Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence

We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin.     

Intrachain disulfide bridges of bovine seminal ribonuclease

The pairing of the four intrachain disulfide bonds of bovine seminal ribonuclease, a dimeric protein isolated from bovine seminal plasma, has been established by the isolation and characterization of the cystine peptides obtained from a thermolytic-tryptic hydrolysate of the protein. These disulfide bonds involve eight half-cystine residues located in the protein subunit chain at sequence positions identical with those of the eight half-cystine residues of the strictly homologous chain of bovine pancreatic ribonuclease. The results reported show that these eight 'homologous' half-cystine residues pair in seminal ribonuclease exactly as they do in pancreatic ribonuclease. They also indirectly confirm that the remaining two half-cystine residues present in each chain of the seminal enzyme are involved in intersubunit bonds.     

Iron-binding Proteins in Milk and Resistance to Escherichia coli Infection in Infants

Human milk contains large quantities of iron-binding protein, of which the greater proportion is lactoferrin, though small amounts of transferrin are also present. Three samples of human milk with unsaturated iron-binding capacities of between 56 and 89% had a powerful bacteriostatic effect on Escherichia coli O111/B4. The bacteriostatic properties of milk were abolished if the iron-binding proteins were saturated with iron. Purified human lactoferrin, in combination with specific E. coli antibody, strongly inhibited the growth of E. coli, and this effect was also abolished by saturating the lactoferrin with iron.Guinea-pig milk also contains lactoferrin and transferrin. Newly born guinea-pigs fed on an artificial diet and dosed with E. coli O111 had higher counts of E. coli O111 in the intestine than suckled animals. The apparent suppressive effect of guinea-pig milk on E. coli in the intestine could be reversed by feeding the iron compound haematin. It seems that iron-binding proteins in milk may play an important part in resistance to infantile enteritis caused by E. coli.     

Crystal structure of human seminal diferric lactoferrin at 3.4 Angstrom resolution

Lactoferrin was purified from human seminal fluid obtained from the semen bank. The purified samples were saturated with Fe3+ and crystallized by microdialysis method. The crystals belong to orthorhombic space group P21212, with a = 55.9 Angstrom. b = 97.2 Angstrom, c = 156.1 Angstrom and Z = 4. The structure was determined with molecular replacement method and refined to an R factor of 18.7% for all the data to 3.4 Angstrom resolution. The overall structure of seminal lactoferrin is similar to human colostrum lactoferrin. The amino acid sequence of seminal lactoferrin shows that it has one amino acid less than human colostrum lactoferrin and the structure of its N-terminal region is far more ordered than other lactoferrins. The structure of the iron-binding site and its immediate surroundings indicate well defined features.     

Expression,purification, and characterization of equine lactoferrin in Pichia pastoris

Lactoferrin is an 80kDa iron-binding glycoprotein. It is secreted by exocrine glands. Many functions such as iron sequestering, anti-bacterial activity, regulation of gene expression, and immunomodulation are attributed to it. In the present study, we report the production of recombinant equine lactoferrin (ELF) in the methylotropic yeast Pichia pastoris using pPIC9K vector. The recombinant protein was purified by one-step affinity chromatography using heparin-Sepharose column. The purified protein has a molecular weight of 80kDa and reacted with antibody raised against the native equine lactoferrin. Its N-terminal sequence was identical to that of the native ELF. The iron-binding behavior and circular dichroism studies of the purified protein indicate that it has folded properly. The recombinant protein appears to be hyperglycosylated by the host strain, GS115. This is the first heterologous expression of equine lactoferrin and also the first report of intact lactoferrin expression using P. pastoris system. An yield of 40mg/l obtained in shake-flask cultures with this system, which is higher than the reported values for other systems.     

