Lactoferrin Fragments Research Articles

Immunologic Effects of a Novel Bovine Lactoferrin-Derived Peptide on the Gut and Clinical Perspectives.

Bovine lactoferrin is a natural iron-binding glycoprotein known for its antimicrobial, antiviral, antitumor, anti-inflammatory, and immunomodulatory properties. In this study, we artificially recombined a fragment of bovine lactoferrin with immunomodulatory and antimicrobial properties to create a novel peptide named LF-MQL. The primary objective was to investigate the effects of LF-MQL on the intestinal tract and immune cells in animals. First, we assessed the in vitro activation effects of LF-MQL on mouse peritoneal macrophages. The results indicated that LF-MQL enhanced the macrophage phagocytic activity and increased IL-1β mRNA expression without significantly affecting IL-6 mRNA levels. Next, we examined the effects of LF-MQL on mucosal immunity by administering LF-MQL orally at doses of 300 mg/kg, 30 mg/kg, and 3 mg/kg to mice. The results demonstrated that different doses of LF-MQL modulated IL-6 and IL-10 mRNA levels in the small intestine. Low doses enhanced the intestinal immune response, while higher doses reduced the inflammatory response. In conclusion, LF-MQL exerts immunomodulatory effects rather than simply boosting immune activity in animal models.

Testing Antimicrobial Properties of Human Lactoferrin-Derived Fragments

Lactoferrin, an iron-binding glycoprotein, plays a significant role in the innate immune system, with antibacterial, antivirial, antifungal, anticancer, antioxidant and immunomodulatory functions reported. It is worth emphasizing that not only the whole protein but also its derived fragments possess antimicrobial peptide (AMP) activity. Using AmpGram, a top-performing AMP classifier, we generated three novel human lactoferrin (hLF) fragments: hLF 397-412, hLF 448-464 and hLF 668-683, predicted with high probability as AMPs. For comparative studies, we included hLF 1-11, previously confirmed to kill some bacteria. With the four peptides, we treated three Gram-negative and three Gram-positive bacterial strains. Our results indicate that none of the three new lactoferrin fragments have antimicrobial properties for the bacteria tested, but hLF 1-11 was lethal against Pseudomonas aeruginosa. The addition of serine protease inhibitors with the hLF fragments did not enhance their activity, except for hLF 1-11 against P. aeruginosa, which MIC dropped from 128 to 64 µg/mL. Furthermore, we investigated the impact of EDTA with/without serine protease inhibitors and the hLF peptides on selected bacteria. We stress the importance of reporting non-AMP sequences for the development of next-generation AMP prediction models, which suffer from the lack of experimentally validated negative dataset for training and benchmarking.

Targeted EpCAM-binding for the development of potent and effective anticancer proteins

Protein-based cancer therapies are considered an alternative to conventional anticancer regimens, providing multifunctional properties while showing low toxicity. However, its widespread use is limited by absorption and instability issues, resulting in higher dosage requirements and a prolonged onset of bioactivity to elicit the desired response. Here, we developed a non-invasive antitumor treatment using designed ankyrin repeat protein (DARPin)-anticancer protein-conjugate that specifically targets the cancer biomarker, epithelial cell adhesion molecule (EpCAM). The DARPin-anticancer proteins bind to EpCAM-positive cancer cells and improve the in vitro anticancer efficacy by over 100-folds within 24 h, where the DARPin-tagged human lactoferrin fragment (drtHLF4) IC50 value is within the nanomolar range. Orally administered drtHLF4 was readily absorbed into the systemic flow of the HT-29 cancer murine model, exerting its anticancer effect on other tumors in the host body. Orally administered drtHFL4 cleared HT29-colorectal tumors using a single dose, whereas intratumoral injection cleared HT29-subcutaneous tumors within three doses. This approach addresses the limitations of other protein-based anticancer treatments by providing a non-invasive anticancer therapy with improved potency and tumor-specificity.

Evaluation of the Anti-Inflammatory and Anti-Oxidative Effects of Therapeutic Human Lactoferrin Fragments.

