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The present study describes the predicted model and functional characterization of an endochitinase (30 kDa) from corms of Gladiolus grandiflorus. ESI-QTOF-MS generated peptide showed 96% sequence homology with family 18, Class III acidic endochitinase of Gladiolus gandavensis. Purified G. grandiflorus chitinase (GgChi) hydrolyzed 4-methylumbelliferyl β-d-N,N′,N′′-triacetylchitotriose substrate showing specific endochitinase activity. Since no structural details of GgChi were available in the Protein Data Bank (PDB), a homology model was predicted using the coordinate information of Crocus vernus chitinase (PDB ID: 3SIM). Ramachandran plot indicated 84.5% in most favored region, 14.8% in additional and 0.6% in generously allowed region while no residue in disallowed region. The predicted structure indicated a highly conserved (β/α)8 (TIM barrel) structure similar to the family 18, class III chitinases. The GgChi also showed sequence and structural homologies with other active chitinases. The GgChi (50 μg/disc) showed no antibacterial activity, but did provide mild growth inhibition of phytopathogenic fungus Fusarium oxysporum at a concentration of 500 μg/well Similarly, insect toxicity bioassays of GgChi (50 μg) against nymphs of Bemisia tabaci showed 14% reduction in adult emergence and 14% increase in mortality rate in comparison to control values. The GgChi (1.5 mg) protein showed significant reduction in a population of flour beetle (Tribolium castaneum) after 35 days, but lower reactivity against rice weevil (Sitophilus oryzae). The results of this study provide detai.led insight on functional characterization of a family 18 class III acidic plant endochitinase. Keywords: Gladiolus grandiflorus, Endochitinase, TIM barrel, Antibacterial, Antifungal

Oilseeds of the plant family Brassicaceae are important for providing both lipid and protein contents to human nutrition. Cruciferins (12S globulins) are seed storage proteins, which are getting attention due to their allergenic and pathogenicity related nature. This study describes the purification and characterization of a trimeric (∼190 kDa) cruciferin protein from the seeds of Brassica nigra (L.). Cruciferin was first partially purified by ammonium sulfate precipitation (30% saturation constant) and further purified by size exclusion chromatography. The N-terminal amino-acid sequence analysis showed 82% sequence homology with cruciferin from Arabidopsis thaliana. The 50–55 kDa monomeric cruciferin produced multiple bands of two major molecular weight ranges (α-polypeptides of 28–32 kDa and β-polypeptides of 17–20 kDa) under reduced conditions of SDS-PAGE. The 2D gel electrophoretic analysis showed the further separation of the bands into their isoforms with major pI ranges between 5.7 and 8.0 (α-polypeptides) and 5.5–8.5 (β-polypeptides). The Dynamic Light Scattering (DLS) showed the monodisperse nature of the cruciferin with hydrodynamic radius of 5.8 ± 0.1 nm confirming the trimeric nature of the protein. The Circular Dichroism (CD) spectra showed both α-helices and β-sheets in the native conformation of the trimeric protein. The pure cruciferin protein (40 mg/ml) was successfully crystallized; however, the crystals diffracted only to low resolution data (8 A). Small-angle x-ray scattering (SAXS) was applied to gain insights into the three-dimensional structure in solution. SAXS showed that the radius of gyration is 4.24 ± 0.25 nm and confirmed the nearly globular shape. The SAXS based ab initio dummy model of B. nigra cruciferin was compared with 11S globulins (PDB ID: 3KGL ) of B. napus which further confirmed a highly similar molecular weight and globular shape indicating a conserved trimerization of B. nigra cruciferin. The comparison of the scattering patterns of both proteins showed a minimized χ2-value of 1.337 confirming a similar molecular structure. This is the first report describing the purification and characterization of a cruciferin protein from seeds of B. nigra.

Due to changing climate, flooding (waterlogged soils and submergence) becomes a major problem in agriculture and crop production. In the present study, the effect of waterlogging was investigated on peroxidases of maize (Zea mays L.) leaves. The plants showed typical adaptations to flooding stress, i.e., alterations in chlorophyll a/b ratios and increased basal shoot diameter. Seven peroxidase bands could be detected by first dimension modified SDS-PAGE and 10 bands by first dimension high resolution Clear Native Electrophoresis that altered in dependence on plant development and time of waterlogging. Native isoelectric focusing revealed three acidic to neutral and four alkaline guaiacol peroxidases that could be further separated by high resolution Clear Native Electrophorese in the second dimension. One neutral peroxidase (pI 7.0) appeared to be down-regulated within four hours after flooding, whereas alkaline peroxidases (pI 9.2, 8.0 and 7.8) were up-regulated after 28 or 52 h. Second dimensions revealed molecular masses of 133 kDa and 85 kDa for peroxidases at pI 8.0 and 7.8, respectively. Size exclusion chromatography revealed native molecular masses of 30–58 kDa for peroxidases identified as class III peroxidases and ascorbate peroxidases by mass spectrometry. Possible functions of these peroxidases in flooding stress will be discussed.

