Factors contributing to the potency of antimicrobial cationic peptides from the N-terminal region of human lactoferrin

This study investigated the antimicrobial activities of peptides derived from the N-terminal region of human lactoferrin, and examined the contributions of individual residues to the activity of the most potent peptide. Two regions of antimicrobial activity were identified, the first corresponding to a weakly active peptide, HLP-9, comprising residues 1-9, and a second corresponding to a more potent peptide, HLP-10, comprising residues 18-26 and containing the hexapeptide motif, FQWQRN. Inhibitory studies on peptides from the first region confirm the importance of tryptophan residues in enhancing and broadening peptide activity. Inhibitory studies with glycine-substituted homologues of the more potent peptide showed that F21/G and R25/G substitutions resulted in a major reduction or complete loss of activity, while increased peptide cationicity or flexibility had little effect. Our findings demonstrate that F21 and R25 are critical determinants of potency for HLP-10, and that the second aromatic residue may act synergistically with W23 in developing and enhancing the activity of this cationic peptide.     

Tryptophan- and arginine-rich antimicrobial peptides: structures and mechanisms of action

Antimicrobial peptides encompass a number of different classes, including those that are rich in a particular amino acid. An important subset are peptides rich in Arg and Trp residues, such as indolicidin and tritrpticin, that have broad and potent antimicrobial activity. The importance of these two amino acids for antimicrobial activity was highlighted through the screening of a complete combinatorial library of hexapeptides. These residues possess some crucial chemical properties that make them suitable components of antimicrobial peptides. Trp has a distinct preference for the interfacial region of lipid bilayers, while Arg residues endow the peptides with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of bacterial membranes. In combination, these two residues are capable of participating in cation-pi interactions, thereby facilitating enhanced peptide-membrane interactions. Trp sidechains are also implicated in peptide and protein folding in aqueous solution, where they contribute by maintaining native and nonnative hydrophobic contacts. This has been observed for the antimicrobial peptide from human lactoferrin, possibly restraining the peptide structure in a suitable conformation to interact with the bacterial membrane. These unique properties make the Arg- and Trp-rich antimicrobial peptides highly active even at very short peptide lengths. Moreover, they lead to structures for membrane-mimetic bound peptides that go far beyond regular alpha-helices and beta-sheet structures. In this review, the structures of a number of different Trp- and Arg-rich antimicrobial peptides are examined and some of the major mechanistic studies are presented.     

Tryptophan- and arginine-rich antimicrobial peptides: Structures and mechanisms of action

Antimicrobial peptides encompass a number of different classes, including those that are rich in a particular amino acid. An important subset are peptides rich in Arg and Trp residues, such as indolicidin and tritrpticin, that have broad and potent antimicrobial activity. The importance of these two amino acids for antimicrobial activity was highlighted through the screening of a complete combinatorial library of hexapeptides. These residues possess some crucial chemical properties that make them suitable components of antimicrobial peptides. Trp has a distinct preference for the interfacial region of lipid bilayers, while Arg residues endow the peptides with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of bacterial membranes. In combination, these two residues are capable of participating in cation-π interactions, thereby facilitating enhanced peptide-membrane interactions. Trp sidechains are also implicated in peptide and protein folding in aqueous solution, where they contribute by maintaining native and nonnative hydrophobic contacts. This has been observed for the antimicrobial peptide from human lactoferrin, possibly restraining the peptide structure in a suitable conformation to interact with the bacterial membrane. These unique properties make the Arg- and Trp-rich antimicrobial peptides highly active even at very short peptide lengths. Moreover, they lead to structures for membrane-mimetic bound peptides that go far beyond regular α-helices and β-sheet structures. In this review, the structures of a number of different Trp- and Arg-rich antimicrobial peptides are examined and some of the major mechanistic studies are presented.     

