Effect of O-linked glycosylation on the antigenicity, cellular uptake and trafficking in dendritic cells of recombinant Ber e 1.

Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.

Allosteric inhibition of human exonuclease1 (hExo1) through a novel extended ß-sheet conformation.

BACKGROUND: Human Exonuclease1 (hExo1) participates in the resection of DNA double-strand breaks by generating long 3'-single-stranded DNA overhangs, critical for homology-based DNA repair and activation of the ATR-dependent checkpoint. The C-terminal region is essential for modulating the activity of hExo1, containing numerous sites of post-translational modification and binding sites for partner proteins. METHODS: Analytical Ultracentrifugation (AUC), Dynamic Light Scattering (DLS), Circular Dichroism (CD) spectroscopy and enzymatic assays. RESULTS: AUC and DLS indicates the C-terminal region has a highly extended structure while CD suggest a tendency to adopt a novel left-handed ß-sheet structure, together implying the C-terminus may exhibit a transient fluctuating structure that could play a role in binding partner proteins known to regulate the activity of hExo1. Interaction with 14-3-3 protein has a cooperative inhibitory effect upon DNA resection activity, which indicates an allosteric transition occurs upon binding partner proteins. CONCLUSIONS: This study has uncovered that hExo1 consist of a folded N-terminal nuclease domain and a highly extended C-terminal region which is known to interact with partner proteins that regulates the activity of hExo1. A positively cooperative mechanism of binding allows for stringent control of hExo1 activity. Such a transition would coordinate the control of hExo1 by hExo1 regulators and hence allow careful coordination of the process of DNA end resection. SIGNIFICANCE: The assays presented herein could be readily adapted to rapidly identify and characterise the effects of modulators of the interaction between the 14-3-3 proteins and hExo1. It is conceivable that small molecule modulators of 14-3-3 s-hExo1 interaction may serve as effective chemosensitizers for cancer therapy.

Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines.

BACKGROUND: Chondrocytes are exposed to an inflammatory micro-environment in the extracellular matrix (ECM) of articular cartilage in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). In OA, degenerative changes and low-grade inflammation within the joint transform the behaviour and metabolism of chondrocytes, disturb the balance between ECM synthesis and degradation, and alter the osmolality and ionic composition of the micro-environment. We hypothesize that chondrocytes adjust their physiology to the inflammatory microenvironment by modulating the expression of cell surface proteins, collectively referred to as the 'surfaceome'. Therefore, the aim of this study was to characterize the surfaceome of primary equine chondrocytes isolated from healthy joints following exposure to the pro-inflammatory cytokines interleukin-1-beta (IL-1ß) and tumour necrosis factor-alpha (TNF-α). We employed combined methodology that we recently developed for investigating the surfaceome in stem cells. Membrane proteins were isolated using an aminooxy-biotinylation technique and analysed by mass spectrometry using high throughput shotgun proteomics. Selected proteins were validated by western blotting. RESULTS: Amongst the 431 unique cell surface proteins identified, a high percentage of low-abundance proteins, such as ion channels, receptors and transporter molecules were detected. Data are available via ProteomeXchange with identifier PXD014773. A high number of proteins exhibited different expression patterns following chondrocyte stimulation with pro-inflammatory cytokines. Low density lipoprotein related protein 1 (LPR-1), thrombospondin-1 (TSP-1), voltage dependent anion channel (VDAC) 1-2 and annexin A1 were considered to be of special interest and were analysed further by western blotting. CONCLUSIONS: Our results provide, for the first time, a repository for proteomic data on differentially expressed low-abundance membrane proteins on the surface of chondrocytes in response to pro-inflammatory stimuli.

Molecular phenotyping of the surfaceome of migratory chondroprogenitors and mesenchymal stem cells using biotinylation, glycocapture and quantitative LC-MS/MS proteomic analysis.

