Identification of a tumor cell receptor for VGVAPG, an elastin-derived chemotactic peptide

Extracellular matrix proteins and their proteolytic products have been shown to modulate cell motility. We have found that certain tumor cells display a chemotactic response to degradation products of the matrix protein elastin, and to an elastin-derived peptide, VGVAPG. The hexapeptide VGVAPG is a particularly potent chemotaxin for lung-colonizing Lewis lung carcinoma cells (line M27), with 5 nM VGVAPG eliciting maximal chemotactic response when assayed in 48-microwell chemotaxis chambers. Binding of the elastin-derived peptide to M27 cells was studied using a tyrosinated analog (Y-VGVAPG) to allow iodination. Scatchard analysis of [125I]Y-VGVAPG binding to viable M27 tumor cells at both 37 and 4 degrees C indicates the presence of a single class of high affinity binding sites. The dissociation constant obtained from these studies (2.7 X 10(-9) M) is equivalent to the concentration of VGVAPG required for chemotactic activity. The receptor molecule was identified as an Mr 59,000 species by covalent cross-linking of the radiolabeled ligand to the M27 tumor cell surface and subsequent analysis of the cross-linked material by electrophoresis and size-exclusion high performance liquid chromatography. These results suggest that M27 tumor cell chemotaxis to VGVAPG is initiated by high affinity binding of the peptide to a distinct cell surface receptor.     

Membrane-bound protein kinase C modulates receptor affinity and chemotactic responsiveness of Lewis lung carcinoma sublines to an elastin-derived peptide

The M27 and H59 variants of Lewis lung carcinoma differ in their responsiveness to the chemotactic elastin peptide Val-Gly-Val-Ala-Pro-Gly (VGVAPG). M27 cells, selected for metastasis to lung, are highly responsive to a positive gradient of VGVAPG. H59 cells, selected for metastasis to liver, do not migrate in response to VGVAPG. Although both cell types bind radiolabeled VGVAPG, Scatchard analysis of 125I-Tyr-VGVAPG binding reveals that M27 cells bind the chemoattractant with a Kd of 2.7 nM, whereas nonresponsive H59 cells bind the peptide with a Kd of 67 nM. These findings indicate that the failure of H59 cells to migrate in response to VGVAPG may be due to the reduced affinity of their VGVAPG receptors. Both receptor affinity and chemotactic responsiveness to VGVAPG can be modulated in each of these two tumor cell lines by the levels of active membrane-associated protein kinase C. Treatment of nonresponsive H59 cells with 12-O-tetradecanoylphorbol 13-acetate increases the level of membrane-bound protein kinase C activity with a concomitant increase in VGVAPG binding affinity and induction of chemotactic responsiveness to VGVAPG. Treatment of M27 cells with the protein kinase C inhibitor, staurosporine, reduces VGVAPG binding affinity and abrogates the chemotactic response. We conclude that chemotactic responsiveness of M27 and H59 tumor cells is dependent upon high VGVAPG receptor affinity, which is strongly correlated to high levels of membrane-bound protein kinase C activity.     

Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes

Recent studies have demonstrated that tropoelastin and elastin-derived peptides are chemotactic for fibroblasts and monocytes. To identify the chemotactic sites on elastin, we examined the chemotactic activity of Val-Gly-Val-Ala-Pro-Gly (VGVAPG), a repeating peptide in tropoelastin. We observed that VGVAPG was chemotactic for fibroblasts and monocytes, with optimal activity at approximately 10(-8) M, and that the chemotactic activity of VGVAPG was substantial (half or greater) relative to the maximum responses to other chemotactic factors such as platelet-derived growth factor for fibroblasts and formyl-methionyl-leucyl-phenylalanine for monocytes. The possibility that at least part of the chemotactic activity in tropoelastin and elastin peptides is contained in VGVAPG sequences was supported by the following: (a) polyclonal antibody to bovine elastin selectively blocked the fibroblast and monocyte chemotactic activity of both elastin-derived peptides and VGVAPG; (b) monocyte chemotaxis to VGVAPG was selectively blocked by preexposing the cells to elastin peptides; and (c) undifferentiated (nonelastin producing) bovine ligament fibroblasts, capable of chemotaxis to platelet-derived growth factor, did not show chemotactic responsiveness to either VGVAPG or elastin peptides until after matrix-induced differentiation and the onset of elastin synthesis. These studies suggest that small synthetic peptides may be able to reproduce the chemotactic activity associated with elastin-derived peptides and tropoelastin.     

