The protein structure of recombinant human lactoferrin produced in the milk of transgenic cows closely matches the structure of human milk-derived lactoferrin

Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the host defence against infection and excessive inflammation. As the availability of (human milk-derived) natural hLF is limited, alternative means of production of this biopharmaceutical are extensively researched. Here we report the crystal structure of recombinant hLF (rhLF) expressed in the milk of transgenic cows at a resolution of 2.4 Å. To our knowledge, the first reported structure of a recombinant protein produced in milk of transgenic livestock. Even though rhLF contains oligomannose- and hybrid-type N-linked glycans next to complex-type glycans, which are the only glycans found on natural hLF, the structures are identical within the experimental error (r.m.s. deviation of only 0.28 Å for the main-chain atoms). Of the differences in polymorphic amino acids between the natural and rhLF variant used, only the side-chain of Asp⁵⁶¹ could be modeled into the rhLF electron density map. Taken together, the results confirm the structural integrity of the rhLF variant used in this study. It also confirms the validity of the transgenic cow mammary gland as a vehicle to produce recombinant human proteins.Ellen A. J. Thomassen, Harrie A. van Veen - These authors have contributed equally to this paper.The PDB-code of recombinant human lactoferrin is 2BJJ     

Effect of lactoferrin on the growth of a human colon adenocarcinoma cell line-comparison with transferrin

Summary Lactoferrin was examined for its effect on the growth of a human colon adenocarcinoma cell line (HT 29) in culture and its action was compared to that produced by transferrin and two different iron solutions (ferrous sulfate and ferric chloride). When transferrin was replaced by either iron solutions the cell grew in proportion to the quantity added and the maximal effect obtained was identical to that produced by transferrin alone. When transferrin was replaced by lactoferrin the cells were unable to proliferate for a long time. However, in the presence of low-concentration iron solutions, lactoferrin stimulated the cell growth, and the effect was more pronounced with the ferric chloride solution. This work was supported by Grants Inserum 817014 and LA CNRS 202.     

Studies on γ Globulin of Rice Embryo

An improved method has been described for the isolation and purification of γ globulin from rice embryo. The method involves the extraction with phosphate buffer, pH 7.0 and ionic strength 0.1, the fractionation in saline solution of ionic strength 0.31, the removal of nucleic acids by precipitation with ammonium sulfate and the gel filtration chromatography on a Sephadex G-200 column. Although the preparations exhibited homogeneous patterns in sedimentation analysis, the electrophoretic patterns on polyacrylamide gels at pH 8.35 and ionic strength 0.11 exhibited at least two components. Three major components, γ₁, γ₂ and γ₃ globulins, were isolated by ion exchange chromatography on a DEAE Sephadex A-50 column. These components were revealed to be homogeneous in electrophoresis as well as sedimentation. N-Terminal amino acid compositions have also been described.

The molecular weight of γ₁ globulin was determined as 2.0 × 10⁵ by the Archibald method, and the intrinsic viscosity, [η], and the sedimentation coefficient, s_{20, w}^°, were found to be 0.0424 dl/g and 7.26S respectively. These values indicated the large asymmetry of the protein. The protein was composed of 18 residues of hexose, 3 residues of pentose, 6 residues of hexosamine and 1751 residues of amino acids: Lys₅₈, His₄₇, Arg₁₄₈, Asp₁₂₆, Glu₂₇₃, Gly₁₆₁, Ala₁₄₄, Val₁₂₁, Leu₁₀₆, Ile₇₂, Pro₈₃, Ser₁₃₆, Thr₄₈, Hyp₆₈, Cys₁₇, Met₁₆, Tyr₄₄, Trp₈, Phe₇₅ and amide ammonia₁₆₃. The N-terminal amino acid analysis suggested that the protein was composed of ten subunits. The properties and the composition were discussed in comparison with those of the 7S globulin of soybean cotyledon.     

Effect of Lactoferrin on the Ferroxidase Activity of Ceruloplasmin

The effects of various forms of lactoferrin (Lf) interacting with ceruloplasmin (Cp, ferro-O2-oxidoreductase, EC 1.16.3.1) on oxidase activity of the latter were studied. Comparing the incorporation of Fe3+ oxidized by Cp into Lf and serum transferrin (Tf) showed that at pH 5.5 apo-Lf binds the oxidized iron seven times and at pH 7.4 four times faster than apo-Tf under the same conditions. Apo-Lf increased the oxidation rate of Fe2+ by Cp 1.25 times when Cp/Lf ratio was 1 : 1. Lf saturated with Fe3+ or Cu2+ increased the oxidation rate of iron 1.6 and 2 times when Cp to holo-Lf ratios were 1 : 1 and 1 : 2, respectively. Upon adding to Cp the excess amounts of apo-Lf (Cp/apo-Lf < 1 : 1) or of holo-Lf (Cp/holo-Lf < 1 : 2) the oxidation rate of iron no longer changed. Complex Cp-Lf demonstrating ferroxidase activity was discovered in breast milk.     

