Crystal structure of an anti-podoplanin antibody bound to a disialylated O-linked glycopeptide.

Podoplanin (PDPN) is a highly O-glycosylated glycoprotein that is utilized as a specific lymphatic endothelial marker under pathophysiological conditions. We previously developed an anti-human PDPN (hPDPN) monoclonal antibody (mAb), clone LpMab-3, which recognizes the epitope, including both the peptides and the attached disialy-core-l (NeuAcα2-3Galßl-3 [NeuAcα2-6]GalNAcαl-O-Thr) structure at the Thr76 residue in hPDPN. However, it is unclear if the mAb binds directly to both the peptides and glycans. In this study, we synthesized the binding epitope region of LpMab-3 that includes the peptide (-67LVATSVNSV-T-GIRIEDLP84-) possessing a disialyl-core-1 O-glycan at Thr76, and we determined the crystal structure of the LpMab-3 Fab fragment that was bound to the synthesized glycopeptide at a 2.8 Å resolution. The six amino acid residues and two sialic acid residues are directly associated with four complementarity-determining regions (CDRs; H1, H2, H3, and L3) and four CDRs (H2, H3, L1, and L3), respectively. These results suggest that IgG is advantageous for generating binders against spacious epitopes such as glycoconjugates.

J(77 Se,1 H) and J(77 Se,13 C) couplings of seleno-carbohydrates obtained by 77 Se satellite 1D 13 C spectroscopy and 77 Se selective HR-HMBC spectroscopy.

Seleno-carbohydrates are those in which the oxygen of the glycosidic bond or the hydroxyl group is artificially replaced with selenium. This substitution changes 1 H and 13 C chemical shifts and produces spin coupling constants involving 77 Se. Coupling constants, such as 2-3 J(77 Se, 1 H), are likely to be useful for conformational analyses of glycans because such couplings are never observed in natural glycans. Several papers have discussed the relationship between 2-3 J(77 Se, 1 H) and conformation; however, only few reports describe 1-3 J(77 Se, 13 C), which could also be useful. Here, we obtain 77 Se coupling constants of seleno-carbohydrates from 77 Se-selective HR-HMBC and 77 Se satellites in 1D 13 C spectra and examine their conformations using the Newman projection scheme.

Synthesis of seleno-fucose compounds and their application to the X-ray structural determination of carbohydrate-lectin complexes using single/multi-wavelength anomalous dispersion phasing.

Selenium-incorporated fucoses (seleno-fucoses) differing in the position of the seleno-substituent were synthesized and applied to the X-ray structural determination of a carbohydrate-lectin complex using single/multi-wavelength anomalous dispersion (SAD/MAD) phasing. The hydroxyl groups at the C-1, -2, -3 and -4 position of fucose were individually substituted with a methylseleno group via a transacetalization reaction using MeSeCH2OBn or by an SN2 reaction with TolSe- equivalents to afford the corresponding MeSe-fucose. The three-dimensional structures of a fucose-binding lectin complexed with several of these MeSe-fucoses have been determined by SAD/MAD phasing by utilizing the diffraction of selenium in the bound MeSe-fucoses.

Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin.

The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed ß-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity.

Structure of a novel α-glucan substitute with the rare 6-deoxy-D-altrose from Lactarius lividatus (mushroom).

A novel α-glucan substituted rare 6-deoxy-D-altropyranose was isolated from edible fruiting bodies of a mushroom (Lactarius lividatus) grown in Okinawa, Japan. The polysaccharide consists of D-glucose, D-galactose and 6-deoxy-D-altrose in a molar ratio of 3.0:1.0:1.0. The specific rotation [α](589) was estimated as +64.3° (0.2% in water) at 25 °C. Based on results of IR, NMR ((1)H, (13)C, 2D-COSY, 2D-HMQC, 2D-ROESY and 2D-HMBC), and methylation analyses, the structure of the polysaccharide was determined as [formula, see text] This work is the first demonstration of rare 6-deoxy-D-altropyranose moiety on polysaccharides.