In human tumors, glycoproteins often exhibit abnormal glycosylation patterns, e.g. certain Lewis structures, TF antigen, Tn antigen and/or their sialylated forms, creating additional binding sites for glycoreceptors. In the present study, we have analyzed the carbohydrate specificity of the C-type lectin CLEC10A using glycan profiling by enzyme-linked immunosorbent assay (ELISA). In addition to the known ligands, we show binding to two tumor-associated antigens, namely Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn, with an affinity of CLEC10A in the micromolar range. Detailed analyses of the glycan-lectin interactions were carried out by surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR. CLEC10A binds Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn with dissociation constants of 297 and 80 µM, respectively, as determined by SPR. Comparison of the STD nuclear magnetic resonance (NMR) binding epitopes of Tn and Neu5Acα2,6-Tn revealed a constant binding mode of the N-acetylgalactosamine moiety. This finding is in good agreement with binding studies of CLEC10A transfectomas, which show a well-defined interaction of transmembrane CLEC10A with 6-sialylated-Tn structures. Since both Neu5Acα2,6-Tn and Neu5Gcα2,6-Tn together with the previously known Tn antigen are expressed in human tumors such as mammary carcinoma, the interaction with CLEC10A expressed by macrophages and dendritic cells could be of major functional significance in tumor progression.

Malate dehydrogenase (MDH, EC 1.1.1.37) is involved in several cellular processes including plant development, nutrient uptake and oxidative stress. Evidence for a plasma membrane-associated MDH has been presented for maize (Zea mays L.) roots. In the present study isoenzymes of MDH were purified from highly enriched plasma membrane preparations of maize and compared with soluble isoenzymes (Km, pH optima, pI and molecular masses). Modified SDS-PAGE analyses revealed monomers of 41 kDa for membrane-associated MDH, whereas monomers (35 kDa) and dimers (70 kDa) were detected for soluble isoenzymes. Membrane-associated MDH of cauliflower (Brassica oleracea L.) inflorescences and spinach (Spinacia oleracea L.) leaves showed molecular masses similar to the membrane-associated MDH of maize. The specific maize MDH involved was identified by mass spectrometry (ESI-QTOF-MS/MS, MALDI-TOF-MS). The corresponding gene was cloned and the protein was characterised after heterologous expression in Escherichia coli. Enzyme kinetics and properties of the recombinant and native proteins were compared. The function of thiol groups and the presence of disulphide bonds were analysed by the effect of N-ethylmaleimide, diagonal electrophoresis and labelling. Semiquantitative reverse transcription polymerase chain reaction of maize root transcripts demonstrated a constitutive expression of the gene encoding plasma membrane-associated MDH.

Prion protein (PrP) protects neural cells against oxidative stress, hypoxia, ischemia, and hypoglycemia. In the present study we confirm that cultured PrP-deficient neurons are more sensitive to oxidative stress than wild-type neurons and present the novel findings that wild-type, but not PrP-deficient astrocytes protect wild-type cerebellar neurons against oxidative stress and that exosomes released from stressed wild-type, but not from stressed PrP-deficient astrocytes reduce neuronal cell death induced by oxidative stress. We show that neuroprotection by exosomes of stressed astrocytes depends on exosomal PrP but not on neuronal PrP and that astrocyte-derived exosomal PrP enters into neurons, suggesting neuronal uptake of astrocyte-derived exosomes. Upon exposure of wild-type astrocytes to hypoxic or ischemic conditions PrP levels in exosomes were increased. By mass spectrometry and Western blot analysis, we detected increased levels of 37/67 kDa laminin receptor, apolipoprotein E and the ribosomal proteins S3 and P0, and decreased levels of clusterin/apolipoprotein J in exosomes from wild-type astrocytes exposed to oxygen/glucose deprivation relative to exosomes from astrocytes maintained under normoxic conditions. The levels of these proteins were not altered in exosomes from stressed PrP-deficient astrocytes relative to unstressed PrP-deficient astrocytes. These results indicate that PrP in astrocytes is a sensor for oxidative stress and mediates beneficial cellular responses, e.g. release of exosomes carrying PrP and other molecules, resulting in improved survival of neurons under hypoxic and ischemic conditions.

Drastic enrichment of potential disease-specific glycoprotein markers in human plasma can be achieved by the combination of affinity- and immuno-depletion. In the affinity-fractionation step all glycoproteins carrying a certain glycostructure are isolated by lectin affinity chromatography, thus depleting other components. Against the respective glycoprotein fraction isolated from the plasma of healthy individuals antibodies are raised in llamas. The llama heavy chain antibodies (which are particularly stable) directed at the isolated plasma glycoprotein fraction are immobilized and the immunoaffinity column thus obtained is used to deplete the respective glycoprotein fraction of patient plasma samples. Depletion of proteins normally found in human plasma by 99.8-99.9% can be achieved, resulting in a 800-1000-fold enrichment of potential disease-specific proteins in the flow-through of the immunoaffinity column.

