A non-specific lipid transfer protein with antifungal and antibacterial activities from the mung bean

A non-specific lipid transfer peptide (nsLTP) with antimicrobial activity was isolated from the mung bean (Phaseolus mungo) seeds. The procedure entailed aqueous extraction, ion exchange chromatography on CM-Sephadex and high performance liquid chromatography (HPLC) on POROS-HS-20. The peptide exhibited a molecular mass of 9.03 kDa in mass spectrometry. It exerted antifungal action toward Fusarium solani, Fusarium oxysporum, Pythium aphanidermatum and Sclerotium rolfsii, and antibacterial action against Staphylococcus aureus but not against Salmonella typhimurium. The lipid binding of this peptide was very similar to that of a previously described lipid transfer protein extracted from wheat seeds and maize seeds, indicating that it possessed lipid transfer activity. The present findings add to the scarcity of the literature on leguminous nsLTPs.     

Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds.

Two novel classes of antifungal proteins were isolated from radish seeds. The first class consists of two homologous proteins (Rs-AFP1 and Rs-AFP2) that were purified to homogeneity. They are highly basic oligomeric proteins composed of small (5-kDa) polypeptides that are rich in cysteine. Both Rs-AFPs have a broad antifungal spectrum and are among the most potent antifungal proteins hitherto characterized. In comparison with many other plant antifungal proteins, the activity of the Rs-AFPs is less sensitive to the presence of cations. Moreover, their antibiotic activity shows a high degree of specificity to filamentous fungi. The amino-terminal regions of the Rs-AFPs show homology with the derived amino acid sequences of two pea genes specifically induced upon fungal attack, to gamma-thionins and to sorghum alpha-amylase inhibitors. The radish 2S storage albumins were identified as the second novel class of antifungal proteins. All isoforms inhibit growth of different plant pathogenic fungi and some bacteria. However, their antimicrobial activities are strongly antagonized by cations.     

Stress induction and antimicrobial properties of a lipid transfer protein in germinating sunflower seeds

Nonspecific lipid transfer proteins (nsLTPs) belong to a large family of plant proteins whose function in vivo remains unknown. In this research, we studied a LTP previously isolated from sunflower seeds (Ha-AP10), which displays strong antimicrobial activity against a model fungus. The protein is present during at least the first 5 days of germination, and tissue printing experiments revealed the homogeneous distribution of the protein in the cotyledons. Here we report that Ha-AP10 exerts a weak inhibitory effect on the growth of Alternaria alternata, a fungus that naturally attacks sunflower seeds. These data put into question the contribution of Ha-AP10 as an antimicrobial protein of direct effect on pathogenic fungus, and rather suggest a function related to the mobilization of lipid reserves. We also show that the levels of Ha-AP10 in germinating seeds increase upon salt stress, fungal infection and ABA treatment, indicating that it somehow participates in the adaptative responses of germinating sunflower seeds.     

Isolation, purification and characterization of a nonspecific lipid transfer protein from Cuminum cyminum

Cuminum cyminum, an aromatic plant from the family Umbelliferae, is used as a flavoring and seasoning agent in foods. This communication reports the characterization of a nonspecific lipid transfer protein nsLTP1 from its seeds. Plant nsLTPs are small basic proteins involved in transport of lipids between membranes. These proteins are known to participate in plant defense; however, the exact mechanism of their antimicrobial action against fungi or bacteria is still unclear.The cumin nsLTP1 has been purified using a combination of chromatographic procedures and further characterized using mass spectrometry, circular dichroism spectroscopy and Edman degradation. Amino acid sequence has been used to predict homology model of cumin nsLTP1 in complex with myristic acid, and lyso-myristoyl phosphatidyl choline (LMPC). Cumin nsLTP1 is a monomeric protein with a molecular weight of 9.7 kDa as estimated by SDS-PAGE and ESIMS. The protein shows an isoelectric point of 7.8 on 6% PAGE. The primary structure consists of 92 amino acids with eight conserved cysteine residues. The global fold of cumin nsLTP1 includes four α-helices stabilized by four disulfide bonds and a C-terminal tail. The role of internal hydrophobic cavity of the protein in lipid transfer is discussed.     

