Browsing School, Graduate by Subject "Galectins"
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Opposite Roles of Zebrafish Galectins in In Vitro Attachment and Infection by the Infectious Hematopoietic Necrosis Virus (IHNV)IHNV is a rhabdovirus with a high mortality rate that has major economic impacts on the salmonid fisheries and aquaculture industry. The virus uses cell surface fibronectin as a receptor for attachment and infection, but the exact mechanism remains unknown. Previously published work in our lab and preliminary data revealed that β-galactoside-binding lectins, known as galectins, interact directly with the IHNV envelope glycoprotein to either promote or inhibit viral attachment and infection of fish epithelial cells. The zebrafish tandem-repeat galectin 9 isoform 1 (Drgal9-L1) displays two carbohydrate recognition domains (CRD) joined by a linker peptide that are similar but not identical in binding specificity. The goal of this study was to explore the mechanism of Drgal9-L1-mediated enhancement of IHNV attachment to epithelial cells (EPC cell line). We showed that Drgal9-L1 crosslinks IHNV to cell surface glycans in a carbohydrate-dependent and -specific manner. We determined that crosslinking was dependent on two functional CRDs through the development of a C-terminal mutant protein that did not enhance IHNV attachment or infection of EPC cells. Drgal9-L1 crosslinks IHNV to fibronectin on the cell surface, enhancing viral attachment, in a carbohydrate -dependent and -specific manner. In addition, Drgal9-L1 binds to alternative ligands, β1-integrin and CD147, to increase IHNV attachment. Double antibody inhibition and siRNA knockdown of fibronectin and β1-integrin in EPC significantly reduced Drgal9-L1 mediated attachment of IHNV. We also investigated the protective role of epidermal mucus glycans for preventing Drgal9-L1 mediated viral attachment to the epithelium. All three galectin classes were detected in the zebrafish epidermal mucus, and Drgal9-L1 as well as Drgal1-L2 were found to bind to mucus glycans in a carbohydrate-specific manner. In a plaque assay, mucus coating of the cell monolayer reduced the number of IHNV plaques on the EPC cells in a concentration and volume-dependent manner and annulled the Drgal9-L1 enhancement of viral attachment and infectivity. Finally, we identified an alternative mechanism of Drgal1-L2 antiviral protective activity, as binding of Drgal1-L2 to surface glycosylated receptors or mucus glycans significantly inhibits IHNV attachment. This research has wide-ranging applications for aquaculture disease management, vaccine development, and a general model of galectin-modulated viral attachment.
Role of Sialic Acids in XenotransplantationThe sialic acid profile on the surface of cells can dynamically change upon cell activation and such change can dramatically affect various cellular functions. One pathway facilitating such change is through the loss of sialic acid by enzymatic cleavage caused by sialidases. In certain cell types, sialidases were found to mobilize from intracellular reservoirs to the cell surface after activation, however, the mechanism of the translocation is still not clear. This surface expressed sialidase activity causes desialylation of the cell surface either in cis or trans on other cell types in close vicinity. One of the mechanisms by which sialic acid cleavage has been shown to affect cell-cell binding is facilitated by the binding of galectins. When sialic acid is cleaved, underlying galactose molecules are exposed. Galactose is in turn the ligand for the lectin receptors galectins. Galectins are defined by sharing a structural homology in their carbohydrate-recognition domains (CRDS). Galectins are expressed in different tissues and cells including endothelial cells (EC) , alveolar macrophages and neutrophils (PMN). It has been found that a change in the level of galectins can affect PMN recruitment. A main limitation of pig lung xenograft injury is the fact that leukocyte (specifically PMN) and platelet sequestration occur in our ex-vivo human blood perfusion system within minutes, and the mechanisms driving these adhesive interactions are largely unknown. We believe that sialidase activity is increased during an ex-vivo lung xeno-perfusion and this causes an increase in desialylation of lung tissue and PMN. Here we characterize sialidase expression during an ex-vivo lung xeno perfusion and evaluate the role of cellular desialylation in PMN and endothelial cell adhesion under xeno settings, most specifically focusing on NEU1 and NEU3. We demonstrate that when sialic acid is cleaved from pig EC/human PMN this increases human PMN adhesion, and that PMN adhesion is further increased when PMN or EC are pre-activated. We show that the galectin binding is a mechanism behind this discovery/phenomenon. These findings provide the first mechanistic explanation of how galectin binding, consequent of sialic acid cleavage, affects human PMN adhesion during xenotransplantation.