Chronic inflammation is considered a pressing health issue that needs resolving. Inflammatory disease such as inflammatory bowel disease requires a long-term medical regimen to prevent disease progression. Conventionally, lactoferrin is used to treat mild gastrointestinal tract and skin inflammation. Protease-digested lactoferrin fragments often exhibit improved therapeutic properties compared to full-length lactoferrin (flHLF). However, there are no studies on the use of protease-digested lactoferrin fragments to treat inflammation. Herein, we assess the anti-inflammatory properties of engineered recombinant lactoferrin fragments (rtHLF4, rteHLF1, and rpHLF2) on non-malignant colonic fibroblast cells and colorectal cancer cells. We found that rtHLF4 is 10 times more effective to prevent inflammation compared to flHLF. These results were investigated by looking into the reactive oxygen species (ROS) production, angiogenesis activity, and cellular proliferation of the treated cells. We have demonstrated in this study the anti-inflammatory properties of the flHLF and the various lactoferrin fragments. These results complement the anti-cancer properties of these proteins that were demonstrated in an earlier study.

Engineering of Human Lactoferrin for Improved Anticancer Activity.

Protease-digested lactoferrin fragments often exhibit improved therapeutic properties. However, there are limited studies investigating the anticancer properties of these fragments. The fragment with improved anticancer activities is an attractive alternative to chemotherapeutic drugs-presenting severe side effects. Herein, we report the isolation and characterization of recombinant engineered-lactoferrin (rtHLF4), exhibiting up to 100-fold improved anticancer activity compared to the full-length lactoferrin (flHLF). Further, rtHLF4 exerts its anticancer effect in a shorter duration. Through transcriptomic analysis of various cancer biomarkers, rtHLF4 was found to upregulate various pro-apoptotic markers and downregulate signaling proteins involved in angiogenesis and metastasis. We further determined that rtHLF4 showed no hemolytic activity at high concentrations. We believe that this anticancer protein can be further developed as a cancer treatment.

Stability-Indicating Analytical Approach for Stability Evaluation of Lactoferrin.

Lactoferrin is a multifunctional iron-binding glycoprotein in milk. Due to its potential for the treatment of various diseases, interest in products containing lactoferrin is increasing. However, as a protein, it is prone to degradation, which critically affects the quality of products. Therefore, the main purpose of our work was to develop a stability-indicating analytical approach for stability evaluation of lactoferrin. We were focused on two complementary methods: reversed-phase and size-exclusion chromatography. The stability-indicating nature of the selected methods was confirmed. They were successfully validated by following the ICH guidelines and applied to preliminary lactoferrin stability studies. Up to three degradation products, as well as aggregates and fragments of lactoferrin, were detected in various samples using complementary reversed-phase and size-exclusion chromatographic methods. The analytical approach was additionally extended with three spectroscopic techniques (absorbance, intrinsic fluorescence, and bicinchoninic acid method), which may provide valuable complementary information in some cases. The presented analytical approach allows the stability evaluation of lactoferrin in various samples, including the ability to detect differences in its degradation mechanisms. Furthermore, it has the potential to be used for the quality control of products containing lactoferrin.

Lactosmart: A Novel Therapeutic Molecule for Antimicrobial Defense.

The problem of antibiotic resistance has prompted researchers around the globe to search for new antimicrobial agents. Antimicrobial proteins and peptides are naturally secreted by almost all the living organisms to fight infections and can be safer alternatives to chemical antibiotics. Lactoferrin (LF) is a known antimicrobial protein present in all body secretions. In this study, LF was digested by trypsin, and the resulting hydrolysates were studied with respect to their antimicrobial properties. Among the hydrolysates, a 21-kDa basic fragment of LF (termed lactosmart) showed promise as a new potent antimicrobial agent. The antimicrobial studies were performed on various microorganisms including Shigella flexneri, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli as well as fungal pathogens such as Candida albicans, Candida tropicalis, and Candida glabrata. In addition, the lipopolysaccharide (LPS)-binding properties of lactosmart were studied using surface plasmon resonance technique in vitro, along with docking of LPS and molecular dynamics (MD) simulation studies. The results showed that lactosmart had better inhibitory effects against pathogenic microorganisms compared to LF. The results of docking and MD simulation studies further validated the tighter binding of LPS to lactosmart compared to LF. The two LPS-binding sites have been characterized structurally in detail. Through these studies, it has been demonstrated that in native LF, only one LPS-binding site remains exposed due to its location being on the surface of the molecule. However, due to the generation of the lactosmart molecule, the second LPS-binding site gets exposed too. Since LPS is an essential and conserved part of the bacterial cell wall, the pro-inflammatory response in the human body caused by LPS can be targeted using the newly identified lactosmart. These findings highlight the immense potential of lactosmart in comparison to native LF in antimicrobial defense. We propose that lactosmart can be further developed as an antibacterial, antifungal, and antibiofilm agent.