Binding mechanism of nonspecific lipid transfer proteins and their role in plant defense

Plant nonspecific lipid transfer proteins (nsLTPs) are small basic proteins that transport phospholipids between membranes. On the basis of molecular mass, nsLTPs are subdivided into nsLTP1 and nsLTP2. NsLTPs are all helical proteins stabilized by four conserved disulfide bonds. The existence of an internal hydrophobic cavity, running through the molecule, is a typical characteristic of nsLTPs that serves as the binding site for lipid-like substrates. NsLTPs are known to participate in plant defense, but the exact mechanism of their antimicrobial action against fungi or bacteria is still unclear. To trigger plant defense responses, a receptor at the plant surface needs to recognize the complex of a fungal protein (elicitin) and ergosterol. NsLTPs share high structural similarities with elicitin and need to be associated with a hydrophobic ligand to stimulate a defense response. In this study, binding of sterol molecules with rice nsLTPs is analyzed using various biophysical methods. NsLTP2 can accommodate a planar sterol molecule, but nsLTP1 binds only linear lipid molecules. Although the hydrophobic cavity of rice nsLTP2 is smaller than that of rice nsLTP1, it is flexible enough to accommodate the voluminous sterol molecule. The dissociation constant for the nsLTP2/cholesterol complex is approximately 71.21 microM as measured by H/D exchange and mass spectroscopic detection. Schematic models of the nsLTP complex structure give interesting clues about the reason for differential binding modes. Comparisons of NMR spectra of the sterol/rice nsLTP2 complex and free nsLTP2 revealed the residues involved in binding.     

In Vitro Antifungal Activity of a Radish (Raphanus sativus L.) Seed Protein Homologous to Nonspecific Lipid Transfer Proteins

A basic 9-kD protein was purified from seeds of radish (Raphanus sativus L.). The 43 amino-terminal amino acids show extensive sequence identity with nonspecific lipid transfer proteins from other plant species. The radish seed nonspecific lipid transfer protein-like protein inhibits the growth of several fungi in vitro.     

Isolation and characterization of a novel thermostable non-specific lipid transfer protein-like antimicrobial protein from motherwort (Leonurus japonicus Houtt) seeds

In screening for potent antimicrobial proteins from plant seeds, a novel heat-stable antimicrobial protein, designated LJAMP2, was purified from seeds of the motherwort (Leonurus japonicus Houtt), a medicine herb, with a procedure involving cation exchange chromatography on a CM FF column, and reverse phase HPLCs on C8 column and C18 column. LJAMP2 exhibited a molecular mass of 6.2 kDa determined. Automated Edman degradation determined the partial N-terminal sequence of LJAMP2 to be NH2-AIGCNTVASKMAPCLPYVTGKGPLGGCCGGVKGLIDAARTTPDRQAVCNCLKTLAKSYSG, which displays homology with plant non-specific lipid transfer proteins (nsLTPs). In vitro bioassays showed that LJAMP2 inhibits the growth of a variety of microbes, including filamentous fungi, bacteria and yeast. The growth of three phytopathogenic fungi, Alternaria brassicae, Botrytis maydis, and Rhizoctonia cerealis, are inhibited at 7.5 μM of LJAMP2, whereas Bacillus subtilis is about 15 μM. The IC₅₀ of LJAMP2 for Aspergillus niger, B. maydis, Fusarium oxysporum, Penicillium digitatum and Saccharomyces cerevisiae are 5.5, 6.1, 9.3, 40.0, and 76.0 μM, respectively.     

CaMBP-10的cDNA克隆和表达及钙调素结合活性分析

采用RT PCR法 ,从中国大白菜中分离了编码CaMBP 1 0的cDNA克隆 .该cDNA全长 4 96bp ,编码 92个氨基酸 ,3′端含有 2 1 6bp的非编码区和poly A尾 .将此BP 1 0cDNA的成熟蛋白序列导入表达质粒pET1 5b并转化至大肠杆菌E .coliBL2 1 (DE3)condonplus RIL进行表达 .以免疫印迹和钙调素结合分析法对重组BP 1 0进行鉴定 ,证明其保持了与天然BP 1 0相同的钙调素结合活性 .氨基酸和核苷酸序列分析结果显示 ,它与植物转脂蛋白高度同源 ,特别是含有 8个保守半胱氨酸 .BP 1 0与转脂蛋白之间具极为相似的理化性质如分子量、等电点、热稳定性等 .据此认为 ,CaMBP 1 0是转脂蛋白家族的新成员 ,Ca2 + CaM信号系统可能参与植物转脂蛋白功能的调节     

Purification and characterization of non-specific lipid transfer proteins from the leaves of Pandanus amaryllifolius (Pandanaceae)

Two proteins were isolated from the saline extract of mature leaves of Pandanus amaryllifolius, using affinity chromatography on fetuin-agarose and Affi-gel Blue gel, anion exchange chromatography as well as gel filtration. The proteins were demonstrated as non-glycoproteins, with molecular mass of 18 and 13 kDa, respectively, comprising of peptide subunits from 6.5 to 9 kDa in the forms of heterodimer and homodimer. All of them have similar N-terminal amino acid sequences with only minor variations and are matched to non-specific lipid transfer proteins (nsLTPs) of the other plants such as wheat LTP using NCBI Blast searching for short, nearly exact matches. Furthermore, they explicated each other as isoforms originated putatively from a multigene family with various molecular weight, binding affinity, ionic strength, and subunits. However, the potencies for antiproliferation of HL-60 cell line and inhibition of the growth of the bacteria Pseudomonas aeruginosa are different in that those of the fetuin-binding protein are greater than non-fetuin binding proteins. The non-specific lipid transfer proteins of P. amaryllifolius exhibit weak to moderate hemagglutinating activity toward rabbit erythrocytes, but, this activity could not be reversed by mannose. They thus could be easily differentiated from the previously reported mannose-binding lectin isolated from this plant, which has subunits with similar molecular weight.     