Isolation and physico-chemical properties of lactoferrin from swine neutrophils

Lactoferrin, a non-heme iron-binding protein was isolated from pig neutrophils. The purification procedure included initial extraction of the protein in the presence of cetyltrimethylammonium bromide followed by chromatography on carboxymethyl-cellulose and Sephadex G-100. The thus obtained protein was found to be homogeneous on polyacrylamide gel (PAAG) electrophoresis at acidic values of pH. PAAG electrophoresis in the presence of sodium dodecyl sulfate revealed a single component with a molecular weight of 75 000-80 000. The resulting protein is capable of binding two atoms of iron molecule. The absorbance spectra for the pig neutrophil lactoferrin are identical to those for cow milk lactoferrin in the visible region and have a maximum at 465 nm. The amino acid composition of pig lactoferrin was determined. Isoelectric focusing of the protein obtained in a PAAG stabilized pH gradient revealed a component with pI of about 6.8. A single precipitin line was observed with rabbit antipig lactoferrin when examined by immunodiffusion. No immunological cross-reactions were observed between pig lactoferrin and bovine lactoferrin.     

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.     

Seminal plasma of brown trout,Salmo trutta fario (L.) contains a factor able to retain iron at acid pH,typical feature of lactoferrin

Blood and seminal plasma of brown trout Salmo trutta fario were analyzed for their iron binding potential adopting two different methods. Seminal plasma showed an iron binding capacity that was retained even if samples were exposed at acid pH, similarly to mammalian lactoferrin that binds ferric iron also at acid pH. This suggests that the iron binding capacity is determined by a factor having a lactoferrin-like activity. Moreover, trout seminal plasma proteins were also analyzed in their pattern by sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membrane. When seminal plasma was subjected to immunoblotting using goat anti-bovine lactoferrin antibodies as a probe, only a single band having an apparent molecular weight of around 80 kDa was specifically detected, showing that this protein has homology with bovine lactoferrin.     

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.     

Purification of membrane-bound lactoferrin from the human milk fat globule membrane

Although lactoferrin is known as a basic soluble glycoprotein, the presence of the membrane-bound form of this protein has also been demonstrated in human milk. Membrane-bound lactoferrin was extracted from the human milk fat globule membrane with a detergent mixture of 1% Tween-20, 0.5% C12E8, and 0.5 M KCl in 20 mM Tris-HCl (pH 7.4). Lactoferrin in the detergent-soluble fraction was purified by affinity chromatography with Concanavalin A and by hydrophobic chromatography with phenyl-Superose. The purified protein gave a single band of 80 kDa by SDS-PAGE. Its N-terminal amino acid sequence was consistent with that of human lactoferrin.     

Iron-binding proteins with vitreous humour

The soluble protein composition of Macaque monkey vitreous humour was studied in order to understand its iron-binding properties. The protein content of vitreous humour was 217 μg/ml ± 4.6%, 40% of which was serum albumin and 30% an iron-binding protein of hydrodynamic properties identical to that of trasferrin or lactoferrin. Relative to serum, the vitreous humour contained a 13-fold excess of this protein(s). Isoelectric focusing, iron-binding and immunoelectrophoretic studies indicated that both vitreous humour and aqueous humour contained lactoferrin as well as serum transferrin. The iron-binding capacity of these proteins in vitreous humour was equivalent to the mass of haemoglobin iron contained in at least 570 000 monkey erythrocytes. It was concluded that the intraocular lactoferrin originated from within the eye. These iron-binding proteins may play a protective role in ocular disturbances such as viterous haemorrahge, iron foreign body toxicity and infection.     

The amino acid sequence of human beta-microseminoprotein

The complete amino acid sequence of beta-microseminoprotein of human seminal plasma was determined by automated Edman degradation of the protein and peptides which were obtained by enzymatic cleavage with trypsin, chymotrypsin and Staphylococcus aureus V8 proteinase. The carboxyl-terminal sequence of the protein was established with the aid of carboxypeptidase A. The amino acid sequence of this protein proved to be as follows: (sequence; see text) Thus, beta-microseminoprotein consisting of 93 amino acid residues has a molecular mass of 10 652 Da. The linear structure of this protein represents the first complete amino acid sequence of a sperm-coating protein specific to human seminal plasma.     

