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Lactoferrin: structure, function and applications
International Journal of Antimicrobial Agents 33 (2009) 301.e1–301.e8
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Review
Lactoferrin: structure, function and applications Susana A. González-Chávez, Sigifredo Arévalo-Gallegos, Quintín Rascón-Cruz ∗ Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Cd. Universitaria s/n, CP 31170, Chihuahua, Chihuahua, Mexico
a r t i c l e
i n f o
a b s t r a c t
Keywords: Lactoferrin Iron-binding protein Transferrin Functional protein
Lactoferrin (LF) is an 80 kDa iron-binding glycoprotein of the transferrin family that is expressed in most biological fluids and is a major component of the mammalian innate immune system. Its protective effects range from direct antimicrobial activities against a large panel of microorganisms, including bacteria, viruses, fungi and parasites, to anti-inflammatory and anticancer activities. These extensive activities are made possible by mechanisms of action utilising not only the capacity of LF to bind iron but also interactions of LF with molecular and cellular components of both host and pathogens. This review summarises the putative antimicrobial mechanisms, clinical applications and heterologous expression models for LF. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction
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.
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 g/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
∗ Corresponding author. Tel.: +52 614 414 4492. E-mail address: [email protected] (Q. Rascón-Cruz).
2. 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 ␣-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 N lobe and 344–703 for the C lobe and is made up of ␣-helix and -pleated sheet structures that create two domains for each lobe (domains I and II) [1]. Each lobe can bind a metal atom in synergy with the carbonate ion (CO3 2− ). The metals that it binds are the Fe2+ or Fe3+ ions, but it has also been observed bound to Cu2+ , Zn2+ and Mn2+ ions [3].
0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.07.020
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S.A. González-Chávez et al. / International Journal of Antimicrobial Agents 33 (2009) 301.e1–301.e8 Table 1 Bacteria against which lactoferrin (LF) has a reported effect Bacteria
Fig. 1. Three-dimensional structure of biferric bovine lactoferrin at a resolution of 2.8 Angstroms [9].
Because of its ability to reversibly bind Fe3+ , LF can exist free of Fe3+ (apo-LF) or associated with it (holo-LF) [10], and it has a different three-dimensional conformation depending on whether it is binding Fe3+ [1]. Apo-LF has an open conformation, whilst holo-LF is a closed molecule with greater resistance to proteolysis [4]. Amino acids directly involved at the iron-binding site in each lobe are Asp, Tyr and His, whilst Arg is involved in the bond with the CO3 2− ion [1]. LF is a basic, positively charged protein with an isoelectric point of 8.0–8.5 [3]. The primary structure of LF shows the number and position of Cys residues that allow the formation of intramolecular disulphide bridges; Asn residues in the N- and C-terminal lobes provide several potential N-glycosylation sites [1]. 3. 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 activities [6]; and several enzymatic functions [12]. LF plays a key role in maintaining cellular iron levels in the body, which has been demonstrated with several studies, mostly in milk. Several decades ago it was shown that breastfed infants have no iron deficiencies, whilst those fed with ironless paediatric formulas show a high risk of iron deficiency and related diseases later in life [13,14]. Also supporting the involvement of LF in this function is the discovery of LF receptors in the enterocytes of various species [15] and the high affinity of these receptors for protein. However, conflicting results have been presented, showing that the lack of these receptors does not affect intestinal iron absorption [16], which leaves LF’s role in this mechanism uncertain.
Study model
Agent administered
Reference
Gram-positives Bacillus stearothermophilus Bacillus subtilis Clostridium sp. Haemophilus influenzae Gram-Negative Listeria monocytogenes Micrococcus sp. Staphylococcus aureus Streptococcus mutans
In vitro In vitro In vitro In vivo In vitro In vivo In vitro In vivo In vitro
hLF hLF hLF and bLF bLF hLF hLF hLF and bLF bLF hLF
[5] [5] [5] [17] [5] [18] [5] [19] [20]
Gram-negatives Chlamydophila psittaci Enteropathogenic Escherichia coli (EPEC) Enteroaggregative E. coli (EAEC) Diffusely adherent E. coli (DAEC) Helicobacter felis Helicobacter pylori Legionella pneumophila Pseudomonas aeruginosa Shigella spp. Vibrio cholerae
In vitro In vitro In vitro In vitro In vivo In vivo In vitro In vivo In vitro In vitro
hLF and bLF hLF hLF hLF rhLF bLF bLFa hLF hLF hLF
[21] [22] [23] [23] [24] [25] [26] [27] [28] [5]
Acid–alcohol-resistant bacilli Mycobacterium tuberculosis
In vitro
hLF
[29]
hLF, human LF; bLF, bovine LF; rhLF, recombinant human LF. a For this study, LF was not effective while binding Fe2+ ; it only inhibited the pathogen when binding Zn2+ or Mn2+ .
