ABSTRACT
BACKGROUND: Exogenous surfactant has been accepted worldwide as a therapy of RDS in premature and term infants. Exogenous surfactant is usually derived from lung extracts containing phospholipids and the surfactant proteins SP-B and SP-C. Synthetic peptides of SP-B and SP-C are being tested with the aim to develop a completely synthetic surfactant preparation. Nevertheless, the effects of these peptides on the endogenous surfactant metabolism remain unknown. OBJECTIVES: The effect of synthetic SP-B peptides on uptake of surfactant-like liposomes was investigated in alveolar cells. Native SP-B and seven SP-B peptides were included: monomeric and dimeric SP-B(1-25) (Cys-11 --> Ala-11), SP-B(63-78)and Ala-SP-B(63-78) (Cys-71 --> Ala-71;Cys-77 --> Ala-77)and their serine mutants. METHODS: In vitro, alveolar macrophages (AM) and alveolar type II cells (ATII) were incubated with liposomes containing SP-B or one of its peptides. In vivo, rats received intratracheally various SP-B peptides (SP-B/lipid ratio 1:33 w/w) incorporated in fluorescent surfactant-like liposomes. One hour after instillation, AM and ATII were isolated and cell-associated fluorescence was determined using flow cytometry. Confocal laser microscopy was performed to ensure internalization of the liposomes. RESULTS: In vitro uptake by AM or ATII was not influenced by the SP-B peptides. In vivo, SP-B(1-25) and Ser-SP-B(1-25) increased the uptake by AM whereas dSP-B(1-25) decreased the uptake. Neither SP-B(1-25) nor dSP-B(1-25 )affected total uptake by ATII. The overall uptake by SP-B(63-78) variants was not changed. CONCLUSIONS: Surface-active synthetic SP-B peptides do not interfere with the normal uptake of surfactant by ATII.
Subject(s)
Liposomes/pharmacokinetics , Peptide Fragments/pharmacology , Pulmonary Alveoli/metabolism , Pulmonary Surfactant-Associated Protein B/pharmacology , Animals , Biological Transport/drug effects , Flow Cytometry , Humans , Kinetics , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/physiology , Peptide Fragments/chemical synthesis , Pulmonary Alveoli/drug effects , Pulmonary Surfactant-Associated Protein B/chemistry , Pulmonary Surfactant-Associated Protein B/isolation & purification , SwineABSTRACT
The intermolecular packing of a beta-hairpin antimicrobial peptide, PG-1, in lipid bilayers is determined using solid-state NMR distance measurements. Previous spin counting experiments showed that PG-1 associates as dimers in POPC bilayers; however, the detailed dimer structure was unknown. We have now measured several intermolecular 13C-19F, 1H-13C, and 15N-13C distances in site-specifically labeled PG-1 to constrain the structure of the intermolecular interface. The distances are measured using the rotational-echo double-resonance (REDOR) technique under magic-angle spinning. The results indicate that two PG-1 molecules align in a parallel fashion with the C-terminal strand of the hairpin forming the dimer interface. Six hydrogen bonds stabilize this interface, and the Phe12 side chain adopts the g- conformation in the membrane as in solution. The parallel packing of the peptide in the lipid bilayer differs from the antiparallel dimer found in DPC micelles and may be stabilized by its strong amphipathic character, which should facilitate its insertion into the amphipathic lipid bilayer. This study demonstrates the utility of the REDOR NMR technique for the elucidation of the oligomeric structure of membrane proteins.
