Accumulation Of Wheat Germ Agglutinin Research Articles

Abstract 1338: Blockage of nucleocytoplasmic transport by wheat germ agglutinin (WGA) induces autophagy and cell death

Abstract The nuclear pore complex (NPC) is a primary conduit for cellular communication between the nucleus and cytoplasm, which is essential for cellular survival and proliferation. During carcinogenesis, many oncoproteins are activated and have to send their signals into nucleus to regulate gene expression. In response to the signals, many genes are upregulated, which results in transcription of many RNA. Because both inward of transcriptional factors and outward of RNAs require interaction of these molecules with NPC, blockage of NPC may provoke serious cellular distress. Wheat germ agglutinin (WGA), a lectin that specifically binds to N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-neuraminic acid (NeuNAc) residues on the cell surface, was reported to disrupt the function of NPC via interaction with POM121. We found that FITC-WGA had located around the cell membrane 0.5 hour after administration to culture media and reached the paranuclear region 4 hours later. The accumulation of WGA around the nucleus subsequently induced a nucleocytoplasmic transport blockage that prevented export of tRNA and import of signaling proteins (NF-κB and Nrf2) into the nucleus. Beyond the concentration of 5 μg/ml, WGA dose- and time-dependently induced cell death in HeLa, SiHa and CaSKi cell lines. The SiHa cells had more intake of WGA than HeLa and CaSKi cells did and became more sensitive to WGA treatment compared to the other two cells. After treatment with WGA in the three cells, we observed that the expression of autophagic markers (ATG5, ATG7 and LC3) are induced in 24 hours and last to 96 hours. On the contrary, the apoptotic protein caspases 3 and downstream substrates, such as poly(ADP-ribose) polymerase (PARP) were only marginally induced. Furthermore, Transmission Electron Microscopy (TEM) imaging disclosed multi-membrane vesicles (autophagosomes and autolysosomes) inside the cytoplasm after WGA treatment, confirming that the autophagy response was fully induced. These findings suggest that blockage of NPC by WGA may induced autophagy-related cell death and targeting NPC may be warranting further development into a new anti-cancer modality. Citation Format: Tsung-Lin Tsai, Chun-Hua Hung, Hao-Chen Wang, Dar-Bin Shieh, Wu-Chou Su, Chien-Chung Lin. Blockage of nucleocytoplasmic transport by wheat germ agglutinin (WGA) induces autophagy and cell death. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1338. doi:10.1158/1538-7445.AM2014-1338

Site specific silencing of caveolin acylation causes accumulation of Alexa labeled Cholera Toxin B chain at distinct sites in the lipid raft‐caveolae endosomal system in polarized HepG2 cells

We have previously shown that silencing of the three distinct acylation sites in caveolin‐1 by substitution of alanine for cysteine at positions 133 (cav 133), 143 (cav 143) and 156 (cav 156) alter the cholesterol transport activity of 2 distinct cytosolic protein complexes. In this study, we examined the effect of these acylation mutants on endocytosis of fluorescent tagged cholera toxin B chain (CT‐B), which binds to the ganglioside GM1 and marks lipid raft/caveolae endocytosis. We stably expressed wild type caveolin (wt cav) or each of the single site acylation mutants in HepG2 cells, a cell line which lacks detectable expression of caveolin. Cells were cultured with Alexa 488 labeled wheat germ agglutinin (WGA), a marker of the constitutive endocytic pathway, and Alexa 555 labeled CT‐B. Ligand uptake was followed by live confocal microscopy at 37 deg or cells were incubated for time points up to 4 hours at 4 deg or 37 deg, fixed and immunostained for caveolin, organelle markers, or annexins (proteins associated with lipid raft/caveolae trafficking). Expression of each of the acylation mutants showed accumulation of CT‐B in distinct intracellular sites compared to cells expressing wt cav. Expression of the mutants also altered the distribution and accumulation of WGA indicating that alteration in caveolae/lipid raft trafficking impacts the constitutive endocytic system. Supported by NIH.

Lectin binding studies on C-28/I2 and T/C-28a2 chondrocytes provide a basis for new tissue engineering and drug delivery perspectives in cartilage research

The present study was performed to evaluate the applicability of plant lectins as mediators of bioadhesion in cartilage research using human chondrocyte cell lines C-28/I2 and T/C-28a2. The bioadhesive properties of fluorescein-labelled lectins with different carbohydrate specificities were investigated by flow cytometry. Specificity of the lectin–cell interactions was ascertained by competitive inhibition using complementary carbohydrates. As compared to that of other lectins, the interaction between wheat germ agglutinin (WGA) and chondrocytic cells was characterised by remarkable cytoadhesion, adequate binding strength and a high degree of specificity for N-acetyl-glucosamine as contained in hyaluronan chains. We therefore suggest WGA to be a promising candidate for mediating bioadhesion to low-adhesive scaffolds in cartilage tissue engineering. Moreover, the WGA-association rate of C-28/I2 and T/C-28a2 cells was dependent on temperature indicating cellular uptake of membrane-bound WGA. Intracellular enrichment was confirmed by confocal microscopy. Equilibration of intracellular pH gradients with monensin resulted in the reversal of quenching effects indicating accumulation of WGA within acid compartments of chondrocytic cells. Thus, WGA might be internalised into chondrocytes together with hyaluronan via the CD44 receptor-mediated endocytosis pathway and accumulated within lysosomes. This physiological process could represent a feasible pathway to target WGA-functionalised drug delivery devices into chondrocytes.

