Expression of full-length bioactive antimicrobial human lactoferrin in potato plants

A cDNA fragment encoding human lactoferrin (hLF) linked to a plant microsomal retention signal peptide (SEKDEL) was stably integrated into the Solanum tuberosum genome by Agrobacterium tumefaciens-mediated leaf disk transformation methods. The lactoferrin gene was expressed under control of both the auxin-inducible manopine synthase (mas) P2 promoter and the cauliflower mosaic virus (CaMV) 35S tandem promoter. The presence of the hLF cDNA in the genome of regenerated transformed potato plants was detected by polymerase chain reaction amplification methods. Full-length hLF protein was identified by immunoblot analysis in tuber tissue extracts from the transformed plants by immunoblot analysis. The hLF produced in transgenic plant tissues migrated during polyacrylamide gel electrophoresis as a single band with an approximate molecular mass equal to hLF. Auxin activation of the mas P2 promoter increased lactoferrin expression levels in transformed tuber and leaf tissues to approximately 0.1% of total soluble plant protein. Antimicrobial activity against four different human pathogenic bacterial strains was detected in extracts of lactoferrin-containing potato tuber tissues. This is the first report of synthesis of full length, biologically active hLF in edible plants.     

High expression of a human lactoferrin in transgenic tobacco cell cultures

Transgenic Nicotiana tabacum cell lines were developed expressing the human lactoferrin gene driven by the oxidative stress-inducible peroxidase (SWPA2) promoter. Western blot analysis showed the accumulation of both the full-length human lactoferrin protein as well as a immuno-reactive truncated fragment. Accumulation of human lactoferrin as monitored by ELISA increased proportionally to cell growth and reached a maximal (up to 4.3% of total soluble proteins) at the stationary phase of growth. Protein extracts from transgenic tobacco cells exhibited antibacterial activity.     

Structure of a complex of human lactoferrin N-lobe with pneumococcal surface protein a provides insight into microbial defense mechanism

Human lactoferrin, a component of the innate immune system, kills a wide variety of microorganisms including the Gram positive bacteria Streptococcus pneumoniae. Pneumococcal surface protein A (PspA) efficiently inhibits this bactericidal action. The crystal structure of a complex of the lactoferrin-binding domain of PspA with the N-lobe of human lactoferrin reveals direct and specific interactions between the negatively charged surface of PspA helices and the highly cationic lactoferricin moiety of lactoferrin. Binding of PspA blocks surface accessibility of this bactericidal peptide preventing it from penetrating the bacterial membrane. Results of site-directed mutagenesis, in vitro protein binding assays and isothermal titration calorimetry measurements corroborate that the specific electrostatic interactions observed in the crystal structure represent major associations between PspA and lactoferrin. The structure provides a snapshot of the protective mechanism utilized by pathogens against the host's first line of defense. PspA represents a major virulence factor and a promising vaccine candidate. Insights from the structure of the complex have implications for designing therapeutic strategies for treatment and prevention of pneumococcal diseases that remain a major public health problem worldwide.     

Effect of nucleotides on the oligomeric state of human lactoferrin

Lactoferrin (LF) is a multifunctional acute-phase protein involved in nonspecific defense against bacteria, viruses, and cancer diseases and is present in human barrier fluids, blood, and milk. Small-angle X-ray scattering (SAXS) and light scattering (LS) demonstrated for the first time that LF occurs in the form of oligomers, with a high monomer unit number in the solution. The degree of LF oligomerization depends on the LF concentration and the storage period of non-frozen neutral LF solutions. The average inertial radius of scattering particles (R _g) reaches 100–450 Å at LF concentrations comparable with those in human milk, while R _g of LF monomers is 26.7 Å. LF forms complexes with various nucleotides and hydrolyzes them. The addition of ATP or AMP to LF solutions accelerates LF oligomerization and increases R _g to 600–700 Å, regardless of the initial degree of LF oligomerization. According to the different models (sphere, plate, and cylinder) of LF aggregates, its complexes with such R _g presumably contain several tens to thousands of LF monomers. The possible role of oligomeric complexes in multiple biological functions of LF is discussed.     

