Involvement of bovine lactoferrin metal saturation, sialic acid and protein fragments in the inhibition of rotavirus infection.

Although the antiviral activity of lactoferrin is one of the major biological functions of this iron binding protein, the mechanism of action is still under debate. We have investigated the role of metal binding, of sialic acid and of tryptic fragments of bovine lactoferrin (bLf) in the activity towards rotavirus (intestinal pathogen naked virus) infecting enterocyte-like cells. The antiviral activity of bLf fully saturated with manganese or zinc was slightly decreased compared to that observed for apo- or iron-saturated bLf. The antiviral activity of differently metal-saturated bLf towards rotavirus was exerted during and after the virus attachment step. The removal of sialic acid enhanced the anti-rotavirus activity of bLf. Among all the peptidic fragments obtained by tryptic digestion of bLf and characterised by advanced mass spectrometric methodologies, a large fragment (86-258) and a small peptide (324-329: YLTTLK) were able to inhibit rotavirus even if at lower extent than undigested bLf.     

Bacterial lactoferrin receptors: insights from characterizing the Moraxella bovis receptors.

Moraxella bovis is the causative agent of infectious conjunctivitis in cattle. Moraxella bovis isolates were shown to specifically bind bovine lactoferrin (bLf) and bovine transferrin (bTf) and to use these proteins as a source of iron to support the growth of iron-limited cells. Affinity isolation experiments with immobilized bTf yielded two proteins readily resolved by SDS-PAGE analysis, whereas only a single band of approximately 100 kDa was detected when immobilized bLf was used as the affinity ligand. Using a novel cloning strategy, regions containing the genes encoding the lactoferrin (Lf) and transferrin (Tf) receptor proteins were isolated and sequenced, demonstrating that they both consisted of two genes, with the tbpB or lbpB gene preceding the tbpA or lbpA gene. The cloned lbp genes were used to generate isogenic mutants deficient in lactoferrin binding protein A and (or) B, and the resulting strains were tested in growth and binding assays. The isogenic mutants were deficient in their use of bLf for growth and had substantially diminished bLf binding capability. The predicted amino acid sequence from the segment encoding Lf binding protein B revealed an internal amino acid homology suggesting it is a bi-lobed protein, with a C-lobe enriched in acidic amino acids, but without the evident clustering observed in Lf-binding proteins from other species.     

Ovotransferrin possesses SOD-like superoxide anion scavenging activity that is promoted by copper and manganese binding

The embryo of oviparous species is confronted by a highly oxidative stress generating as it grows and must rely on effective antioxidant system for protection. Proteins of avian egg albumin have been suggested to play the major redox-modulatory role during embryo development. Recently, we found that ovotransferrin (OTf) undergoes distinct thiol-linked self-cleavage in a redox-dependent process. In this study, we explore that OTf is SOD mimic protein with a potent superoxide anion (O₂⁻) scavenging activity. The O₂⁻ scavenging activity was investigated using the natural xanthine/xanthine oxidase (X/XOD) coupling system. OTf exhibited O₂⁻ scavenging activity in a dose-dependent manner and showed remarkably higher scavenging activity than the known antioxidants, ascorbate or serum albumin. The isolated half-molecules of OTf exhibited higher scavenging activity than the intact molecule, whereas the N-lobe showed much greater activity. OTf dramatically quenched the O₂⁻ flux but had no effect on the urate production in the X/XOD system, indicating its unique specificity to scavenge O₂⁻ but not oxidase inhibition. Strikingly, metal-bound OTf exhibited greater O₂⁻ dismutation capacity than the apo-protein, ranging from moderate (Zn²⁺-OTf and Fe²⁺-OTf) to high (Mn²⁺-OTf and Cu²⁺-OTf) activity with the Cu²⁺-OTf being the most potent scavenger. In a highly sensitive fluorogenic assay, the metal-bound OTf exhibited significant increase in the rate of H₂O₂ production in the X/XOD reaction than the apo-OTf, providing evidence that Zn²⁺-, Mn²⁺- and Cu²⁺-OTf possess SOD mimic activity. This finding is the first to describe that OTf is an O₂⁻ scavenging molecule, providing insight into its novel SOD-like biological function, and heralding a fascinating opportunity for its potential candidacy as antioxidant drug.     

