Elsevier

Molecular Immunology

Volume 40, Issue 7, November 2003, Pages 395-405
Molecular Immunology

Review
Lactoferrin—a multifunctional protein with antimicrobial properties

https://doi.org/10.1016/S0161-5890(03)00152-4Get rights and content

Abstract

Lactoferrin is a member of the transferrin family of iron-binding proteins. Numerous functions have been reported and continue to be reported for the protein, some of which are related to its iron-binding properties. Its extensive antimicrobial activities were originally attributed to its ability to sequester essential iron, however, it is now established that it possesses bactericidal activities as a result of a direct interaction between the protein or lactoferrin-derived peptides. This article reviews the antimicrobial activities of lactoferrin and discusses the potential mode of action of lactoferrin-derived cationic peptides against Gram-negative bacteria in the light of recent studies.

Introduction

Lactoferrin is an avid iron-binding glycoprotein of the transferrin family that includes serum transferrin and ovotransferrin. It is found on mucosal surfaces, within the specific granules of polymorphonuclear leukocytes, and in biological fluids, including milk, saliva and seminal fluid, indicating that it may play a protective role in the innate immune response.

Milk is by far the most abundant source of lactoferrin with human colostrum, the early milk, containing up to 7 g/l (Masson and Heremans, 1971). There is a great variation in the concentration of lactoferrin in other human body fluids. The concentration in tears is as high as 2 mg/ml whereas that in blood is normally only as high as 1 μg/ml, although it can rise as high as 200 μg/ml in the inflammatory situation (Masson and Heremans, 1971). Although lactoferrin is found in the milk of most mammals its concentration is quite variable and dependent on the stage of lactation.

Lactoferrin is a monomeric, bilobal, glycoprotein with a molecular mass of about 80 kDa. The three-dimensional structure of human diferric lactoferrin was first reported in 1987 (Anderson et al., 1987). Since then the structure of the diferric protein has been refined and further structures reported for human apolactoferrin and apo and iron-loaded forms of other lactoferrins (reviewed by Baker et al., 2002). The three-dimensional structure of diferric human lactoferrin is shown in Fig. 1. The protein comprises two homologous lobes corresponding to its amino- (residues 1–333) and carboxyl- (residues 345–692) terminal halves, connected by a three-turn α-helix at residues 334–344. Each lobe is further subdivided into two domains, with a single iron-binding site situated between the inner faces of the inter-domain cleft. Each iron (Fe3+) atom is co-ordinated to four protein ligands, namely, 2 tyrosines, 1 aspartate, and 1 histidine, and also to a synergistic anion—normally carbonate in vivo. Small angle scattering studies have shown that both lobes undergo a substantial confrontational change as a result of iron binding, consistent with closure of the inter-domain cleft (Grossmann et al., 1992).

The sequence of human lactoferrin has been reported from a number of sources: the primary amino acid sequence, derived from human mammary lactoferrin (Metz-Boutigue et al., 1984), and the cDNA sequence, taken from human myeloid and mammary gland libraries (Rado et al., 1987, Rey et al., 1990). These sequences were found to be almost identical, however, the crystallographic structure of diferric human lactoferrin (Anderson et al., 1989) suggested that three arginine residues (GRRRS) and not four (GRRRRS) are present at the N-terminus of the protein. This discrepancy is a consequence of the flexibility of the protein at its N-terminus end and thus the electron density at this point in the structure is ambiguous.

The two lobes have 125 amino acids in common (37% homology) and exhibit very similar tertiary structures, consistent with the hypothesis that the two lobes arose as a product of gene duplication (Williams, 1982, Metz-Boutigue et al., 1984). Human and bovine lactoferrins share 69% sequence homology and are structurally very similar when viewed at tertiary level (Pierce et al., 1991). The iso-electric point (pI) of lactoferrin was found to be between 8.4 and 9.0, which is higher than that observed for other members of the transferrin family, pI 5.4–5.9 (Moguilevsky et al., 1985, Sanchez et al., 1992, Hovanessian and Awdeh, 1976).

Many roles have been proposed, and continue to be proposed, for lactoferrin (Fig. 2). Although some of these are clearly related to its iron-binding properties, for example its ability to provide bacteria with a source of iron and therefore act as a “promicrobial”, others appear to be independent of iron binding.

The antimicrobial activity of lactoferrin is well established. For many years this activity was attributed to the ability of lactoferrin to sequester iron thereby depriving potential pathogens of this essential nutrient. However, lactoferrin is now known to possess a second type of antimicrobial activity, bactericidal as opposed to bacteriostatic, the result of a direct interaction between the protein and the bacterium. This article will concentrate on the antimicrobial activity of lactoferrin and lactoferrin-derived peptides.

Section snippets

Iron uptake systems of pathogenic bacteria

It has been well established that iron is an essential nutrient for the growth of almost all bacteria and bacteria within the body have developed mechanisms for obtaining iron as they are often exposed to iron-limited conditions. In the normal situation, iron in the body is protein-bound, rather than “free”, in order to minimise the generation of unwanted free radicals as a result of iron-catalysed cascades. In response to iron-limited stress many bacteria synthesise and secrete phenolate

Antimicrobial activity of lactoferrin

It has been widely accepted for many years that lactoferrin displays antimicrobial activity against many different infectious agents. This activity was originally attributed to its ability, in common with transferrin, to sequester iron with a high affinity and, unlike transferrin, retain its bound iron under acidic conditions. More recently, however, it has become apparent that some of the antimicrobial properties of lactoferrin are independent of iron-binding.

