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.     

Disulfide bond assignment, lipid transfer activity and secondary structure of a 7-kDa plant lipid transfer protein, LTP2.

The 7-kDa lipid transfer proteins, LTP2s, share some amino-acid sequence similarities with the 9-kDa isoforms, LTP1s. Both proteins display an identical cysteine motif and, in this regard, LTP2s have been classified as lipid transfer proteins. However, in contrast with LTP1s, no data are available on their structure, cysteine pairings, lipid transfer and lipid binding properties. We reported on the isolation of two isoforms of 7-kDa lipid transfer protein, LTP2, from wheat seeds and showed for the first time that they indeed display lipid transfer activity. Trypsin and chymotrypsin digestions of the native LTP2 afforded the sequence of both isoforms and assignment of disulfide bonds. The cysteine pairings, Cys10--Cys24, Cys25--Cys60, Cys2--Cys34, Cys36--Cys67, revealed a mismatch at the Cys34-X-Cys36 motif of LTP2 compared to LTP1. Moreover, the secondary structure as determined by circular dichroism suggested an identical proportion of alpha helices, beta sheets and random coils. By analogy with the structure of the LTP1, we discussed what structural changes are required to accommodate the LTP2 disulfide pattern.     

Puroindolines form ion channels in biological membranes

Wheat seeds contain different lipid binding proteins that are low molecular mass, basic and cystine-rich proteins. Among them, the recently characterized puroindolines have been shown to inhibit the growth of fungi in vitro and to enhance the fungal resistance of plants. Experimental data, using lipid vesicles, suggest that this antimicrobial activity is related to interactions with cellular membranes, but the underlying mechanisms are still unknown. This paper shows that extracellular application of puroindolines on voltage-clamped Xenopus laevis oocytes induced membrane permeabilization. Electrophysiological experiments, on oocytes and artificial planar lipid bilayers, suggest the formation, modulated by voltage, of cation channels with the following selectivity: Cs(+) > K(+) > Na(+) > Li(+) > choline = TEA. Furthermore, this channel activity was prevented by addition of Ca(2+) ions in the medium. Puroindolines were also able to decrease the long-term oocyte viability in a voltage-dependent manner. Taken together, these results indicate that channel formation is one of the mechanisms by which puroindolines exert their antimicrobial activity. Modulation of channel formation by voltage, Ca(2+), and lipids could introduce some selectivity in the action of puroindolines on natural membranes.     

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.     

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.     

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.     

Intracellular localization of a lipid transfer protein in Vigna unguiculata seeds

Lipid transfer proteins (LTP) facilitate transfer of lipids between membranes in vitro. Up to now, they have been found to be localized basically in the plant cell wall and in compartments linked to lipid metabolism, such as glyoxysomes. Accordingly, LTP are considered to be involved in the plant defence against pathogen microbes and lipid metabolism. We herein show, by immunoelectron microscopy, that besides the cell wall, LTP are localized in the lumen of organelles which we suggest to be the protein storage vacuoles, as well as in vesicles similar to the lipid-containing ones and in the extracellular space of Vigna unguiculata seeds. To further characterize these organelles, we performed subcellular fractionation of membranes isolated from imbibed seeds on a sucrose-density gradient. The analysis of these fractions revealed that the lightest membrane vesicles, derived probably from PSV, contain LTP, α-TIP and K⁺ independent PP_iase, but not γ-TIP and K⁺ stimulated PP_iase. The presence of LTP and vicilins (typical storage protein) in the lumen of these vesicles was confirmed by immunoelectron microscopy. Taken together, the data suggest that the intracellular LTP in the V. unguiculata seeds are localized in protein storage vacuoles and in lipid-containing vesicles.     

An Antifungal Protein Purified from Pearl Millet Seeds Shows Sequence Homology to Lipid Transfer Proteins

In the course of a search for antifungal proteins from plant seeds, we observed inhibition of mycelial growth of Trichoderma viride with extracts of pearl millet. We have identified several proteins with antifungal properties in the seeds of pearl millet. One of these proteins has been purified to homogeneity and characterized. The purified protein has a molecular mass of 25 kDa. The N-terminal sequence of the protein (25 residues) shows homology to non-specific lipid transfer proteins (LTPs) of cotton, wheat and barley. The purified LTP inhibited mycelial growth of T. viride and the rice sheath blight fungus, Rhizoctonia solani in vitro.     