Subject(s)
Antimicrobial Cationic Peptides/chemistry , Membrane Proteins/chemistry , Proteins/chemistry , Dimerization , Hydrogen Bonding , Lipid Bilayers/chemistry , Magnetic Resonance Spectroscopy , Phosphatidylcholines/chemistry , Protein Structure, SecondaryABSTRACT
Surfactant protein B (SP-B) is essential for normal lung surfactant function. Theoretical models predict that the disulfide cross-linked, N- and C-terminal domains of SP-B fold as charged amphipathic helices, and suggest that these adjacent helices participate in critical surfactant activities. This hypothesis is tested using a disulfide-linked construct (Mini-B) based on the primary sequences of the N- and C-terminal domains. Consistent with theoretical predictions of the full-length protein, both isotope-enhanced Fourier transform infrared (FTIR) spectroscopy and molecular modeling confirm the presence of charged amphipathic alpha-helices in Mini-B. Similar to that observed with native SP-B, Mini-B in model surfactant lipid mixtures exhibits marked in vitro activity, with spread films showing near-zero minimum surface tensions during cycling using captive bubble surfactometry. In vivo, Mini-B shows oxygenation and dynamic compliance that compare favorably with that of full-length SP-B. Mini-B variants (i.e. reduced disulfides or cationic residues replaced by uncharged residues) or Mini-B fragments (i.e. unlinked N- and C-terminal domains) produced greatly attenuated in vivo and in vitro surfactant properties. Hence, the combination of structure and charge for the amphipathic alpha-helical N- and C-terminal domains are key to SP-B function.
Subject(s)
Peptides/pharmacology , Protein Precursors/chemistry , Protein Structure, Secondary , Proteolipids/chemistry , Pulmonary Surfactants/pharmacology , Surface-Active Agents/pharmacology , Amino Acid Sequence , Animals , Molecular Sequence Data , Peptides/chemical synthesis , Pulmonary Surfactants/chemical synthesis , Rats , Spectroscopy, Fourier Transform Infrared , Surface-Active Agents/chemical synthesisABSTRACT
The ability of certain theta-defensins, including retrocyclin-1, to protect human cells from infection by HIV-1 marks them as potentially useful molecules. Theta-defensins composed of L-amino acids are likely to be unstable in environments that contain host and microbial proteases. This study compared the properties of two enantiomeric theta-defensins, retrocyclin-1, and RC-112. Although these peptides have identical sequences, RC-112 is composed exclusively of D-amino acids, whereas retrocyclin-1 contains only L-amino acids. We compared the ability of these peptides to protect JC53-BL human cells from infection by 30 primary HIV-1 isolates. JC53-BL cells are modified HeLa cells that express surface CD4, CXCR4, and CCR5. They also contain reporter cassettes that are driven by the HIV-1 LTR, and express beta-galactosidase and luciferase. The HIV-1 isolates varied in co-receptor specificity and included subtypes A, B, C, D, CRF01-AE, and G. RC-112 was several fold more potent than retrocyclin-1 across the entire HIV-1 panel. Although RC-112 bound immobilized gp120 and CD4 with lower affinity than did retrocyclin-1, surface plasmon resonance experiments performed with 1 microg/mL of RC-112 and retrocyclin-1 revealed that both glycoproteins were bound to a similar extent. The superior antiviral performance of RC-112 most likely reflected its resistance to degradation by surface-associated or secreted proteases of the JC53-BL target cells. Theta-defensins composed exclusively of D-amino acids merit consideration as starting points for designing microbicides for topical application to the vagina or rectum.
Subject(s)
Defensins/chemistry , Defensins/pharmacology , HIV-1/drug effects , Amino Acids/chemistry , Animals , Anti-HIV Agents/pharmacology , Defensins/metabolism , HIV Infections/drug therapy , Humans , StereoisomerismABSTRACT
Endogenous lung surfactant, and lung surfactant replacements used to treat respiratory distress syndrome, can be inactivated during lung edema, most likely by serum proteins. Serum albumin shows a concentration-dependent surface pressure that can exceed the respreading pressure of collapsed monolayers in vitro. Under these conditions, the collapsed surfactant monolayer can not respread to cover the interface, leading to higher minimum surface tensions and alterations in isotherms and morphology. This is an unusual example of a blocked phase transition (collapsed to monolayer form) inhibiting bioactivity. The concentration-dependent surface activity of other common surfactant inhibitors including fibrinogen and lysolipids correlates well with their effectiveness as inhibitors. These results show that respreading pressure may be as important as the minimum surface tension in the design of replacement surfactants for respiratory distress syndrome.
