Novel function of tenascin-C, a matrix protein relevant to atherosclerosis, in platelet recruitment and activation under flow.

OBJECTIVE: The identification of platelet-reactive proteins exclusively present in atherosclerotic plaques could provide interesting targets for effective and safe antithrombotic strategies. In this context, we explored platelet adhesion and activation to tenascin-C (TN-C), a matrix protein preferentially found within atheroma. METHODS AND RESULTS: We show that platelets efficiently adhere to TN-C under both static and flow conditions. Videomicroscopy revealed a unique behavior under flow, with platelets exhibiting stationary adhesion to TN-C; in contrast, platelets rolled over von Willebrand factor and detached from fibrinogen. Platelet interaction with TN-C was predominantly supported by integrin α(2)ß(1) under static conditions, whereas under high shear, it was dependent on both the α(2)ß(1) integrin and the glycoprotein Ib-IX complex. Integrin α(IIb)ß(3) appeared to play a secondary role but only at low shear rates. The glycoprotein Ib-IX-dependent interaction was indirect, relying on von Willebrand factor, and increased as a function of wall shear rate. Von Willebrand factor bound directly to TN-C, as shown by ELISA and coimmunoprecipitation, suggesting that it acts as a bridge between TN-C and platelets. The adhesion of platelets to TN-C triggered their activation, as demonstrated by a shape change and increases in intracellular calcium level. CONCLUSIONS: This study provides evidence that TN-C serves as a novel adhesive matrix for platelets in a context that is relevant to atherothrombosis.

The proteome map of spinach leaf peroxisomes indicates partial compartmentalization of phylloquinone (vitamin K1) biosynthesis in plant peroxisomes.

Leaf peroxisomes are fragile, low-abundance plant cell organelles that are difficult to isolate from one of the few plant species whose nuclear genome has been sequenced. Leaf peroxisomes were enriched at high purity from spinach (Spinacia oleracea) and approximately 100 protein spots identified from 2-dimensional gels by a combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and de novo sequencing. In addition to the predominant enzymes involved in photorespiration and detoxification, several minor enzymes were detected, underscoring the high sensitivity of the protein identification. The tryptic peptides of three unknown proteins shared high sequence similarity with Arabidopsis proteins that carry putative peroxisomal targeting signals type 1 or 2 (PTS1/2). The apparent Arabidopsis orthologues are a short-chain alcohol dehydrogenase (SDRa/IBR1, At4g05530, SRL>) and two enoyl-CoA hydratases/isomerases (ECHIa, At4g16210, SKL>; NS/ECHId, At1g60550, RLx(5)HL). The peroxisomal localization of the three proteins was confirmed in vivo by tagging with enhanced yellow fluorescent protein (EYFP), and the targeting signals were identified. The single Arabidopsis isoform of naphthoate synthase (NS) is orthologous to MenB from cyanobacteria, which catalyses an essential reaction in phylloquinone biosynthesis, a pathway previously assumed to be entirely compartmentalized in plastids in higher plants. In an extension of a previous study, the present in vivo targeting data furthermore demonstrate that the enzyme upstream of NS, chloroplastic acyl-CoA activating enzyme isoform 14 (AAE14, SSL>), is dually targeted to both plastids and peroxisomes. This proteomic study, extended by in vivo subcellular localization analyses, indicates a novel function for plant peroxisomes in phylloquinone biosynthesis.

The assembly of CD1e is controlled by an N-terminal propeptide which is processed in endosomal compartments.

CD1e displays unique features in comparison with other CD1 proteins. CD1e accumulates in Golgi compartments of immature dendritic cells and is transported directly to lysosomes, where it is cleaved into a soluble form. In these latter compartments, CD1e participates in the processing of glycolipid antigens. In the present study, we show that the N-terminal end of the membrane-associated molecule begins at amino acid 20, whereas the soluble molecule consists of amino acids 32-333. Thus immature CD1e includes an N-terminal propeptide which is cleaved in acidic compartments and so is absent from its mature endosomal form. Mutagenesis experiments demonstrated that the propeptide controls the assembly of the CD1e alpha-chain with beta(2)-microglobulin, whereas propeptide-deleted CD1e molecules are immunologically active. Comparison of CD1e cDNAs from different mammalian species indicates that the CD1e propeptide is conserved during evolution, suggesting that it may also optimize the generation of CD1e molecules in other species.

Use of tandem Biacore-mass spectrometry to identify platelet membrane targets of novel monoclonal antibodies.

The monoclonal antibodies (mAbs) ALMA.17 and ALMA.7 recognize human platelet membrane proteins. ALMA.17 is directed against alpha(IIb)beta(3) integrin, but the target of ALMA.7 was unknown previously. Tandem Biacore micropurification and mass spectrometry (MS) analysis of a platelet membrane lysate was used to identify the target of ALMA.7. Detergent lysates enriched in membrane proteins were perfused over immobilized ALMA.17 or ALMA.7 in a Biacore system. The captured proteins were eluted, concentrated on C3 magnetic beads, and digested with trypsin before nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Critical adjustments needed to be made in (i) the detergent mixture to preserve protein antigenicity and sensor chip integrity and (ii) the method of trypsin digestion to concentrate the proteins and use elution buffers that do not interfere with MS. The target of ALMA.17 was confirmed to be alpha(IIb)beta(3) integrin, whereas that of ALMA.7 was identified as CD226 (PTA-1, DNAM-1, TLiSa-1). This was confirmed by immunoassays comparing ALMA.7 with a commercial anti-CD226 mAb. Thus, a tandem Biacore and nano LC-MS/MS strategy allowed unambiguous identification of an unknown antigen in a complex medium such as a platelet membrane lysate. This strategy may be employed to identify any protein "capturable" on a sensor chip provided that one uses appropriate experimental conditions.