A longitudinal study of unsaturated iron-binding capacity and lactoferrin in unstimulated parotid saliva

Availability of iron is one important nutritional parameter for microbial growth in saliva. This longitudinal study measured the diurnal and day-to-day variations in the total iron (TI), total ironbinding capacity (TIBC), unsaturated iron-binding capacity (UIBC), and lactoferrin (LF) in unstimulated human parotid saliva. Saliva was collected from 15 young male subjects in the morning and afternoon hours each day for five consecutive days. The TI and TIBC were determined by flameless atomic absorption spectroscopy, and UIBC was determined by subtraction of TI from TIBC. The LF was determined by “sandwich” enzyme-linked immunosorbent assay (ELISA). One peripheral blood sample of each subject was also analyzed for TI, TIBC, and ferritin. The results showed no significant diurnal or day-to-day variation of TI, TIBC, UIBC, or LF in saliva for most subjects. However, significant between-subject variations were observed for most parameters. Variations ranged from subjects with constantly positive UIBC values to subjects with constantly negative UIBC values. The relationship between the LF values and the TI and TIBC values suggests that other iron-binding protein(s) are present in saliva. Also, saliva had significantly lower TIBC values than serum. This finding indicated that iron may be easily available in saliva. However, further studies are required to determine the relationship between UIBC value of saliva and oral and dental diseases, and also to detect the presence of other iron-binding proteins in saliva.     

The synergistic effect of nisin and lactoferrin on the inhibition of Listeria monocytogenes and Escherichia coli O157:H7

AIMS: The goal of this study was to determine whether nisin and lactoferrin would act synergistically to inhibit the growth of Listeria monocytogenes and Escherichia coli O157:H7. METHODS AND RESULTS: Lactoferrin and nisin separately or in combination were suspended in peptone yeast glucose broth and following inoculation with L. monocytogenes or E. coli O157:H7 growth inhibition of each pathogen was determined. At 1000 microg ml(-1) lactoferrin L. monocytogenes was effectively inhibited. However, E. coli O157:H7 initially was inhibited and then grew to cell density similar to the control. A combination of 500 microg ml(-1) of lactoferrin and 250 IU ml(-1) of nisin effectively inhibited the growth of E. coli O157:H7, whereas, 250 microg ml(-1) of lactoferrin and 10 IU ml(-1) of nisin were inhibitory to L. monocytogenes. CONCLUSIONS: The results suggest that lactoferrin and nisin act synergistically to inhibit the growth of L. monocytogenes and E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural preservatives that are active against gram-positive and gram-negative pathogens are desirable to the food industry and consumers. This study demonstrates that lactoferrin and nisin work synergistically reducing the levels required independently inhibiting growth of two major foodborne pathogens. Previous reported results indicated a low level of antimicrobial activity; however, this work was not performed in low divalent cation concentration media. It has been suggested that nondivalent cation-limiting medium such as trypticase soy broth (TSB), can reduce or completely eliminate the inhibitory activity. Further knowledge of these interactions can increase the understanding of the antimicrobial activity of lactoferrin. This should make the use of these compounds by industry more attractive.     

Polymorphic sequence of Korean Native goat lactoferrin exhibiting greater antibacterial activity

Lactoferrin, which exhibits antibacterial activity to protect infants from infectious disease, is a major component of colostrum and milk. Lactoferrin was purified from the colostrum of Korean Native goat, and the cDNA from the mammary gland mRNA of the animal was cloned and sequenced. The nucleotide sequence of the lactoferrin gene of Korean Native goat was found to differ in 15 sites from that of the goat lactoferrin gene reported earlier. This difference in nucleotide sequence resulted in six amino acid substitutions: five in the N-lobe and one in the C-lobe. The antibacterial activity of Korean Native goat lactoferrin was found to be greater than that of Sannen goat lactoferrin.     

Cytokines and legionellosis

Recent studies have led to an enhanced understanding of the role of cell-mediated immunity and cytokines in Legionnaires' disease. In particular, the effect of interferon gamma on human mononuclear phagocyte iron metabolism and the role of iron availability inLegionella pneumophila intracellular multiplication in human monocytes has been elucidated. With this knowledge it is now possible to develop treatment strategies for Legionnaires' disease using interferon gamma and/or agents affecting human mononuclear phagocyte iron metabolism.Abbreviations M-CSF Macrophage colony stimulating factor     

Production of Ethanol from Maltose by Zymobacter palmae Fermentation

The production of ethanol from maltose by Zymobacter palmae T109 in monoculture fermentations, and in co-culture fermentations together with Zymomonas mobilis B69 was studies. Zymobacter palmae T109, produced 5.5% (w/v) of ethanol when co-cultured with Zymomonas mobilis B69, but Zymobacter palmae T109 produced only 4.9% (w/v) ethanol from 15% (w/v) maltose medium in monoculture fermentation.     

