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

Hironobu Yamashita, Meiling Shang, Manish Tripathi, Jerome Jourquin, Walter Georgescu, Shanshan Liu, Brandy Weidow, Vito Quaranta

Research output: Contribution to journalArticle

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Abstract

Laminin-332 (Ln-332) is an extracellular matrix molecule that regulates cell adhesion, spreading, and migration by interaction with cell surface receptors such as α3β1 and a6b4. Previously, we developed a function-blocking monoclonal antibody against rat Ln-332, CM6, which blocks hemidesmosome assembly induced by Ln-332-α6β4 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 α3 chain. To specify the residues involved in this epitope, we produced a series of GST-fused α3 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 1089NERSVR1094 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 α3β1 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 α3β1 and α6β4 are closely spaced in the Ln-332 LG domains where they regulate alternative cell functions, namely adhesion/migration or hemidesmosome anchoring.

Original languageEnglish (US)
Pages (from-to)541-548
Number of pages8
JournalJournal of Cellular Physiology
Volume223
Issue number3
DOIs
StatePublished - Jun 1 2010
Externally publishedYes

Fingerprint

Epitope Mapping
Blocking Antibodies
Integrins
Epitopes
Binding Sites
Monoclonal Antibodies
Hemidesmosomes
Rats
Cell adhesion
Laminin
Cell Adhesion
Assays
Adhesion
kalinin
Mutagenesis
Acids
Structural Models
Cell Surface Receptors
Mutant Proteins
Site-Directed Mutagenesis

All Science Journal Classification (ASJC) codes

  • Physiology
  • Clinical Biochemistry
  • Cell Biology

Cite this

Epitope mapping of function-blocking monoclonal antibody CM6 suggests a "weak" integrin binding site on the Laminin-332 LG2 domain. / Yamashita, Hironobu; Shang, Meiling; Tripathi, Manish; Jourquin, Jerome; Georgescu, Walter; Liu, Shanshan; Weidow, Brandy; Quaranta, Vito.

In: Journal of Cellular Physiology, Vol. 223, No. 3, 01.06.2010, p. 541-548.

Research output: Contribution to journalArticle

Yamashita, Hironobu ; Shang, Meiling ; Tripathi, Manish ; Jourquin, Jerome ; Georgescu, Walter ; Liu, Shanshan ; Weidow, Brandy ; Quaranta, Vito. / Epitope mapping of function-blocking monoclonal antibody CM6 suggests a "weak" integrin binding site on the Laminin-332 LG2 domain. In: Journal of Cellular Physiology. 2010 ; Vol. 223, No. 3. pp. 541-548.
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AU - Jourquin, Jerome

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AU - Liu, Shanshan

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AU - Quaranta, Vito

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