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The first rapid and efficient chemical synthesis of a cyclic Arg-Gly-Asp (RGD) peptide containing a chloroalkene dipeptide isostere (CADI) is reported. By a developed synthetic method, an N-tert-butylsulfonyl protected CADI was obtained utilizing diastereoselective allylic alkylation as a key reaction. This CADI was also transformed into an N-Fmoc protected CADI in a few steps. The CADI was used in Fmoc-based solid-phase peptide synthesis. The first synthesis of a CADI-containing cyclic RGD peptide was successful, and the synthesized CADI-containing peptidomimetic was found to be a more potent inhibitor against integrin-mediated cell attachment than the parent cyclic peptide.

Laminin-511, a major component of endothelial basement membrane, consists of α5, β1, 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.

Human laminin-511 (α5β1γ1) and its truncated protein, laminin-511 E8 fragment, bind to integrin α6β1 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 β1-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.

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, β-alanine, and e-aminocaproic acid has been examined. Active laminin-derived peptides, including a syndecan-binding peptide (AG73: RKRLQVQLSIRT), an integrin αvβ3-binding peptide (A99a: ALRGDN), and an integrin α6β1-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.

Mimicking the biological function of the extracellular matrix is an approach to developing cell adhesive biomaterials. The RGD peptide, derived from fibronectin (Fn), mainly binds to integrin αvβ3 and has been widely used as a cell adhesive peptide on various biomaterials. However, cell adhesion to Fn is thought to be mediated by several integrin subtypes and syndecans. In this study, we synthesized an RGD-containing peptide (FIB1) and four integrin α4β1-binding-related motif-containing peptides (LDV, IDAPS, KLDAPT, and PRARI) and constructed peptide-chitosan matrices. The FIB1-chitosan matrix promoted human dermal fibroblast (HDF) attachment, and the C-terminal elongated PRARI (ePRARI-C)-conjugated chitosan matrix significantly promoted HDF attachment through integrin α4β1 and syndecan binding. Next, we constructed a mixed ePRARI-C- and FIB1-chitosan matrix to develop a Fn mimetic biomaterial. The mixed ePRARI-C/FIB1-chitosan matrix promoted significantly better cell attachment and neurite outgrowth compared to those of either ePRARI-C- or FIB1-chitosan matrices. HDF adhesion to the ePRARI-C/FIB1-chitosan matrix was mediated by integrin, α4β1, α5β1, and αvβ3, similar to HDF adhesion to Fn. These data suggest that an ePRARI-C/FIB1-chitosan matrix can be used as a tool to analyze the multiple functions of Fn and can serve as a Fn-mimetic biomaterial.

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 β1 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.

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.

Extracellular matrix molecules are recognized by several integrin subtypes, making identification of cross-talk among different integrin subtypes difficult. Here, we evaluated the cross-talk of integrin subtypes using four different integrin-binding peptides (FIB1; integrin αvβ3/α5β1, A2G10; integrin α6β1, EF1zz; integrin α2β1, or 531; integrin α3β1) derived from extracellular matrix molecules. Various combinations of two different integrin-binding peptides were mixed and conjugated on a chitosan matrix at various molar ratios and were evaluated for cell attachment activity. FIB1/A2G10 (molar ratio 5:5; total 10 nmol/well)-chitosan matrix significantly enhanced cell attachment activity compared with sum of the cell attachment activity on FIB1 (5 nmol/well)-chitosan matrices and A2G10 (5 nmol/well)-chitosan matrices, respectively. However, none of the other peptides showed a significant activity change when they were mixed and conjugated on a chitosan matrix. We investigated the mechanisms of this enhancement. FIB1/A2G10 (8:2 or 6:4)-chitosan matrix increased the cell spreading, phosphorylation of focal adhesion kinase at Y397, and slightly decreased phosphorylation of caveolin-1 at Y14 in fibroblasts compared with FIB1-chitosan and A2G10-chitosan matrices. These results indicate that FIB1/A2G10 (8:2 or 6:4)-chitosan matrix synergistically enhances cell attachment, suggesting that integrins αvβ3/α5β1 and α6β1 are involved in a cross-talk and synergistically enhance cell attachment. These findings also suggest that the mixed peptide-chitosan matrix system can regulate the ratio of two different peptides and is useful for evaluating cellular functions through receptor-specific cross-talk. Further, FIB1/A2G10 (8:2 or 6:4)-chitosan matrix could be a useful material for tissue engineering.

