Fumihiko Katagiri

Laminin-111-derived peptides and cancer

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.

Biological activity of laminin peptide‐conjugated alginate and chitosan matrices

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.

Cell adhesive peptide screening of the mouse laminin α1 chain G domain

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.

Identification of biologically active sequences in the laminin α2 chain G domain

Laminin alpha2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin alpha2 chain G domain using 110 soluble peptides by the peptide-coated plate and the peptide-conjugated Sepharose bead assays. Fourteen peptides showed cell attachment activity in either or both assays. Cell attachment to A2G94 (YFDGTGFAKAVG) was inhibited by anti-integrin beta1 antibody, suggesting that the peptide promotes an integrin beta1-mediated cell attachment. Five peptides promoted PC12 cell neurite outgrowth. Since A2G10 (SYWYRIEASRTG) promoted strong cell attachment in the bead assay but showed slight activity in the plate assay, we conjugated A2G10 to chitosan membranes which increase cell attachment activity of the peptides via conformational stability. A2G10-chitosan membrane promoted an integrin alpha6beta1-mediated cell attachment and spreading with well-organized actin stress fibers and neurite outgrowth. These active peptides are useful for evaluating the molecular mechanisms of laminin-receptor interactions.

The Lutheran/Basal Cell Adhesion Molecule Promotes Tumor Cell Migration by Modulating Integrin-mediated Cell Attachment to Laminin-511 Protein

Background: 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. Results: Expression of Lu/B-CAM promotes tumor cell migration on LM-511 rather than cell attachment. Conclusion: Tumor cell migration on LM-511 requires that Lu/B-CAM competitively weakens cell attachment through integrins. Significance: The competitive interaction of the laminin receptors may provide a balance between static and migratory cell behaviors. 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.

Reconstitution of laminin-111 biological activity using multiple peptide coupled to chitosan scaffolds

Laminin-111, a multifunctional matrix protein, has diverse biological functions. Previously, we have identified various biologically active sequences in laminin-111 by a systematic peptide screening. We also demonstrated that peptide-conjugated chitosan matrices enhance the biological functions of the active sequences and are useful as a scaffold. Here, we conjugated sixty biologically active laminin-111 peptides onto chitosan matrices. The twenty-nine peptide-chitosan matrices promoted various biological activities, including cell attachment, spreading, and neurite outgrowth. The biological activities of peptide-chitosan matrices depend on the peptide. These peptide-chitosan matrices are categorized into six groups depending on their biological activities. Next, we conjugated five active peptides, which showed strong cell attachment activity in the each group, onto a single chitosan matrix to mimic the multiple activities of laminin-111. The mixed peptides-chitosan matrix significantly promoted cell attachment and cell spreading over that observed with the individual peptides. We also demonstrated that a mixed peptides-chitosan matrix, using four neurite outgrowth-promoting peptides each from a different group, enhanced the activity. These data suggest that the mixed peptides synergistically induce laminin-like biological activities on a chitosan matrix. The active peptides-chitosan matrices described here have potential for use as biomaterial for tissue engineering and regeneration.

Laminin-111-derived peptide-hyaluronate hydrogels as a synthetic basement membrane.

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.

Maintenance of hepatic differentiation by hepatocyte attachment peptides derived from laminin chains.

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.

Cell behavior on protein matrices containing laminin α1 peptide AG73

Collagen has been widely used for tissue engineering. Here, we applied bioactive laminin-derived peptides as an additive for collagen, laminin-111, and fibronectin matrices resulting in peptide/collagen, peptide/laminin-111, and peptide/fibronectin matrices. Several syndecan-binding peptides, including AG73 (RKRLQVQLSIRT), enhanced the cell attachment activity of collagen matrices. AG73 synergistically enhanced not only cell attachment but also cell spreading on collagen matrices. AG73 also enhanced integrin-binding to the collagen matrices, including organization of actin stress fibers and promotion of Tyr397-focal adhesion kinase (FAK) phosphorylation. Additionally, AG73 enhanced neurite outgrowth on collagen matrices. These results suggest that the integrin-mediated biological activity of collagen matrices is synergistically enhanced by the syndecan-mediated cellular function of AG73. Further, cell attachment and spreading activity of laminin-111 and fibronectin matrices was also synergistically enhanced by AG73. The syndecan-binding peptides are useful to enhance the integrin-mediated biological activities of extracellular matrix (ECM) proteins, such as collagen, laminin-111, and fibronectin. The peptide/matrix mixed method is a new concept for biomaterial fabrication and has the potential for wide use in cell and tissue engineering.

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 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.