Multifunctional roles of lactoferrin: a critical overview.

Lactoferrin (LF) is a member of the transferrin family that is expressed and secreted by glandular epithelial cells and is found in the secondary granules of neutrophils. Originally viewed as an iron-binding protein in milk, with bacteriostatic properties, it is becoming increasingly evident that LF is a multifunctional protein to which several physiological roles have been attributed. These include regulation of iron homeostasis, host defense against a broad range of microbial infections, anti-inflammatory activity, regulation of cellular growth and differentiation and protection against cancer development and metastasis. While iron binding is likely central to some of the biological roles of LF, other activities, including specific interactions with mammalian receptors and microbial components, also contribute to the pleoitropic functional nature of this protein. In this article, recent advances in the understanding of these functions at the cellular and molecular level are discussed.

Regulation of epidermal Langerhans cell migration by lactoferrin.

Lactoferrin (LF) is a member of the transferrin family of iron-binding glycoproteins to which several anti-inflammatory functions have been ascribed. LF has been shown to down-regulate expression of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha), although the possibility has been raised that the activity of LF in this regard was indirect and secondary to its ability to bind to and inactivate the bacterial lipopolysaccharide (LPS) used to induce cytokine production. However, the identification of putative membrane receptors for LF raises the possibility that the interaction of LF with its receptor may be one important route through which this protein exerts anti-inflammatory activity. In the present investigations the biological properties of LF have been examined in a model of cutaneous immune function where the allergen-induced migration of epidermal Langerhans cells (LC) from the skin and their subsequent accumulation as dendritic cells (DC) in skin-draining lymph nodes are known to be dependent upon the de novo synthesis of TNF-alpha, but independent of exogenous LPS. Consistent with the protein having direct anti-inflammatory properties, it was found that the intradermal injection of recombinant murine LF (either iron-saturated or iron-depleted LF) inhibited significantly allergen (oxazolone) -induced LC migration and DC accumulation. That these inhibitory effects were secondary to the inhibition of local TNF-alpha synthesis was suggested by the findings that first, LF was unable to inhibit LC migration induced by intradermal injection of TNF-alpha itself, and second, that migration stimulated by local administration of another epidermal cytokine, interleukin 1beta, which is also dependent upon TNF-alpha production, was impaired significantly by prior treatment with LF. Finally, immunohistochemical analyses demonstrated the presence of LF in skin, associated primarily with keratinocytes. Collectively these data support the possession by LF of direct immunomodulatory and/or anti-inflammatory activity, probably associated in this case with inhibition of cytokine production. Furthermore, the results suggest that as a constituent of normal skin, LF may play a role in homeostatic regulation of cutaneous immune function.

Restricted spatiotemporal expression of lactoferrin during murine embryonic development.

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins. Lactoferrin is induced by estrogen in the mouse uterus during early pregnancy. However, the expression and function, if any, of lactoferrin in the preimplantation embryo during this developmental period has not been investigated. In the current study, the spatiotemporal expression of lactoferrin during murine embryogenesis was examined using in situ hybridization and immunohistochemical analyses. Lactoferrin expression was first detected in the 2-4 cell fertilized embryo and continued until the blastocyst stage of development. Interestingly, at the 16-cell stage, coinciding with the first major differentiation step in the embryo, lactoferrin messenger RNA (mRNA) is synthesized by the inner cells, whereas the protein is selectively taken up by the outside cells. This differential pattern of lactoferrin messenger RNA and protein localization continues until the blastocyst stage, with expression almost absent in the hatched blastocyst. Lactoferrin expression does not resume in the embryo until the latter half of gestation, where it is first detected in neutrophils of the fetal liver at embryonic day 11.5 and later in epithelial cells of the respiratory and digestive systems. Our results show that lactoferrin is expressed in a tightly regulated spatiotemporal manner during murine embryogenesis and suggest a novel paracrine role for this protein in the development of the trophoectodermal lineage during preimplantation development.

Restricted spatiotemporal expression of lactoferrin during murine embryogenesis.

