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

Abstract

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. 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 ␣ 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. 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 ␣ 2 ␤ 1 integrin-mediated fibroblast attachment and inhibited fibroblast attachment to a recombinant laminin ␣ 1 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.

Identification of Cell Adhesive Sequences in the N-terminal Region of the Laminin α2 Chain

“…EF-1 is located on the E and F strand and the connecting loop region. We synthesized cyclic-EF-1 and found that cyclic-EF-1 had stronger cell adhesive activity than linear EF-1 (17). It is possible that cyclic-A2-8 may promote strong cell attachment activity and enhance the specificity of integrin ␣2␤1 binding more potently than the linear A2-8 peptide.…”

“…3). AG73, which interacts with syndecans and promotes heparin-dependent cell attachment, and EF-1, which interacts with integrin ␣2␤1 and promotes a divalent cation-dependent cell adhesion (17), were used as controls. To evaluate the inhibitory effect of EDTA and heparin on cell adhesive peptides on the 96-well plates, A2-20 was conjugated with a chitosan membrane in the well.…”

“…Previously, we have screened biologically active sites using synthetic peptides to the laminin ␣ chains and identified several active peptides (15)(16)(17). Many active peptides were derived from the LG modules, and some of them were suggested to play a critical role in the specific binding to cell surface receptors.…”

“…AG73 (RKRLQVQLSIRT, mouse laminin ␣1 chain, residues 2719 -2730) (15,18), A3G756 (KNSFMALYLSKGRLVFALG, mouse laminin ␣3 chain, residues 1411-1429) (19), and A4G82 (TLFLAHGRLVFM, mouse laminin ␣4 chain, residues 1514 -1525) (20) bind to syndecans and promote various biological activities, including cell adhesion (20,21), migration (20,21), neurite outgrowth (22,23), branching morphogenesis (24), and tumor growth and metastasis (25,26). EF-1 (DYATLQ-LQEGRLHFMFDLG, mouse laminin ␣1 chain, residues 2747-2765) specifically interacts with integrin ␣2␤1 and induces cell spreading, focal adhesions, and production of actin stress fibers (17). Recently, we analyzed the mouse laminin ␣2 chain LN and LG domains, respectively.…”

Active Peptide-Conjugated Chitosan Matrices as an Artificial Basement Membrane

“…For example, we previously identified about sixty cell-adhesive sequences from laminin-111 using 673 synthetic peptides covering the entire sequence. These active peptides promote various biological activities, including the promotion of cell spreading, cell differentiation, neurite outgrowth, angiogenesis and wound healing, and many of them bind to distinct cellular receptors [32][33][34][35]37,38].…”