Comparison of lipid binding and transfer properties of two lipid transfer proteins from plants

Plant lipid transfer proteins (LTPs) are soluble proteins which are characterized by their in vitro ability to transfer phospholipids between two membranes. We have compared the functional properties of two LTPs purified from maize and wheat seeds knowing that, despite a high degree of sequence identity, the two proteins exhibit structural differences. It was found that wheat LTP had a lower transfer activity than the maize LTP, consistent with a lower kinetics of fatty acid binding. The lower affinity for the fatty acids of the wheat LTP could be explained by a narrowing occurring in the middle part of the binding site, as revealed by comparing the fluorescence spectra of various anthroyloxy-labeled fatty acids associated with the two LTPs. The affinity for some natural fatty acids was studied by competition with fluorescent fatty acids toward binding to the protein. Again, wheat LTP had a lower affinity for those molecules. All together, these observations reveal the complexity of the LTP family in plants, probably reflecting the multiple roles played by these proteins.     

Solution structure of a tobacco lipid transfer protein exhibiting new biophysical and biological features

Plant lipid transfer proteins are small soluble extracellular proteins that are able to bind and transfer a variety of lipids in vitro. Recently, it has been proposed that lipid transfer proteins may play a key role in plant defence mechanisms, especially during the induction of systemic acquired resistance. However, very little is known about the proteins expressed in developing plants and tissues, since almost all the biophysical and structural data available to date on lipid transfer proteins originate from proteins present in storage tissues of monocot cereal seeds. In this paper, we report the structural and functional characteristics of a lipid transfer protein (named LTP1_1) constitutively expressed in young aerial organs of Nicotiana tabacum (common tobacco). The unlabelled and uniformly labelled proteins were produced in the yeast Pichia pastoris, and we determined the three-dimensional (3D) structure of LTP1_1 using nuclear magnetic resonance (NMR) spectroscopy and molecular modeling techniques. The global fold of LTP1_1 is very close to the previously published structures of LTP1 extracted from cereal seeds, including an internal cavity. However, the chemical shift variations of several NMR signals upon lipid binding show that tobacco LTP1_1 is able to bind only one LysoMyristoylPhosphatidylCholine (LMPC), while wheat and maize LTPs can bind either one or two. Titration experiments using intrinsic tyrosine fluorescence confirm this result not only with LMPC but also with two fatty acids. These differences can be explained by the presence in tobacco LTP1_1 of a hydrophobic cluster closing the second possible access to the protein cavity. This result suggests that LTP1 lipid binding properties could be modulated by subtle changes in a conserved global structure. The biological significance of this finding is discussed in the light of the signalling properties of the tobacco LTP1_1-jasmonate complex described elsewhere.     

Computational design and biochemical characterization of maize nonspecific lipid transfer protein variants for biosensor applications

Lipid transfer proteins (LTPs) are a family of proteins that bind and transfer lipids. Utilizing the maize LTP, we have successfully engineered fluorescent reagentless biosensors for the natural ligand of LTPs; this was achieved by using computational protein design to remove a disulfide bridge and attaching a thio-reactive fluorophore. Conformational change induced by ligand titration is thought to affect the fluorescence of the fluorophore, allowing detection of ligand binding. Fluorescence measurements show that our LTP variants have affinity to palmitate that is consistent with wild-type LTP. These molecules have the potential to be utilized as scaffolds to design hydrophobic ligand biosensors or to serve as drug carriers.     

Homology modelling and molecular dynamics aided analysis of ligand complexes demonstrates functional properties of lipid‐transfer proteins encoded by the barley low‐temperature‐inducible gene family, blt4