Many cell-adhesive peptides have been identified from extracellular matrix (ECM) proteins, such as collagen, fibronectin, laminin, and vitronectin. ECM proteins have various cell-adhesive sequences. Most peptides demonstrate cell-adhesive activity when simply coated on a tissue culture plate, but solubility, conformation, and coating efficiency of the peptides can significantly alter their biological function. Evaluation of peptide cell-adhesive activity using peptide-conjugated polysaccharide constructs is a useful strategy for overcoming peptide solubility and conformation problems. After a simple modification of the polysaccharides, various polysaccharides (chitosan, alginate, and hyaluronate) can fix the peptides on the tissue culture plate quantitatively. The peptide-polysaccharide strategy can be used to fix different active peptides to the polysaccharide at same time, thus, mimicking the biological functions of the ECM. This paper describes the modification of polysaccharides that are suitable for covalently coupling the peptides and evaluation of the cell-adhesive activity of peptide as a peptide-polysaccharide matrix. © 2018 by John Wiley & Sons, Inc.

The basement membrane, a thin extracellular matrix, plays a critical role in tissue development and repair. Laminins are the major component of basement membrane and have diverse biological activities. We have identified various cell-adhesive peptides from laminins and their specific cell surface receptors. Polysaccharides, including chitosan, have been used as scaffolds, which regulate cellular functions for tissue engineering. We have developed laminin-derived active peptide-chitosan matrices as functional scaffolds. The biological activity of the peptides was enhanced when the peptides were conjugated to a chitosan matrix, suggesting that the peptide-chitosan matrix approach has an advantage for an active biomaterial. Further, the laminin peptide-chitosan matrices have the potential to mimic the basement membrane and are useful for tissue engineering as an artificial basement membrane.

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. © 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 465–472, 2014.

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 Glu8 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.

Human induced pluripotent stem cells (hiPSCs) grow indefinitely in culture and have the potential to regenerate various tissues. In the development of cell culture systems, a fragment of laminin-511 (LM511-E8) was found to improve the proliferation of stem cells. The adhesion of undifferentiated cells to LM511-E8 is mainly mediated through integrin α6β1. However, the involvement of non-integrin receptors remains unknown in stem cell culture using LM511-E8. Here, we show that dystroglycan (DG) is strongly expressed in hiPSCs. The fully glycosylated DG is functionally active for laminin binding, and although it has been suggested that LM511-E8 lacks DG binding sites, the fragment does weakly bind to DG. We further identified the DG binding sequence in LM511-E8, using synthetic peptides, of which, hE8A5-20 (human laminin α5 2688–2699: KTLPQLLAKLSI) derived from the laminin coiled-coil domain, exhibited DG binding affinity and cell adhesion activity. Deletion and mutation studies show that LLAKLSI is the active core sequence of hE8A5-20, and that, K2696 is a critical amino acid for DG binding. We further demonstrated that hiPSCs adhere to hE8A5-20-conjugated chitosan matrices. The amino acid sequence of DG binding peptides would be useful to design substrata for culture system of undifferentiated and differentiated stem cells.

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 α2β1), A99a (ALRGDN, binding to integrin αvβ3), and A2G10 (SYWYRIEASRTG, binding to integrin α6β1), 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 β1 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 α6β1 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.

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 β1 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.

Laminin-111 is a large trimeric basement membrane glycoprotein with many active sites. In particular, four peptides active in tumor malignancy studies have been identified in laminin-111 using a systematic peptide screening method followed by various assays. Two of the peptides (IKVAV and AG73) are found on the α1 chain, one (YIGSR) of the β1 chain and one (C16) on the γ1 chain. The four peptides have distinct activities and receptors. Since three of the peptides (IKVAV, AG73 and C16) strongly promote tumor growth, this may explain the potent effects laminin-111 has on malignant cells. The peptide, YIGSR, decreases tumor growth and experimental metastasis via a 32/67 kD receptor while IKVAV increases tumor growth, angiogenesis and protease activity via integrin receptors. AG73 increases tumor growth and metastases via syndecan receptors. C16 increases tumor growth and angiogenesis via integrins. Identification of such sites on laminin-111 will have use in defining strategies to develop therapeutics for cancer.

Laminin-511, a major component of endothelial basement membrane, consists of α5, β1, 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 WileySons, Ltd.

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 NaIO

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.

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 β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 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 α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.

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 β1 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 α3β1- and α6β1-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.