The complement of cell surface proteins, collectively referred to as the surfaceome, is a useful indicator of normal differentiation processes, and the development of pathologies such as osteoarthritis (OA). We employed biochemical and proteomic tools to explore the surfaceome and to define biomarkers in chondrogenic progenitor cells (CPC) derived from human OA knee articular cartilage. These cells have great therapeutic potential, but their unexplored biology limits their clinical application. We performed biotinylation combined with glycocapture and high throughput shotgun proteomics to define the surface proteome of human bone marrow mesenchymal stem cells (MSCs) and human CPCs. We prepared cell surface protein-enriched fractions from MSCs and CPCs, and then a proteomic approach was used to compare and evaluate protein changes between undifferentiated MSCs and CPCs. 1256 proteins were identified in the study, of which 791 (63%) were plasma membrane, cell surface or extracellular matrix proteins. Proteins constituting the surfaceome were annotated and categorized. Our results provide, for the first time, a repository of quantitative proteomic data on the surfaceome of two closely related cell types relevant to cartilage biology and OA. These results may provide novel insights into the transformation of the surfaceome during chondrogenic differentiation and phenotypic changes during OA development.

A comparison of the mitochondrial proteome and lipidome in the mouse and long-lived Pipistrelle bats.

It is accepted that smaller mammals with higher metabolic rates have shorter lifespans. The very few species that do not follow these rules can give insights into interesting differences. The recorded maximum lifespans of bats are exceptional - over 40 years, compared with the laboratory mouse of 4 years. We investigated the differences in the biochemical composition of mitochondria between bat and mouse species. We used proteomics and ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry lipidomics, to interrogate mitochondrial fractions prepared from Mus musculus and Pipistrellus pipistrellus brain and skeletal muscle. Fatty acid binding protein 3 was found at different levels in mouse and bat muscle mitochondria and its orthologues were investigated in Caenorhabditis elegans knock-downs for LBP 4, 5 and 6. In the bat, high levels of free fatty acids and N-acylethanolamine lipid species together with a significantly greater abundance of fatty acid binding protein 3 in muscle (1.8-fold, p=0.037) were found. Manipulation of fatty acid binding protein orthologues in C. elegans suggest these proteins and their role in lipid regulation are important for mitochondrial function.

Antigenic cross-reactivity between Schistosoma mansoni and pollen allergens from the birch tree (Betula verrucosa) and Timothy grass (Phleum pratense): involvement of shared glycan epitopes and implications for the hygiene hypothesis.

Previous studies have shown that schistosome infection can protect against allergic symptoms, but the underlying mechanisms are still not fully understood. Here we have shown that rabbit IgG antibodies raised against Schistosoma mansoni soluble egg antigens (SmSEA) are cross-reactive with a wide array of molecules in Timothy grass pollen (TGP) and birch tree pollen (BTP). Five of the cross-reactive pollen molecules (two from TGP and three from BTP) were selected randomly and identified by tandem mass spectrometric (TMS) analysis to be, respectively, the TGP allergens Phl p 1 and Phl p 5b, and BTP glutathione S-transferase (GST), and the BTP allergens Bet v 1 and Bet v 6.0102. Rabbit anti-SmSEA IgG antibodies that cross-reacted with each of the five allergens were found to be reactive with three major S. mansoni egg antigens, IPSE/alpha-1, omega-1 and kappa-5. Pairwise alignment of the amino acid sequences of each of the five TMS-identified pollen allergens with each of the three egg antigens revealed a low level of amino acid sequence identity. Further experiments indicated that the schistosome antigen/allergen cross-reactivity was mostly due to similar glycans present in helminths and plants, but not in mammals: so called cross-reactive carbohydrate determinants (CCDs). Previously, CCDs have been implicated in the cross-reactivity between many plants and invertebrates. Furthermore, pollen-induced anti-CCD IgGs have been found in sera of patients undergoing allergen-specific immunotherapy (SIT) and implicated in the treatment of the allergy. Thus, our finding provides not only possible explanations for the allergy-protective effect of helminth/schistosome infections as explained by the hygiene hypothesis, but also a potential starting point for improved SIT.

Treatment of Schistosoma mansoni with miltefosine in vitro enhances serological recognition of defined worm surface antigens.

