FtsZ Reorganization Facilitates Deformation of Giant Vesicles in Microfluidic Traps*.

The geometry of reaction compartments can affect the local outcome of interface-restricted reactions. Giant unilamellar vesicles (GUVs) are commonly used to generate cell-sized, membrane-bound reaction compartments, which are, however, always spherical. Herein, we report the development of a microfluidic chip to trap and reversibly deform GUVs into cigar-like shapes. When trapping and elongating GUVs that contain the primary protein of the bacterial Z ring, FtsZ, we find that membrane-bound FtsZ filaments align preferentially with the short GUV axis. When GUVs are released from this confinement and membrane tension is relaxed, FtsZ reorganizes reversibly from filaments into dynamic rings that stabilize membrane protrusions; a process that allows reversible GUV deformation. We conclude that microfluidic traps are useful for manipulating both geometry and tension of GUVs, and for investigating how both affect the outcome of spatially-sensitive reactions inside them, such as that of protein self-organization.

Osmoregulation in the Halophilic Bacterium Halomonas elongata: A Case Study for Integrative Systems Biology.

Halophilic bacteria use a variety of osmoregulatory methods, such as the accumulation of one or more compatible solutes. The wide diversity of compounds that can act as compatible solute complicates the task of understanding the different strategies that halophilic bacteria use to cope with salt. This is specially challenging when attempting to go beyond the pathway that produces a certain compatible solute towards an understanding of how the metabolic network as a whole addresses the problem. Metabolic reconstruction based on genomic data together with Flux Balance Analysis (FBA) is a promising tool to gain insight into this problem. However, as more of these reconstructions become available, it becomes clear that processes predicted by genome annotation may not reflect the processes that are active in vivo. As a case in point, E. coli is unable to grow aerobically on citrate in spite of having all the necessary genes to do it. It has also been shown that the realization of this genetic potential into an actual capability to metabolize citrate is an extremely unlikely event under normal evolutionary conditions. Moreover, many marine bacteria seem to have the same pathways to metabolize glucose but each species uses a different one. In this work, a metabolic network inferred from genomic annotation of the halophilic bacterium Halomonas elongata and proteomic profiling experiments are used as a starting point to motivate targeted experiments in order to find out some of the defining features of the osmoregulatory strategies of this bacterium. This new information is then used to refine the network in order to describe the actual capabilities of H. elongata, rather than its genetic potential.

Novel sulfated phosphoglycolipids from Natronomonas moolapensis.

Polar lipid pattern determination is often used for the taxonomic classification of halophilic Archaea in addition to a genomic characterization. During the analysis of polar lipid extracts from the recently described haloarchaeon Natrononomonas moolapensis, an unknown glycolipid was detected. Fragmentation patterns observed from preliminary mass spectrometric analysis initially suggested the presence of a sulfo-hexosyl-phosphatidylglycerol. However, by NMR spectroscopy and enzymatic assays the existence of two isomeric molecules with different hexoses (1-(6-sulfo-d-glcp/galf-ß1,2-glycero)-phospho-2,3-diphytanylglycerol) could be shown. The structural origin from phosphatidylglycerol distinguishes these glycolipids within Archaea, because all other characterized haloarchaeal glycolipids consist of diphytanylglycerol directly linked to an oligoglycosyl moiety. Now the door is open to investigate the physical and functional consequences of these architectural differences of the head groups.

The protein interaction network of a taxis signal transduction system in a halophilic archaeon.