In addition to conventional antibodies, cartilaginous fish have evolved a distinctive type of immunoglobulin, designated as IgNAR, which lacks the light polypeptide chains and is composed entirely by heavy chains. IgNAR molecules can be manipulated by molecular engineering to produce the variable domain of a single heavy chain polypeptide (vNARs). These, together with the VHH camel domains, constitute the smallest naturally occurring domains able to recognize an antigen. Their special features, such as small size, long extended finger-like CDR3, and thermal and chemical stability, make them suitable candidates for biotechnological purposes. Here we describe the generation of two mouse monoclonal antibodies (MAbs), MAb 370-12 and MAb 533-10, that both specifically react with vNAR domains of the horn shark Heterodontus francisci. While the former recognizes a broad spectrum of recombinant vNAR proteins, the latter is more restricted. MAb 370-12 precipitated a single band from whole shark serum, which was identified as IgNAR by mass spectrometry. Additionally, we used MAb 370-12 to follow the IgNAR-mediated immune response of sharks during immunization protocols with two different antigens (complete cells and a synthethic peptide), thus corroborating that MAb 370-12 recognizes both isolated vNAR domains and whole IgNAR molecules. Both MAbs represent an affordable molecular, biochemical, and biotechnological tool in the field of shark single-domain antibodies.

A chitinase has been isolated and purified from Crocus vernus corms. N-­terminal amino-acid sequence analysis of the approximately 30 kDa protein showed 33% identity to narbonin, a seed protein from Vicia narbonensis L. The C. vernus chitinase was crystallized by the hanging-drop vapour-diffusion method using PEG 8000 as the main precipitant. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 172.3, b = 37.1, c = 126.4 A, β = 127° and two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.1 A.

Type II transmembrane serine proteases (TTSPs) are involved in important physiological processes, such as pro-hormone processing, cellular signaling, host immune defense, and cancer development. The diversity of functions is reflected by the multidomain architecture of these proteases, which are composed of a variety of functional domains in addition to the catalytic domain. Recently, we identified rat DESC4, a member of the HAT/DESC1-like subfamily of TTSPs. Intriguingly, DESC4 gene expression is confined to few tissues including gustatory papillae. In the current publication we present the purification of the catalytic domain of recombinant rat DESC4. Subsequently, the catalytic domain was subjected to a refolding procedure. During refolding we observed endogenous catalytic activity leading to smaller fragments, which were analyzed by peptide sequencing. The identified cleavage-sites are typical for trypsin-like serine proteases. For further analyses a homology-based model of the DESC4 catalytic domain was generated enabling us to investigate protease-substrate interaction in more detail.

Yersinia outer protein M (YopM) is a crucial immunosuppressive effector of the plaque agent Yersinia pestis and other pathogenic Yersinia species. YopM enters the nucleus of host cells but neither the mechanisms governing its nucleocytoplasmic shuttling nor its intranuclear activities are known. Here we identify the DEAD-box helicase 3 (DDX3) as a novel interaction partner of Y. enterocolitica YopM and present the three-dimensional structure of a YopM:DDX3 complex. Knockdown of DDX3 or inhibition of the exportin chromosomal maintenance 1 (CRM1) increased the nuclear level of YopM suggesting that YopM exploits DDX3 to exit the nucleus via the CRM1 export pathway. Increased nuclear YopM levels caused enhanced phosphorylation of Ribosomal S6 Kinase 1 (RSK1) in the nucleus. In Y. enterocolitica infected primary human macrophages YopM increased the level of Interleukin-10 (IL-10) mRNA and this effect required interaction of YopM with RSK and was enhanced by blocking YopM's nuclear export. We propose that the DDX3/CRM1 mediated nucleocytoplasmic shuttling of YopM determines the extent of phosphorylation of RSK in the nucleus to control transcription of immunosuppressive cytokines., Author Summary Diarrheagenic bacteria like Salmonella, Shigella and Yersinia employ a molecular injection apparatus (termed type III secretion system) to introduce toxins into host immune cells. This strategy strongly suppresses the immune response and promotes infection. Once within the host cell the bacterial toxins distribute to different subcellular compartments including the nucleus. The major immunosuppressive protein YopM from pathogenic Yersinia species, which has homologues in Salmonella and Shigella, enters the nucleus of host cells. So far it has been unclear what regulates the shuttling of YopM in and out of the nucleus and which molecular activities related to immunosuppression YopM may exert in the nucleus. Here we identify a protein named DDX3 that transports YopM out of the nucleus and thereby adjusts the nuclear level of YopM. We also show that the nuclear level of YopM controls nuclear phosphorylation/activation of the multifunctional kinase RSK1, a known regulator of gene transcription. Furthermore, association of YopM with RSK was required for increased expression of the immunosuppressive cytokine IL-10. Thus we describe a mechanism by which a bacterial virulence factor shuttles between cytosol and nucleus and demonstrate that this influences transcription of immune mediator genes.

