Separation and Characterization of the C-Terminal Half Molecule of Bovine Lactoferrin

Shimazaki, K.; Tanaka, Tetsuya; Kon, Hiroshi; Oota, Kazunary; Kawaguchi, Ayano; Maki, Yoshiyuki; Sato, Tetsuya; Ueda, Yasuyuki; Tomimura, Toshio; Shimamura, Seiichi

doi:10.3168/jds.s0022-0302(93)77421-4

Cited by 28 publications

(23 citation statements)

References 22 publications

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“…The nonequivalence of iron binding to the amino-and carboxyl-terminal lobes of lactoferrin has been reported previously (36,41,42). Studies using a cloned NH 2 -terminal fragment of human lactoferrin (41) and a proteolytically derived COOHterminal fragment from bovine lactoferrin (42) have shown a similar disparity in pH dependence of iron binding as reported in this study.…”

Section: Discussionsupporting

confidence: 87%

Cooperative Interactions between the Amino- and Carboxyl-terminal Lobes Contribute to the Unique Iron-binding Stability of Lactoferrin

Ward

Zhou

Conneely

1996

Journal of Biological Chemistry

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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 aminoand carboxyl-terminal iron-binding lobes of lactoferrin. Further, we show that the unique iron-binding stability of the protein is imparted primarily by the carboxylterminal domain which functions cooperatively to stabilize iron-binding to the amino-terminal domain of lactoferrin.Lactoferrin is a member of the transferrin family of nonheme iron-binding glycoproteins (1) which includes transferrin, the major iron-transport protein in blood (2), ovotransferrin, an avian egg white protein (3), and melanotransferrin, a membrane bound form of this family found in human melanocytes (4). Lactoferrin has a broad distribution, present in both external secretions that bathe the body surfaces (5-9) and also in the secondary granules of polymorphonuclear neutrophils where it can be released into the bloodstream upon neutrophil activation (10). The functions proposed for this protein include iron binding and delivery to the small intestine (11-17), antimicrobial activity against a wide range of Gram-negative and Grampositive bacteria (18 -22), cellular growth promotion (23, 24), regulation of myelopoiesis (25-27), and immunomodulatory properties (28 -30).Lactoferrin shares a high degree of structural homology with other members of the transferrin family. All of these proteins are monomeric glycoproteins with a molecular mass of ϳ80 kDa (1, 31). The three-dimensional structure of lactoferrin (32) and transferrin (33) have been precisely defined by x-ray crystallographic analysis. The proteins are folded into two globular lobes corresponding to the amino-and carboxyl-terminal halves of the protein. This bilobal structure, with ϳ40% conservation between the NH 2 -and COOH-terminal halves, is thought to have evolved by intragenic duplication from a common ancestral gene (34). Each of these lobes can reversibly bind iron with high affinity and w...

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Section: Discussionsupporting

confidence: 87%

Cooperative Interactions between the Amino- and Carboxyl-terminal Lobes Contribute to the Unique Iron-binding Stability of Lactoferrin

Ward

Zhou

Conneely

1996

Journal of Biological Chemistry

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“…on immune cells will probably exist in cats and may act as an immunomodurator in feline immune system. In present study, the binding of bLF to ConA could partly explain its inhibitory effect on the ConA-induced cytokine expressions [29]. However, our results showed that bLF inhibited expression of ConA-induced IFN-γ and IL-2 regardless of the time of addition of bLF to the cells.…”

Section: Cycle Numbercontrasting

confidence: 55%

Effect of Bovine Lactoferrin on Functions of Activated Feline Peripheral Blood Mononuclear Cells During Chronic Feline Immunodeficiency Virus Infection