In vitro study of antimicrobial activity of lactoferrins from various sources

Comparative antimicrobial activity of lactoferrins from various sources (native lactoferrin from Laprot, human hololactoferrin, recombinant human lactoferrin isolated from the cultural medium of permissive cell culture transfected using pseudoadenovirus nanostructure with the human lactoferrin gene, and native bovine lactoferrin) was studied to prove the possibility of their use for development of antimicrobial drugs. It was shown that all the substances were active against the Bacillus standard strains. The antibacterial activity was almost independent of the degree of saturation the lactoferrin molecules with Fe3+. The native human lactoferrin was more active than hololactoferrin against Candida when evaluated by the minimum inhibitory concentration (MIC). Fe(3+)-Non aturated recombinant human lactoferrin demonstrated the antimicrobial activity (by MIC) similar to that of the native human lactoferrin. The results showed that native and recombinant human lactoferrins might be used for the development of intravenous and intracavitary dosage forms, while the native bovine lactoferrin could be useful in development of oral drugs.     

Antimicrobial peptide of korean native goat lactoferrin and identification of the part essential for this activity

The antimicrobial activity of lactoferrin isolated from Korean native goat (KN goat) milk was studied and its antimicrobial domain was identified using synthetic peptides. Antimicrobial activity was assayed by a micro-method using 96-well microplates and a microplate reader. The amino acid sequence of the antimicrobial domain was suggested to be YQWQRRMRKLGAPSIT and this sequence corresponds to amino acid residues 20 to 35 of KN goat lactoferrin. Five peptides with certain amino acid residues deleted were synthesized in an effort to identify the residues essential for antimicrobial activity and it was found that the part with the sequence RRMRK (24-28) is the region most important for this activity. On the other hand, the conformation of the peptides did not influence the antimicrobial activity.     

Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin

The antimicrobial activity of bovine lactoferrin is attributed to lactoferricin, situated in the N1-domain. Based on common features of antimicrobial peptides, a second putative antimicrobial domain was identified in the N1-domain of lactoferrin, designated lactoferrampin. This novel peptide exhibited candidacidal activity, which was substantially higher than the activity of lactoferrin. Furthermore, lactoferrampin was active against Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa, but not against the fermenting bacteria Actinomyces naeslundii, Porphyromonas gingivalis, Streptococcus mutans and Streptococcus sanguis. Notably, lactoferrampin is located in the N1-domain in close proximity to lactoferricin, which plays a crucial role in membrane-mediated activities of lactoferrin.     

Antibacterial activity of 15-residue lactoferricin derivatives.

Lactoferricins are a class of antibacterial peptides isolated after gastric-pepsin digest of the mammalian iron-chelating-protein lactoferrin. For investigation of antibacterial activity, we prepared short synthetic derivatives of bovine, human, caprine, murine and porcine lactoferricins with 15-amino-acid residues of high sequence homology. The peptides corresponded to amino-acid residues 17-31 of the mature bovine lactoferrin. Only the bovine and caprine derivatives displayed measurable antibacterial activity, with the bovine one having a minimal inhibitory concentration of 24 microM and being 10 times more active than the caprine one against Escherichia coli. An alanine-scan of the bovine lactoferricin derivative was performed to identify specific amino acids that were important for the antibacterial activity. We found that neither of the two tryptophan residues (Trp 6 and Trp 8) present in the bovine lactoferricin derivative could be replaced by alanine without a major loss of antibacterial activity. The other lactoferricin derivatives tested contained only one tryptophan residue (Trp 6). Modified human, caprine and porcine lactoferricin derivatives containing two tryptophan residues (Trp 6 and Trp 8) displayed minimal inhibitory concentrations of 74, 174 and 219 microM, respectively, which represented up to a six-fold increase in antibacterial activity. The alanine-scan also revealed that the antibacterial activity was increased when acetamidomethyl-protected cysteine and unprotected glutamine (Cys 3 and Gln 7) were replaced with alanine. Only the bovine lactoferricin derivative and a few of its alanine-modified derivatives displayed measurable activity against Staphylococcus aureus.     