MIZIP was originally identified as a highly conserved zinc-finger protein from human brain interacting with the C-terminus of the melanin-concentrating hormone receptor 1. However, the cellular functions of MIZIP are still not known. Here, we focussed on the identification of associated proteins using affinity purification from human cells. This resulted in the identification of alpha- and beta-tubulin. The interaction was confirmed in vitro and in vivo using GST pull-down and immunoprecipitation assays, and was mapped to the MYND zinc-finger of MIZIP and to the N-terminus of tubulin. Immunoprecipitation and immunocytochemistry analyses demonstrate that MIZIP binds to tubulin but not to cellular microtubules in vivo and that ectopic expression of MIZIP does not interfere with the overall structure of the microtubular cytoskeleton. Our results suggest that MIZIP might play an important role in mammalian cells by associating with tubulin and thus might provide a link between MCHR1 and tubulin functions.

The cell adhesion molecule close homolog of L1 (CHL1) plays important functional roles in the developing and adult nervous system. In search of the binding partners that mediate the diverse and sometimes opposing functions of CHL1, the extracellular matrix-associated proteins vitronectin and plasminogen activator inhibitor-2 (PAI-2) were identified as novel CHL1 interaction partners and tested for involvement in CHL1-dependent functions during mouse cerebellar development. CHL1-induced cerebellar neurite outgrowth and cell migration at postnatal days 6–8 were inhibited by a CHL1-derived peptide comprising the integrin binding RGD motif, and by antibodies against vitronectin or several integrins, indicating a vitronectin-dependent integrin-mediated pathway. A PAI-2-derived peptide, or antibodies against PAI-2, urokinase type plasminogen activator (uPA), uPA receptor, and several integrins reduced cell migration. CHL1 colocalized with vitronectin, PAI-2, and several integrins in cerebellar granule cells, suggesting an association among these proteins. Interestingly, at the slightly earlier age of 4–5 d, cerebellar neurons did not depend on CHL1 for neuritogenesis and cell migration. However, differentiation of progenitor cells into neurons at this stage was dependent on homophilic CHL1–CHL1 interactions. These observations indicate that homophilic CHL1trans-interactions regulate differentiation of neuronal progenitor cells at early postnatal stages, while heterophilictrans-interactions of CHL1 with vitronectin, integrins, and the plasminogen activator system regulate neuritogenesis and neuronal cell migration at a later postnatal stage of cerebellar morphogenesis. Thus, within very narrow time windows in postnatal cerebellar development, distinct types of molecular interactions mediated by CHL1 underlie the diverse functions of this protein.

We cloned the mouse TRH receptor type 2 (mTRH-R2) gene, which is 92% identical with rat TRH-R2 and 50% identical with mTRH-R1 at the amino acid level, and identified an intron within the coding sequence that is not present in the TRH-R1 gene structure. Similar to its rat homolog, mTRH-R2 binds TRH with an affinity indistinguishable from mTRH-R1, signals via the phosphoinositide pathway like mTRH-R1, but exhibits a higher basal signaling activity than mTRH-R1. We found that regulator of G protein signaling 4 (RGS4), which differentially inhibits signaling by other receptors that couple to Gq, inhibits TRH-stimulated signaling via mTRH-R1 and mTRH-R2 to similar extents. In contrast, other RGS proteins including RGS7, RGS9, and GAIP had no effect on signaling by mTRH-R1 or mTRH-R2 demonstrating the specificity of RGS4 action. Interestingly, RGS4 markedly inhibited basal signaling by mTRH-R2. Inhibition of basal signaling of mTRH-R2 by RGS4 suggests that modulation of agonist-independent signaling may be an i...

Cytokinins induce two specific morphological alterations in mosses: (i) the differentiation of a tip-growing cell into a three-faced apical cell (the so-called bud), and (ii) the division of chloroplasts. In a developmental mutant of the moss Physcomitrella patens (Hedw.) B.S.G. (mutant PC22) impeded in both cellular differentiation (bud production) and chloroplast division, addition of cytokinin (N6-delta 2-isopentenyladenine) led to bud production after 3 d in the wild type and after 7 d in the mutant. Hormone induced a division of the mutant macrochloroplasts starting within 24 h and ongoing for 72 h. During this period the abundances of several plastid proteins changed in both genotypes as judged by two-dimensional-protein gel electrophoresis, silver staining and subsequent quantification with novel computer software. Eight of these polypeptides were isolated independently, subjected to microsequencing and thus identified, resulting in the first protein sequence data from a moss. Three polypeptides (24 kDa, 22 kDa, 20 kDa) were found to be homologous to enhancer protein OEE2 of the oxygen-evolving complex, four to represent isoforms of phosphoglycerate kinase (EC 2.7.2.3), and one was identified as the beta-chain of chloroplast ATPase (EC 3.6.1.34). Possible involvement of these key enzymes of the chloroplast energy-conversion machinery in organelle division and in cellular differentiation is discussed. Further sequence information was obtained from both subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39). Amounts of these polypeptides were not appreciably affected by cytokinin in moss chloroplasts.

