Screening of integrin-binding peptides from the laminin a4 and a5 chain G domain peptide library Fumihiko Katagiri, Masaya Ishikawa, Yuji Yamada, Kentaro Hozumi, Yamato Kikkawa, Motoyoshi Nomizu ⇑ Laboratory of Clinical Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan article info Article history: Received 24 January 2012 and in revised form 22 February 2012 Available online 3 March 2012 Keywords: Basement membrane Laminin Synthetic peptide Cell attachment Integrin abstract Laminins, a multifunctional protein family of extracellular matrix, interact with various types of integrin. Here, integrin-mediated cell adhesive peptides have been systematically screened in the laminin a4 and a5 chain G domain peptide library consisting of 211 peptides by both the peptide-coated plastic plates and peptide-conjugated Sepharose bead assays using human dermal fibroblasts. Thirteen peptides promoted cell spreading and the activity was specifically inhibited by EDTA. Cell attachment to 11 peptides was inhibited by anti-integrin b1 antibody. Additionally, cell attachment to the A5G81 (AGQWHRVSVRWG) and A5G84 (TWSQKALHHRVP) peptides was specifically inhibited by anti-integrin a3 and a6 antibodies. These results suggest that the A5G81 and A5G84 peptides promote integrin a3b1- and a6b1-mediated cell attachment. Further, most of the integrin-mediated cell adhesive peptides are located in the loop regions in the G domains, suggesting that structure is important for the integrin specific recognition. Integrin bind- ing peptides are useful for understanding laminin functions and have a potential to use for biomaterials and drug development. Ó 2012 Elsevier Inc. All rights reserved. Introduction Integrins are a family of receptors for cell attachment to extracel- lular matrix (ECM). 1 They also play a role in cell signaling and there- by define cell shape, mobility, and regulate the cell cycle [1]. Integrins are obligate heterodimers containing two distinct chains, called a and b subunits. In mammals, 18 a and eight b subunits have been characterized [1]. To date, 24 integrins have been identified and each integrin has tissue-specific expression and induces various signals [2,3]. The cell surface has multiple types of integrins, which play a vital in tissue structure and maintenance; e.g., differentiation and growth of cells (integrin a1b1) [4], wound healing, and aggluti- nation of platelets (integrin a2b1) [5], renal and pulmonary organ- ogenesis (integrin a3b1) [6], neurite outgrowth, and invasion and metastasis of cancers (integrin a6b1) [7–9]. Integrins function alongside other proteins, such as cadherins, immunoglobulin superfamily cell-adhesion molecules, selectins, and syndecans to mediate cell–cell and cell–matrix interactions and communication [10]. Integrins recognize specific sequences in the ECM components, including fibronectin, vitronectin, collagen, and laminin [11]. The Arg-Gly-Asp (RGD) motif, which is specific ligand for integrin avb3/aIIbb3, was originally identified in the fibronectin sequence [11]. RGD-containing motif derivatives are clinically used as anti- thrombogenic drugs [12]. Laminins are a major glycoprotein component of basement membranes, a thin ECM, and are biologically active and interact with various types of integrin. Laminins consist of three different subunits, a, b, and c chains, and so far, five a, three b, and three c chains have been identified [13]. The C-terminal globular do- mains (G domains) of the laminin a chains, consisting of five lam- inin G domain-like (LG) modules (LG1–5), play a critical role in the biological activities of laminins [14]. We have constructed a syn- thetic peptide library derived from the laminin sequences and sys- tematically screened cell adhesive peptides by the peptide-coated plastic and peptide-conjugated Sepharose bead assays using vari- ous cells [15]. Nineteen cell adhesive peptides were identified from the a1 chain G domain [16]. Fourteen and eleven cell adhesive pep- tides were also identified from the G domains of the a2 and a3 chains, respectively [17,18]. Further, we have identified 10 active peptides, which promote integrin-mediated cell adhesion, from the laminin a1–3 chains G domain sequences [16–18]. In contrast, the laminin a4 and a5 chain G domains have been screened using 0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2012.02.017 ⇑ Corresponding author. Fax: +81 42 676 5662. E-mail address: nomizu@toyaku.ac.jp (M. Nomizu). 1 Abbreviations used: ECM, extracellular matrix; G domains, globular domains; LG, laminin G; RBC, red blood cell; Fmoc, fluorenylmethoxycarbonyl; TFA, trifluoroacetic acid; HPLC, high performance liquid chromatography; HDFs, human neonatal dermal fibroblasts; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; SDS, sodium dodecyl sulfate; CNBr, cyanogen bromide; EDTA, ethylenediaminetetra- acetic acid; HRP, horse radish peroxidase; TBS, Tris-buffered saline; PBS, phosphate- buffered saline; DAPI, 4,6-diamidino-2-phenylindole; HSPGs, heparan sulfate proteoglycans; ES, embryo-stem; iPS, induced pluripotent stem. Archives of Biochemistry and Biophysics 521 (2012) 32–42 Contents lists available at SciVerse ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi