Embryonic heart mesenchymal cell migration in laminin

Laminin, a large glycoprotein and major component of basement membranes, influences cell adhesion, migration, morphology, and differentiation. A peptide sequence, YIGSR, from the B1 chain of laminin has been found to correspond to an active site for cell adhesion. We report here that cardiac mesenchymal cells migrate vigorously within three-dimensional gels of laminin and that the YIGSR peptide will completely abolish this migratory activity. In contrast, migration of the mesenchymal cells into three-dimensional gels composed of collagen or collagen + laminin is not effected by YIGSR or other peptides (GRGDS, GRGDTP) reported to mediate cellular adhesion.     

Synthetic pentapeptide from the B1 chain of laminin promotes B16F10 melanoma cell migration

Laminin is a basement membrane-specific glycoprotein that promotes cell adhesion, proliferation, differentiation, and tumor cell migration. Synthetic peptides from the amino acid sequence deduced from a cDNA clone of the B1 chain of laminin were tested for their ability to promote the migration of B16F10 melanoma cells. A peptide, CDPGYIGSR, that is able to mediate epithelial cell attachment to laminin was found to promote migration, and the constituent pentapeptide YIGSR was also active but to a lesser degree. This nine-amino acid peptide blocked migration of melanoma cells to laminin but had no effect on migration to fibronectin. These data suggest that the cell-binding site and migration site on laminin share a common sequence that is unique to laminin.     

Towards understanding structure-stability and surface properties of laminin peptide YIGSR and mutants

Properties of laminin peptide YIGSR and its mutated sequences YIGSD, YIGSS, YIGSN and YIGSQ have been investigated using molecular dynamics simulations (MDS) and Langmuir films at air/water interface. Simulation studies on laminin peptide YIGSR were performed in the isothermal-isobaric (N, P, T) ensemble, with run up to 5 ns in water as well as lipid environment at 298 K. From different initial configurations, shape transformations of the peptides on the timescale of nanoseconds were observed. The results showed YIGSR to be the most stable peptide with the order of minimized energy being YIGSR相似文献   

Identification of a second active site in laminin for promotion of cell adhesion and migration and inhibition of in vivo melanoma lung colonization

Previously we reported that a pentapeptide (Tyr-Ile-Gly-Ser-Arg or YIGSR) from domain III of the B1 chain of laminin is a cell attachment site with the ability to stimulate cell adhesion and migration and to block experimental metastases. Here we report studies on the activities of synthetic peptides that cover domain III and report a second biologically active peptide PDSGR from this domain with activities similar to YIGSR. We also show that cyclic YIGSR is more potent in these assays than the linear peptide as expected since this sequence on laminin is bracketed by cysteines. Due to their proximity and similar spectrum of activities, it is possible that these sequences act in concert in the native laminin molecule.     

A CHARMM analysis of the conformations of the metastasis-inhibiting laminin pentapeptide

The metastatic invasion of basement membrane by tumor cells involves the binding of tumor cells to laminin. Laminin, a glycoprotein, is a major component of basement membrane. Both tumor and normal cells express a high-affinity receptor for laminin; however, the expression is more pronounced with tumor cells. The pentapeptide, Tyr-Ile-Gly-Ser-Arg, (YIGSR), an amino acid sequence from the B1 chain of laminin, was found to compete with laminin for binding to the laminin receptor. The binding of tumor cells to laminin can be inhibited competitively by YIGSR and, in mice, this has been shown to be translated into a reduction in metastasis. Reports of structural modifications of YIGSR leading to molecules with enhanced activity led us to attempt to learn more about the secondary structure of YIGSR. Through the use of CHARMM, a molecular mechanics program, we were able to discover a conformation of N-acetyl-YIGSR-NHCH₃ that is stable over a wide range of dielectric constants. In this conformation the arginine side chain acts to hold Tyr, Ile, and Gly in a partial right-handed alpha helix. We speculate that this partial alpha helical structure is necessary for binding to the lamin receptor and thereby its antimetastatic activity.     

