Expression of inducible nitric oxide synthase in human burn wounds

A water-soluble synthetic peptide with only nine amino acid residues, comprising the 131-139 sequence region of the cytotoxic protein alpha-sarcin (secreted by the mold Aspergillus giganteus), interacts with large unilamellar vesicles composed of acid phospholipids. It promotes lipid mixing between bilayers and leakage of vesicle aqueous contents, and it also abolishes the phospholipid phase transition. Other larger peptides containing such an amino acid sequence also produce these effects. These peptides acquire alpha-helical conformation in the presence of trifluoroethanol, but display beta-strand conformation in the presence of sodium dodecyl sulfate. The interaction of these peptides with the lipid vesicles also results in beta-structure. The obtained data are discussed in terms of the involvement of the 131-139 stretch of alpha-sarcin in its interaction with lipid membranes.     

Responsiveness of atiii and coagulation factors V and VII to a standardized oral fat load

We have isolated selective ligands to the cell surface receptors of fibronectin (alpha 5 beta 1 integrin), vitronectin (alpha v beta 3 and alpha v beta 5 integrins) and fibrinogen (alpha IIb beta 3 integrin) from phage libraries expressing cyclic peptides. A mixture of libraries was used that express a series of peptides flanked by a cysteine residue on each side (CX5C, CX6C, CX7C) or only on one side (CX9) of the insert. A majority of the integrin-binding sequences derived from the CX9 library contained another cysteine, indicating preferential selection of conformationally constrained cyclic peptides. Each of the four integrins studied primarily selected RGD-containing phage sequences but favored different ring sizes and different flanking residues around the RGD motif. A cyclic peptide ACRGDGWCG was synthesized based on a phage sequence that bound particularly avidly to the alpha 5 beta 1 integrin. This peptide inhibited cell attachment to fibronectin at about 5-fold lower concentrations than the most potent cyclic peptides described earlier. The most interesting structure appeared to contain two disulphide bonds. One such peptide, ACDCRGDCFCG, was synthetized and shown to be at least 20-fold more potent inhibitor of alpha v beta 5- and alpha v beta 3-mediated cell attachment to vitronectin than similar peptides with a single disulphide bond and 200-fold more potent than commonly used linear RGD peptides. These results emphasize the importance of conformational restriction as a means of improving the potency of integrin-binding peptides and point to a new way of designing effective peptides by resticting the peptide conformation with more than one cyclizing bond.     

Investigation of conformational specificity at GPIIb/IIIa: evaluation of conformationally constrained RGD peptides

RGD-containing proteins and peptides are known to bind to the platelet GPIIb/IIIa receptor and inhibit platelet aggregation. That a conformational component to the specificity exists is suggested by significantly lower activity of linear RGD analogs relative to closely related cyclic peptides and small proteins containing the RGD sequence. Recently, conformations for a suite of RGD containing cyclic peptides have been defined by NMR-based methods and, for one molecule, by X-ray diffraction. We report here the NMR-based conformational analysis of an additional cyclic peptide, cyclo(Pro-Arg-Gly-Asp-D-Pro-Gly), and compare the conformational variations in the suite of peptides and related analogs. Biological activity data for these peptides shows a preference of the platelet GPIIb/IIIa receptor for one conformation of the RGD sequence, but suggests its ability to bind a second, distinct conformation.     

Interaction of a bZip oligopeptide model with oligodeoxyribonucleotides modelling DNA binding sites. The effect of flanking sequences

A leucine zipper (bZip) binding peptide BP1 was constructed based on the DNA binding sequence of the GCN4 protein, slightly modified to make it more similar to the sequence of other bZip proteins (Jun) with related DNA binding specificity. Self-complementary DNA hexadecanucleotides containing ATF/CRE, AP-1 and C/EPB target sites were used to study peptide-DNA complex formation. Conformation changes in both components that occur on complex formation were studied by circular dichroism (CD) spectroscopy. The results show that the amount of alpha-helix formed in the peptide strongly depends not only on the target site present, but also on the type of the sequence flanking the ATF/CRE target site. Highest amount of the alpha-helix induced in the peptide was observed when homopurine homopyrimidine flanking sequences were present, whereas the presence of alternating sequences, especially of the CA/TG type, showed considerably lower effects. The change in DNA conformation on complex formation was generally small, but also depended on the type of the flanking sequence. It appears that the sequences flanking the target site can considerably modify the ability of the target sequence to bind specifically the bZip peptide, probably by slightly varying the overall DNA conformation.     

