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.

Lipid-transfer proteins from plants: structure and binding properties.

Plant cells contain lipid-transfer proteins (LTPs) able to transfer phospholipids between membranes in vitro. Plant LTPs share in common structural and functional features. Recent structural studies carried out by NMR and X-ray crystallography on an LTP isolated from maize seeds have showed that this protein involves four helices packed against a C-terminal region and stabilized by four disulfide bridges. A most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule and able to accommodate acyl chains. It was thus of interest to study the ability of maize LTP to bind hydrophobic ligands such as acyl chains or lysophosphatidylcholine and to determine the effect of this binding on phospholipid transfer. The binding abilities of maize LTP, presented in this paper, are discussed and compared to those of lipid-binding proteins from animal tissues.

Characterisation of acyl binding by a plant lipid-transfer protein.

Maize lipid-transfer protein (LTP) is a small soluble protein which is able to transfer in vitro phospholipids between membranes and to bind fatty acids or lysoderivatives. In the studies reported here, fluorescent-labelled fatty acids were used to characterise the nature of the binding site on LTP. A fluorescent analogue of 12 carbons with a pyrene moiety attached at the end, alone or in conjunction with an anthroyloxy analogue, indicated that LTP could bind two fatty acids although with a marked difference in affinity. The binding capacity was strongly affected after reduction of the protein by dithiothreitol, showing that the four S-S bonds of LTP are essential for its lipid binding property. Other analogues used were 16-carbon or 18-carbon fatty acids with an anthracene moiety attached at different points of the hydrocarbon chain. Emission maxima of these molecules varied with the analogue and suggested a motional constraint for the bound fatty acid which is more important around the middle of the chain than at its extremities. Binding displacement studies were carried out with a wide range of fatty acids or fatty acyl derivatives. Fatty acids of 16 to 19 carbons were found to be the preferred ligands. The presence of one double bond did not change appreciably the affinity of LTP, although the presence of two or three double bonds or of a hydroxyl moiety significantly reduced the affinity. Fatty acyl-CoA or lysoderivatives bound as well as the corresponding fatty acid.

A potent antimicrobial protein from onion seeds showing sequence homology to plant lipid transfer proteins.

An antimicrobial protein of about 10 kD, called Ace-AMP1, was isolated from onion (Allium cepa L.) seeds. Based on the near-complete amino acid sequence of this protein, oligonucleotides were designed for polymerase chain reaction-based cloning of the corresponding cDNA. The mature protein is homologous to plant nonspecific lipid transfer proteins (nsLTPs), but it shares only 76% of the residues that are conserved among all known plant nsLTPs and is unusually rich in arginine. Ace-AMP1 inhibits all 12 tested plant pathogenic fungi at concentrations below 10 micrograms mL-1. Its antifungal activity is either not at all or is weakly affected by the presence of different cations at concentrations approximating physiological ionic strength conditions. Ace-AMP1 is also active on two Gram-positive bacteria but is apparently not toxic for Gram-negative bacteria and cultured human cells. In contrast to nsLTPs such as those isolated from radish or maize seeds, Ace-AMP1 was unable to transfer phospholipids from liposomes to mitochondria. On the other hand, lipid transfer proteins from wheat and maize seeds showed little or no antimicrobial activity, whereas the radish lipid transfer protein displayed antifungal activity only in media with low cation concentrations. The relevance of these findings with regard to the function of nsLTPs is discussed.

Selective modifications of the phospholipid fatty acid composition in human platelet membranes using nonspecific and specific lipid transfer proteins.

In order to specifically modify the fatty acid composition of cell membrane phospholipids, we have developed an original method based on the transfer of pure phospholipid molecular species to membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) subclasses containing 18:2n-6 and 22:6n-3 at the sn-2 position were incorporated into human platelet membranes using the endogenous phosphatidylinositol/PC transfer protein (PI/PC-TP) and the phospholipid transfer protein from maize (L-TP), respectively. PI/PC-TP was shown to catalyze a strict exchange of phospholipids between platelet membranes and unilamellar vesicles containing 1,2-diacylglycerophosphocholine (diacyl-GPC; 16:0/18:2-GPC, or 16:0/22:6-GPC). The proportions of 18:2n-6 and 22:6n-3 in diacyl-GPC of platelet membranes were gradually increased from 10.7 to 16.9% and from 0.8 to 10.1%, respectively, whereas the PE and PI fatty acid compositions were not changed. The diacyl-GPC enrichment in 22:6n-3 and 18:2n-6 did not induce changes in membrane fluidity parameters measured by electron-spin resonance of 5- and 16-nitroxy stearic acids. Similarly, 18:2n-6 and 22:6n-3 esterified in 1,2-diacylglycerophosphoethanolamine (diacyl-GPE) have been incorporated in platelet membranes by an apparent exchange process under conditions where donor vesicles had a phospholipid composition equivalent to that of platelet membranes. The proportions of 18:2n-6 and 22:6n-3 were selectively and progressively increased from 6.0 to 21.2% and from 2.2 to 17.2%, respectively, in diacyl-GPE of platelet membranes. Thus, the L-TP- and PI/PC-TP-catalyzed enrichment can be used for studying the modulation of membrane biological activities by defined changes of fatty acid composition of specific phospholipid classes or subclasses.