The role of metal ions in chemical evolution: Polymerization of alanine and glycine in a cation-exchanged clay environment

Summary The effect of the exchangeable cation on the condensation of glycine and alanine was investigated using a series of homoionic bentonites. A cycling procedure of drying, warming and wetting was employed. Peptide bond formation was observed, and the effectiveness of metal ions to catalyze the condensation was Cu²⁺ > Ni²⁺ Zn²⁺ > Na⁺. Glycine showed 6% of the monomer incorporated into oligomers with the largest detected being the pentamer. Alanine showed less peptide bond formation (a maximum of 2%) and only the dimer was observed.     

Molecular dynamics of water and monovalent‐ions transportation mechanisms of pentameric sarcolipin

The Sarcolipin (SLN) is a transmembrane protein that can form a self‐assembled pentamer. In this work, the homology modeling and all‐atom molecular dynamic (MD) simulation was performed to study the model of SLN pentamer in POPC (1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) membrane. The potential of mean force (PMF) was calculated for transmembrane transportation of Na⁺, Cl^? and water molecule along the pore channel of penta‐SLN complex. The root mean square deviation (RMSD) of the SLN pentamer in POPC membrane showed that the stabilized SLN protein complex could exist in the membrane and that the Na⁺ and Cl^? could not permeate through the channel when the pore was under the vacuum state, but the water could permeate through from cytoplasm to lumen. Under the aqueous state, our simulation demonstrated that hydrated state of Na⁺ and Cl^? could pass through the channel. The PMF and radii of the pore showed that the channel had a gate at Leu²¹ that is a key hydrophobicity residue in the channel. Our simulations help to clarify and to understand better the SLN pentamer channel that had a hydrophobic gate and could switch Na⁺ and Cl^? ion permeability by hydrated and vacuum states. Proteins 2016; 84:73–81. © 2015 Wiley Periodicals, Inc.     

The crystal structure of XC847 from Xanthomonas campestris: a 3'-5' oligoribonuclease of DnaQ fold family with a novel opposingly shifted helix

The mechanism by which ligand‐activated EGFR induces autophosphorylation via dimerization is not fully understood. Structural studies have revealed an extracellular loop mediated receptor dimerization. We have previously presented experimental data showing the involvement of a positive 13 amino acid peptide (R645–R657; P13⁺) from the intracellular juxtamembrane domain (JM) of EGFR important for intracellular dimerization and autophosphorylation. A model was presented that suggest that P13⁺ interacts with a negative peptide (D979–E991; P13^?) positioned distal to the tyrosine kinase domain in the opposite EGFR monomer. The present work shows additional data strengthening this model. In fact, by analyzing protein sequences of 21 annotated ErbB proteins from 9 vertebrate genomes, we reveal the high conservation of peptides P13⁺ and P13^? with regard to their sequence as well as their position relative to the tyrosine kinase (TK) domain. Moreover in silico structure modeling of these ErbB intracellular domains supports a general electrostatic P13⁺/P13^? interaction, implying that the C‐terminal of one receptor monomer is facing the TK domain of the other monomer in the receptor dimer and vice versa. This model provides new insights into the molecular mechanism of ErbB receptor activation and suggests a new strategy to pharmacologically interfering with ErbB receptor activity. Proteins 2006. © 2005 Wiley‐Liss, Inc.     

Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1

The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T₁ has two disulfide bonds, one of which appears to be necessary for the full enzyme activity. This depolymerase hydrolyzed not only hydrophobic poly(3-hydroxybutyrate) but also water-soluble trimer and larger oligomers of D-(−)-3-hydroxybutyrate, regardless of their solubilities in water. Kinetic analyses with oligomers of various sizes indicated that the substrate cleaving site of the enzyme consisted of four subsites with individual affinities for monomer units of the substrate. Analyses of the hydrolytic products of oligomers, which had labeled D-(−)-3-hydroxybutyrate at the hydroxy terminus, showed that the enzyme cleaved only the second ester linkage from the hydroxy terminus of the trimer and tetramer, and acted as an endo-type hydrolase toward the pentamer and higher oligomers. The enzyme appeared to have a hydrophobic site which interacted with poly(3-hydroxybutyrate) and determined the affinity of the enzyme toward the hydrophobic substrate.     

Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling.