3.1. Antibacterial activity The antibacterial activity of LF has been widely documented both in vitro and in vivo for Gram-positive and Gram-negative bacteria and in some acid–alcohol-resistant bacteria. Table 1 shows the bacteria against which LF has shown an inhibitory effect and the type of LF used. Some of the bacteria listed in Table 1 are specially categorised as antimicrobial-resistant, such as the strains of Staphylococcus aureus, Listeria monocytogenes and meticillinresistant Klebsiella pneumoniae. LF has also been shown to be effective against strains of Haemophilus influenzae and Streptococcus mutans, which can attach themselves to the host cell. LF’s bacteriostatic function is due to its ability to take up the Fe3+ ion, limiting use of this nutrient by bacteria at the infection site and inhibiting the growth of these microorganisms as well as the expression of their virulence factors [30]. LF’s bactericidal function has been attributed to its direct interaction with bacterial surfaces (Fig. 2). In 1988 it was shown that LF damages the external membrane of Gram-negative bacteria through an interaction with lipopolysaccharide (LPS) [31]. The positively charged N-terminus of LF prevents the interaction between LPS and the bacterial cations (Ca2+ and Mg2+ ), causing a release of LPS from the cell wall, an increase in the membrane’s permeability and ensuing damage to the bacteria [32]. The interaction of LF and LPS also potentiates the action of natural antibacterials such as lysozyme, which is secreted from the mucosa at elevated concentrations along with LF [33]. LF’s mechanism of action against Gram-positive bacteria is based on binding due to its net positive charge to anionic molecules on the bacterial surface, such as lipoteichoic acid, resulting in a reduction of negative charge on the cell wall and thus favouring contact between lysozyme and the underlying peptidoglycan over which it exerts an enzymatic effect [34]. In vitro and in vivo studies have shown that LF has the ability to prevent the attachment of certain bacteria to the host cell. Attachment-inhibiting mechanisms are unknown, but it has been
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Fig. 2. Mechanism of antibacterial action of lactoferrin (LF). (A) Gram-positive bacteria: LF is bound to negatively charged molecules of the cell membrane such as lipoteichoic acid, neutralising wall charge and allowing the action of other antibacterial compounds such as lysozyme. (B) Gram-negative bacteria: LF can bind to lipid A of lipopolysaccharide, causing liberation of this lipid with consequent damage to the cell membrane.
suggested that LF’s oligomannoside glycans bind bacterial adhesins, preventing their interaction with host cell receptors [10]. 3.2. Antiviral activity LF possesses antiviral activity against a broad range of RNA and DNA viruses that infect humans and animals [3]. Human respiratory syncytial virus is inhibited by LF at concentrations ten times lower than those found in human milk. LF also acts against non-enveloped viruses such as adenoviruses and enteroviruses [35]. Human immunodeficiency virus (HIV) remains a major medical challenge, since current treatment of the syndrome that it causes is not completely effective. In vitro studies show that, among human plasma and milk proteins, LF exerts a strong activity against HIV. This effect is due to inhibition of viral replication in the host cell [36]. The antiviral mechanisms of LF have not yet been characterised. LF can block the internalisation of certain viruses into the host cell, such as poliovirus type 1 which causes poliomyelitis in humans [37], herpes simplex virus types I and II [38] and cytomegalovirus [39]. For other viruses, such as hepatitis C virus (HCV) [40] and
rotavirus [41], rather than preventing entry LF inhibits viral replication in the host cell. Several mechanisms of action have been proposed for LF’s antiviral effects (Fig. 3). One of the most widely accepted hypotheses is that LF binds to and blocks glycosaminoglycan viral receptors, especially heparan sulfate (HS). The binding of LF and HS prevents the first contact between virus and host cell and therefore prevents the infection [3]. The antiviral effect of LF has also been observed in viruses that infect animals, such as the Friend virus complex, which causes erythroleukaemia in rodents [42], the feline calicivirus [43] and feline immunodeficiency virus [44]. 3.3. Antifungal activity LF also has antifungal activity. In 1971, Kirkpatrick et al. conducted the first studies with Candida spp. and attributed the antifungal effect of LF to its ability to sequester Fe3+ [45]. Later it was observed that LF can kill both Candida albicans and Candida krusei by altering the permeability of the cell surface, as it does with bacteria [46]. Further studies have confirmed LF’s antifungal activities. In 2003 it was shown that oral LF treatment of oral can-
Fig. 3. Mechanism of antiviral action of lactoferrin (LF). LF can be linked to the viral particle and to glycosaminoglycans, specific viral receptors or heparan sulfate to prevent internalisation of the virus into the host cell.
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didiasis caused by C. albicans reduces the level of the pathogen and promotes a cure [47]. Although the antifungal mechanism of action of LF is through a direct interaction with the pathogen, Fe3+ sequestration is another important mechanism. In 2007, Zarember et al. showed that Fe3+ sequestration by neutrophil apo-LF is important for host defence against Aspergillus fumigatus [48]. LF shows an interesting antifungal effect on body tineas caused by Trichophyton mentagrophytes, against which it acts from a distance. Treatment of guinea pigs with bovine LF (bLF) reduces fungal infection on the skin of the back and limbs in tinea corpus and tinea pedis, respectively [49]. 3.4. Antiparasitic activity Most of the studies on LF’s antiparasitic activity have been performed in vitro, assaying molecular associations in the presence or absence of Fe3+ . This activity has also been shown using peptides derived from the full molecule. Intestinal amoebiasis is caused by a protozoan infection and is one of the leading causes of diarrhoea in children under 5 years of age and the fourth leading cause of death in the world. The infection is caused by Entamoeba histolytica, which uses complex mechanisms to invade the intestinal mucosa and cause amoebic colitis [50]. Apo-LF is the milk protein with the greatest amoebicidal effect against E. histolytica in vitro, as it can bind the lipids on the trophozoite’s membrane causing membrane disruption and damage to the parasite [51]. Other in vitro studies show that hLF can bind the intracellular parasite Toxoplasma gondii, which causes toxoplasmosis and affects both humans and animals. However, LF cannot prevent the parasite from entering the host. Its mechanism of action in this case is inhibition of intracellular growth of T. gondii within host cells [52]. In the case of the haemoparasites Babesia caballi and Babesia equi, LF’s effect depends on whether or not it is bound to Fe3+ [53]. Babesia caballi was found to be significantly suppressed by apo-LF but was not inhibited by the other types of LF; for B. equi none of the LF types showed an inhibitory effect [54].