BACKGROUND: The taxis signaling system of the extreme halophilic archaeon Halobacterium (Hbt.) salinarum differs in several aspects from its model bacterial counterparts Escherichia coli and Bacillus subtilis. We studied the protein interactions in the Hbt. salinarum taxis signaling system to gain an understanding of its structure, to gain knowledge about its known components and to search for new members. RESULTS: The interaction analysis revealed that the core signaling proteins are involved in different protein complexes and our data provide evidence for dynamic interchanges between them. Fifteen of the eighteen taxis receptors (halobacterial transducers, Htrs) can be assigned to four different groups depending on their interactions with the core signaling proteins. Only one of these groups, which contains six of the eight Htrs with known signals, shows the composition expected for signaling complexes (receptor, kinase CheA, adaptor CheW, response regulator CheY). From the two Hbt. salinarum CheW proteins, only CheW1 is engaged in signaling complexes with Htrs and CheA, whereas CheW2 interacts with Htrs but not with CheA. CheY connects the core signaling structure to a subnetwork consisting of the two CheF proteins (which build a link to the flagellar apparatus), CheD (the hub of the subnetwork), two CheC complexes and the receptor methylesterase CheB. CONCLUSIONS: Based on our findings, we propose two hypotheses. First, Hbt. salinarum might have the capability to dynamically adjust the impact of certain Htrs or Htr clusters depending on its current needs or environmental conditions. Secondly, we propose a hypothetical feedback loop from the response regulator to Htr methylation made from the CheC proteins, CheD and CheB, which might contribute to adaptation analogous to the CheC/CheD system of B. subtilis.

Life-style changes of a halophilic archaeon analyzed by quantitative proteomics.

Quantitative proteomics based on isotopic labeling has become the method of choice to accurately determine changes in protein abundance in highly complex mixtures. Isotope-coded protein labeling (ICPL), which is based on the nicotinoylation of proteins at lysine residues and free N-termini was used as a simple, reliable and fast method for the comparative analysis of three different cellular states of the halophilic archaeon Halobacterium salinarum through pairwise comparison. The labeled proteins were subjected to SDS-PAGE, in-gel digested and the proteolytic peptides were separated by LC and analyzed by MALDI-TOF/TOF MS. Automated quantitation was performed by comparing the MS peptide signals of (12)C and (13)C nicotinoylated isotopic peptide pairs. The transitions between (i) aerobic growth in complex versus synthetic medium and (ii) aerobic versus anaerobic/phototrophic growth, both in complex medium, provide a wide span in nutrient and energy supply for the cell and thus allowed optimal studies of proteome changes. In these two studies, 559 and 643 proteins, respectively, could be quantified allowing a detailed analysis of the adaptation of H. salinarum to changes of its living conditions. The subtle cellular response to a wide variation of nutrient and energy supply demonstrates a fine tuning of the cellular protein inventory.

Identification of Archaea-specific chemotaxis proteins which interact with the flagellar apparatus.

BACKGROUND: Archaea share with bacteria the ability to bias their movement towards more favorable locations, a process known as taxis. Two molecular systems drive this process: the motility apparatus and the chemotaxis signal transduction system. The first consists of the flagellum, the flagellar motor, and its switch, which allows cells to reverse the rotation of flagella. The second targets the flagellar motor switch in order to modulate the switching frequency in response to external stimuli. While the signal transduction system is conserved throughout archaea and bacteria, the archaeal flagellar apparatus is different from the bacterial one. The proteins constituting the flagellar motor and its switch in archaea have not yet been identified, and the connection between the bacterial-like chemotaxis signal transduction system and the archaeal motility apparatus is unknown. RESULTS: Using protein-protein interaction analysis, we have identified three proteins in Halobacterium salinarum that interact with the chemotaxis (Che) proteins CheY, CheD, and CheC2, as well as the flagella accessory (Fla) proteins FlaCE and FlaD. Two of the proteins belong to the protein family DUF439, the third is a HEAT_PBS family protein. In-frame deletion strains for all three proteins were generated and analyzed as follows: a) photophobic responses were measured by a computer-based cell tracking system b) flagellar rotational bias was determined by dark-field microscopy, and c) chemotactic behavior was analyzed by a swarm plate assay. Strains deleted for the HEAT_PBS protein or one of the DUF439 proteins proved unable to switch the direction of flagellar rotation. In these mutants, flagella rotate only clockwise, resulting in exclusively forward swimming cells that are unable to respond to tactic signals. Deletion of the second DUF439 protein had only minimal effects. HEAT_PBS proteins could be identified in the chemotaxis gene regions of all motile haloarchaea sequenced so far, but not in those of other archaeal species. Genes coding for DUF439 proteins, however, were found to be integral parts of chemotaxis gene regions across the archaeal domain, and they were not detected in other genomic context. CONCLUSION: Altogether, these results demonstrate that, in the archaeal domain, previously unrecognized archaea-specific Che proteins are essential for relaying taxis signaling to the flagellar apparatus.

