pH-abhängige strukturelle und funktionelle Variationen des ß-Galactosid-bindenden Proteins Galectin-3

The cell has a complex apparatus to control the directed transport of proteins and lipids in the epithelium. To maintain this order controllers have been developed which assign a cargo protein into the right direction. One of these controllers is galectin-3, which belongs to the group of ß-galactoside-binding proteins and binds lactose or N-acetyllactosamine-containing glycoligands, preferably. As an important sorting signal different glycosylation sites have been identified that are critical for the apical or basolateral assignment. It is believed that after binding to a ligand galectin-3 forms a complex and associates within certain membrane areas to introduce sorting of the protein. A direct evidence for this process has not been provided yet. In this work, the binding to complex glycoligands and the association with specific membrane domains could be detected. These measurements were performed in a native plasma membrane in the form of giant plasma membrane vesicles (GPMVs) for the first time. GPMVs as model membranes originated from the apical part of MDCK II cells and have been established for different methodological issues. The vesicles showed a cell-like lipid- and protein-composition, which could be used to analyze physiological processes close to the native cellular system. With this model the effect of galectin-3 on the complex-glycosylated ligands asialofetuin and neurotrophinrezeptor p75 was specified, wherein the sorting process of proteins in the membrane and the binding capacity of galectin-3 as an oligomer have been observed. As a unique feature in the galectin family galectin-3 can form high-molecular clusters with its ligands. In in vitro studies, the size of the cluster with a free ligand and the direct influence of the avidity to the interaction with complex sugars have been described. The oligomerization started in a ligand-initiated fashion and the oligomer can be formed or dissolved dependent on the concentration of the ligand. Since galectin-3 traverses endosomal compartments, the influence of a more acidic environment in the interaction of the protein was analyzed for various sugar ligands. It was found that complex ligands lost the binding to galectin-3 at reduced pH generally, whereas binding to simple sugars such as lactose remained. Also the glycolipid sulfatide has been identified as a new potential interaction partner that could bind to the protein at reduced pH, solely. These specificities give new insights into the effect of galectin-3, as they can describe the exchange between binding of a cargo protein at the plasma membrane and its release into acidic endosomal compartments. In summary, this work opened new avenues in the mechanistic view of galectin-3-dependent sorting processes in epithelial cells.