LF is secreted to the medium, where it shows anti-inflammatory activity [4] through the inhibition of pro-inflammatory cytokines such as interferon-gamma, tumour necrosis factor-alpha and interleukin (IL)-1, IL-2 and IL-6 [61]. At a cellular level, LF increases the number of natural killer (NK) cells [62], boosts the recruitment of polymorphonuclear cells in the blood [63], induces phagocytosis [64] and can modulate the myelopoietic process [65]. 3.6. Anticarcinogenic activity As in inflammation, LF has the ability to modulate the production of cytokines in cancer. LF can induce apoptosis and arrest tumour growth in vitro. It can also block the transition from G1 to S in the cell cycle of malignant cells [4]. Treating tumours in mice with recombinant hLF (rhLF) inhibits their growth by 60% compared with a placebo and increases the levels of anticarcinogenic cytokines such as IL-18, in addition to activating NK cells and CD8+ T-lymphocytes [66]. LF’s anticancer effect was recently observed through the immunoexpression of LF in human kidney cell carcinomas and in adjacent healthy tissue [67]. In vivo studies show that oral administration of LF results in the inhibition of a T-cell-dependent tumour in head and neck squamous cell carcinoma [68]. 3.7. Enzymatic activity LF has the ability to function as an enzyme in some reactions. LF is the milk protein with the highest levels of amylase, DNAse, RNAse and ATPase activities [12,69]. However, these are not the only enzymatic activities of LF. The basis for LF’s various enzymatic activities is unknown. However, the variety of activities can be attributed to variations in the nature of the protein: multiple isoforms; degrees of glycosylation; tertiary structure (holo- or apoLF); and the degree of oligomerisation. For instance, the LF molecule capable of hydrolysing RNA has an isoform that is incapable of binding Fe3+ [70]. The discovery of LF’s enzymatic activities has helped to explain several of its physiological mechanisms, such as protection against microbial pathogens, where LF might inhibit growth partly through hydrolysis of viral, bacterial, fungal and parasitic nucleic acids.
3.5. Immunomodulatory and anti-inflammatory activity 4. Bioactive peptides derived from lactoferrin LF is a modulator of the innate and acquired immune systems [55]. Its relationship with the immune system is evident from the fact that people with congenital or acquired LF deficiency have recurring infections [56]. In 2006, Wakabayashi et al. used the quantitative reverse transcriptase polymerase chain reaction (RTPCR) method to assay the expression of 20 immune-related genes (antimicrobial proteins, pattern recognition receptors and lymphocyte movement related proteins) in the small intestine of mice administered bLF (2.5 g/kg) and observed that LF can both specifically and non-specifically modulate the expression of those genes [57]. LF’s positive charge allows it to bind to negatively charged molecules on the surface of various cells of the immune system [58] and it has been suggested that this association can trigger signalling pathways that lead to cellular responses such as activation, differentiation and proliferation. LF has been observed transported into the nucleus, where it can bind DNA [59,60] and activate different signalling pathways [4]. In addition to inducing systemic immunity, LF can promote skin immunity and inhibit allergic responses. It induces the immune system against skin allergens, causing dose-dependent inhibition of Langerhans cell migration and the accumulation of dendritic cells in lymph nodes [6].
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, Proteus vulgaris, Pseudomonas aeruginosa and Streptococcus bovis. Bellamy et al. [72] identified a region of amino acids at the N-terminus that retains its biological activity when separated from the full molecule. This molecule, called lactoferricin B (LFc B), shows greater antimicrobial activity than LF. This region corresponds to residues 17–41 of bLF [72] and it is now known that it corresponds to residues 12–48 in several species of mammals with highly homologous sequences [5]. In characterising the various peptides generated by LF hydrolysis, it was found that minimal variations in the amino acid sequence change the antimicrobial activity of the peptide. For example, LFampin 268–284 and LFampin 265–284, chemically synthesised fragments from the N-terminal sequence of bLF, differ in only three amino acids (265Asp-Leu-267Ile) but exhibit a different strength of antimicrobial activity [73].
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When isolated from the native molecule, the N- and C-termini show physiological activity through mechanisms independent of iron sequestration [74]. 5. 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 [75–78]. 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 2008, Kang et al. analysed 60 sequences of LF genes with full coding regions. They found that the length of the gene varies widely from species to species, from 2055 to 2190 residues, owing to deletions, insertions and mutations in the stop codon [78]. LF is expressed both constitutively and inducibly. It is constitutively expressed on mucosal surfaces whilst in some tissues it
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is induced by external agents. In 2002, oestrogen response elements (EREs) were identified in the promoter of this gene in humans and mice. In these promoters the ERE overlaps the binding sites of other transcription factors such as COUP-TF, an oestrogenresponsive negative regulator [82]. Other regulatory factors include the repressor of oestrogen receptor activity, whose absence has been proven to increase the expression of oestrogen-induced LF by up to 100-fold [83]. LF can also be induced by compounds other than oestrogens, such as retinoic acid, which stimulates gene expression in embryonic cells [84]. Evidence to date suggests the involvement of multiple signalling pathways in the regulation of LF expression, and further study may reveal an even greater complexity of regulatory mechanisms. 6. 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
Table 2 Expression of lactoferrin (LF) in various organisms Organism Bacteria Escherichia coli Rhodococcus erythropolis Yeasts Saccharomyces cerevisiae Pichia pastoris
LF origin
Expression system
Levels of expression
Size
Characteristics
Reference
bLfc Lfc bLF C lobe
pET32a vector Fusion of Lfc and anionic protein genes pTip LCH1.2 vector
