The signals that prompt the axons to send out processes in peripheral nerves after axotomy are not well understood. Here, we report that galectin-1 can play an important role in this initial stage. We developed an in vitro nerve regeneration model that allows us to monitor the initial axon and support cell outgrowth from the proximal nerve stump, which is comparable to the initial stages of nerve repair. We isolated a factor secreted from COS1 cells that enhanced axonal regeneration, and we identified the factor as galectin-1. Recombinant human galectin-1 (rhGAL-1) showed the same activity at low concentrations (50 pg/ml) that are two orders of magnitude lower than those of lectin activity. A similarly low concentration was also effective in in vivo experiments of axonal regeneration with migrating reactive Schwann cells to a grafted silicone tube after transection of adult rat peripheral nerve. Moreover, the application of functional anti-rhGAL-1 antibody strongly inhibited the regeneration in vivo as well as in vitro. The same effect of rhGAL-1 was confirmed in crush/freeze experiments of the adult mouse sciatic nerve. Because galectin-1 is expressed in the regenerating sciatic nerves as well as in both sensory neurons and motor neurons, we suggest that galectin-1 may regulate initial repair after axotomy. This high activity of the factor applied under nonreducing conditions suggests that galectin-1 may work as a cytokine, not as a lectin.

Galectin-1 has recently been identified as a factor that regulates initial axonal growth in peripheral nerves after axotomy. Although galectin-1 is a well-known beta-galactoside-binding lectin, its potential to promote axonal regeneration as a lectin has not been reported. It is essential that the process of initial repair in peripheral nerves after axotomy is well clarified. We therefore undertook to investigate the relation between the structure and axonal regeneration-promoting activity of galectin-1. Recombinant human galectin-1 secreted into the culture supernatant of transfected COS1 cells (rhGAL-1/COS1) was purified under nonreducing conditions and subjected to structural analysis. Mass spectrometric analysis of peptide fragments from rhGAL-1/COS1 revealed that the secreted protein exists as an oxidized form containing three intramolecular disulfide bonds (Cys2-Cys130, Cys16-Cys88 and Cys42-Cys60). Recombinant human galectin-1 (rhGAL-1) and a galectin-1 mutant in which all six cysteine residues were replaced by serine (CSGAL-1) were expressed in and purified from Escherichia coli for further analysis; the purified rhGAL-1 was subjected to oxidation, which induced the same pattern of disulfide linkages as that observed in rhGAL-1/COS1. Oxidized rhGAL-1 enhanced axonal regeneration from the transected nerve sites of adult rat dorsal root ganglion explants with associated nerve stumps (5.0-5000 pg. mL-1), but it lacked lectin activity. In contrast, CSGAL-1 induced hemagglutination of rabbit erythrocytes but lacked axonal regeneration-promoting activity. These results indicate that galectin-1 promotes axonal regeneration only in the oxidized form containing three intramolecular disulfide bonds, not in the reduced form which exhibits lectin activity.

Glycosylation inhibiting factor (GIF) and macrophage migration inhibitory factor (MIF) share an identical structure gene. Here we unravel two steps of posttranslational modifications in GIF͞MIF molecules in human suppressor T (Ts) cell hybridomas. Peptide mapping and MS analysis of the affinity-purified GIF from the Ts cells revealed that one modification is cysteinylation at Cys-60, and the other is phosphorylation at Ser-91. Cysteinylated GIF, but not the wild-type GIF͞MIF, possessed immunosuppressive effects on the in vitro IgE antibody response and had high affinity for GIF receptors on the T helper hybridoma cells. In vitro treatment of wild-type recombinant human GIF͞MIF with cystine resulted in preferential cysteinylation of Cys-60 in the molecules. The cysteinylated recombinant human GIF and the Ts hybridoma-derived cysteinylated GIF were comparable both in the affinity for the receptors and in the immunosuppressive activity. Polyclonal antibodies specific for a stretch of the amino acid sequence in ␣2-helix of GIF bound bioactive cysteinylated GIF but failed to bind wildtype GIF͞MIF. These results strongly suggest that cysteinylation of Cys-60 and consequent conformational changes in the GIF͞MIF molecules are responsible for the generation of GIF bioactivity.

