Epitope Mapping Of Function-Blocking Monoclonal Antibody Cm6 Suggests A "Weak" Integrin Binding Site On The Laminin-332 Lg2 Domain

Laminin-332 (Ln-332) is an extracellular matrix molecule that regulates cell adhesion, spreading, and migration by interaction with cell surface receptors such as alpha 3 beta 1 and alpha 6 beta 4. Previously, we developed a function-blocking monoclonal antibody against rat Ln-332, CM6, which blocks hemidesmosome assembly induced by Ln-332-alpha 6 beta 4 interactions. However, the location of its epitope on Ln-332 has remained unclear. In this study, we show that the CM6 epitope is located on the laminin G-like (LG)2 module of the Ln-332 alpha 3 chain. To specify the residues involved in this epitope, we produced a series of GST-fused alpha 3 LG2 mutant proteins in which rat-specific acids were replaced with human acids by a site-directed mutagenesis strategy. CM6 reactivity against these proteins showed that CM6 binds to the (NERSVR1094)-N-1089 sequence of rat Ln-332 LG2 module. In a structural model, this sequence maps to an LG2 loop sequence that is exposed to solvent according to predictions, consistent with its accessibility to antibody. CM6 inhibits integrin-dependent cell adhesion on Ln-332 and inhibits cell spreading on both Ln-332 and recombinant LG2 (rLG2; but not rLG3), suggesting the presence of an alpha 3 beta 1 binding site on LG2. However, we were unable to show that rLG2 supports adhesion in standard assays, suggesting that LG2 may contain a "weak" integrin binding site, only detectable in spreading assays that do not require washes. These results, together with our previous findings, indicate that binding sites for alpha 3 beta 1 and alpha 6 beta 4 are closely spaced in the Ln-332 LG domains where they regulate alternative cell functions, namely adhesion/migration or hemidesmosome anchoring. J. Cell. Physiol. 223: 541-548, 2010. (C) 2010 Wiley-Liss, Inc.