Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.     

Structure-activity relationship of an antibacterial peptide, maculatin 1.1, from the skin glands of the tree frog, Litoria genimaculata.

Maculatin 1.1 (Mac) is a cationic antibacterial peptide isolated from the dorsal glands of the tree frog, Litoria genimaculata, and has a sequence of GLFGVLAKVAAHVVPAIAEHF-NH2. A short peptide lacking the N-terminal two residues of Mac was reported to have no activity. To investigate the structure-activity relationship in detail, several analogs and related short peptides of Mac were synthesized. CD measurement showed that all the peptides took more or less an alpha-helical structure in the presence of anionic lipid vesicles. Analogs which are more basic than Mac had strong antibacterial and hemolytic activities, while short peptides lacking one or two terminal residues exhibited weak or no activity. Outer and inner membrane permeabilization activities of the peptides were also reduced with shortening of the peptide chain. These results indicate that the entire chain length of Mac is necessary for full activity, and the basicity of the peptides greatly affects the activity.     

Antibacterial activities of peptides designed as hybrids of antimicrobial peptides

Hybrid peptides (HP-MA, HP-ME), each of 20 residues and incorporating 2–9 residues of Helicobacter pylori ribosomal protein L1 (HP) and 1–12 residues of magainin 2 and melittin, were designed. The antibiotic activities of these peptides were evaluated using bacterial, tumor and human erythrocyte cells. HP-MA had a stronger antibacterial activity against Gram-positive bacteria and Gram-negative bacteria than HP (2-20) and magainin 2, and HP-ME was similar to melittin. None of the hybrids had anti-tumor or hemolytic activity. These peptides were further investigated using an artificial liposomal vesicle and 1,6-diphenyl-1,3,5-hexatriene as a membrane probe, and confirmed to have similar antibacterial activities. The antibacterial effect of these hybrids is probably caused by their ability to damage the bacterial plasma membrane. Additional circular dichroism spectra suggested that the -helical structure of these peptides plays an important role in their antibiotic effect but that -helical property is less connected with the enhanced antibiotic activity.     

Mechanistic and functional studies of the interaction of a proline-rich antimicrobial peptide with mammalian cells

Mammalian antimicrobial peptides provide rapid defense against infection by inactivating pathogens and by influencing the functions of cells involved in defense responses. Although the direct antibacterial properties of these peptides have been widely characterized, their multiple effects on host cells are only beginning to surface. Here we investigated the mechanistic and functional aspects of the interaction of the proline-rich antimicrobial peptide Bac7(1-35) with mammalian cells, as compared with a truncated analog, Bac7(5-35), lacking four critical N-terminal residues (RRIR) of the Bac7(1-35) sequence. By using confocal microscopy and flow cytometry, we showed that although the truncated analog Bac7(5-35) remains on the cell surface, Bac7(1-35) is rapidly taken up into 3T3 and U937 cells through a nontoxic energy- and temperature-dependent process. Cell biology-based assays using selective endocytosis inhibitors and spectroscopic and surface plasmon resonance studies of the interaction of Bac7(1-35) with phosphatidylcholine/cholesterol model membranes collectively suggest the concurrent contribution of macropinocytosis and direct membrane translocation. Structural studies with model membranes indicated that membrane-bound Bac7(5-35) is significantly more aggregated than Bac7(1-35) due to the absence of the N-terminal cationic cluster, thus providing an explanation for hampered cellular internalization of the truncated form. Further investigations aimed to reveal functional implications of intracellular uptake of Bac7(1-35) demonstrated that it correlates with enhanced S phase entry of 3T3 cells, indicating a novel function for this proline-rich peptide.     

Antibacterial activity of peptides derived from the C-terminal region of a hemolytic lectin, CEL-III, from the marine invertebrate Cucumaria echinata

Several synthetic peptides derived from the C-terminal domain sequence of a hemolytic lectin, CEL-III, were examined as to their action on bacteria and artificial lipid membranes. Peptide P332 (KGVIFAKASVSVKVTASLSK-NH(2)), corresponding to the sequence from residue 332, exhibited strong antibacterial activity toward Gram-positive bacteria. Replacement of each Lys in P332 by Ala markedly decreased the activity. However, when all Lys were replaced by Arg, the antibacterial activity increased, indicating the importance of positively charged residues at these positions. Replacement of Val by Leu also led to higher antibacterial activity, especially toward Gram-negative bacteria. The antibacterial activity of these peptides was correlated with their membrane-permeabilizing activity toward the bacterial inner membrane and artificial lipid vesicles, indicating that the antibacterial action is due to perturbation of bacterial cell membranes, leading to enhancement of their permeability. These results also suggest that the hydrophobic region of CEL-III, from which P332 and its analogs were derived, may play some role in the interaction with target cell membranes to trigger hemolysis.     