BACKGROUND: Miltefosine, an anti-cancer drug that has been successfully repositioned for treatment of Leishmania infections, has recently also shown promising effects against Schistosoma spp targeting all life cycle stages of the parasite. The current study examined the effect of treating Schistosoma mansoni adult worms with miltefosine on exposure of worm surface antigens in vitro. METHODOLOGY/PRINCIPAL FINDINGS: In an indirect immunofluorescence assay, rabbit anti-S.mansoni adult worm homogenate and anti-S. mansoni infection antisera gave strong immunofluorescence of the S. mansoni adult worm surface after treatment with miltefosine, the latter antiserum having previously been shown to synergistically enhance the schistosomicidal activity of praziquantel. Rabbit antibodies that recognised surface antigens exposed on miltefosine-treated worms were recovered by elution off the worm surface in low pH buffer and were used in a western immunoblotting assay to identify antigenic targets in a homogenate extract of adult worms (SmWH). Four proteins reacting with the antibodies in immunoblots were purified and proteomic analysis (MS/MS) combined with specific immunoblotting indicated they were the S. mansoni proteins: fructose-1,6 bisphosphate aldolase (SmFBPA), Sm22.6, alkaline phosphatase and malate dehydrogenase. These antibodies were also found to bind to the surface of 3-hour schistosomula and induce immune agglutination of the parasites, suggesting they may have a role in immune protection. CONCLUSION/SIGNIFICANCE: This study reveals a novel mode of action of miltefosine as an anti-schistosome agent. The immune-dependent hypothesis we investigated has previously been lent credence with praziquantel (PZQ), whereby treatment unmasks parasite surface antigens not normally exposed to the host during infection. Antigens involved in this molecular mechanism could have potential as intervention targets and antibodies against these antigens may act to increase the drug's anti-parasite efficacy and be involved in the development of resistance to re-infection.

RadB acts in homologous recombination in the archaeon Haloferax volcanii, consistent with a role as recombination mediator.

Homologous recombination plays a central role in the repair of double-strand DNA breaks, the restart of stalled replication forks and the generation of genetic diversity. Regulation of recombination is essential since defects can lead to genome instability and chromosomal rearrangements. Strand exchange is a key step of recombination - it is catalysed by RecA in bacteria, Rad51/Dmc1 in eukaryotes and RadA in archaea. RadB, a paralogue of RadA, is present in many archaeal species. RadB has previously been proposed to function as a recombination mediator, assisting in RadA-mediated strand exchange. In this study, we use the archaeon Haloferax volcanii to provide evidence to support this hypothesis. We show that RadB is required for efficient recombination and survival following treatment with DNA-damaging agents, and we identify two point mutations in radA that suppress the ΔradB phenotype. Analysis of these point mutations leads us to propose that the role of RadB is to act as a recombination mediator, which it does by inducing a conformational change in RadA and thereby promoting its polymerisation on DNA.

Antigenic cross-reactivity between Schistosoma mansoni and peanut: a role for cross-reactive carbohydrate determinants (CCDs) and implications for the hygiene hypothesis.

The antigenic reactivity of constituents of Schistosoma mansoni and peanut (Arachis hypogaea) was investigated to determine whether identical antigenic epitopes possessed by both organisms provided a possible explanation for the negative correlation between chronic schistosome infection and atopy to allergens. Aqueous extracts of peanuts were probed in Western immunoblots with rabbit IgG antibodies raised against the egg, cercarial and adult worm stages of S. mansoni. Several molecules in the peanut extract were antigenically reactive with antibodies from the various rabbit anti-schistosome sera. A pair of cross-reactive peanut molecules at ~30 000-33 000 molecular weight was purified and both proteins were identified by mass spectrometric analysis as the peanut allergen Ara h 1. Anti-S. mansoni soluble egg antigen antibodies that were eluted off the peanut molecules reacted with two S. mansoni egg antigens identified by mass spectrometry as IPSE/α-1 and κ-5. Alignments of the amino acid sequences of Ara h 1 and either IPSE/α-1 or κ-5 revealed a low level of peptide sequence identity. Incubation of nitrocellulose paper carrying electrophoresed peanut molecules, six constituents of other allergic plants and S. mansoni egg antigens in a mild solution of sodium metaperiodate before probing with antibodies, inhibited most of the cross-reactivities. The results are consistent with the antigenic cross-reactive epitopes of S. mansoni egg antigens, peanut and other allergic plants being cross-reactive carbohydrate determinants (CCDs). These findings are novel and an explanation based on 'blocking antibodies' could provide an insight for the inverse relationship observed between schistosome infection and allergies.