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.     

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.     

A novel lipid transfer protein from the dill Anethum graveolens L.: isolation,structure, heterologous expression,and functional characteristics

A novel lipid transfer protein, designated as Ag‐LTP, was isolated from aerial parts of the dill Anethum graveolens L. Structural, antimicrobial, and lipid binding properties of the protein were studied. Complete amino acid sequence of Ag‐LTP was determined. The protein has molecular mass of 9524.4 Da, consists of 93 amino acid residues including eight cysteines forming four disulfide bonds. The recombinant Ag‐LTP was overexpressed in Escherichia coli and purified. NMR investigation shows that the Ag‐LTP spatial structure contains four α ‐helices, forming the internal hydrophobic cavity, and a long C‐terminal tail. The measured volume of the Ag‐LTP hydrophobic cavity is equal to ~800 A³, which is much larger than those of other plant LTP1s. Ag‐LTP has weak antifungal activity and unpronounced lipid binding specificity but effectively binds plant hormone jasmonic acid. Our results afford further molecular insight into biological functions of LTP in plants. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Diversity of puroindolines as revealed by two-dimensional electrophoresis

Puroindolines are endosperm lipid binding proteins, which are separated by reversed phase-high-performance liquid chromatography or cation exchange chromatography into two isoforms, puroindoline-a (PIN-a) and puroindoline-b (PIN-b). Being very basic and close in molecular weight, PIN-a and PIN-b have never been separated using conventional isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A two-dimensional electrophoresis method, linear immobiline pH gradient (IPGxSDS-PAGE), was developed, using 6-11 linear immobiline Dry Strips in the first dimension, which allowed the puroindolines to be focused between isoelectric point 10.5 and 11. Immunoblotting revealed that both PIN-a and PIN-b were each composed of several spots. Two-dimensional patterns from unrelated wheat varieties revealed that several spots can be highlighted among varieties. Matrix-assisted laser desorption/ionization-time of flight spectrometry allowed the majority of the spots revealed in the puroindoline zone to be identified. The two-dimensional IPGxSDS-PAGE of these very basic wheat endosperm proteins, puroindolines and related grain softness proteins should facilitate the identification of the proteins associated with wheat endosperm texture that have a strong effect on milling, dough properties and end-uses of wheats.     

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.     

Specific adduction of plant lipid transfer protein by an allene oxide generated by 9-lipoxygenase and allene oxide synthase

Lipid transfer proteins (LTPs) are ubiquitous plant lipid-binding proteins that have been associated with multiple developmental and stress responses. Although LTPs typically bind fatty acids and fatty acid derivatives in a non-covalent way, studies on the LTPs of barley seeds have identified an abundantly occurring covalently modified form, LTP1b, the lipid ligand of which has resisted clarification. In the present study, this adduct was identified as the alpha-ketol 9-hydroxy-10-oxo-12(Z)-octadecenoic acid. Further studies on the formation of LTP1b demonstrated that the ligand was introduced by nucleophilic attack of the free carboxylate group of the Asp-7 residue of the protein at carbon-9 of the allene oxide fatty acid 9(S),10-epoxy-10,12(Z)-octadecadienoic acid. This reactive oxylipin was produced in barley seeds by oxygenation of linoleic acid by 9-lipoxygenase followed by dehydration of the resulting hydroperoxide by allene oxide synthase. The generation of protein-oxylipin adducts represents a new function for plant allene oxide synthases, enzymes that have earlier been implicated mainly in the biosynthesis of the jasmonate family of plant hormones. Additionally, the LTP-allene oxide synthase interaction opens new perspectives regarding the roles of LTPs in the signaling of plant defense and development.     

Binding of two mono-acylated lipid monomers by the barley lipid transfer protein, LTP1, as viewed by fluorescence, isothermal titration calorimetry and molecular modelling.

