Lactoferrin

Lactoferrin (LF), also known as lactotransferrin (LTF), is a multifunctional is_associated_with::protein of the is_associated_with::transferrin family. Lactoferrin is a globular is_associated_with::glycoprotein with a molecular mass of about 80 kDa that is widely represented in various secretory fluids, such as is_associated_with::milk, is_associated_with::saliva, is_associated_with::tears, and nasal secretions. Lactoferrin is also present in secondary granules of PMN and is secreted by some acinar cells. Lactoferrin can be purified from milk or produced recombinantly. Human is_associated_with::colostrum ("first milk") has the highest concentration, followed by human milk, then cow milk (150 mg/L).

Lactoferrin is one of the components of the is_associated_with::immune system of the body; it has antimicrobial activity (is_associated_with::bacteriocide, is_associated_with::fungicide) and is part of the innate defense, mainly at mucoses. In particular, lactoferrin provides antibacterial activity to human infants. Lactoferrin interacts with is_associated_with::DNA and is_associated_with::RNA, is_associated_with::polysaccharides and is_associated_with::heparin, and shows some of its biological functions in complexes with these is_associated_with::ligands.

History
Occurrence of iron-containing red protein in bovine milk was reported as early as in 1939; however, the protein could not be properly characterized because it could not be extracted with sufficient purity. Its first detailed studies were reported around 1960. They documented the molecular weight, is_associated_with::isoelectric point, optical absorption spectra and presence of two iron atoms per protein molecule. The protein was extracted from milk, contained iron and was structurally and chemically similar to serum is_associated_with::transferrin. Therefore, it was named lactoferrin in 1961, though the name lactotransferrin was used in some earlier publications, and later studies demonstrated that the protein is not restricted to milk. The antibacterial action of lactoferrin was also documented in 1961, and was associated with its ability to bind iron.

Molecular structure
Lactoferrin is one of the transferrin proteins that transfer is_associated_with::iron to the cells and control the level of free iron in the blood and external secretions. It is present in the milk of humans and other mammals, in the is_associated_with::blood plasma and is_associated_with::neutrophils and is one of the major proteins of virtually all exocrine secretions of mammals, such as is_associated_with::saliva, is_associated_with::bile, is_associated_with::tears and is_associated_with::pancreas. Concentration of lactoferrin in the milk varies from 7 g/L in the is_associated_with::colostrum to 1 g/L in mature milk.

X-ray diffraction reveals that lactoferrin is based on one is_associated_with::polypeptide chain that contains about 700 amino acids and forms two homologous globular domains named N-and C-lobes. N-lobe corresponds to amino acid residues 1–333 and C-lobe to 345–692, and the ends of those domains are connected by a short α-helix. Each lobe consists of two subdomains, N1, N2 and C1, C2, and contains one iron binding site and one is_associated_with::glycosylation site. The degree of glycosylation of the protein may be different and therefore the molecular weight of lactoferrin varies between 76 and 80 kDa. The stability of lactoferrin has been associated with the high glycosylation degree.

Lactoferrin belongs to the basic proteins, its is_associated_with::isoelectric point is 8.7. It exists in two forms: iron-rich hololactoferrin and iron-free apolactoferrin. Their tertiary structures are different; apolactoferrin is characterized by "open" conformation of the N-lobe and the "closed" conformation of the C-lobe, and both lobes are closed in the hololactoferrin.

Each lactoferrin molecule can reversibly bind two ions of iron, is_associated_with::zinc, is_associated_with::copper or other metals. The binding sites are localized in each of the two protein globules. There, each ion is bonded with six ligands: four from the polypeptide chain (two is_associated_with::tyrosine residues, one is_associated_with::histidine residue and one is_associated_with::aspartic acid residue) and two from is_associated_with::carbonate or is_associated_with::bicarbonate ions.