Hepatocytes rapidly lose hepatic functions upon isolation from liver, perhaps due to disrupted cell/matrix interactions. The matrix macromolecule laminin-111 consists of three chains, α1, β1, and γ1; it is a major component of Matrigel, which can maintain hepatic differentiation. We previously showed that the A13 peptide (RQVFQVAYIIIKA, α1 chain 121–133) derived from mouse laminin α1 exhibits hepatocyte attachment activity and maintains hepatic differentiation. Here, we sought to identify hepatocyte adhesive sequences from the mouse laminin β1 and γ1 chains using 22 synthetic peptides that show biological activity for fibrosarcoma cells. Nine peptides showed hepatocyte attachment activity. Of these, B160 (VILQQSAADIAR, β1 chain 1607–1618), and C16 (KAFDITYVRLKF γ1 chain 139–150) exhibited the most potent activity. Hepatocytes cultured on both peptides also maintained expression of albumin, tyrosine aminotransferase, tryptophan-2,3-dioxygenase, and cytochrome P450. The morphology of hepatocytes on both peptides was a rounded shape typical for hepatic differentiation. We also characterized the nature of adhesion to the peptides. Heparin and EDTA inhibited cell attachment to both peptides, suggesting that hepatocyte attachment to the peptides was mediated by multiple receptors. The identification of active sequences regulating hepatic functions may facilitate the design of hepatocyte culture substrata that can regulate specific cellular behaviors in the context of a bioartificial liver. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2011.

Cell adhesive peptides have been widely applied for therapeutic drugs, drug delivery systems, and biomaterials. Previously, we identified various cell adhesive sequences in the G domains of four laminin α chains (α2-α5) by the systematic soluble peptide screening. We also identified five cell-binding sequences in the laminin α1 chain G domain using synthetic peptide-polystyrene beads. Here, we re-screened cell adhesive peptides in the laminin α1 chain G domain by the systematic soluble peptides screening. The 110 soluble peptides were evaluated for their cell adhesive activities using human fibrosarcoma HT1080 cells and human dermal fibroblasts. Fourteen peptides were newly identified as a cell adhesive. Additionally, four peptides (AG22: SSFHFDGSGYAM, AG42: TFDLLRNSYGVRK, AG76: HQNQMDYATLQLQ, AG86: LGGLPSHYRARNI) promoted integrin-mediated cell adhesion. Further, neurite outgrowth activity with rat pheochromocytoma PC12 cells was evaluated and two peptides (AG20: SIGLWNYIEREGK, AG26: SPNGLLFYLASNG) were newly identified for neurite outgrowth activity. These results suggested that the systematic soluble peptides screening approach is an accurate and powerful strategy for finding biologically active sequences. The active sequences newly identified here could be involved in the biological functions of this domain. The active peptides are useful for evaluating molecular mechanisms of laminin-receptor interactions and for developing cell adhesive biomaterials.

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.

Peptide B133 (DSITKYFQMSLE), derived from mouse laminin beta1 chain (residues 1298-1309), promotes cell attachment, neurite outgrowth, and amyloid-like fibril formation. Previously, we showed that the N-terminal Asp-deleted peptide B133a (SITKYFQMSLE) promotes integrin alpha2beta1-mediated cell attachment and spreading but does not form amyloid-like fibrils, and that the C-terminal Glu-deleted peptide B133g (DSITKYFQMSL) attaches cells without cell spreading and forms amyloid-like fibrils. In this study, we further investigated the amino acid sequence requirements of B133 for biological function using a set of truncated and Ala-substituted peptides. Attachment of cells to B133g was inhibited by only heparin, and Congo Red analysis indicated that the amyloid-like fibril formation activity of B133g was stronger than that of B133. Alanine scan analysis for the B133g peptide indicated that Asp and Ile residues are essential for cell attachment. Additionally, the N-terminal Asp residue was required for neurite outgrowth. Further, amyloid-like fibril formation required Asp and Ile residues. These data suggest that the amyloid-like fibril formation of B133g is required for cell attachment activity. We also evaluated the attachment of cells to the peptides using syndecan- and glypican-overexpressing cells. B133g attached to syndecan-overexpressing cells but not to glypican-overexpressing cells, suggesting that the amyloidogenic peptides promote syndecan-mediated cell attachment. These findings were useful for clarifying the mechanism of amyloid-like fibril formation and biological functions. The B133 peptide promotes amyloid-like fibril formation, syndecan-mediated cell attachment, and neurite outgrowth and has the potential for use as a biomaterial for tissue engineering.