Recently, a new protein containing a disintegrin domain, alternagin-C (Alt-C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt-C an ECD amino acid mogif takes the place of the RGD sequence. Most disintegrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investigated the effects of Alt-C on human neutrophil chemotaxis in vitro and the activation of integrin-mediated pathways. Alt-C showed a potent chemotactic effect for human neutrophils when compared to N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt-C significantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt-C and fMLP. A significant increase of F-actin content was observed in cells treated with Alt-C, showing that the chemotactic activity of Alt-C on neutrophils is driven by actin cytoskeleton dynamic changes. Furthermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular signal-regulated kinase 2 nuclear translocation. The chemotactic activity of Alt-C was partially inhibited by LY294002, a specific phosphatidylinositol 3-kinase inhibitor, and by PD98056, a Map kinase kinase inhibitor. These findings suggest that Alt-C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin-activated pathways and that these effects could be related to the ECD mogif present in disintegrin-like domain.     

The active site of yeast endo-polygalacturonase contains seven subsites

The rate of hydrolysis of oligomers by the endopolygalacturonase of yeast is in the order: heptamer > hexamer > pentamer > tetramer. This suggests that the active site accommodates at least 7 units. Since the heptamer disappears concurrently with the bulk of larger oligomers, the maximum number of units appears to be 7. The release of labelled (unsaturated, or ³H labelled and reduced) end units from larger substrate is interpreted to indicate that the enzyme interacts with 3 saccharide units toward the reducing end from the bond to be broken, and with 4 units toward the non-reducing end. The relative affinities for the enzyme of saccharide units in various positions are unequal, as indicated by the very low relative rate of monomer production from the hydrolysis of hexamer and pentamer, and the apparently unequal probability of two other modes of hexamer hydrolysis [(tetramer + dimer) = 2.5 (trimer + trimer)].     

Liquid-chromatographic analysis of the depolymerization of (1→4)-β-d-mannuronan by an extracellular alginate lyase from a marine bacterium

The extracellular alginate lyase activity from a fermentative marine bacterium isolated from actively growing tissues of Sargassum fluitans has been purified and studied with respect to substrate specificity and mechanism. The enzyme endolytically depolymerizes (1→4)-β-d-mannuronan derived from alginate to oligomeric products possessing 4,5-unsaturated, nonreducing termini. Reversed-phase liquid chromatography has established that early in the reaction the tri-, tetra-, and pentameric oligomers are the predominant species. The pentamer and larger products that at first accumulate in the reaction are later degraded to smaller products. The trimer is the major product late in the reaction, at which time the dimer and tetramer are also present in significant amounts. By incubating purified oligomers with enzyme, the trimer is shown to be completely refractory to further depolymerization and therefore represents a limit product of the reaction catalyzed by this enzyme. The tetramer is slowly converted into trimer and monomer, whereas the pentamer is readily converted into trimer and dimer.     

Identification of a chemotactic epitope in human transforming growth factor-β1 spanning amino acid residues 368–374

TGF-b?1 plays a critical role in inflammatory and repair processes due in part to its ability to provide a potent chemotactic stimulus for inflammatory cells such as neutrophils and monocytes and for fibroblasts which initiate the fibrogenic response. In the present study, we have used synthetic oligopeptides representing the amino acid sequence of the 12.1 kDa monomer of human TGF-b?1 in an effort to identify a chemotactic epitope on the molecule. A seven residue peptide containing residues 368-374, Val Tyr Tyr Val Gly Arg Lys, was demonstrated to be capable of inducing chemotactic migration of human peripheral blood neutrophils, monocytes, monocyte leukemia cell line THP-1, and infant foreskin fibroblasts. Furthermore, larger peptides from the carboxy-terminal portion of TGF-b?1 that contained residues 368–374 also induced migration of these cell types. None of the peptides representing the complete amino acid of TGF-b?1 monomer were able to compete with [¹²⁵I]hrTGF-b?1 for binding to TGF-b? cell surface receptors or fibroblasts or THP-1 cells. Implications of these observations are discussed. © 1995 Wiley-Liss, Inc.     

