Novel forms of protein glycosylation.

A large number of new glycans, derived from glycoproteins, has been characterized in the past few years. O-linked fucose was found in epidermal growth factor-like domains of several proteins. For the N-linked glycans of Helix pomatia hemocyanin, novel types of antennae were identified. The positions of noncarbohydrate substituents were established in N-glycans. C-mannosylation of a tryptophan residue was discovered in human ribonuclease 2 and is the first example of C-glycosylation in glycoproteins.

Transferred nuclear Overhauser enhancement (NOE) and rotating-frame NOE experiments reflect the size of the bound segment of the Forssman pentasaccharide in the binding site of Dolichos biflorus lectin.

A complex between the Forssman pentasaccharide alpha-D-GalNAc-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Gal-(1-->4)-beta-D- Gal-(1-->4)-D-Glc and the seed lectin from Dolichos biflorus was studied using transfer-NOESY and transfer rotating frame NOE spectroscopy (ROESY) experiments. The evolution of transferred NOEs and ROEs as a function of the pentasaccharide/lectin ratio was different for the non-reducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNac compared to the rest of the molecule, which reflects distinct relaxation properties and effects of exchange broadening of the corresponding ligand resonances. Significantly, several intermolecular transferred NOEs were observed between protons of the nonreducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and aliphatic as well as aromatic amino acid side chain protons in the binding pocket of the lectin. It is concluded that the non-reducing disaccharide fragment is buried in the lectin-binding pocket, whereas the reducing trisaccharide portion alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-D-Glc has no immediate contacts with the protein. The experimental transfer NOE data were qualitatively compared to theoretical proton-proton distances from a model that was based on a previous homology modeling study of a complex between the disaccharide fragment alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and D. biflorus lectin. It was found that all intermolecular transferred NOEs matched short interatomic distances between ligand protons and aliphatic or aromatic amino acid side chain protons predicted by the theoretical model.

Conformational analysis of blood group A trisaccharide in solution and in the binding site of Dolichos biflorus lectin using transient and transferred nuclear Overhauser enhancement (NOE) and rotating-frame NOE experiments.

The present study is concerned with the elucidation of the conformation of the blood group A trisaccharide (alpha-D-GalNAc(1-3)[alpha-L-Fuc(1-->2)] beta-D-Gal-O-R) in the combining site of Dolichos biflorus seed lectin by use of 400-MHz and 600-MHz NMR spectroscopy. D. biflorus lectin displays a unique specificity for GalNAc residues. It occurs in solution as a tetrameric assembly having a molecular mass of 110 kDa, with two carbohydrate-binding sites per molecule. First, NOE build-up curves were obtained for the free blood group A trisaccharide from one-dimensional transient NOE experiments. Simulated NOE build-up curves were constructed from an ensemble of low-energy conformers derived from previous investigations. The comparison of theoretical and experimental data indicates that an equilibrium between two families of low-energy conformers most likely reflects the solution behavior of the trisaccharide in solution. Two-dimensional transferred NOE and rotating-frame enhancements (ROE) were subsequently measured for the trisaccharide complexed with the D. biflorus seed lectin. In addition to the NOEs observed for the free trisaccharide, the transferred NOESY spectrum showed several new NOEs that were identified as spin diffusion using a rotating-frame NOESY (ROESY) experiment. Experimental interglycosidic transferred nuclear Overhauser effect (TRNOE) build-up curves were compared to theoretical curves calculated for both low-energy conformers located in the D. biflorus lectin-binding site. Calculations of theoretical TRNOE were performed using a combination of the full relaxation matrix and the protein-ligand exchange matrix. Comparison between experimental and simulated TRNOE volumes leads to the conclusion that one conformation of blood group A trisaccharide is selected upon binding by D. biflorus lectin.

NMR, molecular modeling, and crystallographic studies of lentil lectin-sucrose interaction.

The conformational features of sucrose in the combining site of lentil lectin have been characterized through elucidation of a crystalline complex at 1.9-A resolution, transferred nuclear Overhauser effect experiments performed at 600 Mhz, and molecular modeling. In the crystal, the lentil lectin dimer binds one sucrose molecule per monomer. The locations of 229 water molecules have been identified. NMR experiments have provided 11 transferred NOEs. In parallel, the docking study and conformational analysis of sucrose in the combining site of lentil lectin indicate that three different conformations can be accommodated. Of these, the orientation with lowest energy is identical with the one observed in the crystalline complex and provides good agreement with the observed transferred NOEs. These structural investigations indicate that the bound sucrose has a unique conformation for the glycosidic linkage, close to the one observed in crystalline sucrose, whereas the fructofuranose ring remains relatively flexible and does not exhibit any strong interaction with the protein. Major differences in the hydrogen bonding network of sucrose are found. None of the two inter-residue hydrogen bonds in crystalline sucrose are conserved in the complex with the lectin. Instead, a water molecule bridges hydroxyl groups O2-g and O3-f of sucrose.