The biological activities of the laminin alpha2 chain LG4-5 module result from interactions with cell surface receptors, such as heparan sulfate proteoglycans and alpha-dystroglycan. In this study, heparin and alpha-dystroglycan binding sequences were identified using 42 overlapping synthetic peptides from the LG4-5 module and using recombinant LG4-5 protein (rec-alpha2LG4-5). Physiological activities of the active peptides were also examined in explants of submandibular glands. Heparin binding screens showed that the A2G78 peptide (GLLFYMARINHA) bound to heparin and prevented its binding to rec-alpha2LG4-5. Furthermore, alanine substitution of the arginine residue in the A2G78 site on rec-alpha2LG4-5 decreased heparin binding activity. When alpha-dystroglycan binding of the peptides was screened, two peptides, A2G78 and A2G80 (VQLRNGFPYFSY), bound alpha-dystroglycan. A2G78 and A2G80 also inhibited alpha-dystroglycan binding of rec-alpha2LG4-5. A2G78 and A2G80 specifically inhibited end bud formation of submandibular glands in culture. These results suggest that the A2G78 and A2G80 sites play functional roles as heparan sulfate- and alpha-dystroglycan-binding sites in the module. These peptides are useful for elucidating molecular mechanisms of heparan sulfate- and/or alpha-dystroglycan-mediated biological functions of the laminin alpha2 chain.

Laminin active peptide-conjugated chitosan mambranes have been previously demonstrated as a useful biomaterial for tissue engineering. Here, three laminin active peptides, A99 (AGTFALRGDNPQG), AG73 (RKRLQVQLSIRT), and EF1zz (ATLQLQEGRLHFXFDLGKGR, X: Nle), which interact with integrin αvβ3, syndecans, and integrin α2β1, respectively, were conjugated to alginate and evaluated the biological activities. A99-alginate (3-3000 ng/mm(2)) promoted cell attachment depending on the amount of alginate. More than 300 ng/mm(2) of the A99-alginate matrices effectively promoted cell attachment, cell spreading with well-organized actin stress fibers, and neurite outgrowth. AG73- and EF1zz-alginates promoted strong cell attachment at the all amounts (3-3000 ng/mm(2)). A99-alginate (30-3000 ng/mm(2)) promoted strong neurite outgrowth but lower amounts of A99-alginate (3 ng/mm(2)) showed weak activity. In contrast, AG73-alginates (3-30 ng/mm(2)) showed strong neurite outgrowth activity but higher amounts of AG73-alginate (300-3000 ng/mm(2)) decreased the activity. These data indicate that neurite outgrowth activity of peptide-alginate matrices is peptide specific and the activity is dependent on the amount of alginate. Further, biological activities of the peptides on alginate and chitosan matrices were different, suggesting that the integrin- and syndecan-mediated cellular functions on the peptide-matrices are highly influenced by the scaffold structure including polysaccharide types and amounts. The laminin active peptide-conjugated alginate and chitosan matrices can control receptor type specific functions and are useful for tissue engineering.

Dermatopontin, an extracellular matrix component initially purified from bovine dermis, promoted cell adhesion of the human epidermal keratinocyte cell line (HaCaT cells). HaCaT cells spread on dermatopontin and formed actin fibers. Adhesion of HaCaT cells to dermatopontin was inhibited by both EDTA and heparin and was mediated in part by alpha3beta1 integrin. A synthetic peptide (DP-4, PHGQVVVAVRS; bovine dermatopontin residues 33-43) specifically inhibited adhesion of cells to dermatopontin, and when the DP-4 peptide was coated on the well, it promoted cell adhesion in a dose-dependent manner. An active core sequence of the DP-4 peptide was localized to an eight-amino acid sequence (GQVVVAVR). These results indicate that dermatopontin is a novel epidermal cell adhesion molecule and suggest that the DP-4 sequence is critical for the cell adhesive activity of dermatopontin. Adhesion of cells to DP-4 was strongly inhibited by heparin. When HaCaT cells were treated with heparitinase I, the cells failed to adhere to DP-4 but chondroitinase ABC treatment did not influence the adhesion activity. DP-4 specifically interacted with biotinylated heparin, and this interaction was inhibited by unlabeled heparin. DP-4 peptide significantly promoted the adhesion of cells overexpressing syndecans, and syndecan bound to a DP-4 peptide affinity column. These results suggest that HaCaT cells adhere to dermatopontin through alpha3beta1 integrin and a heparan sulfate proteoglycan-type receptor, which is likely a syndecan. We conclude that dermatopontin plays a role as a multifunctional adhesion molecule for epidermal cells.

