Fibulin-7 C-terminal fragment and its active synthetic peptide suppress choroidal and retinal neovascularization.

Wet age-related macular degeneration (AMD) and diabetic retinopathy are the leading causes of blindness through increased angiogenesis. Although VEGF-neutralizing proteins provide benefit, inconsistent responses indicate a need for new therapies. We previously identified the Fibulin-7 C-terminal fragment (Fbln7-C) as an angiogenesis inhibitor in vitro. Here we show that Fbln7-C inhibits neovascularization in vivo, in both a model of wet AMD involving choroidal neovascularization (CNV) and diabetic retinopathy involving oxygen-induced ischemic retinopathy. Furthermore, a short peptide sequence from Fbln7-C is responsible for the anti-angiogenic properties of Fbln7-C. Our work suggests Fbln7-C as a therapeutic candidate for wet AMD and ischemic retinopathy.

Identification of active sequences in human laminin α5 G domain.

Human laminin-511 (α5ß1γ1) and its truncated protein, laminin-511 E8 fragment, bind to integrin α6ß1 and have been widely used for embryonic stem cell and induced pluripotent stem cell culture under feeder-free conditions. In this study, we focused on human laminin α5 chain G domain, which is thought to be critical for the biological functions of laminin-511, and screened its biologically active sequences using a synthetic peptide library. We synthesized 115 peptides (hA5G1-hA5G115) covering the entire laminin α5 chain G domain and evaluated cell attachment activity using both the peptide-coated plate and peptide-chitosan matrix (peptide-ChtM) assays. Seventeen peptides demonstrated cell attachment activity in the assays. Both hA5G18 and hA5G26-coated plates and hA5G74-ChtMs promoted integrin ß1-mediated cell attachment. These findings are useful for the study of molecular mechanisms of laminin-511, and the active peptides have a potential for use as a molecular probe for cell adhesion receptors.

Mixed Peptide-Conjugated Chitosan Matrices as Multi-Receptor Targeted Cell-Adhesive Scaffolds.

Biomaterials are important for cell and tissue engineering. Chitosan is widely used as a scaffold because it is easily modified using its amino groups, can easily form a matrix, is stable under physiological conditions, and is inactive for cell adhesion. Chitosan is an excellent platform for peptide ligands, especially cell adhesive peptides derived from extracellular matrix (ECM) proteins. ECM proteins, such as collagen, fibronectin, and laminin, are multifunctional and have diverse cell attachment sites. Various cell adhesive peptides have been identified from the ECM proteins, and these are useful to design functional biomaterials. The cell attachment activity of peptides is influenced by the solubility, conformation, and coating efficiency to solid materials, whereas immobilization of peptides to a polysaccharide such as chitosan avoids these problems. Peptide⁻chitosan matrices promote various biological activities depending on the peptide. When the peptides are immobilized to chitosan, the activity of the peptides is significantly enhanced. Further, mixed peptide⁻chitosan matrices, conjugated with more than one peptide on a chitosan matrix, interact with multiple cellular receptors and promote specific biological responses via receptor cross-talk. Receptor cross-talk is important for mimicking the biological activity of ECM and the proteins. The mixed peptide⁻chitosan matrix approach is useful to develop biomaterials as a synthetic ECM for cell and tissue engineering.

Biological activity of peptide-conjugated polyion complex matrices consisting of alginate and chitosan.

Peptide-conjugated polysaccharide matrices using bioactive laminin-derived peptides are useful biomaterials for tissue and cell engineering. Here, we demonstrate an easy handling preparation method for peptide-polysaccharide matrices using polyion complex with both alginate and chitosan. First, aldehyde-alginate was synthesized by oxidization of alginate using NaIO4 , and then, reacted with Cys-peptides. Next, the peptide-alginate solution was added to a chitosan-coated plate, and the peptide-polyion complex matrices (peptide-PCMs) were prepared. The peptide-PCMs using an integrin αvß3-binding peptide (A99a: ALRGDN, mouse laminin α1 chain 1145-1150) and an integrin α2ß1-binding peptide (EF1XmR: RLQLQEGRLHFXFD, X = Nle, mouse laminin α1 chain 2751-2763) showed strong cell attachment activity in a dose-dependent manner. When we examined the effect of various spacers on the biological activity of A99a-PCM, hydrophobic and long spacers enhanced the cell attachment activity. Further, the A99a-PCM with the spacers strongly promoted neurite outgrowth. The polyion complex method is an easy way to obtain insolubilized matrix and is widely applicable for various polysaccharides. The peptide-PCM is useful as a biomaterial for cell and tissue engineering.