Role of Cathelicidin Peptides in Bovine Host Defense and Healing

The in vitro antimicrobial activities and biological effects on host cells were compared for the bovine cathelicidins BMAP-28, an alpha-helical AMP, and Bac5 and Bac7, proline-rich AMPs. Our results confirm that the broad-spectrum activity of BMAP-28 correlates with a high capacity to interact with and permeabilize bacterial membranes, whereas the proline-rich AMPs selectively internalize into the cytoplasm of susceptible Gram-negative bacteria with a non-lytic mechanism. All peptides efficiently translocated into mammalian fibroblastic cells, but while Bac5 and Bac7(1–35) localized to nuclear structures and induced cellular proliferation, BMAP-28 associated with mitochondria and did not induce proliferation. Moreover, BMAP-28 was considerably more cytotoxic than the proline-rich peptides due to cytolytic and pro-apoptotic effects. Our results highlight important functional differences among the bovine cathelicidins and suggest that they contribute to an integrated response of the host to infection, with distinct but complementary activities.     

牛抗菌肽Bac7-Bac5融合基因在大肠杆菌中的过表达，纯化及抑菌活性

牛抗菌肽Bac7和Bac5是一种线性阳离子小分子多肽,在机体天然免疫和获得性免疫中都发挥着重要作用。本研究根据Gen bank中公布的牛抗菌肽bac7和bac5成熟肽基因序列,人工合成了融合基因Bac7-Bac5片段,克隆于原核表达载体pET32(a+)中构建了重组表达载体（pET-B7-B5）,将其转化于E coli BL21(DE3) 中实现了重组蛋白B7-B5（rB7-B5）的过表达,表达的rB7-B5以包涵体形式存在,rB7-B5表达量约占细菌总蛋白的36.6％,分子量大小为33kD,与预测大小相符。以经Ni亲和层析柱纯化和多步透析法复性的rB7-B5,对猪胸膜肺炎放线杆菌和耐药性大肠杆菌具有很好的抑菌活性,本研究为新型抗菌制剂的研制和开发奠定了基础。     

Addition and omission analogs of the 13-residue antibacterial and hemolytic peptide PKLLKTFLSKWIG: structural preferences, model membrane binding and biological activities.

The consequences of selective addition or deletion of polar amino acids in a 13-residue antibacterial peptide PKLLKTFLSKWIG on structure, membrane binding and biological activities have been investigated. The variants generated are (a) S and T residues replaced by K, (b) S and T residues deleted individually and together, (c) introduction of two additional K and (d) deletion of L and L with T. In the aqueous environment all the peptides were unordered. In trifluoroethanol, the spectra of peptides belonging to groups (a-c) suggest distorted helical conformation. Peptides in group (d) appear to adopt beta-sheet conformation. The peptides bind to zwitterionic and negatively charged lipid vesicles, although to different extents. With the exception of peptides in group (d), all the other peptides exhibited comparable antibacterial activity against Escherichia coli and Staphylococcus aureus. However, the changes made in the peptides in groups (a-c) resulted in reduction of hemolytic activity compared to the parent peptide. Extent of binding to lipid vesicles composed of phosphatidylcholine and cholesterol appears to correlate with hemolytic activity. It appears that polar and charged residues play a major role in modulating the biological activities of the 13-residue peptide PKLLKTFLSKWIG. The 11-residue peptide-like PKLLKFLKWIG has selective antibacterial activity. Thus, by judicious engineering it should be possible to generate short peptides with selective antibacterial activity.     