Induction of deflagellation by various local anesthetics in Chlamydomonas reinhardtii Dangeard (Chlamydomonadales,Chlorophyceae)

Dibucaine, a local anesthetic, is known to induce flagellar excision in Chlamydomonas reinhardtii. Herein, we investigate whether other local anesthetics have similar effects. Tetracaine, bupivacaine, procaine, and lidocaine also caused flagellar excision, although their potencies were lower than that of dibucaine. Bupivacaine, procaine, and lidocaine induced a morphological change in flagella from a rod‐like shape to a disk‐like shape before flagellar excision. Except for lidocaine, these local anesthetics caused cell‐wall shedding in addition to flagellar excision. The anesthetics in order of their median effective concentration (1‐h EC₅₀) for flagellar excision are as follows: dibucaine (1.37 × 10^?5 M) < tetracaine (3.16 × 10^?5 M) < bupivacaine (4.25 × 10^?4 M) < procaine (2.02 × 10^?3 M) < lidocaine (3.61 × 10^?3 M). In all cases, Ca²⁺ depletion from the solution inhibited flagellar excision. However, Ca²⁺‐channel blockers, IP3 receptor antagonists, and inhibitors of phospholipase C did not prevent excision. We suggest that the local anesthetics induce flagellar excision by increasing the fluidity of the flagellar/cell membrane, thereby allowing extracellular Ca²⁺ to flow into the cell and cause flagellar excision.     

Cholera toxin B subunits assemble into pentamers--proposition of a fly-casting mechanism

The cholera toxin B pentamer (CtxB(5)), which belongs to the AB(5) toxin family, is used as a model study for protein assembly. The effect of the pH on the reassembly of the toxin was investigated using immunochemical, electrophoretic and spectroscopic methods. Three pH-dependent steps were identified during the toxin reassembly: (i) acquisition of a fully assembly-competent fold by the CtxB monomer, (ii) association of CtxB monomer into oligomers, (iii) acquisition of the native fold by the CtxB pentamer. The results show that CtxB(5) and the related heat labile enterotoxin LTB(5) have distinct mechanisms of assembly despite sharing high sequence identity (84%) and almost identical atomic structures. The difference can be pinpointed to four histidines which are spread along the protein sequence and may act together. Thus, most of the toxin B amino acids appear negligible for the assembly, raising the possibility that assembly is driven by a small network of amino acids instead of involving all of them.     

Gln54Leu of the conserved polar residue inthe interfacial coiled coil position (d) results in significant stabilization of the original structure of the COMP pentamerization domain

Summary The assembly domain of cartilage oligomeric matrix protein (COMP) forms an α-helical coiled coil homopentamer with a conserved polar glutamine in the interior (d) position. We substituted Gln⁵⁴ for apolar Leu in the recombinant fragment of the rat COMP domain. Biochemical studies and circular dichroism (CD) spectroscopy showed that the mutant, similarly to the wild-type (w.t.) peptide, forms spontaneously an α-helical pentamer. Thermal transitions of the w.t. and mutant pentamers were analyzed by CD spectroscopy and differential scanning calorimetry. The Gln⁵⁴Leu mutation increased the thermal stability of the pentamer with reduced disulfide bonds from 73°C to 104°C. The denaturation of the disulfide bonded w.t. pentamer was observed at 108°C while the mutant pentamer cannot be denatured up to 120°C (the apparatus limit). Thus, by Gln⁵⁴Leu mutation we found a way to significantly stabilize the coiled coil pentamer, making this peptide even more attractive as an oligomerization tool for various biotechnological applications.     

Collagen oligomers modulate physical and biological properties of three‐dimensional self‐assembled matrices

Elucidation of mechanisms underlying collagen fibril assembly and matrix‐induced guidance of cell fate will contribute to the design and expanded use of this biopolymer for research and clinical applications. Here, we define how Type I collagen oligomers affect in‐vitro polymerization kinetics as well as fibril microstructure and mechanical properties of formed matrices. Monomers and oligomers were fractionated from acid‐solubilized pig skin collagen and used to generate formulations varying in monomer/oligomer content or average polymer molecular weight (AMW). Polymerization half‐times decreased with increasing collagen AMW and closely paralleled lag times, indicating that oligomers effectively served as nucleation sites. Furthermore, increasing AMW yielded matrices with increased interfibril branching and had no correlative effect on fibril density or diameter. These microstructure changes increased the stiffness of matrices as evidenced by increases in both shear storage and compressive moduli. Finally, the biological relevance of modulating collagen AMW was evidenced by the ability of cultured endothelial colony forming cells to sense associated changes in matrix physical properties and alter vacuole and capillary‐like network formation. This work documents the importance of oligomers as another physiologically‐relevant design parameter for development and standardization of polymerizable collagen formulations to be used for cell culture, regenerative medicine, and engineered tissue applications. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 77–93, 2011.     

