乳铁素——来源于乳铁蛋白的多功能抗菌肽

乳铁素是乳铁蛋白在酸性环境条件下经胃蛋白酶水解从N-端释放的多功能活性多肽．乳铁素不仅保持了完整乳铁蛋白的大部分生物学活性,而且乳铁素的某些生物学活性比乳铁蛋白更强．乳铁素具有抗细菌、抗真菌、抗病毒、抗肿瘤、免疫调节和抗炎症等多种生物学功能．然而,乳铁素的生物学作用大部分是通过体外试验发现和验证的,乳铁素的体内生物学效应还需更多的试验加以评价和证实,现代基因组学和蛋白组学分析方法和技术将有助于深入了解乳铁素体内生物学作用机制．本文就乳铁素的结构、生物学功能及其作用机制、制备和应用前景作一综述．     

Hydrolytic breakdown of lactoferricin by lactic acid bacteria

Lactoferricin is a 25-amino acid antimicrobial peptide fragment that is liberated by pepsin digestion of lactoferrin present in bovine milk. Along with its antibacterial properties, lactoferricin has also been reported to have immunostimulatory, antiviral, and anticarcinogenic effects. These attributes provide lactoferricin and other natural bioactive peptides with the potential to be functional food ingredients that can be used by the food industry in a variety of applications. At present, commercial uses of these types of compounds are limited by the scarcity of information on their ability to survive food processing environments. We have monitored the degradation of lactoferricin during its incubation with two types of lactic acid bacteria used in the yogurt-making industry, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, with the aim of assessing the stability of this milk protein-derived peptide under simulated yogurt-making conditions. Analysis of the hydrolysis products isolated from these experiments indicates degradation of this peptide near neutral pH by lactic acid bacteria-associated peptidases, the extent of which was influenced by the bacterial strain used. However, the data also showed that compared to other milk-derived bioactive peptides that undergo complete degradation under these conditions, the 25-amino acid lactoferricin is apparently more resistant, with approximately 50% of the starting material remaining after 4 h of incubation. These findings imply that lactoferricin, as a natural milk protein-derived peptide, has potential applications in the commercial production of yogurt-like fermented dairy products as a multi-functional food ingredient.     

抗菌肽Lactoferricin生物学功能及其应用研究进展

概述了乳铁蛋白活性多肽(lactofericin)所具有的广谱抗菌、抗寄生虫、抗病毒、抗癌、抗氧化等多种生物学活性,讨论了lactnferricin的制备方法,并对lactnferricin作为饲料添加剂的应用前景作了初步探讨。     

Twenty-five years of research on bovine lactoferrin applications

Lactoferrin (LF) was identified as a milk protein in 1960. Large-scale manufacturing of bovine LF (bLF) was established more than 20 years ago. Using this commercially available material, research for bLF applications has advanced from basic studies to clinical studies, and bLF has been applied to commercial food products for the last 25 years. During this period, it was found that LF is digested by gastric pepsin to generate a multi-potent peptide, lactoferricin. It was also demonstrated that oral administration of bLF augments host protection against infections via antimicrobial action and immunomodulation of the host. In addition, researchers have demonstrated that oral administration of bLF prevents cancer development. In this review, we look back on 25 years of bLF research and development.     

Perspectives on interactions between lactoferrin and bacteria.

Lactoferrin has long been recognized for its antimicrobial properties, initially attributed primarily to iron sequestration. It has since become apparent that interaction between the host and bacteria is modulated by a complex series of interactions between lactoferrin and bacteria, lactoferrin and bacterial products, and lactoferrin and host cells. The primary focus of this review is the interaction between lactoferrin and bacteria, but interactions with the lactoferrin-derived cationic peptide lactoferricin will also be discussed. We will summarize what is currently known about the interaction between lactoferrin (or lactoferricin) and surface or secreted bacterial components, comment on the potential physiological relevance of the findings, and identify key questions that remain unanswered.     