Interaction of fibronectin (FN) cell binding fragments and interleukin-8 (IL-8) in regulating neutrophil chemotaxis

This study investigated the possible interaction of FN fragments in regulating IL-8-mediated neutrophil chemotaxis in vitro using Neuroprobe microchambers. Human neutrophil suspensions were incubated with purified FN fragments or an RGD-containing peptide and allowed to migrate in response to chemotactically active concentrations of human recombinant IL-8. The 120-kD fragment of FN containing the RGD sequence or an RGD peptide (GRGDSP) inhibited IL-8-mediated neutrophil chemotaxis; however, these RGD peptides did not inhibit neutrophil chemotaxis in response to other chemotactic agents. Furthermore, FN fragments not containing the RGD sequence had no effect on IL-8-mediated chemotaxis. These data suggest that directed migration of neutrophils in response to IL-8 is inhibited in the presence of cell-binding fragments of FN and may represent a local mechanism for terminating neutrophil migration at areas of tissue injury.     

A new approach to secondary structure evaluation: secondary structure prediction of porcine adenylate kinase and yeast guanylate kinase by CD spectroscopy of overlapping synthetic peptide segments

A new approach for evaluating the secondary structure of proteins by CD spectroscopy of overlapping peptide segments is applied to porcine adenylate kinase (AK1) and yeast guanylate kinase (GK3). One hundred seventy-six peptide segments of a length of 15 residues, overlapping by 13 residues and covering the complete sequences of AK1 and GK3, were synthesized in order to evaluate their secondary structure composition by CD spectroscopy. The peptides were prepared by solid phase multiple peptide synthesis method using the 9-fluorenylmethoxycarbonyl/tert-butyl strategy. The individual peptide secondary structures were studied with CD spectroscopy in a mixture of 30% trifluoroethanol in phosphate buffer (pH 7) and subsequently compared with x-ray data of AK1 and GK3. Peptide segments that cover alpha-helical regions of the AK1 or GK3 sequence mainly showed CD spectra with increasing and decreasing Cotton effects that were typical for appearing and disappearing alpha-helical structures. For segments with dominating beta-sheet conformation, however, the application of this method is limited due to the stability and clustering of beta-sheet segments in solution and due to the difficult interpretation of random-coiled superimposed beta-sheet CD signals. Nevertheless, the results of this method especially for alpha-helical segments are very impressive. All alpha-helical and 71% of the beta-sheet containing regions of the AK1 and GK3 could be identified. Moreover, it was shown that CD spectra of consecutive peptide content reveal the appearance and disappearance of alpha-helical secondary structure elements and help localizing them on the sequence string.     

Cell binding and internalization by filamentous phage displaying a cyclic Arg-Gly-Asp-containing peptide

Ligands that bind mammalian cell surface integrins with high affinity can mediate cellular internalization. We show that particles of the bacteriophage fd that display the cyclic integrin-binding peptide sequence GGCRGDMFGC in a proportion of their major coat protein subunits bind to cells and are efficiently internalized. In the displayed peptide the conformation of the RGD motif is restricted within a hairpin loop formed by a disulfide bridge between the 2 cysteine residues. Cellular internalization of phage was demonstrated by confocal and non-confocal immunofluorescence microscopy of tissue-cultured cells incubated with phage particles. The phage were contained in juxtanuclear vesicles in the same serial sections as transferrin receptor but were not colocalized with the cell surface marker alkaline phosphatase. Cell binding and internalization was inhibited by preincubation of cells with the integrin-binding peptide GRGDSP, whereas the control peptide GRGESP had no inhibitory effect. These results indicate that cyclic integrin-binding peptides can be used to target and enter cells and that it should be possible to exploit such peptides for the introduction of DNA, drugs, or other macromolecules.     