10 mg/L 60 mg/L 3.6 mg/mL
Several 80 kDa 38 kDa
Antimicrobial activity Antimicrobial activity Antimicrobial activity
[92] [93] [93]
hLF
Chelatin promoter
2.0 mg/L
80 kDa
[94]
hLF cLF
PIC 3.5 K vector pGAPZalphaC vector
115 mg/L 2 mg/L
80 kDa 80 kDa
pLF
12 mg/L
78 kDa
bLF
Glyceraldehyde-3-phosphate dehydrogenase promoter NR
Antimicrobial activity, ability to bind Fe3+ Not identified Ability to bind Fe3+ , thermal stability Ability to bind Fe3+ Antimicrobial activity, ability to bind Fe3+ Ability to bind Fe3+
[98]
Antimicrobial activity, ability to bind Fe3+ Immunoreactive, ability to bind Fe3+
[100]
76 kDa
[95] [96] [97]
eLF
pPIC 9 K vector
40 mg/L
80 kDa
Fungi Aspergillus awamori
hLF
Fusion with the glucoamylase gene
2.0 g/L
80 kDa
Aspergillus oryzae
hLF
Alpha amylase promoter of A. oryzae
25 mg/L
78 kDa
hLF
9.5 mg/L
80 kDa
Ability to bind Fe3+
[102]
hLF pLF
Infection with nucleopolyhedrosis virus, P8HLfc vector Infection with baculovirus vBm-hLF Infection with baculovirus
65 mg/mL 205 g/pupa
78 kDa 80 kDa
Antimicrobial activity Biological activity
[103] [104]
Mammals Goat
hLF
Microinjection
0.756 mg/L milk
78 kDa
[105]
Mice
hLF hLF
2 g/L milk 13 mg/mL
80 kDa 80 kDa
Rabbit Bovine
hLF hLF
Infection with attenuated adenovirus Fusion with regulatory elements of the bovine alphaS1 casein gene Adenovirus infection Microinjection
2.3 mg/mL milk 1 g/L milk
80 kDa 80 kDa
Ability to bind Fe3+ , temperature and proteolysis stable NR Antibacterial and anti-inflammatory activity NR Antimicrobial activity, ability to bind Fe3+
Potexvirus infection pLACMODC/18.1 Agroinfection with Agrobacterium tumefaciens plasmid: pIG200 and pIG211 Infection with A. tumefaciens pUC 18 vector
0.6 % SP NR 1.6 mg/g seeds
40 kDa Mixed 78 kDa
Antibacterial activity Antibacterial activity Antibacterial activity
[110] [111] [112]
0.1% SP NR
80 kDa 85 kDa
Antimicrobial activity Adequate glycosylation level
[113] [114]
Insects Spodoptera frugiperda Bombyx mori
Plants Nicotiana benthamiana Nicotiana tabacum Rice
Potato Maize
hLF N lobe hLF hLF
hLF hLF
b, bovine; h, human; c, caprine; p, porcine; e, equine; Lfc, lactoferricin; NR, not reported; SP, soluble protein.
[99]
[101]
[106] [107] [108] [109]
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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 Bifidobacterium and Lactobacillus, which protect the host from harmful bacteria [85]. The activity of LF and its bioactive peptides has been documented both in vitro and in vivo against a large variety of pathogens. Studies at a clinical level have considered LF as a possible treatment or prophylactic for a number of diseases. For example, LF was tested as a second treatment against Helicobacter pylori in patients with recurring infection. Patients supplemented with bLF showed a greater recovery from infection [86]. As with antibacterials, LF has shown synergy with antiviral drugs: ribavirin to treat HCV [87]; cidofovir against cytomegalovirus [3] and zidovudine (an AZT analogue) against HIV [36]. It has also been observed that LF added in subinhibitory concentrations to antifungal agents such as clotrimazole, ketoconazole, fluconazole and itraconazole reduces the minimum inhibitory concentrations of these agents against C. albicans. That is, these combinations synergistically inhibit the growth of the pathogen, with the most resistant strains being the most sensitive to this combination. The preventive effect of LF has also been tested. An example is the use of LF as an adjuvant for the BCG (Bacille Calmette-Guérin) vaccine, where LF enhances the delayed hypersensitivity response and limits the pathology caused by Mycobacterium tuberculosis by increasing class II molecule levels on the surface of antigen-presenting cells and thereby increasing IL-12 and IL-10 expression [88].
7. 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 [89]. Furthermore, since LF is a glycosylated protein it can also be purified by concanavalin A affinity chromatography [91]. However, the need for larger amounts of LF has led to the development of strategies to obtain a recombinant form of the protein and, to date, several LF expression systems have been used (Table 2), including both prokaryotic and eukaryotic organisms. One of the first expression systems was built in filaments of the Aspergillus fungus, where both hLF and murine LF have been expressed [100,101]. Later, as shown in Table 2, expression systems were developed in yeasts and bacteria, where the following proteins have been produced: rhLF [94,95]; caprine LF [96]; bLF [98]; equine LF [99]; and porcine (pLF) [97], as well as LF peptides, including LFc [92,93], reaching expression levels of 115 mg of hLF/L of fermentate from Pichia pastoris grown with a high-density batch fermenter [95]. Biotechnological tools have made it possible to use viral vectors for the expression of LF, mostly through insect infection either in cell culture or directly in the organism, where the expression of both hLF and pLF has been successful, resulting in transgenic Bombyx mori insects expressing 205 g of pLF per infected pupa [103]. LF has also been expressed in higher eukaryotic organisms, both in plants and animals. Using microinjection and direct infection with viral vectors in the mammary gland, transgenic animals have been created that produce milk with rLF. These animals include goats [105], mice [107], rabbits [108] and cows [109], with expression levels of up to 2 g of hLF/L of milk in transgenic goats. Table 2 also shows the expression systems in plants where hLF expression
has been attained; in rice, 1.6 mg of protein per gram of seed was produced. Most of the expression systems that have been developed produce LF or LF peptides in a recombinant form with physical, biochemical and biological characteristics similar and often indistinguishable from those of native LF, including molecular weight, degree of glycosylation, antimicrobial and anti-inflammatory activity, thermostability and ability to bind Fe3+ . These strategies are useful for producing rLF at high levels of expression for efficient economical large-scale production.
8. 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 and purify LF from cells as a potential defence against pathogens. Funding: This work was supported in part by an internal grant from Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, México. SAG-C thanks CONACYT for the MC studies grant. Competing interests: None declared. Ethical approval: Not required.