Proteomic profiling of mature leaves from oil palm (Elaeis guineensis Jacq.).

Oil palm is one of the most productive oil bearing crops grown in Southeast Asia. Due to the dwindling availability of agricultural land and increasing demand for high yielding oil palm seedlings, clonal propagation is vital to the oil palm industry. Most commonly, leaf explants are used for in vitro micropropagation of oil palm and to optimize this process it is important to unravel the physiological and molecular mechanisms underlying somatic embryo production from leaves. In this study, a proteomic approach was used to determine protein abundance of mature oil palm leaves. To do this, leaf proteins were extracted using TCA/acetone precipitation protocol and separated by 2DE. A total of 191 protein spots were observed on the 2D gels and 67 of the most abundant protein spots that were consistently observed were selected for further analysis with 35 successfully identified using MALDI TOF/TOF MS. The majority of proteins were classified as being involved in photosynthesis, metabolism, cellular biogenesis, stress response, and transport. This study provides the first proteomic assessment of oil palm leaves in this important oil crop and demonstrates the successful identification of selected proteins spots using the Malaysian Palm Oil Board (MPOB) Elaeis guineensis EST and NCBI-protein databases. The MS data have been deposited in the ProteomeXchange Consortium database with the data set identifier PXD001307.

Mitochondrial proteomic profiling reveals increased carbonic anhydrase II in aging and neurodegeneration.

Carbonic anhydrase inhibitors are used to treat glaucoma and cancers. Carbonic anhydrases perform a crucial role in the conversion of carbon dioxide and water into bicarbonate and protons. However, there is little information about carbonic anhydrase isoforms during the process of ageing. Mitochondrial dysfunction is implicit in ageing brain and muscle. We have interrogated isolated mitochondrial fractions from young adult and middle aged mouse brain and skeletal muscle. We find an increase of tissue specific carbonic anhydrases in mitochondria from middle-aged brain and skeletal muscle. Mitochondrial carbonic anhydrase II was measured in the Purkinje cell degeneration (pcd5J) mouse model. In pcd5J we find mitochondrial carbonic anhydrase II is also elevated in brain from young adults undergoing a process of neurodegeneration. We show C.elegans exposed to carbonic anhydrase II have a dose related shorter lifespan suggesting that high CAII levels are in themselves life limiting. We show for the first time that the mitochondrial content of brain and skeletal tissue are exposed to significantly higher levels of active carbonic anhydrases as early as in middle-age. Carbonic anhydrases associated with mitochondria could be targeted to specifically modulate age related impairments and disease.

Purification and characterization of an extracellular ß-xylosidase from Pseudozyma hubeiensis NCIM 3574 (PhXyl), an unexplored yeast.

This paper reports on the production of ß-xylosidase from an unexplored yeast, Pseudozyma hubeinsis. The expression of this enzyme could be induced by beech wood xylan when the yeast was grown at 27 °C. The enzyme was purified to homogeneity as a glycoprotein with 23 % glycosylation. The purification protocol involved ammonium sulphate precipitation, QAE-Sephadex A50 ion exchange chromatography and sephacryl-200 column chromatography which resulted in 8.3-fold purification with 53.12 % final recovery. The purified enzyme showed prominent single band on SDS-PAGE. It is a monomeric protein of 110 kDa molecular weight confirmed by SDS-PAGE followed by MALDI-TOF mass spectrometry (112.3 kDa). The enzyme was optimally active at 60 °C and pH 4.5 and stable at pH range (4-9) and at 50 °C for 4 h. Chemical modification studies revealed that active site of the purified enzyme comprised of carboxyl, tyrosine and tryptophan residues. The carboxyl residue is involved in catalysis and tryptophan residue is solely involved in substrate binding. The best match from the search of the NCBInr database was with gi|808364558 glycoside hydrolase of Pseudozyma hubeiensis SY62 with 26 % sequence coverage confirming that it is a glycoside hydrolase/beta-glucosidase. From the search of customized SWISSPROT database, it was revealed that SWISSPROT does not contain any entries that are similar to the purified enzyme.