Identification of laminin α5 short arm peptides active for endothelial cell attachment and tube formation.

Laminin-511, a major component of endothelial basement membrane, consists of α5, ß1, and γ1 chains. The short arm region of the α5 chain is a structural feature of endothelial laminins. In this study, we identified active sequences for human umbilical vein endothelial cells (HUVECs) using recombinant proteins and synthetic peptides. The short arm of the α5 chain contains three globular domains [laminin N-terminal globular domain, laminin 4 domain a, and laminin 4 domain b (LN, L4a, and L4b)] and three rod-like elements [laminin epidermal growth factor-like domain a, b, and c (LEa, LEb, and LEc)]. The cell attachment assay using recombinant proteins showed that RGD-independent cell attachment sites were localized in the α5LN-LEa domain. Further, we synthesized 70 peptides covering the amino acid sequences of the α5LN-LEa domain. Of the 70 peptides, A5-16 (mouse laminin α5 230-243: LENGEIVVSLVNGR) potently exhibited endothelial cell attachment activity. An active sequence analysis using N-terminally and C-terminally truncated A5-16 peptides showed that the nine-amino acid sequence IVVSLVNGR was critical for the endothelial cell attachment activity. Cell adhesion to the peptides was dependent on both cations and heparan sulfate. Further, the A5-16 peptide inhibited the capillary-like tube formation of HUVECs with the cells forming small clumps with short tubes. The eight-amino acid sequence EIVVSLVN in the A5-16 peptide was critical to inhibit HUVEC tube formation. This amino acid sequence could be useful for grafts and thus modulate endothelial cell behavior for vascular surgery. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Down-regulation of cell adhesion via rho-associated protein kinase (ROCK) pathway promotes tumor cell migration on laminin-511.

Epithelial cells, both normal and precancerous, stably anchor to basement membranes, whereas malignant tumors pass through them to achieve metastasis. Of basement membrane components, laminin-511 (α5, ß1, γ1; LM-511) has been found to be a major isoform in many adult basement membranes. Several studies have shown that LM-511 promotes not only cell adhesion but also tumor cell migration. Thus, LM-511 can be viewed like two distinct molecules in normal vs. tumor cells; tumor cells seem to be able to alter their response (adhesive vs. migratory) to LM-511. In this study we examined the effects of biologically active molecules on A549 lung adenocarcinoma cell adhesion to LM-511. Of them, phorbol 12-myristate 13-acetate (PMA) induced transition to a rounded cell shape and significantly promoted cell migration on LM-511. The attachment of PMA-treated A549 cells to LM-511 was weaker than that of control cells. PMA-stimulated signaling pathway reduced the binding of integrin α3ß1 to LM-511. Cell migration assays using inhibitors for signal transduction and cytoskeletal organization showed that suppression of cell adhesion via the rho-associated protein kinase (ROCK) pathway promoted tumor cell migration on LM-511. Our results suggest that the ROCK pathway is involved in the transition from static to migratory cell behaviors on LM-511.

Effect of spacer length and type on the biological activity of peptide-polysaccharide matrices.