Design, synthesis and antibacterial activity studies of model peptidess of proline/arginine-rich region in bactenecin7

Bactenecin 7 (Bac7), a cationic antibacterial peptide, contains a repeating region of Xaa-Pro-Arg-Pro (Xaa = hydrophobic residue). To investigate the structure and property of a Pro/Arg-rich region, e synthesized a series of peptides, Xaa-Pro-Arg-Pro (Xaa = Gly, Arg, Leu, Ile, and Phe) as models and characterized . The conformational preferences of these peptides in water and trifluoroethanol were examined by circular dichroism. The results suggest the presence of largely poly(Pro)-II helical conformation in aqueous and trifluoroethanol solutions. Their antibacterial activity against gram-negative bacteria such as Escherichia coli, Klebsiella Pneumoniae, Pseudomonas aeruginosa, and Escherichia coliHB101, and gram-positive bacteria such as Staphylococcus aureus were measured at various peptide concentrations. Two of our synthetic tetrapeptide fragments containing Gly and Arg were efficiently killed with gram-positive bacteria, Staphylococcus aureus, at the concentration level of 200 microg/mL.     

Correlation of three-dimensional structures with the antibacterial activity of a group of peptides designed based on a nontoxic bacterial membrane anchor

To understand the functional differences between a nontoxic membrane anchor corresponding to the N-terminal sequence of the Escherichia coli enzyme IIA(Glc) and a toxic antimicrobial peptide aurein 1.2 of similar sequence, a series of peptides was designed to bridge the gap between them. An alteration of a single residue of the membrane anchor converted it into an antibacterial peptide. Circular dichroism spectra indicate that all peptides are disordered in water but helical in micelles. Structures of the peptides were determined in membrane-mimetic micelles by solution NMR spectroscopy. The quality of the distance-based structures was improved by including backbone angle restraints derived from a set of chemical shifts ((1)H(alpha), (15)N, (13)C(alpha), and (13)C(beta)) from natural abundance two-dimensional heteronuclear correlated spectroscopy. Different from the membrane anchor, antibacterial peptides possess a broader and longer hydrophobic surface, allowing a deeper penetration into the membrane, as supported by intermolecular nuclear Overhauser effect cross-peaks between the peptide and short chain dioctanoyl phosphatidylglycerol. An attempt was made to correlate the NMR structures of these peptides with their antibacterial activity. The activity of this group of peptides does not correlate exactly with helicity, amphipathicity, charge, the number of charges, the size of the hydrophobic surface, or hydrophobic transfer free energy. However, a correlation is established between the peptide activity and membrane perturbation potential, which is defined by interfacial hydrophobic patches and basic residues in the case of cationic peptides. Indeed, (31)P solid state NMR spectroscopy of lipid bilayers showed that the extent of lipid vesicle disruption by these peptides is proportional to their membrane perturbation potential.     

Structure-activity relationships of fowlicidin-1, a cathelicidin antimicrobial peptide in chicken

Cationic antimicrobial peptides are naturally occurring antibiotics that are actively being explored as a new class of anti-infective agents. We recently identified three cathelicidin antimicrobial peptides from chicken, which have potent and broad-spectrum antibacterial activities in vitro (Xiao Y, Cai Y, Bommineni YR, Fernando SC, Prakash O, Gilliland SE & Zhang G (2006) J Biol Chem281, 2858-2867). Here we report that fowlicidin-1 mainly adopts an alpha-helical conformation with a slight kink induced by glycine close to the center, in addition to a short flexible unstructured region near the N terminus. To gain further insight into the structural requirements for function, a series of truncation and substitution mutants of fowlicidin-1 were synthesized and tested separately for their antibacterial, cytolytic and lipopolysaccharide (LPS)-binding activities. The short C-terminal helical segment after the kink, consisting of a stretch of eight amino acids (residues 16-23), was shown to be critically involved in all three functions, suggesting that this region may be required for the peptide to interact with LPS and lipid membranes and to permeabilize both prokaryotic and eukaryotic cells. We also identified a second segment, comprising three amino acids (residues 5-7) in the N-terminal flexible region, that participates in LPS binding and cytotoxicity but is less important in bacterial killing. The fowlicidin-1 analog, with deletion of the second N-terminal segment (residues 5-7), was found to retain substantial antibacterial potency with a significant reduction in cytotoxicity. Such a peptide analog may have considerable potential for development as an anti-infective agent.     