We report five novel monoclonal antibodies (Ki-S1, Ki-S4, Ki-S6, Ki-S7, and Ki-S8) reactive with a proliferation-related nuclear antigen. In immunoprecipitation and Western blot experiments using crude nuclear extracts, they recognized a protein of 170 kD that, after proteolytic digestion of the immunoprecipitate and sequencing of the resulting peptides, was identified as the α-isoform of human topoisomerase II. This was confirmed by testing the antibodies on a highly purified enzyme preparation. Crossreactivity with topoisomerase IIβ was ruled out by testing the antibodies on crude extracts from yeast cells expressing the β-isoform exclusively. The antibodies bind the antigen with different affinities and at different epitopes, apparently located within the carboxyl third of the enzyme. All five antibodies are suitable for archival material after adequate antigen retrieval, thereby enabling retrospective studies. This report illustrates the tissue and subcellular distribution of the antigen through the cell cycle by immunohistochemistry and confocal fluorescence microscopy. The antibodies will be useful tools in further analysis of morphological and functional aspects of topoisomerase II and may serve diagnostic purposes, as well as providing prognostic information in tumor pathology.

A novel apoprotein of an apparent molecular mass of 86 kDa in its unreduced form was identified in human triglyceride-rich lipoproteins. This protein was purified and the amino acid sequence of six proteolytic fragments was found to overlap with that of the factor H-related proteins. In parallel we identified the cDNA encoding a new complement factor H-related protein, termed FHR-4. The sequences of the new apoprotein overlapped with that of the FHR-4 protein. Similar to the previously described factor H-related proteins, FHR-4 contains a hydrophobic signal sequence followed by a stretch of five repetitive elements termed short consensus repeats. Recombinant FHR-4 protein was expressed in the baculovirus system and has an apparent molecular mass of 42 kDa. In addition a 84-kDa dimeric form of the recombinant FHR-4 was detected. Using an immunoaffinity column with antibodies raised against the recombinant FHR-4, we isolated a 86-kDa protein from human plasma. The different molecular mass of the recombinant FHR-4 and the dimeric FHR-4 in plasma is due to different carbohydrate moieties. The 86-kDa plasma protein and the novel apolipoprotein had identical mobility on SDS-polyacrylamide gel electrophoresis analysis and reacted with antisera raised against the reFHR-4 and the purified apoprotein. In conclusion, we have identified a novel factor H-related protein, FHR-4, in human plasma and demonstrate that this protein is present in triglyceride-rich lipoproteins in a dimeric form. This observation provides an intriguing new aspect on possible function(s) of this novel protein and the other factor H-related proteins.

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer's disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca(2+)- and N-glycan-independent interaction is mediated by amino acids 330-344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β42 levels in culture supernatants, while transfection with the P-domain increases amyloid-β40 levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330-344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β40 and amyloid-β42 levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer's disease.

Von Ebner's glands (VEG) are small lingual salivary glands. Their ducts open into trenches of circumvallate and foliate papillae, thus influencing the milieu where the interaction between taste receptor cells and sapid molecules takes place. The major secretions of human VEG is a protein with a molecular mass of 18 kDa. The human VEG protein crossreacts with antibodies raised against the rat VEG protein, indicating sequence similarity between the rat and human VEG proteins. This was subsequently confirmed by N-terminal protein sequencing. A cDNA clone, isolated from a human VEG library, contained an insert of 735 bp including an open reading frame that encodes the human VEG protein of 176 amino acids. Comparison of the human and rat VEG proteins revealed an overall identity of 60%. Immunocytochemistry, in situ hybridization and in vitro translation studies demonstrated the human VEG protein to be highly and exclusively expressed in VEG. The VEG proteins are members of the lipocalin protein superfamily and, together with the rat odorant binding protein II, they constitute a new subfamily. Sequence similarity to proteins such as the retinol binding protein and the odorant binding protein which are lipophilic ligand carriers, suggests a possible function for the human VEG protein in taste perception.