Galectin-1 has recently been identified as a factor that regulates initial axonal growth in peripheral nerves after axotomy. Although galectin-1 is a well-known b-galactoside-binding lectin, its potential to promote axonal regeneration as a lectin has not been reported. It is essential that the process of initial repair in peripheral nerves after axotomy is well clarified. We therefore undertook to investigate the relation between the structure and axonal regeneration-promoting activity of galectin-1. Recombinant human galectin-1 secreted into the culture supernatant of transfected COS1 cells (rhGAL-1/COS1) was purified under nonreducing conditions and subjected to structural analysis. Mass spectrometric analysis of peptide fragments from rhGAL-1/COS1 revealed that the secreted protein exists as an oxidized form containing three intramolecular disulfide bonds (Cys2±Cys130, Cys16±Cys88 and Cys42±Cys60). Recombinant human galectin-1 (rhGAL-1) and a galectin-1 mutant in which all six cysteine residues were replaced by serine (CSGAL-1) were expressed in and purified from Escherichia coli for further analysis; the purified rhGAL-1 was subjected to oxidation, which induced the same pattern of disulfide linkages as that observed in rhGAL-1/COS1. Oxidized rhGAL-1 enhanced axonal regeneration from the transected nerve sites of adult rat dorsal root ganglion explants with associated nerve stumps (5.0±5000 pg´mL 21 ), but it lacked lectin activity. In contrast, CSGAL-1 induced hemagglutination of rabbit erythrocytes but lacked axonal regeneration-promoting activity. These results indicate that galectin-1 promotes axonal regeneration only in the oxidized form containing three intramolecular disulfide bonds, not in the reduced form which exhibits lectin activity.Keywords: axonal regeneration; dorsal root ganglion; lectin; oxidized galectin-1; structure/activity relationship.Successful nerve regeneration requires the concerted interplay of non-neuronal cells, growth factors, cell adhesion molecules, extracellular matrix materials, regenerating axons and recruiting macrophages [1±3]. However, although various neurotrophins have been investigated to promote axonal regeneration in vivo, it is still unclear what factor initiates axonal regeneration response to nerve injury. We found one of these factors from the culture supernatant of COS1 cells in a previous study [4]. The factor enhanced axonal regeneration in the in vitro nerve regeneration model employed, allowing for initial axon outgrowth from the proximal nerve stump to be monitored, which is comparable with the initial stages of nerve repair. Analysis of the purified protein indicated that it was identical to galectin-1. Recombinant human galectin-1 (rhGAL-1) confirmed that the protein promotes axonal regeneration not only in the in vitro experiment, but also in two other types of in vivo acellular nerve regeneration model. Furthermore, antibodies to galectin-1 clearly inhibited axonal regeneration in vivo as well as in vitro. These observations suggest that galectin-1 mi...

We report here that the delivery of both alpha-galactosylceramide (alphaGalCer), a representative ligand for invariant natural killer T (iNKT) cells, and an antigenic polypeptide to marginal zone B cells induces the differentiation of regulatory cells in vivo, and suppresses the secondary antibody responses in mice. Splenic CD21+ CD23- B cells of mice treated with alphaGalCer-liposomes produce IL-10 when co-cultured with iNKT cells, whereas the cells treated with aqueous alphaGalCer fail to do so. Adoptive transfer of the B cells into syngenic mice leads to the expansion of splenic CD11c(low) CD45RB(high) cells, which convert naive CD4+ T cells from RAG2-deficient DO11.10 mice to CD4+ CD25(high) Foxp3+ T cells in the presence of OVA323-339 peptide. Administration of alphaGalCer-OVA-liposomes into OVA-primed mice causes the development of CD4+ CD25(high) Foxp3+ T cells that produce both IL-10 and IFN-gamma, and induced the antigen-specific suppression of the secondary antibody responses when boosted with OVA alone. These results indicate that antigen-containing alphaGalCer-liposomes can facilitate the development of tolerogenic antigen-presenting cells and inducible regulatory T cells that are involved in the suppression of immune responses to antigens.