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Review
Lactoferrin: structure, function and applications Susana A. González-Chávez, Sigifredo Arévalo-Gallegos, Quintín Rascón-Cruz ∗ Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Cd. Universitaria s/n, CP 31170, Chihuahua, Chihuahua, Mexico
a r t i c l e
i n f o
a b s t r a c t
Keywords: Lactoferrin Iron-binding protein Transferrin Functional protein
Lactoferrin (LF) is an 80 kDa iron-binding glycoprotein of the transferrin family that is expressed in most biological fluids and is a major component of the mammalian innate immune system. Its protective effects range from direct antimicrobial activities against a large panel of microorganisms, including bacteria, viruses, fungi and parasites, to anti-inflammatory and anticancer activities. These extensive activities are made possible by mechanisms of action utilising not only the capacity of LF to bind iron but also interactions of LF with molecular and cellular components of both host and pathogens. This review summarises the putative antimicrobial mechanisms, clinical applications and heterologous expression models for LF. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction
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.
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 g/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
∗ Corresponding author. Tel.: +52 614 414 4492. E-mail address: [email protected] (Q. Rascón-Cruz).
2. 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 ␣-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 N lobe and 344–703 for the C lobe and is made up of ␣-helix and -pleated sheet structures that create two domains for each lobe (domains I and II) [1]. Each lobe can bind a metal atom in synergy with the carbonate ion (CO3 2− ). The metals that it binds are the Fe2+ or Fe3+ ions, but it has also been observed bound to Cu2+ , Zn2+ and Mn2+ ions [3].
0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.07.020
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S.A. González-Chávez et al. / International Journal of Antimicrobial Agents 33 (2009) 301.e1–301.e8 Table 1 Bacteria against which lactoferrin (LF) has a reported effect Bacteria
Fig. 1. Three-dimensional structure of biferric bovine lactoferrin at a resolution of 2.8 Angstroms [9].
Because of its ability to reversibly bind Fe3+ , LF can exist free of Fe3+ (apo-LF) or associated with it (holo-LF) [10], and it has a different three-dimensional conformation depending on whether it is binding Fe3+ [1]. Apo-LF has an open conformation, whilst holo-LF is a closed molecule with greater resistance to proteolysis [4]. Amino acids directly involved at the iron-binding site in each lobe are Asp, Tyr and His, whilst Arg is involved in the bond with the CO3 2− ion [1]. LF is a basic, positively charged protein with an isoelectric point of 8.0–8.5 [3]. The primary structure of LF shows the number and position of Cys residues that allow the formation of intramolecular disulphide bridges; Asn residues in the N- and C-terminal lobes provide several potential N-glycosylation sites [1]. 3. 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 activities [6]; and several enzymatic functions [12]. LF plays a key role in maintaining cellular iron levels in the body, which has been demonstrated with several studies, mostly in milk. Several decades ago it was shown that breastfed infants have no iron deficiencies, whilst those fed with ironless paediatric formulas show a high risk of iron deficiency and related diseases later in life [13,14]. Also supporting the involvement of LF in this function is the discovery of LF receptors in the enterocytes of various species [15] and the high affinity of these receptors for protein. However, conflicting results have been presented, showing that the lack of these receptors does not affect intestinal iron absorption [16], which leaves LF’s role in this mechanism uncertain.
Study model
Agent administered
Reference
Gram-positives Bacillus stearothermophilus Bacillus subtilis Clostridium sp. Haemophilus influenzae Gram-Negative Listeria monocytogenes Micrococcus sp. Staphylococcus aureus Streptococcus mutans
In vitro In vitro In vitro In vivo In vitro In vivo In vitro In vivo In vitro
hLF hLF hLF and bLF bLF hLF hLF hLF and bLF bLF hLF
[5] [5] [5] [17] [5] [18] [5] [19] [20]
Gram-negatives Chlamydophila psittaci Enteropathogenic Escherichia coli (EPEC) Enteroaggregative E. coli (EAEC) Diffusely adherent E. coli (DAEC) Helicobacter felis Helicobacter pylori Legionella pneumophila Pseudomonas aeruginosa Shigella spp. Vibrio cholerae
In vitro In vitro In vitro In vitro In vivo In vivo In vitro In vivo In vitro In vitro
hLF and bLF hLF hLF hLF rhLF bLF bLFa hLF hLF hLF
[21] [22] [23] [23] [24] [25] [26] [27] [28] [5]
Acid–alcohol-resistant bacilli Mycobacterium tuberculosis
In vitro
hLF
[29]
hLF, human LF; bLF, bovine LF; rhLF, recombinant human LF. a For this study, LF was not effective while binding Fe2+ ; it only inhibited the pathogen when binding Zn2+ or Mn2+ .