Subject(s)
Pulmonary Surfactants/chemistry , Pulmonary Surfactants/pharmacology , Serum Albumin/pharmacology , Biophysical Phenomena , Biophysics , Dose-Response Relationship, Drug , Fibrinogen/pharmacology , Humans , Immunoglobulin G/metabolism , Infant, Newborn , Lipids/pharmacology , Microscopy, Fluorescence , Respiratory Distress Syndrome, Newborn/metabolism , Temperature , Time FactorsABSTRACT
BACKGROUND: Surfactant protein B (SP-B) is an essential component of pulmonary surfactant, but shorter SP-B sequences may exert equivalent surface activity. METHODS: We synthesised a peptide based on the amino-terminal domain of SP-B (SP-B1-25), a full length SP-B1-78, and a full length palmitoylated SP-C peptide (SP-C1-35) and compared the in vivo function and sensitivity to plasma inhibition of preparations consisting of mixtures of phospholipids with SP-B1-25 or SP-B1-78 and/or SP-C1-35 to Survanta. Preterm rabbits born at 27 days of gestation were treated at birth with surfactant and ventilated for 60 minutes. At 15 minutes half of them received plasma intratracheally. Dynamic compliance was monitored every 15 minutes and postmortem pressure-volume curves were measured to define lung mechanics. RESULTS: Dynamic compliance and postmortem lung volumes were highest after treatment with a surfactant consisting of an SP-B peptide and SP-C1-35 or Survanta. Plasma instillation decreased dynamic compliance and lung volumes sharply, but the most effective activity was by prior instillation of surfactants containing SP-B1-25. CONCLUSION: These experiments suggest that the N-terminal domain of SP-B (SP-B1-25) exhibits in vitro and in vivo surface activity and is relatively insensitive to plasma inhibition.
Subject(s)
Protein Precursors/pharmacology , Proteolipids/pharmacology , Pulmonary Surfactants/pharmacology , Analysis of Variance , Animals , Animals, Newborn , Biomechanical Phenomena , Lung Compliance/drug effects , Protein Precursors/chemistry , Proteolipids/chemistry , Pulmonary Surfactants/chemistry , RabbitsABSTRACT
We purified two new minidefensins (RTD-2 and RTD-3) from the bone marrow of rhesus monkeys. Both were circular octadecapeptides that contained three intramolecular disulfide bonds and were homologous to RTD-1, a circular (theta) defensin previously described by Tang et al. (Science, 286, 498-502, 1999). However, whereas the 18 residues of RTD-1 represent spliced nonapeptide fragments derived from two different demidefensin precursors, RTD-2 and -3 comprise tandem nonapeptide repeats derived from only one of the RTD-1 precursors. Thus, circular minidefensins are products of a novel posttranslational system that generates effector molecule diversity without commensurate genome expansion. A system wherein two demidefensin genes can produce three circular minidefensins might allow n such genes to produce (n/2)(n+1) peptides.
Subject(s)
Defensins/genetics , Defensins/metabolism , Macaca mulatta/metabolism , Amino Acid Sequence , Animals , Bone Marrow/metabolism , Cloning, Molecular , Defensins/chemistry , Disulfides/chemistry , Genetic Variation , Humans , Macaca mulatta/genetics , Molecular Sequence Data , Oxidation-Reduction , Peptides, Cyclic/chemical synthesis , Polymerase Chain Reaction , Protein Processing, Post-Translational , RNA, Messenger/genetics , Sequence Homology, Amino AcidABSTRACT
We recently described a basic technology to efficiently combine compacted DNA with phospholipids and hydrophobic peptides, to produce homogenous complexes that are completely resistant to nuclease. We have developed this technology further to form gene delivery complexes that transfect cells effectively in vitro. In addition to plasmid DNA, the complexes contained two basic components: (i) a DNA compacting peptide (-CGKKKFKLKH), either conjugated to lipid or extended to contain (WLPLPWGW-) and (ii) either phosphatidylethanolamine or phosphatidylcholine. Complexes containing a 5.5-fold charge equivalence (peptide charge/DNA charge) of WLPLPWGWCGKKKFKLKH and 5 nmol dimyristoleoylphosphatidylethanolamine/microg DNA produced the highest luciferase gene expression, exceeding 1 x 10(9) relative light units/s/mg protein (>3 microg luciferase per mg protein). These complexes transfected OVCAR-3, COS-7 and HeLa cells at either similar or superior levels when compared to polyethylenimine or lipofectamine complexes. With green fluorescent protein reporter gene, >50% of HeLa cells were positive 30 h after addition of these complexes. Furthermore, these optimal complexes were the least sensitive to pre-treatment of cells with chloroquine, indicating efficient endosomal escape. Our results indicated that self-assembling complexes of plasmid DNA, amphiphilic peptide and phosphatidylethanolamine are highly effective non-viral gene delivery systems.