Dual mode of action of Bac7, a proline-rich antibacterial peptide

Proline-rich peptides are a unique group of antimicrobial peptides that exert their activity selectively against Gram-negative bacteria through an apparently non-membranolytic mode of action that is not yet well understood. We have investigated the mechanism underlying the antibacterial activity of the proline-rich cathelicidin Bac7 against Salmonella enterica and Escherichia coli. The killing and membrane permeabilization kinetics as well as the cellular localization were assessed for the fully active N-terminal fragment Bac7(1-35), its all-D enantiomer and for differentially active shortened fragments. At sub-micromolar concentrations, Bac7(1-35) rapidly killed bacteria by a non-lytic, energy-dependent mechanism, whereas its D-enantiomer was inactive. Furthermore, while the L-enantiomer was rapidly internalized into bacterial cells, the D-enantiomer was virtually excluded. At higher concentrations (>or=64 microM), both L- and D-Bac7(1-35) were instead able to kill bacteria also via a lytic mechanism. Overall, these results suggest that Bac7 may inactivate bacteria via two different modes of action depending on its concentration: (i) at near-MIC concentrations via a mechanism based on a stereospecificity-dependent uptake that is likely followed by its binding to an intracellular target, and (ii) at concentrations several times the MIC value, via a non-stereoselective, membranolytic mechanism.     

Antibacterial activities of synthetic peptides corresponding to the carboxy-terminal region of human beta-defensins 1-3

The antibacterial activities of synthetic human beta-defensin analogs, constrained by a single disulfide bridge and in the reduced form, have been investigated. The peptides span the carboxy-terminal region of human beta-defensins (HBD-1-3), which have a majority of cationic residues present in the native defensins. The disulfide constrained peptides exhibited activity against Escherichia coli and Staphylococcus aureus whereas the reduced forms were active only against E. coli. The antibacterial activities were attenuated in the presence of increasing concentrations of NaCl and divalent cations such as Ca(2+) and Mg(2+). The site of action was the bacterial membrane. Peptides spanning the carboxy-terminal region of human beta-defensins could be of help in understanding facets of antimicrobial activity of beta-defensins such as salt sensitivity and mechanisms of bacterial membrane damage.     

Conformation and activity of delta-lysin and its analogs

Delta-Lysin is a 26-residue hemolytic peptide secreted by Staphylococcus aureus. Unlike the bee venom peptide melittin, delta-lysin does not exhibit antibacterial activity. We have synthesized delta-lysin and several analogs wherein the N-terminal residues of the toxin were sequentially deleted. The toxin has three aspartic acids, four lysines and no prolines. Analogs were also generated in which all the aspartic acids were replaced with lysines. A proline residue was introduced in the native sequences as well as in the analogs where aspartic acids were replaced with lysines. We observed that 20- and 22-residue peptides corresponding to residues 7-26 and 5-26 of delta-lysin, respectively, had greater hemolytic activity than the parent peptide. These shorter peptides, unlike delta-lysin, did not self-associate to adopt alpha-helical conformation in water, at lytic concentrations. Introduction of proline or substitution of aspartic acids by lysines resulted in loss in propensity to adopt helical conformation in water. When proline was introduced in the peptides corresponding to the native toxin sequence, loss of hemolytic activity was observed. Substitution of all the aspartic acids with lysines resulted in enhanced hemolytic activity in all the analogs. However, when both proline and aspartic acid to lysine changes were made, only antibacterial activity was observed in the shorter peptides. Our investigations on delta-lysin and its analogs provide insights into the positioning of anionic, cationic residues and proline in determining hemolytic and antibacterial activities.     

Maximins S, a novel group of antimicrobial peptides from toad Bombina maxima

Amphibian skin secretions are rich in antimicrobial peptides acting as important components of innate defense system against invading microorganisms. A novel type of peptide, designated as maximin S, was deduced by random sequencing of 793 clones from a constructed Bombina maxima skin cDNA library. The putative primary structures of maximin S peptides can be grouped into five species, in which maximin S1 has 14 amino acid residues and the rest of maximin S peptides (S2-S5) all have 18 amino acid residues. Unlike most of the amphibian antimicrobial peptides so far identified, the newly characterized four maximin S precursors are composed of maximin S1 and different combinations of tandem repeated maximin S2-S5 linked by internal peptides. Except maximin S1, the predicted secondary structures of maximin S2-S5 show a similar amphipathic alpha-helical structure. MALDI-TOF mass spectrometry analysis of partially isolated skin secretions of the toad indicates that most of the deduced maximin S peptides are expressed. Two deduced maximin S peptides (S1, S4) were synthesized and their antimicrobial activities were tested. Maximin S4 only had an antibiotic activity against mycoplasma and had no antibacterial or antifungal activity toward tested strains. Maximin S1 had no activity under the same conditions.     

