A synthetic peptide deduced from the sequence in the cross-region of laminin A chain mediates neurite outgrowth, cell attachment and heparin binding.
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ABSTRACT:
SUBMITTER: Tashiro K
PROVIDER: S-EPMC1137192 | biostudies-other | 1994 Aug
REPOSITORIES: biostudies-other
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Project description:We synthesized and characterized several peptides containing the IKLLI sequence in the alpha1 chain of laminin-1. The IKLLI-containing peptides, such as LA4 (CSRNLSEIKLLISRARK), LA5 (EIKLLIS) and LA5L (SEIKLLIS), were found to mediate heparin binding and cell adhesion, while also promoting neurite outgrowth in PC12 cells. Furthermore, peptides LA4 and LA5 also mediated proliferation. However, a scrambled peptide, LA5S (ILEKSLI), did not show any of these activities. Anti-LA4 antibodies inhibited laminin- and LA5-mediated cell adhesion and neurite outgrowth, and anti-(integrin alpha3) and anti-(integrin beta1) antibodies inhibited LA5-mediated cell adhesion and neurite outgrowth. Heparin and heparan sulphate inhibited LA5-mediated heparin binding and PC12 cell adhesion in a dose- dependent manner. The IC50 for inhibition of heparin binding and cell adhesion was observed with 9 microM and 8 microM heparin/heparan sulphate respectively. Furthermore, heparan sulphate proteoglycan also inhibited LA5-mediated PC12 cell adhesion with an IC50 of 100 micrograms/ml. However, chondroitin sulphate (dermatan sulphate) did not inhibit cell adhesion. These data suggest that an IKLLI-containing peptide derived from the laminin alpha1 chain may be an active site of laminin and that its cell adhesion may thus interact with both integrin alpha3beta1 and cell- surface heparan sulphate proteoglycan.
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Project description:Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Several pathogens, including spirochetes, have been shown to express surface proteins that interact with the extracellular matrix (ECM). This adhesin-mediated binding process seems to be a crucial step in the colonization of host tissues. This study examined the interaction of putative leptospiral outer membrane proteins with laminin, collagen type I, collagen type IV, cellular fibronectin, and plasma fibronectin. Six predicted coding sequences selected from the Leptospira interrogans serovar Copenhageni genome were cloned, and proteins were expressed, purified by metal affinity chromatography, and characterized by circular dichroism spectroscopy. Their capacity to mediate attachment to ECM components was evaluated by binding assays. We have identified a leptospiral protein encoded by LIC12906, named Lsa24 (leptospiral surface adhesin; 24 kDa) that binds strongly to laminin. Attachment of Lsa24 to laminin was specific, dose dependent, and saturable. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. Triton X-114-solubilized extract of L. interrogans and phase partitioning showed that Lsa24 was exclusively in the detergent phase, indicating that it is a component of the leptospiral membrane. Moreover, Lsa24 partially inhibited leptospiral adherence to immobilized laminin. This newly identified membrane protein may play a role in mediating adhesion of L. interrogans to the host. To our knowledge, this is the first leptospiral adhesin with laminin-binding properties reported to date.
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Project description:Bidirectional signals mediated by Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, play pivotal roles in the formation of neural networks by induction of both collapse and elongation of neurites. However, the downstream molecular modules to deliver these cues are largely unknown. We report here that the interaction of a Rac1-specific guanine nucleotide-exchanging factor, Tiam1, with ephrin-B1 and EphA2 mediates neurite outgrowth. In cells coexpressing Tiam1 and ephrin-B1, Rac1 is activated by the extracellular stimulation of clustered soluble EphB2 receptors. Similarly, soluble ephrin-A1 activates Rac1 in cells coexpressing Tiam1 and EphA2. Cortical neurons from the E14 mouse embryos and neuroblastoma cells significantly extend neurites when placed on surfaces coated with the extracellular domain of EphB2 or ephrin-A1, which were abolished by the forced expression of the dominant-negative mutant of ephrin-B1 or EphA2. Furthermore, the introduction of a dominant-negative form of Tiam1 also inhibits neurite outgrowth induced by the ephrin-B1 and EphA2 signals. These results indicate that Tiam1 is required for neurite outgrowth induced by both ephrin-B1-mediated reverse signaling and EphA2-mediated forward signaling.
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Project description:Laminin-1, an extracellular matrix molecule, promotes neurite outgrowth through the interaction of integrin and actin. Monosialoganglioside GM1 in the lipid rafts associates with and activates the NGF receptor TrkA, and enhances neurite outgrowth. However, the role of GM1 in laminin-1-induced neurite outgrowth was still unclear. Here, we describe that laminin-1 binds to GM1 through a carbohydrate moiety and a specific conformation of GM1, induces focal formation of large clusters of GM1, and enhances the relocation of TrkA in the membrane of dorsal root ganglion (DRG) and PC12 cells. We found that laminin-1-mediated clustering of GM1 causes the translocation and enrichment of beta1 integrin in lipid rafts--where TrkA colocalizes with beta1 integrin--and the activation of Lyn, Akt and MAPK to promote the outgrowth of neurites. Our results suggest that the binding of laminin-1 to GM1 facilitates the formation of a focal microdomain in the membrane, and enhances signal transduction that promotes neurite outgrowth by linking NGF-TrkA signaling with the laminin-integrin signaling pathways.