The interaction of Nanoarchaeum equitans with Ignicoccus hospitalis: proteins in the contact site between two cells.

The two archaea Ignicoccus hospitalis and Nanoarchaeum equitans form a unique intimate association, the character of which is not yet fully understood. Electron microscopic investigations show that at least two modes of cell-cell interactions exist: (i) the two cells are interconnected via thin fibres; and (ii) the two cell surfaces are in direct contact with each other. In order to shed further light on the molecules involved, we isolated a protein complex, by using detergent-induced solubilization of cell envelopes, followed by a combination of chromatography steps. Analysis by MS and comparison with databases revealed that this fraction contained two dominant proteins, representing the respective major envelope proteins of the two archaea. In addition, a considerable set of membrane proteins is specifically associated with these proteins. They are now the focus of further biochemical and ultrastructural investigations.

Chlamydia causes fragmentation of the Golgi compartment to ensure reproduction.

The obligate intracellular bacterium Chlamydia trachomatis survives and replicates within a membrane-bound vacuole, termed the inclusion, which intercepts host exocytic pathways to obtain nutrients. Like many other intracellular pathogens, C. trachomatis has a marked requirement for host cell lipids, such as sphingolipids and cholesterol, produced in the endoplasmic reticulum and the Golgi apparatus. However, the mechanisms by which intracellular pathogens acquire host cell lipids are not well understood. In particular, no host cell protein responsible for transporting Golgi-derived lipids to the chlamydial inclusions has yet been identified. Here we show that Chlamydia infection in human epithelial cells induces Golgi fragmentation to generate Golgi ministacks surrounding the bacterial inclusion. Ministack formation is triggered by the proteolytic cleavage of the Golgi matrix protein golgin-84. Inhibition of golgin-84 truncation prevents Golgi fragmentation, causing a block in lipid acquisition and maturation of C. trachomatis. Golgi fragmentation by means of RNA-interference-mediated knockdown of distinct Golgi matrix proteins before infection enhances bacterial maturation. Our data functionally connect bacteria-induced golgin-84 cleavage, Golgi ministack formation, lipid acquisition and intracellular pathogen growth. We show that C. trachomatis subverts the structure and function of an entire host cell organelle for its own advantage.

Physiological sites of deamidation and methyl esterification in sensory transducers of Halobacterium salinarum.

In Halobacterium salinarum, up to 18 sensory transducers (Htrs) relay environmental stimuli to an intracellular signaling system to induce tactic responses. As known from the extensively studied enterobacterial system, sensory adaptation to persisting stimulus intensities involves reversible methylation of certain transducer glutamate residues, some of which originate from glutamine residues by deamidation. This study analyzes the in vivo deamidation and methylation of membrane-bound Htrs under physiological conditions. Electrospray ionization tandem mass spectrometry of chromatographically separated proteolytic peptides identified 19 methylation sites in 10 of the 12 predicted membrane-spanning Htrs. Matrix-assisted laser desorption/ionization mass spectrometry additionally detected three sites in two soluble Htrs. Sensory transducers contain a cytoplasmic coiled-coil region, composed of hydrophobic heptads, seven-residue repeats in which the first and the fourth residues are mostly hydrophobic. All identified Htr methylations occurred at glutamate residues at the second and/or third position of such heptads. In addition to singly methylated pairs of glutamate and/or glutamine residues, we identified singly methylated aspartate-glutamate and alanine-glutamate pairs and doubly methylated glutamate pairs. The largest methylatable regions detected in Htrs comprise six heptads along the coiled coil. One methylated glutamate residue was detected outside of such a region, in the signaling region of Htr14. Our analysis produced evidence supporting the predicted methyltransferase and methylesterase activities of halobacterial CheR and CheB, respectively. It furthermore demonstrated that CheB is required for Htr deamidations, at least at a specific glutamine-glutamate pair in Htr2 and a specific aspartate-glutamine pair in Htr4. Compared to previously reported methods, the described approach significantly facilitates the identification of physiological transducer modification sites.

