Pauline P. Ward

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.

Lactoferrin: Role in iron homeostasis and host defense against microbial infection

The transferrin family of non-heme iron binding glycoproteins are believed to play a central role in iron metabolism and have been implicated in iron transport, cellular iron delivery and control of the level of free iron in external secretions. Lactoferrin (LF) is a member of this family that is widely localized in external fluids including milk and mucosal secretions, in addition to being a prominent component of the secondary granules of neutrophils. Although structurally related to transferrin, LF appears to have a broader functional role mediated by both iron dependent and iron independent mechanisms. In this review, we will focus on our current understanding on the role of LF in regulating iron homeostasis and its role in host protection against microbial infection at the mucosal surface. In addition, recent insights obtained from analyzing the phenotypic consequences of LF ablation in lactoferrin knockout mice (LFKO), which challenge the long held dogma that LF is required for intestinal iron absorption in the neonate, are summarized.

Iron Status in Mice Carrying a Targeted Disruption of Lactoferrin

ABSTRACT Lactoferrin is a member of the transferrin family of iron-binding glycoproteins present in milk, mucosal secretions, and the secondary granules of neutrophils. While several physiological functions have been proposed for lactoferrin, including the regulation of intestinal iron uptake, the exact function of this protein in vivo remains to be established. To directly assess the physiological functions of lactoferrin, we have generated lactoferrin knockout (LFKO−/−) mice by homologous gene targeting. LFKO−/− mice are viable and fertile, develop normally, and display no overt abnormalities. A comparison of the iron status of suckling offspring from LFKO−/− intercrosses and from wild-type (WT) intercrosses showed that lactoferrin is not essential for iron delivery during the postnatal period. Further, analysis of adult mice on a basal or a high-iron diet revealed no differences in transferrin saturation or tissue iron stores between WT and LFKO−/− mice on either diet, although the serum iron levels were slightly elevated in LFKO-/- mice on the basal diet. Consistent with the relatively normal iron status, in situ hybridization analysis demonstrated that lactoferrin is not expressed in the postnatal or adult intestine. Collectively, these results support the conclusion that lactoferrin does not play a major role in the regulation of iron homeostasis.

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‐α (TNF‐α), 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‐α, 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‐α synthesis was suggested by the findings that first, LF was unable to inhibit LC migration induced by intradermal injection of TNF‐α itself, and second, that migration stimulated by local administration of another epidermal cytokine, interleukin 1β, which is also dependent upon TNF‐α 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.

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.

Stimulus-Dependent Impairment of the Neutrophil Oxidative Burst Response in Lactoferrin-Deficient Mice

Lactoferrin (LF) is an iron-binding protein found in milk, mucosal secretions, and the secondary granules of neutrophils in which it is considered to be an important factor in the innate immune response against microbial infections. Moreover, LF deficiency in the secondary granules of neutrophils has long been speculated to contribute directly to the hypersusceptibility of specific granule deficiency (SGD) patients to severe, life-threatening bacterial infections. However, the exact physiological significance of LF in neutrophil-mediated host defense mechanisms remains controversial and has not yet been clearly established in vivo using relevant animal models. In this study, we used lactoferrin knockout (LFKO) mice to directly address the selective role of LF in the host defense response of neutrophils and to determine its contribution, if any, to the phenotype of SGD. Neutrophil maturation, migration, phagocytosis, granule release, and antimicrobial response to bacterial challenge were unaffected in LFKO mice. Interestingly, a stimulus-dependent defect in the oxidative burst response of LFKO neutrophils was observed in that normal activation was seen in response to opsonized bacteria whereas an impaired response was evident after phorbol myristate-13-acetate stimulation. Taken together, these results indicate that although LF deficiency alone is not a primary cause of the defects associated with SGD, this protein does play an immunomodulatory role in the oxidative burst response of neutrophils.

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

SummaryFour acid phosphatase (phosphomonoesterase E.C.3.1.3.2) genes, werecloned by polymerase chain reaction (PCR). These were pho3, pho5 and pho11 fromSaccharomyces cerevisiae and the gene for a phosphate-respressible acid phosphatase fromAspergillus niger. The individual genes were subcloned into anA. oryzae expression vector downstream from a starch-inducible α-amylase promoter and the resulting expression constructs were transformed into a mutant strain ofA. 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 fermentationmedia. 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, untransformedA. oryzae. Sufficient quantities ofA. 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. Data indicated an increase in available phosphorus of 1 g kg−1 obtained with yeast acid phosphatase andA. niger acid phosphatase representing 40% utilization of unavailable dietary P compared to 48% utilization for commercial phytase.

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.

Regulation by Lactoferrin of Epidermal Langerhans Cell Migration

Lactoferrin is an iron-binding glycoprotein that shares structural homology with transferrin and which is found in milk, other epithelial secretions and the secondary granules of neutrophils1. One function ascribed to lactoferrin is that of antibacterial activity which is effected primarily through the sequestration of iron necessary for microbial growth1,2. It is recognized, however, that in addition to its bacteriostatic properties, lactoferrin may serve as a modulator of immune and inflammatory responses. Of particular interest is the observation that this glycoprotein may influence the production of some cytokines, including proinflammatory cytokines 1–3. Among the cytokines shown to be regulated negatively by lactoferrin are tumour necrosis factor a (TNF-α), interleukin 1β (IL-1β) and granulocyte/macrophage colony-stimulating factor (GM-CSF)2–4. It has been demonstrated that (bovine) lactoferrin administered intravenously to mice prior to an injection of bacterial lipopolysaccharide (LPS) inhibited significantly the production of TNF-α normally induced by LPS5. It has been argued, however, that the ability of lactoferrin to inhibit the stimulation of TNF-α by LPS may be secondary to its capacity to bind LPS and thereby compromise the signal for cytokine production3.

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.