Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.     

Solution structures and model membrane interactions of lactoferrampin, an antimicrobial peptide derived from bovine lactoferrin

Bovine lactoferrampin (LFampinB) has been identified as a novel antimicrobial peptide, which is derived from the N-terminal lobe of bovine lactoferrin. In this study, the solution structure of LFampinB bound to negatively charged sodium dodecyl sulphate micelles and zwitterionic dodecyl phosphocholine micelles was determined using 2-dimensional nuclear magnetic resonance (NMR) spectroscopy. The interaction between LFampinB and multilamellar phospholipid vesicles, containing choline and glycerol head groups, was examined using differential scanning calorimetry (DSC). In addition, the interaction between the N-terminal tryptophan residue and model membranes of varying composition was analyzed by fluorescence spectroscopy. LFampinB adopts an amphipathic alpha-helical conformation across the first 11 residues of the peptide but remains relatively unstructured at the C-terminus. The hydrophobic surface of the amphipathic helix is bordered by the side chains of Trp1 and Phe11, and is seen in both micelle-bound structures. The fluorescence results suggest that Trp1 inserts into the membrane at the lipid/water interface. The phenyl side chain of Phe11 is oriented in the same direction as the indole ring of Trp1, allowing these two residues to serve as anchors for the lipid bilayer. The DSC results also indicate that LFampinB interacts with glycerol head groups in multilamellar vesicles but has little effect on acyl chain packing. Our results support a two step model of antimicrobial activity where the initial attraction of LFampinB is mediated by the cluster of positive charges on the C-terminus followed by the formation of the N-terminal helix which binds to the surface of the bacterial lipid bilayer.     

Structure-activity analysis of the dermcidin-derived peptide DCD-1L, an anionic antimicrobial peptide present in human sweat

Dermcidin encodes the anionic amphiphilic peptide DCD-1L, which displays a broad spectrum of antimicrobial activity under conditions resembling those in human sweat. Here, we have investigated its mode of antimicrobial activity. We found that DCD-1L interacts preferentially with negatively charged bacterial phospholipids with a helix axis that is aligned flat on a lipid bilayer surface. Upon interaction with lipid bilayers DCD-1L forms oligomeric complexes that are stabilized by Zn(2+). DCD-1L is able to form ion channels in the bacterial membrane, and we propose that Zn(2+)-induced self-assembly of DCD-1L upon interaction with bacterial lipid bilayers is a prerequisite for ion channel formation. These data allow us for the first time to propose a molecular model for the antimicrobial mechanism of a naturally processed human anionic peptide that is active under the harsh conditions present in human sweat.     

Structure-activity analysis of SMAP-29, a sheep leukocytes-derived antimicrobial peptide

SAMP-29 is a cathelecidin-derived antimicrobial peptide deduced from sheep myeloid mRNA. To elucidate the structural-activity relationship of SMAP-29, several analogues were synthesized and their antibiotic activity was investigated. Compared to parental SMAP-29, SMAP-29(1-17) and [K(22,25,27)]-SMAP-29 retained relatively effective antimicrobial activity (MIC: 1.0-8.0 microM), but resulted in a complete loss of hemolytic activity. Pro-19 --> Ala substitution ([A19]-SMAP-29) in SMAP-29 induced a significant reduction in antibacterial activity. These results suggested that the N-terminal amphipathic alpha-helical region and the C-terminal hydrophobic region of SMAP-29 are responsible for antimicrobial activity and hemolytic activity, respectively, and the central Pro-19 in SMAP-29 plays a critical role in showing improved antibacterial activity. In particular, [K(2,7,13)]-SMAP-29(1-17) showed potent antimicrobial activity under high salt conditions without hemolytic activity. Thus, this short peptide could serve as an attractive candidate for the development of therapeutic antimicrobial drugs. Structural analysis by circular dichroism suggested that SMAP-29 seems to adopt a helix-bend/turn-extended random conformation.     