Insight into the proteome of the hyperthermophilic Crenarchaeon Ignicoccus hospitalis: the major cytosolic and membrane proteins.

Ignicoccus hospitalis, a hyperthermophilic, chemolithoautotrophic Crenarchaeon, is the host of Nanoarchaeum equitans. Together, they form an intimate association, the first among Archaea. Membranes are of fundamental importance for the interaction of I. hospitalis and N. equitans, as they harbour the proteins necessary for the transport of macromolecules like lipids, amino acids, and cofactors between these organisms. Here, we investigated the protein inventory of I. hospitalis cells, and were able to identify 20 proteins in total. Experimental evidence and predictions let us conclude that 11 are soluble cytosolic proteins, eight membrane or membrane-associated proteins, and a single one extracellular. The quantitatively dominating proteins in the cytoplasm (peroxiredoxin; thermosome) antagonize oxidative and temperature stress which I. hospitalis cells are exposed to at optimal growth conditions. Three abundant membrane protein complexes are found: the major protein of the outer membrane, which might protect the cell against the hostile environment, forms oligomeric complexes with pores of unknown selectivity; two other complexes of the cytoplasmic membrane, the hydrogenase and the ATP synthase, play a key role in energy production and conversion.

Perlinhibin, a cysteine-, histidine-, and arginine-rich miniprotein from abalone (Haliotis laevigata) nacre, inhibits in vitro calcium carbonate crystallization.

We have isolated a 4.785 Da protein from the nacreous layer of the sea snail Haliotis laevigata (greenlip abalone) shell after demineralization with acetic acid. The sequence of 41 amino acids was determined by Edman degradation supported by mass spectrometry. The most abundant amino acids were cysteine (19.5%), histidine (17%), and arginine (14.6%). The positively charged amino acids were almost counterbalanced by negatively charged ones resulting in a calculated isoelectric point of 7.86. Atomic-force microscopy studies of the interaction of the protein with calcite surfaces in supersaturated calcium carbonate solution or calcium chloride solution showed that the protein bound specifically to calcite steps, inhibiting further crystal growth at these sites in carbonate solution and preventing crystal dissolution when carbonate was substituted with chloride. Therefore this protein was named perlinhibin. X-ray diffraction investigation of the crystal after atomic-force microscopy growth experiments showed that the formation of aragonite was induced on the calcite substrate around holes caused by perlinhibin crystal-growth inhibition. The strong interaction of the protein with calcium carbonate was also shown by vapor diffusion crystallization. In the presence of the protein, the crystal surfaces were covered with holes due to protein binding and local inhibition of crystal growth. In addition to perlinhibin, we isolated and sequenced a perlinhibin-related protein, indicating that perlinhibin may be a member of a family of closely related proteins.

Large-scale identification of N-terminal peptides in the halophilic archaea Halobacterium salinarum and Natronomonas pharaonis.

