Identification of a second active site in laminin for promotion of cell adhesion and migration and inhibition of in vivo melanoma lung colonization (original) (raw)
Related papers
Identification of Cell Adhesive Sequences in the N-terminal Region of the Laminin α2 Chain
Journal of Biological Chemistry, 2012
Background: The N-terminal region of the laminin ␣2 chain promotes laminin assembly and cell adhesion. Results: Screening of 218 peptides revealed that 11 sequences derived from N-terminal laminin ␣2 chain promote cell adhesion. Conclusion: Three amino acids in the N terminus LN domain are critical sites for integrin ␣21 binding. Significance: Eleven active peptides may be useful as tools for the study of laminin-receptor interactions. The laminin ␣2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of ␣2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN؉, and rec-a2N) by both the peptide-or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin ␣2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin 1 and anti-integrin ␣21 antibodies. These results suggest that A2-8 promotes an integrin ␣21-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin ␣21 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN؉ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin ␣21-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin ␣21 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.
Cell Adhesive Sequences in Mouse Laminin β1 Chain
Archives of Biochemistry and Biophysics, 2000
Laminin-1, a major component of the basement membrane, consists of three different chains, ␣1, 1, and ␥1. We sought to identify cell adhesive sequences from the mouse laminin 1 chain by testing HT-1080 fibrosarcoma and B16-F10 melanoma cells for binding to 187 overlapping synthetic peptides which covered the entire chain. Fourteen peptides showed cell adhesive activities with either peptide-conjugated Sepharose beads or peptide-coated plates or both. Additional cells, including neuronal, endothelial, and salivary gland cells, showed biological responses in a cell type-specific manner. B-7, B-133, and B-160 showed the most potent cell attachment. Cell binding on three peptides (B-34, B-133, and B-160) was inhibited by EDTA. Cell adhesion to 11 of the 12 active peptides was inhibited to varying degrees by heparin. Of the 17 active peptides identified in the laminin 1 chain in this and other studies, 8 are clustered on the amino terminal globular domain, suggesting a possible important role in cell binding for this domain that may be multifunctional. These data demonstrate that the laminin 1 chain has multiple active sites for cell adhesion, some of which are cell-type specific.
Identification of Cell Binding Sites in the Laminin α5-Chain G Domain
Experimental Cell Research, 2002
The laminins consist of at least 11 polypeptides (5 ␣-chains, 3 -chains, and 3 ␥-chains) specific to basement membranes. Here we investigate the biological activity associated with the G domain of the newly identified laminin ␣5-chain using 113 overlapping synthetic peptides (positions 2679-3635). Using HT-1080 cells, 21 peptides showed attachment activity either on peptide-coated tissue culture plates or to peptideconjugated Sepharose beads. Heparin inhibited cell attachment to 16 peptides, while ethylenediaminetetraacetic acid exhibited no inhibitory activity. Peptides A5G-27, A5G-65, and A5G-71 showed the strongest cell attachment, with the minimum active core sequences of the peptides being GIIFFL, HQNMGSVN-VSV, and YLQFVG, respectively. Furthermore, these 16 peptides were tested for their ability to stimulate neurite outgrowth in the PC12 cells. A5G-3, A5G-33, A5G-71, A5G-73, A5G-81, and A5G-101 were the only peptides of the 16 that demonstrated the ability to promote neurite outgrowth. These results demonstrate that synthetic peptides with ␣5-chain G domain primary amino acid sequences possess some of the same biological activities attributable to the whole laminin and the ␣5-chain G domain. Therefore, these peptides may be useful in the investigation of lamininreceptor interactions and possibly mechanisms of laminin signal transduction.