Structure and dynamics of peptide-amphiphiles incorporating triple-helical proteinlike molecular architecture

Organized polymeric assemblies that incorporate bioactive sequences and structures are finding important applications for the study of protein structure-function relationships. We have recently described a heteropolymeric peptide-amphiphile system that forms organized structures in solution and on surfaces. While the overall three-dimensional features of peptide-amphiphiles have been studied previously, the precise environment of specific residues, particularly those within biologically active regions, have not been examined in detail. In the present study, we have used heteronuclear single quantum coherence (HSQC) and inverse-detected 1H-15N NMR spectroscopy to examine the structure and dynamics of a peptide and peptide-amphiphile that incorporate the alpha1(IV)1263-1277 ([IV-H1]) amino acid sequence from type IV collagen. Three variants of the sequence (Gly-Pro-Hyp)4-[IV-H1]-(Gly-Pro-Hyp)4 were constructed with a single 15N-labeled Gly placed in the middle of the N-terminal (Gly-Pro-Hyp)4 region (residue Gly7), in the middle of the [IV-H1] sequence (residue Gly19), or in the middle of the C-terminal (Gly-Pro-Hyp)4 region (residue Gly34). These peptides were also N-terminally acylated with hexanoic acid to create an analogous series of 15N-labeled peptide-amphiphiles. HSQC spectra indicated that both the peptide and the peptide-amphiphile were in triple-helical conformation at low temperature, supporting prior circular dichroism (CD) spectroscopic results. The intensities of the triple-helical cross-peaks were stronger for the peptide-amphiphile, consistent with an enhanced triple-helical thermal stability within the peptide-amphiphile construct compared to that of the peptide alone. Relative relaxation values for the peptide-amphiphile monomeric and trimeric species were consistent with those reported previously for other triple-helical peptides. Relaxation measurements indicated that the triple-helical [IV-H1] region did not appear to be dramatically more flexible than the Gly-Pro-Hyp regions. The angle between Gly N-H bonds and the helix dyad axis, determined from the relaxation data, was within the range expected for triple helices. Overall, the peptide headgroup of the C6-(Gly-Pro-Hyp)4-[IV-H1]-(Gly-Pro-Hyp)4 peptide-amphiphile appears to form a continuous triple helix that behaves similarly, in a dynamic sense, to a triple-helical peptide. The enhanced thermal stability of the peptide-amphiphile compared to the analogous triple-helical peptide, along with the multitude of organized structures formed by lipidlike compounds, suggest that peptide-amphiphiles could be utilized as targeted liposomes, sensors, receptors, or enzymes.     

Interactions between growth factors and integrins: latent forms of transforming growth factor-beta are ligands for the integrin alphavbeta1

The multipotential cytokine transforming growth factor-beta (TGF-beta) is secreted in a latent form. Latency results from the noncovalent association of TGF-beta with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-beta-binding protein (LTBP) produces the most common form of latent TGF-beta, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-beta. LTBP and the LAP propeptides of TGF-beta (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. To increase our understanding of latent TGF-beta function in the ECM, we determined whether latent TGF-beta1 interacts with integrins. A549 cells adhered and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion was blocked by an RGD peptide, and cells were unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion was also blocked by mAbs to integrin subunits alphav and beta1. We purified LAP-binding integrins from extracts of A549 cells using LAP bound to Sepharose. alphavbeta1 eluted with EDTA. After purification in the presence of Mn2+, a small amount of alphavbeta5 was also detected. A549 cells migrated equally on fibronectin- and LAP-coated surfaces; migration on LAP was alphavbeta1 dependent. These results establish alphavbeta1 as a LAP-beta1 receptor. Interactions between latent TGF-beta and alphavbeta1 may localize latent TGF-beta to the surface of specific cells and may allow the TGF-beta1 gene product to initiate signals by both TGF-beta receptor and integrin pathways.     