Members of the 14-3-3 protein family have been implicated in neuronal migration, synaptic plasticity and learning. Using affinity chromatography followed by mass spectrometry analysis, we show here that the cytoskeletal protein alphaII spectrin is a novel ligand of 14-3-3beta. We found that 14-3-3beta interacts with alphaII spectrin via the mode 2 14-3-3 binding motif RLIQS(1302)HP. Binding required phosphorylation of Ser(1302) by casein kinase II and was enhanced in the presence of calmodulin. Co-immunoprecipitation of alphaII spectrin and 14-3-3beta with the neural cell adhesion molecule NCAM suggested that the 14-3-3-spectrin-interaction affects NCAM function. Indeed, disruption of the 14-3-3beta/alphaII spectrin interaction by mutating Ser(1302) to Ala enhanced NCAM-dependent neurite outgrowth. Our results indicate that the phosphorylation-dependent interaction between 14-3-3beta and alphaII spectrin acts as a switch between positive and negative regulation of neurite outgrowth stimulated by NCAM, representing a novel and acute mechanism preventing uncontrolled elongation of neuronal processes.

Virulence of nosocomial pathogen Staphylococcus epidermidis is essentially related to formation of adherent biofilms, assembled by bacterial attachment to an artificial surface and subsequent production of a matrix that mediates interbacterial adhesion. Growing evidence supports the idea that proteins are functionally involved in S. epidermidis biofilm accumulation. We found that in S. epidermidis 1585v overexpression of a 460 kDa truncated isoform of the extracellular matrix-binding protein (Embp) is necessary for biofilm formation. Embp is a giant fibronectin-binding protein harbouring 59 Found In Various Architectures (FIVAR) and 38 protein G-related albumin-binding (GA) domains. Studies using defined Embp-positive and -negative S. epidermidis strains proved that Embp is sufficient and necessary for biofilm formation. Further data showed that the FIVAR domains of Embp mediate binding of S. epidermidis to solid-phase attached fibronectin, constituting the first step of biofilm formation on conditioned surfaces. The binding site in fibronectin was assigned to the fibronectin domain type III12. Embp-mediated biofilm formation also protected S. epidermidis from phagocytosis by macrophages. Thus, Embp is a multifunctional cell surface protein that mediates attachment to host extracellular matrix, biofilm accumulation and escape from phagocytosis, and therefore is well suited for promoting implant-associated infections.

Dissemination of primary cancer cells to distant sites is an early event in breast cancer. These cells can invade the bone marrow, rest there, and many years later disseminated tumor cells (DTC) can grow out to form overt metastases. Epithelium specific cytokeratins are commonly used as marker proteins for sensitive detection of metastatic lesions. However, due to difficulties in the detection of DTC, the question arises if DTC necessarily have the same protein expression profile as advanced tumors. On that account, we analyzed the previously uncharacterized breast cancer DTC cell line BC-M1 by 2-D DIGE. Special protein concentration and purification protocols for 2-DE were developed which resulted in high recovery rates and increased display of alkaline proteins. A broad range reference map of metastasis relevant proteins was compiled including the cytokeratins 5, 7, 8, 17, 18, and 19 and several classes of cytoskeleton proteins involved in metastasis like ezrin, gelsolin, vinculin, or vimentin. BC-M1 shows the rare and highly metastatic vimentin/cytokeratin 5 positive and cytokeratin 8/18 negative breast cancer phenotype and expresses Her-2, which is also found in stem cells/progenitor cells of primary tumors. Supported by the detection of several other epithelium-derived proteins, the example BC-M1 indicates that the protein expression profile of DTC might be reminiscent of the expression profile of the early tumor, which differs from the advanced tumor. Hence, DTC from breast cancer patients' bone marrow expressed cytokeratin 5, which further supports our hypothesis.

Production of reactive oxygen species (ROS) is a widely reported response of plants to wounding. However, the nature of enzymes responsible for ROS production and metabolism in the apoplast is still an open question. We identified and characterized the proteins responsible for the wound-induced production and detoxification of ROS in the apoplast of wheat roots (Triticum aestivum L.). Compared to intact roots, excised roots and leachates derived from them produced twice the amount of superoxide (O2(*-)). Wounding also induced extracellular peroxidase (ECPOX) activity mainly caused by the release of soluble peroxidases with molecular masses of 37, 40 and 136 kD. Peptide mass analysis by electrospray ionization-quadrupole time-of-flight-tandem mass spectrometry (ESI-QTOF-MS/MS) following lectin affinity chromatography of leachates showed the presence of peroxidases in unbound (37 kD) and bound (40 kD) fractions. High sensitivity of O2(*-)-producing activity to peroxidase inhibitors and production of O2(*-) by purified peroxidases in vitro provided evidence for the involvement of ECPOXs in O2(*-) production in the apoplast. Our results present new insights into the rapid response of roots to wounding. An important component of this response is mediated by peroxidases that are released from the cell surface into the apoplast where they can display both oxidative and peroxidative activities.