Subject(s)
Transfection/methods , Amino Acid Sequence , Animals , COS Cells , Cation Exchange Resins/chemistry , Chloroquine/pharmacology , DNA/chemistry , DNA/genetics , Dose-Response Relationship, Drug , Gene Expression , Glycerophospholipids/chemistry , HeLa Cells , Humans , Lipids/chemistry , Luciferases/drug effects , Luciferases/genetics , Luciferases/metabolism , Molecular Sequence Data , Oligopeptides/chemistry , Phosphatidylcholines/chemistry , Phospholipids/chemistry , Plasmids/chemistry , Plasmids/genetics , Polyethyleneimine/chemistry , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
The human airways are protected from pathogenic colonization by a blanket of fluid impregnated with innate antimicrobial effector molecules. Among several previously uncharacterized components, we isolated a peptide that had activity primarily targeting Gram-negative bacteria. We named the peptide 'calcitermin' since its amino acid sequence and mass were equivalent to the 15 C-terminal residues of the S100 protein, calgranulin C. The antimicrobial activity of calcitermin was enhanced in acidic buffers (pH 5.4) and in the presence of micromolar concentrations of ZnCl(2). Analysis revealed a putative zinc-binding consensus sequence as well as an alpha-helical conformation in structure-promoting solvents.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Nasal Mucosa/metabolism , Peptide Fragments/isolation & purification , S100 Proteins/isolation & purification , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides , Circular Dichroism , Escherichia coli/drug effects , Humans , Hydrogen-Ion Concentration , Listeria monocytogenes/drug effects , Microbial Sensitivity Tests , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Peptides , Protein Conformation , S100 Proteins/chemistry , S100 Proteins/pharmacology , Sequence Homology, Amino Acid , Spectrophotometry, Atomic , Zinc/pharmacologyABSTRACT
Here we report the characterization and optimization of a peptide/non-cationic lipid gene delivery system that successfully produces high levels of gene expression when delivered by microinjection into chicken embryos in vivo. In addition to plasmid DNA, the delivery complex consisted of four components: 1) a "condensing" peptide with both hydrophobic and cationic amino acid segments; 2) a "fusogenic" peptide with both membrane insertion and amphipathic helical segments; 3) a relatively short-chain phosphatidylcholine (14:1 cis-9); and 4) polyethyleneglycol conjugated to dioleoylphosphatidylethanolamine through a disulfide linkage. Optimum amounts of each component were determined by measuring expression of a luciferase reporter gene following a 24-hour incubation with chick embryo fibroblast (CEF) cells in culture. When relatively low amounts of condensing peptide, fusogenic peptide, or lipid were assembled into the complexes, relatively large concentrations of complex were required to reach maximum gene expression. When the amounts of peptide or lipid were increased, less complex was required to achieve maximum expression, but expression fell substantially with higher amounts of added complex. The polyethyleneglycol component significantly increased gene expression. With some preparations, luciferase activities in the CEF cells reached 1x10(10) relative light units per second per mg protein within 24 hours. Following the optimization experiments with the CEF cells, formulations containing low levels, intermediate levels, and high levels of the delivery system components were assembled with green fluorescent protein plasmid DNA, then microinjected into somite regions of chicken embryos in vivo. It was found that intermediate levels of the components gave the most reliable formulations for inducing localized gene expression in the somitic cells.