Uptake of lactoferrin by mononuclear phagocytes inhibits their ability to form hydroxyl radical and protects them from membrane autoperoxidation.

Human mononuclear phagocytes do not contain the iron-binding protein lactoferrin that we have previously demonstrated inhibits the potential for human neutrophils to generate hydroxyl radical in the presence of an exogenous iron catalyst of the Haber-Weiss reaction. Previous work by other investigators has suggested that mononuclear phagocytes (monocytes and monocyte-derived macrophages (MDM] have the capacity to bind exogenous lactoferrin via lactoferrin-specific membrane surface receptors. Accordingly, we examined the possibility that uptake of iron-free (apo) lactoferrin by human mononuclear phagocytes could play a role in limiting the potential for generation of hydroxyl radical during the monocyte/MDM respiratory burst. When monocytes or MDM were incubated in the presence of apo-lactoferrin, cell-associated lactoferrin increased in proportion to the concentration of lactoferrin provided. Similar results were obtained with iron-loaded (diferric) milk lactoferrin. Consistent with the in vivo importance of these findings, we found that lactoferrin was intimately associated with human alveolar macrophages obtained by bronchoalveolar lavage. The fucose polymer fucoidan inhibited lactoferrin uptake whereas exogenous transferrin or MDM exposure to IFN-gamma was without effect. Scatchard binding analysis confirmed the presence of a lactoferrin-specific receptor with a calculated kDa of 3.56 x 10(-6) M and 3.4 x 10(7) binding sites per cell. Subcellular fractionation studies indicated that twofold more of the lactoferrin which became cell-associated over the 1-h incubation time could be found in the cytoplasmic fraction compared to the plasma membrane-containing fraction, consistent with previous evidence by others for internalization of lactoferrin by mononuclear phagocytes. When lactoferrin-loaded monocytes/MDM were incubated in lactoferrin-free media, evidence for release of lactoferrin was obtained by SDS-PAGE and immunoblot analysis, suggesting the presence of a recyclable pool of cell-associated lactoferrin. To assess the impact of lactoferrin loading on monocyte/MDM hydroxyl radical formation, lactoferrin-loaded phagocytes were stimulated with PMA in the presence of catalytic iron. Hydroxyl radical generation by lactoferrin-loaded cells was decreased to about 50% of control cells. Similarly, monocytes that had been lactoferrin-loaded demonstrated a 28% decrease in autooxidation of their membrane when stimulated in the presence of catalytic iron. These data suggest that lactoferrin binding may play an important role in maintaining optimal mononuclear phagocyte function and protecting adjacent tissue from untoward phagocyte-associated hydroxyl radical generation.     

A structural comparison of human serum transferrin and human lactoferrin

The transferrins are a family of proteins that bind free iron in the blood and bodily fluids. Serum transferrins function to deliver iron to cells via a receptor-mediated endocytotic process as well as to remove toxic free iron from the blood and to provide an anti-bacterial, low-iron environment. Lactoferrins (found in bodily secretions such as milk) are only known to have an anti-bacterial function, via their ability to tightly bind free iron even at low pH, and have no known transport function. Though these proteins keep the level of free iron low, pathogenic bacteria are able to thrive by obtaining iron from their host via expression of outer membrane proteins that can bind to and remove iron from host proteins, including both serum transferrin and lactoferrin. Furthermore, even though human serum transferrin and lactoferrin are quite similar in sequence and structure, and coordinate iron in the same manner, they differ in their affinities for iron as well as their receptor binding properties: the human transferrin receptor only binds serum transferrin, and two distinct bacterial transport systems are used to capture iron from serum transferrin and lactoferrin. Comparison of the recently solved crystal structure of iron-free human serum transferrin to that of human lactoferrin provides insight into these differences.     