Cationic amphipathic peptides, derived from bovine and human lactoferrins, with antimicrobial activity against oral pathogens

Peptides derived from the N-terminal domain that comprises an amphipathic alpha-helix in human lactoferrin (LFh 18-31 and LFh 20-38) and bovine lactoferrin (LFb 17-30 and LFb 19-37) were chemically synthesised. Since many positively charged amphipathic alpha-helices contain antimicrobial activity, the peptides were tested for their antimicrobial activity against various oral pathogens. Both peptides from bovine lactoferrin had more potent antimicrobial activities than the human equivalents. Peptide LFb 17-30, containing the largest number of positively charged amino acids, showed the highest antimicrobial activity to both Gram-positive and Gram-negative bacteria. Since native lactoferrin molecules had no killing activity, release of these peptides from the native protein should be investigated to explore the use in oral care products.     

Lactoferrampin, an antimicrobial peptide of bovine lactoferrin, exerts its candidacidal activity by a cluster of positively charged residues at the C-terminus in combination with a helix-facilitating N-terminal part

The antimicrobial activity of bovine lactoferrin (bLF) is attributed to lactoferricin, which is situated in the N1-domain of bLF. Recently, another antimicrobial domain consisting of residues 268-284, designated lactoferrampin (LFampin), has been identified in the N1-domain of bLF, which exhibited antimicrobial activity against Candida albicans and several bacteria. In the present study, the candidacidal activity of a series of peptides spanning this antimicrobial domain was investigated in relation to the charge and the capacity to form a helical conformation in hydrophobic environments. C-Terminal truncation of LFampin resulted in a drastic decrease in candidacidal activity. Positively charged residues clustered at the C-terminal side of the LFampin domain appeared to be crucial for the candidacidal activity. The ability to adopt helical conformations did not change when LFampin was truncated at the C-terminal side. N-Terminally truncated LFampin peptides, truncated up to the sequence 270-284, were more reluctant to adopt a helical conformation. Therefore, we conclude that the C-terminal part of LFampin 265-284, which is the most active peptide, is crucial for its candidacidal activity, due to the presence of clustered positive charges, and that the N-terminal part is essential for activity as it facilitates helix formation.     

Expression and characterization of recombinant bovine lactoferrin in E. coli

Lactoferrin is a member of the transferrin family of iron-binding proteins with a number of properties, including antibacterial activity against a broad spectrum of Gram-negative and Gram-positive bacteria. bovine lactoferrin cDNA was isolated, cloned and expressed as a fusion protein. The amino acid sequence of the fusion was analyzed and compared with other species. Crystallographic data were used to compare structural differences between bovine and human lactoferrin in 3-D models. A thioredoxin fusion protein was expressed and shown to have a different molecular weight compared with native bLf. After purification using Ni-NTA, the yield of recombinant bovine lactoferrin was 15.3 mg/l with a purity of 90.3 %. Recombinant bLf and pepsin-digested rbLf peptides demonstrated antibacterial activity of 79.8 and 86.9 %, respectively. The successful expression of functional, active and intact rbLf allows us to study the biochemical interactions of antimicrobial proteins and peptides and will facilitate their study as immunomodulators.     

Novel lactoferrampin antimicrobial peptides derived from human lactoferrin

Human lactoferrampin is a novel antimicrobial peptide found in the cationic N-terminal lobe of the iron-binding human lactoferrin protein. The amino acid sequence that directly corresponds to the previously characterized bovine lactoferrin-derived lactoferrampin peptide is inactive on its own (WNLLRQAQEKFGKDKSP, residues 269-285). However, by increasing the net positive charge near the C-terminal end of human lactoferrampin, a significant increase in its antibacterial and Candidacidal activity was obtained. Conversely, the addition of an N-terminal helix cap (sequence DAI) did not have any appreciable effect on the antibacterial or antifungal activity of human lactoferrampin peptides, even though it markedly influenced that of bovine lactoferrampin. The solution structure of five human lactoferrampin variants was determined in SDS micelles and all of the structures display a well-defined amphipathic N-terminal helix and a flexible cationic C-terminus. Differential scanning calorimetry studies indicate that this peptide is capable of inserting into the hydrophobic core of a membrane, while fluorescence spectroscopy results suggest that a hydrophobic patch encompassing the single Trp and Phe residues as well as Leu, Ile and Ala side chains mediates the interaction between the peptide and the hydrophobic core of a phospholipid bilayer.     