Subject(s)
Gene Transfer Techniques , Lipids , Microinjections , Peptides , Animals , Cells, Cultured , Chick Embryo , Disulfides , Fibroblasts , Gene Expression , Genes, Reporter , Luciferases/genetics , Luciferases/metabolism , Phosphatidylcholines/administration & dosage , Phosphatidylcholines/chemistry , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/administration & dosage , Phosphatidylethanolamines/chemistry , Phosphatidylethanolamines/metabolism , Plasmids/administration & dosage , Plasmids/metabolism , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/chemistry , Polyethylene Glycols/metabolism , Structure-Activity Relationship , TransfectionABSTRACT
Langmuir isotherms, fluorescence microscopy, and atomic force microscopy were used to study lung surfactant specific proteins SP-B and SP-C in monolayers of dipalmitoylphosphatidylglycerol (DPPG) and palmitoyloleoylphosphatidylglycerol (POPG), which are representative of the anionic lipids in native and replacement lung surfactants. Both SP-B and SP-C eliminate squeeze-out of POPG from mixed DPPG/POPG monolayers by inducing a two- to three-dimensional transformation of the fluid-phase fraction of the monolayer. SP-B induces a reversible folding transition at monolayer collapse, allowing all components of surfactant to remain at the interface during respreading. The folds remain attached to the monolayer, are identical in composition and morphology to the unfolded monolayer, and are reincorporated reversibly into the monolayer upon expansion. In the absence of SP-B or SP-C, the unsaturated lipids are irreversibly lost at high surface pressures. These morphological transitions are identical to those in other lipid mixtures and hence appear to be independent of the detailed lipid composition of the monolayer. Instead they depend on the more general phenomena of coexistence between a liquid-expanded and liquid-condensed phase. These three-dimensional monolayer transitions reconcile how lung surfactant can achieve both low surface tensions upon compression and rapid respreading upon expansion and may have important implications toward the optimal design of replacement surfactants. The overlap of function between SP-B and SP-C helps explain why replacement surfactants lacking in one or the other proteins often have beneficial effects.
Subject(s)
Lung , Membranes, Artificial , Phospholipids/chemistry , Phospholipids/metabolism , Proteolipids/metabolism , Pulmonary Surfactants/metabolism , Amino Acid Sequence , Anions/metabolism , Humans , Microscopy, Atomic Force , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , Phosphatidylglycerols/chemistry , Phosphatidylglycerols/metabolism , Protein Binding , Proteolipids/chemistry , Proteolipids/genetics , Pulmonary Surfactants/chemistry , Pulmonary Surfactants/genetics , Surface Tension , TemperatureABSTRACT
This work reports the first x-ray scattering measurements to determine the effects of SP-B(1-25), the N-terminus peptide of lung surfactant-specific protein SP-B, on the structure of palmitic acid (PA) monolayers. In-plane diffraction shows that the peptide fluidizes a portion of the monolayer but does not affect the packing of the residual ordered phase. This implies that the peptide resides in the disordered phase, and that the ordered phase is essentially pure lipid, in agreement with fluorescence microscopy studies. X-ray reflectivity shows that the peptide is oriented in the lipid monolayer at an angle of approximately 56 degrees relative to the interface normal, with one end protruding past the hydrophilic region into the fluid subphase and the other end embedded in the hydrophobic region of the monolayer. The quantitative insights afforded by this study lead to a better understanding of the lipid/protein interactions found in lung surfactant systems.
Subject(s)
Lipid Metabolism , Membranes, Artificial , Proteolipids/chemistry , Proteolipids/metabolism , Pulmonary Surfactants/chemistry , Pulmonary Surfactants/metabolism , X-Ray Diffraction/instrumentation , X-Ray Diffraction/methods , Amino Acid Sequence , Molecular Conformation , Molecular Sequence Data , Palmitic Acid/metabolism , Protein Structure, Tertiary , Sodium Chloride/metabolism , Synchrotrons , Temperature , Water/metabolismABSTRACT
Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome.
