: In an inflammatory response, recruitment of leukocytes from the bloodstream into lymphatic tissues is initiated by rolling of the cells across high endothelial venules (HEVs). Rolling exposes the leukocytes to chemokines, and induction of adhesion molecules on leukocytes and the endothelium arrests rolling, enabling the cells to traverse the endothelium. Constitutively expressed on leukocytes, L-selectin is one of three selectin molecules which mediate rolling by binding carbohydrate moieties of ligands. To facilitate rolling, the bonds between L-selectin and its ligands must be rather transient while simultaneously withstanding the hydrodynamic force of the bloodstream. Hence, understanding the dynamics of this interaction is central to elucidating the mechanism of rolling. Recently it has been shown that binding of a mAb specific for the lectin domain induces a conformational change, possibly involving the EGF-like domain of L-selectin. In addition, determination of the structures of the lectin and EGF-like domains of P-selectin, in its high and low-affinity binding conformations, suggests that L-selectin undergoes an analogous change. To investigate this, full-length and soluble, recombinant L-selectin molecules stabilized in the two conformations will be produced. Comparative and computational modeling will be used to predict the structures, and intramolecular mobility will be restricted by either introduction of disulfide bonds or changes in amino acids which will favor a particular protein fold. To elucidate how the different states of L-selectin mediate rolling, binding affinities, kinetics, and the mechanical properties of the bonds between the L-selectin variants and their ligands will be measured. In addition, rolling and cytoskeletal association mediated by the L-selectin variants will be examined.
| Phan, Uyen T; Waldron, Travis T; Springer, Timothy A (2006) Remodeling of the lectin-EGF-like domain interface in P- and L-selectin increases adhesiveness and shear resistance under hydrodynamic force. Nat Immunol 7:883-9 |