Effects of antimicrobial peptides of neutrophils on tumor and normal host cells in culture

We carried out a study of the effects of two structurally different cationic antimicrobial peptides of cathelicidin family, porcine protegrin 1 (PG1) and caprine Bac5 on selected tumor and normal mammalian cells in vitro. Protegrins are amphiphilic β-hairpin molecules having broad-spectrum antimicrobial activity due to their marked membranolytic effects. Bac5 belongs to a group of proline-rich peptides, which adopt a polyproline type II extended helix and kill microorganisms rather by a nonlytic mechanism. We have shown that while PG1 exerted distinct and fast cytotoxic effects towards most of used tumor cells being in a lesser degree toxic for nontransformed host cells; the proline-rich peptide Bac5 possessed modest cytotoxic activity for all tested cells. The toxic effects of PG1 were partially declined in the presence of 10% fetal calf serum. It was revealed that PG1 was able to interact with proteins of serpin family (as was previously established for human defensins by Panyutich at al., 1995). Pre-incubation of PG1 with α1-antitrypsin caused the decrease of the cytotoxic activity of the peptide and, on the other hand, the antiprotease activity of α1-antitrypsin was reduced after the interaction of the serpin with PG1 (while Bac5 did not affect the antiprotease activity of α1-antitrypsin). We used BODIPY FL-tagged PG1 and Bac5 to study the internalization of the labeled peptides into target cells and their intracellular distribution by confocal microscopy. Bac5-BODIPY (at 5 μ M) was rapidly taken into the cells. PG1-BODIPY at non-toxic concentrations (1—3 μM) was also able to enter the cells without their damaging. By using flow cytometry we showed that lowering a temperature to 4°C caused a significant decrease in the uptake into K562 and U937 cells for both Bac5-BODIPY and PG1-BODIPY. A decline of target cells metabolism also diminished the process of both peptides internalization but for a lesser degree. In the presence of endocytosis inhibitors the penetration of Bac5-BODIPY and PG1-BODIPY into K562 cells was also reduced, but not completely abolished, suggesting that along with endocytosis process some direct penetration of the peptides across cell membranes takes place. The ability of the peptides to internalize into eukaryotic cells may contribute to the idea of participation of AMPs in varied intracellular events, occurring in normal or malignant host cells, for instance, in the modulation of intracellular serpins activity.     

Design and synthesis of cyclic disulfide-bonded antibacterial peptides on the basis of the alpha helical domain of Tenecin 1, an insect defensin

We synthesized cyclic disulfide-bonded (i, i+4) peptides with various net positive charges (+2-+5) from linear peptides derived from the alpha helical domain of Tenecin 1, an insect defensin, and investigated the effect of the intradisulfide bridge (i, i+4) on hydrophobicity, secondary structure, leakage activity and binding activity for large unilamellar vesicles, antimicrobial activity, and hemolytic activity. Intradisulfide bridge formation of the peptides resulted in the increase of amphiphilicity and hydrophobicity. Cyclic forms of the peptides did not deeply penetrate into PG/PC (1:1, mole ratio) large unilamellar vesicles and had a decreased lipid membrane perturbation activity for PG/PC LUVs. When the peptides interacted with PG/CL (2:1, mole ratio) LUVs, cyclic peptides with a high net positive charge (+4-+5) showed similar binding affinities and leakage activities for vesicles to those of linear forms, whereas cyclic peptides with a low net positive charge (+2-+3) exhibited lower leakage activity than their linear forms. CD spectra indicate that the intradisulfide bridge (i, i+4) provided little conformational constraint to linear peptides in buffer solution but resulted in the decrease of alpha helicity of the peptides in lipid membrane mimic conditions. The cyclic peptide with the highest net positive charge had a similar antibacterial activity to that of the linear peptide, whereas the cyclic peptides with a low net positive charge (+3-+4) exhibited lower antibacterial activity than their linear forms. The cyclic peptides of an appropriate net charge showed more potent activities against some bacteria than those of linear forms under high salt conditions.     

The antibiotic activity of cationic linear amphipathic peptides: lessons from the action of leucine/lysine copolymers on bacteria of the class Mollicutes.

