Production of stable isotope enriched antimicrobial peptides in Escherichia coli: An application to the production of a 15N-enriched fragment of lactoferrin

A method is described for the production of recombinant isotopically enriched peptides in E. coli. Peptides are produced in high yield as fusion proteins with ketosteroid isomerase which form insoluble inclusion bodies. This insoluble form allows easy purification, stabilizes the peptide against degradation and prevents bactericidal activity of the peptide. Cyanogen bromide cleavage released peptide which was conjugated with alkylamines to form lipopeptide. An important advantage of this system is that it allows production of peptides that are toxic to bacteria, which we have demonstrated on a dodecapeptide based on residues 21–31 of human bactericidal protein lactoferrin.     

Crystal structure of a complex formed between proteolytically-generated lactoferrin fragment and proteinase K

Lactoferrin is an iron binding glycoprotein with a molecular weight of 80 kDa. The molecule is divided into two lobes representing the N-terminal and C-terminal halves of the polypeptide chain, each containing an iron binding site. The serine proteinases such as trypsin, chymotrypsin, and pepsin hydrolyze lactoferrin into two unequal halves while proteinase K divides this protein into two equal halves. In the first step of hydrolysis by proteinase K, the C- and N-lobes, each having a molecular weight of approximately 40 kDa, are generated. In the next step, the lobes are further hydrolyzed into small molecular weight peptides. The proteinase K isolated from the hydrolyzed product does not show enzymatic activity suggesting that the enzyme is inhibited. Furthermore, the hydrolysis experiments on N-lobe and C-lobe showed that the inhibitory fragment came from the C-lobe. The purified lactoferrin fragment was found to be a decapeptide with an amino acid sequence of H₂N-Val-Ala-Gln-Gly-Ala-Ala-Gly-Leu-Ala-COOH. The complex formed between proteinase K and lactoferrin fragment was crystallized by microdialysis. The crystals belonged to the monoclinic space group P2₁with cell dimensions a = 44.4 Å, b = 38.6 Å, c = 79.2 Å, β = 105.8^o and Z = 2. The crystal structure has been determined at 2.4 Å resolution. It has been refined to an R factor of 0.163 for 9044 reflections. The Lf-fragment forms several intermolecular interactions with proteinase K. The Ser-224 Oγ and His-57 Nϵ2 move away to a distance of 3.68 Å in the complex. In the crystal structure, Gln-3I (I indicates inhibitor i.e., lactoferrin fragment) is involved in a direct intermolecular interaction with a symmetry related proteinase K molecule through a strong hydrogen bond with Asp-254. The mode of intermolecular interactions in the complex conformational features of the enzyme and placement of the fragment with respect to the enzyme resemble with the molecular complex of proteinase K with its natural inhibitor PKI3 from wheat. Proteins 33:30–38, 1998. © 1998 Wiley-Liss, Inc.     

Structural characterization of the ceruloplasmin: lactoferrin complex in solution

Ceruloplasmin is a copper protein found in vertebrate plasma, which belongs to the family of multicopper oxidases. Like transferrin of the blood plasma, lactoferrin, the iron-containing protein of human milk, saliva, tears, seminal plasma and of neutrophilic leukocytes tightly binds two ferric ions. Human lactoferrin and ceruloplasmin have been previously shown to interact both in vivo and in vitro forming a complex. Here we describe a study of the conformation of the human lactoferrin/ceruloplasmin complex in solution using small angle X-ray scattering. Our ab initio structural analysis shows that the complex has a 1:1 stoichiometry and suggests that complex formation occurs without major conformational rearrangements of either protein. Rigid-body modeling of the mutual arrangement of proteins in the complex essentially yields two families of solutions. Final discrimination is possible when integrating in the modeling process extra information translating into structural constraints on the interaction between the two partners.     

The effect of bovine lactoferrin and lactoferricin B on the ability of feline calicivirus (a norovirus surrogate) and poliovirus to infect cell cultures