Journal of Biological Chemistry
Laminin is a basement membrane glycoprotein which consists of A, B1, and B2 chains. Laminin has diverse biological activities including promoting cell adhesion, migration, differentiation, growth, and neurite extension. Synthetic peptides from the active region of the A chain were prepared and tested for their biological activity. A 19-mer peptide (designated PA22-2), from just above the carboxyl globule on the long arm of the A chain, was found to promote cell adhesion, spreading, migration, and neurite outgrowth. By testing smaller sequences within the 19mer peptide, a constituent pentapeptide, IKVAV (Ile-Lys-Val-Ala-Val), was identified as the active site for cell adhesion and neurite outgrowth. These data suggest that this sequence is one of the principle sites in laminin which regulate cellular behavior. Laminin is a large glycoprotein (Mr = 900,000) found only in basement membranes (1,2). Laminin has a cross-like shape when examined by electron microscopy, with one long arm and three short arms (Fig. 1) (3). Each of the short arms contains at least two globules whereas the long arm terminates with a large globule at the carboxyl end. Current models suggest that a predominant form of laminin contains one A chain (Mr = 440,000), one B1 chain (Mr = 225,000), and one B2 chain (Mr = 205,000). Part of each chain forms a short arm, with the remainder of each chain projecting down the long arm (4-7). Laminin binds to other basement membrane constituents including collagen IV, heparan sulfate proteoglycan, and nidogen/entactin, and it mediates cellular interactions with basal lamina (8-14). Diverse biological activities have been attributed to laminin, including stimulation of epithelial cell growth, migration, and proliferation (15). Laminin also promotes the adhesion of epithelial cells, myoblasts, Schwann cells, and certain tumor cells (15-17). Laminin alters cell morphology, including inducing neurite formation (18-20). In vitro studies using proteolytic digests of laminin suggest that a binding domain is present in a fragment which includes the three short arms * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adoertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Structure-activity study of a laminin α1 chain active peptide segment Ile-Lys-Val-Ala-Val (IKVAV)
FEBS Letters, 1995
The IKVAV sequence, one of the most potent active sites of laminin-1, has been shown to promote cell adhesion, neurite outgrowth, and tumor growth. Here we have determined the structural requirements of the IKVAV sequence for cell attachment and neurite outgrowth using various 12-mer synthetic peptide analogs. All-Land alI-D-IKVAV peptides showed cell attachment and neurite outgrowth activities. In contrast, all-Land all-D-reverse-sequence peptides were not active. Some of the analogs, in which the lysine and isolencine residues of the IKVAV peptide were substituted with different amino acids, promoted cell attachment, but none of the analog peptides showed neurite outgrowth activity comparable to that of the IKVAV peptide. These results suggest that the lysine and isoleucine residues are critical for the biological functions of the IKVAV peptide.
Identification of Cell Binding Sequences in Mouse Laminin γ1 Chain by Systematic Peptide Screening
Journal of Biological Chemistry, 1997
Laminin-1, a major component of basement membranes, consists of three different chains designated ␣1, 1, and ␥1 and has diverse biological functions. We have identified cell binding sites on the mouse laminin ␥1 chain, using systematic screening of 165 overlapping synthetic peptides covering the entire chain. We identified 12 cell binding sequences using HT-1080 human fibrosarcoma and B16-F10 mouse melanoma cells in two independent assays employing peptide-conjugated Sepharose beads and peptide-coated dishes. Four peptides (C-16, C-28, C-64, and C-68) located on the globular domains of the ␥1 chain were the most active and showed dose-dependent cell attachment. Cell attachment to C-68 was inhibited by EDTA and by anti-␣ 2  1 integrin antibodies. Cell attachment to C-16 and C-64 was partially inhibited by EDTA but was not inhibited by anti-integrin antibodies. EDTA and anti-integrin antibodies did not affect cell attachment to C-28. The four peptides were tested in adhesion and differentiation assays with endothelial, neuronal, and human salivary gland cells. C-16 was the most active for all of the cells, whereas the other three peptides showed cell type specificity in their activities. The active core sequences of C-16, C-28, C-64, and C-68 are YVRL, IRVTLN, TTVKYIFR, and SIKIRGTY, respectively. These sequences are highly conserved among the different species and in the laminin ␥2 chain. These results suggest that the specific sequences on the laminin ␥1 chain are biologically active and interact with distinct cell surface receptors. Laminin-1, a major component of the basement membrane matrix, has multiple biological activities including promotion of cell adhesion, spreading, growth, neurite outgrowth, tumor metastasis, and collagenase IV secretion (1-4). There are at least 11 isoforms of laminin, each consisting of three different chains (5). The most extensively characterized laminin, laminin-1 (M r ϭ 900,000) from the mouse Engelbreth-Holm-Swarm tumor consists of ␣1, 1, and ␥1 chains, which assemble into a triple-stranded cross-like structure with a coiled-coil domain at the long arm (6). Several active sequences on laminin-1 have been identified using proteolytic fragments, recombinant proteins and syn