Physico-chemical characterization and application for drug carrier of soybean-derived sterols and their glucosides-containing liposomes

With a rapid demand to decrease the side effect of drug, a variety of drug delivery systems have been developed. This review will focus on the development of liposomes with soybean-derived sterols and their glucosides for drug carriers. Current status and further perspectives in this research field are reviewed mainly based on the results obtained in our laboratory. First we studied the different physicochemical properties of dipalmitoylphos-phatidylcholine (DPPC) liposomes with soybean-derived sterols (SS) and their glucosides (SG). SS rigidifies the liposomal membrane but SG fluidizes it. SS stabilizes liposomes more than cholesterol that is conventionally used as stabilizing agents in liposomes. On the basis of this information, we developed liposomes with SS and SG for a drug carrier. Secondly we studied the stability of liposomes in the blood and biodistribution and found that liposomes with SS were stable as expected in vitro results. In particular, DPPC: SS (7:4, molar ratio) size 0.2 micron showed long circulation. Thirdly successful targeting of the drugs to the liver was achieved by liposomes with SG. Finally, we succeeded in developing liposomal erythropoietin and doxorubicin using liposomes with SS for sustained release of drugs. Liposomal drugs increased the pharmacological effect compared with free drugs, suggesting a decrease of side effect and long circulation. The attempt for oral administration using liposomes of peptide drugs was carried out successfully. We have established that the study of physicochemical properties of liposomes is needed rationally as the distribution of drugs in liposomes and the rigidity of liposomal membrane, prior to the development of the drug carrier of liposomes. SS is useful to stabilize liposomes and SG to targeting to the liver parenchymal cells. This information can be useful and practical for the development of liposomes for drug carriers.     

Identification of a domain on the integrin alpha5 subunit implicated in cell spreading and signaling

The alpha5 beta1 integrin is a cell surface receptor for fibronectin implicated in several cellular activities including cell proliferation, differentiation, and migration. The primary site at which the alpha5 beta1 integrin interacts with fibronectin is the RGD (Arg-Gly-Asp) amino acid sequence. In general, the sites on the integrin alpha subunits involved in ligand binding are not well characterized. Based on previous cross-linking studies, sequence alignment, predicted conformation, and intron-exon boundaries, we identified a 144-residue region (positions 223-367) on the alpha5 subunit as a putative binding region and divided it into four subdomains named domains I, II, III, and IV. Chimeric receptors were prepared in which sequences on the alpha5 subunit were exchanged with the corresponding sequences on the alpha6 subunit, which is specific for laminin and does not bind via an RGD sequence. The mutated human alpha5 integrin gene was transfected into CHO B2 cells, which are deficient in alpha5 expression. Only chimeras of domain III or IV express on the cell surface. Both of these chimeras decreased the adhesion, spreading, focal adhesion assembly, and migration on fibronectin. The adhesion of the chimeric receptors to fibronectin remained sensitive to the RGD peptide, and antibodies that inhibit interaction with the fibronectin synergy site and RGD loop remain inhibitory for the chimeras, indicating that our chimeras do not inhibit binding to either the RGD or synergy sites. Finally, the affinity of soluble fibronectin to cells via the alpha5 beta1 receptor decreased only about 3-fold. This decrease is substantially less than the observed effects on migration and spreading, which were not altered by changes in substrate concentration. Thus, the alteration in binding sites does not easily account for the changes in cell spreading and focal adhesion assembly. The tyrosine phosphorylation and focal adhesion assembly that are seen when cells expressing the wild type alpha5 receptor adhere to fibronectin were inhibited in cells expressing the chimeric receptors. Therefore, our results suggest that the chimeras of these domains likely interrupt alpha5-mediated conformational signaling.     

Amphiphilic alpha-helices are important structural motifs in the alpha and beta peptides that constitute the bacteriocin lactococcin G--enhancement of helix formation upon alpha-beta interaction

Lactococcin G (LcnG) is an antimicrobial substance (bacteriocin) consisting of two peptides, LcnG-alpha and LcnG-beta. The structures of intact LcnG-alpha and LcnG-beta as well as various fragments of these peptides were studied by circular dichroism (CD) under several conditions. All peptides had a non-structured conformation in aqueous solutions. In the presence of trifluoroethanol, dodecylphosphocholine micelles and (negatively charged) dioleoylglycerophosphoglycerol (Ole2GroPGro) liposomes, varying amounts of alpha-helical structure were induced. Comparisons of the various fragments showed that helicity was concentrated in those parts of LcnG-alpha and LcnG-beta that would become amphiphilic if an alpha-helical structure was adopted. In the presence of zwitterionic dioleoylglycerophosphocholine (Ole2GroPCho) liposomes, the peptides were much less (if at all) structured, suggesting that the excess of positive charge on the antimicrobial peptides needs to be compensated by an excess of negative charge on the membrane. The structuring of LcnG-alpha and LcnG-beta in the presence of Ole2GroPGro liposomes was considerably enhanced when both peptides were presented simultaneously to the membranes. Consecutive addition of the two peptides to Ole2GroPGro liposomes did not give this additional structuring, indicating that the individual LcnG-alpha and LcnG-beta peptides associate with the membrane in a virtually irreversible manner that makes them inaccessible for interaction with the complementary peptide. The results suggest that upon arrival at and interaction with the target membrane, LcnG-alpha and LcnG-beta form a complex that consists of approximately 50% amphiphilic alpha-helices.     