Efficient and Short Method for Conjugation of 1-Nitro-9-Aminoacridine to Important Peptidyl Fragments by a Solid Support Synthesis.

The efficient and short techniques for conjugation of 9-aminoacridine with different peptidyl fragments are necessary for the development of active pharmaceutical ingredients (API). They need to be adopted to generate a new branch of acridine conjugates, enhancing their bioavailability for the examination in biological systems. The branch of developing acridine conjugates, built via different linkers and synthesized in this study, are expected as potential effective chemotherapeutics with dual mechanism of action. Recently, the methodology based on a solid-phase technique has been successfully demonstrated in preparing a number of promising compounds. However, the reaction conditions for amide bond formation between 1-nitro-9-aminoacridine and peptidyl fragments need to be optimized. In this study, the optimization of amide bond formation was demonstrated with the use of the solid-phase synthesis to build a new promising group of 1-nitro-9-aminoacridines conjugated to lactoferrin fragments via especially carboxy linker length.

Resolution-Associated Lactoferrin Peptides Limit LPS Signaling and Cytokine Secretion from Human Macrophages.

The neutrophil granule protein lactoferrin is cleaved and accumulates in efferocytic macrophages as inflammation is resolved. Two peptides present within a resolution-associated 17 kDa fragment of lactoferrin promote the termination of inflammation in vivo by enhancing murine macrophage reprogramming. Here, we report that these two bioactive tripeptides, phenylalanine-lysine-aspartic acid and phenylalanine-lysine-glutamic acid (FKD and FKE, respectively), inhibit ERK and cJun activation following human macrophage exposure to LPS. In addition, these peptides at low concentrations (1–10 μM) modulate human macrophage reprogramming to an anti-inflammatory/pro-resolving phenotype. This was reflected by inhibition of LPS-induced TNF-α and IL-6 secretion and increased IL-10 levels. Moreover, we found naturally occurring FKE analogs (FKECH and FKECHLA) can recapitulate the activity of the short peptide in regulating macrophage cytokine secretion, whereas a reversed EKF peptide was inert in this respect. Curiously, FKD and FKE also regulated cytokine production by bone marrow-derived mouse macrophages, but in a very different fashion than their effect on human macrophages. Thus, lactoferrin peptides limit pro-inflammatory signaling and cytokine production by LPS-activated human macrophages and thereby enhance the resolution of inflammation.

A 17-kDa Fragment of Lactoferrin Associates With the Termination of Inflammation and Peptides Within Promote Resolution.

During the resolution of inflammation, macrophages engulf apoptotic polymorphonuclear cells (PMN) and can accumulate large numbers of their corpses. Here, we report that resolution phase macrophages acquire the neutrophil-derived glycoprotein lactoferrin (Lf) and fragments thereof in vivo and ex vivo. During the onset and resolving phases of inflammation in murine peritonitis and bovine mastitis, Lf fragments of 15 and 17 kDa occurred in various body fluids, and the murine fragmentation, accumulation, and release were mediated initially by neutrophils and later by efferocytic macrophages. The 17-kDa fragment contained two bioactive tripeptides, FKD and FKE that promoted resolution phase macrophage conversion to a pro-resolving phenotype. This resulted in a reduction in peritoneal macrophage numbers and an increase in the CD11blow subset of these cells. Moreover, FKE, but not FKD, peptides enhanced efferocytosis of apoptotic PMN, reduced TNFα and interleukin (IL)-6, and increased IL-10 secretion by lipopolysaccharide-stimulated macrophages ex vivo. In addition, FKE promoted neutrophil-mediated resolution at high concentrations (100 µM) by enhancing the formation of cytokine-scavenging aggregated NETs (tophi) at a low cellular density. Thus, PMN Lf is processed, acquired, and “recycled” by neutrophils and macrophages during inflammation resolution to generate fragments and peptides with paramount pro-resolving activities.