Journal of Biological Chemistry, 1989
Laminin is a basement membrane glycoprotein which consists of A, B1, and B2 chains. Laminin has diverse biological activities including promoting cell adhesion, migration, differentiation, growth, and neurite extension. Synthetic peptides from the active region of the A chain were prepared and tested for their biological activity. A 19-mer peptide (designated PA22-2), from just above the carboxyl globule on the long arm of the A chain, was found to promote cell adhesion, spreading, migration, and neurite outgrowth. By testing smaller sequences within the 19mer peptide, a constituent pentapeptide, IKVAV (Ile-Lys-Val-Ala-Val), was identified as the active site for cell adhesion and neurite outgrowth. These data suggest that this sequence is one of the principle sites in laminin which regulate cellular behavior. Laminin is a large glycoprotein (Mr = 900,000) found only in basement membranes (1,2). Laminin has a cross-like shape when examined by electron microscopy, with one long arm and three short arms (Fig. 1) (3). Each of the short arms contains at least two globules whereas the long arm terminates with a large globule at the carboxyl end. Current models suggest that a predominant form of laminin contains one A chain (Mr = 440,000), one B1 chain (Mr = 225,000), and one B2 chain (Mr = 205,000). Part of each chain forms a short arm, with the remainder of each chain projecting down the long arm (4-7). Laminin binds to other basement membrane constituents including collagen IV, heparan sulfate proteoglycan, and nidogen/entactin, and it mediates cellular interactions with basal lamina (8-14). Diverse biological activities have been attributed to laminin, including stimulation of epithelial cell growth, migration, and proliferation (15). Laminin also promotes the adhesion of epithelial cells, myoblasts, Schwann cells, and certain tumor cells (15-17). Laminin alters cell morphology, including inducing neurite formation (18-20). In vitro studies using proteolytic digests of laminin suggest that a binding domain is present in a fragment which includes the three short arms * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adoertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The Journal of Cell Biology, 1991
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 -20 amino acids long and containing multiple positively charged amino acids . In direct 3 H-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 3 H-heparin, and GD-5 bound essentially no 3H-heparin . The binding of 3 H-heparin to peptides GD-1 and GD-2 appeared to be of high affinity, since significant binding of 3 H-heparin to these two peptides was still observed even when the NaCl concentration was raised to 1 .0 M . Four of the T HE basement membrane glycoprotein laminin isolated from the murine Engelbreth-Holm-Swarm (EHS) tumor consists ofone 400-kD A chain and two B chains, each of ti 200 kD. Laminin promotes the adhesion and spreading of a variety of cells and binds many different types of molecules, such as glycosaminoglycans or proteoglycans . Studies using enzymatic digests of laminin or monoclonal antibodies raised against laminin have defined some of the biologically active regions ofthe 400-kD A chain of murine laminin . The large carboxy-terminal globular domain (G domain) on the long arm of the A chain of laminin is reported to be the major heparin binding domain of laminin . In early studies, enzymatic digests oflaminin were passed over heparin-affinity columns and proteolytic fragments containing the G domain were found to adhere . Later studies using monoclonal antibodies in combination with heparin-affinity chromatography and a solid-phase radioligand binding assay showed that the G domain, also termed This work previously appeared in part in abstract form (
Journal of Cell Biology, 1988
Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of a heparin-binding domain between the inner globule of a lateral short arm and the cross region of laminin. Using the information from the amino acid sequence of the B1 chain of laminin, several peptides were synthesized from areas with a low hydropathy index and a high density of lysines and/or arginines. One of these, peptide F-9 (RYVVLPRPVCFEKGMNYTVR), which is derived from the inner globular domain of the lateral short arm, demonstrated specific binding to heparin. This was tested in direct solid-phase binding assays by coating the peptide either on nitrocellulose or on polystyrene and in indirect competition assays where the peptide was in solution and either laminin or heparin was immobilized on a solid support. The binding of [3H]heparin to peptide F-9 was dramatically reduced when heparin but not other glycosaminoglycans other than heparin (dextran sulfate, dermatan sulfate) were...