1 research outputs found
Galectin‑3 Binding to α<sub>5</sub>β<sub>1</sub> Integrin in Pore Suspended Biomembranes
Galectin-3 (Gal3)
is a β-galactoside binding lectin that
mediates many physiological functions, including the binding of cells
to the extracellular matrix for which the glycoprotein α5β1 integrin is of critical importance. The
mechanisms by which Gal3 interacts with membranes have not been widely
explored to date due to the complexity of cell membranes and the difficulty
of integrin reconstitution within model membranes. Herein, to study
their interaction, Gal3 and α5β1 were purified, and the latter reconstituted into pore-suspended
lipid bilayers comprised eggPC:eggPA. Using electrochemical impedance
and fluorescence lifetime correlation spectroscopy, we found that
on incubation with low nanomolar concentrations of wild-type Gal3,
the membrane’s admittance and fluidity, as well as integrin’s
lateral diffusivity, were enhanced. These effects were diminished
in the following conditions: (i) absence of integrin, (ii) presence
of lactose as a competitive inhibitor of glycan–Gal3 interaction,
and (iii) use of a Gal3 mutant that lacked the N-terminal oligomerization
domain (Gal3ΔNter). These findings indicated that WTGal3 oligomerized
on α5β1 integrin in a glycan-dependent
manner and that the N-terminal domain interacted directly with membranes
in a way that is yet to be fully understood. At concentrations above
10 nM of WTGal3, membrane capacitance started to decrease and very
slowly diffusing molecular species appeared, which indicated the formation
of protein clusters made from WTGal3−α5β1 integrin assemblies. Overall, our study demonstrates the
capacity of WTGal3 to oligomerize in a cargo protein-dependent manner
at low nanomolar concentrations. Of note, these WTGal3 oligomers appeared
to have membrane active properties that could only be revealed using
our sensitive methods. At slightly higher WTGal3 concentrations, the
capacity to generate lateral assemblies between cargo proteins was
observed. In cells, this could lead to the construction of tubular
endocytic pits according to the glycolipid–lectin (GL–Lect)
hypothesis or to the formation of galectin lattices, depending on
cargo glycoprotein stability at the membrane, the local Gal3 concentration,
or plasma membrane intrinsic parameters. The study also demonstrates
the utility of microcavity array-suspended lipid bilayers to address
the biophysics of transmembrane proteins