Omarmosley1570

In the 2010s, a novel lectin family with β-trefoil folding has been identified in marine mussels from the family Mytilidae (phylum Mollusca). "MytiLec-1," the lectin described in this chapter, was the first member of this family to be isolated and characterized from the Mediterranean mussel Mytilus galloprovincialis, a commercially and ecologically important species, spread in marine coastal areas worldwide. MytiLec-1 bound to the sugar moiety of globotriose (Gb3 Galα1-4Galβ1-4Glc), an α-galactoside, leading to apoptosis of Gb3-expressing Burkitt's lymphoma cells. Although the primary structure of MytiLec-1 was quite unusual, its three-dimensional structure was arranged as a β-trefoil fold, which is the typical architecture of "Ricin B chain (or R)-type" lectins, which are found in a broad range of organisms. To date, MytiLec-1-like lectins have been exclusively found in a few species of the mollusk family Mytilidae (M. galloprovincialis, M. trossulus, M. californianus, and Crenomytilus grayanus) and in the phylum Brachiopoda. Transcriptome data revealed the presence of different structural forms of mytilectin in mussels, which included prototype and chimera-type proteins. The primary sequence of these lectins did not match any previously described known protein family, leading to their assignment to the new "mytilectin family." We here report the method of purification of this lectin and describe its use in cell biology.Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is the most potent toxin and produced as a complex with non-toxic components. Food-borne botulism is caused by the ingestion of these BoNT complexes. Hemagglutinin (HA), one of the non-toxic components, is known to have lectin (carbohydrate binding) activity and E-cadherin-binding activity. These activities promote the intestinal absorption of BoNT. To elucidate the mechanism of the onset of food-borne botulism, we focused on the role of HA in the intestinal absorption of BoNT. We describe the functional analysis methods for HA, including the expression of recombinant proteins, binding to glycoproteins and epithelial cells, and localization in mouse intestinal tissue.Botulinum hemagglutinin (HA) is one of the auxiliary protein components of the botulinum neurotoxin (BoNT) complex, the most lethal toxin known. HA promotes the intestinal absorption of BoNT by at least two mechanisms, resulting in high oral toxicity. PP1 nmr One of the mechanisms is the attachment of large progenitor toxin complexes (L-PTCs) to the cell surface of the intestinal epithelium by the carbohydrate-binding activity of HA. The other is epithelial barrier disruption by the E-cadherin-binding activity of HA. The carbohydrate-binding activity of HA also promotes attachment to the basolateral cell surface, which increases the frequency of contact between HA and E-cadherin. Together, the carbohydrate-binding activity of HA is critical for the intestinal absorption of BoNTs. The trimeric triskelion-shaped structure of HA confers the multivalent binding to its ligands and increases the pathogenic biological activities of HA.Antibodies are useful for localizing glycoprotein antigens in histochemistry, but they do not differentiate glycoforms in tissue sections because conventional antibodies recognize only protein epitopes rather than glycans. Glycan epitopes are recognized by lectins, which are found, occasionally, to inhibit antigen-antibody reaction in a glycoform-specific manner (lectin inhibition). Here we describe the application of lectin inhibition to immunohistochemistry for visualizing a glycoform in a tissue section.Glycoforms are otherwise identical proteins with different glycosylation. A lectin, Sambucus sieboldiana agglutinin (SSA), specifically binds glycoforms having α2,6-sialyl residues. The binding is found to inhibit antigen-antibody reaction; e.g., SSA inhibits anti-transferrin antibody binding to α2,6-sialylated transferrin (Tf) (SSA inhibition). SSA inhibition is not observed with other Tf glycoforms, indicating that the inhibition is glycoform-specific. Here we describe the application of SSA inhibition to ELISA as a specific assay for quantifying α2,6-sialylated Tf.CEL-III is a Ca2+-dependent and galactose-specific lectin purified from the sea cucumber, Cucumaria echinata; it exhibits hemolytic and hemagglutinating activities. CEL-III consists of the following three distinct domains two N-terminal carbohydrate-binding domains (1 and 2), which adopt β-trefoil folds such as the B-chain of ricin and are members of the (QXW)3 motif family, and domain 3, an oligomerization domain. After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores composed of CEL-III heptamers, leading to cell lysis or death. In this chapter, the purification and carbohydrate-coated microplate binding assay of CEL-III are described.Quality control of newly synthesized glycoproteins is tightly regulated by sugar processing of N-glycans and by recognition of specific glycan structures by lectins in the endoplasmic reticulum (ER). Mannose trimming and its recognition determine the targeting of misfolded glycoproteins for ER-associated degradation. ER degradation-enhancing α-mannosidase-like (EDEM) proteins in mammals and their homologue Htm1p/Mnl1p in Saccharomyces cerevisiae are involved in this process. To analyze the function of EDEM proteins, we expressed and purified recombinant EDEM3 from HEK293 cells and assessed its mannose-trimming activity in vitro.Studies on the effects of components derived from the human pathogenic fungi Paracoccidioides brasiliensis have identified paracoccin (PCN), as a bifunctional protein with lectin (GlcNAc-binding) and enzymatic (chitinase) activities, able to induce modulation of host immune response. Endogenous PCN acts as a fungal virulence factor, whereas exogenous purified PCN, administered to the host, confers protective immunity in a murine model of paracoccidioidomycosis. The immunomodulation induced by purified-PCN injection has characterized it as an agent applicable in the therapy and vaccine against paracoccidioidomycosis. This section describes methods for PCN purification and validation of its lectin and enzymatic activities. It includes detailed protocols to obtain homogeneous PCN from P. brasiliensis yeasts, as well as to purify recombinant PCN from transformed heterologous microorganisms.