Lactoferrin forms reddish complex with iron; its affinity for iron is 300 times higher than that of is_associated_with::transferrin. The affinity increases in weakly acidic medium. This facilitates the transfer of iron from transferrin to lactoferrin during is_associated_with::inflammations, when the pH of tissues decreases due to accumulation of lactic and other acids. The saturated iron concentration in lactoferrin in is_associated_with::human milk is estimated as 10 to 30% (100% corresponds to all lactoferrin molecules containing 2 iron atoms). It is demonstrated that lactoferrin is involved not only in the transport of iron, zinc and copper, but also in the regulation of their intake. Presence of loose ions of zinc and copper does not affect the iron binding ability of lactoferrin, and might even increase it.

Polymeric forms
Both in blood plasma and in secretory fluids lactoferrin can exist in different polymeric forms ranging from is_associated_with::monomers to is_associated_with::tetramers. Lactoferrin tends to polymerize both in vitro and in vivo, especially at high concentrations. Several authors found that the dominant form of lactoferrin in physiological conditions is a tetramer, with the monomer:tetramer ratio of 1:4 at the protein concentrations of 10−5 M.

It is suggested that the is_associated_with::oligomer state of lactoferrin is determined by its concentration and that is_associated_with::polymerization of lactoferrin is strongly affected by the presence of Ca2+ ions. In particular, monomers were dominant at concentrations below 10−10−10−11 M in the presence of Ca2+, but they converted into tetramers at lactoferrin concentrations above 10−9−10−10 M. is_associated_with::Titer of lactoferrin in the blood corresponds to this particular "transition concentration" and thus lactoferrin in the blood should be presented both as a monomer and tetramer. Many functional properties of lactoferrin depend on its oligomeric state. In particular, monomeric, but not tetrameric lactoferrin can strongly bind to DNA.

Biological functions
Lactoferrin belongs to the is_associated_with::innate immune system. Apart from its main biological function, namely binding and transport of iron ions, lactoferrin also has antibacterial, antiviral, is_associated_with::antiparasitic, catalytic, anti-cancer, anti-allergic and radioprotecting functions and properties.

Antibacterial activity
Lactoferrin's primary role is to sequester free iron, and in doing so remove essential substrate required for bacterial growth. Antibacterial action of lactoferrin is also explained by the presence of specific receptors on the cell surface of microorganisms. Lactoferrin binds to lipopolysaccharide of bacterial walls, and the oxidized iron part of the lactoferrin oxidizes bacteria via formation of is_associated_with::peroxides. This affects the membrane permeability and results in the cell breakdown (is_associated_with::lysis).

Although lactoferrin also has other antibacterial mechanisms not related to iron, such as stimulation of phagocytosis, the interaction with the outer bacterial membrane described above is the most dominant and most studied. Lactoferrin not only disrupts the membrane, but even penetrates into the cell. Its binding to the bacteria wall is associated with the specific is_associated_with::peptide is_associated_with::lactoferricin, which is located at the N-lobe of lactoferrin and is produced by in vitro cleavage of lactoferrin with another protein, is_associated_with::trypsin. A mechanism of the antimicrobial action of lactoferrin has been reported as lactoferrin targets H(+)-ATPase and interferes with proton translocation in the cell membrane, resulting in a lethal effect in vitro.

Lactoferrin prevents the attachment of is_associated_with::H. pylori in the stomach, which in turn, aids in reducing digestive system disorders. Bovine lactoferrin has more activity against is_associated_with::H. pylori than human lactoferrin.

Antiviral activity
Lactoferrin acts, mostly in vitro, on a wide range of human and animal viruses based on DNA and RNA is_associated_with::genomes, including the is_associated_with::herpes simplex virus 1 and 2, is_associated_with::cytomegalovirus, is_associated_with::HIV, is_associated_with::hepatitis C virus,  is_associated_with::hantaviruses, is_associated_with::rotaviruses, is_associated_with::poliovirus type 1, is_associated_with::human respiratory syncytial virus and is_associated_with::murine leukemia viruses.

The most studied mechanism of antiviral activity of lactoferrin is its diversion of virus particles from the target cells. Many viruses tend to bind to the is_associated_with::lipoproteins of the cell membranes and then penetrate into the cell. Lactoferrin binds to the same lipoproteins thereby repelling the virus particles. Iron-free apolactoferrin is more efficient in this function than hololactoferrin; and lactoferricin, which is responsible for antimicrobial properties of lactoferrin, shows almost no antiviral activity.

