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Galectin-3 Binding to α 5 β 1 Integrin in Pore Suspended Biomembranes
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Edité par CCSD ; American Chemical Society -
International audience. 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 51 integrin is of critical importance. To study their interaction, Gal3 and 51 integrin were purified, and the latter reconstituted into microcavity supported lipid bilayers composed of physiological eggPC:eggPA, which enabled multimodal interrogation of the membrane using electrochemical impedance and fluorescence lifetime correlation spectroscopies. Upon incubation with wild-type Gal3 (WTGal3) at low nanomolar concentrations, the membrane electrical resistance of 51 integrin-containing membranes decreased, while membrane capacitance, fluidity, and the lateral diffusivity of the integrin increased. These effects were much reduced or even absent when a Gal3 deletion mutant lacking the N-terminal oligomerization domain (Gal3∆Nter) was used, when lactose was present as a competitive inhibitor of glycan-WTGal3 interaction, or when WTGal3 or Gal3∆Nter were incubated with membranes in the absence of 51 integrin. These findings indicated that Gal3 oligomerized on 51 integrin in a glycandependent manner, and that the N-terminal domain interfered 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 a protein layer made from WTGal3-51 integrin assemblies. In conclusion, 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 had not been revealed before using our sensitive methods. At slightly higher WTGal3 concentrations, the capacity to generate lateral assemblies between cargo proteins was observed. In cells, this would 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 specific or plasma membrane intrinsic parameters. Significance statement Upon expression from animal tissue, galectins are synthesized in the cytosol and translocated to the extracellular milieu by unconventional secretion. Galectins drive the endocytosis of glycoproteins such as α5β1 integrin but also organize them into lattices. Although these notions are in apparent contradiction, we now envision a model in which galectin lattices and galectin-driven endocytosis cooperate to control the homeostasis of glycoproteins at the plasma membrane. The intricacies of the interactions between oligomerization competent galectins and glycoproteins that usually carry several glycans are still incompletely understood. In this work, we employed pore-suspended lipid bilayers as an in vitro platform to address the multivalent and specific carbohydrate-dependent interactions of galectin-3 with α5β1 integrin.
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