et al. 2008

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ABSTRACT. Feline immunodeficiency virus (FIV) infection is characterized by chronic overactivation of immune and inflammatory system, resulting in anergic state and dysfunction of immune cells. Lactoferrin (LF), a glycoprotein present in exocrine secretions and neutrophils, plays an important role in host defense system. Our previous study showed that oral administration of bovine LF (bLF) suppressed oral inflammation, improved the clinical symptoms and decreased serum γ-globulin as a marker of inflammation in FIV-infected cats with intractable stomatitis. The anti-inflammatory effect was partly involved in regulation of neutrophil function by bLF. In this study, to clarify the relationship between anti-inflammatory effects of bLF and peripheral blood mononuclear cells (PBMC), we examined the effect of bLF on proliferation, cell cycle progression and cytokine expression in mitogen-activated PBMC. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay showed that bLF inhibited the concanavalin A (ConA)-induced cell proliferation in FIVinfected cats with the asymptomatic carrier and AIDS-related complex (ARC) phase. Bovine LF restored ConA-induced cell cycle progression and resulted in suppression of the induced apoptosis in feline PBMC. Real-time RT-PCR showed that bLF suppressed ConAinduced expression of interferon-gamma and interleukin-2 in cells of the ARC group regardless of the time of its addition to the medium. These results suggest the hypothesis that therapy with bLF may have the potential to improve and protect functions of overactivated lymphocytes by modulating the cell proliferation, cell cycle and cytokines expression in cats in terminal stage of FIV infection. KEY WORDS: anti-inflammatory activity, bovine lactoferrin, FIV-infected cat, persistent immune activation.

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“…Apoprotein residual iron charge was checked by absorption spectroscopy, where the ratio of the absorbance at 465 nm was 0.9% that at 280 nm, indicating a lack of iron in the protein (Ainscough, 1980;Chung and Raymond, 1993). Protein purity was confirmed by the absence of an absorbance peak at 412 nm, which is the indicator of a haemopexin impurity (Shimazaki et al, 1993). Purity and iron load were further checked by uredpol yacrylamide gel electrophoresis according to Makey and Seal (1976).…”

Section: Methodsmentioning

confidence: 99%

Transferrin

Pakdaman

Chahine

1997

European Journal of Biochemistry

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The interaction of apolactoferrin with hydrogen carbonate (bicarbonate) has been investigated in the pH range 6.5-9.2. In the absence of bicarbonate apolactoferrin loses a single proton with pK,, of 8.10. This proton loss is independent of the interaction with the synergistic anion. The C-site of apolactoferrin interacts with bicarbonate with a very low affinity (K;' = 3.2 M-'). This process is accompanied by a proton loss, which is probably provided by the bicarbonate in interaction with the protein. This proton loss can possibly be the result of a shift in the proton dissociation constant, pK,, of the bicarbonate/ carbonate acid/base equilibrium, which would decrease from pK, 10.35 to pK,, 6.90 in the bicarbonatelactoferrin adduct. The N-site of the protein interacts with bicarbonate with an extremely low affinity, which excludes the presence of the N-site-synergistic anion adduct in neutral physiological media. Contrary to serum transferrin, the concentration of the apolactoferrin in interaction with bicarbonate is pH dependent. Between pH 7.4 and pH 9 with [HCO,] about 20 mM, the concentration of the serum transferrin-bicarbonate adduct is always about 30 %, whereas that of the apolactoferrin-synergistic anion adduct varies from 25% at pH 7.5 to 90% at pH 9. This implies that, despite an affinity for bicarbonate two orders of magnitude lower than that of serum transferrin, lactoferrin interacts better with the synergistic anion. This can be explained by the possible interaction of lactoferrin with carbonate in neutral media, whereas transferrin only interacts with bicarbonate.Keywords; transferrin ; lactoferrin; synergistic anion ; iron metabolism; iron transport.The transferrin family constitutes the most important irontransport system in vertebrates and in some invertebrates, mainly worms and insects (Aisen, 1989;Kurama et al., 1995). These proteins transport iron from the biological fluid into the cytoplasm via the plasma membrane by receptor-mediated endocytosis. With the exception of melanotransferrin, which is overexpressed in melanocytes, the other transferrins of the family are soluble glycoproteins (Baker and Lindley, 1992). These are represented by serum transferrin, responsible for iron transport in mammals, egg white ovotransferrin and milk lactoferrin, which is also present in tears, genital secretion, seminal fluid, sweat, etc. (Aisen, 1989). In addition to their iron-transport function, some transferrins, such as lactoferrins, are involved in the immune response and are bactericidal and bacteriostatic (FurmanCorrespondence to J

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Separation and Characterization of the C-Terminal Half Molecule of Bovine Lactoferrin

Cited by 28 publications

References 22 publications

Cooperative Interactions between the Amino- and Carboxyl-terminal Lobes Contribute to the Unique Iron-binding Stability of Lactoferrin

Cooperative Interactions between the Amino- and Carboxyl-terminal Lobes Contribute to the Unique Iron-binding Stability of Lactoferrin

Effect of Bovine Lactoferrin on Functions of Activated Feline Peripheral Blood Mononuclear Cells During Chronic Feline Immunodeficiency Virus Infection

Transferrin

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