RGD and YIGSR synthetic peptides facilitate cellular adhesion identical to that of laminin and fibronectin but alter the physiology of neonatal cardiac myocytes

In the mammalian heart, the extracellular matrix plays an important role in regulating cell behavior and adaptation to mechanical stress. In cell culture, a significant number of cells detach in response to mechanical stimulation, limiting the scope of such studies. We describe a method to adhere the synthetic peptides RGD (fibronectin) and YIGSR (laminin) onto silicone for culturing primary cardiac cells and studying responses to mechanical stimulation. We first examined cardiac cells on stationary surfaces and observed the same degree of cellular adhesion to the synthetic peptides as their respective native proteins. However, the number of striated myocytes on the peptide surfaces was significantly reduced. Focal adhesion kinase (FAK) protein was reduced by 50% in cardiac cells cultured on YIGSR peptide compared with laminin, even though ₁-integrin was unchanged. Connexin43 phosphorylation increased in cells adhered to RGD and YIGSR peptides. We then subjected the cardiac cells to cyclic strain at 20% maximum strain (1 Hz) for 48 h. After this period, cell attachment on laminin was reduced to 50% compared with the unstretched condition. However, in cells cultured on the synthetic peptides, there was no significant difference in cell adherence after stretch. On YIGSR peptide, myosin protein was decreased by 50% after mechanical stimulation. However, total myosin was unchanged in cells stretched on laminin. These results suggest that RGD and YIGSR peptides promote the same degree of cellular adhesion as their native proteins; however, they are unable to promote the signaling required for normal FAK expression and complete sarcomere formation in cardiac myocytes. cell adhesion; connexin43; focal adhesion kinase; surface chemistry     

Antibody to integrin alpha 6 subunit specifically inhibits cell-binding to laminin fragment 8

A large number of cell lines which attach and spread on laminin show a comparable binding either to both laminin fragments P1 and E8 or exclusively to E8. Adhesion to fragment E8 was with one exception completely inhibited by a monoclonal antibody to the alpha 6 integrin subunit, indicating that VLA-6 or a related structure is the major cellular receptor for laminin. It is not involved in fragment P1 adhesion. Synthetic peptides possessing RGD or YIGSR sequences were without inhibitory activity for alpha 6-mediated adhesion to fragment E8.     

High affinity binding between laminin and laminin binding protein of Leishmania is stimulated by zinc and may involve laminin zinc-finger like sequences.

In the course of trying to understand the pathogenesis of leishmaniasis in relation to extracellular matrix (ECM) elements, laminin, a major ECM protein, has been found to bind saturably and with high affinity to a 67-kDa cell surface protein of Leishmania donovani. This interaction involves a single class of binding sites, which are ionic in nature, conformation-dependent and possibly involves sulfhydryls. Binding activity was significantly enhanced by Zn2+, an effect possibly mediated through Cys-rich zinc finger-like sequences on laminin. Inhibition studies with monoclonals against polypeptide chains and specific peptides with adhesive properties revealed that the binding site was localized in one of the nested zinc finger consensus sequences of B1 chain containing the specific pentapeptide sequence, YIGSR. Furthermore, incubation of L. donovani promastigotes with C(YIGSR)3-NH2 peptide amide or antibody directed against the 67-kDa laminin-binding protein (LBP) induced tyrosine phosphorylation of proteins with a molecular mass ranging from 115 to 130 kDa. These studies suggest a role for LBP in the interaction of parasites with ECM elements, which may mediate one or more downstream signalling events necessary for establishment of infection.     