The laminin alpha3 chain is mainly expressed at the skin, and its C-terminal G domain has a critical role in multiple biological functions. We screened for biologically active sites on the mouse laminin alpha3 chain G domain using 107 synthetic peptides on coated plates and conjugated to Sepharose beads with HT1080 human fibrosarcoma cells, HaCaT human skin keratinocyte cells, and human dermal fibroblasts (HDFs). Eleven peptides exhibited cell attachment activity with respect to the peptide-coated plates and/or peptide-Sepharose beads. MA3G28 (WTIQTTVDRGLL) strongly binds to HaCaT cells. Four peptides promoted PC12 cell neurite outgrowth. Heparin inhibited attachment of HDFs to eight peptides on the coated plates. In contrast, EDTA significantly inhibited attachment of HDFs to MA3G27 (NAPFPKLSWTIQ) and MA3G28 but had no effect on the attachment of the other peptides. HDF cells formed well-organized actin stress fibers and focal contacts with vinculin accumulation on MA3G27. Additionally, attachment of HDFs to MA3G27 was inhibited by anti-alpha6 and anti-beta1 integrin antibodies, suggesting that MA3G27 promotes alpha6beta1 integrin-mediated cell adhesion. MA3G57 (NQRLASFSNAQQS) exhibited cell attachment activity only in the peptide bead assay. MA3G57 conjugated to a chitosan membrane promoted HDF attachment and spreading with well-organized actin stress fibers. The anti-beta1 integrin antibody partially inhibited attachment of HDFs to the MA3G57-chitosan membrane, suggesting that the MA3G57 site is involved in beta1 integrin-mediated cell attachment. These active sites are likely important in the biological activities of the laminin alpha3 chain G domain and would be useful for the study of molecular mechanisms of laminin-receptor interactions.

Laminin alpha chains contain five tandem globular modules (LG1-5) at the C-terminus. Here, we focused on the LG45 module, which play a critical biological role via binding to heparin/heparan sulfate, and examined their chain-specific heparin-binding affinity. The relative heparin-binding affinity of recombinant laminin alpha chain LG45 proteins was as follows: alpha5 > alpha4 > alpha1 > alpha2 and alpha3. The alpha5 chain LG45 module also promoted the strongest cell attachment. We screened heparin-binding sequences using the recombinant alpha5LG45 protein and 43 synthetic peptides. Four peptides, A5G71 (GPLPSYLQFVGI) (IC(50) = 91.8 microM), A5G77 (LVLFLNHGHFVA) (IC(50) = 7.0 microM), A5G81 (AGQWHRVSVRWG) (IC(50) = 5.9 microM), and A5G94 (KMPYVSLELEMR) (IC(50) = 0.84 microM), inhibited the heparin-binding of rec-alpha5LG45. Additionally, the same four peptides exhibited dose-dependent heparin-binding activity in a solid-phase assay. We found that the alpha5 chain LG45 module contains four heparin-binding sequences, and this number is higher than that of the other LG45 modules (alpha2 and alpha3, one sequence; alpha1 and alpha4, two sequences). The data suggest that the active sequences identified from the synthetic peptide screening contribute to the heparin-binding activity of the LG45 module. Most of the heparin-binding sequences in the LG45 modules are located in the N-terminal regions of the LG4 module within the loop regions in the proteins. The data suggest that the N-terminal loop regions of the LG4 module are mainly involved in the heparin/heparan sulfate-mediated biological functions.

The Pro-His-Ser-Arg-Asn (PHSRN) sequence in fibronectin is a second cell-binding site that synergistically affects with Arg-Gly-Asp. The PHSRN peptide also induces cell invasion and accelerates wound healing. Here, we examined the sequence specificity of PHSRN on corneal epithelial migration using various synthetic peptides. Elongation and deletion analyses of Ac-PHSRN-NH(2) suggest that the five amino acid length was a minimum and essential sequence for promotion of rabbit corneal epithelial migration ex vivo. Additionally, alanine substituted analysis indicated that the Ser- and Arg-residues are critical for the biological activities. The Ser-Arg motif is involved in various biologically active peptides, suggesting that the unique sequence interacts with cellular receptor(s) and regulates biological functions. Further, the N-acetyl and C-amide of Ac-PHSRN-NH(2) contributed effectively for the chemical stability in tears. The Ac-PHSRN-NH(2) peptide has potential to use as a therapeutic reagent especially for corneal wound healing.