牛乳铁蛋白素及其衍生物的研究进展

牛乳铁蛋白素是牛乳铁蛋白经胃蛋白酶水解后释放出来的一段小肽,是牛乳铁蛋白的活性中心。通过对不同动物来源乳铁蛋白素活性的研究发现牛乳铁蛋白素的抗菌活性最强。进一步的丙氨酸突变实验研究表明,在牛乳铁蛋白素活性最强的15个氨基酸序列中,色氨酸在抗菌过程中起着重要作用。牛乳铁蛋白素正是因为含有两个色氨酸,其活性才会比只含有一个色氨酸的其它来源的乳铁蛋白素活性要高。很多实验室围绕着牛乳铁蛋白素中的色氨酸、碱性氨基酸和其他一些芳香族氨基酸展开了一系列的突变研究,本文综述了这些研究及在氨基酸改变后活性的变化,为以后研究及开发牛乳铁蛋白素提供理论基础。     

Variation in antimicrobial activity of lactoferricin-derived peptides explained by structure modelling

Antimicrobial peptides bovine lactoferricin (LfcinB) and human lactoferricin (LfcinH) are produced from the respective lactoferrin, but are more active than their precursors. Despite sequence homology, the bovine peptide and its derivatives are more active than their human homologs. Such differences between not only the peptides and their precursor but also between the bovine and the human peptides could relate to structural differences. Upon sequence alignment of both peptides with their parental proteins, the structural differences observed between the bovine lactoferrin (BLf) and LfcinB were also found between the human lactoferrin (HLf) and the LfcinH. The helical structures in HLf are replaced by beta-strands separated by a strong turn in LfcinH suggesting an antiparallel beta-sheet structure similar to LfcinB. MIC assays with HLP-2 and BLP-2, 11-residue peptides derived from the active core of both Lfcins, against Escherichia coli, showed that the bovine derivative, BLP-2, is more active than its human homolog HLP-2. Both 3D models for HLP-2 and BLP-2 showed that the beta-strand is centred between the aromatic residues giving both side chains the same orientations. The displacement towards the N-terminus observed for the beta-strand in HLP-2, compared with its central location in BLP-2, could be less favourable to membrane interaction and therefore responsible for the decrease in activity. Such a model suggests for LfcinH a mechanism similar to the one observed for LfcinB, where the absence of long-range interaction, present in lactoferrin, destabilises the first alpha helix, as observed in solution and, upon interaction with the membrane, could result in the formation of a beta-strand, as observed in the presence of LPS. The location of the beta-strand in relation to the positive charges, seems to define the efficiency of the activity of the peptide and may explain the difference in activity obtained between HLP-2 and BLP-2.     

The structure of the antimicrobial active center of lactoferricin B bound to sodium dodecyl sulfate micelles

Lactoferricin B (LfcinB) is a 25-residue antimicrobial peptide released from bovine lactoferrin upon pepsin digestion. The antimicrobial center of LfcinB consists of six residues (RRWQWR-NH2), and it possesses similar bactericidal activity to LfcinB. The structure of the six-residue peptide bound to sodium dodecyl sulfate (SDS) micelles has been determined by NMR spectroscopy and molecular dynamics refinement. The peptide adopts a well defined amphipathic structure when bound to SDS micelles with the Trp sidechains separated from the Arg residues. Additional evidence demonstrates that the peptide is oriented in the micelle such that the Trp residues are more deeply buried in the micelle than the Arg and Gln residues.     

