Mayumi Mochizuki

Laminin-1 peptide-conjugated chitosan membranes as a novel approach for cell engineering

Laminin, a major component of the basement membrane, has diverse biological activities. Recently, we identified various biologically active sequences on laminin‐1 by using a large set of synthetic peptides. Chitosan, a polysaccharide, is biodegradable and has been used as a biomaterial. Here, we conjugated several biologically active laminin peptides onto chitosan membranes and measured the cell attachment activity of peptide‐conjugated chitosan membranes with various cell types. The active laminin peptide‐conjugated chitosan membranes promoted cell attachment with cell type specificity. A99 (AGTFALRGDNPQG)‐chitosan membrane promoted cell attachment with well‐organized actin stress fibers. This adhesion was inhibited by EDTA but not by heparin. AG73 (RKRLQVQLSIRT)‐chitosan membrane promoted cell attachment with filopodia formation, and this adhesion was inhibited by heparin but not by EDTA. These data suggest that the A99‐chitosan membrane interacted with an integrin cellular receptor and that the AG73‐chitosan membrane promoted proteoglycan‐mediated cell attachment, as previously reported. Furthermore, both AG73‐chitosan and A99‐chitosan membranes effectively promoted neurite outgrowth with PC12 rat pheochromocytoma cells. We conclude that conjugation on a chitosan membrane is applicable for testing quantitatively the biological activity of synthetic peptides and that these constructs have a potential ability to serve as bioadhesive materials for tissue regeneration and engineering.

Identification of an Active Site on the Laminin α5 Chain Globular Domain That Binds to CD44 and Inhibits Malignancy

The laminin α5 chain is a component of laminin-10 (α5β1γ1) and -11 (α5β2γ1). In this study, we have screened 113 overlapping synthetic peptides from the laminin α5 globular domain (G-domain) for cell attachment activity with B16-F10 cells using peptide-coated dishes. Eleven attachment-active peptides were identified. In vivo experimental B16-F10 pulmonary metastasis and primary tumor growth assays found that 4 of the 11 peptides inhibited tumor metastasis and growth and increased apoptosis. These four peptides also blocked tumor cell migration, invasion, and angiogenesis. Two of the peptides were highly homologous and showed significant similarity to sequences in collagens. We sought to identify the B16-F10 cell surface receptors for each of the four active peptides using peptide affinity chromatography. Only one peptide recognized a cell surface protein. Peptide A5G27 (RLVSYNGIIFFLK, residues 2892–2904) bound a diffuse Mr ∼120,000–180,000 band that eluted with 2 m NaCl. Glycosidase digestion of the 2 m eluate yielded protein bands of Mr 90,000 and 60,000 that reacted in Western blot analysis with antibodies to CD44. Immunoprecipitation of the A5G27-bound membrane proteins with various cell surface proteoglycan antibodies confirmed CD44 as the surface receptor for A5G27. Finally, attachment assays to A5G27 in the presence of soluble glycosaminoglycans (GAGs) identified the GAGs of CD44 as the binding sites for A5G27. Our results suggest that A5G27 binds to the CD44 receptor of B16-F10 melanoma cells via the GAGs on CD44 and, thus, inhibits tumor cell migration, invasion, and angiogenesis in a dominant-negative manner.

Ile-Lys-Val-Ala-Val (IKVAV)-containing laminin α1 chain peptides form amyloid-like fibrils

The Ile‐Lys‐Val‐Ala‐Val (IKVAV) sequence derived from laminin‐1 promotes cell adhesion, neurite outgrowth, and tumor growth and metastasis. Here, we examined amyloid formation of an IKVAV‐containing peptide (LAM‐L: AASIKVAVSADR, mouse laminin α1 chain 2097–2108). The LAM‐L peptide was stained with Congo red and exhibited fibrils in electron microscopy with a characteristic cross‐β X‐ray diffraction pattern. Further, infrared spectra of LAM‐L suggested a β‐sheet structure. These results indicate that LAM‐L forms amyloid‐like fibrils. We also examined amyloid‐like fibril formation of LAM‐L analogs. The neurite outgrowth activity of the LAM‐L analogs was closely related to their amyloid‐like fibril formation.

Laminin α5 Chain Metastasis- and Angiogenesis-Inhibiting Peptide Blocks Fibroblast Growth Factor 2 Activity by Binding to the Heparan Sulfate Chains of CD44

Recently, we reported that the laminin alpha5 synthetic peptide A5G27 (RLVSYNGIIFFLK, residues 2,892-2,904) binds to the CD44 receptor of B16-F10 melanoma cells via the glycosaminoglycans on CD44 and inhibits tumor cell migration, invasion, and angiogenesis in a dominant-negative manner. Here, we have identified the potential mechanism of A5G27 activity using WiDr human colorectal carcinoma cells. WiDr cells bound to the laminin A5G27 peptide via the heparin-like and chondroitin sulfate B glycosaminoglycan side chains of CD44. Cell binding to fibroblast growth factor (FGF2) was blocked by laminin peptide A5G27 but not by either a scrambled version of this peptide or by another laminin peptide known to bind cell surface proteoglycans. FGF2 signaling involving tyrosine phosphorylation was also blocked by laminin peptide A5G27 but was not affected by peptide controls. Finally, we have shown that peptide A5G27 directly blocks FGF2 binding to heparin. Peptide A5G27 has sequence homology to a region on FGF2 that binds heparin and the FGF receptor and is important in FGF2 central cavity formation. We conclude that peptide A5G27 inhibits metastasis and angiogenesis by blocking FGF2 binding to the heparan sulfate side chains of CD44 variant 3, thus decreasing FGF2 bioactivity.

