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Galectin-1 is a ␤-galactoside-binding protein with potent anti-inflammatory and immunoregulatory effects. However, its expression and function have not been assessed in the context of an infectious disease. The present study documents, for the first time, the regulated expression of galectin-1 in the context of an infectious process and its influence in the modulation of macrophage microbicidal activity and survival. A biphasic modulation in parasite replication and cell viability was observed when macrophages isolated from Trypanosoma cruziinfected mice were exposed to increasing concentrations of galectin-1. While low concentrations of this protein increased parasite replication and did not affect macrophage survival, higher inflammatory doses of galectin-1 were able to commit cells to apoptosis and inhibited parasite replication. Furthermore, galectin-1 at its lowest concentration was able to down-regulate critical mediators for parasite killing, such as interleukin 12 (IL-12) and nitric oxide, while it did not affect IL-10 secretion. Finally, endogenous galectin-1 was found to be up-regulated and secreted by the J774 macrophage cell line cultured in the presence of trypomastigotes. This result was extended in vivo by Western blot analysis, flow cytometry, and reverse transcription-PCR using macrophages isolated from T. cruzi-infected mice. This study documents the first association between galectin-1's immunoregulatory properties and its role in infection and provides new clues to the understanding of the mechanisms implicated in host-parasite interactions during Chagas' disease and other parasite infections.

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Galectins, a family of soluble beta-galactosyl-binding lectins, are believed to mediate cell-cell and cell-extracellular matrix interactions during development, inflammation, apoptosis, and tumor metastasis. However, neither the detailed mechanisms of their function(s) nor the identities of their natural ligands have been unequivocally elucidated. Of the several galectins present in the nematode Caenorhabditis elegans, the 16-kDa "proto" type and the 32-kDa "tandem-repeat" type are the best characterized so far, but their carbohydrate specificities have not been examined in detail. Here, we report the carbohydrate-binding specificity of the recombinant C. elegans 16-kDa galectin and the structural analysis of its binding site by homology modeling. Our results indicate that unlike the galectins characterized so far, the C. elegans 16-kDa galectin interacts with most blood group precursor oligosaccharides (type 1, Galbeta1,3GlcNAc, and type 2, Galbeta1,4GlcNAc; Talpha, Galbeta1,3GalNAcalpha; Tbeta, Galbeta1,3GalNAcbeta) and gangliosides containing the Tbeta structure. Homology modeling of the C. elegans 16-kDa galectin CRD revealed that a shorter loop containing residues 66-69, which enables interactions of Glu(67) with both axial and equatorial -OH at C-3 of GlcNAc (in Galbeta1,4GlcNAc) or at C-4 of GalNAc (in Galbeta1,3GalNAc), provides the structural basis for this novel carbohydrate specificity.

Galectin-1 (Gal1) and galectin-3 (Gal3) are two members of a family of carbohydrate-binding proteins that are found in the nucleus and that participate in pre-mRNA splicing assayed in a cellfree system. When nuclear extracts (NE) of HeLa cells were subjected to adsorption on a fusion protein containing glutathione S-transferase (GST) and Gal3, the general transcription factor II-I (TFII-I) was identified by mass spectrometry as one of the polypeptides specifically bound. Lactose and other saccharide ligands of the galectins inhibited GST-Gal3 pull-down of TFII-I while nonbinding carbohydrates failed to yield the same effect. Similar results were also obtained using GSTGal1. Site-directed mutants of Gal1, expressed and purified as GST fusion proteins, were compared with the wild-type (WT) in three assays: (a) binding to asialofetuin-Sepharose as a measure of the carbohydrate-binding activity; (b) pull-down of TFII-I from NE; and (c) reconstitution of splicing in NE depleted of galectins as a test of the in vitro splicing activity. The binding of GST-Gal1(N46D) to asialofetuin-Sepharose was less than 10% of that observed for GST-Gal1(WT), indicating that the mutant was deficient in carbohydrate-binding activity. In contrast, both GST-Gal1(WT) and GSTGal1(N46D) were equally efficient in pull-down of TFII-I and in reconstitution of splicing activity in the galectin-depleted NE. Moreover, while the splicing activity of the wild-type protein can be inhibited by saccharide ligands, the carbohydrate-binding deficient mutant was insensitive to such inhibition. Together, all of the results suggest that the carbohydrate-binding and the splicing activities of Gal1 can be dissociated and therefore, saccharide-binding, per se, is not required for the splicing activity.