LACTOFERIN in clinical practice
LACTOFERIN in clinical practice
M. Nikolova, P. Nikolov, M. Baleva
Medical University of Sofia
1. Introduction
Lactoferrin (lactotransferrin) is a glycoprotein belonging to the family of transferrins, which are able to bind and transport ferric (Fe3+) ions[2,7,9]. It was first isolated in 1939 by Sørensen and Sørensen[9], and in 1960 it was determined by three independent laboratories to be the major iron-binding protein in human milk[2,7,12]. It was subsequently demonstrated in exocrine gland secretions and in specific neutrophil granules. The main source of lactoferrin in plasma is the degranulation of neutrophils[2,12]. Due to an increase in its levels in the course of inflammatory reactions (both in the peripheral blood and in the focus of inflammation), many authors also classify it as an acute-phase protein. .
2. Structure and characteristic
Lactoferrin is a glycoprotein with a molecular weight of about 80 kDa, belonging to the transferrin family, with a high affinity for iron[2]. Its molecular structure was revealed in 1984. Three different isoforms have been isolated, tentatively named lactoferrin-alpha, lactoferrin-beta and lactoferrin-gamma; the former has iron-binding properties but no ribonuclease activity, and the latter two forms have ribonuclease activity but lack iron-binding and transport functions [2,12].
The lactoferrin molecule is composed of one polypeptide chain containing 703 amino acids spatially located in two globular domains – N- and C-amino-terminal, connected by an alpha-axis. Each domain consists of two regions - C1, C2 and N1, N2. In each domain there is one non-Fe3+ binding site (each lactoferrin molecule can bind two ferric ions).
Lactoferrin molecules contain a varying number of glycolysis sites, such as on the surface of the molecule. The most common saccharide in the molecule is mannose, about 3% hexasines about 1% hexosamines[2]. The extent of glycolysis varies and is directly determined by the degree of resistance to proteases and pH levels.
The ability of lactoferrin to bind ferric ions is about twice that of trasferia (the latter can act as an ion donor for lactoferrin). Depending on the binding to iron, three forms of lactoferrin are described - apolactoferrin (unbound), monoferrin form (bound to one ferric ion) and hololactoferrin (bound to two iron ions). The tertiary structure of hololactoferrin and apolactoferrin is different. Four amino acid residues are particularly important for iron binding (histidine, two tyrosine residues and aspartic acid).
In addition to iron, lactoferrin also binds other substances, such as lipopolysaccharides, heparin, glycosaminoglycans, DNA, other metal ions (aluminum, magnesium, zinc, etc., and its affinity for these ions is lower than for Fe3+). oxalates, carboxylates and others. Thus, lactoferrin affects the metabolism and transport of various substances in the body.
The ability of lactoferrin to bind iron ions even at low pH is important, especially at sites of inflammation where pH levels are very low. In such cases, lactoferrin also binds Fe3+ donated by serum transferrin and blocks bacterial proliferation[2,12].
Lactoferrin is resistant to the proteolytic activity of trypsin and trypsin-like enzymes, the degree of resistance being proportional to the degree of iron saturation.
3. Sources of lactoferrin in the body
Lactoferrin proves already in the period of embryonic development, it is not detected during implantation until mid-gestation, subsequently it is proved in neutrophils and in epithelial cells forming the reproductive and digestive systems. It is synthesized in the cells of the myeloid order (which store it in specific granules, it is not synthesized by mature neutrophils) and by all exocrine glands, it is found in high levels in breast milk and colostrum, in mucous secretions, tears, etc.[2, 3,10-12]It is also synthesized in kidney tissue [1].
In peripheral blood, lactoferrin levels are quite variable, but are known to increase in the course of infections, inflammatory diseases, increased iron intake, and tumor growth.[2,12]
4. Regulation of lactoferrin synthesis
The regulation of lactoferrin synthesis is carried out by the factors and hormones controlling the secretion of the relevant exocrine glands - prolactin in the mammary glands, sex hormones in the reproductive organs, estrogens and epidermal growth factor in the endometrium, etc. [2,3,10-12].
5. Lactoferrin receptors
The biological functions of lactoferrin are mediated by specific receptors expressed on the surface of various target cells (mucosal epithelial cells, hepatocytes, monocytes, macrophages, polymorphonuclear leukocytes, lymphocytes, platelets, fibroblasts, as well as the surface of various bacteria)[2,3,10 -12]. Some cells even have receptors that allow them to bind not only lactoferrin, but also other transferrins. In addition to membrane receptors, nuclear receptors for lactoferrin have also been described.
