Screening of integrin-binding peptides in a laminin peptide library derived from the mouse laminin β chain short arm regions.
Laminins, major components of basement membrane, consist of three different subunits, α, β, and γ chains, and so far, five α, three β, and three γ chains have been identified. We have constructed synthetic peptide libraries derived from the laminin sequences and identified various cell-adhesive peptides. Ten active peptides from the laminin α chain sequences (α1-α5) were found to promote integrin-mediated cell adhesion. Previously, we found fourteen cell-adhesive peptides from the β1 chain sequence but their receptors have not been analyzed. Here, we expanded the synthetic peptide library to add peptides from the short arm regions of the laminin β2 and β3 chains and screened for integrin-binding peptides. Twenty-seven peptides promoted human dermal fibroblast (HDF) attachment in a peptide-coated plate assay. The morphological appearance of HDFs on the peptide-coated plates differed depending on the peptides. B34 (REKYYYAVYDMV, mouse laminin β1 chain, 255-266), B67 (IPYSMEYEILIRY, mouse laminin β1 chain, 604-616), B2-105 (APNFWNFTSGRG, mouse laminin β2 chain, 1081-1092), and B3-19 (GHLTGGKVQLNL, mouse laminin β3 chain, 182-193) promoted HDF spreading and HDF attachment was inhibited by EDTA, suggesting that the peptides interact with integrins. Immunostaining analyses revealed that B67 induced well-organized actin stress fibers and focal contacts containing vinculin, however, B34, B2-105, and B3-19 did not exhibit stress fiber formation or focal contacts. The inhibition assay using anti-integrin antibodies indicated that B67 interacts with α3, α6, and β1 integrins, and B34 and B3-19 interact with β1 integrin. Based on adhesion analysis of peptides modified with an alanine scan and on switching analysis with the homologous inactive sequence B2-64 (LPRAMDYDLLLRW, mouse laminin β2 chain, 618-630), the Glu(8) residue in the B67 peptide was critical for HDF adhesion. These findings are useful for identifying an integrin binding motif. The B67 peptide has potential for use as a molecular probe for integrins.
Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Fumihiko
F
Katagiri
Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Masaharu
M
Takagi
Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Minako
M
Nakamura
Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Yoichiro
Y
Tanaka
Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Kentaro
K
Hozumi
Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Yamato
Y
Kikkawa
Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Electronic address: nomizu@toyaku.ac.jp.
Motoyoshi
M
Nomizu
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020405:Dermis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007797:Laminin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D022502:Stress Fibers / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016596:Vinculin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000097794:Kalinin
D000906:Antibodies; D015815:Cell Adhesion Molecules; D004492:Edetic Acid; D016023:Integrins; D007797:Laminin; D019151:Peptide Library; D010455:Peptides; D016596:Vinculin; D000097794:Kalinin; C513277:Lamb1 protein, mouse; C488955:VCL protein, human; C058783:laminin beta2
Human dermal fibroblast; Integrin; Laminin; Synthetic peptide library