Characterization of protein N-terminal peptides supports the quality assessment of data derived from genomic sequences (e.g., the correct assignment of start codons) and hints to in vivo N-terminal modifications such as N-terminal acetylation and removal of the initiator methionine. The current work represents the first large-scale identification of N-terminal peptides from prokaryotes, of the two halophilic euryarchaeota Halobacterium salinarum and Natronomonas pharaonis. Two methods were used that specifically allow the characterization of protein N-terminal peptides: combined fractional diagonal chromatography (COFRADIC) and strong cation exchange chromatography (SCX), both known to enrich for N-terminally blocked peptides. In addition to these specific methods, N-terminal peptide identifications were extracted from our previous genome-wide proteomic data. Combining all data, 606 N-terminal peptides from Hbt. salinarum and 328 from Nmn. pharaonis were reliably identified. These results constitute the largest available dataset holding identified and characterized protein N-termini for prokaryotes (archaea and bacteria). They allowed the validation/improvement of start codon assignments as automatic gene finders tend to misassign start codons for GC-rich genomes. In addition, the dataset allowed unravelling N-terminal protein maturation in archaea, showing that 60% of the proteins undergo methionine cleavage and that-in contrast to current knowledge-Nalpha-acetylation is common in the archaeal domain of life with 13-18% of the proteins being Nalpha-acetylated. The protein sets described in this paper are available by FTP and might be used as reference sets to test the performance of new gene finders.

The low molecular weight proteome of Halobacterium salinarum.

Systematic investigation of low molecular weight proteins (LMW, below 20 kDa) in the archaeon Halobacterium salinarum resulted in a 6-fold enhancement of the identification rate, reaching 35% of the theoretical proteome in that size range. This was achieved by optimization of common protocols for protein analysis with general applicability. LMW proteins were rapidly and effectively enriched by filter membrane centrifugation followed by tricine SDS-PAGE. Without staining and with significantly shortened digestion protocols, LMW proteins were identified using an FT-ICR mass spectrometer which allows reliable protein identification by MS3 of a single peptide. In addition to a series of technical challenges, small proteins may show low gene expression levels as suggested by their low average codon adaptation index. Twenty functionally uncharacterized proteins contain a characteristic DNA/RNA binding zinc finger motif which underlines the biological relevance of the small proteome and the necessity of their analysis for systems biology.

Genome-wide proteomics of Natronomonas pharaonis.

The aerobic, haloalkaliphilic archaeon Natronomonas pharaonis is able to survive in salt-saturated lakes of pH 11. According to genome analysis, the theoretical proteome consists of 2843 proteins. To reach further conclusions about its cellular physiology, the cytosolic protein inventory of Nmn. pharaonis has been analyzed using MS/MS on an ESI-Q-TOF mass spectrometer coupled on-line with a nanoLC system. The efficiency of this shotgun approach is illustrated by the identification of 929 proteins of which 886 are soluble proteins representing 41% of the cytosolic proteome. Cell lysis under denaturing conditions in water with subsequent separation by SDS-PAGE prior to nanoLC-MS/MS resulted in identification of 700 proteins. The same number (but a different subset) of proteins was identified upon cell lysis under native conditions followed by size fractionation (retaining protein complexes) prior to SDS-PAGE. Additional size fractionation reduced sample complexity and increased identification reliability. The set of identified proteins covers about 60% of the cytosolic proteins involved in metabolism and genetic information processing. Many of the identified proteins illustrate the high genetic variability among the halophilic archaea.

Proteomics analysis of Thermoplasma acidophilum with a focus on protein complexes.

Two-dimensional gel electrophoresis (2DE) and MALDI-TOF MS were used to obtain a global view of the cytoplasmic proteins expressed by Thermoplasma acidophilum. In addition, glycerol gradient ultracentrifugation coupled to 2DE-MALDI-TOF MS analysis was used to identify subunits of macromolecular complexes. With the 2DE proteomics approach, over 900 spots were resolved of which 271 proteins were identified. A significant number of these form macromolecular complexes, among them the ribosome, proteasome, and thermosome, which are expressed at high levels. In the glycerol gradient heavy fractions, 10 as yet uncharacterized proteins (besides the well known ribosomal subunits, translation initiation factor eIF-6-related protein, elongation factor 1, and DNA-dependent RNA polymerase) were identified that are putative building blocks of protein complexes. These proteins belong to the categories of hypothetical or conserved hypothetical proteins, and they are present in the cytosol at low concentrations. Although these proteins exhibit homology to known sequences, their structures, subunit compositions, and biological functions are not yet known.