Cross-Reactivity between Schistosoma mansoni Antigens and the Latex Allergen Hev b 7: Putative Implication of Cross-Reactive Carbohydrate Determinants (CCDs).

IgG antibodies produced by rabbits immunized against S. mansoni antigens cross-reacted with aqueous soluble constituents of a variety of allergens. The antibody cross-reactivity was largely sensitive to degradation by treatment of the target antigens with sodium meta-periodate, suggesting the cross-reactivity was due to carbohydrate determinants that were common to both the schistosome and the allergens (CCDs). The reaction between the rabbit antibodies and a 43 kDa molecule in a rubber latex extract was analysed further: tandem mass spectrometry identified the latex molecule as allergen Hev b 7. Rabbit anti-schistosome IgG antibodies purified by acid-elution from solid-phase latex Hev b 7 reacted with the S. mansoni egg antigens IPSE/alpha-1 and kappa-5 and cercarial antigens SPO-1 and a fatty acid-binding protein. Moreover, purified anti-S. mansoni egg, latex cross-reactive antibodies reacted with antigenic constituents of some fruits, a result of potential relevance to the latex-fruit syndrome of allergic reactions. We propose that IgG anti-schistosome antibodies that cross-react with allergens may be able to block IgE-induced allergic reactions and thus provide a possible explanation for the hygiene hypothesis.

Differential proteomic analysis of embryogenic lines in oil palm (Elaeis guineensis Jacq).

UNLABELLED: Oil palm tissue culture is one way to produce superior oil palm planting materials. However, the low rate of embryogenesis is a major hindrance for the adoption of this technology in oil palm tissue culture laboratories. In this study, we use proteomic technologies to compare differential protein profiles in leaves from palms of high and low proliferation rates in tissue culture in order to understand the underlying biological mechanism for the low level of embryogenesis. Two protein extraction methods, namely trichloroacetic acid/acetone precipitation and polyethylene glycol fractionation were used to produce total proteins and fractionated protein extracts respectively, with the aim of improving the resolution of protein species using two-dimensional gel electrophoresis. A total of 40 distinct differential abundant protein spots were selected from leaf samples collected from palms with proven high and low proliferation rates. The variant proteins were subsequently identified using mass spectrometric analysis. Twelve prominent protein spots were then characterised using real-time polymerase chain reaction to compare the mRNA expression and protein abundant profiles. Three proteins, namely triosephosphate isomerase, l-ascorbate peroxidase, and superoxide dismutase were identified to be potential biomarker candidates at both the protein abundant and mRNA expression levels. BIOLOGICAL SIGNIFICANCE: In this study, proteomic analysis was used to identify abundant proteins from total protein extracts. PEG fractionation was used to reveal lower abundant proteins from both high and low proliferation embryogenic lines of oil palm samples in tissue culture. A total of 40 protein spots were found to be significant in abundance and the mRNA levels of 12 of these were assessed using real time PCR. Three proteins namely, triosephosphate isomerase, l-ascorbate peroxidase and superoxide dismutase were found to be concordant in their mRNA expression and protein abundance. Triosephosphate isomerase is a key enzyme in glycolysis. Both l-ascorbate peroxidase and superoxide dismutase play a role in anti-oxidative scavenging defense systems. These proteins have potential for use as biomarkers to screen for high and low embryogenic oil palm samples.

Interrupting peptidoglycan deacetylation during Bdellovibrio predator-prey interaction prevents ultimate destruction of prey wall, liberating bacterial-ghosts.