Novel peptide-conjugated chitosan membranes were fabricated and used to deliver keratinocytes to dermal wounds in mice. Three active peptides of 12 or 13 amino acids each, RLVSYNGIIFFLK (A5G27), ASKAIQVFLLAG (A5G33), and AGTFALRGDNPQG (A99) were selected from a cell-adhesive peptide library of laminin, a major constituent of basement membrane. The peptides were synthesized and coupled to chitosan membranes, and the resulting peptide-chitosan membranes were tested for keratinocyte attachment. Two of the peptides that bind to cell surface heparin-like receptors (A5G27 and A5G33) were found to promote strong keratinocyte attachment, whereas the one that binds to integrin (A99) was inactive. Subsequently, A5G27- and A5G33-chitosan membranes were tested as vehicles for keratinocyte delivery in a wound model. We found that keratinocytes were delivered into the full-thickness wound with either membrane. Using the A5G33-chitosan membrane, we further evaluated the activity of the delivered keratinocytes in wound healing. Immunohistochemistry for granulation tissue markers, including tenascin and alpha-smooth muscle actin, showed that keratinocyte delivery by the present peptide-chitosan membranes in the wound bed provided a favorable condition for keratinocyte migration along the wound surface and reduced granulation tissue formation.

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, β1, 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 α3β1 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.

We identified a new extracellular protein, TM14, by differential hybridization using mouse tooth germ cDNA microarrays. TM14 cDNA encodes 440 amino acids containing a signal peptide. The protein contains 3 EGF modules at the center, a C-terminal domain homologous to the fibulin module, and a unique Sushi domain at the N terminus. In situ hybridization revealed that TM14 mRNA was expressed by preodontoblasts and odontoblasts in developing teeth. TM14 mRNA was also expressed in cartilage, hair follicles, and extraembryonic tissues of the placenta. Immunostaining revealed that TM14 was localized at the apical pericellular regions of preodontoblasts. When the dentin matrix was fully formed and dentin mineralization occurred, TM14 was present in the predentin matrix and along the dentinal tubules. We found that the recombinant TM14 protein was glycosylated with N-linked oligosaccharides and interacted with heparin, fibronectin, fibulin-1, and dentin sialophosphoprotein. We also found that TM14 preferentially bound dental mesenchyme cells and odontoblasts but not dental epithelial cells or nondental cells such as HeLa, COS7, or NIH3T3 cells. Heparin, EDTA, and anti-integrin beta1 antibody inhibited TM14 binding to dental mesenchyme cells, suggesting that both a heparan sulfate-containing cell surface receptor and an integrin are involved in TM14 cell binding. Our findings indicate that TM14 is a cell adhesion molecule that interacts with extracellular matrix molecules in teeth and suggest that TM14 plays important roles in both the differentiation and maintenance of odontoblasts as well as in dentin formation. Because of its protein characteristics, TM14 can be classified as a new member of the fibulin family: fibulin-7.

The AG73 peptide (RKRLQVQLSIRT, mouse laminin alpha 1 chain 2719-2730) promotes cell adhesion and tumor metastasis, and interacts with transmembrane syndecan proteoglycans. Here, we demonstrate AG73 peptide angiogenic activity using in vitro, ex vivo, and in vivo models. AG73 induced murine endothelial cell (SVEC4-10) tube formation on Cultrex Basement Membrane Extract (Cultrex BME) and stimulated sprouting of aortic rings. None of the homologous sequences from the laminin alpha2, alpha3, alpha4, or alpha5 chains was as active as AG73 in promoting sprouting formation. AG73 also mediated angiogenesis in the chick chorioallantonic membrane (CAM) assay. Using subcutaneously injected Cultrex BME supplemented with AG73, we observed a large angiogenic response. Furthermore, AG73-conjugated to a chitosan membrane promoted a strong angiogenic response in the CAM assay. These results indicate that the AG73 peptide is a potent syndecan-binding angiogenesis stimulator and may be useful for therapeutic application to treat ischemic injuries.

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, β-alanine, and ε-aminocaproic acid has been examined. Active laminin-derived peptides, including a syndecan-binding peptide (AG73: RKRLQVQLSIRT), an integrin αvβ3-binding peptide (A99a: ALRGDN), and an integrin α6β1-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.

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 β1 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.

