Molecular, Cell and Tissue Studies of Galectin-3:CD98 Interactions in IPF and COVID Pneumonitis

Galectin-3 critically mediates experimental fibrosis and is implicated in idiopathic pulmonary fibrosis (IPF). It can bind multiple proteins through protein:glycan as well as protein:protein interactions, including self-oligomerisation. It binds to TGF-β receptor (TGF-βR) II and potentiates TGF-β1 signalling. It also interacts with CD98, a mediator of innate inflammatory responses in such cells. I tested the gal-3-fibrosome hypothesis, that galectin-3 mediates IPF pathogenesis by nucleating a macromolecular complex of pro-fibrotic factors including CD98:integrin complex and TGF-βRII at the cell surface, in lung tissue, cell lines, and primary human lung cells.

Co-localisation of galectin-3, β1-integrin and CD98 was supported by proximity ligation assay and immunohistochemistry in human lung tissue, and confocal microscopy and co-immunoprecipitation studies in cells. TGF-β1 stimulation increased expression and co-localisation of all three proteins in A549 cells. CD98 silencing abrogated epithelial wound healing similarly to galectin-3 knockdown. In myofibroblasts TGF-β1 stimulation increased protein levels of β1-integrin and CD98 whilst reducing galectin-3. However, co-localisation of galectin-3 with β1-integrin and CD98 appeared relatively preserved in IPF myofibroblasts. Galectin-3 oligomerisation was most tractable to electron microscopy (EM) analysis by cross-linking ligand-induced oligomers and peptide N-glycosidase treatment, but further optimisation is required ahead of cryo-EM studies. I successfully extracted endogenous CD98-containing complexes from cell membranes within nanodiscs, using styrene maleic acid (SMA). CD98 co-immunoprecipitated with galectin-3 in nanodiscs. Both CD98 and galectin-3 were essential for interleukin (IL)-6 and IL-8 responses to SARS-CoV-2 Spike receptor binding domain in lung epithelial and macrophage cell lines.

Together, my data support a gal-3-fibrosome response to disease-relevant stimuli in tissue and cells. In myofibroblasts, they suggest a negative feedback loop that is overridden in IPF. Extensive work has defined promising conditions for future structural studies. CD98-dependent inflammatory responses are observed across cell types as well as diverse injury stimuli. Galectin-3 interactions with CD98:β1-integrin may transduce acute injury responses to pro-fibrotic behaviour in lung tissue, e.g. in acute exacerbations of IPF.