Production of human lactoferrin in animal milk

Genetic constructs containing the human lactoferrin (hLf) gene were created within a joint program of Russian and Belorussian scientists. Using these constructs, transgenic mice were bred (the maximum hLf concentration in their milk was 160 g/L), and transgenic goats were also generated (up to 10 g/L hLf in their milk). Experimental goatherds that produced hLf in their milk were also bred, and the recombinant hLf was found to be identical to the natural protein in its physical and chemical properties. These properties included electrophoretic mobility, isoelectric point, recognition by polyclonal and monoclonal antibodies, circular dichroic spectra, interaction with natural ligands (DNA, lipopolysaccharides, and heparin), the binding of iron ions, the sequence of the 7 terminal amino acids, and its biological activity. The latter was assessed by the agglutination of Micrococcus luteus protoplasts, bactericidal activity against Escherichia coli and Listeria monocytogenes , and fungicidal activity against Candida albicans . We also demonstrated a significant increase in the activity of antibiotics when used in combination with Lf.     

Detection of 14-kDa group II phospholipase A2 in human seminal plasma

About 90% of phospholipase A2 activity detected in human seminal plasma reacted with monoclonal antibodies raised against human synovial fluid phospholipase A2. The crude seminal plasma yielded a pure immuno-cross-reactive phospholipase A2 preparation in a single purification step using immuno-affinity chromatography. The amino acid sequence of the N-terminal 20 residues of this seminal enzyme was determined and found to be identical with that of human synovial phospholipase A2. Thus, it is suggested that human seminal plasma contains phospholipase A2, belonging to the 14-kDa group II enzyme family, as the major isoenzyme.     

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.     

Lactoferrin binding molecules in human seminal plasma

During ejaculation, the iron binding protein lactoferrin binds to sperm and forms a major component of sperm-coating antigens. Physicochemical properties of lactoferrin in seminal plasma (SP) and on sperm differ from those of purified lactoferrin. These differences have been attributed to the binding of unknown seminal macromolecules to lactoferrin. We have studied lactoferrin binding molecules in SP. The SP samples were coated onto microtiter plates and tested for binding of biotinylated lactoferrin. SP was found to specifically bind biotinylated lactoferrin. This binding was competitively inhibited by coincubation with unlabeled lactoferrin but was not affected by control incubations done with human IgG or transferrin. Lactoferrin binding molecules in SP were biochemically characterized by using SDS-PAGE and ligand blotting. Biotinylated lactoferrin bound to SP molecules of approximately 120, 60 and 30 kDa. No binding was observed with biotinylated transferrin. The presence of molecules that associate with lactoferrin in SP was further studied by using crossed immunoelectrophoresis. Lactoferrin in SP immunoprecipitated as two peaks, one of which corresponded to purified lactoferrin. These results suggest that some lactoferrin molecules in SP are free and that others are associated with lactoferrin binding molecules. Binding of lactoferrin to lactoferrin binding molecules appears to change its physicochemical properties and thus could influence its biologic activity and its affinity to sperm.     

Complete amino-acid sequence of bovine seminal ribonuclease, a dimeric protein from seminal plasma

The complete amino-acid sequence of BS-RNAse, a dimeric ribonuclease isolated from bovine seminal plasma, was determined. The reduced and S-carboxymethylated subunit chain of the enzyme was cleaved by trypsin and chymotrypsin. The resulting peptides, purified by cation-exchange chromatography were sequenced by dansyl-Edman, subtractive Edman degradation and carboxypeptidase A and B digestion. Chymotryptic peptides were used for the alignment. Automated Edman degradation of the native protein, through the N-terminal 41 amino-acid residues, completed the sequence information. The subunit chain of BS-RNAse, composed of 124 amino-acid residues, with a molecular mass of 13,610 Da, is highly homologous (81%) to pancreatic ribonuclease A. A good degree of homology (31%) was also found with human angiogenin. No N-linked carbohydrate-attachment sites, such as Asn-X-Ser/Thr, were found in the protein.