LaNt PLOSOne 2025 numerical data.

Laminin N-terminus α31 (LaNt α31) is a netrin-like protein generated by alternative splicing of the laminin α3 gene. While previously shown to regulate vascular permeability in vivo, its role in epithelial tissues remains less defined. Here, we demonstrate that LaNt α31 modulates epithelial cell behavior by altering laminin 332 (LM332) organization and hemidesmosome (HD) maturation. Adenoviral driven overexpression of LaNt α31 in corneal epithelial cells led to premature HD assembly, marked by enhanced recruitment of collagen XVII and BPAG1e to β4 integrin, and reduced cell migration. LaNt α31 expression reorganized LM332 from diffuse arcs into tight clusters and co-localized with LMβ3 during matrix deposition. Notably, phenotypes were rescued by precoated extracellular matrix, indicating a matrix-dependent mechanism. Furthermore, LaNt α31 increased matrix metalloproteinase (MMP) activity and LMα3 proteolytic processing, both essential for its effects, as MMP inhibition reversed LM clustering and HD maturation. These findings identify LaNt α31 as a regulator of epithelial homeostasis through modulation of LM332 architecture and cell–matrix adhesion.