Comparative proteomic analysis with postmortem prefrontal cortex tissues of suicide victims versus controls.

BACKGROUND: The origin of suicidal behaviour is multifactorial including genetic, neurobiological and psychosocial correlates. Although there is no doubt that serotonin has a central role, the overall genetic findings with candidate genes of the serotonergic pathway are relatively inconsistent and suggests that other, yet unidentified, genes and gene products are also contributing to the vulnerability of suicidality. Proteomics is a powerful method to investigate modifications in protein expression. METHODS: We performed comparative proteomic analysis with prefrontal cortex tissues of 17 suicide victims and 9 controls. RESULTS: Applying two dimensional gel electrophoresis and image analysis we detected five protein spots to differ significantly in intensities between both groups. Three of them appeared only in suicide victims and could be identified by means of MALDI-TOF-MS analysis and protein database search as alpha crystallin chain B (CRYAB), glial fibrillary acidic protein (GFAP) and manganese superoxide dismutase (SOD2). CRYAB belongs to the low molecular heat shock proteins and GFAP is known as a marker of astrocytic activation in gliosis. SOD2 is a major antioxidant enzyme protecting cells against oxidative injury. Two further spots revealed higher intensities in the control group but had no unambiguous protein to match. CONCLUSIONS: Our findings suggest that proteins, being involved in glial function, neurodegeneration and oxidative stress neuronal injury, might also have an impact upon the neurobiological cascade leading to suicidality. As animal data provide evidence for an up-regulation of GFAP synthesis in astrocytes due to alterations in 5-HT levels, similar mechanisms of interaction might also be relevant in humans.

Archaeal N-terminal protein maturation commonly involves N-terminal acetylation: a large-scale proteomics survey.

We present the first large-scale survey of N-terminal protein maturation in archaea based on 873 proteomically identified N-terminal peptides from the two haloarchaea Halobacterium salinarum and Natronomonas pharaonis. The observed protein maturation pattern can be attributed to the combined action of methionine aminopeptidase and N-terminal acetyltransferase and applies to cytosolic proteins as well as to a large fraction of integral membrane proteins. Both N-terminal maturation processes primarily depend on the amino acid in penultimate position, in which serine and threonine residues are over represented. Removal of the initiator methionine occurs in two-thirds of the haloarchaeal proteins and requires a small penultimate residue, indicating that methionine aminopeptidase specificity is conserved across all domains of life. While N-terminal acetylation is rare in bacteria, our proteomic data show that acetylated N termini are common in archaea affecting about 15% of the proteins and revealing a distinct archaeal N-terminal acetylation pattern. Haloarchaeal N-terminal acetyltransferase reveals narrow substrate specificity, which is limited to cleaved N termini starting with serine or alanine residues. A comparative analysis of 140 ortholog pairs with identified N-terminal peptide showed that acetylatable N-terminal residues are predominantly conserved amongst the two haloarchaea. Only few exceptions from the general N-terminal acetylation pattern were observed, which probably represent protein-specific modifications as they were confirmed by ortholog comparison.

Quantitative profiling of the membrane proteome in a halophilic archaeon.

