# Wheat Germ Agglutinin--From Toxicity to Biomedical Applications
## 4 WGA Biological Activity
### Cytotoxicity  
Since the early discovery of lectins, they were considered toxic due to the obvious toxicity of first-identified lectins, such as abrin [32] or ricin [33]. Even though only a few lectins are highly toxic in low doses, cytotoxic activity is typically dose-dependent. Lectin cytotoxicity can occur in a lectin-dependent cell-mediated manner or as direct toxicity to cells [34]. Lectins interact with immune cells as antigens and exhibit their cytotoxic activities and induce a humoral response. Concentration-dependent increases with IFN-\(\gamma\), and TNF-\(\alpha\) release were observed after whole blood samples were treated with WGA [35]. Another study has found that WGA binds to glycosylated T3 receptor, which is followed by the formation of inositol phosphate--an intracellular secondary messenger that takes part in the regulation of cell functions, such as apoptosis--in human blood T cells [36]. On the other hand, WGA inhibits T lymphocyte proliferation by inhibiting the responsiveness of the lymphocytes to interleukin 2 (IL-2) rather than interfering with IL-2 production and IL-2 receptor expression [37; 38].  
Direct lectin cytotoxicity depends on interaction time and affinity to glycoconjugates on the cell surface. WGA demonstrates high specificity to GlcNAc, but interaction with sialic acid is important for WGA cell-killing activity. WGA-induced acute myeloid leukemia cell death is dependent on both GlcNAc binding and interaction with sialic acids. The authors noticed a reduction in WGA cell binding and killing after sialic acid was removed by neuraminidase treatment [39]. Sialic acid specificity plays a crucial role because most of the cancer cells are hyper sialylated [40; 41]. Experiments show that WGA is endocytosed into the Chinese hamster ovary (CHO) cells after 30 min of treatment [42]. Later research confirmed the same time-dependent endocytosis with leukemic Jurkat cell line [43]. WGA is endocytosed into cells by clathrin-independent endocytosis mechanism depending on interactions with N-acetylineuraminic acid or N-acetylglucosamine (GlcNAcNAc) on the cell surface of normal and malignant cells [44; 45]. WGA binds high molecular weight glycoproteins (HMWAG) expressed on the cell surface. WGA-HMWAG complexes rapidly become localized within endosomes and then move more slowly to a tubular endocytic network within the perinuclear region of the cell [42]. Endocytosed WGA interacts with the cell nuclear envelope pore membrane protein POM 121 and blocks RNA export [46]. This interaction with nuclear pore proteins depends on GlcNAc glycosylation moieties in other cell models of the nuclear transport blockade [47; 48]. Endocytosed WGA can suppress rat pancreatic acinar tumor cell line proliferation by blocking auto stimulation and prevention of the trophic effect of glycosylated cholecystkinin or gastrin-like peptides [49].  
WGA is shown to exert cytotoxic effects to differing extents with a final outcome (apoptosis or necrosis) depending on the cell line used in toxicity assays. WGA induced paradosis in L92 murine fibroblast cell line. Vaculation, loss of cell architecture, and upregulation of the apoptosis-related proteins Bax and caspase-3 were observed. However, internucleosomal DNA fragmentation, apoptotic bodies, and related apoptotic phenotypes were not detected [50]. Meanwhile, WGA intake into pancreatic carcinoma cell lines caused chromatin condensation, nuclear fragmentation, and DNA release consistent with apoptosis [51]. Interaction with the leukemic Jurkat cell line induced a loss of transmembrane potential, disruption of the inner mitochondrial membrane, and release of cytochrome c and caspase-9 activation of apoptosis [43]. These variances of outcomes might also be caused by differences between cell glycosylation patterns [52].