The micelle-bound structure of an antimicrobial peptide derived from the alpha-chain of bovine hemoglobin isolated from the tick Boophilus microplus

Hemoglobin is known to be a source of peptides involved in several functions. The peptide FLSFPTTKTYFPHFDLSHGSAQVKGHGAK (Hb33-61) is a proteolytic product of the bovine hemoglobin alpha-chain found in the gut content of the cattle tick, Boophilus microplus, and it possesses antimicrobial activity. Since in the past we showed that the amidated form of Hb33-61, Hb33-61a, is active against a few Gram-positive bacteria and fungi strains at micromolar concentration [Fogaca et al. (1999) J. Biol. Chem. 274, 25330-25334], we have been prompted to shed more light on its functional and structural features. Here we show that the peptide is able to disrupt the bacterial membrane ofMicrococcus luteus A270. As for its structure, it has a random conformation in water, and it does not interact with zwitterionic micelles. On the other hand, it binds to negatively charged micelles acquiring a finite structural organization. The 3D structure of Hb33-61a bound to SDS micelles exhibits a nonconventional conformation for an antimicrobial peptide. The backbone is characterized by the presence of a beta-turn in the N-terminus and by a beta-turn followed by a alpha-helical stretch in the C-terminus. A hinge, whose spatial organization is stabilized by side-chain-side-chain interactions, joins these two regions. Interestingly, it preserves structural features present in the corresponding segment of the bovine hemoglobin alpha-chain. Hb33-61a does not possess a well-defined amphipathic nature, and H/D exchange experiments show that while the C-terminal region is embedded in the SDS micelle, one face of the N-terminal half is partly exposed to the solvent.     

Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma

The protein heterogeneity of fractions isolated by immunoaffinity chromatography on anti-apolipoprotein A-I and anti-apolipoprotein A-II affinity columns was analyzed by high resolution two-dimensional gel electrophoresis. The two-dimensional gel electrophoresis profiles of the fractions were analyzed and automatically compared by the computer system MELANIE. Fractions containing apolipoproteins A-I + A-II and only A-I as the major protein components have been isolated from plasma and from high density lipoproteins prepared by ultracentrifugation. Similarities between the profiles of the fractions, as indicated by two-dimensional gel electrophoresis, suggested that those derived from plasma were equivalent to those from high density lipoproteins (HDL), which are particulate in nature. The established apolipoproteins (A-I, A-II, A-IV, C, D, and E) were visible and enriched in fractions from both plasma and HDL. However, plasma-derived fractions showed a much greater degree of protein heterogeneity due largely to enrichment in bands corresponding to six additional proteins. They were present in trace amounts in fractions isolated from HDL and certain of the proteins were visible in two-dimensional gel electrophoresis profiles of the plasma. These proteins are considered to be specifically associated with the immunoaffinity-isolated particles. They have been characterized in terms of Mr and pI. Computer-assisted measurements of protein spot-staining intensities suggest an asymmetric distribution of the proteins (as well as the established apolipoproteins), with four showing greater prominence in particles containing apolipoprotein A-I but no apolipoprotein A-II.     

In vivo antimicrobial evaluation of an alanine-rich peptide derived from Pleuronectes americanus

In several organisms, the first barrier against microbial infections consists of antimicrobial peptides (AMPs) which are molecules that act as components of the innate immune system. Recent studies have demonstrated that AMPs can perform various functions in different tissues or physiological conditions. In this view, this study was carried out in order to evaluate the multifunctional activity in vivo of an alanine-rich peptide, known as Pa-MAP, derived from the polar fish Pleuronectes americanus. Pa-MAP was evaluated in intraperitoneally infected mice with a sub-lethal concentration of Escherichia coli at standard concentrations of 1 and 5 mg kg⁻¹. At both concentrations, Pa-MAPs exhibited an ability to prevent E. coli infection and increase mice survival, similar to the result observed in mice treated with ampicillin at 2 mg kg⁻¹. In addition, mice were monitored for weight loss. The results showed that mice treated with Pa-MAPs at 1 mg kg⁻¹ gained 0.8% of body weight during the 72 h of experiment. The same was observed with Pa-MAP at 5 mg kg⁻¹, which had a gain of 0.5% in body weight during the treatment. Mice treated with ampicillin at 2 mg kg⁻¹ show a significant weight loss of 5.6% of body weight. The untreated group exhibited a 5.5% loss of body weight. The immunomodulatory effects were also evaluated by the quantification of IL-10, IL-12, TNF-α, IFN-γ and nitric oxide cytokines in serum, but no immunomodulatory activity was observed. Data presented here suggest that Pa-MAP should be used as a novel antibiotic against infection control.     