Gastrulation in the Sea Urchin,Strongylocentrotus purpuratus,Is Disrupted by the Small Laminin Peptides YIGSR and IKVAV

Laminin is present on the apical and basolateral sides of epithelial cells of very early sea urchin blastulae. We investigated whether small laminin-peptides, known to have cell binding activities, alter the development of sea urchin embryos. The peptide YIGSR-NH₂ (850 μM) and the peptide PA22-2 (5 μM), which contains the peptide sequence IKVAV (Tashiro et al., J. Biol. Chem. 264, 16174, 1989), typically blocked archenteron formation when added to the sea water soon after fertilization. At lower doses, the YIGSR peptide allowed invagination of the archenteron but blocked archenteron extension and differentiation and evagination of the feeding arms. The effect of YIGSR and PA22-2 peptides declined when added to progressively older stages until no effect was seen when added at the mesenchyme blastula stage (24 hours after fertilization). Control peptides GRGDS, YIGSE, and SHA22, a dodeca-peptide with a scrambled IKVAV sequence, had no effect on development. The YIGSK peptide containing a conserved amino acid modification had only a small effect on gastrulation. The results suggest that YIGSR and IKVAV peptides specifically disrupt cell/extracellular matrix interactions required for normal development of the archenteron and feeding arms. Our recent finding that YTGIR is at the cell binding site of the B1 chain of S. purpuratus laminin supports this conclusion. Evidently, laminin or other laminin-like molecules are among the many extracellular matrix components needed for the invagination and extension of the archenteron during the gastrulation movements of these embryos.     

The RGD containing site of the mouse laminin A chain is active for cell attachment, spreading, migration and neurite outgrowth

The laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD-containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA-6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.     

Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta 1 integrin subunit

The large carboxy-terminal globular domain (G domain; residues 2,110-3,060) of the A chain of murine-derived laminin has been shown to promote heparin binding, cell adhesion, and neurite outgrowth. This study was conducted to define the potential sequence(s) originating from the G domain of laminin with any of these functional activities. A series of peptides were synthesized from the G domain, termed GD peptides, each approximately 20 amino acids long and containing multiple positively charged amino acids. In direct 3H-heparin binding assays, peptides GD-1 and GD-2 bound high levels of 3H-heparin, while peptides GD-3 and GD-4 bound lower levels of 3H-heparin, and GD-5 bound essentially no 3H-heparin. The binding of 3H-heparin to peptides GD-1 and GD-2 appeared to be of high affinity, since significant binding of 3H-heparin to these two peptides was still observed even when the NaCl concentration was raised to 1.0 M. Four of the peptides, GD-1, GD-2, GD-3, and GD-4, directly promoted the adhesion and spreading of HT-1080 human fibrosarcoma cells as well as the outgrowth of neurites from chick spinal cord and dorsal root ganglia neurons. In addition, solutions of these peptides or antibodies generated against these peptides inhibited laminin-mediated HT-1080 cell adhesion. Antibodies against the beta 1 integrin subunit inhibited HT-1080 cell adhesion and neurite outgrowth on surfaces adsorbed with peptides GD-3 and GD-4. Therefore, laminin appears to have multiple, independent sequences in the G domain that serve a similar cell adhesion promoting function for different cell types. Furthermore, these results suggest that the sequences comprising peptides GD-3 and GD-4 use an integrin as a receptor, of which the beta 1 integrin subunit is a component for these various cell types.     

The Presence of Arginine in the Pro-Arg-Pro Motif Augments the Lethality of Proline Rich Antimicrobial Peptides of Insect Source

Antimicrobial peptides are emerging as alternate drug candidates over couple of decades. The diversity exists in amino acid sequence, conformation and mechanism of action of these peptides. Cationic antimicrobial peptides constitute major class which is further classified depending on abundance of amino acid. Insect originated cationic proline rich antimicrobial peptides do not destabilize bacterial cell membrane but have intracellular targets. Some of the peptides belonging to proline rich class such as Apidaecins and Drosocin have unique tripeptide, Pro-Arg-Pro motif which is absent in Formaecin I of same group. Earlier we have designed a non-glycosylated analog of Formaecin I which contains a Pro-Lys-Pro motif. In this report, we have shown that substitution of lysine to arginine in Pro-Lys-Pro motif increases lethal action of this peptide against all the tested bacterial strains without affecting its structural, cytotoxic and membrane permeabilization properties. Importance of arginine in Pro-Arg-Pro motif is reemphasized when substitution of arginine to lysine in Pro-Arg-Pro, tripeptide sequence of Apidaecin and Drosocin results into decrease in their activity. Maintaining overall charge, this substitution indicates the role of arginine beyond providing cationicity.     