Physicochemical characterization and functional site analysis of lactoferrin gene of Vechur cow

The Bos indicus Vechur breed cow milk is known for its medicinal value and the breed is listed under the category of critically maintained breeds by the Food and Agriculture Organization. The lactoferrin protein in milk is known for its nutritional value. Gene polymorphisms have been reported for Bovine lactoferrin. Mutations in the evolutionarily conserved sites tend to impair protein function and are related with the physicochemical difference between the known variants with 11 SNPs within the wild type. Structural differences are located due to these SNPs that may lead to functional variations. The structural variation is seen primarily in the first 48 residues at 5' end in all the samples modelled. Out of 11 SNPs 5 amino acid variations fall under alpha helix and beta sheet region, this might be of functional significance. This result may provide evidence that the SNPs detected in lactoferrin gene might have potential effects on milk composition. Our result demonstrates one major domain that could be a common binding pocket to all the samples, and important as an active site common to all the breeds that could be utilized for effective drug designing. Moreover, at some SNP positions in Vechur breed, antimicrobial peptides were located indicating importance of those residues for enhanced antimicrobial activity in lactoferrin of Vechur breed. Second binding pocket found in N- lobe region with the three required residues aspartic acid, histidine and tyrosine for iron binding, was considered as major binding site.     

Anti-complement effects of lactoferrin-derived peptides

Lactoferrin is an important biological molecule with many functions such as modulation of the inflammatory response, iron metabolism and antimicrobial defense. One effect of lactoferrin is the inhibition of the classical complement pathway. This study reports that antimicrobial peptides derived from the N-terminal region from both human and bovine lactoferrin, lactoferricin H and lactoferricin B, respectively, inhibit the classical complement pathway. No inhibitory effect of these peptides was observed on the alternative complement pathway in an AP50 assay. However, lactoferricin B reduced the inhibitory properties of serum against Escherichia coli in a concentration dependent manner. These results suggest that the N-terminal region of lactoferrin is the important part in the inhibition of complement activation and that these peptides possess other important properties than their antimicrobial effect.     

Conformational analysis of the broad-spectrum antibacterial peptide, ranatuerin-2CSa: identification of a full length helix-turn-helix motif

Design of clinically valuable antibacterial agents based upon naturally occurring peptides requires the use of spectroscopic methods, particularly NMR, to determine the three-dimensional structure of the native peptide so that analogues with improved therapeutic properties can be made. Ranatuerin-2CSa (GILSSFKGVAKGVAKDLAG KLLETLKCKITGC), first isolated from skin secretions of the Cascades frog, Rana cascadae, represents a promising candidate for drug development. The peptide shows potent growth inhibitory activity against Escherichia coli (MIC=5 microM) and Staphylococcus aureus (MIC=10 microM) but displays haemolytic activity against human erythrocytes (LC(50)=160 microM). The solution structure of ranatuerin-2CSa was investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, the peptide lacks secondary structure but, in a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O solvent mixture, the structure is characterised by a full length helix-turn-helix conformation between residues I(2)-L(21), L(22)-L(25) and K(26)-T(30) respectively. This structural information will facilitate the design of novel therapeutic agents based upon the ranatuerin-2CSa structure with improved antimicrobial potencies but decreased cytolytic activities against mammalian cells.     

Influence of specific amino acid side-chains on the antimicrobial activity and structure of bovine lactoferrampin

Lactoferrin is an 80 kDa iron binding protein found in the secretory fluids of mammals and it plays a major role in host defence. An antimicrobial peptide, lactoferrampin, was identified through sequence analysis of bovine lactoferrin and its antimicrobial activity against a wide range of bacteria and yeast species is well documented. In the present work, the contribution of specific amino acid residues of lactoferrampin was examined to evaluate the role that they play in membrane binding and bilayer disruption. The structures of all the bovine lactoferrampin derivatives were examined with circular dichroism and nuclear magnetic resonance spectroscopy, and their interactions with phospholipids were evaluated with differential scanning calorimetry and isothermal titration calorimetry techniques. From our results it is apparent that the amphipathic N-terminal helix anchors the peptide to membranes with Trp 268 and Phe 278 playing important roles in determining the strength of the interaction and for inducing peptide folding. In addition, the N-terminal helix capping residues (DLI) increase the affinity for negatively charged vesicles and they mediate the depth of membrane insertion. Finally, the unique flexibility in the cationic C-terminal region of bovine lactoferrampin does not appear to be essential for the antimicrobial activity of the peptide.     