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Project description:With no lysine kinase 1 (WNK1) phosphorylates and activates STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress responsive kinase 1 (OSR1) to regulate ion homeostasis in the kidney. Mutations in WNK1 result in dysregulation of the WNK1-SPAK/OSR1 pathway and cause pseudohypoaldosteronism type II (PHAII), a form of hypertension. WNK1 is also involved in the autosomal recessive neuropathy, hereditary sensory and autonomic neuropathy type II (HSANII). Mutations in a neural-specific splice variant of WNK1 (HSN2) cause HSANII. However, the mechanisms underlying HSN2 regulation in neurons and effects of HSN2 mutants remain unclear. Here, we found that HSN2 regulated neurite outgrowth through OSR1 activation and glycogen synthase kinase 3β (GSK3β). Moreover, HSN2-OSR1 and HSN2-GSK3β signalling induced expression of LIM homeobox 8 (Lhx8), which is a key regulator of cholinergic neural function. The HSN2-OSR1/GSK3β-LHX8 pathway is therefore important for neurite outgrowth. Consistently, HSN2 mutants reported in HSANII patients suppressed SPAK and OSR1 activation and LHX8 induction. Interestingly, HSN2 mutants also suppressed neurite outgrowth by preventing interaction of between wild-type HSN2 and GSK3β. These results indicate that HSN2 mutants cause dysregulation of neurite outgrowth via GSK3β in the HSN2 and/or WNK1 pathways.
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Project description:Peptides with cell attachment activity are beneficial component of biomaterials for tissue engineering. Conformational structure is one of the important factors for the biological activities. The EF1 peptide (DYATLQLQEGRLHFMFDLG) derived from laminin promotes cell spreading and cell attachment activity mediated by ?2?1 integrin. Although the sequence of the EF2 peptide (DFATVQLRNGFPYFSYDLG) is homologous sequence to that of EF1, EF2 does not promote cell attachment activity. To determine whether there are structural differences between EF1 and EF2, we performed replica exchange molecular dynamics (REMD) simulations and conventional molecular dynamics (MD) simulations. We found that EF1 and EF2 had ?-sheet structure as a secondary structure around the global minimum. However, EF2 had variety of structures around the global minimum compared with EF1 and has easily escaped from the bottom of free energy. The structural fluctuation of the EF1 is smaller than that of the EF2. The structural variation of EF2 is related to these differences in the structural fluctuation and the number of the hydrogen bonds (H-bonds). From the analysis of H-bonds in the ?-sheet, the number of H-bonds in EF1 is larger than that in EF2 in the time scale of the conventional MD simulation, suggesting that the formation of H-bonds is related to the differences in the structural fluctuation between EF1 and EF2. From the analysis of other non-covalent interactions in the amino acid sequences of EF1 and EF2, EF1 has three pairs of residues with hydrophobic interaction, and EF2 has two pairs. These results indicate that several non-covalent interactions are important for structural stabilization. Consequently, the structure of EF1 is stabilized by H-bonds and pairs of hydrophobic amino acids in the terminals. Hence, we propose that non-covalent interactions around N-terminal and C-terminal of the peptides are crucial for maintaining the ?-sheet structure of the peptides.
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Project description:Mitochondrial superoxide dismutase 2 (SOD2) is a major antioxidant defense enzyme. Here we provide evidence that SOD2 plays critical roles in maintaining calcium homeostasis in newly generated embryonic cerebral cortical neurons, which is essential for normal mitochondrial function and subcellular distribution, and neurite outgrowth. Primary cortical neurons in cultures established from embryonic day 15 SOD2+/+ and SOD2-/- mice appear similar during the first 24 h in culture. During the ensuing two days in culture, SOD2-/- neurons exhibit a profound reduction of neurite outgrowth and their mitochondria become fragmented and accumulate in the cell body. The structural abnormalities of the mitochondria are associated with reduced levels of phosphorylated (S637) dynamin related protein 1 (Drp1), a major mitochondrial fission-regulating protein, whereas mitochondrial fusion regulating proteins (OPA1 and MFN2) are relatively unaffected. Mitochondrial fission and Drp1 dephosphorylation coincide with impaired mitochondrial Ca2+ buffering capacity and an elevation of cytosolic Ca2+ levels. Treatment of SOD2-/- neurons with the Ca2+ chelator BAPTA-AM significantly increases levels of phosphorylated Drp1, reduces mitochondrial fragmentation and enables neurite outgrowth.
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Project description:The laminins are large heterotrimeric glycoproteins with fundamental roles in basement membrane architecture and function. The C-terminus of the laminin alpha chain contains a tandem of five laminin G-like (LG) domains. We report the 2.0 A crystal structure of the laminin alpha2 LG4-LG5 domain pair, which harbours binding sites for heparin and the cell surface receptor alpha-dystroglycan, and is 41% identical to the laminin alpha1 E3 fragment. LG4 and LG5 are arranged in a V-shaped fashion related by a 110 degrees rotation about an axis passing near the domain termini. An extended N-terminal segment is disulfide bonded to LG5 and stabilizes the domain pair. Two calcium ions, one each in LG4 and LG5, are located 65 A apart at the tips of the domains opposite the polypeptide termini. An extensive basic surface region between the calcium sites is proposed to bind alpha-dystroglycan and heparin. The LG4-LG5 structure was used to construct a model of the laminin LG1-LG5 tandem and interpret missense mutations underlying protein S deficiency.
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