3.1. Antibacterial activity The antibacterial activity of LF has been widely documented both in vitro and in vivo for Gram-positive and Gram-negative bacteria and in some acid–alcohol-resistant bacteria. Table 1 shows the bacteria against which LF has shown an inhibitory effect and the type of LF used. Some of the bacteria listed in Table 1 are specially categorised as antimicrobial-resistant, such as the strains of Staphylococcus aureus, Listeria monocytogenes and meticillinresistant Klebsiella pneumoniae. LF has also been shown to be effective against strains of Haemophilus influenzae and Streptococcus mutans, which can attach themselves to the host cell. LF’s bacteriostatic function is due to its ability to take up the Fe3+ ion, limiting use of this nutrient by bacteria at the infection site and inhibiting the growth of these microorganisms as well as the expression of their virulence factors [30]. LF’s bactericidal function has been attributed to its direct interaction with bacterial surfaces (Fig. 2). In 1988 it was shown that LF damages the external membrane of Gram-negative bacteria through an interaction with lipopolysaccharide (LPS) [31]. The positively charged N-terminus of LF prevents the interaction between LPS and the bacterial cations (Ca2+ and Mg2+ ), causing a release of LPS from the cell wall, an increase in the membrane’s permeability and ensuing damage to the bacteria [32]. The interaction of LF and LPS also potentiates the action of natural antibacterials such as lysozyme, which is secreted from the mucosa at elevated concentrations along with LF [33]. LF’s mechanism of action against Gram-positive bacteria is based on binding due to its net positive charge to anionic molecules on the bacterial surface, such as lipoteichoic acid, resulting in a reduction of negative charge on the cell wall and thus favouring contact between lysozyme and the underlying peptidoglycan over which it exerts an enzymatic effect [34]. In vitro and in vivo studies have shown that LF has the ability to prevent the attachment of certain bacteria to the host cell. Attachment-inhibiting mechanisms are unknown, but it has been
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Fig. 2. Mechanism of antibacterial action of lactoferrin (LF). (A) Gram-positive bacteria: LF is bound to negatively charged molecules of the cell membrane such as lipoteichoic acid, neutralising wall charge and allowing the action of other antibacterial compounds such as lysozyme. (B) Gram-negative bacteria: LF can bind to lipid A of lipopolysaccharide, causing liberation of this lipid with consequent damage to the cell membrane.
suggested that LF’s oligomannoside glycans bind bacterial adhesins, preventing their interaction with host cell receptors [10]. 3.2. Antiviral activity LF possesses antiviral activity against a broad range of RNA and DNA viruses that infect humans and animals [3]. Human respiratory syncytial virus is inhibited by LF at concentrations ten times lower than those found in human milk. LF also acts against non-enveloped viruses such as adenoviruses and enteroviruses [35]. Human immunodeficiency virus (HIV) remains a major medical challenge, since current treatment of the syndrome that it causes is not completely effective. In vitro studies show that, among human plasma and milk proteins, LF exerts a strong activity against HIV. This effect is due to inhibition of viral replication in the host cell [36]. The antiviral mechanisms of LF have not yet been characterised. LF can block the internalisation of certain viruses into the host cell, such as poliovirus type 1 which causes poliomyelitis in humans [37], herpes simplex virus types I and II [38] and cytomegalovirus [39]. For other viruses, such as hepatitis C virus (HCV) [40] and
rotavirus [41], rather than preventing entry LF inhibits viral replication in the host cell. Several mechanisms of action have been proposed for LF’s antiviral effects (Fig. 3). One of the most widely accepted hypotheses is that LF binds to and blocks glycosaminoglycan viral receptors, especially heparan sulfate (HS). The binding of LF and HS prevents the first contact between virus and host cell and therefore prevents the infection [3]. The antiviral effect of LF has also been observed in viruses that infect animals, such as the Friend virus complex, which causes erythroleukaemia in rodents [42], the feline calicivirus [43] and feline immunodeficiency virus [44]. 3.3. Antifungal activity LF also has antifungal activity. In 1971, Kirkpatrick et al. conducted the first studies with Candida spp. and attributed the antifungal effect of LF to its ability to sequester Fe3+ [45]. Later it was observed that LF can kill both Candida albicans and Candida krusei by altering the permeability of the cell surface, as it does with bacteria [46]. Further studies have confirmed LF’s antifungal activities. In 2003 it was shown that oral LF treatment of oral can-
Fig. 3. Mechanism of antiviral action of lactoferrin (LF). LF can be linked to the viral particle and to glycosaminoglycans, specific viral receptors or heparan sulfate to prevent internalisation of the virus into the host cell.
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didiasis caused by C. albicans reduces the level of the pathogen and promotes a cure [47]. Although the antifungal mechanism of action of LF is through a direct interaction with the pathogen, Fe3+ sequestration is another important mechanism. In 2007, Zarember et al. showed that Fe3+ sequestration by neutrophil apo-LF is important for host defence against Aspergillus fumigatus [48]. LF shows an interesting antifungal effect on body tineas caused by Trichophyton mentagrophytes, against which it acts from a distance. Treatment of guinea pigs with bovine LF (bLF) reduces fungal infection on the skin of the back and limbs in tinea corpus and tinea pedis, respectively [49]. 3.4. Antiparasitic activity Most of the studies on LF’s antiparasitic activity have been performed in vitro, assaying molecular associations in the presence or absence of Fe3+ . This activity has also been shown using peptides derived from the full molecule. Intestinal amoebiasis is caused by a protozoan infection and is one of the leading causes of diarrhoea in children under 5 years of age and the fourth leading cause of death in the world. The infection is caused by Entamoeba histolytica, which uses complex mechanisms to invade the intestinal mucosa and cause amoebic colitis [50]. Apo-LF is the milk protein with the greatest amoebicidal effect against E. histolytica in vitro, as it can bind the lipids on the trophozoite’s membrane causing membrane disruption and damage to the parasite [51]. Other in vitro studies show that hLF can bind the intracellular parasite Toxoplasma gondii, which causes toxoplasmosis and affects both humans and animals. However, LF cannot prevent the parasite from entering the host. Its mechanism of action in this case is inhibition of intracellular growth of T. gondii within host cells [52]. In the case of the haemoparasites Babesia caballi and Babesia equi, LF’s effect depends on whether or not it is bound to Fe3+ [53]. Babesia caballi was found to be significantly suppressed by apo-LF but was not inhibited by the other types of LF; for B. equi none of the LF types showed an inhibitory effect [54].