Liposomes enhance delivery and expression of an RGD-oligolysine gene transfer vector in human tracheal cells

Nonviral gene delivery systems consist predominantly of lipoplexes or receptor-targeting and nontargeting polyplexes. We examined integrin-mediated gene delivery using an Arg-Gly-Asp/oligo-L-lysine ([K]16RGD) cyclic peptide and investigated its gene transfer efficiency when associated with a cationic liposome. We demonstrated that human cystic fibrosis and noncystic fibrosis tracheal epithelial cells in culture express integrins that recognise the RGD integrin-binding motif. We found a 10-fold (P < 0.01) increased expression of a luciferase encoding plasmid in these cells when complexing the plasmid to the [K]16RGD peptide as compared with plasmid alone. This increase was specific to the [K]16RGD peptide since neither a [K]16RGE nor a [K]16 peptide gave a comparable increase. Expression was further enhanced 30-fold (P < 0.01) with lipofectamine and the ratio of DNA/peptide/lipofectamine was critical for specificity and expression. Fluorescence and radioactive labelling of the complex showed that the [K]16RGD peptide increased the endocytic uptake of DNA into cells. The cell association of both DNA and peptide increased even further with lipofectamine. Confocal microscopy showed that the [K]16RGD peptide and the DNA internalised together within 30 min and localised to vesicles in the perinuclear region. These results show that an integrin-binding ligand can deliver genetic material to airway cells and that a cationic liposome can enhance the efficacy of this nonviral vector system.     

Conformational studies on murein-lipoprotein from the outer membrane of Escherichia coli

Conformational studies on an isolated integral membrane protein are reported. Lipoprotein of Escherichia coli outer membrane was released from murein by treatment with either lysozyme or trypsin. The isolated lysozyme-released lipoprotein (lipoprotein I) contained 2 or 3 muropeptides covalently linked at the C-terminal end, while the trypsin-released lipoprotein (lipoprotein II) was free of muropeptides and lacked the C-terminal peptide Tyr-Arg-Lys. Circular dichroism spectra of the two preparations were essentially identical, and they show an alpha-helix content of about 80%. According to calculations based on the Chou-Fasman rules for proteins of known sequence, lipoprotein is 64% alpha-helix and 15% beta-structure. Infrared spectroscopy qualitatively supports these values. The conformation was stable in the pH range of 5 - 12. Danaturation of lipoprotein by heat, 8 M urea, or sodium dodecylsulphate was a fully reversible, cooperative process. The thermal denaturation of lipoprotein occurs in two steps with transition points at 79.4 degrees C for lipoprotein I and at 85.1 degrees C for lipoprotein II. Lioprotein markedly changes conformation at dodecylsulphate concentrations where micelle formation sets in. The unusual behaviour of the lipoprotein convormation in sodium dodecylsulphate is discussed in relation to the lipoprotein conformation and aggregation within the membrane.     