The occurrence of three plasma membrane-bound class III peroxidases has been demonstrated for maize (Zea mays L.) roots [Mika and Luthje (2003) Plant Physiol. 132:1489-1498]. In the present work a novel PM-bound peroxidase (pmPOX3) was partially purified. The experimental molecular mass of the heme protein was 38 kDa after size exclusion, and 57 kDa in non-reducing SDS-PAGE stained with the peroxidase substrates tetramethylbenzidine and H(2)O(2). The glycosylation of pmPOX1, pmPOX2b and pmPOX3 was shown by different approaches. The full length sequences of pmPOX1, pmPOX2b and pmPOX3 were identified by ESI-MS/MS and MALDI-TOF MS analysis in combination with in silico and in vivo cloning. Thus, we report the first sequence analysis of membrane-bound class III peroxidases. A partial gene analysis revealed two or three introns. Experimental and theoretical isoelectric points and molecular masses were compared. Targeting signals, the putative protein structures and the localization of the active center of the enzymes on the outside of the plasma membrane were deduced of the amino acid sequences. In contrast to other class III peroxidases, pmPOX1 seems to have a dimeric structure. Predictions of hydrophobic domains in comparison with solubilization experiments suggest an N-terminal transmembrane domain for the isoenzymes.

Prion-related protein (PrP) is a neural cell adhesion molecule involved in neurite outgrowth, neuronal survival, and synaptic function. In search of novel binding partners for PrP, we identified the α2/β2-Na+/K+-ATPase and showed that this astroglial ATPase interacts directly with the immunoglobulin superfamily adhesion molecule basigin. In cultured astrocytes, PrP is involved in regulating lactate transport via the astroglial monocarboxylate transporter 1 (MCT1) and in conjunction with α2/β2-ATPase and basigin. Lactate transport via MCT1 is glutamate dependent and regulated by glutamate receptor 2 (GluR2)-containing AMPA receptors with which PrP interacts. The functional interplay between PrP, GluR2, α2/β2-ATPase, basigin, and MCT1 in regulating lactate transport of astrocytes may be functional in the metabolic cross talk between astrocytes and neurons, most likely under stress.

Snails from the family Helicidae produce in their albumen glands a highly branched galactan, which consists almost exclusively of D- and L-galactose. The D-Gal residues are glycosydically beta(1-->6)- or beta(1-->3)-linked, whereas the L-Gal moieties are attached alpha(1-->2). Up until the present time, two beta(1-->6)-D-galactosyl transferases and one alpha(1-->2)-L-galactosyl transferase have been identified in a membrane preparation of these glands. These were used to synthesise various oligosaccharides by successive addition of the NDP-activated (NDP=nucleoside-5'-diphosphate) D-Gal or L-Fuc moieties, up to a heptasaccharide by starting from the disaccharide D-Gal-beta(1-->3)-D-Gal-beta(1-->OMe. Even larger oligosaccharides up to a tridecasaccharide were obtained by starting with the hexasaccharide D-Gal-[beta(1-->3)-D-Gal]4-beta(1-->4)-D-Glc as an acceptor substrate. This tandem exploitation process has high potential for the easy introduction of D-Gal and L-Fuc residues into a great variety of oligosaccharides, which can be used in ligand/acceptor studies.

Purpose: The monoclonal antibody Ki-A10 (IgG1) generated after immunization of mice with Hodgkin's lymphoma cell line L428 detects a nuclear antigen in human tissues with a restricted distribution pattern similar to cancer/testis antigens. The aim of this study was to characterize the antigen and to determine the expression profile in Hodgkin's lymphoma.Experimental Design: The half-life and phosphorylation of the antigen were determined by radiolabeling. The antigen was characterized by immunopurification and sequencing. Demethylation of genes is used to induce cancer/testis antigens. Ki-A10-negative cells were treated with 5-aza-2′-deoxycytidine. The Ki-A10 expression in paraffin-embedded tumors was determined immunohistochemically.Results: Immunopurification of the 25/22-kDa antigen and sequencing revealed a peptide of 14 amino acids corresponding to the gene product of the newly described gene family MGC27005, located on chromosome Xq26.3, now termed CT45. CT45 is significantly phosphorylated and down-regulated during mitosis. Demethylation of CT45-negative HeLa cells and stimulated peripheral blood lymphocytes induced CT45 expression. Except testis, immunohistochemical stainings of normal tissues, reactive lymphoid lesions, and most malignant tumors were negative. In comparison, 54 of 99 (55%) samples from pediatric and adolescent Hodgkin's lymphoma patients enrolled in the multicenter trial HD-95 stained Ki-A10 positive. Ki-A10 expression correlated with histologic subtypes (nodular sclerosis Hodgkin's lymphoma 68% versus mixed cellularity Hodgkin's lymphoma 40% versus nodular lymphocyte predominant Hodgkin's lymphoma 9%; P < 0.001).Conclusions: Ki-A10 is the first monoclonal antibody that detects CT45. As benign lymphoid lesions did not express CT45, the use of Ki-A10 antibody will facilitate the discrimination of Hodgkin's lymphoma from reactive lymphadenopathies.