Subject(s)
Lung/metabolism , Palmitic Acid/pharmacology , Proteolipids/metabolism , Pulmonary Surfactants/metabolism , Surface-Active Agents/chemistry , Adsorption , Amino Acid Sequence , Animals , Biophysical Phenomena , Biophysics , Cattle , Chromatography, High Pressure Liquid , Electromagnetic Phenomena , Humans , Membranes, Artificial , Microscopy, Atomic Force , Microscopy, Fluorescence , Molecular Sequence Data , Peptides/chemistry , Protein Folding , TemperatureABSTRACT
Structural and functional studies were performed to assess the membrane actions of peptides based on HIV-1 glycoprotein 41,000 (gp41). Previous site-directed mutagenesis of gp41 has shown that amino acid changes in either the N-terminal fusion or N-leucine zipper region depressed viral infection and syncytium formation, while modifications in the C-leucine zipper domain both increased and decreased HIV fusion. Here, synthetic peptides were prepared corresponding to the N-terminal fusion region (FP-I; gp41 residues 519-541), the nearby N-leucine zipper domain (DP-107; gp41 residues 560-597), and the C-leucine zipper domain (DP-178; gp41 residues 645-680). With erythrocytes, FP-I or DP-107 induced dose-dependent hemolysis and promoted cell aggregation; FP-I was more hemolytic than DP-107, but each was equally effective in aggregating cells. DP-178 produced neither hemolysis nor aggregation, but blocked either FP-I- or DP-107-induced hemolysis and aggregation. Combined with previous nuclear magnetic resonance and Fourier transform infrared spectroscopic results, circular dichroism (CD) spectroscopy showed that the alpha-helicity for these peptides in solution decreased in the order: DP-107 >> DP-178 > FP-I. CD analysis also indicated binding of DP-178 to either DP-107 or FP-I. Consequently, DP-178 may inhibit the membrane actions mediated by either FP-I or DP-107 through direct peptide interactions in solution. These peptide results suggest that the corresponding N-terminal fusion and N-leucine zipper regions participate in HIV infection, by promoting membrane perturbations underlying the merging of the viral envelope with the cell surface. Further, the C-leucine zipper domain in "prefusion" HIV may inhibit these membrane activities by interacting with the N-terminal fusion and N-leucine zipper domains in unactivated gp41. Last, exogenous DP-178 may bind to the N-terminal and N-leucine zipper domains of gp41 that become exposed on HIV stimulation, thereby preventing the fusogenic actions of these gp41 regions leading to infection.
Subject(s)
Erythrocytes/drug effects , HIV Envelope Protein gp41/chemistry , Membrane Fusion/drug effects , Peptides/chemical synthesis , Peptides/pharmacology , Amino Acid Sequence , Circular Dichroism , Erythrocyte Aggregation/drug effects , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/metabolism , Hemolysis/drug effects , Humans , Molecular Sequence Data , Peptides/chemistryABSTRACT
Cysteine-rich antimicrobial peptides are abundant in animal and plant tissues involved in host defense. In insects, most are synthesized in the fat body, an organ analogous to the liver of vertebrates. From human urine, we characterized a cysteine-rich peptide with three forms differing by amino-terminal truncation, and we named it hepcidin (Hepc) because of its origin in the liver and its antimicrobial properties. Two predominant forms, Hepc20 and Hepc25, contained 20 and 25 amino acid residues with all 8 cysteines connected by intramolecular disulfide bonds. Reverse translation and search of the data bases found homologous liver cDNAs in species from fish to human and a corresponding human genomic sequence on human chromosome 19. The full cDNA by 5' rapid amplification of cDNA ends was 0.4 kilobase pair, in agreement with hepcidin mRNA size on Northern blots. The liver was the predominant site of mRNA expression. The encoded prepropeptide contains 84 amino acids, but only the 20-25-amino acid processed forms were found in urine. Hepcidins exhibited antifungal activity against Candida albicans, Aspergillus fumigatus, and Aspergillus niger and antibacterial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and group B Streptococcus. Hepcidin may be a vertebrate counterpart of cysteine-rich antimicrobial peptides produced in the fat body of insects.