Peptide-polysaccharide matrices can mimic extracellular matrix structure and function and are useful for tissue and cell engineering. The spacer between the peptide and the polysaccharide is important for both peptide conformation and the interaction between the peptide and receptors. Here, the effect of a spacer on the biological activity of peptide-polysaccharide matrices using various lengths of spacers consisting of glycine, ß-alanine, and ε-aminocaproic acid has been examined. Active laminin-derived peptides, including a syndecan-binding peptide (AG73: RKRLQVQLSIRT), an integrin αvß3-binding peptide (A99a: ALRGDN), and an integrin α6ß1-binding peptide (A2G10: SYWYRIEASRTG), were used as the peptide ligands and chitosan was used as a polysaccharide matrix. The spacers did not influence the biological activity of the AG73-chitosan matrix. In contrast, the integrin-binding peptide-chitosan matrices showed spacer-dependent activity. Hydrophobic spacers enhanced the cell attachment activity of the A99a-chitosan matrix. A four-glycine spacer showed the strongest effect for the biological activity of the A2G10-chitosan matrix. These results suggested that spacer-optimization for each peptide is important for designing effective peptide-polysaccharide matrices. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 512-520, 2016.

Identification of peptides derived from the C-terminal domain of fibulin-7 active for endothelial cell adhesion and tube formation disruption.

Despite the research done on pathological angiogenesis, there is still a need for the development of new therapies against angiogenesis-related diseases. Fibulin-7 (Fbln7) is a member of the extracellular matrix fibulin protein family. The Fbln7 C-terminal fragment, Fbln7-C, binds to endothelial cells and inhibits their tube formation in culture. In this study, we screened 12 synthetic peptides, covering the fibulin-globular domain of Fbln7-C, to identify active sites for endothelial cell adhesion and in vitro antiangiogenic activity. Three peptides, fc10, fc11, and fc12, promoted Human Umbilical Vein Endothelial Cells (HUVECs) adhesion, and the morphology of HUVECs on fc10 was similar to that on Fbln7-C. EDTA and the anti-integrin ß1 function-blocking antibody inhibited HUVECs adhesion to both fc10 and fc12, and heparin inhibited HUVECs adhesion to both fc11 and fc12. fc10 and fc11 inhibited HUVECs tube formation. Our results suggest that three peptides from Fbln7-C are biologically active for endothelial cell adhesion and disrupt the tube formation, suggesting a potential therapeutic use of these peptides for angiogenesis-related diseases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 184-195, 2016.

Soluble Lutheran/basal cell adhesion molecule is detectable in plasma of hepatocellular carcinoma patients and modulates cellular interaction with laminin-511 in vitro.

Lutheran (Lu), an immunoglobulin superfamily transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a subunit of laminin-511 (LM-511) that is a major component of basement membranes in various tissues. Our previous study showed that Lu/B-CAM was cleaved by MT1-MMP and released from cell surfaces. In this study we examined the soluble Lu/B-CAM in culture media and in plasma of mice bearing HuH-7 hepatocellular carcinoma (HCC) cells and patients with HCC. Two HCC cell lines, HepG2 and HuH-7, released Lu/B-CAM into the culture media. Although Lu/B-CAM was cleaved by MT1-MMP in HuH-7 cells, HepG2 cells released Lu/B-CAM in a MMP-independent manner. The concentration of Lu/B-CAM released into mouse plasma correlated with tumor size. Moreover the soluble Lu/B-CAM in plasma of HCC patients was significantly decreased after resection of the tumor. Immunohistochemical studies showed that although the expression of Lu/B-CAM was observed in most HCCs, MT1-MMP was not always expressed in tumor tissues, suggesting that a part of Lu/B-CAM in plasma of HCC patients was also released in a MMP-independent manner. In vitro studies showed that the soluble Lu/B-CAM released from HCC cells bound to LM-511. Moreover the soluble Lu/B-CAM influenced cell migration on LM-511. These results suggest that soluble Lu/B-CAM serves as not only a novel marker for HCC but also a modulator in tumor progression.

Biological activities of the homologous loop regions in the laminin α chain LG modules.

