Biological Activities of Homologous Loop Regions in the Laminin α Chain G Domains

Nobuharu Suzuki,Hiroko Nakatsuka,Mayumi Mochizuki,Norio Nishi,Yuichi Kadoya,Atsushi Utani,Shinya Oishi,Nobutaka Fujii,Hynda K Kleinman,Motoyoshi Nomizu

doi:10.1074/jbc.m304667200

Abstract

Laminin alpha chains (alpha1-alpha5 chains) have diverse chain-specific biological functions. The LG4 modules of laminin alpha chains consist of a 14-stranded beta-sheet (A-N) sandwich structure. Several biologically active sequences have been identified in the connecting loop regions. Here, we evaluated the biological activities of the loop regions of the E and F strands in the LG4 modules using five homologous peptides from each of the mouse alpha chains (EF-1: DYATLQLQEGRLHFMFDLG, alpha1 chain 2747-2765; EF-2: DFGTVQLRNGFPFFSYDLG, alpha2 chain 2808-2826; EF-3: RDSFVALYLSEGHVIFALG, alpha3 chain 2266-2284; EF-4: DFMTLFLAHGRLVFMFNVG, alpha4 chain 1511-1529; EF-5: SPSLVLFLNHGHFVAQTEGP, alpha5 chain 3304-3323). These homologous peptides showed chain-specific cell attachment and neurite outgrowth activities. Well organized actin stress fibers and focal contacts with vinculin accumulation were observed in fibroblasts attached on EF-1, whereas fibroblasts on EF-2 and EF-4 showed filopodia with ruffling. Fibroblast attachment to EF-2 and EF-4 was mediated by syndecan-2. In contrast, EF-1 promoted alpha2beta1 integrin-mediated fibroblast attachment and inhibited fibroblast attachment to a recombinant laminin alpha1 chain LG4-5. The receptors for EF-3 and EF-5 are unknown. Further, when the active core sequence of EF-1 was cyclized, utilizing two additional cysteine residues at both the N and C termini through a disulfide bridge, the cyclic peptide significantly enhanced integrin-mediated cell attachment. These results indicate that integrin-mediated cell attachment to the EF-1 sequence is conformation-dependent and that the loop structure is important for the activity. The homologous peptides, which promote either integrin- or syndecan-mediated cell attachment, may be useful for understanding the cell type- and chain-specific biological activities of the laminins.

Highlights

Laminin ␣ chains (␣1–␣5 chains) have diverse chainspecific biological functions
We evaluated the biological activities of the loop regions of the E and F strands in the LG4 modules using five homologous peptides from each of the mouse ␣ chains (EF-1: DYATLQLQEGRLHFMFDLG, ␣1 chain 2747–2765; EF-2: DFGTVQLRNGFPFFSYDLG, ␣2 chain 2808 –2826; EF-3: RDSFVALYLSEGHVIFALG, ␣3 chain 2266 –2284; EF-4: DFMTLFLAHGRLVFMFNVG, ␣4 chain 1511–1529; EF-5: SPSLVLFLNHGHFVAQTEGP, ␣5 chain 3304 –3323)
Organization of Actin Filaments and Localization of Vinculin—we focused on the EF-1, EF-2, EF-4, and EF-5 peptides, which promoted fibroblast attachment, and examined the organization of actin filaments and localization of vinculin on the peptide-coated plates (Fig. 4)

Summary

Introduction

Laminin ␣ chains (␣1–␣5 chains) have diverse chainspecific biological functions. The LG4 modules of laminin ␣ chains consist of a 14-stranded ␤-sheet (A-N) sandwich structure. We evaluated the biological activities of the loop regions of the E and F strands in the LG4 modules using five homologous peptides from each of the mouse ␣ chains (EF-1: DYATLQLQEGRLHFMFDLG, ␣1 chain 2747–2765; EF-2: DFGTVQLRNGFPFFSYDLG, ␣2 chain 2808 –2826; EF-3: RDSFVALYLSEGHVIFALG, ␣3 chain 2266 –2284; EF-4: DFMTLFLAHGRLVFMFNVG, ␣4 chain 1511–1529; EF-5: SPSLVLFLNHGHFVAQTEGP, ␣5 chain 3304 –3323) These homologous peptides showed chainspecific cell attachment and neurite outgrowth activities. The homologous peptides, which promote either integrin- or syndecan-mediated cell attachment, may be useful for understanding the cell type- and chain-specific biological activities of the laminins. The A4G82 (TLFLAHGRLVFM, mouse laminin ␣4 chain 1514 –1525) sequence showed heparin binding and cell attachment activity [31, 32] These active sequences are in the homologous region of the LG4 modules of the ␣3 and ␣4 chains. Mutagenesis analysis using recombinant LG4 proteins with serine or alanine substitutions of basic residues (Lys1421 and Arg1423, human laminin ␣3 chain; His1519 and Arg1521, mouse laminin ␣4 chain) in the E-F loop regions suggested that the loop regions are critical for the biological activity of the recombinant proteins [29, 31]

Methods

Results

Conclusion