乳铁蛋白的理化性质及其重组表达系统的研究进展

乳铁蛋白(Lactoferrin, Lf)是分子量大小约为80 kDa的铁离子结合糖蛋白,是转铁蛋白(Transferrin, Tf)家族的成员之一。其理化性质独特,具有抑菌、抗病毒、抗癌、免疫调节、调节铁离子的吸收等诸多生物学功能。获得高产且有生物活性的重组乳铁蛋白,并用于临床治疗,一直是研究热点。随着基因工程技术的发展,已获得多个可表达重组乳铁蛋白的表达系统。本文对乳铁蛋白的理化性质、生物学活性、临床研究以及目前的重组表达系统进行综述,以期为乳铁蛋白的临床应用提供参考。     

Acanthamoeba castellanii Proteases are Capable of Degrading Iron‐Binding Proteins as a Possible Mechanism of Pathogenicity

Acanthamoeba castellanii, a free‐living amoeba, is an amphizoic organism that can behave as an opportunistic pathogen, causing granulomatous amoebic encephalitis in immunocompromised patients or infecting immunocompetent individuals via cutaneous lesions, sinusoidal infections, or amoebic keratitis. Therefore, this amoeba could be in contact with different iron‐binding proteins, such as lactoferrin in tears and mucosa and transferrin and hemoglobin in blood. Iron is a vital and necessary element for host metabolism but also for parasite survival. Accordingly, parasites have developed iron uptake mechanisms, one of which is the utilization of proteases to degrade host iron‐binding proteins. In this work, we performed a partial biochemical characterization of A. castellanii proteases at different pHs and utilizing protease inhibitors with 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and copolymerized with different iron‐binding proteins. We describe for the first time the presence of several cysteine proteases in a total A. castellanii crude extract and in conditioned culture medium precipitated with ethanol. These amoebic peptidases degraded human holo‐lactoferrin, holo‐transferrin, hemoglobin, and horse spleen ferritin; some of these proteases were substrate specific, and others degraded multiple substrates. These proteases could be considered virulence factors that promote iron acquisition from the host.     

A Facile Synthesis of 4,8-Dimethyldecanal,Aggregation Pheromone of Flour Beetles,and Its Analogues

A Rhodococcus erythropolis expression system for the bovine lactoferrin C-lobe was constructed. The DNA fragments encoding the BLF C-lobe were amplified and cloned into vector pTip LCH1.2. R. erythropolis carrying the pTip-C-lobe was cultured at 30 °C with shaking, and expression of the rBLF C-lobe was induced by adding 1 μg/ml (final concentration) thiostrepton. The rBLF C-lobe was isolated in native and denatured (8 M urea) form by Ni-NTA affinity chromatography. To obtain a bioactive rBLF C-lobe, the protein isolated in the denatured form was refolded by stepwise dialysis against refolding buffers. The antibacterial activity of the rBLF C-lobe was tested by the filter-disc plate assay method. The refolded rBLF C-lobe demonstrated antibacterial activity against selected strains of Escherichia coli.     

Potassium efflux induced by a new lactoferrin-derived peptide mimicking the effect of native human lactoferrin on the bacterial cytoplasmic membrane

A 31-amino acid synthetic peptide (NH₂-FFSASCVPGADKGQFPNLCRLCAGTGENKCA-COOH) was chemically synthesized based on the amino acid sequence of a region of human lactoferrin homologous to other sequences present in the N- and C-lobes of all members of the transferrin family proteins. The peptide, termed kaliocin-1, and lactoferrin showed a bactericidal effect in assays performed in low-ionic-strength conditions. This is the first time that it is shown that the antimicrobial effect of lactoferrin depends on the extracellular cation concentration. The antimicrobial effect of kaliocin-1 was lower than that of human lactoferrin, but their activities were inhibited by Na⁺ or K⁺ in a cation concentration-dependent manner. In addition, the peptide was able to mimic native lactoferrin, inducing K⁺-efflux and a selective dissipation of the transmembrane electrical potential of Escherichia coli cells without causing extensive damage to the outer and inner bacterial membranes. In contrast, the peptide, but not lactoferrin, was able to permeabilize different ions through liposomal membranes. The hypothetical interaction of kaliocin-1 with a bacterial membrane compound is discussed based in the different ion flux induced on cellular and artificial membranes as well as data from circular dichroism assays. Kaliocin-1 was not cytotoxic and could be a suitable model for the design of analogs able to mimic the antibacterial effect of human lactoferrin.     