Each laminin α chain (α1-α5 chains) has chain-specific diverse biological functions. The C-terminal globular domain of the α chain consists of five laminin-like globular (LG1-5) modules and plays a critical role in biological activities. The LG modules consist of a 14-stranded ß-sheet (A-N) sandwich structure. Previously, we described the chain-specific biological activities of the loop regions between the E and F strands in the LG4 modules using five homologous peptides (G4EF1-G4EF5). Here, we further analyze the biological activities of the E-F strands loop regions in the rest of LG modules. We designed 20 homologous peptides (approximately 20 amino acid length), and 17 soluble peptides were used for the cell attachment assay. Thirteen peptides promoted cell attachment activity with different cell morphologies. Cell attachment to peptides G1EF1, G1EF2, G2EF1, G3EF4, and G5EF4 was inhibited by heparin, and peptides G1EF1, G1EF2, and G2EF1 specifically bound to syndecan-overexpressing cells. Cell attachment to peptides G2EF3, G3EF1, G3EF3, G5EF1, G5EF3, and G5EF5 was inhibited EDTA. Further, cell attachment to peptides G3EF3, G5EF1, and G5EF5 was inhibited by both anti-integrin α2 and ß1 antibodies, whereas cell attachment to peptide G5EF3 was inhibited by only anti-integrin ß1 antibody. Cell attachment to peptides G1EF4, G3EF4, and G5EF4 was inhibited by both heparin and EDTA and was not inhibited by anti-integrin antibodies. The active peptide sequence alignments suggest that the syndecan-binding peptides contain a "basic amino acid (BAA)-Gly-BAA" motif in the middle of the molecule and that the integrin-binding peptides contain an "acidic amino acid (AAA)"-Gly-BAA motif. Core-switched peptide analyses suggested that the "BAA-Gly-BAA" motif is critical for binding to syndecans and that the "AAA-Gly-BAA" motif has potential to recognize integrins. These findings are useful for understanding chain-specific biological activities of laminins and to evaluate receptor-specific binding mechanisms.

The lutheran/basal cell adhesion molecule promotes tumor cell migration by modulating integrin-mediated cell attachment to laminin-511 protein.

Cell-matrix interactions are critical for tumor cell migration. Lutheran (Lu), also known as basal cell adhesion molecule (B-CAM), competes with integrins for binding to laminin α5, a subunit of LM-511, a major component of basement membranes. Here we show that the preferential binding of Lu/B-CAM to laminin α5 promotes tumor cell migration. The attachment of Lu/B-CAM transfectants to LM-511 was slightly weaker than that of control cells, and this was because Lu/B-CAM disturbed integrin binding to laminin α5. Lu/B-CAM induced a spindle cell shape with pseudopods and promoted cell migration on LM-511. In addition, blocking with an anti-Lu/B-CAM antibody led to a flat cell shape and inhibited migration on LM-511, similar to the effects of an activating integrin ß1 antibody. We conclude that tumor cell migration on LM-511 requires that Lu/B-CAM competitively modulates cell attachment through integrins. We suggest that this competitive interaction is involved in a balance between static and migratory cell behaviors.

Screening of integrin-binding peptides in a laminin peptide library derived from the mouse laminin ß chain short arm regions.

Laminins, major components of basement membrane, consist of three different subunits, α, ß, and γ chains, and so far, five α, three ß, 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 ß1 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 ß2 and ß3 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 ß1 chain, 255-266), B67 (IPYSMEYEILIRY, mouse laminin ß1 chain, 604-616), B2-105 (APNFWNFTSGRG, mouse laminin ß2 chain, 1081-1092), and B3-19 (GHLTGGKVQLNL, mouse laminin ß3 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 ß1 integrins, and B34 and B3-19 interact with ß1 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 ß2 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.

Double-stranded DNA stereoselectively promotes aggregation of amyloid-like fibrils and generates peptide/DNA matrices.