6. Метаболизъм на лактоферина
After being synthesized and performing its biological functions, lactoferrin is eliminated from the body in several ways - through phagocytic cells and transfer of non-iron to ferritin; by uptake by Kupffer cells in the liver, hepatic endothelial cells, and hepatocytes; with the urine (including it is proven in the urine of naturally fed infants)[2,3,10-12].
7. Biological functions of lactoferrin
Lactoferrin has a wide range of biological functions, incl. participation in the exchange of iron in the body, fight against infections and infestations, regulation of cell growth and differentiation, bone exchange and metabolism of nucleic acids, etc. The main biological functions of lactoferrin are presented below [2-6,8-12]:
Lactoferrin and iron metabolism
It has been suggested that the importance of lactoferrin for iron metabolism is related to its similarity to the transferrin molecule. Lactoferrin has been suggested to be important for iron metabolism in the foci of inflammation. The importance of lactoferrin for systemic iron metabolism is supported by the fact that its biliary levels are strongly influenced by oral iron intake, as well as the change in the expression of intestinal lactoferrin receptors in infants depending on the levels of iron in the body. The importance of lactoferrin in the systemic regulation of iron metabolism remains poorly understood, but this glycoprotein is known to have a large number of other biological functions.
Antimicrobial properties
Lactoferrin is considered part of the innate immune response, but is also involved in some specific immune responses. It is expressed on the mucous membranes and is one of the body's first lines of defense against infections. On the one hand, it suppresses the proliferation of a number of bacteria, viruses, fungi and protozoa, and on the other hand, it can be a source of Fe3+ for some iron-requiring bacteria, such as Lactobacillus sp. and Bifidobacterium sp., which belong to the normal bacterial flora. Lactoferrin has a pronounced beneficial effect on the intestinal microflora, which, along with its preventive properties regarding the development of necrotizing enterocolitis in premature infants, is of great importance in the first weeks of a newborn's life.
Some bacteria synthesize siderophores (iron chelators) that compete with lactoferrin for ferric ion binding. Other highly adaptive bacteria develop and express specific receptors on their surface that bind lactoferrin, change its tertiary structure and lead to Fe3+ dissociation.
Lactoferrin also has other inhibitory effects on microorganisms unrelated to Fe3+, due to a change in the permeability of bacterial membranes.
Evidence is a product of the exchange of lactoferrin - a cationic peptide called lactoferricin, which has a pronounced bactericidal effect. This protein has two forms – lactoferricin H, derived from human lactoferrin, and lactoferricin B, from bovine lactoferrin.
Lactoferrin catalyzes the production of free radicals and increases the bactericidal activity of neutrophils.
There are also in vitro data that lactoferrin can suppress the formation of biofilms. The lack of iron in the medium forces the bacteria to move, which is why they cannot attach to surfaces.
Lactoferrin may also help protect against the invasion of facultative intracellular bacteria into cells by preventing adhesion of the invader cells to the cells of the macroorganism.
It is believed that the proteolytic activity of lactoferrin is responsible for fighting bacteria colonizing the body with the help of specific proteins. These proteins are lysed by lactoferrin. This function of lactoferrin is inhibited by serine protease inhibitors.
The antimicrobial activity of lactoferrin is of great importance in premature children, where breastfeeding and the intake of lactoferrin have a pronounced positive effect on the development of necrotizing enterocolitis.
Lactoferrin is of great importance for the development of the systemic inflammatory response in sepsis - it regulates inflammatory processes in the direction of a physiological response without rapid development of systemic inflammatory changes and septic shock.
Furthermore, lactoferrin regulates the expression of mediators of innate immunity, which subsequently influence the adaptive immune response. Therefore, this glycoprotein is believed to play the role of a bridge between the two types of immunity - innate and adaptive.
Lactoferrin has the ability to bind various DNA and RNA viruses and suppress viral replication in cell cultures and increases the expression of anti-inflammatory factors (including interleukin 11) in animal models of inflammation and viral hepatitis, suppresses lipid peroxidation. The main feature of lactoferrin related to its anti-viral action is its ability to bind to glycosaminoglycans in cell membranes, preventing viruses from entering cells and preventing infection in the early phase (HSV, CMV, HIV). Lactoferrin inhibits hepatitis C virus infection of human hepatocyte tissue cultures [13], and cells [14]. due to the binding of lactoferrin to proteins of the virus envelope (E1 and E2), thus preventing the adhesion, entry and replication of the virus in liver cells [14]. In patients with chronic hepatitis C, lactoferrin therapy reduced alanine aminotransferase (ALT) levels and reduced lipid peroxidation resulting from oxidative stress [15]. Oral administration of lactoferrin changes the direction of the immune response to Th1-mediated immune responses (including interleukin 18) [16]. Combined with the reduction in HCV RNA titer, this effect on the immune response supports the effectiveness of combination therapy with interferon and ribavirin [17].