The homology modelling technique was used to predict the tertiary structures of three members of the low-temperature-inducible barley vegetative shoot epidermal lipid-transfer protein (LTP) family, BLT4, on the basis of the X-ray crystallographically determined three-dimensional structure of a maize seedling LTP. Differences between the maize LTP and the BLT4 family include amino acid substitutions around the entrance and inside the predicted hydrophobic binding tunnels of these proteins. Because of the deletion of the loop region corresponding to Val60–Gly62 of the maize LTP from all three BLT4 LTPs, their internal hydrophobic tunnels are longer. Molecular dynamics modelling shows that BLT4.9 can accommodate hexadecanoic acid in its binding tunnel in similar conformation to the maize LTP. However, modelled cis,cis-9,12-octadecandienoic acid had a more favourable interaction with the BLT4.9 LTP than with the maize protein. Di-cis,cis-9,12-octadecandienoyl phosphatidylglycerol and di-cis,cis-9,12-octadecandienoyl phosphatidylcholine were modelled in the BLT4.9 structure with the fatty acyl group at position 1 embedded in the binding tunnel and the group at position 2 located on the solvent accessible surface of the protein. The results of the modelling suggest that the phospholipid headgroup can form hydrogen and salt bridges with polar and charged residues outside the binding tunnel and the exposed hydrocarbon chain interacts with hydrophobic amino acids on the surface. These results are consistent with the proposal that BLT4 LTPs have a lipid-transfer function associated with frost acclimation in barley.     

Role of plant lipid transfer proteins in plant cell physiology-a concise review

Plant lipid transfer proteins (LTP) are cationic peptides, subdivided into two families, which present molecular masses of around 7 and 10 kDa. The peptides were, thus, denominated due to their ability to reversibly bind and transport hydrophobic molecules in vitro. Both subfamilies possess conserved patterns of eight cysteine residues and the three-dimensional structure reveals an internal hydrophobic cavity that comprises the lipid binding site. Based on the growing knowledge regarding structure, gene expression and regulation and in vitro activity, LTPs are likely to play a role in key processes of plant physiology. Although the roles of plant LTPs have not yet been fully determined. This review aims to present comprehensive information of recent topics, cover new additional data, and present new perspectives on these families of peptides.     

Lipid transfer proteins enhance cell wall extension in tobacco

Plant cells are enclosed by a rigid cell wall that counteracts the internal osmotic pressure of the vacuole and limits the rate and direction of cell enlargement. When developmental or physiological cues induce cell extension, plant cells increase wall plasticity by a process called loosening. It was demonstrated previously that a class of proteins known as expansins are mediators of wall loosening. Here, we report a type of cell wall-loosening protein that does not share any homology with expansins but is a member of the lipid transfer proteins (LTPs). LTPs are known to bind a large range of lipid molecules to their hydrophobic cavity, and we show here that this cavity is essential for the cell wall-loosening activity of LTP. Furthermore, we show that LTP-enhanced wall extension can be described by a logarithmic time function. We hypothesize that LTP associates with hydrophobic wall compounds, causing nonhydrolytic disruption of the cell wall and subsequently facilitating wall extension.     

Lipid-transfer proteins: Tools for manipulating membrane lipids

Like other eukaryotic cells, plant cells contain proteins able to bind or to transfer lipids. Since they are able to facilitate movements of various phospholipids between membranes and are also capable of binding fatty acids or acyl-CoAs, they have been termed lipid-transfer proteins (LTP). LTPs are basic proteins containing 90 to 95 residues (molecular mass 9 kDa), eight of them being cysteines found in conserved locations. These proteins have been used to manipulate in vitro the lipid composition of isolated membranes either from plant or mammalian sources. In addition to purified LTPs, recombinant LTPs produced by genes expressed in microorganisms can be used for this purpose. Several genes coding for these proteins have been characterized in various plants with different patterns of expression. However, it remains to be investigated whether these recombinant proteins behave functionally as LTPs. The use of purified or recombinant LTPs is promising for the study of the effect of lipid composition on membrane functional properties.     

Calmodulin binds to maize lipid transfer protein and modulates its lipids binding ability

Although plant non-specific lipid transfer proteins (ns-LTPs) are characterized by their ability to bind and transfer a broad range of hydrophobic ligands in vitro, their biological functions in vivo remain unclear. Recently, it has been proposed that ns-LTPs may play a key role in plant defense mechanisms, particularly during the induction of systemic acquired resistance, however, very little is known about the regulation in this process. We report that the binding of maize non-specific lipid transfer protein (Zm-LTP) to calmodulin (CaM) is in a calcium-independent manner. To better understand the interaction mechanism between Zm-LTP and CaM, the CaM-binding site of Zm-LTP was mapped to the region of amino acids 46-60. Point mutations indicate that four amino acid residues, R46, R47, K54 and R58, in this region are crucial for binding. Furthermore, we tested the effects of CaM on the lipid-binding activity of Zm-LTP in the presence of Ca(2+), EGTA, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide and trifluoperazine respectively. We also investigated the structural features of CaM-binding motifs in LTPs from different species and strong differences were observed. Taken together, our results suggest that the interaction with CaM could be a common feature of plant LTPs. The identification and characterization of CaM-binding domain of LTPs should provide new insights into the mechanism by which the physiological functions of LTPs are regulated.     

