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.

Suppression of cell adhesion through specific integrin crosstalk on mixed peptide-polysaccharide matrices.

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 αvß3), EF1zz (integrin α2ß1), and 531 (integrin α3ß1), 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.

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.

Cell surface receptor-specific scaffold requirements for adhesion to laminin-derived peptide-chitosan membranes.

Scaffolds are used for bioengineering to regulate cellular functions. Previously, we developed laminin-derived peptide-conjugated chitosan membranes for cell engineering. Here, we determined whether changes in the chitosan scaffold altered the cellular response. When an alphavbeta3 integrin-binding peptide A99a (ALRGDN) was conjugated on chitosan membranes of varying density (1.5-1500 ng/mm(2)), cell adhesion was altered depending on the amount of chitosan. 3 or 30 ng/mm(2) of the A99a-chitosan membrane effectively promoted cell attachment, cell spreading with well-organized actin stress fibers, phosphorylation of FAK Tyr397, and neurite outgrowth. In contrast, syndecan-binding peptide AG73 (RKRLQVQLSIRT) conjugated chitosan membranes density (1.5-1500 ng/mm(2)) promoted similar biological activities at all of the concentrations tested. These results suggest that integrin-mediated cell adhesion is sensitive to the scaffold condition. To improve the function of integrin-mediated biological activities on a large amount of scaffold, we designed an A99a/AG73 mixed peptide-chitosan membrane. The mixed peptide-chitosan membrane promoted the strongest biological activities at 150-1500 ng/mm(2) of chitosan membrane. We conclude that the A99a/AG73 mixed peptide-chitosan membrane effectively interacts with both integrins and syndecans and is a useful multi-functional biomaterial.

Biologically active sequences in the mouse laminin alpha3 chain G domain.

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.

Mixed peptide-chitosan membranes to mimic the biological activities of a multifunctional laminin alpha1 chain LG4 module.

Laminin alpha1 chain LG4 module is multifunctional and interacts with syndecans and integrin alpha2beta1 via AG73 (RKRLQVQLSIRT) and EF-I (DYATLQLQEGRLHFMFDLG) sites, respectively. Here, we conjugated the AG73 and EF1zz (ATLQLQEGRLHFXFDLGKGR, X: Nle) peptides on a chitosan membrane in various ratios to develop an LG4 mimic biomaterial. The AG73-chitosan membrane promoted strong cell attachment with membrane ruffling and the EF1zz-chitosan membrane promoted integrin-mediated cell adhesion with well-organized actin stress fibers. When AG73 and EF1zz were conjugated on a chitosan membrane with 1:9 molar ratio, the mixed peptide-chitosan membrane promoted the strong cell attachment and neurite outgrowth similar to that on the recombinant LG4 protein. Well-organized actin stress fibers and vinculin accumulated focal contacts were observed in the cells attached on the AG73:EF1zz (molar ratio=1:9)-chitosan membrane. These results suggest that the mixed peptide-chitosan membrane interacts with both syndecans and integrin alpha2beta1 and mimics the cell adhesion of a multifunctional LG4 protein. The mixed peptide-chitosan approach has potential as a multifunctional biomaterial for cell and tissue engineering.