Barley Lipid Transfer Protein Research Articles

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.

The maize major allergen, which is responsible for food-induced allergic reactions, is a lipid transfer protein

Background: Cereals are the most important nutritional component in the human diet. Food-induced allergic reactions to these substances therefore have serious implications, and exhaustive diagnosis is required. Such diagnosis is still difficult because of the incomplete knowledge about major cereal allergens. In particular, few food-induced allergic reactions to maize have been reported, and no information on the allergenic proteins is available. Objective: Having observed several anaphylactic reactions to maize, we planned a study to identify maize major allergens and cross-reactivity with other cereals, as well as to peach because the majority of patients also reacted to Prunoideae fruits. Methods: Twenty-two patients with systemic symptoms after maize ingestion and positive skin prick test responses and serum-specific IgE antibodies to maize were selected. The IgE-reactivity pattern was identified by SDS-PAGE and immunoblotting. The major allergen identified was then purified by HPLC and characterized by mass spectrometry, determination of the isoelectric point value, and N-terminal amino acid sequencing. Results: Sera from 19 (86%) of the 22 patients recognized a 9-kd protein, thus confirming this as the maize major allergen. This protein had an isoelectric point of greater than 9, a molecular mass of 9047.0 d, and no glycosylation. Determination of its N-terminal sequence showed that it was a lipid transfer protein (LTP). By using immunoblotting-inhibition experiments, we demonstrated that the LTP cross-reacts completely with rice and peach LTPs but not with wheat or barley LTPs. N-terminal sequence of the 16-kd allergen (recognized by 36% of patients) showed it to be the maize inhibitor of trypsin. This protein cross-reacts completely with grass, wheat, barley, and rice trypsin inhibitors. Conclusion: The major allergen of maize is an LTP with a molecular weight of 9 kd that is highly homologous with the peach LTP, the major allergen of the Prunoideae subfamily. (J Allergy Clin Immunol 2000;106:744-51.)

Purification and structural characterization of LTP1 polypeptides from beer.

We report on the purification of lipid transfer proteins (LTP) from barley seeds and beer with the aim of investigating the chemical modifications that occur during the brewing process. In seeds, the well-known LTP of 9 kDa (LTP1) has been found together with a second form named LTPb that displays comparable amino acid composition but was not fully sequenced. These two forms have been recovered in beer with marked chemical modifications including disulfide bond reduction and rearrangement and especially glycation by Maillard reaction. The glycation is heterogeneous with variable amounts of hexose units bound to LTPs. Circular dichroism shows that glycated LTP1 having all their disulfide bridges reduced are totally unfolded. These results provide a first basis for understanding how barley LTPs become foam-promoting agents during the malting and brewing process.

LTP is not a Cysteine Endoprotease Inhibitor in Barley Grains

Several different protease inhibitors have been identified in the mature barley grain, which are proposed to play a defensive role against potential barley pathogens. Cysteine protease inhibitors have been detected in mature grain and in the early stages of germination. The nature of these inhibitors has recently been investigated, and barley lipid transfer protein (LTP1) has been identified as an effective inhibitor of both cysteine and serine endoprotease activity expressed in germinating grain. We show that barley LTP1, in its native state, is not a cysteine protease inhibitor, but in a denatured state becomes a preferred substrate for the barley endoprotease EP-B and, as such, behaves as a competitive inhibitor for poorer substrates of EP-B. The presence of significant amounts of LTP1 in barley malt beer suggests that this very compact protein is highly resistant to proteolytic attack during malting and mashing and its denaturation during wort boiling coincides with inactivation of the malt endoproteases. Analysis of the cleavage products of denatured LTP1, generated by EP-B, provides further evidence for the cleavage site specificity of this barley cysteine endoprotease, where a hydrophobic residue in the P2position is strongly preferred.

Nucellar-cell-specific expression of a lipid transfer protein gene in barley ( Hordeum vulgare L.)

Plant lipid transfer proteins (LTPs) are thought to participate in many aspects of cell growth and development. We have identified a novel LTP gene (LTP-ne) from barley (Hordeum vulgare) through differential screening of an ovary cDNA library. LTP-ne was mapped to the distal region of the long arm of barley chromosome 4, distinct from the genomic location of other known LTPs. Sequence comparison indicated that LTP-ne was more closely related to the 7-kDa LTPs than to the 9-kDa LTPs in barley. In Northern hybridization, LTP-ne transcripts were detected in ovary, but not in other tissues, and were most abundant 3–4 days after pollination. Furthermore, RNA in situ hybridization showed that within the ovary, the LTP-ne gene was specifically expressed in the outer cell layer of the nucellus. These results showed that the newly identified barley LTP was ovular cell specific and regulated by pollination/fertilization.

Development of Arabidopsis thaliana leaves at low temperatures releases the suppression of photosynthesis and photosynthetic gene expression despite the accumulation of soluble carbohydrates.