Conformations and time-resolved fluorescence of oligomers of (−)-epicatechin with 4β → 8 interflavan bonds

Oligomers of (?)-epicatechin with 4β → 8 interflavan bonds, and as many as five monomer units, have been studied by conformational analysis and time-resolved fluorescence. The conformational analysis yields 2^x?1 conformations for each oligomer with x monomer units. There are two conformations accessible at each interflavan bond. These conformations are denoted by + and ?. The dominant conformations for the trimer and higher oligomers have an interaction between the phenolic hydroxyl groups on monomers i and i+2. This interaction involves the hydroxyl group at C(5) on monomer i, and either C(13) or C(8) of monomer i+2, depending on whether the conformation of the two intervening interflavan bonds is + + or + ?, respectively. Minor contributions to the ensemble for the tetramer and pentamer are made by conformations that contain the sequence of successive interflavan bonds denoted by ?+ or ??. In ?+ the interaction between monomer units i and i + 2 involves an aliphatic hydroxyl with a phenolic hydroxyl, and there are no hydroxyl-hydroxyl interactions between units iand i + 2 in ??. The onset of a different decay law for the fluorescence when x increases from 3 to 4 may be associated with the appearance of the ?+ and ?? conformations as minor constituents in the ensemble.     

The investigation of the ultrastructural neutrophil changes in alloxan-induced diabetes in rats: response to a chemotactic challenge

Experimental diabetes is one of the most popular conditions in which to study the relation between neutrophil leukocyte activity and periodontal destruction. The aetiology of neutrophil dysfunction in the gingival tissue associated with diabetes has yet to be clarified. Diabetes in rats decreases neutrophil chemotactic activity in proportion to the severity of this systemic disorder. The present study was carried out to evaluate the relationship between the severity of diabetes and the neutrophil response to two chemotactic agents, and to correlate the observed neutrophil defects with the degree of diabetes. In this study two chemotactic agents, casein (0.2 μl, 2 mg ml^?1) or N‐formylmethionylleucylphenylalanine (FMLP; 0.2 μl, 10^?4 M ), were placed into the gingival crevices of alloxan‐induced diabetic rats. Gingival biopsies were taken 15 min later and then at 5‐min intervals up to 45 min and investigated by electron microscopy. Adherence and migration were observed in the rats with moderate diabetes 30 min after the application of casein. There was chemotaxis after 35 min of administration of the peptide FMLP. By 40 min neutrophils with pyknotic nuclei were observed. At 45 min neutrophils with a decreased number of granules were present. As the severity of the diabetes increased, the neutrophils degenerated and were structurally distorted. In the rats which had alloxan‐induced diabetes there was abnormal periodontal damage. This damage is thought to be related to dysfunctional neutrophils. These findings many contribute to an answer to the following question: why is there an apparent variability in the susceptibilty of periodontal breakdown in diabetics? Copyright © 2003 John Wiley & Sons, Ltd.     

Ghrelin inhibits AGEs-induced apoptosis in human endothelial cells involving ERK1/2 and PI3K/Akt pathways

Endothelial dysfunction caused by cell apoptosis is thought to be a major cause of diabetic vascular complications. Advanced glycation end products (AGEs) play an important role in the pathogenesis of diabetic vascular complications by inducing apoptosis of endothelial cells. The aim of this study was to explore the effect of ghrelin on AGEs‐induced apoptosis in cultured human umbilical vein endothelial cells (HUVECs) and the potential mechanisms involved in this process. Exposure to AGEs (200 mg l^?1) for 48 h caused a significant increase in cell apoptosis, while pretreatment with ghrelin eliminated AGEs‐induced apoptosis in HUVECs, as evaluated by MTT assays, flow cytometry and Hoechst 33258 staining. The induction of caspase‐3 activation was also prevented by ghrelin in cells incubated with AGEs. Exposure to ghrelin (10^?6 M) resulted in a rapid activation of extracellular signal‐regulated protein kinase (ERK)1/2 and Akt. The inhibitory effect of ghrelin on caspase‐3 activity was attenuated by inhibitors of ERK1/2 (PD98059), PI3K/Akt (LY294002) and growth hormone secretagogue receptor (GHSR)‐1a (D ‐Lys³‐growth hormone releasing peptide‐6). The results of this study indicated that ghrelin could inhibit AGEs‐mediated cell apoptosis via the ERK1/2 and PI3K/Akt pathways and GHSR‐1a was also involved in the protective action of ghrelin in HUVECs. As such, ghrelin demonstrates significant potential for preventing diabetic cardiovascular complications. Copyright © 2011 John Wiley & Sons, Ltd.