Peptides composed of leucyl and lysyl residues ('LK peptides') with different compositions and sequences were compared for their antibacterial activities using cell wall-less bacteria of the class Mollicutes (acholeplasmas, mycoplasmas and spiroplasmas) as targets. The antibacterial activity of the amphipathic alpha-helical peptides varied with their size, 15 residues being the optimal length, independent of the membrane hydrophobic core thickness and the amount of cholesterol. The 15-residue ideally amphipathic alpha helix with a +5 positive net charge (KLLKLLLKLLLKLLK) had the strongest antibacterial activity, similar to that of melittin. In contrast, scrambled peptides devoid of amphipathy and the less hydrophobic beta-sheeted peptides [(LK)nK], even those 15-residue long, were far less potent than the helical ones. Furthermore, the growth inhibitory activity of the peptides was correlated with their ability to abolish membrane potential. These data are fully consistent with a predominantly flat orientation of LK peptides at the lipid/water interface and strongly supports that these peptides and probably the linear polycationic amphipathic defence peptides act on bacterial membranes in four main steps according to the 'carpet' model: (a) interfacial partitioning with accumulation of monomers on the target membrane (limiting step); (b) peptide structural changes (conformation, aggregation, and orientation) induced by interactions with the lipid bilayer (as already shown with liposomes and erythrocytes); (c) plasma membrane permeabilization/depolarization via a detergent-like effect; and (d) rapid bacterial cell death if the extent of depolarization is maintained above a critical threshold.     

Single disulfide and linear analogues corresponding to the carboxy-terminal segment of bovine beta-defensin-2: effects of introducing the beta-hairpin nucleating sequence d-pro-gly on antibacterial activity and Biophysical properties

Mammalian defensins (alpha as well as beta forms) have a beta-hairpin structural motif spanning approximately 20 residues at the carboxy-terminal end. We have investigated the antibacterial activity and biophysical properties of synthetic peptides corresponding to the carboxy-terminal segment of bovine beta-defensin-2 (BNBD-2): VRNHVTC(1)RINRGFC(2)VPIRC(3)PGRTRQIGTC(4)FGPRIKC(5)C(6)RSW (positions of disulfide bonds are C(1)[bond]C(5), C(2)[bond]C(4), and C(3)[bond]C(6)). The parent sequence chosen was RCPGRTRQIGTIFGPRIKCRSW (P1), which spans the carboxy-terminal region of BNBD-2. Since the dipeptide sequence D-Pro-Gly favors nucleation of beta-hairpin structures even in acyclic peptides, analogues of P1 with one D-Pro-Gly at the central portion and two D-Pro-Gly segments near the N- and C-terminal ends were generated. An analogue in which GP (residues 14 and 15) in P1 was switched to PG was also synthesized. It was observed that the cyclic form as well as their linear forms exhibited antibacterial activity. Circular dichroism and theoretical studies indicated that while the beta-hairpin conformation is populated, there is conformational plasticity in the cyclic and linear peptides. The mode of bacterial killing was by membrane permeabilization. The entire mammalian defensin sequence does not appear to be essential for manifestation of antibacterial activity. Hence, short peptides corresponding to the C-terminal segments of mammalian defensins could have potential as therapeutic agents.     

Amino acid sequences of two proline-rich bactenecins. Antimicrobial peptides of bovine neutrophils

Bactenecins are highly cationic polypeptides of the large granules of bovine neutrophils, exerting in vitro a potent antimicrobial activity. Two bactenecins, with an approximate molecular weight of 7000 and 5000, called Bac7 and Bac5, are characterized by a high content of proline (greater than 45%) and arginine (greater than 23%) residues. Their complete amino acid sequences were determined by automated Edman degradation combined, in the case of Bac5, with plasma desorption mass spectrometry. Bac7 comprises 59 residues and includes three tandem repeats of a tetradecamer characterized by several Pro-Arg-Pro triplets spaced by single hydrophobic amino acids. Resolution of the primary structure of Bac5 required fragmentation with N-bromosuccinimide as well as digestion of the obtained C-terminal fragment with carboxypeptidases P and Y directly in the mass spectrometer. Bac5 comprises 42 amino acid residues with a repeated motif of Arg-Pro-Pro triplets also alternating with single apolar residues.