Cationic peptides

A wide variety of organisms produce antimicrobial peptides as a primary innate immune strategy (Hancock and Lehrer, 1998). To date, hundreds of such peptides have been isolated throughout nature, from single celled micro-organisms, mammals, amphibians, birds, fish and plants (Hancock and Chapple, 1999), indicating their importance in the innate immune system (Bevins, 1994, Hancock and Diamond, 2000). Typically, these peptides are relatively short (less than 100 amino acids), positively charged,

Conclusion

It is now more than 40 years since Groves (1960) and Johansson (1960) independently reported the isolation of a red protein from milk, a protein we now know to be lactoferrin, a member of the transferrin family of iron-binding proteins. The X-ray structure of diferric human lactoferrin (Anderson et al., 1987) provided us with the first information on the nature of the iron-binding sites in the transferrin family. We now have extensive high-resolution structural data on a number of different

Acknowledgements

We are grateful for financial support from the Wellcome Trust.

References (83)

  • T.A Rado et al.

    Isolation of lactoferrin cDNA from a human myeloid library and expression of mRNA during normal and leukemic myelopoiesis

    Blood

    (1987)
  • H Saito et al.

    Potent bactericidal activity of bovine lactoferrin hydrolysate produced by heat treatment at acidic pH

    J. Dairy Sci.

    (1991)
  • D.J Schibli et al.

    The structure of the antimicrobial active centre of lactoferricin B bound to sodium dodecyl sulfate micelles

    FEBS Lett.

    (1999)
  • Y Shai

    Mechanism of the binding, insertion and destabilization of the phospholipid bilayer membrane by α-helical antimicrobial and cell non-selective membrane-lytic peptides

    Biochim. Biophys. Acta

    (1999)
  • J Tachezy et al.

    Trichomonus foetus: iron acquisition from lactoferrin and transferrin

    Exp. Parasitol.

    (1996)
  • J Williams

    The evolution of transferrins

    TIBS

    (1982)
  • G.F Ames et al.

    Protein composition of the outer membrane of Salmonella typhimurium: effect of lipopolysaccharide mutations

    J. Bacteriol.

    (1974)
  • B.F Anderson et al.

    Structure of human lactoferrin at 3.2-Å resolution

    Proc. Natl. Acad. Sci. U.S.A.

    (1987)
  • B.J Appelmelk et al.

    Lactoferrin is a lipid A-binding protein

    Infect. Immun.

    (1994)
  • R.R Arnold et al.

    A bactericidal effect for human lactoferrin

    Science (Washington, DC)

    (1977)
  • R.R Arnold et al.

    Bactericidal activity of human lactoferrin: sensitivity of a variety of microorganisms

    Infect. Immun.

    (1980)
  • R.R Arnold et al.

    Bactericidal activity of human lactoferrin: influence of physical conditions and metabolic state of the target microorganism

    Infect. Immun.

    (1981)
  • E.N Baker et al.

    Lactoferrin and transferrin: functional variations on a common structural framework

    Biochem. Cell Biol.

    (2002)
  • W Bellamy et al.

    Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin

    J. Appl. Bacteriol.

    (1992)
  • W.R Bellamy et al.

    Role of cell-binding in the antibacterial mechanism of lactoferricin B

    J. Appl. Microbiol.

    (1993)
  • C Bevins

    Antimicrobial peptides as agents of mucosal immunity

    Ciba Found. Symp.

    (1994)
  • S.E Blondelle et al.

    The antimicrobial activity of hexapeptides derived from synthetic combinatorial libraries

    J. Appl. Bacteriol.

    (1995)
  • H.G Boman

    Peptide antibiotics and their role in innate immmunity

    Ann. Rev. Immun.

    (1995)
  • C.A Bortner et al.

    Bactericidal effect of lactoferrin on Legionella pneumophila

    Infect. Immun.

    (1986)
  • C.A Bortner et al.

    Bactericidal effect of lactoferrin on Legionella pneumophila: effect of the physiological state of the organism

    Can. J. Microbiol.

    (1989)
  • J.H Brock

    The physiology of lactoferrin

    Biochem. Cell Biol.

    (2002)
  • D.S Chapple et al.

    Structure-function relationship of antibacterial synthetic peptides homologous to a helical surface region on human lactoferrin against Escherichia coli serotype O111

    Infect. Immun.

    (1998)
  • O Cirioni et al.

    Inhibition of growth of Pneumocystis carinii by lactoferrins alone and in combination with pyrimethamine, clarithromycin and minocycline

    J. Antimicrob. Chemother.

    (2000)
  • E Elass-Rochard et al.

    Lactoferrin-lipopolysaccharide interaction: involvement of the 28–34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide

    Biochem. J.

    (1995)
  • R.T Ellison et al.

    Killing of Gram negative bacteria by lactoferin and lysozyme

    J. Clin. Invest.

    (1991)
  • R.T Ellison et al.

    Damage of the outer membrane of enteric Gram-negative bacteria by lactoferrin and transferrin

    Infect. Immun.

    (1988)
  • R.T Ellison et al.

    Lactoferrin and transferrin damage of the Gram-negative outer membrane is modulated by Ca2+ and Mg2+

    J. Gen. Microbiol.

    (1990)
  • J.J Esposito et al.

    Enterovirus type 70 virion and intracellular proteins

    J. Virol.

    (1976)
  • R.W Evans et al.

    Transferrin-mediated iron acquisition by pathogenic Neisseria

    Biochem. Soc. Trans.

    (2002)
  • E Gazit et al.

    Mode of action of the antimicrobial cecropin B2: a spectrofluorometric study

    Biochemistry

    (1994)
  • G.C Gonzales et al.

    Identification and characterisation of a porcine-specific transferring receptor in Actinobacillus pleuropneumoniae

    Mol. Microbiol.

    (1990)
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