Ki-Mcm6, a New Monoclonal Antibody Specific to Mcm6: Comparison of the Distribution Profile of Mcm6 and the Ki-67 Antigen

mRNA localization is a complex pathway. Besides mRNA sorting per se , this process includes aspects of regulated translation. It requires protein factors that interact with defined sequences (or sequence motifs) of the transcript, and the protein/RNA complexes are finally guided along the cytoskeleton to their ultimate destinations. The mRNA encoding the vasopressin (VP) precursor protein is localized to the nerve cell processes in vivo and in primary cultured nerve cells. Sorting of VP transcripts to dendrites is mediated by the last 395 nucleotides of the mRNA, the dendritic localizer sequence, and it depends on intact microtubules. In vitro interaction studies with cytosolic extracts demonstrated specific binding of a protein, enriched in nerve cell tissues, to the radiolabeled dendritic localizer sequence probe. Biochemical purification revealed that this protein is the multifunctional poly(A)-binding protein (PABP). It is well known for its ability to bind with high affinity to poly(A) tails of mRNAs, prerequisite for mRNA stabilization and stimulation of translational initiation, respectively. With lower affinities, PABP can also associate with non-poly(A) sequences. The physiological consequences of these PABP/RNA interactions are far from clear but may include functions such as translational silencing. Presumably, the translational state of mRNAs subject to dendritic sorting is influenced by external stimuli. PABP thus could be a component required to regulate local synthesis of the VP precursor and possibly of other proteins.

Editing of apolipoprotein B (apoB) mRNA requires the catalytic component APOBEC-1 together with "auxiliary" proteins that have not been conclusively characterized so far. Here we report the purification of these additional components of the apoB mRNA editing enzyme-complex from rat liver and the cDNA cloning of the novel APOBEC-1-stimulating protein (ASP). Two proteins copurified into the final active fraction and were characterized by peptide sequencing and mass spectrometry: KSRP, a 75-kDa protein originally described as a splicing regulating factor, and ASP, a hitherto unknown 65-kDa protein. Separation of these two proteins resulted in a reduction of APOBEC-1-stimulating activity. ASP represents a novel type of RNA-binding protein and contains three single-stranded RNA-binding domains in the amino-terminal half and a putative double-stranded RNA-binding domain at the carboxyl terminus. Purified recombinant glutathione S-transferase (GST)-ASP, but not recombinant GST-KSRP, stimulated recombinant GST-APOBEC-1 to edit apoB RNA in vitro. These data demonstrate that ASP is the second essential component of the apoB mRNA editing enzyme-complex. In rat liver, ASP is apparently associated with KSRP, which may confer stability to the editing enzyme-complex with its substrate apoB RNA serving as an additional auxiliary component.

Myotoxicity and membrane damage play a central role in the life-threatening effects of the viper envenomation. Myotoxins are an important part of the viper venomics. A Ser49 PLA2-like myotoxin from the venom of Vipera ammodytes meridionalis, the most venomous snake in Europe, was crystallized and its three-dimensional structure determined. The toxin is devoid of phospholipolytic activity. The structure demonstrates a formation of dimers. In the dimers functionally important peptide segments, located on the protein surface, point in the same direction which can strengthen the pharmacological effect. This supports the hypothesis about the physiological importance of the toxin oligomerization for the myotoxicity and membrane damage. The crystallographic model revealed that the structural determinants of myotoxicity (a positively charged C-terminal region and a hydrophobic knuckle) are fully exposed on the protein surface and accessible for interactions with target membranes. Distortion of the catalytic site region explains the absence of enzymatic activity. The structure reveals anion-binding sites which can be considered as possible sites of interactions of the toxin with a negatively charged membrane surface. The high structural similarity of the Ser49 myotoxin and Asp49 PLA2 from the same venom suggests an evolutionary relationship: probably, the Ser49 myotoxin is a product of evolution of the catalytically active phospholipase A2. The first toxin lost the enzymatic activity which is not necessary for the myotoxicity but preserved the cytotoxicity and membrane damaging activity as important components of the venom toxicity.

A cytosolic protein specifically binding to and degrading atrial natriuretic peptide (ANP) was purified from rat brain homogenate. Based on partial amino acid sequences and enzymatic properties, this protein with an apparent molecular mass of 112 kDa has been identified as the rat insulin-degrading enzyme (IDE). In addition to the known substrates, insulin and transforming-growth-factor alpha IDE binds also with high affinity (apparent Kd 60 nM) to ANP. Competition studies with structural variants of ANP demonstrate that both the C terminus and the disulfide loop of the molecule are essential for high-affinity binding. The data suggest that IDE might be involved in the cellular processing and/or metabolic clearance of ANP.