Plant lipid binding proteins: properties and applications

Lipid transfer proteins (LTP) and puroindolines are abundant lipid binding proteins of plant seeds. While LTP are ubiquitous plant proteins, puroindolines are only found in the seeds of plants from the Triticae and Avenae tribes. These proteins display a similar overall folding pattern but different lipid binding properties. The unique and diverse biological and technological functions of LTPs and puroindolines are closely related to their structural and lipid binding properties. These proteins are attractive to improve the agronomic performances and food quality of crops. Heterologous expression and genetic engineering should allow industrial production and enlarge applications of these lipid binding proteins.     

转脂蛋白CaMBP10的胶体金标记及对白菜中CaMBP10结合蛋白的鉴定

植物转脂蛋白 (LTP)是一类广泛存在于高等植物中的空间结构高度保守的碱性小分子蛋白,其确切功能和调节机制至今仍不清楚.本室从白菜中分离的钙调素结合 蛋白10 (CaMBP10),经序列分析被鉴定为植物转脂蛋白家族成员.近期研究结果表明 ,CaMBP10 参与了植物的生物与非生物胁迫反应.为了深入探讨CaMBP10的抗性机制,确定植物中与其相互作用的蛋白质,本文拟建立胶体金标记CaMBP10 的方法,通过凝胶覆盖分析,检测植物样品中的CaMBP10 结合蛋白为此,对标记反应的最适条件进行了优化,确定最佳条件为：交联剂戊二醛用量为0.034%,交联反应pH值为7 .0,交联反应时间为40 min,胶体金颗粒度为10 nm,胶体金溶液的pH为7.0. 本文确定建立了植物样品中CaMBP10结合蛋白的分析与鉴定方法.     

Modulation of the biological activity of a tobacco LTP1 by lipid complexation

Plant lipid transfer proteins (LTPs) are small, cysteine-rich proteins secreted into the extracellular space. They belong to the pathogenesis-related proteins (PR-14) family and are believed to be involved in several physiological processes including plant disease resistance, although their precise biological function is still unknown. Here, we show that a recombinant tobacco LTP1 is able to load fatty acids and jasmonic acid. This LTP1 binds to specific plasma membrane sites, previously characterized as elicitin receptors, and is shown to be involved in the activation of plant defense. The biological properties of this LTP1 were compared with those of LTP1-linolenic and LTP1-jasmonic acid complexes. The binding curve of the LTP1-linolenic acid complex to purified tobacco plasma membranes is comparable to the curve obtained with LTP1. In contrast, the LTP1-jasmonic acid complex shows a strongly increased interaction with the plasma membrane receptors. Treatment of tobacco plants with LTP1-jasmonic acid resulted in an enhancement of resistance toward Phytophthora parasitica. These effects were absent upon treatment with LTP1 or jasmonic acid alone. This work presents the first evidence for a biological activity of a LTP1 and points out the crucial role of protein-specific lipophilic ligand interaction in the modulation of the protein activity.     

Effects of acylation on the structure,lipid binding,and transfer activity of wheat lipid transfer protein

Study of the effect of protein chemical acylation on their functional properties or activity often brings valuable information regarding structure-function relationships. We performed such work on wheat lipid transfer protein, LTP1, to investigate the role of grafted acyl chains on the lipid binding and transfer properties. LTP1 was acylated by using anhydride derivatives of various chain lengths from C2 to C6. Only the chemical modifications with hexanoic acid yielded a marked effect on the tertiary structure and a slight change in the secondary structure. The affinity of the modified proteins for myristoyl-lysophosphatidylcholine was similar to that of the native protein accompanied by a slight decrease in stoichiometry. Interestingly, the acylation of LTP1 enhanced the lipid transfer activity by at least a factor of 10 for hexanoic chain length. Finally, the grafting of acyl chains was investigated by means of molecular modelling, and an attempt is made to correlate with our experimental data.     

