Ikuko Okazaki

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 Biologically Active Sequences in the Laminin α4 Chain G Domain

Laminins are a family of trimeric extracellular matrix proteins consisting of α, β, and γ chains. So far five different laminin α chains have been identified. The laminin α4 chain, which is present in laminin-8/9, is expressed in cells of mesenchymal origin, such as endothelial cells and adipocytes. Previously, we identified heparin-binding sites in the C-terminal globular domain (G domain) of the laminin α4 chain. Here we have focused on the biological functions of the laminin α4 chain G domain and screened active sites using a recombinant protein and synthetic peptides. The rec-α4G protein, comprising the entire G domain, promoted cell attachment activity. The cell attachment activity of rec-α4G was completely blocked by heparin and partially inhibited by EDTA. We synthesized 116 overlapping peptides covering the entire G domain and tested their cell attachment activity. Twenty peptides showed cell attachment activity, and 16 bound to heparin. We further tested the effect of the 20 active peptides in competition assays for cell attachment and heparin binding to rec-α4G protein. A4G6 (LAIKNDNLVYVY), A4G20 (DVISLYNFKHIY), A4G82 (TLFLAHGRLVFM), and A4G83 (LVFMFNVGHKKL), which promoted cell attachment and heparin binding, significantly inhibited both cell attachment and heparin binding to rec-α4G. These results suggest that the four active sites are involved in the biological functions of the laminin α4 chain G domain. Furthermore, rec-α4G, A4G6, and A4G20 were found to interact with syndecan-4. These active peptides may be useful for defining of the molecular mechanism laminin-receptor interactions and laminin-mediated cellular signaling pathways.

High and low affinity heparin-binding sites in the G domain of the mouse laminin alpha 4 chain

G domains of the mouse laminin α1 and α4 chains consisting of its five subdomains LG1–LG5 were overexpressed in Chinese hamster ovary cells and purified by heparin chromatography. α1LG1–LG5 and α4LG1–LG5 eluted at NaCl concentrations of 0.30 and 0.47 m, respectively. In solid phase binding assays with immobilized heparin, half-maximal concentrations of 14 (α1LG1–LG5) and 1.4 nm(α4LG1–LG5) were observed. N-Glycan cleavage of α4LG1–LG5 did not affect affinity to heparin. The affinity of α4LG1–LG5 was significantly reduced upon denaturation with 8m urea but could be recovered by removing urea. Chymotrypsin digestion of α4LG1–LG5 yielded high and low heparin affinity fragments containing either the α4LG4–LG5 or α4LG2–LG3 modules, respectively. Trypsin digestion of heparin-bound α4LG1–LG5 yielded a high affinity fragment of about 190 residues corresponding to the α4LG4 module indicating that the high affinity binding site is contained within α4LG4. Competition for heparin binding of synthetic peptides covering the α4LG4 region with complete α4LG1–LG5 suggests that the sequence AHGRL1521 is crucial for high affinity binding. Introduction of mutation of H1518A or R1520A in glutathioneS-transferase fusion protein of the α4LG4 module produced in Escherichia coli markedly reduced heparin binding activity of the wild type. When compared with the known structure of α2LG5, this sequence corresponds to the turn connecting strands E and F of the 14-stranded β-sheet sandwich, which is opposite to the proposed binding sites for calcium ion, α-dystroglycan, and heparan sulfate.

Amino acids and peptides. Part 39: A bivalent poly(ethylene glycol) hybrid containing an active site (RGD) and its synergistic site (PHSRN) of fibronectin

Fibronectin contains the active sequence Arg-Gly-Asp (RGD), along with its synergic site Pro-His-Ser-Arg-Asn (PHSRN). However, the PHSRN peptide does not show synergic activity when it is mixed with the RGD peptide, indicating that a spatial array between RGD and PHSRN in fibronectin may be necessary for synergic activity. Here, we have used an amino acid type poly(ethylene glycol) derivative (aaPEG) to design a bivalent PEG hybrid of fibronectin active peptides. We prepared the aaPEG hybrid peptides PHSRN-aaPEG, aaPEG-RGD, and PHSRN-aaPEG-RGD, and tested their biological activity. Whereas aaPEG-RGD promoted cell spreading activity, PHSRN-aaPEG had no activity. The PHSRN-aaPEG-RGD hybrid strongly promoted cell spreading compared with aaPEG-RGD. These results suggest that the PHSRN sequence in the PHSRN-aaPEG-RGD molecule synergistically enhances the cell spreading activity of the RGD sequence, and that the bivalent aaPEG hybrid method may be useful for conjugating functionally active peptides.

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].