A study of effects of peptide fragments of bovine and human lactoferrins on activities of three key HIV-1 enzymes

The intent of this study was to examine human and bovine lactoferrin fragments including lactoferrin (1-11), lactoferricin and lactoferrampin, all of which did not demonstrate hemolytic activity toward rabbit erythrocytes at 1mM concentration, for possible inhibitory effects on the activities of HIV-1 reverse transcriptase, protease and integrase. The data showed that human lactoferricin was the most potent in inhibiting HIV-1 reverse transcriptase (IC50=2μM). Bovine lactoferricin (IC50=10μM) and bovine lactoferrampin (IC50=150μM) were less potent. Human lactoferrampin and human and bovine lactoferrin (1-11) at 1mM concentration did not exhibit any inhibitory effect on HIV-1 reverse transcriptase. All peptides showed only a slight inhibitory effect (from slightly below 2% to 6% inhibition) on HIV-1 protease. Human lactoferrampin and bovine lactoferrampin showed obvious inhibitory effect on HIV-1 integrase at 37μM and 18.5μM, respectively. The HIV-1 integrase inhibitory activity of human lactoferrampin and bovine lactoferrampin was dose-dependent. The other peptides were devoid of HIV-1 integrase inhibitory activity. Thus, it is concluded that some lactoferrin fragments exert an inhibitory action on HIV-1 reverse transcriptase and HIV-1 integrase.

Preparation and Antimicrobial Action of Three Tryptic Digested Functional Molecules of Bovine Lactoferrin

Lactoferrin is an 80 kDa bilobal, iron binding glycoprotein which is primarily antimicrobial in nature. The hydrolysis of lactoferrin by various proteases in the gut produces several functional fragments of lactoferrin which have varying molecular sizes and properties. Here, bovine lactoferrin has been hydrolyzed by trypsin, the major enzyme present in the gut, to produce three functional molecules of sizes approximately 21 kDa, 38 kDa and 45 kDa. The molecules have been purified using ion exchange and gel filtration chromatography and identified using N-terminal sequencing, which reveals that while the 21 kDa molecule corresponds to the N2 domain (21LF), the 38 kDa represents the whole C-lobe (38LF) and the 45 kDa is a portion of N1 domain of N-lobe attached to the C-lobe (45LF). The iron binding and release properties of 21LF, 38LF and 45LF have been studied and compared. The sequence and structure analysis of the portions of the excision sites of LF from various species have been done. The antibacterial properties of these three molecules against bacterial strains, Streptococcus pyogenes, Escherichia coli, Yersinia enterocolitica and Listeria monocytogenes were investigated. The antifungal action of the molecules was also evaluated against Candida albicans. This is the first report on the antimicrobial actions of the trypsin cleaved functional molecules of lactoferrin from any species.

Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

Antimicrobial and DNA-binding activities of the peptide fragments of human lactoferrin and histatin 5 against Streptococcus mutans

ObjectiveTo investigate the killing effect of two salivary antimicrobial peptides, hLF1–11 and P-113, and identify the antibacterial mechanism of the peptides. MethodsThe antimicrobial activities of hLF1–11 and P-113 against oral Streptococci strains were determined using the broth microdilution method. The effects of hLF1–11 and P-113 on the bacterial plasma membrane were visualized by scanning electron microscopy. Cell membrane permeability was monitored using the intracellular dye calcein. The subcellular localization of hLF1–11 and P-113 in bacteria was measured by fluorescence light microscopy. An electrophoretic mobility shift assay (EMSA) was performed to evaluate the DNA binding capabilities of hLF1–11, P-113 and MUC7 12-mer. ResultsBoth hLF1–11 and P-113 exerted potent bactericidal activities against all selected oral Streptococci. Streptococcus mutans UA 159 was the most susceptible of the oral bacterial species tested to the antimicrobial effects of the three peptides. The cell membranes of bacteria treated with hLF1–11 or P-113 were still intact after 30min. hLF1–11 and P-113 could penetrate the bacterial cell membranes and accumulate in the cytoplasm in S. mutans. Both hLF1–11 and P-113 showed DNA binding affinity. ConclusionsTogether, our results demonstrate that hLF1–11 and P-113 display antibacterial activity against dental cavity-inducing S. mutans through an intracellular mechanism that could involve DNA binding. Thus, these peptides might be attractive and valuable candidates for development into effective antimicrobial therapies to combat dental caries.

