Regulation of Biological Activity and Matrix Assembly of Laminin-5 by COOH-terminal, LG4–5 Domain of α3 Chain

Abstract The basement membrane protein laminin-5 (LN5; α3β3γ2) undergoes specific proteolytic processing of the 190-kDa α3 chain to the 160-kDa form after the secretion, releasing its COOH-terminal, LG4–5 domain. To clarify the biological significance of this processing, we tried to express a recombinant precursor LN5 with a 190-kDa α3 chain (pre-LN5), in which the cleavage sequence Gln-Asp was changed to Ala-Ala by point mutation. When the wild-type and mutated LN5 heterotrimers were expressed in HEK293 cells, the wild-type α3 chain was completely cleaved, whereas the mutated α3 chain was partially cleaved at the same cleavage site (Ala-Ala). pre-LN5 was preferentially deposited on the extracellular matrix, but this deposition was effectively blocked by exogenous heparin. This suggests that interaction between the LG4–5 domain and heparan sulfate proteoglycans on the cell surface and/or extracellular matrix is important in the matrix assembly of LN5. Next, we purified both pre-LN5 and the mature LN5 with the processed, 160-kDa α3 chain (mat-LN5) from the conditioned medium of the HEK293 cells and compared their biological activities. mat-LN5 showed higher activities to promote cell adhesion, cell scattering, cell migration, and neurite outgrowth than pre-LN5. These results indicate that the proteolytic removal of LG4–5 from the 190-kDa α3 chain converts the precursor LN5 from a less active form to a fully active form. Furthermore, the released LG4–5 fragment stimulated the neurite outgrowth in the presence of mat-LN5, suggesting that LG4–5 synergistically enhances integrin signaling as it is released from the precursor LN5.