Bioconjugation of laminin peptide YIGSR with poly(styrene co-maleic acid) increases its antimetastatic effect on lung metastasis of B16-BL6 melanoma cells

A comb-shaped polymeric modifier, SMA [poly(styrene comaleic anhydride)], which binds to plasma albumin in blood was used to modify the synthetic cell-adhesive laminin peptide YIGSR, and its inhibitory effect on experimental lung metastasis of B16-BL6 melanoma cells was examined. YIGSR was chemically conjugated with SMA via formation of an amide bond between the N-terminal amino group of YIGSR and the carboxyl anhydride of SMA. The antimetastatic effect of SMA-conjugated YIGSR was approximately 50-fold greater than that of native YIGSR. When injected intravenously, SMA-YIGSR showed a 10-fold longer plasma half-life than native YIGSR in vivo. In addition, SMA-YIGSR had the same binding affinity to plasma albumin as SMA, while native YIGSR did not bind to albumin. These findings suggested that the enhanced antimetastatic effect of SMA-YIGSR may be due to its prolonged plasma half-life by binding to plasma albumin, and that bioconjugation of in vivo unstable peptides with SMA may facilitate their therapeutic use.     

The minimal essential sequence for a major cell type-specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine-aspartic acid-valine

Fibronectin contains at least two major domains that support cell adhesion. One is the central cell-binding domain that is recognized by a variety of cell types via the integrin alpha 5 beta 1. The second, originally identified by its ability to support melanoma cell adhesion, is located in the alternatively spliced type III connecting segment (IIICS). A dominant cell type-specific adhesion site within the IIICS has been localized to a peptide designated as CS1 comprising its amino-terminal 25 residues. The receptor for CS1 is the integrin alpha 4 beta 1. We have synthesized a variety of peptides with overlapping sequences in order to identify the minimum active amino acid sequence of this major cell adhesion site. A peptide comprising the carboxyl-terminal 8 amino acids of CS1, EILDVPST, was found to support melanoma cell spreading, while all peptides without this sequence had little or no activity. Two smaller overlapping pentapeptides, EILDV and LDVPS, were also active, whereas EILEV, containing a conservative substitution of Glu for Asp, was inactive. These data suggested that the minimum sequence for cell adhesion activity is Leu-Asp-Val, the tripeptide sequence common to both active peptides. This prediction was confirmed by the observed ability of the Leu-Asp-Val peptide itself to block spreading on fibronectin, whereas Leu-Glu-Val was inactive. Interspecies amino acid sequence comparison also supports the importance of the LDV sequence, since it is completely conserved in the IIICS regions of human, rat, bovine, and avian fibronectins.     

肝癌细胞-胞外基质粘附性与粘附识别序列的相关性

以微管吸吮技术研究了人肝癌细胞在ＩＶ型胶原／层粘连蛋白（ＬＮ）／纤维连结蛋白（ＦＮ）裱衬表面的粘附性。进一步,用四种人工合成肽精－甘－天冬－丝（ＲＧＤＳ）、甘－精－甘－天冬－苏－脯ＧＲＧＤＴＰ）、酪－异亮－甘－丝－精（ＹＩＧＳＲ０和半胱－天冬－脯－甘－酪－异亮－甘－丝－精（ＣＤＰＧＹＩＧＳＲ）研究了肝癌细胞粘附性对两种粘附识别序列ＲＧＤ和ＹＩＧＳＲ的依赖性。为了归纳和整理实验结果,根据竞争性抑制的     

