On the other hand the presence of an affinity tag can be useful for immobilization of the lectin onto a solid surface for subsequent binding studies. acknowledgement and signaling processes throughout biology. To exactly control and regulate myriad physical relationships, lectinCcarbohydrate interactions must be extremely specific and exact (Gabius, Andre, Jimenez-Barbero, Romero, & Solis, 2011). Both lectins and carbohydrates can be viewed as multivalent molecules with lectins often containing more than one carbohydrate-binding site per monomeric subunit as well as assembling as oligomers, and carbohydrates often existing as branched or long-chain polymers. These attributes combined give rise to enormous variability; nonetheless through layers of acknowledgement that start at the monosaccharide level and increase to include factors such as valency, denseness of surface-displayed glycans or receptors, and distances and orientations of binding interfaces, high examples of specificity are accomplished. To fully understand the chemical and structural basis for carbohydrate-mediated events in biology, it is necessary to characterize each coating of recognition. To achieve this, multiple complementary techniques must be used. Among surface-displayed glycoproteins, the HIV envelope glycoprotein gp120 (120 kDa) is one of the most enigmatic. Asn-linked glycans make up approximately half of its molar mass (60 kDa) with the majority displayed by high-mannose oligosaccharides that form a so-called glycan shield. While this glycan coating is necessary for folding and oligomerization of gp120 into fusion-competent trimers, it also appears like a main epitope of, or is definitely accommodated by, a growing number of anti-HIV antibodies (Burton et al., 2012; Doores, 2015; Stewart-Jones et al., 2016). HIV gp120 represents a logical target for HIV inhibitors as it facilitates computer virus entry into target cells by a direct association with cellular receptors such as CD4 and CCR5, and viral transport by membrane lectins such as DC- and L-SIGN (Wilen, Tilton, & Doms, 2012), and is the only target of HIV-neutralizing antibodies (Burton et al., 2012; Doores, 2015). As fresh approaches to obstructing HIV infection remain a priority, desire for carbohydrate-binding providers (including lectins, antibodies, natural products, and synthetic receptors) as antivirals offers continued to rise. Carbohydrate-binding agents capable of binding the gp120 glycan shield have been shown to block computer virus infection, preventing connection with the sponsor (Acharya, Lusvarghi, Bewley, & Kwong, 2015). In particular, lectins that are specific for high-mannose oligosaccharides are encouraging candidates for microbicide development as PF 06465469 they can block HIV illness with amazing breadth and potency (Balzarini, 2007). The mannose-binding lectins cyanovirin-N and griffithsin (GRFT) are among the most potent HIV PF 06465469 inhibitors explained to day (Boyd et al., 1997; Mori et al., 2005). Their relationships with soluble mannosides have been studied quite thoroughly and three-dimensional constructions of those complexes have been solved (Bewley, 2001; Zi?kowska et al., 2006). Detailed descriptions of their relationships with their biological targets, such as Man9GlcNAc2Asn and gp120, have been more challenging in part due to limitations that arise from formation of cross-linked products. In this chapter, we use the well-studied model system of HIV-1 envelope glycoprotein gp120 and an HIV-binding restorative lectin GRFT to present different strategies and a general workflow utilizing complementary chemical and biophysical methods that allow for precise characterization of these types of relationships in the context of individual oligosaccharides, as part of a glycoprotein, and closing with visualization of relationships with whole virions (Fig. 1). Open in a separate windows Fig. 1 Schematic showing the increasing level of intermolecular relationships covered with this chapter. They range from detecting and characterizing a single sugars bound to a lectin, up to complex macromolecular relationships between PF 06465469 networks of lectins and viral particles, all mediated by proteinCcarbohydrate relationships. 2.?SELECTION AND PRODUCTION OF THE LECTIN Many of the anti-HIV lectins described HILDA to day are of nonhuman ori gin and were isolated from algae, cyanobacteria, or bacteria (Hoorelbeke et al., 2010; Ziolkowska & Wlodawer, 2006). These lectins are generally amenable to heterologous manifestation in well-proven bacterial manifestation systems utilizing commercial plasmids such as the pET vectors. For many studies, lectins may be indicated by subcloning the encoding gene into an inducible manifestation vector. To assist with appropriate folding and/or excretion, solubility, and purification, it may be desired to fuse the protein to a periplasmic secretion transmission, a solubility tag, or an affinity tag, respectively. It is important to note that for structural and biophysical studies discussed here, the presence of a protein fusion tag attached to the lectin may be detrimental to some of the methods because of the launch of artifacts in binding attributed.