Lactoferrin is a member of the transferrin family of iron-binding proteins to which several physiological functions have been ascribed. While there is a wealth of evidence about the distribution and function of this protein in the adult, the expression and function, if any, of lactoferrin during embryogenesis has not been investigated. In the current study, the spatiotemporal distribution of lactoferrin was analyzed during normal murine embryonic development. This analysis demonstrated that lactoferrin is expressed in three distinct patterns during embryogenesis. First, lactoferrin is expressed at the 2-cell stage in the preimplantation embryo where it continues to be expressed until the blastocyst stage when expression ceases. The second phase of lactoferrin expression is not detected until the latter half of gestation when the protein is detected in the myeloid cells, beginning in the fetal liver at embryonic day 11 and later in the spleen and bone marrow coinciding with the onset and diversification of myelopoiesis in these organs during embryogenesis. Finally, lactoferrin is detected in a variety of glandular epithelial cells and/or their secretions, including respiratory and oral epithelia which is consistent with the expression pattern observed for this protein in the adult where it plays an important role in host defense at the mucosal surface. Taken together, these analyses indicate that the role of lactoferrin in the developing embryo is restricted to the preimplantation stage and development of first and second line host defense systems.

Expression and characterization of recombinant murine lactoferrin.

Lactoferrin (Mw=78 kDa) is a member of the transferrin family of iron-binding glycoproteins. Previous studies carried out primarily in vitro indicate that the protein has multifunctional properties and may be involved in regulation of iron homeostasis, inhibition of bacterial growth and regulation of immune responses. However, the significance and species specificity of these proposed functions in vivo have not been adequately addressed due to lack of sufficient purified homospecies lactoferrin for analysis in small animal models. We previously reported the successful production of biologically active recombinant human lactoferrin using an Aspergillus expression system. In the present study, we report the production of recombinant murine lactoferrin using a similar expression strategy. Recombinant murine lactoferrin was purified to homogeneity and was similar in size and immunoreactivity to native murine milk lactoferrin. The recombinant protein was correctly processed at its N-terminus and was glycosylated. Interestingly, while both human and murine lactoferrin bind iron in a 2:1 molar ratio, iron bound to recombinant murine lactoferrin was more acid labile than human lactoferrin, demonstrating species-specific variation in the stability of iron-binding to this protein. Finally, the availability of recombinant murine lactoferrin will now facilitate the study of the species specificity of lactoferrin action in a mouse model system.

Direct activity of recombinant human lactoferrin against Helicobacter pylori.

We report the activity of recombinant human lactoferrin against Helicobacter pylori. Lactoferrin exerted a time- and dose-dependent action against 8 of the 13 clinical isolates of H. pylori tested in vitro. These results highlight a potential therapeutic use for lactoferrin against H.pylori infection.

Cooperative interactions between the amino- and carboxyl-terminal lobes contribute to the unique iron-binding stability of lactoferrin.

Lactoferrin is a member of the transferrin family of iron-binding proteins. Several functions have been ascribed to lactoferrin, including regulation of iron homeostasis, antibacterial properties, and regulation of myelopoiesis. However, the structural features of lactoferrin that are required for most of these functions are unknown. Previously, we reported the development of an efficient fungal expression system to produce recombinant human lactoferrin. The availability of this production system demonstrated the feasibility of producing mutant lactoferrins to address the structure/function relationship of the protein. In the present study, we used a site-directed mutagenesis approach to address the contribution of the bilobal structure of lactoferrin to its unique iron-binding stability. Like transferrin, lactoferrin consists of two repeated iron-binding lobes that bind one iron atom each. However, unlike transferrin, lactoferrin retains iron over a broad pH range, a key property that contributes to the unique iron-binding functions of the protein. Using mutants that selectively ablate the iron-binding function in either lobe, we demonstrate differential iron-binding stability of the amino- and carboxyl-terminal iron-binding lobes of lactoferrin. Further, we show that the unique iron-binding stability of the protein is imparted primarily by the carboxyl-terminal domain which functions cooperatively to stabilize iron-binding to the amino-terminal domain of lactoferrin.

Molecular cloning, expression and evaluation of phosphohydrolases for phytate-degrading activity.