Activity optimization of an undecapeptide analogue derived from a frog-skin antimicrobial peptide

While natural antimicrobial peptides are potential therapeutic agents, their physicochemical properties and bioactivity generally need to be enhanced for clinical and commercial development. We have previously developed a cationic, amphipathic α-helical, 11-residue peptide (named herein GA-W2: FLGWLFKWASK-NH₂) with potent antimicrobial and hemolytic activity, which was derived from a 24-residue, natural antimicrobial peptide isolated from frog skin. Here, we attempted to optimize peptide bioactivity by a rational approach to sequence modification. Seven analogues were generated from GA-W2, and their activities were compared with that of a 12-residue peptide, omiganan, which is being developed for clinical and commercial applications. Most of the modifications reported here improved antimicrobial activity. Among them, the GA-K4AL (FAKWAFKWLKK-NH₂) peptide displayed the most potent antimicrobial activity with negligible hemolytic activity, superior to that of omiganan. The therapeutic index of GA-K4AL was improved more than 53- and more than 31-fold against Gram-negative and Gram-positive bacteria, respectively, compared to that of the starting peptide, GA-W2. Given its relatively shorter length and simpler amino acid composition, our sequence-optimized GA-K4AL peptide may thus be a potentially useful antimicrobial peptide agent.     

Neurite outgrowth-stimulating peptide derived from bovine kappa-casein

We found a novel peptide that stimulates neurite outgrowth in Neuro-2a mouse neuroblastoma cells, from the pepsin-pancreatin digest of bovine kappa-casein. The amino acid sequence of this peptide is Phe-Leu-Pro-Tyr-Pro-Tyr (FLPYPY), corresponding to peptidic sequence 76-81 of bovine kappa-casein. The neurite outgrowth-stimulating activity of FLPYPY was seen over 10(-9) M. On the other hand, FLPYP and FLPYPYY, which corresponded to sequences 76-80 and 76-82 of bovine kappa-casein respectively, were ineffective.     

New antibacterial peptide derived from bovine hemoglobin

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration.     

Peptidomics-based discovery of an antimicrobial peptide derived from insulin-like growth factor-binding protein 5

Antimicrobial peptides (AMPs) are effector molecules that are able to kill or inactivate microbial pathogens. However, most AMPs harbor multiple basic amino acids that hamper current proteomic identification. In our peptidomic survey of endogenous peptides, we identified a novel intramolecular disulfide-linked 22-residue amidated peptide. This peptide, designated AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5), showed antimicrobial activity against six of the eight microorganisms tested at concentrations comparable to or lower than those for well-characterized AMPs cathelicidin and β-defensin-2. AMP-IBP5 is identical at the amino acid level between human, mouse, rat, pig, and cow. Natural occurrence of this peptide as the originally isolated form was demonstrated in the rat brain and intestine, using mass spectrometric characterization of major immunoreactivity. The peptide is flanked N-terminally by a single arginine and C-terminally by a common amidation signal, indicating that insulin-like growth factor-binding protein 5 (IGFBP-5) undergoes specific cleavage by a defined set of processing proteases. Furthermore, the intramolecular linkage C199-C210 reveals itself as a correct disulfide pairing in the precursor protein, the finding not inferred from closely related family members IGFBP-4 and -6. In principle, neither conventional proteomics nor bioinformatics would achieve the identification of this AMP. Our study exemplifies the impact of peptidomics to study naturally occurring peptides.     