Lectin-mediated Drug Delivery: Discrimination Between Cytoadhesion and Cytoinvasion and Evidence for Lysosomal Accumulation of Wheat Germ Agglutinin in the Caco-2 Model

Lectin-mediated drug delivery may become a promising strategy to improve the efficacy of poorly permeable drugs by utilising active high-capacity transport pathways of epithelial tissues. This requires the elucidation of the basic mechanisms of lectin uptake prior to their practical use. We studied the interaction between the dietary lectin wheat germ agglutinin (WGA) and Caco-2 cells (single cells and monolayers) by a newly established assay design that is able to discriminate between cellular binding and uptake as well as by confocal microscopy: (i) All binding sites available for WGA at the cell membrane were occupied within 10 min of incubation. (ii) Cytoadhesion was followed by immediate uptake. After 20 min, 60% (single cells) or 30% (monolayers) of the membrane bound lectin were internalised. However, regardless of cell arrangement, 80% of the surface bound lectin was taken up into the cells during the course of the experiment. (iii) About 50% of the internalised lectin accumulated within the lysosomes after 1 h. This was confirmed by assays in the presence of monensin, an inhibitor of endosomal acidification, and by colocalisation with lysosomal cathepsin followed by semiquantitative image analysis. Further analysis by immunocytochemistry suggested that the trans-Golgi complex and the caveoli were not involved. Due to cytoadhesion, cytoinvasion and partial lysosomal accumulation, WGA-mediated drug delivery may provide for improved intracellular availability of conjugated drugs or colloidal carrier systems.

Stimulation of wheat germ agglutinin gene expression in root seedlings by 24-epibrassinolide

The effects of 24-epibrassinolide (EB) on the growth of four-day-old seedlings of Triticum aestivum L. roots, the accumulation of wheat germ agglutinin (WGA) in them, and its gene expression were studied. EB stimulated growth with two optimum concentrations, 0.4 nM and 0.4 μM. Both concentrations enhanced WGA accumulation in roots, which resulted from the stimulation of its gene expression. The latter was shown by dot-blot hybridization of transcripts with a DNA probe corresponding to WGA. Since EB did not affect the level of endogenous ABA, its direct effect on WGA-gene transcription in wheat roots is suggested.

Induction of wheat germ agglutinin synthesis by abscisic and gibberellic acids in roots of wheat seedlings

The dynamics of ABA and wheat germ agglutinin (WGA) content and WGA-geneexpression have been studied in roots of 4-d-old wheat seedlings treated with 4 ABA and 0.3 GA. Both hormones caused rapidand significant effects on WGA accumulation, though GA might control thisprocess independently of ABA, since the content of endogenous ABA did not changeafter GA-treatment. The results of dot-blot analysis of transcripts of RNA usingthe 32P labelled WGA-gene as a probe showed that a two-fold increasein WGA content resulted from ABA- and GA-induced activation of WGA-geneexpression.

Multiple Hormonal Control of the Lectin Content in Roots of Wheat Seedlings

The effects of 0.2 μM GA, 4.4 μM benzyladenine (BA), and 5.7 μM IAA on the contents of wheat germ agglutinin (WGA) and ABA in the roots of four-day-old wheat (Triticum aestivumL.) seedlings were studied. All phytohormones tested almost doubled the level of WGA. BA and IAA evidently stimulated the WGA accumulation by inducing ABA accumulation, whereas GA affected the level of the WGA in an ABA-independent way. The authors conclude that phytohormones control the WGA level via several pathways.

Drought-induced increase in wheat germ agglutinin (WGA) accumulation in developing wheat embryos appears to be independent of ABA

Accumulation of wheat germ agglutinin (WGA) in the developing embryos of three different wheat (Triticum aestivum L.) cultivars, PBW-138, PBW-299 and C-306, was studied in relation to abscisic acid (ABA) accumulation under water stress conditions at 18, 24 and 30 days post anthesis (DPA) under natural conditions. Imposition of water stress in all three cultivars resulted in enhanced ABA levels in the embryos at all stages of grain development. On the contrary, the increase in WGA accumulation in the embryos in response to drought was stage- and cultivar-dependent. Our results suggest that apart from ABA, other factors that are temporally expressed may be involved in drought-induced regulation of the WGA gene.