Role for laminin-α5 chain LG4 module in epithelial branching morphogenesis

Laminin-alpha5 chain was localized in all epithelial basement membranes (BMs) of mouse submandibular gland (SMG) from the onset of branching morphogenesis and became restricted to BMs of epithelial ducts in the adult. To investigate whether the laminin-alpha5 chain plays a role in branching morphogenesis, a set of cell-adhesive peptides from the C-terminal globular domains (LG1-5) was tested for their effects in SMG organ cultures. One peptide, LVLFLNHGH (A5G77f), which represents a sequence located in the connecting loop between strands E and F of LG4. perturbed branching morphogenesis and resulted in irregularities in the contours of epithelial structures, with formation of deep clefts. The data suggest a role for the laminin-alpha5 LG4 module in the development of the duct system, rather than in the bifurcation of epithelial clusters. The epithelial BM of A5G77f-peptide-treated explants was continuous, which was in contrast to our previous finding of impaired epithelial BM assembly in explants treated with the larninin-alpha1 LG4 module peptide, or with a monoclonal antibody against this domain. A5G77f also perturbed in vitro development of lung and kidney. These results suggest a crucial role for the LG4 module of larninin-alpha5 in epithelial morphogenesis that is distinct from that of the laminin-alpha1 LG4

A novel cell‐adhesive scaffold material for delivering keratinocytes reduces granulation tissue in dermal wounds

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 α‐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.

Peptide-chitosan matrix: a new multifunctional biomaterial.

Introduction Tissue engineering requires biomaterials for delivering transplanted cells to organ sites needing repair/regeneration. Extracellular matrix (ECM) plays a critical role in tissue regeneration by promoting cell adhesion, migration, proliferation, and differentiation. ECM mimetics are of importance for tissue engineering because of their functions as scaffolds for cells. Previously, we developed bioactive lamininderived peptide-conjugated chitosan membranes and demonstrated their celland peptide-type specific functions [1]. We also demonstrated that a most active laminin peptide (AG73: RKRLQVQLSIRT)-conjugated chitosan membrane can deliver cells and is applicable for keratinocyte transferring to wound bed [2]. Here, we conjugated four integrin-binding peptides derived from ECM proteins onto chitosan membranes and examined biological activity. FIB1 (YAVTGRGDSPAS; from fibronectin), EF1zz (ATLQLQEGRLHFXFDLGKGR; from laminin α1 chain), A99 (AGTFALRGDNPQG; from laminin α1 chain) [3], and 531 (GEFYFDLRLKGDKY; from collagen α1 (IV) chain) [4] conjugated chitosan membranes promoted integrin-dependent cell adhesion. Various integrins, including αv, β1, and β3, were involved in the cell adhesion to the peptide-chitosan membranes. Further, only the FIB1and A99-chitosan membranes promoted neurite outgrowth with PC12 rat pheochromocytoma cells. These data demonstrate that integrin binding peptide-chitosan membranes can regulate specific integrin-mediated cell responses.

Cell Attachment and Neurite Outgrowth Activities of Laminin Peptide-Conjugated Chitosan Membrane

Basement membranes have been found to play a critical role in tissue development and repair. Laminin, a major cell adhesive protein of the basement membrane matrix, has multiple biological activities [1]. There are at least twelve isoforms of laminin (laminin-1 to-12), each consisting of three different chains α, β, and γ [1]. The most extensively characterized laminin, laminin-1 (Mr = 900,000), consists of α1, β1, and yl chains, which assemble into a triple-stranded coiled-coil structure at the long arm to form a cross-like structure [1], Laminin-1 has multiple biological activities including promotion of cell adhesion, spreading, proliferation, neurite outgrowth, angiogenesis, and tumor metastasis [1]. Recently, we demonstrated a systematic peptide screening for identification of cell binding sites from the laminin-1 molecule using 673 overlapping peptides [2–5]. Approximately twenty different cell-binding sequences with various biological functions were identified. Five peptides (A13, A99, A208, AG73, and CI6) showed unusually strong cell attachment activity as well as additional biological functions (Figure 1). Al3 and C16 inhibited laminin-mediated endothelial cell tube formation and promoted aortic sprouting and tumor metastasis suggesting it as a potent angiogenic sequence [6,7]. A99, located on the short arm of the ocl chain and containing the RGD sequence [8], was found to interact with integrins and to promote cell adhesion and migration [9]. A208, on the C-terminus of the coiled-coil region of the laminin ocl chain and containing the IKVAV sequence, promoted cell adhesion, neurite outgrowth, angiogenesis, and tumor metastasis [10].