Lactoferrin binds CpG-containing oligonucleotides and inhibits their immunostimulatory effects on human B cells

Unmethylated CpG dinucleotide motifs in bacterial DNA, as well as oligodeoxynucleotides (ODN) containing these motifs, are potent stimuli for many host immunological responses. These CpG motifs may enhance host responses to bacterial infection and are being examined as immune activators for therapeutic applications in cancer, allergy/asthma, and infectious diseases. However, little attention has been given to processes that down-modulate this response. The iron-binding protein lactoferrin is present at mucosal surfaces and at sites of infection. Since lactoferrin is known to bind DNA, we tested the hypothesis that lactoferrin will bind CpG-containing ODN and modulate their biological activity. Physiological concentrations of lactoferrin (regardless of iron content) rapidly bound CpG ODN. The related iron-binding protein transferrin lacked this capacity. ODN binding by lactoferrin did not require the presence of CpG motifs and was calcium independent. The process was inhibited by high salt, and the highly cationic N-terminal sequence of lactoferrin (lactoferricin B) was equivalent to lactoferrin in its ODN-binding ability, suggesting that ODN binding by lactoferrin occurs via charge-charge interaction. Heparin and bacterial LPS, known to bind to the lactoferricin component of lactoferrin, also inhibited ODN binding. Lactoferrin and lactoferricin B, but not transferrin, inhibited CpG ODN stimulation of CD86 expression in the human Ramos B cell line and decreased cellular uptake of ODN, a process required for CpG bioactivity. Lactoferrin binding of CpG-containing ODN may serve to modulate and terminate host response to these potent immunostimulatory molecules at mucosal surfaces and sites of bacterial infection.     

Towards a structure-function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides.

The iron-binding protein lactoferrin is a multifunctional protein that has antibacterial, antifungal, antiviral, antitumour, anti-inflammatory, and immunoregulatory properties. All of these additional properties appear to be related to its highly basic N-terminal region. This part of the protein can be released in the stomach by pepsin cleavage at acid pH. The 25-residue antimicrobial peptide that is released is called lactoferricin. In this work, we review our knowledge about the structure of the peptide and attempt to relate this to its many functions. Microcalorimetry and fluorescence spectroscopy data regarding the interaction of the peptide with model membranes show that binding to net negatively charged bacterial and cancer cell membranes is preferred over neutral eukaryotic membranes. Binding of the peptide destabilizes the regular membrane bilayer structure. Residues that are of particular importance for the activity of lactoferricin are tryptophan and arginine. These two amino acids are also prevalent in "penetratins", which are regions of proteins or synthetic peptides that can spontaneously cross membranes and in short hexapeptide antimicrobial peptides derived through combinatorial chemistry. While the antimicrobial, antifungal, antitumour, and antiviral properties of lactoferricin can be related to the Trp/Arg-rich portion of the peptide, we suggest that the anti-inflammatory and immunomodulating properties are more related to a positively charged region of the molecule, which, like the alpha- and beta-defensins, may act as a chemokine. Few small peptides are involved in as wide a range of host defense functions as bovine and human lactoferricin.     

The lactoferrin receptor complex in gram negative bacteria

Bacteria that inhabit the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment on the mucosal surface where iron is complexed by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic anti-microbial peptide lactoferricin is produced by proteolysis of lactoferrin. Several members of the Neisseriaceae and Moraxellaceae families express surface receptors, capable of specifically binding host lactoferrin and extracting the iron from lactoferrin as a source of iron for growth. The receptor is comprised of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a largely exposed surface lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for mediating growth using lactoferrin as a sole iron source whereas LbpB only plays a facilitating role. LbpB, with the presence of a large tract of negatively charged residues, appears to protect the bacterial cell from the bactericidal effects of the lactoferricin. The lactoferrin receptors in these species appear to be essential for survival and thus may serve as potential vaccine targets.     

The effect of bovine lactoferrin and lactoferricin B on the ability of feline calicivirus (a norovirus surrogate) and poliovirus to infect cell cultures

