ReviewLactoferrin: structure, function and applications
Introduction
Lactoferrin (LF) is a non-haem iron-binding protein that is part of the transferrin protein family, along with serum transferrin, ovotransferrin, melanotransferrin and the inhibitor of carbonic anhydrase [1], whose function is to transport iron in blood serum. LF is produced by mucosal epithelial cells in various mammalian species, including humans, cows, goats, horses, dogs and several rodents. Recent studies have shown that LF is also produced by fish, as it has been identified in rainbow trout eggs using molecular biology techniques [2]. This glycoprotein is found in mucosal secretions, including tears, saliva, vaginal fluids, semen [3], nasal and bronchial secretions, bile, gastrointestinal fluids, urine [4] and most highly in milk and colostrum (7 g/L) [5], making it the second most abundant protein in milk [6], after caseins. It can also be found in bodily fluids such as blood plasma and amniotic fluid. LF is also found in considerable amounts in secondary neutrophil granules (15 μg/106 neutrophils) [7], where it plays a significant physiological role. LF possesses a greater iron-binding affinity and is the only transferrin with the ability to retain this metal over a wide pH range [8], including extremely acidic pH. It also exhibits a greater resistance to proteolysis. In addition to these differences, LF’s net positive charge and its distribution in various tissues make it a multifunctional protein. It is involved in several physiological functions, including: regulation of iron absorption in the bowel; immune response; antioxidant, anticarcinogenic and anti-inflammatory properties; and protection against microbial infection, which is the most widely studied function to date. The antimicrobial activity of LF is mostly due to two mechanisms. The first is iron sequestration in sites of infection, which deprives the microorganism of this nutrient, thus creating a bacteriostatic effect. The other mechanism is the direct interaction of the LF molecule with the infectious agent. The positive amino acids in LF can interact with anionic molecules on some bacterial, viral, fungal and parasite surfaces, causing cell lysis.
Considering the physiological capabilities of LF in host defence, in addition to current pharmaceutical and nutritional needs, LF is considered to be a nutraceutical and for several decades investigators have searched for the most convenient way to produce it. Today, we can obtain it as native LF isolated mostly from the milk and colostrum of several mammals, or as recombinant LF (rLF) generated from bacterial, fungal and viral expression systems. The expression of this protein has also been attained in higher organisms such as plants and mammals.
Section snippets
Structure and properties
LF (Fig. 1) is an 80 kDa glycosylated protein of ca. 700 amino acids with high homology among species. It is a simple polypeptide chain folded into two symmetrical lobes (N and C lobes), which are highly homologous with one another (33–41% homology). These two lobes are connected by a hinge region containing parts of an α-helix between amino acids 333 and 343 in human LF (hLF) [1], which provides additional flexibility to the molecule [3]. The polypeptide chain includes amino acids 1–332 for the
Biological functions of lactoferrin
Several functions have been attributed to LF. It is considered a key component in the host’s first line of defence, as it has the ability to respond to a variety of physiological and environmental changes [6]. The structural characteristics of LF provide functionality in addition to the Fe3+ homeostasis function common to all transferrins: strong antimicrobial activity against a broad spectrum of bacteria, fungi, yeasts, viruses [5] and parasites [11]; anti-inflammatory and anticarcinogenic
Bioactive peptides derived from lactoferrin
Since it was first isolated in 1960 [71], LF has been widely studied for its antimicrobial characteristics. One of the main mechanisms used by LF is Fe3+ sequestration. However, it is known that LF may also interact directly with the pathogen [10]. Enzymatic treatment of bLF with pepsin produced a low-molecular-weight peptide with antibacterial properties against a large number of Gram-positive and Gram-negative bacteria, including Escherichia coli, Salmonella enteritidis, K. pneumoniae,
Lactoferrin gene regulation
LF has been identified in several tissues both in humans and animals and it has extensive homology among species. Its mRNA levels vary by tissue, suggesting tissue- or cell-specific regulation [75], [76], [77], [78].
To date, the LF gene has been found at the chromosome level in a set of different species (human chromosome 3 [79] and mouse chromosome 9 [80]) and its size ranges from 23 kb to 35 kb. The LF gene is organised in 17 exons, 15 of which are identical in cows, pigs and mice [81]. In
Clinical applications of lactoferrin
On account of LF’s many functions, it has been tested for clinical use in disease prevention, treatment and diagnosis.
One of the first applications of LF was in infant formula. Several studies showed that infants fed with infant formulas had less intestinal iron absorption than breastfed infants [13], [14]. Most of the LF is absorbed intact by the infant’s bowel, and thus LF is distributed by the bloodstream. LF also promotes the proliferation of lactic acid bacteria in the bowel such as
Production of native and recombinant lactoferrin
Because of the functional characteristics of LF, attempts have been made to produce or purify this protein for use as a food additive or therapeutic. Protein purification strategies are based on the properties of the molecule and depend on three types of chromatography. Since LF has a net positive charge [3], it is efficiently absorbed on cation exchange resins and is eluted with saline solutions [89] at >95% purity [90]. LF binds Fe3+ so it can be purified by metal ion affinity chromatography
Concluding remarks
A wide spectrum of functions have been described for LF. The beneficial effect of LF in the treatment of various infectious diseases caused by bacteria, fungi, protozoa and viruses in animals and humans is described above. Despite extensive literature available on LF, the molecular interactions of this protein with regulatory elements and other proteins for antimicrobial function require further investigation. The great utility of this functional protein has motivated scientists to overexpress
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