Recombinant human lactoferrin treatment for global health issues: iron deficiency and acute diarrhea

Iron deficiency and diarrhea are two of the most significant issues for global health. Iron deficiency anemia is the most common nutritional deficiency in the world, affecting nearly 25% of the world population (UNICEF/WHO 1999). The prevalence of iron deficiency in developing countries is illustrated by comparison with other deficiencies: iron deficiency affects 3.5 billion people, while vitamin A and iodine deficiency affect 0.3 billion people and 0.8 billion people, respectively. The prevalence is highest among young children and women of childbearing age (particularly pregnant women). It is estimated that national productivity levels could be raised as much as 20% by correcting iron deficiency in developing countries. Recombinant human lactoferrin (rhLF), expressed and extracted from rice seed, is being evaluated by Ventria Bioscience for use as a dietary supplement to treat iron deficiency and/or iron deficiency anemia. Diarrhea is also a major world health issue. Sixty percent of children who die under age five die of pneumonia, diarrhea or measles. World Health Organization oral rehydration solution (WHO-ORS) is one of the major medical advances in the past 50 years, saving the lives of 1 to 2 million children annually. Many studies have demonstrated similar efficacy of rice-based ORS. There are studies documenting the reduced frequency of diarrhea in breast-fed children and this health improvement is attributed to the antimicrobial action of the human milk proteins lactoferrin and lysozyme. In vitro data document the growth inhibition of the diarrheal associated organisms: rotavirus, ETEC, cholera, salmonella, and shigella by human lactoferrin (hLF) and human lysozyme. Using Ventria's ExpressTec system, we have expressed human lactoferrin and human lysozyme in rice. In a rice-based ORS formulation, these proteins have the potential to provide not only the benefits of reduced stool volume and improved weight gain, but also shorten the course of diarrheal episodes via antimicrobial activity against the causative agent.     

DNA and oligosaccharides stimulate oligomerization of human milk lactoferrin

Lactoferrin (LF) is a Fe³⁺-transferring glycoprotein and is contained in human barrier fluids, blood, and milk. LF is an acute phase protein, is involved in nonspecific defense, and displays a unique set of biological functions. Small-angle X-ray scattering and light scattering experiments demonstrated that DNA and oligosaccharides added to LF with various levels of initial oligomerization increased the oligomerization rate. Almost complete dissociation into monomers was observed when 1 M NaCl was added to LF oligomers obtained in the presence of DNA, oligosaccharides, and nucleotides, previously identified as oligomerization effectors. LF complexes obtained with different oligomerization effectors differed in stability. Incubation with 50 mM MgCl₂ completely destructed LF complexes formed in the presence of ATP and oligosaccharides but only partly destructed AMP- and d(pT)₁₀-dependent complexes, which was followed by the formation of new complexes with a higher salt stability. A possible role of oligomerization in various LF functions is discussed.     

Expression of a human lactoferrin N-lobe in Nicotiana benthmiana with potato virus X-based agroinfection

A DNA fragment encoding the N-terminal half (N-lobe) of the human lactoferrin (hLfN) gene has now been cloned into recombinant Potexvirus potato virus X (PVX) vector and expressed in Nicotiana benthmiana using agroinfection. Western blot analysis showed the recombinant protein with an apparent molecular mass on electrophoresis of ca. 40 kDa, corresponding to the predicted size of the hLfN. The yield of hLfN reached a maximum (up to 0.6% of total soluble proteins) when recombinant PVX systemically infected an entire plant. Protein extracts from infected plants had antibacterial activity.     

乳腺生物反应器表达的重组人乳铁蛋白的分离纯化及生物活性鉴定

以国产高交联度的快流速琼脂糖为基质,合成了不同配基密度的SP(Sulfopropyl,磺酸基)离子交换介质,建立了乳腺生物反应器表达重组人乳铁蛋白(Recombinant Human Lactoferrin,rHLF)的纯化方法。以溶菌酶为模型蛋白考察了不同配基密度离子交换介质的静态和动态吸附行为,结果表明介质具有良好的吸附性能。不同配基密度离子交换介质均可纯化得到rHLF,其中,高配基密度(0.24mol/L)的离子交换介质每毫升可以处理50mL rHLF牛乳,rHLF收率为86.5%,纯度为98.5%。圆二色谱的测定结果表明纯化的rHLF二级结构与天然人乳铁蛋白一致。生物学功能实验结果表明,rHLF的铁结合与释放活性与天然人乳铁蛋白相似,浓度为5g/L的rHLF对大肠杆菌的生长具有明显的抑制作用。     

Comparative analysis of the site-specific N-glycosylation of human lactoferrin produced in maize and tobacco plants.