Effect of specific phospholipid molecular species incorporated in human platelet membranes on thromboxane A2/prostaglandin H2 receptors.

The incorporation of albumin-bound docosahexaenoic acid (22:6n-3), but not linoleic acid (18:2n-6), into cellular phospholipids inhibits platelet aggregation induced by the thromboxane analogue U46619. [3H]U46619 specific binding to thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, as well as specific binding of the antagonist [3H]SQ29548 to these sites were also decreased in these modified cells (P. G., Swann et al. 1990. J. Biol. Chem. 265: 21692-21697). More than 80% of the 22:6n-3 incorporated in these cells was esterified in the various endogenous phospholipid classes and the remaining was found in neutral lipids and in the unesterified fatty acid pool. In this study, we determined whether the effects observed could be attributed to the esterification of 22:6n-3 in phospholipids and whether the 22:6n-3 biological activity might depend on its esterification in specific phospholipid classes. Therefore, pure phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecular species were transferred to platelet membranes, using lipid transfer proteins. PC and PE containing palmitate (16:0) and 22:6n-3 or 16:0 and 18:2n-6 at position sn-1 and sn-2, respectively, were incorporated into membranes only at the expense of the corresponding endogenous phospholipid class, by an apparent exchange process. When such modified membranes were tested for specific binding of U46619 and SQ29548, a significant decrease of the receptor site affinity was only observed in membranes highly enriched with 1-palmitoyl-2-docosahexaenoyl-glycerophosphocholine (16:0/22:6-GPC). Fluidity parameters measured by electron spin resonance of 5- and 16-nitroxy-stearic acids were not significantly different in membranes enriched with 16:0/22:6-GPC relative to those enriched with 16:0/18:2n-6-GPC, arguing against a generalized perturbation of the membrane due to 22:6n-3 incorporation. We conclude that molecular species of PC with 22:6n-3 at the sn-2 position can affect TXA2/PGH2 receptors. The selectivity of the inhibitory effect of PC containing 22:6n-3 is discussed.

Four 9-kDa proteins excreted by somatic embryos of grapevine are isoforms of lipid-transfer proteins.

Four 9-kDa small extracellular proteins produced by embryogenic cultures in the absence of auxin have been purified from the extracellular medium of grapevine somatic embryo cultures through cation-exchange chromatography and hydrophobic-interaction chromatography. The partial amino-acid sequences reflect high similarities between the four proteins as well as with the sequences established for carrot, spinach, millet and maize nonspecific lipid-transfer proteins. All these sequences show conservation of three cysteines at positions 4, 14 and 30-32, as well as glycine, valine, tyrosine and lysine residues at positions 5, 7, 17 and 37, respectively. In-vitro lipid-transfer assays reveal that the four proteins catalyze the transfer of phosphatidylcholine from liposomes towards mitochondria with an efficiency similar or higher than that of a purified maize lipid-transfer protein.

Control of maize lipid transfer protein activity by oxido-reducing conditions.

The activity of lipid transfer proteins (LTPs) isolated from maize, able to facilitate phospholipid movement between membranes, was studied under various oxido-reducing conditions. A progressive inactivation of LTP transfer activity was observed with increasing concentrations of reduced dithiothreitol (DTTred). This inactivation was accompanied by an increase in SH titer as well as by changes of the protein conformation deduced from its higher mobility in SDS-PAGE. By contrast, DTTred did not affect the formation of lipid-LTP complex. Transfer activity and original electrophoretic mobility were partially restored under reoxidation by air or oxidized DTT. Together, these results demonstrate the critical role of correct S-S bondings on LTP activity and suggest a possible in vivo regulation, according to the specific oxido-reducing conditions prevailing in different cell compartments.

Spatial and temporal expression of a maize lipid transfer protein gene.

