2.1.1 General Considerations
Carbohydrates are part of important biological molecules like nucleic acids, glycoproteins or glycolipids where they are covalently bound. Yet there are also many non-covalent interactions that are mediated by carbohydrates. The binding partners for the saccharides are usually anti-carbohydrate antibodies, carbohydrate processing enzymes or another class of proteins called lectins.[36] A lectin has been defined as “a carbohydrate binding protein of nonimmune origin that agglutinates cells or precipitates polysaccharides or glycoconjugates”[37]. Lectins bind specifically to certain carbohydrates and are characterized according to which monosaccharide they exhibit the highest affinity. The five monosaccharides that act as main binding partners are: N-acetylglucosamine (GlcNAc), N-acetylneuraminic acid (NeuNAc), fucose, galactose (Gal)/N-acetylgalacosamine (GalNAc), or mannose (Man). Based on their structural features, lectins can be further categorized in simple lectins which are rather small structures, mosaic lectins in which the lectin is part of a bigger assembly of protein domains, and macromolecular examples where many copies of proteins form macromolecular structures like bacterial fimbriae.[36] Many lectins exist as oligomers of homologous domains. This leads to more than one carbohydrate binding site, making the lectin multivalent. The binding affinity of monosaccharides to the corresponding lectins is usually low. However, if the monosaccharides are displayed in a multivalent fashion, several lectin-monosaccharide interactions can be in action simultaneously. This may result in a considerably stronger binding. Lectins participate in a plethora of processes. Many can be termed cell recognition events. A lot of lectins and carbohydrate structures are situated at the outside of the cell membrane which makes them easily accessible to each other. As already indicated they take part in many different pathologic events. Not only viral surface lectins mediate the infection of cells but also many bacteria take advantage of lectin carbohydrate interactions to interact with their host cells.
Adhesion of Pseudomonas aeruginosa, a pathogen that can lead to acute and chronic infections especially in the respiratory tract, to the host epithelial cells is mediated by the lectins LecA and LecB.[38]
E. coli possess fimbriae with affinity for carbohydrate structures that allows the adhesion on host surfaces.[39] Galectins which are binding specifically to galactosides are associated with cancer[40], galectin-3 for example can promote metastasis[41-42]. Also various toxins carry a lectin subunit that enables the toxin to bind to the membrane of its target, followed by endocytosis and the toxic effect of the toxin subunit. Examples are cholera toxin[43-44], shiga toxin[6, 45], or ricin[46]. Being involved in many diseases, lectins could be a potent medical target to interfere or inhibit the above mentioned processes.
Lectins
phagocytosis.[47] Selectins, C-type lectins that need Ca2+ for the carbohydrate binding, are found on leukocytes and allow the adhesion of the latter to endothelial cells of the blood vessels. That enables the direction of leukocytes to cites of inflammation.[7] P-type lectins participate in transport of lysosomal enzymes to their destination.[48]. The manifold functions of lectins offer many opportunities for research in this field.
2.1.2 Wheat Germ Agglutinin
One example of a multivalent lectin is wheat germ agglutinin. It was first discovered when tumor cells were treated with wheat germ lipase which lead to an aggregation of the tumor cells.[49] This could be traced back to the presence of an impurity in the lipase samples. The impurity was identified as a lectin that was termed wheat germ agglutinin (WGA)[50] and could be isolated in pure form[51]. Subsequent studies established the affinity of the lectin for N-acetylglucosamine (GlcNAc)[52] 1, its oligomers, for example N,N-diacetylchitobiose[51, 53] 2, and N-acetylneuraminic acid (NeuNAc)[54] 3 (Figure 1). Soon after, the first crystal structure of WGA could be obtained[55-56] and paved way for many following studies describing the binding of the saccharides to the lectin[54, 57-59].
Figure 1 GlcNAc 1, N,N-diacetylchitobiose 2, and NeuNAc 3 that can bind to wheat germ agglutinin.
At neutral pH, WGA exists as a homodimer with a mass of 34 kDa (Figure 2).[60] The monomers are built of four domains (A, B, C, and D) each containing four disulfide bridges. The dimer is very stable in solution and can be dissociated only at low or high pH.[56] It contains eight binding sites for carbohydrates which are located at the interface of the two monomers and are formed by contribution of two domains each. The binding sites are grouped in primary and secondary binding sites. The primary binding sites, which are termed B1C2, B2C1, C1B2 and C2B1 after the domains that are involved, exhibit higher affinities for the ligands. On the contrary the secondary binding sites A1, A2, D1A2 and D2A1 often remained unoccupied in crystal structures of the protein with its ligands.[57, 59] Each domain contributes by aromatic or polar residues to the binding with the exception of binding sites A1 and A2 where the polar contributions of domains D1 and D2 are lacking.[61]
Figure 2 Crystal structure of WGA. The protein is shown as surface representation with domains of monomer 1 colored red to yellow and domains of monomer 2 colored cyan to dark blue, with the binding sites marked as yellow ellipses. Picture created using USCF Chimera 1.10.1[62] based on PDB ID: 2X52.[11]
The binding affinity of WGA for GlcNAc has first been determined by an fluorescence assay.[63] The obtained Kd was 1.4 mM which is comparable to the later assessed Kd of 2.6 mM derived from isothermal titration calorimetry.[64] For the oligomers of GlcNAc the affinity rises with each monomer up to a Kd of 81 µM for the trisaccharide.[64]
The role of WGA in wheat has been in discussion for a long time. Early studies showed evidence for the inhibition of the growth of certain fungi by WGA.[65-66] Others reported increased WGA levels in cells under stress conditions and proposed a role as radical scavenger due to the high cystine content.[67]
It has also been suggested that WGA plays a beneficial role in the interaction of wheat with nitrogen fixing bacteria.[68] A more recent study revealed increased bacterial adhesion to wheat roots treated with WGA.[69] In summary, the role of WGA in wheat remains still vague and cannot be clearly defined. Yet its eight binding sites make it a very useful and often used model to study multivalent carbohydrate-lectin interactions.
Multivalency