Structure-activity relationships and physico-chemical properties of synthetic lipopeptide inhibitors of PKC

Four synthetic lipopeptides, (K-pm 19,31), (K-pm 19,21,31), (K-pm 19,28,31) and (K-pm 19,21,28,31) with the lysine-palmitoyl (K-pm) residue as a lipophilic moiety, based on the pseudosubstrate sequence 19RFARKGALRQKNV31 (R19-V31), were found to be potent protein kinase C (PKC) inhibitors. However, the lipopeptides (K-pm 19,21,31), (K-pm 19,28,31) and (K-pm 19,21,28,31) were also found to act as protein kinase cAMP-dependent (PKA) inhibitors. Peptide (K-pm 19,31), the least water soluble, is marginally selective towards PKC, unlike the other palmitoyl derivatives studied here. Since the non-palmitoylated analogues (K 19,31), (K-ac 19,31), (K 19,21,31) and (K-ac 19,21,31) were inhibitors of PKC but not of PKA, the palmitoyl moiety must play a role in the specificity of protein kinase inhibition. In vitro, the lipophilic peptides showed greater stability to protease-mediated hydrolysis than the pseudosubstrate peptide depending upon the number of lipophilic (K-pm) residues. CD studies showed that in comparison with the peptide analogues, the remarkable resistance of the pseudosubstrate (R19-V31) to adopt an alpha-helix conformation in TFE, known to be strongly alpha-helix inducing, rules out this structure as the peptide binding conformation to PKC. By contrast, in aqueous media all the peptides show an extended conformation that correlates well with their inhibitory activity. This is in compliance with the crystallographic observation that an extended structure has been observed for the (5-24) PKI peptide inhibitor bound to PKA.     

NMR and molecular dynamics studies of tachykinins: conformation of the C-terminal pentapeptide of substance P(SP7-11)

Substance P belongs to the tachykinin family of neuropeptides which exhibit diverse pharmacological activity. The conformation of Phe1-Phe2-Gly3-Leu4-Met5-NH2 the C-terminal pentapeptide of substance P (SP7-11) has been studied by NMR and molecular dynamics (MD) methods. NMR studies were carried out both in DMSO-d6 and 95% H2O. Based on the observed chemical shifts, 3JNH alpha coupling constants, temperature coefficients of chemical shifts of NH resonances and the pattern of inter- and intraresidue NOE's, a predominantly extended backbone conformation has been deduced for the peptide in both DMSO and H2O. MD calculations carried out in vacuo indicate that the global minimum energy conformation of the molecule is folded with an intramolecular hydrogen bond between the protonated N-terminal and the C-terminal CONH2 group. The simulation shows that beta-turns are energetically unfavourable, while alpha-helices are seen to be unstable for the peptide. gamma-Bends at either Gly3 or Leu4 are the most preferred ones. Simulations carried out in DMSO as well as in water show a preference for a nearly extended conformation.     

Modification of delivery system enhances MHC nonrestricted immunogenicity of V3 loop region of HIV-1 gp120

A successful peptide vaccine for AIDS is desired to elicit T-helper and cytotoxic T lymphocyte responses besides neutralizing antibodies. The V3 loop peptide of HIV-1 has been shown to contain the principal neutralizing domain, one of the most immunodominant regions, having both B-cell and T-cell determinants. In this study, the tip of the V3 loop region was mutated from GPGR to GPGQ based on the sequence of Indian isolates (CKRKIHIGPGQAFYT). To further enhance the immunogenicity of this epitope, two delivery systems of immune stimulating complexes (ISCOMs) and liposomes were used to incorporate the peptide. Mice of differing haplotypes, H-2b, H-2d, H-2k and H-2s, showed no MHC restriction when immunized with these formulations. The IgG levels as assessed by ELISA were found to be significantly higher (P < 0.05 to P < 0.001) for even five-fold lower doses of the peptide in ISCOMs and liposomes as compared to the conventional alum-based preparation. The major subtype elicited was IgG2a/IgG2b, suggestive of a Th1-like response for all the formulations. Thus, it would appear that the same peptide incorporated in ISCOMs and liposomes selects a Th1 response and may therefore be important not only for neutralization but also for virus clearance.     