Accessibility of tobacco lipid transfer protein cavity revealed by 15N NMR relaxation studies and molecular dynamics simulations

Plant LTP1 are small helical proteins stabilized by four disulfide bridges and are characterized by the presence of an internal cavity, in which various hydrophobic ligands can be inserted. Recently, we have determined the solution structure of the recombinant tobacco LTP1_1. Unexpectedly, despite a global fold very similar to the structures already known for cereal seed LTP1, its binding properties are different: Tobacco LTP1_1 is able to bind only one monoacylated lipid, whereas cereal LTP1 can bind either one or two. The 3D structure of tobacco LTP1_1 revealed the presence of a hydrophobic cluster, not observed on cereal LTP1 structures, which may hinder one of the two entrances of the cavity defined for wheat LTP1. To better understand the mechanism of lipid entrance for tobacco LTP1_1 and to define the regions of the protein monitoring the accessibility of the cavity, we have complemented our structural data by the study of the internal dynamics of tobacco LTP1_1, using (15)N magnetic relaxation rate data and MD simulations at room and high temperatures. This work allowed us to define two regions of the protein experiencing the largest motions. These two regions delineate a portal that opens up during the simulation constituting a unique entrance of the hydrophobic cavity, in contrast with wheat LTP1 where two routes were detected. The hydrophobic interactions resulting from a few point mutations are strong enough to completely block the second portal so that the accessibility of the cavity is restricted to one entrance, explaining why this particular LTP1 binds only one lipid molecule.     

The crystal structure of oxylipin-conjugated barley LTP1 highlights the unique plasticity of the hydrophobic cavity of these plant lipid-binding proteins

The barley lipid transfer protein (LTP1) adducted by an α-ketol, (9-hydroxy-10-oxo-12(Z)-octadecenoic acid) exhibits an unexpected high lipid transfer activity. The crystal structure of this oxylipin-adducted LTP1, (LTP1b) was determined at 1.8 Å resolution. The covalently bound oxylipin was partly exposed at the surface of the protein and partly buried within the hydrophobic cavity. The structure of the oxylipidated LTP1 emphasizes the unique plasticity of the hydrophobic cavity of these plant lipid-binding proteins when compared to the other members of the family. The plasticity of the hydrophobic cavity and increase of its surface hydrophobicity induced by the oxylipin account for the improvement of the lipid transfer activity of LTP1b. These observations open new perspectives to explore the different biological functions of LTPs, including their allergenic properties.     

Use of a Fluorimetric Method to Assay the Binding and Transfer of Phospholipids by Lipid Transfer Proteins from Maize Seedlings and Arabidopsis Leaves

A fluorimetric method has been used to study the binding andtransfer of phospholipids mediated by a lipid transfer protein(LTP) from maize seedlings and by proteins extracted from Arabidopsisleaves. This method is based on the use of donor vesicles preparedby sonication or injection and consisting of either self quenchingvesicles of (1-palmitoyl 2-{12-{(7-nitrobenzoxadiazol-4-yl)amino}dodecanoyl}-Sn-glycero-3-phosphocholine(NBD-PC) or trinitrophenylphosphate phosphatidyl ethanolamine(TNP-PE) quenched vesicles of l-palmitoyl-2(l-pyredecanoyl)-Snglycero-3-phosphocholine(Pyr-PC). Acceptor vesicles consisted in a mixture (90/10; v/v)of dioleoylphosphatidylcholine and phosphatidyl inositol (DOPC-PI).The use of injected vesicles of Pyr-PC allowed to achieve sensitiveand qualitative tests of binding and transfer since 0.05 µMLTP were detected. By contrast, when sonicated vesicles of NBD-PCwere used, quantitative determinations of phospholipid-LTP complexas well as measurement of the initial rate of phospholipid transferon a large range of protein concentration (0.5 to 20 µM)were performed. This fluorimetric method has been successfullyused to study the activity of maize LTP during its purificationor the activity of LTPs present in Arabidopsis extracts. (Received December 5, 1993; Accepted December 7, 1993)     

