Elsevier

Matrix Biology

Volume 25, Issue 2, March 2006, Pages 112-117
Matrix Biology

Probing the integrin-binding site within the globular domain of laminin-511 with the function-blocking monoclonal antibody 4C7

https://doi.org/10.1016/j.matbio.2005.10.003Get rights and content

Abstract

In an attempt to elucidate the integrin-binding site within laminin-511 (α5β1γ1), we mapped the epitope for mAb 4C7, which recognizes the globular (G) domain of the laminin α5 chain and inhibits binding of integrin α6β1 to laminin-511, using a series of recombinant laminin-511 mutants with deletions or substitutions in the G domain. Deletion of the LG2–5 modules only partially compromised the 4C7 binding activity, while deletion of all 5 LG modules completely abrogated the activity, indicating that the epitope for 4C7 resides in the LG1 module. In support of this conclusion, 4C7 reactivity was abolished when the LG1 module of laminin-511 was swapped with the corresponding module of laminin-111, but the reactivity was retained after swapping the LG2 or LG3 module. Despite the requirement of LG1 for 4C7 binding, a recombinant LG1 module failed to bind to 4C7 when expressed alone or in tandem with LG2, but exhibited significant 4C7 binding activity when expressed as an array of LG1–3. These results indicate that 4C7 recognizes an epitope in the LG1 module, whose active conformation is stabilized in the context of the LG1–3 modules. Despite their 4C7 binding activities, neither the recombinant LG1–3 fragment nor the LG2 and LG3 swap mutants were capable of binding to integrin α6β1. Thus, the integrin binding activity does not necessarily parallel the 4C7 reactivity, and possibly requires a strictly defined conformation of the LG1 module which can only be attained within an array of the intact LG1–3 modules connected to the preceding coiled-coil domain.

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.

References (24)

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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.

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