Though plant lectins have been widely used for the enrichment of glycoproteins/glycopeptides, mammalian lectins have rarely been explored for this purpose. Using the conserved sequence of the carbohydrate recognition domain (CRD) of the human galectin-3 protein, we engineered the following two novel human galectins:Gal3C (containing one CRD) and Tetra-Gal3C (containing four tandem CRDs). Moreover, we designed a galectin affinity column that could be used for glycoprotein/glycopeptide enrichment, by taking advantage of the ability of these recombinant galectins to become immobilized onto streptavidin agarose beads. SDS-PAGE, western blotting, and mass spectrometry were used to characterize the biological features and glycoprotein/glycopeptide enrichment abilities of both these recombinant galectins. Our results showed that both Gal3C and Tetra-Gal3C predominantly enrich glycoproteins/glycopeptides and that this enrichment process has a high specificity and sensitivity. Because Tetra-Gal3C contains three more CRD structural domains, it exhibited a better ability to achieve enrichment than Gal3C. Moreover, the immobilized Gal3C and Tetra-Gal3C were successfully used for the enrichment of glycoproteins from HepG2 cells, which indicated the selective glycoprotein/glycopeptide enrichment potential of recombinant galectins in complex samples.
Keywords: carbohydrate recognition domain (CRD); galectin-3 (Gal-3); glycoprotein enrichment; liquid chromatography-mass spectrometry (LC-MS).