Hepatocyte attachment to laminin is mediated through multiple receptors

The interaction of hepatocytes with the basement membrane glycoprotein laminin was studied using synthetic peptides derived from laminin sequences. Rat hepatocytes bind to laminin and three different sites within the A and B1 chains of laminin were identified. Active laminin peptides include the PA22-2 peptide (close to the carboxyl end of the long arm in the A chain), the RGD-containing peptide, PA21 (in the short arm of the A chain) and the pentapeptide YIGSR (in the short arm of the B1 chain). PA22-2 was the most potent peptide, whereas the other two peptides had somewhat lower activity. Furthermore, hepatocyte attachment to laminin was inhibited by the three peptides, with PA22-2 being the most active. Various proteins from isolated membranes of cell-surface iodinated hepatocytes bound to a laminin affinity column including three immunologically related binding proteins : Mr = 67,000, 45,000, and 32,000. Several proteins--Mr = 80,000, 55,000, and 38,000-36,000--with a lower affinity for laminin were also identified. Affinity chromatography on peptide columns revealed that the PA22-2 peptide specifically bound the Mr = 80,000, 67,000, 45,000, and 32,000 proteins, the PA21 peptide bound the Mr = 45,000 and 38,000-36,000 proteins and the YIGSR peptide column bound the 38,000-36,000 protein. Antisera to a bacterial fusion protein of the 32-kD laminin-binding protein (LBP-32) reacted strongly with the three laminin-binding proteins, Mr = 67,000, 45,000, and 32,000, showing that they are immunologically related. Immunoperoxidase microscopy studies confirmed that these proteins are present within the plasma membrane of the hepatocyte. The antisera inhibited the adhesion of hepatocytes to hepatocytes to laminin by 30%, supporting the finding that these receptors and others mediate the attachment of hepatocytes to several regions of laminin.     

Increasing the activity of cell adherent cyclic NGR peptides by optimizing the peptide length and amino acid character

Cyclic peptides are an attractive modality for the development of therapeutics and the identification of functional cyclic peptides that contribute to novel drug development. The peptide array is one of the optimization methods for peptide sequences and also useful to understand sequence–function relationship of peptides. Cell adherent cyclic NGR peptide which selectively binds to the aminopeptidase N (APN or CD13) is known as an attractive tumor marker. In this study, we designed and screened a library of different length and an amino acid substitution library to identify stronger cell adhesion peptides and to reveal that the factor of higher binding between CD13 and optimized cyclic peptides. Additionally, we designed and evaluated 192 peptide libraries using eight representative amino acids to reduce the size of the library. Through these optimization steps of cyclic peptides, we identified 23 peptides that showed significantly higher cell adhesion activity than cKCNGRC, which was previously reported as a cell adhesion cyclic peptide. Among them, cCRHNGRARC showed the highest activity, that is, 1.65 times higher activity than cKCNGRC. An analysis of sequence and functional data showed that the rules which show higher cell adhesion activity for the three basic cyclic peptides (cCX₁HNGRHX₂C, cCX₁HNGRAX₂C, and cCX₁ANGRHX₂C) are related with the position of His residues and cationic amino acids.     

Conformational studies of antimetastatic laminin-1 derived peptides in different solvent systems, using solution NMR spectroscopy.

Due to its critical role in cancer progression, interactions between laminin-1 and the 67 kDa Laminin-Binding Protein (the 67 kDa LBP) have been the focus of a number of structural and biological studies. As laminin-1 is such a large and complex molecule, research interests have turned to the investigation of bioactive peptides derived from binding domains of laminin-1. Two peptides of interest, CDPGYIGSR (peptide 11) and YIGSR, both derived from the beta1 chain of laminin-1, have been shown to block invasion of basement membranes by tumor cells. Substituting the C-terminal arginine to lysine, a conservative substitution, results in a loss of peptide antimetastatic activity. This difference in bioactivity has been attributed, based on numerous modeling studies of free peptide conformations, to structural differences between YIGSR and YIGSK. Yet the nature of the 'active' free peptide backbone conformation has been a matter of debate and controversy. In order to test the validity of the structural modeling claims, we have undertaken detailed conformational studies of the two laminin-1 derived peptides YIGSR and CDPGYIGSR along with the biologically inactive YIGSK analog by two-dimensional solution 1H NMR spectroscopy in three different solvent systems. Herein we report that although both the active (YIGSR, CDPGYIGSR) and the inactive (YIGSK) peptides can adopt several closely related conformations in solution, the two peptides share similar conformational preferences, and there are no significant structural differences between the active and inactive peptides, contrary to previously reported modeling data. We conclude that the basis of the peptide biological activity, in contrast to published models, cannot be attributed to well-defined structural preferences of the free peptides. We infer that the difference in bioactivity observed between YIGSR and YIGSK originates primarily from the chemical nature of the arginine versus lysine sidechain substitution, rather than being due to a structural change in the free peptide conformations.     