The laminin alpha1 chain is a subunit of laminin-1, a heterotrimeric basement membrane protein. The LG4-5 module at the C terminus of laminin alpha1 contains major binding sites for heparin, sulfatide, and alpha-dystroglycan and plays a critical role in early embryonic development. We previously identified active synthetic peptides AG73 and EF-1 from the sequence of laminin alpha1 LG4 for binding to syndecan and integrin alpha2beta1, respectively. However, their activity and functional relationship within the laminin-1 and LG4 as well as the functional relation between these sites and alpha-dystroglycan binding sites in LG4 are not clear. To address these questions, we created mutant recombinant LG4 proteins containing alanine substitutions within the AG73 (M1), EF-1 (M2, M3), and alpha-dystroglycan binding sites (M4, M5) and analyzed their activities. We found that recombinant proteins rec-M1 and rec-M5, containing mutations within M1 and M5, respectively, did not bind heparin or lymphoid cell lines expressing syndecans. These results suggest that LG4 binds to heparin and syndecans through M1 and M5. Rec-M1 and rec-M5 reduced fibroblast attachment, whereas mutant rec-M2 and rec-M3 retained cell attachment activity but did not promote cell spreading. Fibroblast attachment to rec-LG4 was inhibited by heparin but not by integrin antibodies. Spreading of fibroblasts on rec-LG4 was inhibited by anti-integrin alpha2 and beta1 but not by anti-integrin alpha1 and alpha6. These results suggest that the M1 and M5 sites are necessary for cell attachment on LG4 through syndecans and that the EF-1 site is for cell spreading activity through integrin alpha2beta1. In contrast, laminin-1-mediated fibroblast attachment and spreading were not inhibited by heparin or anti-integrin alpha2. Our findings indicate that LG4 has a unique function distinct from laminin-1 and suggest that laminin alpha1 LG4-5 may also be produced by a proteolytic cleavage in certain tissues where it exerts its activity.

It is known that the process of amyloid fibril formation, including amyloid β (Aβ) and islet amyloid polypeptide, is generally associated with diseases, such as Alzheimer’s disease, type II diabetes, and prion diseases. However, many proteins form amyloid-like fibrils but do not associate with amyloidrelated diseases. Amyloid fibrils are elongated, insoluble structures of 7-10 nm in diameter found in extracellular plaques. Structural studies have shown that mature amyloid fibrils involve the alignment of short peptide segments, usually between 6 and 12 residues in length, from many protein monomers. Together, these aligned polypeptide stretches form the characteristic core structure of the amyloid fibril, the cross β sheet, in which β strands run perpendicular to the main axis of the fibril. Amyloid fibrils possess a characteristic UV absorbing spectrum at the peak of 540 nm with Congo red and a characteristic X-ray diffraction pattern. Thus, specific pattern of molecular interactions, rather than nonspecific hydrophobic interactions, lead to ordered structures. Nevertheless, common structural elements responsible for such organized structures have not been identified and the mechanism of amyloid fibril formation is unclear. Previously, we screened the synthetic peptide library derived from the laminin-111 sequence, and identified five amyloidgenic peptides, A119 (LSNIDYILIKAS, mouse laminin α1 chain, 1321-1332), A208 (AASIKVAVSADR, mouse laminin α1 chain, 2097-2108), AG97 (SAKVDAIGLEIV, mouse laminin α1 chain, 2942-2953), B133 (DISTKYFQMSLE, mouse laminin β1 chain, 1367-1378), and B160 (VILQQSAADIAR, mouse laminin β1 chain, 1607-1618) [1]. Laminins, major components of basement membranes, regulate multiple biological functions, including cell adhesion, migration, differentiation, neurite outgrowth, wound healing, angiogenesis, and tumor progression. Laminins are a family of heterotrimeric glycoproteins that consist of α, β, and γ chains. So far, five α, three β, and three γ chains have been identified and at least 16 laminin isoforms have been reported that are formed by various combinations of each chains. Here, we focused on the AG97 and B160 peptide, which exhibits amyloid-like fibril formation, heparin-dependent cell attachment activity, and neurite outgrowth activity. Our approach was to investigate the essential residues for fibril formation and cell behavior using alanine-substituted peptides.

Nucleoplasmin, isolated from the Xenopus laevis egg, was the first protein defined as a molecular chaperone. DNA forms a nucleosome structure with histones in eucaryotic somatic cells. Nucleoplasmin promotes the assembly of the nucleosome structure. We have now determined the effect of nucleoplasmin on the structure of the nuclease digested-nucleohistone (d-NH). The d-NH was prepared from nucleohistone by treatment with micrococcal nuclease. The DNA of the d-NH showed 200 bp ladder bands on an agarose gel electrophoresis. The Circular dichroism spectra of nucleohistone demonstrated an α-helical structure with spectra minima at 208 and 222 nm, while that of the d-NH showed a minimum peak at 228 nm. The turbidity of d-NH was found to be higher than that of nucleohistone, suggesting that d-NH was aggregated. The aggregation was inhibited by an addition of NaCl. Furthermore, guanidine HCl reduced the d-NH aggregation, while urea did not affect the aggregation. These results suggest that hydrogen bonds and/or ionic charges were mainly involved in the aggregation. When nucleoplasmin was added to a solution of d-NH, the mixtures possessed an α-helical conformation. Poly-l-glutamic acid was also found to inhibit the aggregation and to promote the formation of α-helical structures, however, this was not as effective as of nucleoplasmin. The conformational thermal stability of the nucleoplasmin/d-NH mixture was higher than that of d-NH alone. These results indicate that nucleoplasmin inhibits aggregation of d-NH and increases the α-helical structure of the core histones. Nucleoplasmin seems to play a critical role in the nucleosome assembly as a molecular chaperone by inhibiting the aggregation of histone and DNA. These data define the molecular mechanisms of the nucleosome assembly by nucleoplasmin.