Subject(s)
Anti-Infective Agents/isolation & purification , Antimicrobial Cationic Peptides/isolation & purification , Liver/metabolism , Amino Acid Sequence , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Cloning, Molecular , Hepcidins , Humans , Molecular Sequence DataABSTRACT
We cloned the precursor of a novel peptide from a cDNA library prepared from pharyngeal tissues of the tunicate, Styela clava. Its sequence predicted a histidine-rich, amidated 23-residue peptide (FLRF(IG)SVIHGIGHLVHHIGVAL-NH2) that we named clavaspirin. A synthetic clavaspirin was prepared and it was found that it killed Gram-positive and Gram-negative bacteria, permeabilized the outer and inner membranes of Escherichia coli, lysed phosphatidylglycerol (POPG) liposomes, and was potently haemolytic towards human and bovine erythrocytes. Each of these activities was performed more effectively at an acidic pH. Circular dichroism measurements of synthetic clavaspirin revealed a largely alpha-helical structure and polarized and residue-specific FTIR spectrometry showed that its association with phospholipid membranes was influenced by pH. Peptides such as clavaspirin may equip tunicate haemocytes to mediate cytotoxicity and participate in antimicrobial defence.
Subject(s)
Anti-Bacterial Agents/pharmacology , Peptides/genetics , Peptides/pharmacology , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Bacteria/drug effects , Base Sequence , Blood Proteins/chemistry , Blotting, Northern , Cell Membrane Permeability , Circular Dichroism , Cloning, Molecular , Hemolysis , Humans , Liposomes/metabolism , Microbial Sensitivity Tests/methods , Molecular Sequence Data , Peptides/chemistry , Sequence Homology, Amino Acid , Spectroscopy, Fourier Transform Infrared , UrochordataABSTRACT
Mammalian lung surfactant is a mixture of phospholipids and four surfactant-associated proteins (SP-A, SP-B, SP-C, and SP-D). Its major function is to reduce surface tension at the air-water interface in the terminal airways by the formation of a surface-active film highly enriched in dipalmitoyl phosphatidylcholine (DPPC), thereby preventing alveolar collapse during expiration. SP-A and SP-D are large hydrophilic proteins, which play an important role in host defense, whereas the small hydrophobic peptides SP-B and SP-C interact with DPPC to generate and maintain a surface-active film. Surfactant replacement therapy with bovine and porcine lung surfactant extracts, which contain only polar lipids and SP-B and SP-C, has revolutionized the clinical management of premature infants with respiratory distress syndrome. Newer surfactant preparations will probably be based on SP-B and SP-C, produced by recombinant technology or peptide synthesis, and reconstituted with selected synthetic lipids. The development of peptide analogues of SP-B and SP-C offers the possibility to study their molecular mechanism of action and will allow the design of surfactant formulations for specific pulmonary diseases and better quality control. This review describes the hydrophobic peptide analogues developed thus far and their potential for use in a new generation of synthetic surfactant preparations.
Subject(s)
Proteolipids/chemistry , Pulmonary Surfactants/chemistry , Amino Acid Sequence , Animals , Cattle , Drug Design , Humans , Infant, Newborn , Models, Molecular , Molecular Sequence Data , Proteolipids/genetics , Proteolipids/pharmacology , Pulmonary Surfactants/genetics , Pulmonary Surfactants/pharmacology , Respiratory Distress Syndrome, Newborn/drug therapyABSTRACT
A difficult problem concerning the interaction of DNA with amphiphiles of opposite charge above their critical micelle concentration is the propensity for aggregation of the condensed DNA complexes. In this study, this problem was addressed by attenuating amphiphile charge density within a cholate micelle environment. The amphiphile consisted of a cationic peptide, acetyl-CWKKKPKK-amide, conjugated to dilaurylphos-phatidylethanolamine. In the presence of cholate, multiple equivalents of cationic charge were required to bring about the completion of DNA condensation. At the end point of condensation, stable, soluble DNA-micelle complexes were formed, which by dynamic light scattering exhibited apparent hydro-dynamic diameters between 30 and 60 nm. Aggregation, as measured by static light scattering at 90 degrees and by turbidity, was not observed until further additions of peptide-lipid conjugate were made beyond the end point of DNA condensation. Liposome complexes containing the non-aggregated, compacted DNA were formed by adding dioleoylphosphatidylcholine followed by removing the cholate by dialysis. The resulting complexes were distributed within a narrow density range, the DNA was quantitatively assembled into the liposomes, and liposomes without DNA were not detected. Small particles were formed with a mean hydrodynamic diameter of 77 nm. The liposomal DNA showed complete retention of its supercoiled form and no detectable sensitivity to DNase (25 U/10 microg DNA, 1.5 h, 37 degrees C). The use of an anionic, dialyzable amphiphile to attenuate charge inter-actions between DNA and cationic amphiphiles is a useful technology for the quantitative assembly of compacted DNA into conventional liposomes, with complete protection against nuclease activity.
