Carbohydrate Analogue Microarrays for Identification of Lectin-Selective Ligands.

Fifty-five mono- and disaccharide analogues were prepared and used for the construction of microarrays to uncover lectin-selective ligands. The microarray study showed that two disaccharide analogues, 28' and 44', selectively bind to Solanum tuberosum lectin (STL) and wheat germ agglutinin (WGA), respectively. Cell studies indicated that 28' and 44' selectively block the binding of STL and WGA to mammalian cells, unlike the natural ligand LacNAc, which suppresses binding of both STL and WGA to cells.

The role of wheat germ agglutinin in the attachment of Pseudomonas sp. WS32 to wheat root.

Wheat germ agglutinin (WGA), which is secreted on the surface of wheat root, has been defined as a protein that reversibly and non-enzymatically binds to specific carbohydrates. However, little attention has been paid to the function of WGA in the attachment of bacteria to their host plants. The aim of this study was to investigate the role of WGA in the attachment of Pseudomonas sp. WS32 to wheat roots. Wheat roots were initially treated with double-distilled water, WGA-H (WGA solution that was heated at 100°C for 15 min) and WGA, independently. Subsequently, the roots were co-incubated with cell solutions (109 cells/ml). A dilution plate method using a solid nutrient medium was employed to determine the adsorption of WS32 to wheat roots. WGA was labeled with fluorescein isothiocyanate and detected using the fluorescent in situ hybridization (FISH) technique. The number of adsorptive WS32 cells on wheat roots was significantly increased when the wheat roots were pretreated with WGA, compared with the control treatment (p = 0.01). However, WGA-H failed to increase the amount of bacterial cells that attached to the wheat roots because of the loss of its physiological activity. The FISH assay also revealed that more cells adhered to WGA-treated wheat roots than to control or WGA-H-treated roots. The results indicated that WGA can mediate Pseudomonas strain WS32's adherence to wheat seedling roots. The findings of this study provide a better understanding of the processes involved in plant-microbe interactions.

Efficient wheat germ agglutinin purification with a chitosan-based affinity chromatographic matrix.

An efficient affinity chromatographic matrix based on chitosan for wheat germ agglutinin (WGA) purification was developed. The matrices assayed consisted of chitosan mini-spheres cross-linked with epichlorhydrin 45, 250 or 500 mM. The maximum adsorption capacity of pure WGA - calculated from the corresponding isotherms - was between 43.2 and 48.9 mg/g at pH 5.0 and between 16.6 and 27.6 mg/g at pH 8.5. However, the adsorption of agglutinin from wheat germ extract was higher at pH 8.5. In addition, 0.5 g of mini-spheres cross-linked with epichlorhydrin 250 mM adsorbed 94.5% of the WGA present in 5 mL of the concentrated extract. Acetic acid was able to elute 100% of the adsorbed WGA. The purity of the WGA obtained was greater than 95% and the purification factor was 56.8. The matrix was able to maintain an efficient performance of the purification process for three consecutive cycles. A new method to monitor the purification process by RP-HPLC was developed.

Egg-matrix for large-scale single-step affinity purification of plant lectins with different carbohydrate specificities.

Hen eggs represent an easily available and inexpensive source of glycoproteins expressing a variety of sugars. Egg glycoproteins might therefore be exploited to purify by affinity chromatography carbohydrate-binding proteins (lectins) with different specificities. A method to generate an affinity matrix from hen eggs is described. The matrix was assayed for its ability to purify in a single step biologically active phytohemagglutinin, wheat germ agglutinin, lentil lectin, and peanut agglutinin. Milligrams of purified lectins per gram of matrix was obtained, with the only exception of peanut agglutinin that was not efficiently retained into the affinity column. Hen egg chromatography is a relatively simple, fast, and reproducible method to purify high amount of plant lectins.

Identification of human placental wheat germ agglutinin-immunoreactive protein by mass spectrometry.

A preparation of polyclonal antibodies to human placental calcium-dependent, carbohydrate-binding glycoprotein, previously identified as wheat germ agglutinin-immunoreactive protein, was applied as the ligand in an immunoaffinity procedure. Following electrophoretic separation of purified material, the specific 66-kDa antigen band was excised and subjected to in-gel protein cleavage. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the tryptic digest yielded 42 isotopically resolved peptide fragments from mass 860 to 2,690. Empirical data from MALDI-MS were analyzed by computer assistance using ProteoMetrics PROFOUND software. Protein candidates reported in the specified identification database search are discussed in relation to the biochemical characteristics of the protein analyzed and its possible antigenic relatedness to wheat germ agglutinin. We speculate that a fibulin-like member of the epidermal growth factor-repeat protein family might be selected as a positive match.

A specific wheat germ agglutinin-immunoreactive protein in human placenta.

In this study we examined human placenta for the presence of molecules antigenically related to a plant lectin, wheat germ agglutinin. The initial results of immunolocalization using polyclonal antibodies against wheat germ agglutinin showed that human placenta contains protein(s) recognized specifically. Staining of syncytiotrophoblast brush border and cytotrophoblast, granular in appearance was observed in first trimester human placenta. Specific binding was also seen in trophoblast-derived JAr and BeWo carcinoma cells. Isolation of wheat germ agglutinin-immunoreactive material from human placenta was achieved by ion-exchange- and affinity-chromatography on anti-wheat germ agglutinin-immunoglobulin G-Sepharose. The placental protein having molecular mass of 66 kD was identified as specific. The protein of 66 kD was characterized as a calcium-dependent, asialofetuin-binding molecule.