Prediction of antibiotic activity and synthesis of new pentadecapeptides based on lactoferricins.

The antibacterial activity against Escherichia coli and Staphylococcus aureus has been studied for a number of modified pentadecapeptides based on lactoferricins of different origin. The peptides were classified by multivariate methods and quantitative structure-activity relationships (QSAR) were developed using theoretically derived variables for the amino acids. For the modified peptides based on bovine lactoferricin (LFB) a model was calculated and used for prediction of new peptides that were then tested for antibacterial activity in order to improve peptide activity and to check the validity of the model. Models were also calculated including lactoferricins of different origin. Theories of the mechanism of action of the peptides are briefly discussed.     

Synthesis and iron-binding properties of quinolobactin,a siderophore from a pyoverdine-deficient <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Quinolobactin is a new siderophore produced by a pyoverdine deficient mutant of Pseudomonas fluorescens. A simple and efficient synthesis of quinolobactin is described, starting from xanthurenic acid. The protonation constants of quinolobactin were determined by potentiometric titrations as pK_a2=5.50 ± 0.07, pK_a1=10.30 ± 0.05. The equilibria of the metal complexes were studied by means of spectrophotometric and potentiometric titrations. The overall stability constants of the quinolobactin-Fe^III complexes was found to be log ₁₁₁=18.60 ± 0.10, log ₁₂₁=32.60 ± 0.20, log ₁₂₀=28.20 ± 0.25 resulting in a pFe^III value of 18.2 at pH 7.4. The UV-visible spectral parameters of the [FeL₂] are in agreement with a complex containing two ligands coordinated to one Fe³⁺ cation through the oxygen and nitrogen quinoline atoms.     

植物内生菌研究进展

内生菌由于生长于植物体内且不引起植物病害所以不易被发现,它是一类具有高实用价值的植物内生微生物。经过人们的不断研究,越来越多的植物内生菌被发现,其活性代谢产物的优势也得到了更多研究者的认可。综述了国内外有关植物内生菌研究的主要成果和进展,并从植物内生菌的发现、研究历史、多样性、生理生化特性以及植物内生菌与宿主植物间的作用关系等多个层次进行了概述,以期为植物内生菌相关研究提供参考。     

Differential Glycosylation of rhLf Expressed in the Mammary Gland of Transgenic Mice

Differential glycosylation of natural hLf and rhLf from hLf-transgenic mice, which harbored a 146 Kb BAC insert that includes the intact hLf gene sequence, was studied in the present report. There were significant differences between the immunoblotting results of rhLf and natural hLf, which were denatured with nonreducing SDS sample buffer. The differences disappeared after rhLf and natural hLf samples were digested with N-glycosidase F, respectively. The results showed that there were significant differences (P < 0.01) between the glycosylation of natural hLf and rhLf that were purified, respectively, from milk samples of seven hLf-transgenic mouse lines.     

乳酸乳球菌表达重组牛乳铁蛋白肽的抑菌活性分析

牛乳铁蛋白肽是由牛乳铁蛋白经消化酶水解产生的一类具有广谱抑菌活性的短肽;乳酸乳球菌作为食品级微生物,既有天然的益生作用,又是理想的表达牛乳铁蛋白肽的载体。【目的】探究重组乳酸乳球菌pAMJ399-LFcinBA/MG1363表达牛乳铁蛋白肽的抑菌活性。【方法】利用牛乳铁蛋白肽标准品绘制定量标准曲线来确定重组牛乳铁蛋白肽的含量,利用牛津杯法及微量肉汤稀释法测定重组牛乳铁蛋白肽对大肠杆菌、金黄色葡萄球菌等35株细菌的抑菌活性及最小抑菌浓度,利用扫描电镜、透射电镜、荧光显微镜、凝胶阻滞试验、黏附试验来探究重组牛乳铁蛋白肽对菌体结构、细菌DNA及黏附力的影响,利用CCK-8检测其对RAW 264.7细胞的毒性作用,并对小鼠红细胞溶血率进行测定。【结果】重组乳酸乳球菌上清中牛乳铁蛋白肽的浓度为24.39μg/mL,重组牛乳铁蛋白肽对测试的25株致病菌均有不同程度的抑制作用,抑菌浓度范围在16–128μg/mL,但对9株乳酸菌以及粪肠球菌没有明显的抑制作用,对大肠杆菌、金黄色葡萄球菌、多杀性巴氏杆菌、鸡白痢沙门菌的菌体完整性具有不同程度的破坏作用,其主要作用靶点为细菌的细胞膜,可以与细菌DNA结合...