Subject(s)
DNA/chemistry , Liposomes/chemistry , Micelles , Phosphatidylcholines , Anions , Cations , Chemical Phenomena , Chemistry, Physical , Cholates/pharmacology , Light , Peptides/chemistry , Phosphatidylcholines/chemistry , Scattering, RadiationABSTRACT
Surfactant protein B (SP-B) is a 17-kDa dimeric protein produced by alveolar type II cells. Its main function is to lower the surface tension by inserting lipids into the air/liquid interface of the lung. SP-B's function can be mimicked by a 25-amino acid peptide, SP-B(1-25), which is based on the N-terminal sequence of SP-B. We synthesized a dimeric version of this peptide, dSP-B(1-25), and the two peptides were tested for their surface activity. Both SP-B(1-25) and dSP-B(1-25) showed good lipid mixing and adsorption activities. The dimeric peptide showed activity comparable to that of native SP-B in the pressure-driven captive bubble surfactometer. Spread surface films led to stable near-zero minimum surface tensions during cycling while protein free, and films containing SP-B(1-25) lost material from the interface during compression. We propose that dimerization of the peptide is required to create a lipid reservoir attached to the monolayer from which new material can enter the surface film upon expansion of the air/liquid interface. The dimeric state of SP-B can fulfill the same function in vivo.
Subject(s)
Peptide Fragments/chemistry , Proteolipids/chemistry , Pulmonary Surfactants/chemistry , 1,2-Dipalmitoylphosphatidylcholine/chemistry , Amino Acid Sequence , Calcium/chemistry , Dimerization , Fluorescence , Humans , Lipids/chemistry , Liposomes/chemistry , Membranes, Artificial , Molecular Sequence Data , Phosphatidylcholines/chemistry , Phosphatidylglycerols/chemistry , Solubility , Surface Properties , Surface TensionABSTRACT
Synthetic peptides based on the N-terminal domain of human surfactant protein B (SP-B1-25; 25 amino acid residues; NH2-FPIPLPYCWLCRALIKRIQAMIPKG) retain important lung activities of the full-length, 79-residue protein. Here, we used physical techniques to examine the secondary conformation of SP-B1-25 in aqueous, lipid and structure-promoting environments. Circular dichroism and conventional, 12C-Fourier transform infrared (FTIR) spectroscopy each indicated a predominate alpha-helical conformation for SP-B1-25 in phosphate-buffered saline, liposomes of 1-palmitoyl-2-oleoyl phosphatidylglycerol and the structure-promoting solvent hexafluoroisopropanol; FTIR spectra also showed significant beta- and random conformations for peptide in these three environments. In further experiments designed to map secondary structure to specific residues, isotope-enhanced FTIR spectroscopy was performed with 1-palmitoyl-2-oleoyl phosphatidylglycerol liposomes and a suite of SP-B1-25 peptides labeled with 13C-carbonyl groups at either single or multiple sites. Combining these 13C-enhanced FTIR results with energy minimizations and molecular simulations indicated the following model for SP-B1-25 in 1-palmitoyl-2-oleoyl phosphatidylglycerol: beta-sheet (residues 1-6), alpha-helix (residues 8-22) and random (residues 23-25) conformations. Analogous structural motifs are observed in the corresponding homologous N-terminal regions of several proteins that also share the 'saposin-like' (i.e. 5-helix bundle) folding pattern of full-length, human SP-B. In future studies, 13C-enhanced FTIR spectroscopy and energy minimizations may be of general use in defining backbone conformations at amino acid resolution, particularly for peptides or proteins in membrane environments.