Antiparasitic properties
It is believed that lactoferrin disrupts the integrity of the cell membrane of parasites (eg Toxoplasma gondii) and thus inactivates them, moreover some parasites compete with the host for iron (eg Pneumocystis carinii). There are, however, parasites that manage to use lactoferrin as an iron donor.
Lactoferrin and the immune system
Lactoferrin affects cells of the immune system and cells of inflammation: it affects antigen-presenting cells (macrophages, dendritic cells in the skin), cells of the adaptive immune response (T- and B-lymphocytes), has an immunomodulatory effect and prevents the development of an acute inflammatory response in sepsis, modulates chemokine recognition and lymphocyte migration. On the one hand, it stimulates the proliferation, differentiation and activation of cells of the immune system and the immune response, but on the other hand it also has anti-inflammatory properties.
Lactoferrin leads to suppression of the production of some inflammatory cytokines (tumor necrosis factor alpha[TNFa], interleukins[IL]IL-1b and IL-6) and enhanced production of the anti-inflammatory IL-10 and IL-11.
In addition, taken before birth, lactoferrin leads to a reduction in the levels of interleukin 6 in the cervicovaginal fluid and the risk of preterm birth.
Lactoferrin suppresses skin allergic reactions and skin inflammatory reactions, and this effect is independent of its iron-binding properties. There is a similar effect in the gastro-intestinal tract – suppression of inflammatory reactions, possibly mediated suppression of the TNFα-mediated immune response. Oral administration of lactoferrin has been shown to reduce Helicobacter felis colonization and associated gastric changes. It has been proven that exogenously applied lactoferrin (including bovine) has a beneficial effect in inflammatory changes of the periodontium, in severe infections with a systemic inflammatory response, in anemia.
Lactoferrin and tumor growth
In experimental mouse models, lactoferrin suppresses tumor growth and metastasis. It has been shown to suppress the development of neoplastic cells in mammary carcinoma by inhibiting the transition of the cell from G1 to S phase, possibly due to the alteration of the expression and activity of regulatory proteins.
Lactoferrin-dependent, cytokine-mediated stimulation of NK cell activity and CD4+ and CD8+ lymphocytes represents an important protective factor against tumor development. After oral administration of lactoferrin, the number of these cells increases in both peripheral blood and lymphoid tissue.
Inhibition of tumor growth by lactoferrin is also associated with apoptosis due to induction of Fas-dependent signal transduction.
Lactoferrin and cell proliferation and differentiation
In the past, lactoferrin was thought to promote cell proliferation due to its ability to deliver iron inside cells. Lactoferrin was later shown to act as a growth factor activator. The effect of lactoferrin on the growth of epithelial cells of the gastro-intestinal tract is more pronounced than that of epidermal growth factor. Lactoferrin per se is able to stimulate the proliferation of endometrial cells. Furthermore, it is a proven transcription factor, able to enter cells and activate the transcription of specific DNA sequences.
Lactoferrin and bone turnover
Lactoferrin is a potential anabolic factor affecting osteocytes. It stimulates osteoblastic proliferation, improves thymidine incorporation into osteocytes, and reduces osteoblast apoptosis by between 50% and 70%. Similar effects have been demonstrated for chondrocytes. Lactoferrin suppresses osteoclastogenesis (dose-dependent effect), but does not affect bone resorption by osteoclasts. In addition to direct effects, this glycoprotein can indirectly affect bone tissue by inhibiting osteolytic cytokines such as TNFa and IL-1b, whose levels increase during inflammation.
Ribonuclease activity
It exists extensively in lactoferrin and ribonuclease A molecules. There is evidence that lactoferrin can hydrolyze RNA, and this activity depends on both the type of lactoferrin and the RNA itself.
Conclusion
Lactoferrin is an iron-binding glycoprotein with a wide range of actions, incl. influencing inflammatory processes, cell proliferation and differentiation, immune response, neoplastic growth, induction of cytokine synthesis and associated inflammatory changes, premature birth, septic response, etc. Due to its unique immunomodulatory, antimicrobial and even antineoplastic properties, it has an outstanding potential in clinical practice.
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