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.
Takeuchi
Tomoharu
T
Department of Biological Chemistry, School of Pharmaceutical Science, Teikyo University, Sagamiko, Sagamihara, Kanagawa 229-0195, Japan. t-take@pharm.teikyo-u.ac.jp
Hayama
Ko
K
Hirabayashi
Jun
J
Kasai
Ken-ichi
K
D000818:Animals; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000378:metabolism*; D004187:Disaccharides / Q000737:chemistry* / Q000378:metabolism; D037161:Galectins / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D029742:Caenorhabditis elegans Proteins; D004187:Disaccharides; D037161:Galectins; C534045:LEC-6 protein, C elegans; D011134:Polysaccharides