Glycosaminoglycans are not indispensable for the anti-herpes simplex virus type 2 activity of lactoferrin

Lactoferrin has been recognized as a potent inhibitor of human herpetic viruses, such as herpes simplex type 1 (HSV-1) and 2 (HSV-2). In particular, bovine lactoferrin (bLf) has been found to prevent viral infection by binding to heparan sulphate (HS) glycosaminoglycans (GAGs) that in turn can act as cell receptors for human herpetic viruses. In this study we further investigate the mechanism of inhibiting activity of both human lactoferrin (hLf) and bLf against HSV-2. The antiviral effect of these proteins towards HSV-2 strain 333 and its glycoprotein C (gC)-truncated derivative HSV-2 gC-neg1 has been tested in monkey kidney cells. Our results indicate that the antiviral activity of bLf does not involve gC-HS interaction as there was no difference in its effectiveness towards wild type and mutant virus. As regards hLf, the mutant virus HSV-2 gC-neg1 was more sensitive compared to the wild type, suggesting that the human protein might interact with some viral structures that in wild-type viruses are masked by gC. When the modulation of HSV-2 infection by bLf and hLf was investigated under different experimental conditions, the bovine protein proved more effective than the human protein. Moreover, we found that, differently from what observed with HSV-1, bLf inhibited HSV-2 plaque-forming activity also in cells devoid of GAG expression. These results suggest that bLf may block a virus receptor of non-GAG nature and add new information on the anti-herpes virus activity of this protein, confirming it as an outstanding candidate for the treatment of herpetic infections.     

Ca2+ binding to bovine lactoferrin enhances protein stability and influences the release of bacterial lipopolysaccharide.

Bovine lactoferrin (bLf) is known to damage the outer membrane of Gram-negative bacteria by binding to bacterial lipopolysaccharide (LPS). We report that LPS is released from bacterial outer membranes also when apo- or metal-saturated Lf is separated from bacterial cells by a dialysis membrane. This process occurs in phosphate-buffered saline with no added Ca2+ and Mg2+ and is hindered by addition of these cations. The effect of bLf is similar to that induced by EDTA and has been ascribed to chelation of Ca2+. In fact, it may be envisaged that Ca2+-binding sites on LPS have different affinities and that bLf can remove those ions that are more weakly bound. Ca2+ binding does not alter Lf iron-binding properties significantly or its UV and CD spectral features but brings about changes in the FT-IR bands due to carboxylate residues. Ca2+ binding is characterized by an apparent dissociation constant of 6 microM and a stoichiometry of 1.55 Ca2+ per Lf molecule; it enhances bLf stability towards chemical and thermal denaturation. The increase in stability takes place in both the apo- and iron-saturated forms but not in the desialilated protein, indicating that the carboxylate groups of the sialic acid residues present on two of the glycan chains are involved in Ca2+ binding.     

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.     

The bovine lactoferrin region responsible for promoting the collagen gel contractile activity of human fibroblasts

We have reported that bovine lactoferrin (bLf) promotes the contractile activity of collagen gels by WI-38 human fibroblasts via the phosphorylation of myosin light chain (MLC). To identify the region of bLf that is responsible for this activity, we prepared bLf fragments by limited proteolysis using trypsin and investigated the effects of each fragment on gel contractile activity. Lf consists of a single polypeptide chain containing two lobes that are independent globular structures termed the N- and C-lobes.The fragment corresponding to the C-lobe of bLf (amino acids 341-689) had a more prominent effect on collagen gel contractile activity than did that of either native bLf or its N-lobe (1-284). Further hydrolysis of the C-lobe with either pepsin or trypsin resulted in a loss of this activity. The effect of the C-lobe on collagen gel contraction by fibroblasts was dose-dependent and was associated with the elevation of MLC phosphorylation.     

The binding of lactoferrin to glycosaminoglycans on enterocyte-like HT29-18-C1 cells is mediated through basic residues located in the N-terminus

Although lactoferrins (Lfs) isolated from milk of various mammals exhibit a close structural relationship, they show species-specific binding to cells. To define the specificity of recognition of human (hLf), bovine (bLf) and murine (mLf) lactoferrin by human intestinal cells, we analysed the binding of the three proteins to a subclone derived from human carcinoma cell line HT29. We observed that hLf and bLf interact with two types of binding sites (K_d: 63±22 nM; 0.7±0.2 μM) while mLf was recognized only by the lowest affinity binding sites with a lower number of binding sites. Using N-terminal deleted human Lf variants, we found that the sequence G¹RRRR⁵ is mainly responsible for the interactions with HT29 cells. Lactoferrin-binding sites on the surface of HT29 cells were further identified as heparan sulphate and chondroitin sulphate glycosaminoglycans. We conclude that the presence of the sequence A¹PRK⁴ in bLf and K¹ATT⁴ in mLf provides an insight into why the interaction of bLf with cell membrane-associated glycosaminoglycans is similar to that of hLf and why binding of these lactoferrin species differs from that of murine Lf.     

