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

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 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.

SUBMITTER: Yamashita H

PROVIDER: S-EPMC2874318 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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

Yamashita Hironobu H Shang Meiling M Tripathi Manisha M Jourquin Jerome J Georgescu Walter W Liu Shanshan S Weidow Brandy B Quaranta Vito V

Journal of cellular physiology 20100601 3

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 ...[more]

PMID: 20301201

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Project description:BackgroundMucous membrane pemphigoid is a group of chronic subepithelial autoimmune blistering diseases that mainly affect mucous membranes. Laminin 332-specific autoantibodies are present in approximately 1/3 of the patients, being associated with an increased risk of malignancy. Because of the severe complications, an early recognition of the disease allowing a timely therapy is essential. The gold standard methods for detection of laminin 332-specific autoantibodies, including the immunoprecipitation and immunoblotting are non-quantitative, laborious and restricted to a few specialized laboratories worldwide. In addition, the use of radioimmunoassays, although highly sensitive and specific, are laborious, expensive and tightly regulated. Therefore, there is a stringent need for a quantitative immunoassay for the routine detection of laminin 332-specific autoantibodies more broadly available to diagnostic laboratories. The aim of this study was to compare different antigenic substrates, including native, recombinant laminin 332 and laminin 332-rich keratinocyte extracellular matrix, for development of an ELISA to detect autoantibodies in mucous membrane pemphigoid.ResultsUsing a relatively large number of sera from MMP patients with well-characterized autoantibody reactivity we show the suitability of ELISA systems using laminin 332 preparations as adjunct diagnostic tools in MMP. While glycosylation of laminin 332 does not appear to influence its recognition by MMP autoantibodies, ELISA systems using both purified, native and recombinant laminin 332 demonstrated a high sensitivity and good correlation with the detection of autoantibodies by immunoblotting. ELISA systems using different laminin 332 preparations represent a feasible and more accessible alternative for a broad range of laboratories.ConclusionsOur findings qualify the use of immunoassays with the laminin 332-rich preparations as an ancillary diagnostic tool in mucous membrane pemphigoid.

Dataset Information

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

Publications

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

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