Plant defensins and virally encoded fungal toxin KP4 inhibit plant root growth

Plant defensins are small, highly stable, cysteine-rich antimicrobial proteins that are thought to constitute an important component of plant defense against fungal pathogens. There are a number of such defensins expressed in various plant tissues with differing antifungal activity and spectrum. Relatively little is known about the modes of action and biological roles of these proteins. Our previous work on a virally encoded fungal toxin, KP4, from Ustilago maydis and subsequently with the plant defensin, MsDef1, from Medicago sativa demonstrated that some of these proteins specifically blocked calcium channels in both fungi and animals. The results presented here demonstrate that KP4 and three plant defensins, MsDef1, MtDef2, and RsAFP2, all inhibit root growth in germinating Arabidopsis seeds at low micromolar concentrations. We have previously demonstrated that a fusion protein composed of Rab GTPase (RabA4b) and enhanced yellow fluorescent protein (EYFP) is dependent upon calcium gradients for localization to the tips of the growing root hairs in Arabidopsis thaliana. Using this tip-localized fusion protein, we demonstrate that all four proteins rapidly depolarize the growing root hair and block growth in a reversible manner. This inhibitory activity on root and root hair is not directly correlated with the antifungal activity of these proteins and suggests that plants apparently express targets for these antifungal proteins. The data presented here suggest that plant defensins may have roles in regulating plant growth and development. A. Allen and A.K. Snyder contributed equally.     

A new family of plant antifungal proteins.

Plant seeds contain high concentrations of many antimicrobial proteins. These include chitinases, beta-1,3-glucanases, proteinase inhibitors, and ribosome-inactivating proteins. We recently reported the presence in corn seeds of zeamatin, a protein that has potent activity against a variety of fungi but has none of the above activities. Zeamatin is a 22-kDa protein that acts by causing membrane permeabilization Using a novel bioautography technique, we found similar antifungal proteins in the seeds of 6 of 12 plants examined. A polyclonal antiserum was raised against zeamatin and was used in immunoblots to confirm the presence of zeamatinlike proteins in these seeds. N-terminal amino acid sequencing was carried out on the antifungal proteins from corn, oats, sorghum, and wheat, and these sequences revealed considerable homology with each other. Interestingly, these N-terminal sequences are also similar to those of thaumatin, a pathogenesis-related protein from tobacco, and two salt stress-induced proteins. These results indicate that zeamatin is not unique but is a member of a previously unrecognized family of plant defense proteins that may include some species of pathogenesis-related proteins.     

Expression of a lipid transfer protein in Escherichia coli and its phosphorylation by a membrane-bound calcium-dependent protein kinase

Ha-AP10 is a basic antifungal peptide from sunflower seeds (Helianthus annuus antifungal peptide of 10 kDa) belonging to the family of plant lipid transfer proteins. We report here its expression in E. coli [Glutathione S-transferase (GST) system] and its phosphorylation by endogenous membrane-bound calcium-dependent protein kinases.     

Characterization and expression of an nsLTPs-like antimicrobial protein gene from motherwort (Leonurus japonicus)

In screening for potent antimicrobial proteins (AMPs) from plant seeds, we had purified a heat-stable AMP, LJAMP2, from the seeds of a medicine herb, motherwort (Leonurus japonicus Houtt). In an in vitro assay, the protein can inhibit the growth of both fungi and bacteria. Then a cDNA encoding LJAMP2 was cloned by the rapid amplification of cDNA ends based on the N-terminal amino acid sequence determined. The deduced amino acid sequences of this cDNA show similarity to plant non-specific lipid transfer proteins. Northern blotting assay revealed that this nsLTP-like gene, designated LJAMP2, was expressed in seeds. Overexpression of LJAMP2 in tobacco enhanced resistance to the fungal pathogen Alternaria alternata and the bacterial pathogen Ralstonia solanacearum, significantly, while no visible alteration in plant growth and development. Our data confirm the antifungal and antibacterial function of LJAMP2 from motherwort seeds and suggest the potential of LJAMP2 in improving disease resistance in plants.     