Improved autoprocessing efficiency of mutant subtilisins E with altered specificity by engineering of the pro-region.

Modification of substrate specificity of an autoprocessing enzyme is accompanied by a risk of significant failure of self-cleavage of the pro-region essential for activation. Therefore, to enhance processing, we engineered the pro-region of mutant subtilisins E of Bacillus subtilis with altered substrate specificity. A high-activity mutant subtilisin E with Ile31Leu replacement (I31L) as well as the wild-type enzyme show poor recognition of acid residues as the P1 substrate. To increase the P1 substrate preference for acid residues, Glu156Gln and Gly166Lys/Arg substitutions were introduced into the I31L gene based upon a report on subtilisin BPN' [Wells et al. (1987) Proc. Natl. Acad. Sci. USA 84, 1219-1223]. The apparent P1 specificity of four mutants (E156Q/G166K, E156Q/G166R, G166K, and G166R) was extended to acid residues, but the halo-forming activity of Escherichia coli expressing the mutant genes on skim milk-containing plates was significantly decreased due to the lower autoprocessing efficiency. A marked increase in active enzyme production occurred when Tyr(-1) in the pro-region of these mutants was then replaced by Asp or Glu. Five mutants with Glu(-2)Ala/Val/Gly or Tyr(-1)Cys/Ser substitution showing enhanced halo-forming activity were further isolated by PCR random mutagenesis in the pro-region of the E156Q/G166K mutant. These results indicated that introduction of an optimum arrangement at the cleavage site in the pro-region is an effective method for obtaining a higher yield of active enzymes.     

Apoptotic effects of bovine apo‐lactoferrin on HeLa tumor cells

Lactoferrin (Lf), a cationic iron‐binding glycoprotein of 80 kDa present in body secretions, is known as a compound with marked antimicrobial activity. In the present study, the apoptotic effect of iron‐free bovine lactoferrin (apo‐bLf) on human epithelial cancer (HeLa) cells was examined in association with reactive oxygen species and glutathione (GSH) levels. Apoptotic effect of iron‐free bovine lactoferrin inhibited the growth of HeLa cells after 48 hours of treatment while the diferric‐bLf was ineffective in the concentration range tested (from 1 to 12.5 μM). Western blot analysis showed that key apoptotic regulators including Bax, Bcl‐2, Sirt1, Mcl‐1, and PARP‐1 were modulated by 1.25 μM of apo‐bLf. In the same cell line, apo‐bLf induced apoptosis together with poly (ADP‐ribose) polymerase cleavage, caspase activation, and a significant drop of NAD⁺. In addition, apo‐bLf–treated HeLa cells showed a marked increase of reactive oxygen species level and a significant GSH depletion. On the whole, apo‐bLf triggered apoptosis of HeLa cells upon oxygen radicals burst and GSH decrease.     

Oral administration of lactoferrin increases hemoglobin and total serum iron in pregnant women.

Iron deficiency anemia (IDA) during pregnancy continues to be of world-wide concern. IDA is a risk factor for preterm delivery and subsequent low birth weight, and possibly for poor neonatal health. Iron supplementation in pregnancy is a widely recommended practice, yet intervention programs have met with many controversies. In our study, 300 women at different trimesters of pregnancy were enrolled in a trial of oral administration of ferrous sulfate (520 mg once a day) or 30% iron-saturated bovine lactoferrin (bLf) (100 mg twice a day). Pregnant women refusing treatment represented the control group. In this group hemoglobin and total serum iron values measured after 30 d without treatment decreased significantly, especially in women at 18-31 weeks of pregnancy. In contrast, after 30 d of oral administration of bLf, hemoglobin and total serum iron values increased and to a greater extent than those observed in women treated orally for 30 d with ferrous sulfate, independently of the trimester of pregnancy. Unlike ferrous sulfate, bLf did not result in any side effects. These findings lead us to hypothesize that lactoferrin could influence iron homeostasis directly or through other proteins involved in iron transport out of the intestinal cells into the blood.     

