Abstract
BackgroundPrevious work, by us and others, has shown that mammalian galectins-1 have a growth-inhibitory activity for mammalian cells which is apparently independent of their β-galactoside binding site.ResultsWe have made recombinant human galectin-1 as a bacterial fusion protein with an N-terminal hexahistidine tag. This protein displays both haemagglutination and growth-inhibitory activities, even in the presence of the hexahistidine tag. Site-directed mutagenesis of this protein has confirmed the independent nature of the protein sites responsible for the two biological activities. Mutant proteins were created, which displayed each activity in the absence of the other.ConclusionsHuman galectin-1 possesses a growth-inhibitory site, which is not part of the β-galactoside binding site. A surface loop, comprising amino acid residues 25–30, and joining two internal β-strands, forms part of the growth-inhibitory site. This region is relatively close to the N-terminus of the protein, and N-terminal substitutions or extensions also affect growth-inhibitory activity. Further experiments will be necessary to fully define this site.
Highlights
Previous work, by us and others, has shown that mammalian galectins-1 have a growth-inhibitory activity for mammalian cells which is apparently independent of their βgalactoside binding site
The concept of a galactose-binding protein as a negative growth regulator was first seriously proposed when a protein from mouse fibroblast cultures, the murine homologue of galectin-1, was shown to have both properties [4]. These workers went on to demonstrate that growth-inhibitory activity of this mGBP was present even when it bound a glycan at its galactose-binding site [5], suggesting that the two properties were independent functions of galectin-1
Specific mutagenesis of the recombinant galectin molecule is an obvious way of confirming this conclusion. Consideration of both tertiary structure and mutagenesis studies suggests that histidine-45, asparagine-47, arginine-49, tryptophan-69, glutamate-72 and arginine-74 are all involved in, or influence, sugar binding [11,12,13,14]. (In numbering galectin-1 amino acid residues, we have found it more convenient to number from the N-terminal methionine, rather than to use the convention of Abbott and Feizi [15], who numbered from alanine-2, which is the N-terminus of the mature, natural protein)
Summary
By us and others, has shown that mammalian galectins-1 have a growth-inhibitory activity for mammalian cells which is apparently independent of their βgalactoside binding site. The concept of a galactose-binding protein as a negative growth regulator was first seriously proposed when a protein from mouse fibroblast cultures, the murine homologue of galectin-1, was shown to have both properties [4] These workers went on to demonstrate that growth-inhibitory activity of this mGBP was present even when it bound a glycan at its galactose-binding site [5], suggesting that the two properties were independent functions of galectin-1. In an attempt to test this hypothesis, recombinant human galectin-1 was prepared as a bacterial fusion protein with glutathione-S-transferase [10] Both the fusion protein, and the galectin-1 derived from it by proteolysis and repurification, functioned as multivalent lectins, and dis-
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