Inhibition of vesicular secretion in both neuronal and nonneuronal cells by a retargeted endopeptidase derivative of Clostridium botulinum neurotoxin type A.

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types by a mechanism that involves cleavage of specific components of the vesicle docking/fusion complex, the SNARE complex. A derivative of the type A neurotoxin from Clostridium botulinum (termed LH(N)/A) that retains catalytic activity can be prepared by proteolysis. The LH(N)/A, however, lacks the putative native binding domain (H(C)) of the neurotoxin and is thus unable to bind to neurons and effect inhibition of neurotransmitter release. Here we report the chemical conjugation of LH(N)/A to an alternative cell-binding ligand, wheat germ agglutinin (WGA). When applied to a variety of cell lines, including those that are ordinarily resistant to the effects of neurotoxin, WGA-LH(N)/A conjugate potently inhibits secretory responses in those cells. Inhibition of release is demonstrated to be ligand mediated and dose dependent and to occur via a mechanism involving endopeptidase-dependent cleavage of the natural botulinum neurotoxin type A substrate. These data confirm that the function of the H(C) domain of C. botulinum neurotoxin type A is limited to binding to cell surface moieties. The data also demonstrate that the endopeptidase and translocation functions of the neurotoxin are effective in a range of cell types, including those of nonneuronal origin. These observations lead to the conclusion that a clostridial endopeptidase conjugate that can be used to investigate SNARE-mediated processes in a variety of cells has been successfully generated.

Fusarium sp. growth inhibition by wheat germ agglutinin.

The antifungal role of wheat germ agglutinin (WGA) isolated from a Romanian dihaploid variety of wheat against two pathogenic fungal species of Fusarium, F. graminearum and F. oxysporum, is demonstrated. WGA was prepared from unprocessed wheat germs by a new purification procedure using chitin and fetuin-Sepharose as affinity chromatography supports. SDS-PAGE and chitinase assay showed that the WGA preparation migrated as a single protein band and was devoid of any contaminating enzyme chitinase, well known for its antifungal effects. Based on its affinity for N-acetylglucosamine residues, WGA binding to the chitin-containing walls of the fungi was detected by fluorescence microscopy using WGA coupled with fluorescein isothiocyanate (FITC). In vitro testing of WGA action on early developmental stages of both fungal strains resulted in various modifications of the germ tubes, visualised by light microscopy: swelling, vacuolation of the cellular content and lysis of cell walls. Viability tests performed on potato tuber slices showed that the microbial infection was prevented from spreading by pretreatment of the fungal suspension with WGA.

Expression and secretion of wheat germ agglutinin by Saccharomyces cerevisiae.

Genes encoding pre-protein and prepro-protein of wheat germ agglutinin isolectin 2 (WGA2) were chemically synthesized and expressed in the yeast Saccharomyces cerevisiae under the control of the ENO1 promoter. Yeast harboring either a pre-WGA2 or a prepro-WGA2 gene expression plasmid secreted a mature form of WGA2 into the culture medium. The amount of WGA2 secreted by the strain KS58-2Ddel, which has a ssl1 mutation causing a supersecretion of human lysozyme [Suzuki, K., Ichikawa, K. & Jigami, Y. (1989) Mol. Gen. Genet. 219, 58-64], was 20-fold greater than that secreted by the wild-type strain KK4. The recombinant WGA2 from the cells containing the prepro-WGA2 gene expression plasmid was purified to homogeneity by a three-step ion-exchange chromatography scheme. As in wheat, the N-terminal signal peptide of recombinant WGA2 purified from yeast culture was processed to form an N-terminal 5-oxoprolyl (pyroglutamyl) residue. Likewise, we found that the C-terminal pro-region of recombinant WGA2 had also been processed in yeast. Using electrospray ionization mass spectrometry, we found the processed C-terminus to be heterogeneous in both recombinant WGA2 purified from yeast and in authentic WGA2. The major component of the recombinant WGA2 contained two additional amino acids at its C-terminus compared to that of authentic WGA2. In spite of this difference in the C-terminus, the recombinant WGA2 exhibited a sugar binding activity that was indistinguishable from that of authentic WGA2.

Bead cellulose derivatives as supports for immobilization and chromatographic purification of proteins.

Characteristic data are presented for Divicell, a macroporous bead cellulose with excellent flow parameters. The preparation of Divicell derivatives and their properties are described with respect to their application as chromatographic supports. The ion exchangers Divicell DEAE and Divicell CM were manufactured in two types with different exclusion limits and an available capacity for proteins of up to 100 mg/ml gel. Divicell Blue is a bead cellulose with covalently bound Cibacron Blue F3G-A and was found to be a very suitable adsorbent for the selective separation and purification of human serum albumin. Activation of Divicell with sodium periodate, epichlorohydrin and 5-norbornene-2,3-dicarboximido carbonochloridate provided activated supports used for immobilization of ligands in organic solvents and in aqueous solutions. Coupling of amines, diamines, amino acids, carbohydrates and proteins is described. The immobilized ligands retained their biological activity as determined by their specific adsorption of proteins. Divicell alkyl derivatives were tested in hydrophobic interaction chromatography with bovine serum albumin as a model. Examples are presented of the application of Divicell derivatives to the purification of biomacromolecules such as immunoglobulins and lectins by affinity chromatography. The results were comparable to those obtained using the corresponding Sepharose-derived absorbents.