Bovine lactoferrin inhibits Influenza A virus induced programmed cell death in vitro

Influenza is one of the main plagues worldwide. The statistical likelihood of a new pandemic outbreak, together with the alarming emergence of influenza virus strains that are resistant to available antiviral medications, highlights the need for new antiviral drugs. Lactoferrin, a 80 kDa bi-globular iron-binding glycoprotein, is a pleiotropic factor with potent antimicrobial and immunomodulatory activities. Although the antiviral effect of lactoferrin is one of its major biological functions, the mechanism of action is still under debate. In this research, we have analyzed the effect of bovine lactoferrin (bLf) on Influenza A virus infection in vitro. Our results showed that (i) Influenza virus infected cells died as a result of apoptosis, (ii) bLf treatment inhibited programmed cell death by interfering with function of caspase 3, a major virus-induced apoptosis effector, and (iii) bLf efficiently blocked nuclear export of viral ribonucleoproteins so preventing viral assembly. These results provide further insights on the antiviral activity of bLf and suggest novel strategies for treatment of Influenza virus infection.     

Lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases

Conflicting data are reported on pro- or anti-inflammatory activity of bovine lactoferrin (bLf) in different cell models as phagocytes or epithelial cell lines infected by bacteria. Here we evaluated the bLf effect on epithelial models mimicking two human pathologies characterized by inflammation and infection with specific bacterial species. Primary bronchial epithelium from a cystic fibrosis (CF) patient and differentiated intestinal epithelial cells were infected with Pseudomonas aeruginosa LESB58 isolated from a CF patient and Adherent-Invasive Escherichia coli LF82 isolated from a Crohn’s disease patient. Surprisingly, bLf significantly reduced the intracellular bacterial survival, but differently modulated the inflammatory response. These data lead us to hypothesize that bLf differentially acts depending on the epithelial model and infecting pathogen. To verify this hypothesis, we explored whether bLf could modulate ferroportin (Fpn), the only known cellular iron exporter from cells, that, by lowering the intracellular iron level, determines a non permissive environment for intracellular pathogens. Here, for the first time, we describe the bLf ability to up-regulate Fpn protein in infected epithelial models. Our data suggest that the mechanism underlying the bLf modulating activity on inflammatory response in epithelial cells is complex and the bLf involvement in modulating cellular iron homeostasis should be taken into account.     

A high-throughput, homogeneous, fluorescence polarization assay for inhibitors of hedgehog protein autoprocessing

Hedgehog (Hh) signaling plays an important role in embryonic patterning and adult stem cell renewal but has recently been found also to be involved in certain stem cell cancers. One of the first steps in Hh signaling is the autoprocessing of Hh protein, in which the C-terminal domain (Hh-C) catalyzes a cholesterol-dependent autocleavage reaction that leads to the production of the cholesterol ester of the N-terminal Hh domain (Hh-N), thereby yielding a signaling molecule that activates the Hh pathway by binding to the Patched receptor. This article describes an in vitro, homogeneous assay system that measures changes in fluorescence polarization that accompany the cholesterol-dependent autocleavage of Hh protein. The assay system makes use of a modified Hh protein in which Hh-N, which is not essential for autocleavage, is replaced by a 25-residue peptide containing a tetracysteine motif, complexed with a bisarsenical fluorophore. The assay is quite robust and easily adapted to high-throughput screening in 384-well plates with Z' factors above 0.8. It has been used to screen the National Institutes of Health Clinical Collection, which has led to the identification of 2 compounds that inhibit the cholesterol-dependent autocleavage of Hh protein at micromolar concentrations.     

Structural constraints on autoprocessing of the human nucleoporin Nup98

Nucleoporin Nup98, a 98-kDa protein component of the nuclear pore complex, plays an important role in both protein and RNA transport. During its maturation process, Nup98 undergoes post-translational autoproteolysis, which is critical for targeting to the NPC. Here we present high-resolution crystal structures of the C-terminal autoproteolytic domains of Nup98 (2.3 A for the wild type and 1.9 A for the S864A precursor), and propose a detailed autoproteolysis mechanism through an N-O acyl shift. Structural constraints are found at the autocleavage site, and could thus provide a driving force for autocleavage at the scissile peptide bond. Such structural constraints appear to be generated, at least in part, by anchoring a conserved phenylalanine side chain into a highly conserved hydrophobic pocket at the catalytic site. Our high-resolution crystal structures also reveal that three highly conserved residues, Tyr866, Gly867, and Leu868, provide most of the interactions between the autoproteolytic domain and the C-terminal tail. These results suggest that Nup98 may represent a new subtype of protein that utilizes autoprocessing to control biogenesis pathways and intracellular translocation.     

cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N-linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin-like (convertase) site and a C-terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure-fertilization function predictions for this phylogenetically conserved family of glycoproteins.