AIMS: To characterize the effect of bovine lactoferrin and lactoferricin B against feline calicivirus (FCV), a norovirus surrogate and poliovirus (PV), as models for enteric viruses. METHODS AND RESULTS: Crandell-Reese feline kidney (CRFK) cells were used for the propagation of FCV and monkey embryo kidney (MEK) cells for PV. The assays included visual assessment of cell lines for cytopathic effects and determination of the percentage cell death using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] dye reduction assay. Incubation of bovine lactoferrin with CRFK cells either prior to or together with FCV inoculation substantially reduced FCV infection. In contrast, the interference of lactoferrin with the infection of cells with PV was demonstrated only when lactoferrin was present with cell lines and virus for the entire assay period. Using indirect immunofluorescence, lactoferrin was detected on the surface of both CRFK and MEK cells, suggesting that the interference of viral infection may be attributed to lactoferrin binding to the surfaces of susceptible cells, thereby preventing the attachment of the virus particles. Lactoferricin B, a cationic antimicrobial peptide derived from the N-terminal domain of bovine lactoferrin, reduced FCV but not PV infection. CONCLUSION: Lactoferrin was shown to interfere with the infection of cells for both FCV and PV. However, lactoferricin B showed no interference of infection with PV and interference with infection for FCV required the presence of lactoferricin B together with the cell line and virus. SIGNIFICANCE AND IMPACT OF THE STUDY: An in vitro basis is provided for the effects of bovine lactoferrin and lactoferricin B in moderating food-borne infections of enteric viruses.     

Lactoferrin Is the Major Deoxyribonuclease of Human Milk

Lactoferrin is the major iron-transferring protein of human barrier fluids such as blood and milk. It is a polyfunctional protein capable of binding DNA exposed on the surface of various cells. Electrophoretically homogenous lactoferrin was prepared by sequential chromatography of human milk proteins on DEAE-cellulose, heparin-Sepharose, and Sepharose containing immobilized anti-lactoferrin antibodies. By subsequent chromatography on Blue Sepharose the resulting lactoferrin was fractionated into several subfractions with different affinity for the sorbent, and this was associated with separation of additional lactoferrin peaks with DNase activity from the main peak. By various techniques, in particular, by in situ testing the DNase activity of lactoferrin in a DNA-containing gel after SDS-electrophoresis, hydrolysis of DNA was for the first time shown to be an intrinsic property of lactoferrin. The substrate specificity of lactoferrin in hydrolysis of DNA was different from specificities of known human DNases. Hydrolysis of DNA was activated by bivalent metal ions and also by ATP and NAD. Unlike the main fraction of lactoferrin with the highest affinity for Blue Sepharose, all protein subfractions with DNase activity were cytotoxic and suppressed growth of human and mouse tumor cell lines.     

Proton NMR Spectra of Peracetylated Derivatives of α,α-Trehalose and Sucrose in the Presence of a Europium Shift Reagent

The possibility of using recombinant human lactoferrin from rice (rhLF) makes it necessary to study its differences from the protein of milk. In this work, the binding of different iron-saturated forms of rhLF to Caco-2 cells was studied. Iron-saturated rhLF bound in higher proportion than the apo-form, but, the data obtained for specific binding were not compatible with receptor-mediated binding. Competition assays showed the same binding capacity for human milk lactoferrin as for rhLF to Caco-2 cells. Another basic protein of milk, lactoperoxidase, was found to compete with rhLF for binding to Caco-2 cell membranes, suggesting an electrostatic interaction. The transport of iron (⁵⁹Fe) bound to rhLF and to citrate and the transport of rhLF (¹²⁵I-labeled) were studied on Caco-2 monolayers. Transport of iron was found to be significantly greater when bound to citrate than to rhLF. The amount of intact lactoferrin that traversed the Caco-2 monolayers was very low, suggesting degradation of it across these cells.     

重组人乳铁蛋白和重组人溶菌酶对小鼠溃疡性结肠炎的改善作用研究

溃疡性结肠炎（ulcerative colitis,UC）是一种发生于直肠和结肠的慢性非特异性炎症疾病,近年来发病率明显上升。为探究转基因牛乳中提取的重组人乳铁蛋白和重组人溶菌酶对改善UC的作用,采用葡聚糖硫酸钠（dextran sulfate sodium salt,DSS）构建小鼠UC模型,30只雄性C57BL/6N小鼠随机分为空白对照组（CON组）、模型对照组（DSS组）、低浓度乳铁蛋白组（L-rLF组,50 mg·kg^-1·BW^-1）、高浓度乳铁蛋白组（H-rLF组,100 mg·kg^-1·BW^-1）、低浓度溶菌酶组（L-rLZM组,50 mg·kg^-1·BW^-1）和高浓度溶菌酶组（H-rLZM组,100 mg·kg^-1·BW^-1）。造模后用重组乳铁蛋白和溶菌酶分别灌胃1周,取小鼠血清及结肠,观察器官病理变化,测定炎症因子以及肠道菌群等相关指标。研究结果显示,与模型组相比,低浓度乳铁蛋白组和低浓度溶菌酶组小鼠疾病活动指数（disease activity index,DAI）、结肠缩短量、组织病理学评分均显著降低,且结肠组织中促炎因子（IL-6、lL-1β、TNF-α）表达量、血清和肝脏中脂多糖（lipopolysaccharide,LPS）浓度显著降低,高浓度乳铁蛋白处理组小鼠肠道菌群结构显著改善。表明重组人乳铁蛋白和重组人溶菌酶均可以不同程度地改善小鼠UC,为重组人乳铁蛋白和重组人溶菌酶的未来应用提供了新的思路和理论支持。     

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.     

