Epitope mapping of function-blocking monoclonal antibody CM6 suggests a "weak" integrin binding site on the laminin-332 LG2 domain.

Yamashita H, Shang M, Tripathi M, Jourquin J, Georgescu W, Liu S, Weidow B, Quaranta V
J Cell Physiol. 2010 223 (3): 541-8

PMID: 20301201 · PMCID: PMC2874318 · DOI:10.1002/jcp.22107

Laminin-332 (Ln-332) is an extracellular matrix molecule that regulates cell adhesion, spreading, and migration by interaction with cell surface receptors such as alpha3beta1 and alpha6beta4. Previously, we developed a function-blocking monoclonal antibody against rat Ln-332, CM6, which blocks hemidesmosome assembly induced by Ln-332-alpha6beta4 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 alpha3 chain. To specify the residues involved in this epitope, we produced a series of GST-fused alpha3 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 (1089)NERSVR(1094) 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 alpha3beta1 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 alpha3beta1 and alpha6beta4 are closely spaced in the Ln-332 LG domains where they regulate alternative cell functions, namely adhesion/migration or hemidesmosome anchoring.

(c) 2010 Wiley-Liss, Inc.

MeSH Terms (18)

Amino Acid Sequence Animals Antibodies, Blocking Antibodies, Monoclonal Antibody Specificity Binding Sites Cell Adhesion Molecules Cell Line Cell Movement Epitope Mapping Humans Integrins Laminin Molecular Sequence Data Mutagenesis, Site-Directed Peptides Protein Structure, Tertiary Rats

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