Beside interacting with the cell membrane, lactoferrin also directly binds to viral particles, such as the is_associated_with::hepatitis viruses. This mechanism is also confirmed by the antiviral activity of lactoferrin against rotaviruses, which act on different cell types.

Lactoferrin also suppresses virus replication after the virus penetrated into the cell. Such an indirect antiviral effect is achieved by affecting is_associated_with::natural killer cells, is_associated_with::granulocytes and is_associated_with::macrophages – cells, which play a crucial role in the early stages of viral infections, such as is_associated_with::severe acute respiratory syndrome (SARS).

Antifungal activity
Lactoferrin and lactoferricin inhibit in vitro growth of Trichophyton mentagrophytes, which are responsible for several skin diseases such as is_associated_with::ringworm. Lactoferrin also acts against the is_associated_with::Candida albicans – a is_associated_with::diploid is_associated_with::fungus (a form of is_associated_with::yeast) that causes opportunistic oral and is_associated_with::genital infections in humans. is_associated_with::Fluconazole has long been used against Candida albicans, which resulted in emergence of strains resistant to this drug. However, a combination of lactoferrin with fluconazole can act against fluconazole-resistant strains of Candida albicans as well as other types of Candida: C. glabrata, C. krusei, C. parapsilosis and C. tropicalis. Antifungal activity is observed for sequential incubation of Candida with lactoferrin and then with fluconazole, but not vice versa. The antifungal activity of lactoferricin exceeds that of lactoferrin. In particular, synthetic peptide 1–11 lactoferricin shows much greater activity against Candida albicans than native lactoferricin.

Administration of lactoferrin through drinking water to mice with weakened immune systems and symptoms of is_associated_with::aphthous ulcer reduced the number of Candida albicans strains in the mouth and the size of the damaged areas in the tongue. Oral administration of lactoferrin to animals also reduced the number of pathogenic organisms in the tissues close to the is_associated_with::gastrointestinal tract. Candida albicans could also be completely eradicated with a mixture containing lactoferrin, is_associated_with::lysozyme and is_associated_with::itraconazole in HIV-positive patients who were resistant to other antifungal drugs. Such antifungal action when other drugs deem inefficient is characteristic of lactoferrin and is especially valuable for HIV-infected patients. Contrary to the antiviral and antibacterial actions of lactoferrin, very little is known about the mechanism of its antifungal action. Lactoferrin seems to destroy the cell wall and bind the is_associated_with::plasma membrane of C. albicans.

Bone activity
Ribonuclease-enriched lactoferrin has been used to examine how lactoferrin affects bone. Lactoferrin has shown to have positive effects on bone turnover. It has aided in decreasing bone resorption and increasing bone formation. This was indicated by a decrease in the levels of two bone resorption markers (is_associated_with::deoxypyridinoline and N-telopeptide) and an increase in the levels two bone formation markers (is_associated_with::osteocalcin and is_associated_with::alkaline phosphatase). It has reduced osteoclast formation, which signifies a decrease in pro-inflammatory responses and an increase in anti-inflammatory responses which indicates a reduction in bone resorption as well.

Interaction with nucleic acids
One of the important properties of lactoferrin is its ability to bind with nucleic acids. The fraction of protein extracted from milk, contains 3.3% RNA, besides, the protein preferably binds to the double-stranded than to the single-stranded DNA. The ability of lactoferrin to bind DNA is used for the isolation and purification of lactoferrin using is_associated_with::affinity chromatography with columns containing immobilized DNA-containing is_associated_with::sorbents, such as is_associated_with::agarose with the immobilized single-stranded DNA.

Enzymatic activity of lactoferrin
Lactoferrin hydrolyzes is_associated_with::RNA and exhibits the properties of is_associated_with::pyrimidine-specific secretory is_associated_with::ribonucleases. In particular, by destroying the RNA genome, milk RNase inhibits reverse transcription of is_associated_with::retroviruses that cause is_associated_with::breast cancer in mice. is_associated_with::Parsi women in West is_associated_with::India have the milk RNase level markedly lower than in other groups, and their is_associated_with::breast cancer rate is three times higher than average. Thus, is_associated_with::ribonucleases of milk, and lactoferrin in particular, might play an important role in is_associated_with::pathogenesis of diseases caused by various is_associated_with::retroviruses.