Arabidopsis thaliana plants were grown at 23 degrees C and changes in carbohydrate metabolism, photosynthesis and photosynthetic gene expression were studied after the plants were shifted to 5 degrees C. The responses of leaves shifted to 5 degrees C after development at 23 degrees C are compared to leaves that developed at 5 degrees C. Shifting warm developed leaves to 5 degrees C lead to a severe suppression of photosynthesis that correlated with a rapid and sustained accumulation of hexose phosphates and soluble sugars. Associated with the suppression of photosynthesis and the accumulation of soluble sugars was a reduction in the amount of transcript for genes encoding photosynthetic proteins (cab and rbcS). In contrast, leaves that developed at 5 degrees C showed an increase in photosynthesis and control levels of photosynthetic gene expression. This recovery occurred even though leaves that developed at 5 degrees C maintained large pools of soluble sugars. Leaves that developed at 5 degrees C also showed a strong upregulation of the cytosolic pathway for soluble sugar synthesis but not of the chloroplastic pathway for starch synthesis. This was shown at the level of both enzyme activity and the amount of transcript. Thus, development of Arabidopsis leaves at 5 degrees C resulted in metabolic changes that enabled them to produce and accumulate large soluble sugar pools without any associated suppression of photosynthesis or photosynthetic gene expression. These changes were also associated with enhanced freezing tolerance. We suggest that this reprogramming of carbohydrate metabolism associated with development at low temperature is essential to the development of full freezing tolerance and for winter survival of over-wintering herbaceous annuals.

Enhanced tolerance to bacterial pathogens caused by the transgenic expression of barley lipid transfer protein LTP2.

Purified lipid transfer protein LTP2 from barley applied on tobacco leaves eliminated symptoms caused by infiltration of Pseudomonas syringae pv. tabaci 153. Growth of the pathogen in leaves of transgenic tobacco plants was retarded when compared with non-transformed controls. The percentage of inoculation points that showed necrotic lesions was greatly reduced in transgenic tobacco (17-38% versus 78%) and the average size of these lesions was 61-81% that of control. The average total lesion area (necrosis and chlorosis) in the transgenic plants was also reduced (38% of control). Arabidopsis thaliana transgenic plants inoculated with P. syringae pv. tomato DC3000 also had lower percentages of necrotic lesions (22-38% versus 76%), a reduced average area for each lesion (53-67% of control), and a smaller total lesion area per inoculation (43% of control). These results further support the assignment of a defense role for LTPs and highlight their biotechnological potential.

Mechanisms of Beer Colloidal Stabilization

The effects of treatments that influence beer colloidal stability on foam-active protein and haze-active protein and polyphenols were studied. Unchillproofed beer was treated with bentonite, silica gel, and polyvinylpolypyrrolidone (PVPP). Bentonite adsorption removed both haze-active and foam-active protein. Silica gel adsorption removed haze-active protein but almost completely spared foam-active protein. This specificity results because silica gel binds to the proline residues in the protein; these are the same sites where polyphenols attach to create haze. As a result, only those proteins that can participate in haze formation are affected. PVPP removed approximately half of the haze-active polyphenol from beer but had little effect on foam-active or haze-active protein. Fining with gelatin greatly reduced the level of haze-active protein in the beer. Fining with tannic acid reduced the level of haze-active protein and, at higher addition rates, the concentration of haze-active polyphenol. Theoretical aspects of enzymatic chillproofing were considered. Since phenylalanine and tryptophan are present in hordein but not the foam-active barley lipid transfer protein 1 (LTP1), it is theoretically possible that an enzyme could be found that would attack haze-active but not foam-active protein.

Purification, Identification, and Partial Characterization of a Barley Protein that Inhibits Green Malt Endoproteinases

Endoproteinases control the rate of hydrolysis of storage proteins during barley germination and are thus critically important to the malting process. We have shown that endoproteinases comprising all four proteinase classes are present in green malt, with the cysteine proteinases probably being most important for hydrolyzing storage proteins during malting. Compounds from both barley and malt inhibit some of these cysteine proteinases. This article reports the purification and characterization of a 10-kDa barley protein, purified from both seed and beer extracts, that specifically inhibits green malt cysteine endoproteinases. Amino acid composition, matrix-assisted laser desorption/ionization mass spectrometric and N-terminal amino acid sequence data indicated that the inhibitor is identical to barley lipid transfer protein 1 (probable amylase/proteinase inhibitor, PAPI), a nonspecific lipid-transfer protein. The protein did not inhibit the activities of either papain or subtilisin but did suppress the activities of many of the green malt cysteine endoproteinase activities that are separated on a two-dimensional isoelectric focusing and polyacrylamide gel electrophoresis system. Some serine proteinases were also partially inhibited. The purified inhibitor totally inhibited the activity of a purified 31-kDa cysteine endoproteinase from green malt. In the absence of inhibitor, the 31-kDa enzyme rapidly hydrolyzed barley storage proteins. LTP1-PAPI may well play an important role in controlling protein hydrolysis during malting.