Characterization of Chain Order of an Acylated-Lactoferricin Peptide by Solid-State NMR Spectroscopy and All-Atom Molecular Dynamics Simulations

The hexapeptide, LfB6 (RRWQWR-NH2), derived from a cationic antimicrobial 25-residue fragment of bovine lactoferrin, retains broad spectrum activity. The two tryptophans and three arginines are thought to promote membrane interaction. Attachment of a short fatty acid to the N-terminus (C6-LfB6) further enhances membrane binding (Greathouse, et al. 2008. J. Pept. Sci. 14:1103). The mechanism by which antimicrobial peptides interact with bacterial cell membranes is proposed to depend on lipid composition. Mammalian membranes are comprised primarily of neutral lipids, whereas bacterial membranes contain a significant fraction of negatively charged lipids. We have shown using all-atom molecular dynamics simulations that association of C6-LfB6 with negatively charged membranes (3:1 POPE:POPG) begins with the arginines, followed by the tryptophans, with the C6 ‘tails’ inserting last into the membrane (Grossfield, et al. 2010. Biophys J. 98:92a). By contrast, the association with a zwitterionic membrane (POPC) is led by the C6 tail, followed by the tryptophans and arginines. Solid-state 2H NMR experimental results confirmed that the lipid order parameters are not significantly changed when C6-LfB6 is bound to negatively-charged membranes, while a slight decrease in order is observed with zwitterionic membranes. We now present the order parameters for a deuterated 6-carbon acyl chain (C6-d11) attached to either LfB6 or a single glycine control. Solid-state 2H NMR results shown an increase in acyl chain order for C6-LfB6 in POPE:POPG compared to POPC, whereas the chain order for C6-Gly is the same in POPE:POPG and POPC. Additionally, the quadrupolar splittings of individual methylene deuterons are resolved at acyl chain carbons 2-4 in C6-LfB but not in C6-Gly, suggesting restricted motion for the membrane-embedded acyl-peptide. Results from all-atom molecular dynamics simulations will be compared with experimental data from solid-state NMR.

N-Acylation of Antimicrobial Peptides Causes Different Mode of Cell Membrane Damage

Lipopeptides such as polymyxins, octapeptins or daptomycin often show an increased activity against bacteria as compared to their non-acylated analogue. Thus, we have studied N-acylated synthetic peptides derived from a fragment of human lactoferrin (LF-11) to elucidate the interaction of these peptides with Gram-negative and Gram-positive bacteria and membrane mimetic systems using various biophysical and biological methods.Calorimetric studies on liposomes composed of phosphatidylglycerol revealed that the parent peptide induced a phase separation into peptide-enriched and -poor domains, which however consist of a similar domain size as calculated by the cooperative units. In contrast, at the same lipid-to-peptide molar ratio (25:1) the N-acylated derivatives strongly broadened the phase transition range and lowered markedly the main transition temperature. This is indicative for rather small and inhomogeneous domains, which will result in large line defects increasing membrane permeability as observed in intact bacteria. Membrane destabilization of E. coli and S. aureus induced by the peptides was monitored by using the membrane-potential-sensitive dye DiIC1 and the extent of membrane damage caused by the peptides by the cationic dye SYTOX green, which cannot enter intact cells unless its membrane is disrupted by external compounds. In both assays the N-acylated peptides showed a dramatic increase of fluorescence indicating massive membrane damage. This is supported by electron micrographs, which clearly showed a loss of cytoplasmic content and membrane rupture in the presence of the N-acylated peptide. Nevertheless, the extent of cell membrane rupture does not necessarily strongly correlate with the MIC-value of the peptides emphasizing the different mode of interaction of (non)-acylated peptides, which in part may be related to different degree of interaction with cell membrane/wall components such as lipopolysaccharides and lipoteichoic acid.Acknowledgement to EC-projects “ANEPID” and “BIOCONTROL”

In vitro reconstitution of antimicrobial pathogen activity by expressed recombinant bovine lactoferrin N-terminal peptide in Escherichia coli

