phlebotomus papatasi (14,15), it is possible that some galectin-like molecules, which contain the extended carbohydrate binding pocket (like galectin-3), are involved in this interaction. Therefore, further investigation is necessary to address the question of whether other galectins can bind to the L. major-specific polygalactose epitopes. L. major-truncated galectin-3 was subjected to N-terminal protein sequencing. Cleavage sites of bands A, B, and C are indicated in the peptide sequence of human galectin-3, and the sequences obtained by N-terminal protein sequencing are underlined. In the case of band A, N-terminal protein sequence analysis revealed that there were two peptides, a major component and a minor component; the major component is indicated with the larger character.
It has been demonstrated that galectin-3 plays a role in several innate immune responses (19 -21). Moreover, recent work indicates that the presence of multivalent galectin-3glycoprotein lattices on lymphocyte cell surface could actively restrict the lateral mobility of receptors such as the T cell receptor and consequently raise the threshold for ligand-dependent receptor clustering and signal transduction (31,37). Lack of this multivalent galectin-3 lattice induces prolonged delayed-type hypersensitivity (Th1 response) in vivo, suggesting that this lattice represses/reduces the development of Th1 type response (37). The induction of Th1-type immune response is suggested to be one of the critical elements to develop immune responses to L. major infection (56). Because the formation of stable galectin-3-receptor lattices is achieved by the oligomerization through N-terminal domain of galectin-3 (36), it is expected that L. major-truncated dominant negative form of galectin-3 cannot form high orders of galectin-3 lattices. Here we demonstrate that the infection of macrophages with L. major results in the cleavage of galectin-3 on the surface of macrophage in a Leishmania speciesdependent manner, likely leading to the destruction of galectin-3's lattice. Thus, we currently hypothesize that, by disrupting surface galectin-3 lattices of leukocytes, L. major infection decreases the threshold for the initiation of signal transduction pathways that bias the immune response toward a Th1 type profile and/or strong local inflammatory responses observed in L. major infection.
In conclusion, our data suggest that galectin-3 recognizes L. major through L. major-specific polygalactose epitopes. The binding of galectin-3 to L. major leads to the formation of a dominant-negative form of galectin-3. As recent reports indicate the potential of galectin-3 as a novel immunomodulator for the development of innate immune responses, the roles of different forms of galectin-3 in the development of Leishmania species-specific inflammation observed in leishmaniasis are important goals for future work.