Probing the integrin-binding site within the globular domain of laminin-511 with the function-blocking monoclonal antibody 4C7☆
Introduction
Laminins are a family of basement membrane glycoproteins that consist of three chains, termed α, β and γ. To date, five α, three β and three γ chains have been identified, combinations of which yield at least 15 isoforms with distinct subunit compositions (Timpl, 1996, Colognato and Yurchenco, 2000). Laminins mediate many biological functions, including cell adhesion, migration and proliferation, through binding to cell surface receptors, particularly the integrin family of cell adhesion molecules (Miner et al., 1995, Belkin and Stepp, 2000, Gu et al., 2001, Gu et al., 2002, Li et al., 2003b). Integrins play important roles in the control of cell growth and differentiation. Binding sites for integrins have been mapped to the C-terminal globular (G) domain of the laminin α chains (Timpl et al., 2000). The G domain consists of five tandemly repeated LG modules of ∼ 200 amino acid residues, designated LG1 through LG5. Although the integrin-binding sites within the G domain have been extensively studied by examining the cell-adhesive activities of recombinant LG modules expressed individually or in tandem (Talts and Timpl, 1999, Shang et al., 2001, Yu and Talts, 2003), this approach has suffered from the difficulties associated with reproducing the cell-adhesive activities of intact laminins. The basis for these difficulties is currently unclear, and the integrin-binding sites within the G domain remain to be fully defined.
An alternative approach that circumvents these difficulties is to use function-blocking monoclonal antibodies against laminins as probes for the integrin-binding sites, since their epitopes are likely to overlap with or be in close proximity to the integrin-binding sites. One of these antibodies, 4C7, has been shown to recognize the G domain of laminin-511 (α5β1γ1) and inhibit the cell–substrate adhesion mediated by laminin-511 (Engvall et al., 1986, Tiger et al., 1997, Li et al., 2003a). In the present study, we mapped the epitope for 4C7 through the production of a series of recombinant laminin-511 mutants with deletions or substitutions within the G domain as well as recombinant LG modules expressed individually or in tandem, in order to gain an insight into the integrin-binding site within the G domain of laminin-511.
Section snippets
Results and discussion
4C7 has been shown to recognize the G domain of the laminin α5 chain and inhibit cell adhesion to laminin-511 (Engvall et al., 1986, Tiger et al., 1997, Li et al., 2003a). Therefore, it appears likely that 4C7 recognizes a region that overlaps with or is proximal to the integrin-binding site of laminin-511, thereby sterically blocking integrin binding to laminin-511. To explore this possibility, we examined the binding of integrin α6β1 to laminin-511 in the presence of either 4C7 or 5D6,
Antibodies
5D6, a mAb against the human laminin α5 chain, was produced in our laboratory (Fujiwara et al., 2001). The specificity of 5D6 was determined by isolation and sequencing of peptides, from a thermolysin digest of human placenta, that selectively bound to 5D6-conjugated Sepharose 4B. The N-terminal amino acid sequence of the 5D6-binding 44 kDa fragment was IEASNAYSRILQAVQ, identical to the sequence of domain I/II of the human laminin α5 chain (Ile2519–Gln2533). 4C7, another mAb against the human
Acknowledgments
We thank Noriko Sanzen for establishing the hybridoma clones and purifying mAb 5D6. We also thank Dr. Junichi Takagi for providing the recombinant β1 integrin expression vector and anti-ACID/BASE polyclonal antibody. We are grateful to Drs. Shaoliang Li and Masashi Yamada for their valuable comments. This study was partly supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.
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2017, StructureCitation Excerpt :This interpretation would be consistent with the finding that the integrin specificity of laminin heterotrimers is largely determined by the α chain (Nishiuchi et al., 2006). It is also noteworthy that the epitopes of two function-blocking antibodies map to the LG1 and LG2 domains, respectively (Ido et al., 2006; Yamashita et al., 2010). Because no structure of a laminin-binding integrin is available, and because the laminin γ1 tail could reorient substantially upon integrin binding, it is difficult to predict how the LG1 or LG2 domains might contact the integrin.
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A new nomenclature for laminin isoforms (Aumailley et al., 2005) has been used throughout this paper. Laminin-111, laminin-α1β1γ1 (also designated laminin-1); laminin-332, laminin-α3β3γ2 (laminin-5); laminin-511, laminin-α5β1γ1 (laminin-10).
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H.I. and K.H. equally contributed to this work.