Mapping of the Physcomitrella patens proteome.

The moss Physcomitrella patens is unique among land plants due to the high rate of homologous recombination in its nuclear DNA. The feasibility of gene targeting makes Physcomitrella an unrivalled model organism in the field of plant functional genomics. To further extend the potentialities of this seed-less plant we aimed at exploring the P. patens proteome. Experimental conditions had to be adopted to meet the special requirements connected to the investigations of this moss. Here we describe the identification of 306 proteins from the protonema of Physcomitrella. Proteins were separated by two dimensional electrophoresis, excised form the gel and analysed by means of mass spectrometry. This reference map will lay the basis for further profound studies in the field of Physcomitrella proteomics.

Lysosomal-associated transmembrane protein 5 (LAPTM5) is a molecular partner of CD1e.

The CD1e protein participates in the presentation of lipid antigens in dendritic cells. Its transmembrane precursor is transported to lysosomes where it is cleaved into an active soluble form. In the presence of bafilomycin, which inhibits vacuolar ATPase and consequently the acidification of endosomal compartments, CD1e associates with a 27 kD protein. In this work, we identified this molecular partner as LAPTM5. The latter protein and CD1e colocalize in trans-Golgi and late endosomal compartments. The quantity of LAPTM5/CD1e complexes increases when the cells are treated with bafilomycin, probably due to the protection of LAPTM5 from lysosomal proteases. Moreover, we could demonstrate that LAPTM5/CD1e association occurs under physiological conditions. Although LAPTM5 was previously shown to act as a platform recruiting ubiquitin ligases and facilitating the transport of receptors to lysosomes, we found no evidence that LATPM5 controls either CD1e ubiquitination or the generation of soluble lysosomal CD1e proteins. Notwithstanding these last observations, the interaction of LAPTM5 with CD1e and their colocalization in antigen processing compartments both suggest that LAPTM5 might influence the role of CD1e in the presentation of lipid antigens.

Rapid alteration of the phosphoproteome in the moss Physcomitrella patens after cytokinin treatment.

Cytokinin hormones are crucial regulators of a large number of processes in plant development. Recently, significant progress has been made toward the elucidation of the molecular details of cytokinin that has led to a model for signal transduction involving a phosphorylation cascade. However, the current knowledge of cytokinin action remains largely unknown and does not explain the different roles of this hormone. To gain further insights into this aspect of cytokinin action and the inducible phosphorelay, we have produced the first large-scale map of a phosphoproteome in the moss Physcomitrella patens. Using a protocol that we recently published (Heintz, D.; et al. Electrophoresis 2004, 25, 1149-1159) that combines IMAC, MALDI-TOF-MS, and LC-MS/MS, a total of 172 phosphopeptide sequences were obtained by a peptide de novo sequencing strategy. Specific P. patens EST and raw genomic databases were interrogated, and protein homology searches resulted in the identification of 112 proteins that were then classified into functional categories. In addition, the temporal dynamics of the phosphoproteome in response to cytokinin stimulation was studied at 2, 4, 6, and 15 min after hormone addition. We identified 13 proteins that were not previously known targets of cytokinin action. Among the responsive proteins, some were involved in metabolism, and several proteins of unknown function were also identified. We have mapped the time course of their activation in response to cytokinin and discussed their hypothetical biological significance. Deciphering these early induced phosphorylation events has shown that the cytokinin effect can be rapid (few minutes), and the duration of this effect can be variable. Also phosphorylation events can be differentially regulated. Taken together our proteomic study provides an enriched look of the multistep phosphorelay system mediating cytokinin response and suggests the existence of a multidirectional interaction between cytokinin and numerous other pathways.

An efficient protocol for the identification of protein phosphorylation in a seedless plant, sensitive enough to detect members of signalling cascades.

We describe a reproducible protocol to explore for the first time the phosphoproteome of a seedless plant, the moss Physcomitrella patens. Following tryptic digestion of a total protein extract, phosphorylated peptides were isolated using the combination of C18 reverse-phase chromatography (RP-C18), immobilized Fe(3+) metal affinity chromatography (IMAC), capillary zone electrophoresis (CZE), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. The total protein extracts were first prepared as usually made for plant two-dimensional gel electrophoresis, the tryptic digest was desalted and concentrated by reverse phase chromatography, and from this mixture the phosphorylated peptides were captured by IMAC. Subsequently, the complex phosphopeptide mixture was separated into ten fractions by RP-C18-HPLC and each analyzed by CZE. This permitted the detection of 253 distinct phosphopeptides. These were identified by nano-LC-MS/MS and MALDI-TOF-MS analysis in conjunction with alkaline phosphatase treatment to remove covalently bound phosphate to specifically identify the phosphopeptides. Among others, several kinases and a transcription factor were identified. This protocol will be taken as a basis to unravel early events in plant signal transduction known to occur via rapid phosphorylation/dephosphorylation of proteins.