Independent Role of Beer Proteins, Melanoidins and Polysaccharides in Foam Formation

Beer foam proteins, melanoidins, and carbohydrates were individually added to bottles of a solution consisting of a carbonated, ethanolic mixture containing iso-α-acids and the inorganic ion composition typically found in a Pilsner-style beer. Foam properties of the bottled solutions were tested using a pouring-style foam test. By this foam test, a beer protein with excellent foam and physical stability was identified. The protein was identical to barley lipid transfer protein by amino acid sequence homology. The protein's complete amino acid sequence was determined, and the molecular weights of the protein and sequenced peptides were analyzed by mass spectroscopy. A 40-kDa protein, identified in the literature as derived from barley protein Z, had adequate foam stability but poor solubility when isolated from the other proteins and from melanoidins. A 12-kDa protein of unknown origin had no independent foam stability. Melanoidins isolated from beer also formed stable foams. The melanoidins have foam-stabilizing properties that are independent of any role in aiding the foam properties of beer proteins. Beer polysaccharides foamed, but the foam stability was less than that of the proteins and melanoidins.

Lipid transfer protein genes specifically expressed in barley leaves and coleoptiles.

Genes/cDNAs encoding so-called lipid-transfer proteins (LTPs) have been isolated from a variety of tissues from different plants, but the in-vivo function of the LTP proteins is not yet known. In barley (Hordeum vulgare L.), the LTP1 gene (encoding a probable amylase/protease inhibitor, Mundy and Rogers 1986, Planta 169, 51-63) is active in aleurone tissue, and in this paper two LTP-encoding cDNAs isolated from green leaves are described. The encoded proteins start with signal sequences, they are 75% homologous to each other, 60-63% homologous to rice aleurone LTP and maize seed/coleoptile LTP, but only 48% homologous to barley aleurone LTP. Northern hybridization experiments established that the two seedling-specific genes are both highly expressed in leaves and coleoptiles whereas the LTP1 gene is inactive in seedlings. No LTP gene expression was detected in roots using either seedling or aleurone cDNA clones as probes. Tissue-print hybridization indicates that the LTP genes are first expressed in young epidermal cells in leaves and coleoptiles, and subsequently expressed in the vascular strands. Genomic Southern analysis indicates that the barley LTP gene family has four to six members.

Phosphorylation of barley and wheat phospholipid transfer proteins by wheat calcium-dependent protein kinase

Wheat ( Triticum aestivum L.) germ Ca 2+-dependent protein kinase (CDPK) phosphorylates a wheat basic protein (WBP) which is a putative phospholipid transfer protein and one of the best protein substrates yet found for wheat CDPK. Reversed phase high performance liquid chromatography (HPLC) of the tryptic digest of [ 32P]phospho WBP resolved two [ 32P]phosphopeptide peaks. Edman sequencing of one these established that WBP is phosphorylated by CDPK on S17 within the sequence A 13PCISYA. WBP may also be phosphorylated at an additional site. Wheat CDPK also phosphorylates barley ( Hordeum vulgare) lipid transfer protein (LTP1) and [ 32P]phosphoLTP1 exactly comigrates with LTP1 on SDS-PAGE. Unlike wheat WBP, LTP1 is a relatively poor substrate for wheat CDPK and is phosphorylated largely on threonine. Reversed phase high performance liquid chromatography (HPLC) of the tryptic digest of [ 32P]phosphopeptide resolved one [ 32P]phosphopeptide and subsequent Edman sequencing of this phosphopeptide showed that LTP1 is phosphorylated on T15 within the sequence K 11PCLTYVQ. This phosphorylation site amino acid sequence motif differs from the Basic-X-X-Ser(Thr) motif found for a variety of synthetic peptide substrates of wheat germ CDPK but corresponds to the Pro-Cys-X-Ser/Thr phosphorylation site motif found for the phosphorylation by wheat germ CDPK of WBP, which is a homologue of barley LTP1. The phosphorylation sites determined for WBP and LTP1 are similar structurally and are within precisely aligned homologous sequences.

Structure and expression of the barley lipid transfer protein gene Ltp1.

We have characterized a gene (Ltp1) encoding a barley lipid transfer protein. Northern blot analysis showed that Ltp1 mRNA accumulates specifically in the aleurone layer of developing and germinating seeds. Southern blot analysis indicated that LTP1 protein is encoded by a single gene in barley. Sequence analysis of Ltp1 showed that it contains an open reading frame of 351 bp interrupted by a single intron of 133 bp. Transient expression assays indicated that 702 bp of the 5' upstream region of Ltp1 is sufficient to direct aleurone-specific expression during late seed development and early germination.

A 10 kD barley basic protein transfers phosphatidylcholine from liposomes to mitochondria

A small basic 10 kD abundant protein in barley seeds can convey up to 7% of the phosphatidylcholine in liposomes to potato mitochondria whereas cholesteryloleate is not transported. The demonstration of this activity combined with a somewhat more than 50% homology of its primary structure to that of other plant phospholipid transfer proteins are the bases for our naming it a barley lipid transfer protein (LTP).