Properties of Succinylated Wheat‐Germ Agglutinin

Abstract

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a p l of 4.0 0.2 while the native lectin is basic, p l of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 pg/ml with both lectins and the concentrations of AT-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins.The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins.The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.Wheat germ agglutinin is a plant lectin which is capable of agglutinating erythrocytes and some other types of cells [l] (for recent reviews see [2,3]).Agglutination is inhibited by N-acetylglucosamine and its (gl-4)oligomers [4-61. Wheat germ agglutinin is a basic protein with an isoelectric point of 8.7 & 0.3 [7]. At neutral pH, wheat germ agglutinin is a dimer; in acidic medium, it dissociates into two subunits of M , 18000 [8,9].Subunit dissociation also occurs upon acetylation [7] or tryptophan oxidation with N-bromosuccinimide [lo]; this dissociation is concommitent with a total loss of agglutination properties. However upon succinylation, neither subunit dissociation nor loss of A+ human red-blood-cell agglutinating activity occur [7]. In the present paper, the physicochemical properties and the sugar binding properties of succinylated wheat germ agglutinin are compared with those of the unmodified lectin. MATERIALS AND METHODS MaterialsWheat germ agglutinin prepared as in [ l l ] was purchased from Pharmindustrie-IBF-Reactifs, Clichy (France). Tri-N-acetylchitotriose and tetra-N-acetylchitotetraose were prepared by acetolysis of chitin, isolation of the per-0,N-acetylated oligosaccharides by silica gel column chromatography [12], de-0-acetylation by catalytic methanolysis with 0.01 M sodium methoxide in methanol at 4°C for 24 h. The tri-Nacetylchitotriose and the tetra-N-acetylchitotetraose were obtained by crystallization from their methanolic solution. Biogel P-60 was purchased from Biorad. Ultrogel ACA 54, Ultrogel A4 and HA-Ultrogel (hydroxyapatite) were purchased from Pharmindustrie-IBF-Reactifs. Fluorescein isothiocyanate was obtained from Research Organics Inc. (Clevela...

The 17-residue transmembrane domain of beta-galactoside alpha 2,6-sialyltransferase is sufficient for Golgi retention

“…These results demonstrate that S33D had been modified by glycosyltransferases of the trans-Golgi cisternae and the TGN and suggest that it is localized to the trans-Golgi cisternae and/or TGN . WGA binds both sialic acid and N-acetylglucosamine while succinylated WGA (sWGA) binds only N-acetylglucosamine (Monsigny et al, 1979(Monsigny et al, , 1980 . The binding of 110-kD S33D to WGA but not sWGA not only confirmed the presence of sialic acid on the N-linked glycans but also demonstrated that no N-acetylglucosamine residues were exposed on the glycans .…”

Wheatgerm agglutinin-mediated toxicity in pancreatic cancer cells

“…Wheatgerm agglutinin (WGA, specific for sialic acid and GlcNAc), Concanavalin A (ConA, specific for α-D-mannose and α-D-glucose), and phytohaemagglutinin L (PHA-L, specific for the β1-6 branch at the trimannosyl core of N-linked carbohydrate) were obtained from Sigma Chemicals. Succinyl-WGA (succWGA, specific only for GlcNAc but not for sialic acid (Monsigny et al, 1979)) was purchased from Wheatgerm agglutinin-mediated toxicity in pancreatic cancer cells Vector Laboratories. Control lectins for sialic acid binding were Maackia amurensis agglutinin (MAA), Sambucus nigra agglutinin (SNA) and Limulus polyphemus agglutinin (LPA), all purchased from Sigma Chemicals.…”

Wheat germ agglutinin blocks the acrosome reaction in Strongylocentrotus purpuratus sperm by binding a 210,000-mol-wt membrane protein.

“…Some batches of WGA were modified by suecinylation (13) and/or conjugated to tetramethylrhodamine isothiocyanate (6). Lectin solutions were made fresh each day and stored at 0*C.…”