The cathelicidin-like peptide derived from panda genome is a potential antimicrobial peptide

A novel cathelicidin-like antimicrobial peptide was identified by mining genome of panda. This peptide (cathelicidin-AM) was synthesized. It showed potential antimicrobial activities against wide spectrum of microorganisms including Gram-negative and -positive bacteria, and fungi. It had similar antimicrobial abilities against both standard and clinically isolated drug-resistant strains. Cathelicidin-AM could rapidly exert its antibacterial activities. It just took less than 1 h to kill all Staphylococcus sciuri at the concentration of 2, 4 or 10 times of minimal inhibitory concentration (MIC) while clindamycin took 6 h. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis indicated that cathelicidin-AM killed bacteria by directly affecting bacterial cell wall and membrane. Phylogenetic analysis revealed that the panda cathelicidin had the nearest evolution relationship with dog cathelicidin. The current work provides a novel cathelicidin-like peptide with strong antimicrobial abilities.     

Molecular dynamics simulations of a fibrillogenic peptide derived from apolipoprotein C-II

The pathway to amyloid fibril formation in proteins involves specific structural changes leading to the combination of misfolded intermediates into oligomeric assemblies. Recent NMR studies showed the presence of “turns” in amyloid peptides, indicating that turn formation may play an important role in the nucleation of the intramolecular folding and possible assembly of amyloid. Fully solvated all-atom molecular dynamics simulations were used to study the structure and dynamics of the apolipoprotein C-II peptide 56 to 76, associated with the formation of amyloid fibrils. The peptide populated an ensemble of turn structures, stabilized by hydrogen bonds and hydrophobic interactions enabling the formation of a strong hydrophobic core which may provide the conditions required to initiate aggregation. Two competing mechanisms discussed in the literature were observed. This has implications in understanding the mechanism of amyloid formation in not only apoC-II and its fragments, but also in other amyloidogenic peptides.     

A one-enzyme strategy to release an antimicrobial peptide from the LFampin-domain of bovine lactoferrin

Antimicrobial peptides have been found throughout living nature, yet antimicrobial sequences may still lie hidden within a wide variety of proteins. A rational strategy was developed to select interesting domains, based on the presumed common features of antimicrobial peptides, and to release these from accessible and safe proteins. In silico proteolysis simulations of bovine lactoferrin (bLF) with selected endoproteinases predicted the liberation of peptides that encompasses a cationic amphipathic alpha-helix. Three predicted peptides were synthesized and tested for their biological activity, demonstrating that one single enzyme was sufficient to obtain an antimicrobial peptide. The proof of principle demonstrated that a 32-mer fragment isolated from the endoproteinase AspN digestion of bLF possessed strong antimicrobial activity. Moreover, desalted crude digest had improved activity over native bLF. Hence, selective digestion of bLF increases its antimicrobial activity by release of antimicrobial stretches.     

Structure-activity relationships of piscidin 4, a piscine antimicrobial peptide

Piscidin 4, an antimicrobial peptide recently isolated from mast cells of hybrid striped bass (Morone chrysops female × Morone saxatilis male), is unusual in that it is twice as long (44 amino acids) as the typical members of the piscidin family. We previously showed that native piscidin 4 had a modified amino acid at position 20, but synthetic piscidin 4 (having an unmodified Trp at position 20) had similar potent activity against a number of both human and fish bacterial pathogens. In this study, the structure and membrane topology of synthetic piscidin 4 were examined using liposomes as model bilayers. Circular dichroism analyses revealed that it had a disordered structure in aqueous solution and folded to form a relatively weak α-helical structure in both membrane-mimetic trifluoroethanol solutions and liposome suspensions. Fluorescence data (piscidin 4 embedded in liposomes) and leakage experiments indicated that piscidin 4 interacted strongly with the hydrophobic part of the liposome. Binding of piscidin 4 to liposomes induced significant blue shifts of the emission spectra of the single Trp residue (Trp20). Quenching of Trp20 by water-soluble quencher (either acrylamide or I-) indicated that the fluorescence of Trp20 decreased more in the presence of liposomes than in buffer solution, thus revealing that Trp20 is less accessible to the quenchers in the presence of liposomes. The relative leakage abilities of piscidin 4 (1 μM) with liposomes were in the following order: DPPC (100%)≥EYPC (94%)>DPPC/DPPG (65%)>EYPC/EYPG (0%). This high activity against DPPC and EYPC liposomes was contrary to our data suggesting that piscidin 4 has a much weaker tendency to form an α-helix than other piscidins, such as piscidin 1. However, the structural similarity of protozoan membranes to EYPC liposomes might explain our discovery of the potent activity of piscidin 4 against the important skin/gill parasite ich (Ichthyophthirius multifiliis), but its negligible hemolytic activity against vertebrate membranes (hybrid striped bass or human erythrocytes). It also suggests that other conformation(s) in addition to the α-helix of this peptide may be responsible for its selective activity. This differential toxicity also suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass and may be capable of functioning extracellularly.     