Mini-review: Lactoferrin: a bioinspired,anti-biofilm therapeutic

Medically relevant biofilms have gained a significant level of interest, in part because of the epidemic rise in obesity and an aging population in the developed world. The associated comorbidities of chronic wounds such as pressure ulcers, venous leg ulcers, and diabetic foot wounds remain recalcitrant to the therapies available currently. Development of chronicity in the wound is due primarily to an inability to complete the wound healing process owing to the presence of a bioburden, specifically bacterial biofilms. New therapies are clearly needed which specifically target biofilms. Lactoferrin is a multifaceted molecule of the innate immune system found primarily in milk. While further investigation is warranted to elucidate mechanisms of action, in vitro analyses of lactoferrin and its derivatives have demonstrated that these complex molecules are structurally and functionally well suited to address the heterogeneity of bacterial biofilms. In addition, use of lactoferrin and its derivatives has proven promising in the clinic.     

乳铁素——来源于乳铁蛋白的多功能抗菌肽

乳铁素是乳铁蛋白在酸性环境条件下经胃蛋白酶水解从N-端释放的多功能活性多肽．乳铁素不仅保持了完整乳铁蛋白的大部分生物学活性,而且乳铁素的某些生物学活性比乳铁蛋白更强．乳铁素具有抗细菌、抗真菌、抗病毒、抗肿瘤、免疫调节和抗炎症等多种生物学功能．然而,乳铁素的生物学作用大部分是通过体外试验发现和验证的,乳铁素的体内生物学效应还需更多的试验加以评价和证实,现代基因组学和蛋白组学分析方法和技术将有助于深入了解乳铁素体内生物学作用机制．本文就乳铁素的结构、生物学功能及其作用机制、制备和应用前景作一综述．     

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.     

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.     

不同来源乳铁蛋白对幼鼠肠道发育的影响

目的 探究哺乳期乳铁蛋白（lactoferrin，LF）的缺失及不同来源LF补充后对幼鼠肠道发育的影响。方法 以LF基因敲除型雌鼠作为哺乳母鼠造成幼鼠哺乳期无LF的摄入，且从幼鼠出生第3~21天每日人工饲喂100 mg/kg 牛血清白蛋白（BSA）、牛源乳铁蛋白（bovine Lactoferrin，bLF）及重组人源乳铁蛋白（recombinant human Lactoferrin，rhLF），于幼鼠21日龄取样，测定各组小鼠小肠发育指标。结果 在本实验周期下，哺乳期rhLF的补充显著性增加小鼠回肠绒毛长度/隐窝深度值（P<0.05），且上调回肠Occludin和ZO-1基因的表达（P<0.05），增加小鼠十二指肠、空肠和回肠麦芽糖酶酶活/乳糖酶酶活比值（P<0.05），表明哺乳期rhLF的补充能够增强小鼠肠道消化吸收能力和肠屏障功能；哺乳期bLF的补充显著增加小鼠十二指肠及回肠麦芽糖酶活性/乳糖酶活性比值（P<0.05）。结论 对于哺乳期无LF摄入的乳鼠来说，哺乳期间LF的补充能够增强乳鼠肠道对营养物质的消化吸收能力、促进肠道的发育成熟、增强肠道屏障功能，并且，本实验中rhLF表现出比bLF更加有效的作用。     

Bovine lactoferrin region responsible for binding to bifidobacterial cell surface proteins

Bovine lactoferrin promotes bifidobacterial growth. Its binding to bifidobacteria is thought to be responsible for such action. After separating the bovine lactoferrin half molecule and extraction of surface proteins from bifidobacteria, binding profiles were observed by immunoblotting. No binding appeared when lactoferrin C-lobe was reacted with the cell surface proteins on a polyvinylidene difluoride membrane. Conversely, a 50-kDa band appeared when the surface proteins were reacted with either intact or nicked bovine lactoferrin. This result strongly suggests that the binding region could be lactoferrin N-lobe. Interestingly, despite the absence of binding, C-lobe enhanced bifidobacterial growth.     