We studied the temporal and spatial pattern of lipid transfer protein (LTP) gene expression, as well as the localization of this protein, in maize. Using an LTP gene, we observed an accumulation of LTP mRNA in embryos and endosperms during seed maturation. LTP gene expression was also investigated in young seedlings. After germination, the level of LTP mRNA in the coleoptile increased, with a maximum at 7 days, whereas LTP mRNA levels were low in the scutellum and negligible in roots. The high levels of LTP mRNA found in coleoptiles and embryos were confirmed by in situ hybridization. Moreover, LTP gene expression appeared to be localized in the external cellular layers and around the leaf veins. Using immunogold methods, we also observed that LTP was distributed heterogeneously in the different cells of coleoptiles and leaves. The highest concentrations of LTP were found in the outer epidermis of the coleoptiles as well as the leaf veins. Together, our observations indicate that LTP gene expression is not only organ specific and time specific but also cell specific.

Use of spin-labeled and fluorescent lipids to study the activity of the phospholipid transfer protein from maize seedlings.

The transfer of spin-labeled and fluorescent lipids between sonicated vesicles and different host membranes has been measured in the presence or absence of a phospholipid transfer protein purified from maize seedlings. It was found that the protein has little specificity towards the phospholipid head group and allows the transfer of hydrophobic long chain phospholipids. By contrast, no transfer of a cholesterol analogue could be detected. By EPR spectroscopy, evidence is presented that shows that the protein catalyzes the incorporation of labeled phospholipids in the outer monolayer of the acceptor membranes. The efficiency of the transfer depends largely on the nature of the acceptor: erythrocytes are more difficult to label than chromaffin granules or liposomes made with unsaturated lipids. Thus, consistent with the high activation energy observed, the transfer is facilitated when it involves fluid membranes. These results are in favor of a process involving the exchange of phospholipids, facilitated by a shuttle protein rather than a fusion mechanism.

Changes in Level and Activity of Phospholipid Transfer Protein during Maturation and Germination of Maize Seeds.

The variations of the amounts of phospholipid transfer proteins (PLTP), determined by ELISA and immunoblotting methods, were followed during the maturation and germination of maize (Zea mays L.) seeds. Changes of the amounts of PLTP occur during seed maturation. The levels of PLTP, low in the first 3 weeks after fecondation, strongly raised 3 to 5 weeks after, then reached and maintained a high value (10% of total soluble proteins) during the last steps of maturation. These variations, determined by ELISA, are in accordance with the observations made by immunoblotting. Changes in phospholipid transfer activity were also found when protein extracts prepared from seeds at different stages of maturation were assayed for transfer activity. The levels of PLTP were also determined during the germination of maize seeds and the early growth of the plantlets, both in the endosperm and the aerial parts. While no major change was observed in the endosperm, a high increase in PLTP level was found in the aerial part of the plantlet, both by ELISA and immunoblotting. An enhancement of the phospholipid transfer activity was parallely observed in the protein extracts of plantlets at various stages of germination. These results are consistent with an in vivo correlation between the synthesis of phospholipid transfer protein, observed during the maturation and germination of maize seeds, and the biogenesis of membranes which involves intracellular movements of phospholipids.

Synthesis of phospholipid transfer proteins from maize seedlings.

The synthesis of phospholipid transfer proteins has been studied in vitro after isolation of poly(A)+RNAs from maize seedlings and by in vivo labelling of coleoptiles. After immunoprecipitation of translation products in wheat germ or in reticulocyte lysate systems, the analysis by electrophoresis revealed two bands of molecular mass 9 kDa and 12 kDa. The in vitro synthesized 12 kDa protein is a precursor of the 9 kDa purified protein from maize seedlings as suggested by competition experiments with the pure protein. After immunoprecipitation of in vivo labelled proteins, two bands were detected. One of them, having a molecular mass of 7 kDa, could be related to the in vitro synthesized 9 kDa protein, the other corresponding to the purified protein. Furthermore, biosynthesis of both precursors occurs on membrane-bound polysomes. Presumably a post translational process occurs, yielding to the mature forms.

In vitro synthesis of a plant phospholipid transfer protein: a study by high performance liquid chromatography.

In order to study the biosynthesis of a plant phospholipid transfer protein (PLTP), poly (A)+RNAs have been prepared from maize seedlings and translated in vitro with a rabbit reticulocyte lysate. The newly synthesized proteins were then separated by fast protein liquid chromatography (FPLC) followed by SDS-PAGE or by high performance liquid chromatography (HPLC) coupled to a radioactivity detector monitor. It has been showed that a radioactive band comigrating with a 14C methylated pure PLTP was detected by SDS-PAGE. This result, confirmed by direct radioactive monitoring, indicates that PLTP is actively synthesized in vitro.