The binding of two mono-acylated lipid monomers by plant lipid transfer proteins (LTP1s) presents an attractive field of research that could help our understanding of the functional role of this protein family. This task has been investigated in the case of barley LTP1 because it is known to exhibit a small cavity in its free state. The titration with lipids could not be followed by fluorescence with the native protein. Indeed, this LTP1 possesses a tyrosine residue on its C-terminus, Tyr91, which is not sensitive to lipid binding but mainly contributes to the fluorescence signal intensity. However, the binding of 1-myristoylglycerophosphatidylcholine (MyrGro-PCho) could be monitored by fluorescence after removal of Tyr91 by a carboxypeptidase. These experiments returned a dissociation constant of about 1 microM and showed that the protein can indeed bind two monomers. This result was corroborated by molecular modelling where the structure of the complex between barley LTP1 and MyrGro-PCho was derived from that determined in the case of wheat [Charvolin, D., Douliez, J.P., Marion, D., Cohen-addad, C. & Pebay-Peyroula, E. (1999) Eur. J. Biochem. 264, 562-568.]. Results from isothermal titration calorimetry experiments indicated non-classic titration behaviour but also suggested that two lipids could be bound by the protein. Finally, barley LTP1 binds two omega-hydroxypalmitic acid, a compound found in the family of cutin monomers. The fact that the binding of two lipids could be related to the physiological role of this protein family is discussed.     

Proteomes of hard and soft near-isogenic wheat lines reveal that kernel hardness is related to the amplification of a stress response during endosperm development

Wheat kernel texture, a major trait determining the end-use quality of wheat flour, is mainly influenced by puroindolines. These small basic proteins display in vitro lipid binding and antimicrobial properties, but their cellular functions during grain development remain unknown. To gain an insight into their biological function, a comparative proteome analysis of two near-isogenic lines (NILs) of bread wheat Triticum aestivum L. cv. Falcon differing in the presence or absence of the puroindoline-a gene (Pina) and kernel hardness, was performed. Proteomes of the two NILs were compared at four developmental stages of the grain for the metabolic albumin/globulin fraction and the Triton-extracted amphiphilic fraction. Proteome variations showed that, during grain development, folding proteins and stress-related proteins were more abundant in the hard line compared with the soft one. These results, taken together with ultrastructural observations showing that the formation of the protein matrix occurred earlier in the hard line, suggested that a stress response, possibly the unfolded protein response, is induced earlier in the hard NIL than in the soft one leading to earlier endosperm cell death. Quantification of the albumin/globulin fraction and amphiphilic proteins at each developmental stage strengthened this hypothesis as a plateau was revealed from the 500 °Cd stage in the hard NIL whereas synthesis continued in the soft one. These results open new avenues concerning the function of puroindolines which could be involved in the storage protein folding machinery, consequently affecting the development of wheat endosperm and the formation of the protein matrix.     

Lipid transfer proteins

Translocations of various lipid species between membranes have been extensively studied. The transport of water-insoluble lipids is thought to require the participation of lipid transfer proteins (LTP). Several LTP, differing in their physiochemical properties and substrate specificities, have been purified to homogeneity from blood plasma, eucaryotic and procaryotic cells. Depending on their site of activity, they can be classified as extracellular and intracellular LTP. Extracellular LTP are found in the blood plasma and intracellular LTP, which were originally characterized as phospholipid exchange proteins, are ubiquitous in nature. Despite the enormous knowledge about their physicochemical properties and their function in vitro their physiological role has not been clearly demonstrated. However, their ubiquitous occurrence indicates an important role in cellular events. This review gives an overview of this interesting category of proteins, which are able to catalyze inter-membrane transfer and exchange of lipids.     

Cloning and expression of two plant proteins: similar antimicrobial activity of native and recombinant form

Antimicrobial peptides and proteins are being studied with increasing interest because of their broad range antimicrobial activity. Among plant antimicrobial proteins, the wheat seed polypeptides, puroindoline a and puroindoline b, are particularly interesting because of their established antibacterial activity. In this paper we describe different strategies used to clone His tagged and GST tagged puroindolines obtaining 1.5 mg recombinant protein from 1 l culture. The antimicrobial activity of recombinant and native puroindolines was comparable.     

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.