Four acid phosphatase (phosphomonoesterase E.C.3.1.3.2) genes were cloned by polymerase chain reaction (PCR). These were pho3, pho5 and pho11 from Saccharomyces cerevisiae and the gene for a phosphate-respressible acid phosphatase from Aspergillus niger. The individual genes were subcloned into an A. oryzae expression vector downstream from a starch-inducible alpha-amylase promoter and the resulting expression constructs were transformed into a mutant strain of A. oryzae, AO7. Southern hybridization analysis confirmed that the acid phosphatase genes had been integrated into the host genome with estimates of integrated copy numbers ranging from 2 to 20 for individual transformants. Northern hybridization analysis of total RNA from individual transformants revealed the presence of a single transcript of the expected size of 1.8 kb. Production of recombinant protein was induced by the addition of 30 g L-1 of soluble starch in the fermentation media. Active acid phosphatases, not present in control cultures, were detected in the supernatant fractions of transformant cultures by acid phosphatase activity staining of non-denaturing polyacrylamide gels. The ability of the recombinant acid phosphatases to hydrolyze phytate was assessed by referenced phytase (myoinositol hexakisphosphate phosphohydrolase E.C. 3.1.3.8) activity assay procedures. A two- to six-fold increase in phytase activity was measured in transformants compared to control, untransformed A. oryzae. Sufficient quantities of A. niger and pho5 recombinant acid phosphatases were generated from large-scale fermentations to assess the efficacy of these enzymes as phytate-degrading enzymes when included in poultry diets.(ABSTRACT TRUNCATED AT 250 WORDS)

A system for production of commercial quantities of human lactoferrin: a broad spectrum natural antibiotic.

We previously reported the production of limited quantities of biologically active recombinant human lactoferrin in the filamentous fungus Aspergillus oryzae. In the present study, we report a modification of this production system combined with a classical strain improvement program that has enabled production of levels of recombinant human lactoferrin in excess of 2 g/l. The protein was expressed in Aspergillus awamori as a glucoamylase fusion polypeptide which was secreted into the growth medium and processed to mature human lactoferrin by an endogenous KEX-2 peptidase. The recombinant protein retains full biological activity in terms of its ability to bind iron and human enterocyte receptors. Furthermore, the recombinant protein functions as a potent broad spectrum antimicrobial protein.

An inducible expression system for the production of human lactoferrin in Aspergillus nidulans.

The production and secretion of human lactoferrin (hLF) in Aspergillus nidulans is described. The hLF cDNA was expressed under the control of the strong ethanol-inducible alcohol dehydrogenase (alcA) promoter. Recombinant hLF (re-hLF) is produced at levels up to 5 micrograms/ml. Approximately 30% of the re-hLF produced in this system is secreted into the growth medium. The re-hLF is indistinguishable from native hLF with respect to size and immunoreactivity. Furthermore, re-hLF is functional by the criterion of iron-binding capacity. The A. nidulans expression system offers an inexpensive, convenient method for the controlled production of mg amounts of biologically active mammalian glycoproteins.

Production of biologically active recombinant human lactoferrin in Aspergillus oryzae.

We report the production of recombinant human lactoferrin in Aspergillus oryzae. Expression of human lactoferrin (hLF), a 78 kD glycoprotein, was achieved by placing the cDNA under the control of the A. oryzae alpha-amylase promoter and the 3' flanking region of the A. niger glucoamylase gene. Using this system, hLF is expressed and secreted into the growth medium at levels up to 25 mg/l. The recombinant lactoferrin is indistinguishable from human milk lactoferrin with respect to its size, immunoreactivity, and iron-binding capacity. The recombinant protein appears to be appropriately N-linked glycosylated and correctly processed at the N-terminus by the A. oryzae secretory apparatus. Lactoferrin is the largest heterologous protein and the first mammalian glycoprotein expressed in the Aspergillus system to date. Hence, this expression system appears suitable for the large-scale production and secretion of biologically active mammalian glycoproteins.

A mixed carcinoid tumour of the middle ear.

Carcinoid tumours belong to the rarest neoplasms of the middle ear. Murphy et al. (1980) described an interesting carcinoid tumour of the middle ear displaying adenomatous features. The histological similarity of carcinoid tumours to adenomatous tumours of the middle ear has also been noted by Fayemi and Toker (1975). We describe a similar case which caused certain diagnostic difficulties. Light microscopy on frozen sections favoured the diagnosis of a low-grade adenocarcinoma but electron microscopy has revealed the presence of many intracellular membrane-bound neurosecretory granules and the diagnosis was revised to that of a mixed carcinoid tumour.