Reduction of Lectin Valency Drastically Changes Glycolipid
Dynamics in Membranes but Not Surface Avidity
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Abstract
Multivalency is proposed
to play a role in the strong avidity of
lectins for glycosylated cell surfaces and also in their ability to
affect membrane dynamics by clustering glycosphingolipids. Lectins
with modified valency were designed from the β-propeller fold
of Ralstonia solanacearum lectin (RSL)
that presents six fucose binding sites. After identification of key
amino acids by molecular dynamics calculations, two mutants with reduced
valency were produced. Isothermal titration calorimetry confirmed
the loss of three high affinity binding sites for both mutants. Crystal
structures indicated that residual low affinity binding occurred in
W76A but not in R17A. The trivalent R17A mutant presented unchanged
avidity toward fucosylated surfaces, when compared to hexavalent RSL.
However, R17A is not able anymore to induce formation of membrane
invaginations on giant unilamellar vesicules, indicating the crucial
role of number of binding sites for clustering of glycolipids. In
the human lung epithelial cell line H1299, wt-RSL is internalized
within seconds whereas the kinetics of R17A uptake is largely delayed.
Neolectins with tailored valency are promising tools to study membrane
dynamics