Resistance function of rice lipid transfer protein LTP110

Plant lipid transfer proteins (LTPs) are a class of proteins whose functions are still unknown. Some are proposed to have antimicrobial activities. To understand whether LTP110, a rice LTP that we previously identified from rice leaves, plays a role in the protection function against some serious rice pathogens, we investigated the antifungal and antibacterial properties of LTP110. A cDNA sequence, encoding the mature peptide of LTP110, was cloned into the Impact-CN prokaryotic expression system. The purified protein was used for an in vitro inhibition test against rice pathogens, Pyricularia oryzae and Xanthomonas oryzae. The results showed that LTP110 inhibited the germination of Pyricularia oryzae spores, and its inhibitory activity decreased in the presence of a divalent cation. This suggests that the antifungal activity is affected by ions in the media; LTP110 only slightly inhibited the growth of Xanthomonas oryzae. However, the addition of LTP110 to cultured Chinese hamster ovarian cells did not retard growth, suggesting that the toxicity of LTP110 is only restricted to some cell types. Its antimicrobial activity is potentially due to interactions between LTP and microbe-specific structures.     

Complete amino acid sequence determination of the major allergen of peach (Prunus persica) Pru p 1

The major protein allergen of peach (Prunus persica), Pru p 1, has recently been identified as a lipid transfer protein (LTP). The complete primary structure of Pru p 1, obtained by direct amino acid sequence and liquid chromatography-mass spectrometry (LC-MS) analyses with the purified protein, is described here. The protein consists of 91 amino acids with a calculated molecular mass of 9178 Da. The amino acid sequence contains eight strictly conserved cysteines, as do all known LTPs, but secondary structure predictions failed to classify the peach 9 kDa protein as an 'all-alpha type', due to the high frequency of amino acids (nine prolines) disrupting alpha helices. Although the sequence similarity with maize LTP is only 63%, out of the 25 amino acids forming the inner surface of the tunnel-like hydrophobic cavity in maize ns-LTP 16 are identical and 7 similar in the peach homolog, supporting the hypothesis of a similar function.     

The biochemistry and biology of extracellular plant lipid-transfer proteins (LTPs)

Plant lipid-transfer proteins (LTPs) are abundant, small, lipid binding proteins that are capable of exchanging lipids between membranes in vitro. Despite their name, a role in intracellular lipid transport is considered unlikely, based on their extracellular localization. A number of other biological roles, including antimicrobial defense, signaling, and cell wall loosening, have been proposed, but conclusive evidence is generally lacking, and these functions are not well correlated with in vitro activity or structure. A survey of sequenced plant genomes suggests that the two biochemically characterized families of LTPs are phylogenetically restricted to seed plants and are present as substantial gene families. This review aims to summarize the current understanding of LTP biochemistry, as well as the evidence supporting the proposed in vivo roles of these proteins within the emerging post-genomic framework.     

Affinity of phosphatidylcholine molecular species for the bovine phosphatidylcholine and phosphatidylinositol transfer proteins. Properties of the sn-1 and sn-2 acyl binding sites

Both the phosphatidylcholine transfer protein (PC-TP) and the phosphatidylinositol transfer protein (PI-TP) act as carriers of phosphatidylcholine (PC) molecules between membranes. To study the structure of the acyl binding sites of these proteins, the affinity of 32 distinct natural and related PC molecular species was determined by using a previously developed fluorometric competition assay. Marked differences in affinity between species were observed with both proteins. Affinity vs lipid hydrophobicity (determined by reverse-phase HPLC) plots displayed a well-defined maximum indicating that the acyl chain hydrophobicity is an important determinant of binding of a phospholipid molecule by these transfer proteins. However, besides the overall lipid hydrophobicity, steric properties of the individual acyl chains contribute considerably to the affinity, and PC-TP and PI-TP respond differently to modifications of the acyl chain structure. The affinity of PC-TP increased steadily with increasing unsaturation of the sn-2 acyl moiety, resulting in high affinity for species containing four and six double bonds in the sn-2 chain, whereas the affinity of PI-TP first increased up to two to three double bonds and then declined. These data, as well as the distinct effects of sn-2 chain double bond position and bromination, indicate that the sn-2 acyl chain binding sites of the two proteins are structurally quite different. The sn-1 acyl binding sites are dissimilar as well, since variation of the length of saturated sn-1 chain affected the affinity differently. The data are discussed in terms of the structural organization of the sn-1 and sn-2 acyl binding sites of PC-TP and PI-TP.(ABSTRACT TRUNCATED AT 250 WORDS)