Francis Delmotte

Protein—sugar interactions. Association of β-(1→4) linked N-acetyl-D-glucosamine oligomer derivatives with wheat germ agglutinin (lectin)

Wheat germ agglutinin (WGA) is a lectin [l-3] which binds selectively N-acetyl-D-glucosamine, N, N’-diacetyl-chitobiose (GlcNAc)* N,N’,N”-triacelyichitotriose (G~cNAc)~ and Npfl’fl”-tetraacetyl chitotetraose (G~cNAc)~ [4,5]. These monoand oligosaccharides have been shown to be the best inhibitors of the agglutination of erythrocytes and other types of cells [l-3] . Levine et al. [6] reported that WGA has one N-acetyl-D-glucosamine binding site per 23 000 g of the protein, on the basis of equilibrium dialysis experiments. The same conclusion was also deduced from fluorescence experiments in the case of /3(1’4) linked oligomers of N-acetyl-Dglucosamine [4]. We report here that WGA has two independent and homogeneous binding sites for reduced chitotetraose per mole of protein, on the basis of equilibrium dialysis experiments, and that WGA forms a precipitate with chitobiose-bovine serum albumin conjugate, even under conditions where it is known that the protein is in its monomeric form.

Productivity, water-use efficiency and tolerance to moderate water deficit correlate in 33 poplar genotypes from a Populus deltoides × Populus trichocarpa F1 progeny

Genotypic variability for productivity, water-use efficiency and leaf traits in 33 genotypes selected from an F1 progeny of Populus deltoides Bartr. ex Marsh x Populus trichocarpa L. was explored under optimal and moderate water-deficit conditions. Saplings of the 33 genotypes were grown in a two-plot open field at INRA Orléans (France) and coppiced every year. A moderate water deficit was induced during two successive years on one plot by withholding irrigation, while the second one remained irrigated (control). Stem biomass and leaf structure (e.g., specific leaf area and leaf area) were measured in 2004 and 2005 and functional leaf traits (e.g., carbon isotope discrimination, Delta) were measured only in 2004. Tolerance to water deficit was estimated at genotype level as the ability to limit losses in biomass production in water deficit versus control trees. Stem biomass, leaf structure and Delta displayed a significant genotypic variability whatever the irrigation regime. For all traits, genotype ranks remained stable across years for similar irrigation conditions. Carbon isotope discrimination scaled negatively with productivity and leaf nitrogen content in controls. The most productive genotypes were the least tolerant to moderate water deficit. No relationship was evidenced between Delta and the level of tolerance to water deficit. The relationships between traits evidenced in this collection of P. deltoides x P. trichocarpa F1 genotypes contrast with the ones that were previously detected in a collection of P. deltoides x Populus nigra L. cultivars tested in the same field trial.

Protein-sugar Interactions. Association of wheat germ agglutinin (lectin) and O-(4-methyl-umbelliferyl)-glycosides

Wheat germ agglutinin (WGA) is a lectin which agglutinates erythrocytes and other types of cells [l-3]. It has been shown that N-acetyl-D-glucosamine and its /.%( 14) linked oligomers specifically inhibit the agglutination reaction [2,4] and induce fluorescence spectral changes upon binding to WGA [5,6]. The number of binding site(s) per polypeptide chain has been reported to be one by Levine et al. [7], Greenaway and Levine [S] and two by Nagata and Burger [9] on the basis of equilibrium dialysis experiments with N-acetyl-D-glucosamine. Nagata and Burger [9] did not exclude the possibility of having determined two subsites as separate binding sites. In a previous paper [lo] , we reported the presence of two binding sites per polypeptide chain on the basis of equilibrium dialysis experiments with reduced-N, N’, N”, N”‘-tetraacetyl chitotetraose. Recently, Dean and Homer [ 1 l] used 0(4.methyl-umbelliferyl) -a-D-mannoside to investigate carbohydrate-concanavaline A interactions, and found that the fluorescence of this glycoside was quantitatively quenched upon binding to the lectin. In contrast, Delmotte et al. [ 121 showed that the fluorescence of 044-methyl-umbelliferyl)-iV, N’,N”-tri-acetylgchitotrioside was enhanced upon binding to lysozyme. It was of interest to investigate the behaviour of O-(4-methyl-umbelliferyl)-glycosides of N-acetyl-/3Dglucosamine and of its fl-( 1+4) linked oligomers upon binding to WGA. We report here that the fluorescence of these glycosides are completely quenched upon binding to WGA, and that WGA has two independent binding sites per polypeptide chain for 0-(4.methylumbelliferyl)-glycosides.

Interactions glycanne-protéine. Synthése des 4-méthylombelliféryl-(2-acétamido-2-désoxy-β-D-glucopyranoside), -DI-N-acétyl-β-chitobioside et -tri-N-acétyl-β-chitotrioside. Interaction de ces osides avec le lysozyme

4-Methylumbelliferyl 2-acetamido-2-deoxy-β-D-glucopyranoside, 2-acetamido-4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-2-deoxy-β-D-glucopyranoside (di-N-acetyl-β-chitobioside), and O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside (tri-N-acetyl-β-chitotrioside) were obtained in good yield from the corresponding peracetylated glycosyl chlorides by condensation with the sodium salt of 4-methylumbelliferone in N,N-dimethylformamide. The trisaccharide glycoside is hydrolyzed by lysozyme and is, therefore, a convenient substrate for this enzyme; the 4-methylumbelliferone produced can be determined by the increase of the fluorescence intensity at 442 nm. The intensity of the fluorescence of 4-methylumbelliferyl tri-N-acetyl-β-chitotrioside is enhanced upon binding with lysozyme without modification of the position of the absorption maximum. The binding constant and the rate of hydrolysis of the trisaccharide glycoside by lysozyme are higher than those obtained with p-nitrophenyl tri-N-acetyl-β-chitotrioside.

