ReviewLactoferrin—a multifunctional protein with antimicrobial properties
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.
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