Regulation of adult neural progenitor cells by Galectin-1/beta1 Integrin interaction.

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 beta1 Integrin and modulates cell adhesion. Here, we studied the functional interaction between Galectin-1 and beta1 Integrin in the adult mouse SEZ. Beta1 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 beta1 Integrin-expressing cells in the SEZ. Furthermore, in the adult SEZ, the simultaneous infusion of a beta1 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 beta1 Integrin was required for Galectin-1's function in NPC adhesion in vitro. These results suggest that the interaction between beta1 Integrin and Galectin-1 plays an important role in regulating the number of adult NPCs through mechanisms including cell adhesion.

Enrichment strategies for glycopeptides.

In order to understand glycoprotein functionality, information on the structure of both the core proteins and the glycan moieties is necessary. From a practical viewpoint, glycopeptides rather than whole glycoproteins are the general targets for structural analysis, which is primarily carried out by employing mass spectrometry (MS). Using the "glycoproteomics" concept, several techniques have recently been developed to allow the preparation of a series of reference glycopeptides. In this chapter, we describe two selective capturing methods for glycopeptides, i.e., lectin-affinity chromatography and polysaccharide hydrophilic affinity physicochemical chromatography. The combined use of these methods effectively removes non-glycosylated peptides, the inclusion of which substantially interferes with glycopeptide ionization in MS analysis.

Caenorhabditis elegans N-glycans containing a Gal-Fuc disaccharide unit linked to the innermost GlcNAc residue are recognized by C. elegans galectin LEC-6.

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.

Caenorhabditis elegans galectins LEC-1-LEC-11: structural features and sugar-binding properties.

Galectins form a large family of beta-galactoside-binding proteins in metazoa and fungi. This report presents a comparative study of the functions of potential galectin genes found in the genome database of Caenorhabditis elegans. We isolated full-length cDNAs of eight potential galectin genes (lec-2-5 and 8-11) from a lambdaZAP cDNA library. Among them, lec-2-5 were found to encode 31-35-kDa polypeptides containing two carbohydrate-recognition domains similar to the previously characterized lec-1, whereas lec-8-11 were found to encode 16-27-kDa polypeptides containing a single carbohydrate-recognition domain and a C-terminal tail of unknown function. Recombinant proteins corresponding to lec-1-4, -6, and 8-10 were expressed in Escherichia coli, and their sugar-binding properties were assessed. Analysis using affinity adsorbents with various beta-galactosides, i.e., N-acetyllactosamine (Galbeta1-4GlcNAc), lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), and asialofetuin, demonstrated that LEC-1-4, -6, and -10 have a significant affinity for beta-galactosides, while the others have a relatively lower affinity. These results indicate that the integrity of key amino acid residues responsible for recognition of lactose (Galbeta1-4Glc) or N-acetyllactosamine in vertebrate galectins is also required in C. elegans galectins. However, analysis of their fine oligosaccharide-binding properties by frontal affinity chromatography suggests their divergence towards more specialized functions.

A combined strategy for glycan profiling: a model study with pyridylaminated oligosaccharides.

Structural glycomics plays a fundamental role in glycoscience and glycotechnology. In this paper, a novel strategy for the structural characterization of glycans is described, in which MS2 analysis involving a LIFT-TOF/TOF procedure is combined with frontal affinity chromatography (FAC). As model compounds, 20 neutral pyridylaminated (PA) oligosaccharides were chosen, which included four groups of structural isomers differing in sequence, linkage, position, or branching features. By depicting significant diagnostic ions on MS2, most of the analyzed oligosaccharides were successfully differentiated, while two pairs of linkage isomers, i.e., LNT/LNnT, and LNH/LNnH were not. For subsequent analysis by FAC, 14 lectins showing significant affinity to either LNT (type 1) or LNnT (type 2) were screened, and a galectin from the marine sponge Geodia cydonium (GC1) and a plant seed lectin from Ricinus communis (RCA-I) were used for determination of type 1 and 2 chains, respectively. With these specific probes, both of the isomeric pairs were unambiguously differentiated. Furthermore, a pair of triantennary, asparagine-linked oligosaccharide isomers could also be successfully differentiated. Thus, the combination of MS2 and FAC is a practical alternative for the structural characterization of complex glycans.

Affinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans.

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.