Preparation and biological properties of a covalent antitumor drug—arm—carrier (DAC conjugate)

Cancer chemotherapeutic agents are not selective in their action against cancer cells. Therefore, the possibility of using a macromolecule, such as an antibody molecule, as a specific carrier of antitumor drugs has attracted considerable attention [ 1,2]. For instance, daunorubicin was covalently linked to antibodies [3-61 or to Fab dimers [7] or to a lectin [8] by coupling methods using glutaraldehyde, carbodiimide and periodate oxidation of the drug. For technical reasons, and because the activity of a drug is partially or totally lost when it is substituted or chemically modified, we devised a spacer arm such that the drug-carrier conjugate is stable in serum and can be specifically split by lysosomal proteases leading to the free drug inside the target cells.

Osmotic stress sensing in Populus: Components identification of a phosphorelay system

To study the Populus response to an osmotic stress, we have isolated one cDNA encoding a histidine‐aspartate kinase (HK1) and four cDNAs encoding histidine‐containing phosphotransfer proteins (HPts), HPt1–4. The predicted HK1 protein shares a typical structure with ATHK1 and SLN1 osmosensors. The 4 HPTs are characterized by the histidine phosphotransfer domain. We have shown that HK1 is upregulated during an osmotic stress in hydroponic culture. We have detected an interaction between HK1 and HPt2, using the yeast two‐hybrid system. These results suggest the existence of a multi‐step phosphorelay pathway probably involved in osmotic stress sensing in Populus.

Protein-sugar interaction: purification by affinity chromatography of Solanum tuberosum agglutinin (STA-lectin).

Solanum tuberosum tubers contain a lectin which agglutinates erythrocytes [I] . Several procedures of purification of this lectin have been proposed: Singh et al. [2] used an acetone fractionation, Marinkovich [3] used chromatography on an SPcellulose column and electrophoresis on starch block; recently Allen and Neuberger [4] used ammonium sulfate precipitation and several column chromatography steps on DEAE-cellulose, CM-cellulose, Sephadex G100 and SP-Sephadex. The material isolated by Singh et al. [2] was not a protein, the procedure of Marinkovich [3] did not yield a pure product, and the procedure of Allen and Neuberger [4] was time consuming. In order to overcome these difficulties, we have tried to develop ari easier procedure using affinity chromatography.

Alkylsyringamides, new inducers of Agrobacterium tumefaciens virulence genes

The virulence genes of Agrobacterium tumefaciens are specifically activated by plant phenolic compounds and allow this organism to genetically transform plant cells. New types of phenolic compounds, three phenol amides derived from syringic acid, were synthesized. Introduction of an amide group in syringic acid strongly enhances its vir gene inducing activity.

Protein—sugar interactions A nuclear magnetic resonance investigation of the binding of O‐methyl‐di‐N‐acetyl‐β‐chitobioside to wheat germ agglutinin (lectin)

Wheat germ agglutinin (WGA) is a plant lectin which can agglutinate various animal cells, particularly malignant cells and normal protease treated cells [1-3]. Agglutination is inhibited by N-acetyl-glucosamine and its/3-1--4-1inked oligomers [2--4]. Under physiological conditions, WGA is a dimer. In acidic media, it dissociates in 2 subunits mol. wt 18 000 [5,6]. The dimer binds 4 ligands containing the Naeetylglucosamine moiety with equal affinity. In other words, each protomer binds saccharides at 2 spatially different but chemically equivalent locations [5,7,8]. Lectin-sugar interactions have been studied by fluorescence [9-13], absorption [13] and circular dichroism [ 14] techniques. Although nuclear magnetic resonance (NMR) has been used extensively to study the interactions of sugars with lysozyme (see ref in [ 15 ] ) and concanavalin A [16], only one report so far has been devoted to WGA [ 17]. The binding constant ofN-acetyl glucosamine was determined [ 17] and showed that the methyl group resonance was strongly shifted upon binding. In this communication, we present results of an z H NMR investigation of the binding of 1-O-methyl-di-N-acetyl-/~-chitobioside (CBOCH3) and of 1-O-methyl-tri-N-acetyl~-chitotrioside (CTOCHs) to WGA. The use of methyloside derivatives, in effect removing the anomeric equilibrium, greatly simplifies the analysis.

New syntheses of plant aryl glycosides as potential gene inducers

Abstract Aryl β- d -glycopyranosides have been synthesized by coupling acetovanillone (4-hydroxy-3-methoxyacetophenone) with d -glucose, d -galactose, and maltose; acetosyringone (4-hydroxy-3,5-dimethoxyacetophenone) with d -glucose and d -galactose; syringaldehyde (3-methoxyvanillin) with d -glucose; and syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid) with d -glucose. The Mauthners procedure using peracetylated glycosyl bromides and phenolates in aqueous acetone afforded the acetylated β- d -glycosides, which were deacetylated.