We report a detailed structural analysis of the N-glycans of Caenorhabditis elegans recognized by C. elegans galectin LEC-6. Glycoproteins of C. elegans captured by an immobilized LEC-6 affinity adsorbent were isolated. The N-glycans of these glycoproteins were then liberated by hydrazinolysis and labeled with the fluorophore 2-aminopyridine (PA). The derived pyridylaminated (PA)-sugars were further fractionated by rechromatography on immobilized LEC-6 adsorbent and by reversed-phase high-performance liquid chromatography (HPLC). The structures of the PA-sugars thus obtained were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS) in conjunction with glycosidase digestion. We confirmed that all PA-sugars having affinity for LEC-6 contain a Gal-Fuc disaccharide unit, and that this unit is bound to the innermost GlcNAc residue of the N-glycan chain. The dissociation constants of LEC-6 for these glycans were measured by frontal affinity chromatography. LEC-6 exhibited higher affinity for oligosaccharides having a Gal-Fuc unit linked to position 6 of the innermost GlcNAc residue than for those having Galbeta1-4GlcNAc units. Affinity for the former disappeared, however, following treatment with beta-galactosidase. If the glycan contained a Hex-Fuc disaccharide linked to the penultimate GlcNAc residue, the affinity would be diminished. We propose, therefore, that the galectins of C. elegans utilize the Gal-Fuc disaccharide unit for recognition instead of the Gal-GlcNAc unit that is common in vertebrates.
Protein glycosylation is a central issue for post-genomic (proteomic) sciences. We have taken a systematic approach for analyzing soluble glycoproteins produced in the nematode Caenorhabditis elegans. The approach aims at assigning (i) genes that encode glycoproteins, (ii) sites where glycosylation occurs, and (iii) types of attached glycan structures. A soluble extract of C. elegans, as a starting material, was applied first to a concanavalin A (ConA) column (specific for high-mannose type N-glycans), and then the flow-through fraction was applied to a galectin LEC-6 (GaL6) column (specific for complex-type N-glycans). The adsorbed glycoproteins were digested with lysylendopeptidase, and the resultant glycopeptides were selectively recaptured with the same lectin columns. The glycopeptides were separated by reversed-phase chromatography and then subjected to sequence determination. As a result, 44 and 23 glycopeptides captured by the ConA and GaL6 columns, respectively, were successfully analyzed and assigned to 32 and 16 corresponding genes, respectively. For these glycopeptides, 49 N-glycosylation sites were experimentally confirmed, whereas 21 sites remained as potential sites. Of the identified genes, about 80% had apparent homologues in other species, as represented by typical secreted proteins. However, the two sets of genes assigned for the ConA and GaL6-recognized glycopeptides showed only 1 overlap with each other. Proof of the practical applicability of the glyco-catch method to a model organism, C. elegans, directs us to explore more complex multicellular organisms.
J. Neurochem. (2010) 113, 1516–1524.
Abstract
Neural stem cells (NSCs) proliferate and generate new neurons in the adult brain. A carbohydrate‐binding protein (lectin), Galectin‐1, is expressed in the NSCs in the subependymal zone (SEZ) of the adult mouse brain. The infusion and knockout of Galectin‐1 in the SEZ results in an increase and decrease, respectively, of NSCs and subsequently born progenitor cells. The molecular mechanism of this effect, however, has been unknown. Previous studies outside the brain suggest that Galectin‐1 binds to a carbohydrate structure of β1 Integrin and modulates cell adhesion. Here, we studied the functional interaction between Galectin‐1 and β1 Integrin in the adult mouse SEZ. β1 Integrin was purified from adult SEZ tissue by binding to a Galectin‐1 affinity column, and this binding depended on Galectin‐1’s carbohydrate‐binding activity. In adult brain sections, Galectin‐1‐binding activity was detected on β1 Integrin‐expressing cells in the SEZ. Furthermore, in the adult SEZ, the simultaneous infusion of a β1 Integrin‐neutralizing antibody with Galectin‐1 protein reversed the increasing effect of Galectin‐1 on the number of adult neural progenitor cells (NPCs). Finally, intact β1 Integrin was required for Galectin‐1’s function in NPC adhesion in vitro. These results suggest that the interaction between β1 Integrin and Galectin‐1 plays an important role in regulating the number of adult NPCs through mechanisms including cell adhesion.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.