AIMS: To characterize the effect of bovine lactoferrin and lactoferricin B against feline calicivirus (FCV), a norovirus surrogate and poliovirus (PV), as models for enteric viruses. METHODS AND RESULTS: Crandell-Reese feline kidney (CRFK) cells were used for the propagation of FCV and monkey embryo kidney (MEK) cells for PV. The assays included visual assessment of cell lines for cytopathic effects and determination of the percentage cell death using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] dye reduction assay. Incubation of bovine lactoferrin with CRFK cells either prior to or together with FCV inoculation substantially reduced FCV infection. In contrast, the interference of lactoferrin with the infection of cells with PV was demonstrated only when lactoferrin was present with cell lines and virus for the entire assay period. Using indirect immunofluorescence, lactoferrin was detected on the surface of both CRFK and MEK cells, suggesting that the interference of viral infection may be attributed to lactoferrin binding to the surfaces of susceptible cells, thereby preventing the attachment of the virus particles. Lactoferricin B, a cationic antimicrobial peptide derived from the N-terminal domain of bovine lactoferrin, reduced FCV but not PV infection. CONCLUSION: Lactoferrin was shown to interfere with the infection of cells for both FCV and PV. However, lactoferricin B showed no interference of infection with PV and interference with infection for FCV required the presence of lactoferricin B together with the cell line and virus. SIGNIFICANCE AND IMPACT OF THE STUDY: An in vitro basis is provided for the effects of bovine lactoferrin and lactoferricin B in moderating food-borne infections of enteric viruses.     

Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin.

A physiologically diverse range of Gram-positive and Gram-negative bacteria was found to be susceptible to inhibition and inactivation by lactoferricin B, a peptide produced by gastric pepsin digestion of bovine lactoferrin. The list of susceptible organisms includes Escherichia coli, Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris, Yersinia enterocolitica, Pseudomonas aeruginosa, Campylobacter jejuni, Staphylococcus aureus, Streptococcus mutans, Corynebacterium diphtheriae, Listeria monocytogenes and Clostridium perfringens. Concentrations of lactoferricin B required to cause complete inhibition of growth varied within the range of 0.3 to 150 micrograms/ml, depending on the strain and the culture medium used. The peptide showed activity against E. coli O111 over the range of pH 5.5 to 7.5 and was most effective under slightly alkaline conditions. Its antibacterial effectiveness was reduced in the presence of Na+, K+, Mg2+ or Ca2+ ions, or in the presence of various buffer salts. Lactoferricin B was lethal, causing a rapid loss of colony-forming capability in most of the species tested. Pseudomonas fluorescens, Enterococcus faecalis and Bifidobacterium bifidum strains were highly resistant to this peptide.     

The Negatively Charged Regions of Lactoferrin Binding Protein B,an Adaptation against Anti-Microbial Peptides

Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein’s C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides.     

Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin

A physiologically diverse range of Gram-positive and Gram-negative bacteria was found to be susceptible to inhibition and inactivation by lactoferricin B, a peptide produced by gastric pepsin digestion of bovine lactoferrin. The list of susceptible organisms includes Escherichia coli, Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris, Yersinia enterocolitica, Pseudomonas aeruginosa, Campylobacter jejuni, Staphylococcus aureus, Streptococcus mutans, Corynebacterium diphtheriae, Listeria monocytogenes and Clostridium perfringens. Concentrations of lactoferricin B required to cause complete inhibition of growth varied within the range of 0.3 to 150 μg/ml, depending on the strain and the culture medium used. The peptide showed activity against E. coli O111 over the range of pH 5.5 to 7.5 and was most effective under slightly alkaline conditions. Its antibacterial effectiveness was reduced in the presence of Na⁺, K⁺, Mg²⁺ or Ca²⁺ ions, or in the presence of various buffer salts. Lactoferricin B was lethal, causing a rapid loss of colony-forming capability in most of the species tested. Pseudomonas fluorescens, Enterococcus faecalis and Bifidobacterium bifidum strains were highly resistant to this peptide.     

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.     

Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1

Although both lactoferrin (Lf), a component of the innate immune system of living organisms, and its N-terminal pepsin cleavage product lactoferricin (Lfcin) have anti-herpes activity, the precise mechanisms by which Lf and Lfcin bring about inhibition of herpes infections are not fully understood. In the present study, experiments were carried out to characterize the activity of bovine Lf and Lfcin (BLf and BLfcin) against the Herpes simplex virus-1 (HSV-1). HSV-1 cellular uptake and intracellular trafficking were studied by immunofluorescence microscopy. In comparison to the untreated infected control cells, both the BLf- and BLfcin-treated cells showed a significant reduction in HSV-1 cellular uptake. The few virus particles that were internalized appeared to have a delayed intracellular trafficking. Thus, in addition to their interference with the uptake of the virus into host cells, Lf and Lfcin also exert their antiviral effect intracellularly.     