Membrane fusion induced by 11-mer anionic and cationic peptides: a structure-function study

We recently demonstrated that an amphipathic net-negatively charged peptide consisting of 11 amino acids (WAE 11) strongly promotes fusion of large unilamellar liposomes (LUV) when anchored to a liposomal membrane [Pecheur, E. I., Hoekstra, D., Sainte-Marie, J., Maurin, L., Bienvenue, A., and Philippot, J. R. (1997) Biochemistry 36, 3773-3781]. To elucidate a potential relationship between peptide structure and its fusogenic properties and to test the hypothesis that specific structural motifs are a prerequisite for WAE-induced fusion, three 11-mer WAE-peptide analogues (WAK, WAEPro, and WAS) were synthesized and investigated for their structure and fusion activity. Structural analysis of the synthetic peptides by infrared attenuated total reflection spectroscopy reveals a distinct propensity of each peptide toward a helical structure after their anchorage to a liposomal surface, emphasizing the importance of anchorage on conveying a secondary structure, thereby conferring fusogenicity to these peptides. However, whereas WAE and WAK peptides displayed an essentially nonleaky fusion process, WAS- and WAEPro-induced fusion was accompanied by substantial leakage. It appears that peptide helicity as such is not a sufficient condition to convey optimal fusion properties to these 11-mer peptides. Studies of changes in the intrinsic Trp fluorescence and iodide quenching experiments were carried out and revealed the absence of migration of the Trp residue of WAS and WAEPro to a hydrophobic environment, upon their interaction with the target membranes. These results do not support the penetration of both peptides as their mode of membrane interaction and destabilization but rather suggest their folding along the vesicle surface, posing them as surface-seeking helixes. This is in striking contrast to the behavior observed for WAE and WAK, for which at least partial penetration of the Trp residue was demonstrated. These results indicate that subtle differences in the primary sequence of a fusogenic peptide could induce dramatic changes in the way the peptide interacts with a bilayer, culminating in equally drastic changes in their functional properties. The data also reveal a certain degree of sequence specificity in WAE-induced fusion.     

Role of metalloproteinases in the development and healing of acetic acid-induced gastric ulcer in rats

The three main components involved in thrombosis and haemostasis are thrombin, platelets, and plasmin. Almost all inhibitors of thrombosis are focused either on the inhibition of thrombin or on the inhibition of platelets. We designed a construct using the fibrinolytic activity of staphylokinase, fused via a cleavable linker to an antithrombotic peptide of 29 amino acids. The peptide was designed to include three inhibitory regions: (1) the Arg-Gly-Asp (RGD) amino acid sequence to prevent fibrinogen binding to platelets; (2) a part of fibrinopeptide A, an inhibitor of thrombin; and (3) the tail of hirudin, a potent direct antithrombin. The amino acid sequence of the 29 amino acid peptide was reverse translated, and the gene was chemically synthesised and cloned into an expression vector as a 3' fusion to the staphylokinase gene. Gene expression was induced in E. coli Top 10 cells and the fusion protein, designated PLATSAK, was purified using metal affinity chromatography. The purified fusion protein significantly lengthened the activated partial thromboplastin time and thrombin time and inhibited the amidolytic activity of thrombin. The fibrinolytic activity was almost equal to that of recombinant staphylokinase as measured with a thrombelastograph. Platelet aggregation was not markedly inhibited by PLATSAK, probably due to the unfavourable three dimensional structure, with the Arg-Gly-Asp sequence buried inside. Our results confirm that it is feasible to design and produce a hybrid multifunctional protein that targets various components of the haemostatic process.     

CD11b/CD18 mediates the neutrophil chemotactic activity of fibrin degradation product D domain

Coagulation and fibrinolysis universally accompany tissue injury and repair. The accumulation of regionally generated fibrin degradation products (FDP) may modify the local inflammatory response. We have found FDP to be potent neutrophil chemotaxins. We separated plasmin FDP by chromatofocusing and found chemotactic activity limited to fractions containing the fibrinogen D domain (D-D dimer and D monomer). The bioactivity of the D-D dimer did not require an intact cross link site as removal of this sequence with puff adder venom or hypocalcemic plasmic digestion did not decrease chemotaxis. Peptide inhibition studies confirmed that the chemotactic region did not involve terminal gamma chain sequences or alpha chain RGD motifs. The internal gamma chain peptide KYGWTVFQKRLDGSV (P1), known to bind CD11b/CD18, exhibited concentration dependent chemotactic activity. Similarly, monoclonal antibodies directed against CD11b/CD18 blocked PMN migration to FDP without similar inhibition of chemotaxis to IL-8 or LTB4. Thus, neutrophil chemotaxis to FDP is mediated by interactions between the fibrinogen D domain and CD11b/CD18.