Molecular modelling of the interaction between the catalytic site of pig pancreatic alpha-amylase and amylose fragments.

A stereo chemical refinement of the crystalline complex between porcine pancreatic alpha-amylase and a pseudopentasaccharide from the amylostatin family has been performed through molecular mechanics calculations, using a set of parameters appropriate for protein and protein-carbohydrate interactions. The refinement provided a starting point for docking a maltopentaose moiety within the catalytic site, in the absence of water. A thorough exploration of the different orientations and conformations of maltopentaose established the sense of binding of the amylosic substrate in the amylase cleft. After optimising the geometry of the binding site, the conformations adopted by the four contiguous linkages could be rationalised by considering the environment, either hydrophobic or hydrophilic, of the different glucose moieties. Seemingly, details of the non-bonded interactions (hydrogen bonds, van der Waals and stacking interactions) that underlie this molecular recognition have been established. In particular, it was confirmed that the three acidic amino acids of the catalytic site (Asp197, Asp300 and Glu233) are close to their glucosidic target, and that there is no steric reason to propose an alteration of the 4C1 conformation of the glucose residue prior to hydrolysis. However, in the absence of water molecules, it is difficult to elucidate the details of the catalysis. Additional macroscopic information has been gained, such as the impossibility to fit a double-helical arrangement of amylose chains in the amylasic cleft. This explains why some native starches containing such motifs resist amylolytic enzymes. Tentative models involving longer amylosic chains have been elaborated, which extend our knowledge of the interaction and orientation of starch fragments in the vicinity of the hydrolytic sites.

The monosaccharide binding site of lentil lectin: an X-ray and molecular modelling study.

The X-ray crystal structure of lentil lectin in complex with alpha-D-glucopyranose has been determined by molecular replacement and refined to an R-value of 0.20 at 3.0 A resolution. The glucose interacts with the protein in a manner similar to that found in the mannose complexes of concanavalin A, pea lectin and isolectin I from Lathyrus ochrus. The complex is stabilized by a network of hydrogen bonds involving the carbohydrate oxygens O6, O4, O3 and O5. In addition, the alpha-D-glucopyranose residue makes van der Waals contacts with the protein, involving the phenyl ring of Phe123 beta. The overall structure of lentil lectin, at this resolution, does not differ significantly from the highly refined structures of the uncomplexed lectin. Molecular docking studies were performed with mannose and its 2-O and 3-O-m-nitro-benzyl derivatives to explain their high affinity binding. The interactions of the modelled mannose with lentil lectin agree well with those observed experimentally for the protein-carbohydrate complex. The highly flexible Me-2-O-(m-nitro-benzyl)-alpha-D-mannopyranoside and Me-3-O-(m-nitro-benzyl)-alpha-D-mannopyranoside become conformationally restricted upon binding to lentil lectin. For best orientations of the two substrates in the combining site, the loss of entropy is accompanied by the formation of a strong hydrogen bond between the nitro group and one amino acid, Gly97 beta and Asn125 beta, respectively, along with the establishment of van der Waals interactions between the benzyl group and the aromatic amino acids Tyr100 beta and Trp128 beta.

Molecular modelling of the Dolichos biflorus seed lectin and its specific interactions with carbohydrates: alpha-D-N-acetyl-galactosamine, Forssman disaccharide and blood group A trisaccharide.

The three-dimensional structure of Dolichos biflorus seed lectin has been constructed using five legume lectins for which high resolution crystal structures were available. The validity of the resulting model has been thoroughly investigated. Final structure optimization was conducted for the lectin complexed with alpha GalNAc, providing thereby the first three-dimensional structure of lectin/GalNAc complex. The role of the N-acetyl group was clearly evidenced by the occurrence of a strong hydrogen bond between the protein and the carbonyl oxygen of the carbohydrate and by hydrophobic interaction between the methyl group and aromatic amino acids. Since the lectin specificity is maximum for the Forssman disaccharide alpha GalNAc(1-3) beta GalNAc-O-Me and the blood group A trisaccharide alpha GalNAc(1-3)[alpha Fuc(1-2)] beta Gal-O-Me, the complexes with these oligosaccharides have been also modelled.