Identification of metastasis-promoting sequences in the mouse laminin alpha 1 chain.

Laminin-1, a major basement membrane matrix glycoprotein, enhances adhesion, migration, and metastasis of tumor cells. We have screened 208 overlapping synthetic peptides covering the short and long arms of mouse laminin alpha1 chain for their adhesion activity with B16-F10 mouse melanoma cells. Cell adhesion activity was determined using various amounts of peptides coated on plastic dishes and by measuring cell adhesion on peptide-conjugated Sepharose beads. Nineteen peptides showed B16-F10 cell adhesion activity. Three peptides, designated A-13, -24, and -208, showed the strongest attachment activity in the plate assay, whereas 4 peptides, A-13, -51, -99, and -112, demonstrated the strongest cell adhesion when conjugated to beads. The 19 peptides were tested in vivo for their effect on experimental pulmonary metastasis by B16-F10 cells. Four peptides, A-13, -51, -64, and -119, significantly enhanced metastasis, with A-13 showing the strongest dramatic enhancement. The four metastasis-promoting peptides also stimulated migration of B16-F10 cells in the Boyden chamber assay in vitro with A-13 being the most potent stimulator. In addition, the 4 peptides inhibited laminin-induced cell attachment and migration, which indicates that these four sequences are possible functional B16-F10 cell binding sites in laminin-1. All the four sequences are located on the globular domains of the short arm. Other peptides, including strong adhesion-active peptides, A-24, -99, -112, and a scrambled A-13 peptide, did not stimulate either migration or metastasis. Thus, laminin-1 has multiple active sites in the globular domains of the short arm which promote migration and metastasis of B16-F10 cells.     

Peptide inhibitors of fibronectin, laminin, and other adhesion molecules: unique and shared features

Synthetic peptides can specifically inhibit the function of certain adhesive glycoproteins in vitro and in vivo. We have compared the relative activities of a set of six variant synthetic peptides based on the sequence of fibronectin in terms of their ability to inhibit the interactions of fibroblasts with fibronectin, spreading factor/vitronectin, laminin, and native collagen gels. BHK (baby hamster kidney) and chick embryo fibroblasts spreading on these adhesive molecules displayed distinctive patterns of sensitivity to inhibition by this panel of peptides, which depended on the adhesive molecule rather than the cell type. For fibronectin, Gly-Arg-Gly-Asp-Ser was considerably more active than Arg-Gly-Asp-Ser, whereas these two peptides displayed little difference in activity in inhibiting cell adhesion to spreading factor. For both proteins, the inverted peptide sequence Ser-Asp-Gly-Arg was also moderately active, whereas closely related peptides containing a transposition, a deletion, or a single, conserved amino acid substitution were much less active. For inhibiting interactions with laminin or native type I collagen gels, Gly-Arg-Gly-Asp-Ser was only weakly active, but the inverted peptide Ser-Asp-Gly-Arg unexpectedly continued to display inhibitory activity for both attachment proteins in both cell types. Our results indicate that different adhesive processes depend on distinct peptide recognition events by a cell. However, there may be a possible common denominator among attachment proteins in a moderate sensitivity to Ser-Asp-Gly-Arg. Our study also underscores the importance of examining a full set of peptide analogs when these novel inhibitors are used to characterize biological processes.