Antimicrobial peptides from Amaranthus caudatus seeds with sequence homology to the cysteine/glycine-rich domain of chitin-binding proteins.

Two antimicrobial peptides (Ac-AMP1 and Ac-AMP2) were isolated from seeds of amaranth (Amaranthus caudatus), and their physicochemical and biological properties were characterized. On the basis of fast atom bombardment mass spectroscopy, Ac-AMP1 and Ac-AMP2 have monoisotopic molecular masses of 3025 and 3181, respectively. Both proteins have pI values above 10. The amino acid sequence of Ac-AMP1 (29 residues) is identical to that of Ac-AMP2 (30 residues), except that the latter has 1 additional residue at the carboxyl terminus. The sequences are highly homologous to the cysteine/glycine-rich domain occurring in many chitin-binding proteins. Both Ac-AMP1 and Ac-AMP2 bind to chitin in a reversible way. Ac-AMP1 and Ac-AMP2 inhibit the growth of different plant pathogenic fungi at much lower doses than other known antifungal chitin-binding proteins. In addition, they show some activity on Gram-positive bacteria. The antimicrobial effect of Ac-AMP1 and Ac-AMP2 is strongly antagonized by cations.     

骆驼蓬种子中一种具抗肿瘤活性蛋白的分离纯化及鉴定

骆驼蓬种子经浸提、硫酸铵沉淀、CM阳离子交换层析和Superdex 75凝胶过滤层析分离纯化得到一种具有抗肿瘤细胞增殖活性的蛋白(命名为PhLTP),经Tricine-SDS-PAGE检测为单一蛋白条带,高效液相色谱检测其表观分子量为14.8 kDa左右,表明PhLTP是由两条相同的亚基组成的蛋白.采用Edman降解法对该纯化蛋白的N-末端进行氨基酸测序,其N-末端序列与其他植物非特异性脂转移蛋白相似.对PhLTP抗肿瘤活性进行研究,结果表明其对HeLa、Eca-109、MGC-9和BEL-7404细胞都有增殖抑制活性,其中对HeLa细胞增殖的抑制作用较好,并具有浓度和时间依赖性,其IC50为45 μg/mL.通过Hoechst33258染色观察细胞形态,发现PhLTP能诱导HeLa细胞发生凋亡.     

Comparison of solution and crystal structures of maize nonspecific lipid transfer protein: A model for a potential in vivo lipid carrier protein

The three-dimensional solution structure of maize nonspecific lipid transfer protein (nsLTP) obtained by nuclear magnetic resonance (NMR) is compared to the X-ray structure. Although both structures are very similar, some local structural differences are observed in the first and the fourth helices and in several side-chain conformations. These discrepancies arise partly from intermolecular contacts in the crystal lattice. The main characteristic of nsLTP structures is the presence of an internal hydrophobic cavity whose volume was found to vary from 237 to 513 ų without major variations in the 15 solution structures. Comparison of crystal and NMR structures shows the existence of another small hollow at the periphery of the protein containing a water molecule in the X-ray structure, which could play an important structural role. A model of the complexed form of maize nsLTP by α-lysopalmitoylphosphatidylcholine was built by docking the lipid inside the protein cavity of the NMR structure. The main structural feature is a hydrogen bond found also in the X-ray structure of the complex maize nsLTP/palmitate between the hydroxyl of Tyr81 and the carbonyl of the lipid. Comparison of 12 primary sequences of nsLTPs emphasizes that all residues delineating the cavities calculated on solution and X-ray structures are conserved, which suggests that this large cavity is a common feature of all compared plant nsLTPs. Furthermore several conserved basic residues seem to be involved in the stabilization of the protein architecture. Proteins 31:160–171, 1998. © 1998 Wiley-Liss, Inc.     

Five disulfide bridges stabilize a hevein-type antimicrobial peptide from the bark of spindle tree (Euonymus europaeus L.)

A small 45 amino acid residue antifungal polypeptide was isolated from the bark of spindle tree (Euonymus europaeus L.). Though the primary structure of this so-called E. europaeus chitin-binding protein or Ee-CBP is highly similar to the hevein domain, it distinguishes itself from most previously identified hevein-type antimicrobial peptides (AMP) by the presence of two extra cysteine residues that form an extra disulfide bond. Due to these five disulfide bonds Ee-CBP is a remarkably stable protein. Agar diffusion and microtiterplate assays demonstrated that Ee-CBP is a potent antimicrobial protein. IC₅₀-values as low as 1 μg/ml were observed for the fungus Botrytis cinerea. Comparative assays further demonstrated that Ee-CBP is a stronger inhibitor of fungal growth than Ac-AMP2 from Amaranthus caudatus seeds, which is considered one of the most potent antifungal hevein-type plant proteins.