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。     

Molecular dynamics simulations of bovine lactoferricin: turning a helix into a sheet

Bovine lactoferricin is a 25-residue peptide that is excised through pepsin cleavage in the stomach from the intact 80 kDa bovine milk protein lactoferrin. This basic peptide contains a single disulfide crosslink and is considerably more active as an antimicrobial peptide than the intact protein. It has been suggested that the dramatic difference in potency is related to a change in the secondary and tertiary structure of this peptide, moving from a mixed alpha-helical beta-strand region in the protein to an amphipathic twisted antiparallel beta-sheet in the peptide. Here we have used equilibrium and restrained molecular dynamics calculations to compare the stability of the solution structure of the isolated peptide with that excised from the intact protein. Simulations were performed for fully solvated peptides in the absence and presence of 250 mM salt. Our results show that the peptide as released from the protein is relatively unstable, particularly in the absence of salt. However, even though the simulations extended over 60 nsecs, no interconversion could be observed between the crystal and solution structures, unless a relatively small directional force was exerted on the peptide. A pathway for the structural transition from a helical to a sheet structure was identified in this fashion.     

Identification of the bactericidal domain of lactoferrin.

We report the existence of a previously unknown antimicrobial domain near the N-terminus of lactoferrin in a region distinct from its iron-binding sites. A single active peptide representing this domain was isolated following gastric pepsin cleavage of human lactoferrin, and bovine lactoferrin, and sequenced by automated Edman degradation. The antimicrobial sequence was found to consist mainly of a loop of 18 amino acid residues formed by a disulfide bond between cysteine residues 20 and 37 of human lactoferrin, or 19 and 36 of bovine lactoferrin. Synthetic analogs of this region similarly exhibited potent antibacterial properties. The active peptide of bovine lactoferrin was more potent than that of human lactoferrin having effectiveness against various Gram-negative and Gram-positive bacteria at concentrations between 0.3 microM and 3.0 microM, depending on the target strain. The effect of the isolated domain was lethal causing a rapid loss of colony-forming capability. Our studies suggest this domain is the structural region responsible for the bacterial properties of lactoferrin.     

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.     

Microbicidal effect of the lactoferrin peptides Lactoferricin17–30, Lactoferrampin265–284, and Lactoferrin chimera on the parasite Entamoeba histolytica

Entamoeba histolytica is a parasitic protozoan that produces amoebiasis, an intestinal disease characterized by ulcerative colitis and dysentery. In some cases, trophozoites can travel to the liver leading to hepatic abscesses and death. Recently, lactoferrin and lactoferricin B have been shown to be amoebicidal in axenic cultures. The aim of this work was to determine whether the lactoferrin-peptides lactoferricin amino acids 17–30, lactoferrampin amino acids 265–284, and lactoferrin chimera which is a fusion product of the two peptides, are capable of producing a microbicidal effect to trophozoites of E. histolytica. We evaluated the killing effect of these peptides in growth kinetics carried out in axenic culture medium to which different concentrations of peptides were added. At 50 μM of peptide concentration, lactoferricin and lactoferrampin had a moderate amoebicidal effect, since a 45–50% of trophozoites remained viable at 24 h culture. However, at 50 μM of the lactoferrin chimera 75% amoeba were killed whereas at 100 μM all cells died. These data indicate that of lactoferrin-peptides mainly the chimera have amoebicidal activity in a time- and concentration-dependent manner. The lactoferrin-peptides might be useful as therapeutic agents against amoebiasis and thereby diminish the use of metronidazole, which is extremely toxic for the host.     

The role of lactoferrin binding protein B in mediating protection against human lactoferricin

Bacteria that inhabit the mucosal surfaces of the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment because of iron sequestration by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic, antimicrobial peptide lactoferricin is produced by proteolysis of lactoferrin. Several Gram-negative pathogens express a lactoferrin receptor that enables the bacteria to use lactoferrin as an iron source. The receptor is composed of an integral membrane protein, lactoferrin binding protein A (LbpA), and a membrane-bound lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for growth with lactoferrin as the sole iron source, whereas the role of LbpB in iron acquisition is not yet known. In this study, we demonstrate that LbpB from 2 different species is capable of providing protection against the killing activity of a human lactoferrin-derived peptide. We investigated the prevalence of lactoferrin receptors in bacteria and examined their sequence diversity. We propose that the protection against the cationic antimicrobial human lactoferrin-derived peptide is associated with clusters of negatively charged amino acids in the C-terminal lobe of LbpB that is a common feature of this protein.