Recombinant bovine lactoferrin N-terminal polypeptide (rbLF-N) Escherichia coli expression system was constructed and the rbLF-N antimicrobial activity was displayed by enzymatic proteolysis in this study. A 162 bp 5'-terminal fragment of bovine lactoferrin (bLF) gene from bovine liver gDNA was amplified by PCR. The DNA fragment containing exon-2 of the bLF gene was cloned into the expression vector pGEX-4T1 and the glutathione-S-transferase-rbLF-N (GST-rbLF-N) fusion protein was obtained by over-expression in Esch. coli BL21(DE3). After thrombin/pepsin digestion, the rbLF-N was released from the fusion protein. The recombinant peptide was separated and identified by SDS-PAGE, HPLC and LC-MS/MS analysis. A very strong anti-food-born microbial pathogen activity of the rbLF-N peptides was displayed through bio- and kinetic-assays in vitro. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the rbLF-N peptide for bacterial pathogens Staphylococcus aureus, Streptococcus mutans, Esch. coli and Klebsiella pneumoniae were 11.7, 11.7, 11.7, 23.4 microg and 23.4, 11.7, 11.7, 46.4 microg, respectively. This study created a new route for exploring lactoferrin peptide application in food science.

The anti-papillomavirus activity of human and bovine lactoferricin

Human papillomavirus (HPV) cause common warts, laryngeal papilloma and genital condylomata and is necessary for the development of cervical cancer. We have previously found that lactoferrin has antiviral activity against HPV-16 and others have demonstrated that lactoferricin, an N-terminal fragment of lactoferrin, has inhibitory activities against several viruses. Two cell lines and two virus types, HPV-5 and HPV-16, were used to study if lactoferrin and lactoferricin could inhibit HPV pseudovirus (PsV) infection. We demonstrated that bovine lactoferrin (bLf) and human lactoferrin (hLf) were both potent inhibitors of HPV-5 and -16 PsV infections. Among the four lactoferricin derivatives we analyzed, a 15 amino acid peptide from bovine lactoferricin (bLfcin) 17–31 was the most potent inhibitor of both HPV-5 and HPV-16 PsV infection. Among the other derivatives, the human lactoferricin (hLfcin) 1–49 showed some antiviral activity against HPV PsV infection while bLfcin 17–42 inhibited only HPV-5 PsV infection in one of the cell lines. When we studied initial attachment of HPV-16, only bLfcin 17–42 and hLfcin 1–49 had an antiviral effect. This is the first time that lactoferricin was demonstrated to have an inhibitory effect on HPV infection and the antiviral activity differed depending on size, charge and structures of the lactoferricin.

Bovine lactoferrin peptidic fragments involved in inhibition of Echovirus 6 in vitro infection

Bovine lactoferrin is a multifunctional glycoprotein folded in two symmetric globular lobes (N- and C-lobes), each being able to bind one ferric ion. We have previously demonstrated that this protein is able to prevent echovirus-induced apoptosis. In the present study, we have investigated both the role of tryptic fragments of bovine lactoferrin and the mechanism of lactoferrin effect on echovirus infection. Results obtained showed that bovine lactoferrin inhibits echovirus-induced cytopathic effect and antigen synthesis in a dose-dependent manner and that this protein is able to prevent viral replication when added not only during the entire cycle of infection but also before, during or after the viral adsorption step. The N-terminal cationic peptide was sufficient to prevent viral binding. Our data suggest that lactoferrin inhibition of echovirus attachment to cell receptors could be mediated by the cluster of positive charges at its N-terminus (lactoferricin).

Antiviral activity of ovotransferrin derived peptides

Ovotransferrin and lactoferrin are iron-binding proteins with antiviral and antibacterial activities related to natural immunity, showing marked sequence and structural homologies. The antiviral activity of two hen ovotransferrin fragments DQKDEYELL (hOtrf 219–227) and KDLLFK (hOtrf 269–301 and hOtrf 633–638) towards Marek’s disease virus infection of chicken embryo fibroblasts is reported here. These fragments have sequence homology with two bovine lactoferrin fragments with antiviral activity towards herpes simplex virus, suggesting that these fragments could have a role for the exploitation of the antiviral activity of the intact proteins towards herpes viruses. NMR analysis showed that these peptides, chemically synthetized, did not possess any favourite conformation in solution, indicating that both the aminoacid sequence and the conformation they display in the intact protein are essential for the antiviral activity.