Interaction between tachyplesin I,an antimicrobial peptide derived from horseshoe crab,and lipopolysaccharide

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program.CD and NMR measurements revealed that binding to LPS slightly extends the two β-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS.     

Structure-activity relationships for the beta-hairpin cationic antimicrobial peptide polyphemusin I

The solution structure of polyphemusin I was determined using (1)H-NMR spectroscopy. Polyphemusin I was found to be an amphipathic, beta-hairpin connected by a type I' beta-turn. The 17 low-energy structures aligned very well over the beta-sheet region while both termini were poorly defined due in part to a hinge-like region centred in the molecule about arginine residues 6 and 16. Conversely, a linear analogue, PM1-S, with all cysteines simultaneously replaced with serine was found to be dynamic in nature, and a lack of medium and long-range NOEs indicated that this molecule displayed no favoured conformation. Circular dichroism (CD) spectroscopy confirmed that in solution, 50% trifluoroethanol (TFE) and in the presence of liposomes, PM1-S remained unstructured. The antimicrobial activity of PM1-S was found to be 4- to 16-fold less than that of polyphemusin I and corresponded with a 4-fold reduction in bacterial membrane depolarization. Both peptides were able to associate with lipid bilayers in a similar fashion; however, PM1-S was completely unable to translocate model membranes while polyphemusin I retained this activity. It was concluded that the disulfide-constrained, beta-sheet structure of polyphemusin I is required for maximum antimicrobial activity. Disruption of this structure results in reduced antimicrobial activity and completely abolishes membrane translocation indicating that the linear PM1-S acts through a different antimicrobial mechanism.     

A novel peptide derived from human apolipoprotein E is an inhibitor of tumor growth and ocular angiogenesis

Angiogenesis is a hallmark of tumor development and metastasis and now a validated target for cancer treatment. We previously reported that a novel dimer peptide (apoEdp) derived from the receptor binding region of human apolipoprotein E (apoE) inhibits virus-induced angiogenesis. However, its role in tumor anti-angiogenesis is unknown. This study demonstrates that apoEdp has anti-angiogenic property in vivo through reduction of tumor growth in a mouse model and ocular angiogenesis in a rabbit eye model. Our in vitro studies show that apoEdp inhibits human umbilical vein endothelial cell proliferation, migration, invasion and capillary tube formation. We document that apoEdp inhibits vascular endothelial growth factor-induced Flk-1 activation as well as downstream signaling pathways that involve c-Src, Akt, eNOS, FAK, and ERK1/2. These in vitro data suggest potential sites of the apoE dipeptide inhibition that could occur in vivo.This is the first evidence that a synthetic dimer peptide mimicking human apoE has anti-angiogenesis functions and could be an anti-tumor drug candidate.     

Identification of antimicrobial peptide regions derived from genomic sequences of phage lysins

This study was designed to test the possibility that antimicrobial peptides could be derived from the genomic sequences of phage lysins. Using two lysins (D3 and PhiKZ) we selected and produced two putative peptides (X and Z, respectively) believed to possess antimicrobial properties based on their physicochemical characteristics. The data presented support this hypothesis in that the peptides and various analogs displayed antibacterial activity, bacteriostatic or bactericidal, either individually or upon combination. These putative peptides are believed to act by a mechanism of action resembling that of conventional antimicrobial peptides when judged by both structural and functional criteria. Thus, the peptides are shown to have the ability to form a helical structure, to bind to model bacterial membranes and permeabilize model liposomes. They also display rapid bactericidal kinetics and their antibacterial potency is increased upon amidation. The possible relevance of these results in contributing to potency of phage lysins is discussed. Such peptides may be used to design new potent antimicrobial compounds much needed in face of the ever threatening drug resistance problems.