Galectin-3 alters the lateral mobility and clustering of beta 1-integrin receptors

Glycoprotein receptors are influenced by myriad intermolecular interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examined the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the alpha 5 beta 1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor reductions in lateral mobility. Treatment of cells with Gal-3 increased beta 1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics analysis to confirm that cells expressed endogenous Gal-3, and found that addition of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3-integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins

Viability of cells under cell migration conditions.

<p>Viability of cells under cell migration conditions.</p

Model of Gal-3 interactions with integrin.

<p>(<b>a.</b>) Glycosylated receptors, such as the integrins, will have reduced binding sites for Gal-3 if they are heavily sialylated. (<b>b.</b>) Removal of sialic acids by neuraminidase enzymes (or decreased SiaT activity) will increase the number of Gal-3 binding sites present, and should increase oligomerization (only a dimer is shown for clarity). Oligomers likely interact with cytoskeletal regulators, including talin,[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184378#pone.0184378.ref089" target="_blank">89</a>] which lead to increased mobility through active processes. (<b>c.</b>) Addition of exogenous Gal-3C or a competitive binder (e.g. LNnT) will disrupt the formation of oligomers. This will occur either by (<b>d.</b>) competition for dimerization sites or (<b>e.</b>) blocking dimer binding sites.</p

Clustering of integrins is increased on Gal-3 treated cells.

<p>Cells were stained using the same anti-α5-Cy5 conjugate employed for tracking experiments. Ten fields of stained cells were analyzed using ImageJ to identify clusters and measure their size. Treatment with Gal-3 resulted in an increase in the size of integrin clusters. See Table B and Figure A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184378#pone.0184378.s001" target="_blank">S1 File</a>. Data were compared to a PBS control, or PBS containing BME (control) using a student’s t-test to determine p values; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005; ****, p ≤ 0.0001.</p

Viability of cells treated with LNnT, Gal-3C, and Gal-3.

<p>Cells were treated for 21 h with buffer control, Gal-3 (50 μg mL^-1), Gal-3C (50 μg mL^-1), and LNnT (100 mM). Viability of each condition were measured using MTS assay.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184378#pone.0184378.ref088" target="_blank">88</a>] Viability for each condition was normalized and compared to buffer control; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005; ****, p ≤ 0.0001.</p