An amyloidogenic LAM-L peptide (AASIKVAVSADR, all-L configuration) derived from laminin promoted cell adhesion, neurite outgrowth, and angiogenesis. Here, we prepared novel matrices using double-stranded DNA and the LAM-L peptide. Double-stranded DNA promoted aggregation of amyloid-like fibrils and generated a LAM-L/DNA matrix through electrostatic interactions between the phosphate groups of DNA and the amino groups of LAM-L. This formation of peptide/DNA matrix depends on the Ile-Lys-Val-Ala-Val (IKVAV) sequence in the peptide, since LAM-RM peptide (AASVVIAKSADR), which is scrambled peptide of LAM-L, did not form a matrix with DNA. Further, LAM-D (all-D configuration of LAM-L), which forms amyloid-like fibrils and promotes similar biological activities as LAM-L, did not form amyloid-like fibrils with DNA, suggesting that DNA selectively interacts with the L-configured peptide. Moreover, the LAM-L/DNA matrices showed stronger cell attachment activity compared with LAM-L alone, suggesting the LAM-L/DNA matrices have potential for use as a novel biomaterial in tissue engineering.

Identification of cell adhesive sequences in the N-terminal region of the laminin α2 chain.

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin ß1 and anti-integrin α2ß1 antibodies. These results suggest that A2-8 promotes an integrin α2ß1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2ß1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2ß1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2ß1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.

Cell attachment and spreading activity of mixed laminin peptide-chitosan membranes.

Laminins are a multifunctional molecule with numerous active sites that have been identified in short peptide sequences. Mixed peptide-conjugated chitosan membranes using laminin-derived active peptides have been previously demonstrated to be useful as a biomaterial for tissue engineering. In this study, two syndecan-binding peptides, AG73 (RKRLQVQLSIRT) and C16 (KAFDITYVRLKF), and three integrin-binding peptides, EF1zz (ATLQLQEGRLHFXFDLGKGR, X: Nle, binding to integrin α2ß1), A99a (ALRGDN, binding to integrin αvß3), and A2G10 (SYWYRIEASRTG, binding to integrin α6ß1), were mixed in various combinations, conjugated to chitosan membranes, and evaluated for their cell attachment and spreading activities. The cell attachment and spreading activity of EF1zz, A99a, and A2G10 were enhanced by AG73. In contrast, C16 enhanced only the cell attachment and spreading activity of A99a and did not influence the activity of EF1zz and A2G10. As well as previous study, the AG73-chitosan membrane bound to only syndecan. On the other hand, the C16-chitosan membrane interacted with both syndecan and ß1 integrin. These data suggest that interaction of different receptors can cause synergistic effects. Therefore, AG73 is widely applicable as a synergistic agent for mixed peptide-matrices using several types of integrin-binding peptides. Additionally, the A2G10/AG73-chitosan membrane may be useful to investigate detailed biological functions of α6ß1 integrin, which is a major laminin-binding receptor. Using a combination of tissue-appropriate laminin-derived peptides, the mixed peptide-chitosan membranes may serve as functional biomaterials for tissue engineering.

Identification of active sequences in the L4a domain of laminin α5 promoting neurite elongation.

Laminin α5 is an extracellular matrix protein containing multiple domains implicated in various biological processes, such as embryogenesis and renal function. In this study, we used recombinant proteins and synthetic peptides to identify amino acid residues within the short arm region of α5 that were critical for neurite outgrowth activity. The short arm of α5 contains three globular domains (LN, L4a, and L4b) and three rodlike elements (LEa, LEb, and LEc). Recombinant proteins comprised of the α5 short arm fused with a Fc tag produced in 293 cells were assayed for PC12 (pheochromocytoma) cell adhesion and neurite outgrowth activities. Although it did not have cell attachment activity, neurite outgrowth was promoted by the recombinant protein. To narrow the region involved in neurite outgrowth activity, two truncated recombinant proteins were produced in 293 cells. A recombinant protein lacking L4a and LEb lost activity. Furthermore, we synthesized 78 partially overlapping peptides representing most of the amino acid sequences of L4a and LEb. Of the peptides, A5-76 [mouse laminin α5 928-939 (TSPDLFRLVFRY) in L4a] exhibited neurite outgrowth activity. Mutagenesis studies showed that Phe(933) and Arg(934) were involved in neurite outgrowth activity. Moreover, inhibition assays using anti-integrin monoclonal antibodies showed that neurite outgrowth on the α5 short arm was partially mediated by integrin α1ß1. However, the antibodies to integrin α1 and ß1 did not inhibit neurite elongation on the A5-76 peptide. These results suggest that in addition to cellular interactions with the active site in the L4a domain, the binding of integrin α1ß1 seems to modulate neurite elongation on the short arm of α5.