乳铁蛋白的多功能生物活性与临床进展

目前,为应对抗生素耐药性问题,需要探索更多的抗生素替代物用于对抗微生物的感染。乳铁蛋白是一类源自哺乳动物乳汁或其他粘膜分泌的一种糖基化蛋白,具有广谱抗微生物活性、免疫调节以及抗癌作用。乳铁蛋白对于易受感染的早产儿也有很好的耐受作用,基本无不良反应,具有良好的开发前景。本文主要概述乳铁蛋白的基本性质、多功能的生物学活性和作用机制,并探讨牛乳铁蛋白与talactoferrin的临床研究情况。     

Construction and synthesis of lactoferricin derivatives with enhanced antibacterial activity

A series of peptides derived from sequences from human, bovine, murine and caprine lactoferrin has been prepared and investigated for antibacterial effect. Among the four species investigated peptides based on the bovine sequence displayed significant activity. The bovine sequence, bovine lactoferricin, showed a MIC value of 30 μg/mL on E. coli and S. aureus, whereas the three other lactoferricins possessed MIC values above 200 μg/mL. Based on these findings, novel peptides with enhanced antibacterial activities, were prepared with sequences designed by molecular modelling and structure‐activity studies. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Novel surface attachment mechanism of the Streptococcus pneumoniae protein PspA.

Pneumococcal surface protein A (PspA) of Streptococcus pneumoniae has been found to utilize a novel mechanism for anchoring to the bacterial cell surface. In contrast to that of surface proteins from other gram-positive bacteria, PspA anchoring required choline-mediated interactions between the membrane-associated lipoteichoic acid and the C-terminal repeat region of PspA. Release of PspA from the cell surface could be effected by deletion of 5 of the 10 C-terminal repeat units, by high concentrations of choline, or by growth in choline-deficient medium. Other pneumococcal proteins, including autolysin, which has a similar C-terminal repeat region, were not released by these treatments. The attachment mechanism utilized by PspA thus appears to be uniquely adapted to exploit the unusual structure of the pneumococcal cell surface. Further, it has provided the means for rapid and simple isolation of immunogenic PspA from S. pneumoniae.     

Crystal structure of a bacterial albumin-binding domain at 1.4 A resolution

The albumin-binding domain, or GA module, of the peptostreptococcal albumin-binding protein expressed in pathogenic strains of Finegoldia magna is believed to be responsible for the virulence and increased growth rate of these strains. Here we present the 1.4A crystal structure of this domain, and compare it with the crystal structure of the GA-albumin complex. An analysis of protein-protein interactions in the two crystals, and the presence of multimeric GA species in solution, indicate the GA module is "sticky", and is capable of forming contacts with a range of protein surfaces. This might lead to interactions with different host proteins.     

Structural investigation of Borrelia burgdorferi OspB, a bactericidal Fab target

Certain antibody Fab fragments directed against the C terminus of outer surface protein B (OspB), a major lipoprotein of the Lyme disease spirochete, Borrelia burgdorferi, have the unusual property of being bactericidal even in the absence of complement. We report here x-ray crystal structures of a C-terminal fragment of B. burgdorferi OspB, which spans residues 152-296, alone at 2.0-A resolution, and in a complex with the bactericidal Fab H6831 at 2.6-A resolution. The H6831 epitope is topologically analogous to the LA-2 epitope of OspA and is centered around OspB Lys-253, a residue essential for H6831 recognition. A beta-sheet present in the free OspB fragment is either disordered or removed by proteolysis in the H6831-bound complex. Other conformational changes between free and H6831-bound structures are minor and appear to be related to this loss. In both crystal structures, OspB C-terminal fragments form artificial dimers connected by intermolecular beta-sheets. OspB structure, stability, and possible mechanisms of killing by H6831 and other bactericidal Fabs are discussed in light of the structural data.     

N-glycosylation potential of maize: the human lactoferrin used as a model

In order to determine the N-glycosylation potential of maize, a monocotyledon expression system for the production of recombinant glycoproteins, human lactoferrin was used as a model. The human lactoferrin coding sequence was inserted into the pUC18 plasmid under control of the wheat glutenin promoter. Maize was stably transformed and recombinant lactoferrin was purified from the fourth generation seeds. Glycosylation was analysed by gas chromatography, lectin detection, glycosidase digestions and mass spectrometry. The results indicated that both N-glycosylation sites of recombinant lactoferrin are mainly substituted by typical plant paucimannose-type glycans, with 1,2-xylose and 1,3-linked fucose at the proximal N-acetylglucosamine, and that complex-type glycans with Lewis^a determinants are not present in maize recombinant lactoferrin.