Identification and characterization of a phospholipid-binding site of bovine factor Va

Coagulation factor Va is a cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. The phospholipid-binding domain of bovine factor Va has been reported to be located on the light chain of the molecule and more precisely on a fragment of Mr = 30,000 which is obtained after digestion of factor Va light chain by factor Xa. This proteolytic fragment is located in the NH2-terminal part of factor Va light chain (residues 1564-1765). In order to further characterize the lipid-binding domain of bovine factor Va, isolated bovine light chain was preincubated with synthetic phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine) and digested with trypsin, chymotrypsin, and elastase. Two peptide regions protected from proteolytic cleavage were identified and characterized from each proteolytic digestion. A comparison of the NH2-terminal sequence and amino acid composition of the two tryptic peptides with the deduced sequence of human factor V indicates a match with residues 1657-1791 of the light chain of human factor V for one peptide and residues 1546-1656 for the other peptide. When chymotrypsin or elastase were used for digestion, the NH2-terminal sequence of one peptide showed a match with residues 1667-1797 of the light chain, while the other peptide presented an NH2-terminal sequence identical with the previously described for the bovine factor Va light chain. When these peptides were assayed for direct binding to phospholipid vesicles, only the tryptic and the chymotryptic peptides covering the middle region of the A3 domain of the bovine factor Va light chain demonstrated an ability to interact with phospholipid vesicles. Thus, knowing that the factor Xa cleavage site on the factor Va light chain is located between residues 1765 and 1766 of the light chain this lipid-binding region of the bovine factor Va is further localized to amino acid residues 1667-1765.     

Processing of lysozyme at distinct loops by pepsin: a novel action for generating multiple antimicrobial peptide motifs in the newborn stomach

C-type lysozyme (cLZ) is an antimicrobial enzyme that plays a major defense role in many human secretions. Recently, we have identified a helix-loop-helix antimicrobial peptide fragment of cLZ. This finding suggests that processing by coexisting proteases might be a relevant physiological process for generating peptides that contribute to the in vivo mucosal defense role of cLZ. In this study, we found that pepsin, under condition relevant to the newborn stomach (pH 4.0), generated various peptides from cLZ with potent bactericidal activity against several strains of Gram-negative and Gram-positive bacteria. Microsequencing and mass spectral analysis revealed that pepsin cleavage occurred at conserved loops within the alpha-domain of cLZ. We found that the bactericidal domain, which was isolated by gel filtration and reversed-phase HPLC, contains two cationic alpha-helical peptides generated from a helix-loop-helix domain (residues 1-38 of cLZ) by nicking at leucine17. A third peptide consisting of an alpha-helix (residues 18-38) and a two-stranded beta-sheet (residues 39-56) structure was also identified. These peptides share structural motifs commonly found in different innate immune defenses. Functional cellular studies with outer membrane-, cytoplasmic membrane vitality- and redox-specific fluorescence dyes revealed that the lethal effect of the isolated antimicrobial peptides is due to membrane permeabilization and inhibition of redox-driven bacterial respiration. The results provide the first demonstration that pepsin can fine-tune the antimicrobial potency of cLZ by generating multiple antimicrobial peptide motifs, delineating a new molecular switch of cLZ in the mucosal defense systems. Finally, this finding offers a new strategy for the design of antibiotic peptide drugs with potential use in the treatment of infectious diseases.