LF is secreted to the medium, where it shows anti-inflammatory activity [4] through the inhibition of pro-inflammatory cytokines such as interferon-gamma, tumour necrosis factor-alpha and interleukin (IL)-1, IL-2 and IL-6 [61]. At a cellular level, LF increases the number of natural killer (NK) cells [62], boosts the recruitment of polymorphonuclear cells in the blood [63], induces phagocytosis [64] and can modulate the myelopoietic process [65]. 3.6. Anticarcinogenic activity As in inflammation, LF has the ability to modulate the production of cytokines in cancer. LF can induce apoptosis and arrest tumour growth in vitro. It can also block the transition from G1 to S in the cell cycle of malignant cells [4]. Treating tumours in mice with recombinant hLF (rhLF) inhibits their growth by 60% compared with a placebo and increases the levels of anticarcinogenic cytokines such as IL-18, in addition to activating NK cells and CD8+ T-lymphocytes [66]. LF’s anticancer effect was recently observed through the immunoexpression of LF in human kidney cell carcinomas and in adjacent healthy tissue [67]. In vivo studies show that oral administration of LF results in the inhibition of a T-cell-dependent tumour in head and neck squamous cell carcinoma [68]. 3.7. Enzymatic activity LF has the ability to function as an enzyme in some reactions. LF is the milk protein with the highest levels of amylase, DNAse, RNAse and ATPase activities [12,69]. However, these are not the only enzymatic activities of LF. The basis for LF’s various enzymatic activities is unknown. However, the variety of activities can be attributed to variations in the nature of the protein: multiple isoforms; degrees of glycosylation; tertiary structure (holo- or apoLF); and the degree of oligomerisation. For instance, the LF molecule capable of hydrolysing RNA has an isoform that is incapable of binding Fe3+ [70]. The discovery of LF’s enzymatic activities has helped to explain several of its physiological mechanisms, such as protection against microbial pathogens, where LF might inhibit growth partly through hydrolysis of viral, bacterial, fungal and parasitic nucleic acids.
3.5. Immunomodulatory and anti-inflammatory activity 4. Bioactive peptides derived from lactoferrin LF is a modulator of the innate and acquired immune systems [55]. Its relationship with the immune system is evident from the fact that people with congenital or acquired LF deficiency have recurring infections [56]. In 2006, Wakabayashi et al. used the quantitative reverse transcriptase polymerase chain reaction (RTPCR) method to assay the expression of 20 immune-related genes (antimicrobial proteins, pattern recognition receptors and lymphocyte movement related proteins) in the small intestine of mice administered bLF (2.5 g/kg) and observed that LF can both specifically and non-specifically modulate the expression of those genes [57]. LF’s positive charge allows it to bind to negatively charged molecules on the surface of various cells of the immune system [58] and it has been suggested that this association can trigger signalling pathways that lead to cellular responses such as activation, differentiation and proliferation. LF has been observed transported into the nucleus, where it can bind DNA [59,60] and activate different signalling pathways [4]. In addition to inducing systemic immunity, LF can promote skin immunity and inhibit allergic responses. It induces the immune system against skin allergens, causing dose-dependent inhibition of Langerhans cell migration and the accumulation of dendritic cells in lymph nodes [6].
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, Proteus vulgaris, Pseudomonas aeruginosa and Streptococcus bovis. Bellamy et al. [72] identified a region of amino acids at the N-terminus that retains its biological activity when separated from the full molecule. This molecule, called lactoferricin B (LFc B), shows greater antimicrobial activity than LF. This region corresponds to residues 17–41 of bLF [72] and it is now known that it corresponds to residues 12–48 in several species of mammals with highly homologous sequences [5]. In characterising the various peptides generated by LF hydrolysis, it was found that minimal variations in the amino acid sequence change the antimicrobial activity of the peptide. For example, LFampin 268–284 and LFampin 265–284, chemically synthesised fragments from the N-terminal sequence of bLF, differ in only three amino acids (265Asp-Leu-267Ile) but exhibit a different strength of antimicrobial activity [73].
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When isolated from the native molecule, the N- and C-termini show physiological activity through mechanisms independent of iron sequestration [74]. 5. 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 [75–78]. 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 2008, Kang et al. analysed 60 sequences of LF genes with full coding regions. They found that the length of the gene varies widely from species to species, from 2055 to 2190 residues, owing to deletions, insertions and mutations in the stop codon [78]. LF is expressed both constitutively and inducibly. It is constitutively expressed on mucosal surfaces whilst in some tissues it
301.e5
is induced by external agents. In 2002, oestrogen response elements (EREs) were identified in the promoter of this gene in humans and mice. In these promoters the ERE overlaps the binding sites of other transcription factors such as COUP-TF, an oestrogenresponsive negative regulator [82]. Other regulatory factors include the repressor of oestrogen receptor activity, whose absence has been proven to increase the expression of oestrogen-induced LF by up to 100-fold [83]. LF can also be induced by compounds other than oestrogens, such as retinoic acid, which stimulates gene expression in embryonic cells [84]. Evidence to date suggests the involvement of multiple signalling pathways in the regulation of LF expression, and further study may reveal an even greater complexity of regulatory mechanisms. 6. 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
Table 2 Expression of lactoferrin (LF) in various organisms Organism Bacteria Escherichia coli Rhodococcus erythropolis Yeasts Saccharomyces cerevisiae Pichia pastoris
LF origin
Expression system
Levels of expression
Size
Characteristics
Reference
bLfc Lfc bLF C lobe
pET32a vector Fusion of Lfc and anionic protein genes pTip LCH1.2 vector
10 mg/L 60 mg/L 3.6 mg/mL