We present a large scale quantitation study of the membrane proteome from Halobacterium salinarum. To overcome problems generally encountered with membrane proteins, we established a membrane preparation protocol that allows the application of most proteomic techniques originally developed for soluble proteins. Proteins were quantified using two complementary approaches. For gel-based quantitation, DIGE labeling was combined with two-dimensional gel electrophoresis on an improved 16-benzyldimethyl-n-hexadecylammonium chloride/SDS system. MS-based quantitation was carried out by combining gel-free separation with the recently developed isotope-coded protein labeling technique. Good correlations between these two independent quantitation strategies were obtained. From computational analysis we conclude that labeling of free amino groups by isotope-coded protein labeling (Lys and free N termini) is better suited for membrane proteins than Cys-based labeling strategies but that quantitation of integral membrane proteins remains cumbersome compared with soluble proteins. Nevertheless we could quantify 155 membrane proteins; 101 of these had transmembrane domains. We compared two growth states that strongly affect the energy supply of the cells: aerobic versus anaerobic/phototrophic conditions. The photosynthetic protein bacteriorhodopsin is the most highly regulated protein. As expected, several other membrane proteins involved in aerobic or anaerobic energy metabolism were found to be regulated, but in total, however, the number of regulated proteins is rather small.

Amino acid sequences and phosphorylation sites of emu and rhea eggshell C-type lectin-like proteins.

Avian calcified eggshell layers contain in their organic matrix one or two C-type lectin-like proteins. Previously characterized eggshell proteins of this family are chicken ovocleidin-17 (OC-17), goose ansocalcin and ostrich struthiocalcins 1 and 2 (SCA-1, SCA-2). In this report we present the amino acid sequences of two emu (Dromaius novaehollandiae) (dromaiocalcin-1 and -2; DCA-1, DCA-2) and of two rhea (Rhea americana) (rheacalcin-1 and -2; RCA-1, RCA-2) C-type lectin-like eggshell proteins, thus doubling the data set for comparison of these major specific eggshell proteins. The ratite proteins can be divided into two groups. Group 1, comprising SCA-1, DCA-1 and RCA-1, shows by 70--77% identity of sequences, the lack of phosphorylation, and a variable number (7--9) of cysteines. Group 2, consisting of SCA-2, DCA-2 and RCA-2, shows 78--85% identical sequences, 2--3 phosphorylated serines located at almost identical sites, and contains only the common set of six conserved cysteins characteristic for this family of proteins. While goose ansocalcin fits perfectly into group 1 with a sequence identity of 63--70% to the other members, no phosphorylation, and seven cysteines, chicken OC-17 was assigned to group 2 in spite of only 42--47% sequence identity (and 37--39% to group 1) because of its two phosphorylated serines and its regular set of six cysteines. At present it remains unknown why ratites, but not goose or chicken, require two different types of C-type lectin-like proteins to construct their eggshells.

Structural identification of oxidized acyl-phosphatidylcholines that induce platelet activation.

Oxidation of low-density lipoprotein (LDL) generates proinflammatory and prothrombotic mediators that may play a crucial role in cardiovascular and inflammatory diseases. In order to study platelet-activating components of oxidized LDL 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, a representative of the major phospholipid species in LDL, the 1-acyl-phosphatidylcholines (PC), was oxidized by CuCl(2) and H(2)O(2). After separation by high-performance liquid chromatography, three compounds were detected which induced platelet shape change at low micromolar concentrations. Platelet activation by these compounds was distinct from the pathways stimulated by platelet-activating factor, lyso-phosphatidic acid, lyso-PC and thromboxane A(2), as evidenced by the use of specific receptor antagonists. Further analyses of the oxidized phospholipids by electrospray ionization mass spectrometry structurally identified them as 1-stearoyl-2-azelaoyl-sn-glycero-3-phosphocholine (m/z 694; SAzPC), 1-stearoyl-2-glutaroyl-sn- glycero-3-phosphocholine (m/z 638; SGPC), and 1-stearoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (m/z 622; SOVPC). These observations demonstrate that novel 1-acyl-PC which had previously been found to stimulate interaction of monocytes with endothelial cells also induce platelet activation, a central step in acute thrombogenic and atherogenic processes.