We have compared the site-by-site N-glycosylation status of human lactoferrin (Lf) produced in maize, a monocotyledon, and in tobacco, used as a model dicotyledon. Maize and tobacco plants were stably transformed and recombinant Lf was purified from both seeds and leaves. N-glycopeptides were generated by trypsin digestion of recombinant Lf and purified by reverse-phase HPLC. The N-glycosylation pattern of each site was determined by mass spectrometry. Our results indicated that the N-glycosylation patterns of recombinant Lf produced in maize and tobacco share common structural features. In particular, both N-glycosylation sites of each recombinant Lf are mainly substituted by typical plant paucimannose-type N-glycans, with beta1,2-xylose and alpha1,3-linked fucose at the proximal N-acetylglucosamine. However, tobacco Lf shows a significant amount of processed N-glycans with one or two beta1,2GlcNAc linked to the trimannose core, which are weakly expressed in maize Lf. Finally, no Lewisa epitope was observed on tobacco Lf.     

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     

Prophylactic effect of human lactoferrin against Streptococcus mutans bacteremia in lactoferrin knockout mice

Streptococcus mutans is the primary agent of dental caries, which is often detected in transient bacteremia. Lactoferrin is a multifunctional glycoprotein showing antibacterial activities against several Streptococcus species. We reported here the prophylactic effect of human lactoferrin (hLF) in a lactoferrin knockout mouse (LFKO^−/−) bacteremic model. The hLF treatment significantly cleared S. mutans from the blood and organs of bacteremic mice when compared to the non-hLF treated mice. Further, analysis of serum cytokines, spleen and liver cytokine mRNA levels revealed that hLF prophylaxis modulates their release differently when compared to the non-hLF treated group. C-reactive protein level (P = 0.003) also decreased following hLF prophylaxis in S. mutans induced bacteremic mice. Additional quantitative RT-PCR analysis revealed that hLF prophylaxis significantly decreased the expression level of IFN-γ, TNF-α, IL-1β, IL-6, MPO and iNOS in spleen and liver. These results suggested that the hLF protects the host against S. mutans-induced experimental bacteremia.     

Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropriate post-translational modifications. The nuclear transfer of transgenic somatic cells is a powerful method to produce mammary gland bioreactors. We established an efficient gene transfer and nuclear transfer approach in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer and some of reconstructed embryos could develop into blastocyst in vitro. __________ Translated from Hereditas (Beijing), 2006, 28(12): 1513–1519 [译自: 遗传]     

DNA,oligosaccharides, and mononucleotides stimulate oligomerization of human lactoferrin

Using small‐angle X‐ray scattering (SAXS), light scattering (LS), and soft laser ablation we have shown that lactoferrin (LF) in solution at neutral pH is oligomerized in the absence of salt or at physiological salt concentrations. The level of oligomerization depends on the concentration of LF, KCl or NaCl, and on the duration of the protein storage in solution. At the concentrations comparable with those in human milk (1 ÷ 6 mg/ml), the average radius of gyration (R_g) values of LF can attain 400 ÷ 480 Å´, while fresh solution of previously lyophylized LF demonstrate a lower average R_g (50 ÷ 100 Å´), and R_g value characterizing the LF monomer formed at 1 M NaCl is 26.7 Å´. The addition of oligonucleotides, oligosaccharides, or mononucleotides to LF in the presence or in the absence of KCl with different level of initial oligomerization accelerates the oligomerization rate and increases the R_g values up to ～600 ÷ 700 Å´, which correspond to associates containing ten or more protein molecules. During gel filtration on Sepharose 4B, high‐degree LF oligomers dissociate nearly completely forming different degraded complexes, but in some cases it is possible to reveal small amount of a decamer. A possible role for oligomerization of LF, a highly polyfunctional protein, for its different biological activities is discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.     