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.

Crosstalk of different integrins, which bind to distinct types of extracellular matrix proteins, promotes specific functions. This crosstalk has not been investigated in depth. Previously, we demonstrated that integrin-syndecan crosstalk accelerated cell adhesion. Here, we evaluated the crosstalk of two different integrins using mixed peptide-polysaccharide (chitosan or alginate) matrices. Two different integrin binding peptides, FIB1 (integrin αvβ3), EF1zz (integrin α2β1), and 531 (integrin α3β1), were mixed in various molar ratios (9:1, 4:1, 1:1) and conjugated on a polysaccharide matrix. The mixture of FIB1/EF1zz- and FIB1/531-polysaccharide matrices did not show any difference in human dermal fibroblast (HDF) adhesion against the mono polysaccharide matrices. Interestingly, the EF1zz/531-polysaccharide matrix (molar ratio = 1:4) exhibited significantly decreased cell adhesion, but other EF1zz/531-polysaccharide matrices did not show any difference. When we examined the signal transduction of the EF1zz/531(1:4), Y397 phosphorylation of FAK significantly decreased but Y514 phosphorylation of Src did not exhibit any differences. Further investigation revealed that this suppression was mediated by PI3K signaling through the activation of integrin, and PKA signaling modulated suppression of HDF attachment. These findings suggest that a mixed peptide-polysaccharide matrix using receptor specific ligands can regulate cellular functions through receptor-specific crosstalk and is a useful approach to understand receptor specific crosstalk.

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 β-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 β1 antibodies, whereas cell attachment to peptide G5EF3 was inhibited by only anti-integrin β1 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.

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.

Nucleoplasmin was isolated from Xenopus laevis eggs and purified by an improved method using an open column. Its conformation was investigated spectrophotometrically by UV, CD and fluorescence. It was shown that alpha-helix content of nucleoplasmin was 30-40%, and one of the two tryptophan residues in nucleoplasmin located in the hydrophobic surroundings and the other in the relatively hydrophilic surroundings. The isolated nucleoplasmin was found to decondense sperm nuclei of salmon also, suggesting a possibility of the existence of nucleoplasmin-like protein in fish as well. Collapse of the protamine (salmine)-DNA complex as a simple model for fish sperm nuclei by nucleoplasmin was directly observed by measuring OD320 of aqueous protamine-DNA mixtures. This is a molecular level observation for the removal of protamine from DNA-protamine complex.

We have identified a number of cell-adhesive peptides from laminins, a major component of basement membranes. Cell-adhesive peptides derived from basement membrane proteins are potential candidates for incorporating cell-binding activities into scaffold materials for tissue engineering. Our goal is development of a chemically synthetic basement membrane using laminin-derived cell-adhesive peptides and polymeric materials. In this study, we used hyaluronic acid (HA) as a scaffold material and laminin-derived cell-adhesive peptides, A99 (AGTFALRGDNPQG, binds to integrin αvβ3), AG73 (RKRLQVQLSIRT, binds to syndecans), and an A99/AG73 mixture (molar ratio = 9:1) conjugated to two-dimensional (2D) HA matrices. As a result, it was found that the 2D A99/AG73-HA matrices have strong biological functions, such as cell attachment, cell spreading, and neurite outgrowth, similar to that of basement membrane extract (BME)-coated plates. Next, we developed three-dimensional (3D) peptide-HA matrices using the A99/AG73 mixture. The 3D A99/AG73-HA matrices promoted cell spreading and improved cell viability and collagen gene expression. Further, PC12 neurite extension was observed in the 3D A99/AG73-HA matrices. These biological activities of the 3D A99/AG73-HA matrices were similar to those of the 3D BME matrices. These results suggest that the peptide-HA matrices are useful as 2D and 3D matrices and can be applied for tissue engineering as a synthetic basement membrane.

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 α2β1), A99a (ALRGDN, binding to integrin αvβ3), and A2G10 (SYWYRIEASRTG, binding to integrin α6β1), 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 β1 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 α6β1 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.

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 αvβ3-binding peptide (A99a: ALRGDN, mouse laminin α1 chain 1145-1150) and an integrin α2β1-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.