Structural requirement of fibrogenic laminin-derived peptide A119 (LSNIDYILIKAS) for amyloid-like fibril formation and cellular activity.

A119 peptide (LSNIDYILIKAS), derived from the mouse laminin α1 chain sequence (residues 1321-1332), promotes cell attachment, neurite outgrowth, and amyloid-like fibril formation. In this study, we evaluated the structural requirements of A119 for biological activities and amyloid-like fibril formation. The attachment of the cell to A119 was inhibited by heparin, and using syndecan- and glypican-overexpressed cells, it was determined that A119 specifically binds to syndecans. We also evaluated the critical residues for A119 activities using a set of alanine-substituted peptides. Cell attachment activity was significantly reduced in the Leu(1)-, Ser(2)-, Asn(3)-, Ile(4)-, Ile(7)-, Ile(9)-, and Lys(10)-substituted alanine peptides. Residues Ile(4), Ile(7), Ile(9), and Lys(10) were important for neurite outgrowth activity. Congo red staining and electron microscopic examination revealed that the Ile(4), Ile(7), Ile(9), and Ser(12) residues of A119 were required for amyloid-like fibril formation. These data suggest that the Ile residues are critical for the amyloid-like fibril formation, cell attachment, and neurite outgrowth activity of A119. Furthermore, an enantiomer of A119 showed similar amyloid-like fibril formation and increased levels of cell attachment and FAK signal transduction. These findings shed light on the mechanism of amyloid-like fibril formation and demonstrate a relationship between the ability to form amyloid-like fibrils and cell behavior.

Screening of integrin-binding peptides from the laminin α4 and α5 chain G domain peptide library.

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 α4 and α5 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 ß1 antibody. Additionally, cell attachment to the A5G81 (AGQWHRVSVRWG) and A5G84 (TWSQKALHHRVP) peptides was specifically inhibited by anti-integrin α3 and α6 antibodies. These results suggest that the A5G81 and A5G84 peptides promote integrin α3ß1- and α6ß1-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 binding peptides are useful for understanding laminin functions and have a potential to use for biomaterials and drug development.

Construction and activity of a synthetic basement membrane with active laminin peptides and polysaccharides.

Cell-adhesive peptides derived from extracellular matrix (ECM) proteins are potential candidates for incorporating cell-binding activities into materials for tissue engineering. We have identified a number of cell adhesive peptides from laminins, which are major components of basement membrane ECM. Our goal is the development of synthetic basement membranes using the peptides on scaffolds. We review peptide­polysaccharide complexes, which were prepared by conjugation of the peptides to chitosan and alginate, and the biological activities of the resulting matrices. The peptide­polysaccharide matrices can also be used as a biomaterial for cell transplantation. These studies suggest that the peptide­polysaccharide complexes have the potential to mimic the multifunctional basement membrane and may be useful for tissue engineering.

Design and synthesis of amidine-type peptide bond isosteres: application of nitrile oxide derivatives as active ester equivalents in peptide and peptidomimetics synthesis.

Amidine-type peptide bond isosteres were designed based on the substitution of the peptide bond carbonyl (C=O) group with an imino (C=NH) group. The positively-charged property of the isosteric part resembles a reduced amide-type peptidomimetic. The peptidyl amidine units were synthesized by the reduction of a key amidoxime (N-hydroxyamidine) precursor, which was prepared from nitrile oxide components as an aminoacyl or peptidyl equivalent. This nitrile oxide-mediated C-N bond formation was also used for peptide macrocyclization, in which the amidoxime group was converted to peptide bonds under mild acidic conditions. Syntheses of the cyclic RGD peptide and a peptidomimetic using both approaches, and their inhibitory activity against integrin-mediated cell attachment, are presented.