Drought stress induced accumulation of wheat germ agglutinin in the developing embryos of wheat ( Triticum aestivum L.) and its likely role in vivo

Summary The effect of drought stress on wheat germ agglutinin (WGA) accumulation in the developing embryos of different wheat ( Triticum aestivum L.) cultivars was investigated. Although water potential of the grains was relatively less affected by water stress, a substantial increase in WGA accumulation was observed in response to drought, especially at 18 days post anthesis (DPA). After 30 DPA draughted embryos depicted degradation of WGA whereas embryos of irrigated plants maintained higher levels of WGA. Further, a likely role of WGA as scavenger of free radicals has been discussed.

Effect of temperature shock on the dynamics of abscisic acid and wheat germ agglutinin accumulation in wheat cell culture

Abscisic acid (ABA) and lectin content was immunoassayed in wheat cell cultures affected by temperature stress. The elevated temperature (40°C) resulted in a 7-fold increase in the level of ABA and a 10-fold increase in that of lectin. The increase in the lectin content in cells was preceded by ABA accumulation. It is suggested that this ABA increase induces the synthesis of lectin, which in addition to stress proteins, play an important role in controlling mechanisms of plant adaptation to unfavourable environments.

Wheat germ agglutinin in wheat seedling roots: induction by elicitors and fungi

Treatment of wheat (Triticum aestivum L.) seedlings with elicitors originating from either plant or fungal cell walls induces about a 2-fold increase of wheat germ agglutinin (WGA) in the roots. While the WGA content in roots of healthy plants normally decreases as a function of germination time, a transient accumulation of WGA could be observed in plants challenged with different fungi, including Rhizoctonia solani, Fusarium culmorum, Pythium ultimum and Neurospora crassa. Peak levels in challenged roots were 2 to 5 times as high as in control plants. Most of this induced WGA could be released from the roots by soaking them in a solution of the hapten N-acetylglucosamine. On the basis of the results obtained it is postulated that WGA may be involved in the defence of wheat against fungal attack.

Stress-Induced Accumulation of Wheat Germ Agglutinin and Abscisic Acid in Roots of Wheat Seedlings

Wheat germ agglutinin (WGA) levels in roots of 2-day-old wheat seedlings increased up to three-fold when stressed by air-drying. Similar results were obtained when seedling roots were incubated either in 0.5 molar mannitol or 180 grams per liter polyethylene glycol 6000, with a peak level of WGA after 5 hours of stress. Longer periods of osmotic treatment resulted in a gradual decline of WGA in the roots. Since excised wheat roots incorporate more [(35)S]cysteine into WGA under stress conditions, the observed increase of lectin levels is due to de novo synthesis. Measurement of abscisic acid (ABA) levels in roots of control and stressed seedlings indicated a 10-fold increase upon air-drying. Similarly, a five- and seven-fold increase of ABA content of seedling roots was found after 2 hours of osmotic stress by polyethylene glycol 6000 and mannitol, respectively. Finally, the stress-induced increase of WGA in wheat roots could be inhibited by growing seedlings in the presence of fluridone, an inhibitor of ABA synthesis. These results indicate that roots of water-stressed wheat seedlings (a) contain more WGA as a result of an increased de novo synthesis of this lectin, and (b) exhibit higher ABA levels. The stress-induced increase of lectin accumulation seems to be under control of ABA.

Wheat germ agglutinin accumulation in coleoptiles of different genotypes of wheat. Localization by monoclonal antibodies

We have produced a library of 18 monoclonal antibodies (mABs) against wheat germ agglutinin (WGA). It was difficult to establish antibody-producing hybridomas when soluble WGA was used for immunization. The frequency of specific hybridomas was increased, however, by injecting mice with insoluble antigen-antibody complex.We distinguished groups of mABs that are especially efficient for particular immunoassays. One group (mABs 005, 006, 007, 009, 011, 014, 015, 016, 017, 018, 019) strongly immunostains denatured antigen on electroblots of sodium dodecyl sulfate polyacrylamide gels. A second group (all mABs except 012) shows high activity for WGA when native protein is analyzed by enzyme-linked immunosorbent assay. The third group (mABs 002, 005, 008, 009, 010, 011, 014, 016, 018, 019) works well for immunocytochemistry.We used the mABs to localize WGA in wheat varieties of various ploidy and with different ancestral wheat genomes. Whereas lectin is detected in the coleoptile of varieties with hexaploid and DD and SS genomes, WGA is absent in the coleoptile of the diploid Triticum monococcum (AA). Lectin accumulates in the coleoptile of mature embryos of T. monococcum, however, when they are treated with abscisic acid.