Oxidized galectin-1 promotes axonal regeneration in peripheral nerves but does not possess lectin properties

Abstract

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...

Functional analyses of placental protein 13/galectin‐13

“…amounts of both PP13-B and PP13-R induced haemagglutination, and strong agglutination was detected at and above 50 lgAEmL )1 applied protein concentrations ( Fig. 4), which was very similar to the phenomenon seen in cases of other galectins [35]. The pattern and effectiveness of both PP13-B and PP13-R were identical in agglutination of erythrocytes.…”

Mouse galectin‐1 inhibits the toxicity of glutamate by modifying NR1 NMDA receptor expression

“…In axotomy experiments, galectin-1 was shown to promote axonal regeneration and Schwann cell migration (Horie et al, 1999). The protective effects of galectin-1 were suggested to be caused by an oxidized monomeric form, unable to bind to galactose (Inagaki et al, 2000). In contrast, our protein contained both monomeric and dimeric fractions.…”

Key regulators of galectin–glycan interactions

“…However, in 1991 Hirbayashi and Kasai substituted serine for cysteine residues and demonstrated that these mutants exhibited virtually no change in the ability of galectin-1 (Gal-1) to bind to asialofetuin-agarose [20]. These results were corroborated by subsequent studies and demonstrated that the cysteine residues of Gal-1 are not necessary for glycanbinding [20][21][22][23]. Instead, these studies demonstrated that the reduced thiols of cysteine residues are important for maintaining protein stability and that oxidation at such residues eliminates Gal-1 carbohydrate binding activity [20][21][22][23].…”

“…While the unique sensitivity of Gal-1 to oxidative inactivation can complicate the study of Gal-1 in biological systems [29,40], the ability of redox potential to regulate carbohydrate independent activities of Gal-1 represents a relatively unexplored, yet critical factor responsible for Gal-1 function [23,27,36,41,42]. This is especially important when considering that while oxidation appears to impact the carbohydrate binding capacity of Gal-1, subsequent studies suggested that oxidized Gal-1 actually possesses biological activity that occurs through glycan-independent processes [23,36,41,[43][44][45][46]. Indeed, oxidized Gal-1 appears to regulate neurite outgrowth and may be important in overall neuron regeneration following injury [23,36,41,[43][44][45][46][47].…”

“…This is especially important when considering that while oxidation appears to impact the carbohydrate binding capacity of Gal-1, subsequent studies suggested that oxidized Gal-1 actually possesses biological activity that occurs through glycan-independent processes [23,36,41,[43][44][45][46]. Indeed, oxidized Gal-1 appears to regulate neurite outgrowth and may be important in overall neuron regeneration following injury [23,36,41,[43][44][45][46][47]. Whether oxidation of other galectins likewise converts these proteins into alternative products with distinct biologically activity remains unknown.…”