Mid-infrared prediction of lactoferrin content in bovine milk: potential indicator of mastitis

Lactoferrin (LTF) is a milk glycoprotein favorably associated with the immune system of dairy cows. Somatic cell count is often used as an indicator of mastitis in dairy cows, but knowledge on the milk LTF content could aid in mastitis detection. An inexpensive, rapid and robust method to predict milk LTF is required. The aim of this study was to develop an equation to quantify the LTF content in bovine milk using mid-infrared (MIR) spectrometry. LTF was quantified by enzyme-linked immunosorbent assay (ELISA), and all milk samples were analyzed by MIR. After discarding samples with a coefficient of variation between 2 ELISA measurements of more than 5% and the spectral outliers, the calibration set consisted of 2499 samples from Belgium (n = 110), Ireland (n = 1658) and Scotland (n = 731). Six statistical methods were evaluated to develop the LTF equation. The best method yielded a cross-validation coefficient of determination for LTF of 0.71 and a cross-validation standard error of 50.55 mg/l of milk. An external validation was undertaken using an additional dataset containing 274 Walloon samples. The validation coefficient of determination was 0.60. To assess the usefulness of the MIR predicted LTF, four logistic regressions using somatic cell score (SCS) and MIR LTF were developed to predict the presence of mastitis. The dataset used to build the logistic regressions consisted of 275 mastitis records and 13 507 MIR data collected in 18 Walloon herds. The LTF and the interaction SCS × LTF effects were significant (P < 0.001 and P = 0.02, respectively). When only the predicted LTF was included in the model, the prediction of the presence of mastitis was not accurate despite a moderate correlation between SCS and LTF (r = 0.54). The specificity and the sensitivity of models were assessed using Walloon data (i.e. internal validation) and data collected from a research herd at the University of Wisconsin – Madison (i.e. 5886 Wisconsin MIR records related to 93 mastistis events – external validation). Model specificity was better when LTF was included in the regression along with SCS when compared with SCS alone. Correct classification of non-mastitis records was 95.44% and 92.05% from Wisconsin and Walloon data, respectively. The same conclusion was formulated from the Hosmer and Lemeshow test. In conclusion, this study confirms the possibility to quantify an LTF indicator from milk MIR spectra. It suggests the usefulness of this indicator associated to SCS to detect the presence of mastitis. Moreover, the knowledge of milk LTF could also improve the milk nutritional quality.     

人乳铁蛋白cDNA 基因乳腺表达载体的构建与鉴定

为了构建人乳铁蛋白基因 (hLF) 的乳腺表达载体并验证其在乳腺细胞中的表达情况,本载体以山羊β-casein基因上游包括启动子、外显子1、内含子1、部分外显子2作为5′端调控序列,下游包括部分外显子7、内含子7、外显子8、内含子8、外显子9及3′部分基因组片段作为3′端调控序列,长度分别为6.2 kb和7.1 kb,将hLF基因 (目的基因) 和Neo基因 (筛选标记) 分别插入到5′端调控序列和3′端调控序列的下游,构建成pBC1-hLF-Neo载体,其全长为25.348 kb。为了检测该载体的生物学     

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.     

人乳铁蛋白(hLF)转基因山羊的遗传背景分析

以随机整合方式获得的转基因动物外源基因的拷贝数、整合位点及染色体核型等遗传背景并不清楚,可能会存在外源基因的沉默整合、无效整合、毒性整合以及其表达水平不可预测等问题。文中选取了6只原代(F0)及其相对应的子一代(F1)的人乳铁蛋白(hLF)转基因山羊作为研究对象,分别颈静脉采血、提取DNA,通过染色体核型分析、实时荧光定量PCR(qPCR)、ELISA和Westernblotting等检测技术,研究其外源基因的遗传背景与表达水平。结果显示,6只F0代转基因山羊的染色体没有明显的形态变异、数量改变等异常情况。相对拷贝数高低不同(2–16),且能够稳定地遗传给下一代,F0和F1代hLF基因拷贝数一致。F1代转基因山羊表达hLF水平最高可达1.12 g/L(L3-1,拷贝数8)。结果表明,整合的外源基因能够稳定地遗传下一代,也没有对转基因山羊个体的生长发育造成障碍,而且拷贝数高低与hLF表达水平无明显的相关性,这为转基因山羊及其他转基因动物的新品种培育奠定了基础,解析了遗传背景。