Anticarcinogenic activity
The anticancer activity of is_associated_with::bovine lactoferrin (bLF) has been demonstrated in experimental lung, bladder, tongue, colon, and liver carcinogeneses on rats, possibly by suppression of phase I enzymes, such as cytochrome P450 1A2 (is_associated_with::CYP1A2). Also, in another experiment done on is_associated_with::hamsters, bovine lactoferrin decreased the incidence of is_associated_with::oral cancer by 50%. Because bLF by far did not show any toxicity and because it's readily available in milk, bLF offers promise as a potential chemopreventive agent for oral cancer. Currently, bLF is used as an ingredient in is_associated_with::yogurt, is_associated_with::chewing gums, is_associated_with::infant formulas, and is_associated_with::cosmetics.

Genes of lactoferrin
At least 60 gene sequences of lactoferrin have been characterized in 11 species of mammals. In most species, is_associated_with::stop codon is TAA, and TGA in is_associated_with::Mus musculus. Deletions, insertions and mutations of stop codons affect the coding part and its length varies between 2,055 and 2,190 is_associated_with::nucleotide pairs. Gene polymorphism between species is much more diverse than the intraspecific polymorphism of lactoferrin. There are differences in amino acid sequences: 8 in is_associated_with::Homo sapiens, 6 in is_associated_with::Mus musculus, 6 in is_associated_with::Capra hircus, 10 in is_associated_with::Bos taurus and 20 in is_associated_with::Sus scrofa. This variation may indicate functional differences between different types of lactoferrin.

In humans, lactoferrin gene LTF is located on the third is_associated_with::chromosome in the locus 3q21-q23. In is_associated_with::oxen, the coding sequence consists of 17 is_associated_with::exons and has a length of about 34,500 is_associated_with::nucleotide pairs. Exons of the lactoferrin gene in oxen have a similar size to the exons of other genes of the is_associated_with::transferrin family, whereas the sizes of introns differ within the family. Similarity in the size of exons and their distribution in the domains of the protein molecule indicates that the evolutionary development of lactoferrin gene occurred by duplication. Study of polymorphism of genes that encode lactoferrin helps selecting livestock breeds that are resistant to is_associated_with::mastitis.

Lactoferrin receptor
The lactoferrin receptor plays an important role in the is_associated_with::internalization of lactoferrin; it also facilitates absorption of iron ions by lactoferrin. It was shown that is_associated_with::gene expression increases with age in the is_associated_with::duodenum and decreases in the is_associated_with::jejunum. The moonlighting glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (is_associated_with::GAPDH) has been demonstrated to function as a receptor for lactoferrin.

Cystic fibrosis
The human lung and saliva contain a wide range of antimicrobial compound including lactoperoxidase system, producing is_associated_with::hypothiocyanite and lactoferrin, with hypothiocyanite missing in is_associated_with::cystic fibrosis patients. Lactoferrin, a component of innate immunity, prevents bacterial is_associated_with::biofilm development. The loss of microbicidal activity and increased formation of biofilm due to decreased lactoferrin activity is observed in patients with cystic fibrosis. These findings demonstrate the important role of lactoferrin in human host defense and especially in lung.

Lactoferrin with hypothiocyanite has been granted is_associated_with::orphan drug status by the EMEA and the FDA.

Nanotechnology
Lactotransferrin has been used in the synthesis of fluorescent gold quantum clusters, which has potential applications in nanotechnology.

In Diagnosis
Lactoferrin levels in tear fluid have been shown to decrease in dry eye diseases such as Sjogren's syndrome. A rapid, portable test utilizing microfluidic technology has been developed to enable measurement of lactoferrin levels in human tear fluid at the point-of-care with the aim of improving diagnosis of Sjogren's syndrome and other forms of dry eye disease.