Several 80 kDa 38 kDa
Antimicrobial activity Antimicrobial activity Antimicrobial activity
[92] [93] [93]
hLF
Chelatin promoter
2.0 mg/L
80 kDa
[94]
hLF cLF
PIC 3.5 K vector pGAPZalphaC vector
115 mg/L 2 mg/L
80 kDa 80 kDa
pLF
12 mg/L
78 kDa
bLF
Glyceraldehyde-3-phosphate dehydrogenase promoter NR
Antimicrobial activity, ability to bind Fe3+ Not identified Ability to bind Fe3+ , thermal stability Ability to bind Fe3+ Antimicrobial activity, ability to bind Fe3+ Ability to bind Fe3+
[98]
Antimicrobial activity, ability to bind Fe3+ Immunoreactive, ability to bind Fe3+
[100]
76 kDa
[95] [96] [97]
eLF
pPIC 9 K vector
40 mg/L
80 kDa
Fungi Aspergillus awamori
hLF
Fusion with the glucoamylase gene
2.0 g/L
80 kDa
Aspergillus oryzae
hLF
Alpha amylase promoter of A. oryzae
25 mg/L
78 kDa
hLF
9.5 mg/L
80 kDa
Ability to bind Fe3+
[102]
hLF pLF
Infection with nucleopolyhedrosis virus, P8HLfc vector Infection with baculovirus vBm-hLF Infection with baculovirus
65 mg/mL 205 g/pupa
78 kDa 80 kDa
Antimicrobial activity Biological activity
[103] [104]
Mammals Goat
hLF
Microinjection
0.756 mg/L milk
78 kDa
[105]
Mice
hLF hLF
2 g/L milk 13 mg/mL
80 kDa 80 kDa
Rabbit Bovine
hLF hLF
Infection with attenuated adenovirus Fusion with regulatory elements of the bovine alphaS1 casein gene Adenovirus infection Microinjection
2.3 mg/mL milk 1 g/L milk
80 kDa 80 kDa
Ability to bind Fe3+ , temperature and proteolysis stable NR Antibacterial and anti-inflammatory activity NR Antimicrobial activity, ability to bind Fe3+
Potexvirus infection pLACMODC/18.1 Agroinfection with Agrobacterium tumefaciens plasmid: pIG200 and pIG211 Infection with A. tumefaciens pUC 18 vector
0.6 % SP NR 1.6 mg/g seeds
40 kDa Mixed 78 kDa
Antibacterial activity Antibacterial activity Antibacterial activity
[110] [111] [112]
0.1% SP NR
80 kDa 85 kDa
Antimicrobial activity Adequate glycosylation level
[113] [114]
Insects Spodoptera frugiperda Bombyx mori
Plants Nicotiana benthamiana Nicotiana tabacum Rice
Potato Maize
hLF N lobe hLF hLF
hLF hLF
b, bovine; h, human; c, caprine; p, porcine; e, equine; Lfc, lactoferricin; NR, not reported; SP, soluble protein.
[99]
[101]
[106] [107] [108] [109]
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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 Bifidobacterium and Lactobacillus, which protect the host from harmful bacteria [85]. The activity of LF and its bioactive peptides has been documented both in vitro and in vivo against a large variety of pathogens. Studies at a clinical level have considered LF as a possible treatment or prophylactic for a number of diseases. For example, LF was tested as a second treatment against Helicobacter pylori in patients with recurring infection. Patients supplemented with bLF showed a greater recovery from infection [86]. As with antibacterials, LF has shown synergy with antiviral drugs: ribavirin to treat HCV [87]; cidofovir against cytomegalovirus [3] and zidovudine (an AZT analogue) against HIV [36]. It has also been observed that LF added in subinhibitory concentrations to antifungal agents such as clotrimazole, ketoconazole, fluconazole and itraconazole reduces the minimum inhibitory concentrations of these agents against C. albicans. That is, these combinations synergistically inhibit the growth of the pathogen, with the most resistant strains being the most sensitive to this combination. The preventive effect of LF has also been tested. An example is the use of LF as an adjuvant for the BCG (Bacille Calmette-Guérin) vaccine, where LF enhances the delayed hypersensitivity response and limits the pathology caused by Mycobacterium tuberculosis by increasing class II molecule levels on the surface of antigen-presenting cells and thereby increasing IL-12 and IL-10 expression [88].
7. 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 [89]. Furthermore, since LF is a glycosylated protein it can also be purified by concanavalin A affinity chromatography [91]. However, the need for larger amounts of LF has led to the development of strategies to obtain a recombinant form of the protein and, to date, several LF expression systems have been used (Table 2), including both prokaryotic and eukaryotic organisms. One of the first expression systems was built in filaments of the Aspergillus fungus, where both hLF and murine LF have been expressed [100,101]. Later, as shown in Table 2, expression systems were developed in yeasts and bacteria, where the following proteins have been produced: rhLF [94,95]; caprine LF [96]; bLF [98]; equine LF [99]; and porcine (pLF) [97], as well as LF peptides, including LFc [92,93], reaching expression levels of 115 mg of hLF/L of fermentate from Pichia pastoris grown with a high-density batch fermenter [95]. Biotechnological tools have made it possible to use viral vectors for the expression of LF, mostly through insect infection either in cell culture or directly in the organism, where the expression of both hLF and pLF has been successful, resulting in transgenic Bombyx mori insects expressing 205 g of pLF per infected pupa [103]. LF has also been expressed in higher eukaryotic organisms, both in plants and animals. Using microinjection and direct infection with viral vectors in the mammary gland, transgenic animals have been created that produce milk with rLF. These animals include goats [105], mice [107], rabbits [108] and cows [109], with expression levels of up to 2 g of hLF/L of milk in transgenic goats. Table 2 also shows the expression systems in plants where hLF expression
has been attained; in rice, 1.6 mg of protein per gram of seed was produced. Most of the expression systems that have been developed produce LF or LF peptides in a recombinant form with physical, biochemical and biological characteristics similar and often indistinguishable from those of native LF, including molecular weight, degree of glycosylation, antimicrobial and anti-inflammatory activity, thermostability and ability to bind Fe3+ . These strategies are useful for producing rLF at high levels of expression for efficient economical large-scale production.
8. 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 and purify LF from cells as a potential defence against pathogens. Funding: This work was supported in part by an internal grant from Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, México. SAG-C thanks CONACYT for the MC studies grant. Competing interests: None declared. Ethical approval: Not required.
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