The peptidoglycan wall, located in the periplasm between the inner and outer membranes of the cell envelope in Gram-negative bacteria, maintains cell shape and endows osmotic robustness. Predatory Bdellovibrio bacteria invade the periplasm of other bacterial prey cells, usually crossing the peptidoglycan layer, forming transient structures called bdelloplasts within which the predators replicate. Prey peptidoglycan remains intact for several hours, but is modified and then degraded by escaping predators. Here we show predation is altered by deleting two Bdellovibrio N-acetylglucosamine (GlcNAc) deacetylases, one of which we show to have a unique two domain structure with a novel regulatory"plug". Deleting the deacetylases limits peptidoglycan degradation and rounded prey cell "ghosts" persist after mutant-predator exit. Mutant predators can replicate unusually in the periplasmic region between the peptidoglycan wall and the outer membrane rather than between wall and inner-membrane, yet still obtain nutrients from the prey cytoplasm. Deleting two further genes encoding DacB/PBP4 family proteins, known to decrosslink and round prey peptidoglycan, results in a quadruple mutant Bdellovibrio which leaves prey-shaped ghosts upon predation. The resultant bacterial ghosts contain cytoplasmic membrane within bacteria-shaped peptidoglycan surrounded by outer membrane material which could have promise as "bacterial skeletons" for housing artificial chromosomes.

Analysis of Mitochondrial haemoglobin in Parkinson's disease brain.

Mitochondrial dysfunction is an early feature of neurodegeneration. We have shown there are mitochondrial haemoglobin changes with age and neurodegeneration. We hypothesised that altered physiological processes are associated with recruitment and localisation of haemoglobin to these organelles. To confirm a dynamic localisation of haemoglobin we exposed Drosophila melanogaster to cyclical hypoxia with recovery. With a single cycle of hypoxia and recovery we found a relative accumulation of haemoglobin in the mitochondria compared with the cytosol. An additional cycle of hypoxia and recovery led to a significant increase of mitochondrial haemoglobin (p<0.05). We quantified ratios of human mitochondrial haemoglobin in 30 Parkinson's and matched control human post-mortem brains. Relative mitochondrial/cytosolic quantities of haemoglobin were obtained for the cortical region, substantia nigra and cerebellum. In age matched post-mortem brain mitochondrial haemoglobin ratios change, decreasing with disease duration in female cerebellum samples (n=7). The change is less discernible in male cerebellum (n=18). In cerebellar mitochondria, haemoglobin localisation in males with long disease duration shifts from the intermembrane space to the outer membrane of the organelle. These new data illustrate dynamic localisation of mitochondrial haemoglobin within the cell. Mitochondrial haemoglobin should be considered in the context of gender differences characterised in Parkinson's disease. It has been postulated that cerebellar circuitry may be activated to play a protective role in individuals with Parkinson's. The changing localisation of intracellular haemoglobin in response to hypoxia presents a novel pathway to delineate the role of the cerebellum in Parkinson's disease.

Purification of a chymotrypsin-like enzyme present on adult Schistosoma mansoni worms from infected mice and its characterization as a host carboxylesterase.

A serine protease-like enzyme found in detergent extracts of Schistosoma mansoni adult worms perfused from infected mice has been purified from mouse blood and further characterized. The enzyme is approximately 85 kDa and hydrolyses N-acetyl-DL-phenylalanine ß-naphthyl-ester, a chromogenic substrate for chymotrypsin-like enzymes. The enzyme from S. mansoni worms appears to be antigenically and enzymatically similar to a molecule that is present in normal mouse blood and so is seemingly host-derived. The enzyme was partially purified by depleting normal mouse serum of albumin using sodium chloride and cold ethanol, followed by repeated rounds of purification by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified material was subjected to tandem mass spectrometry and its derived peptides found to belong to mouse carboxylesterase 1C. Its ability to hydrolyse α- or ß-naphthyl acetates, which are general esterase substrates, has been confirmed. A similar carboxylesterase was purified and characterized from rat blood. Additional evidence to support identification of the enzyme as a carboxylesterase has been provided. Possible roles of the enzyme in the mouse host-parasite relationship could be to ease the passage of worms through the host's blood vessels and/or in immune evasion.

Label-free proteomic analysis of the hydrophobic membrane protein complement in articular chondrocytes: a technique for identification of membrane biomarkers.

CONTEXT: There is insufficient knowledge about the chondrocyte membranome and its molecular composition. OBJECTIVE: To develop a Triton X-114 based separation technique using nanoLC-MS/MS combined with shotgun proteomics to identify chondrocyte membrane proteins. MATERIALS AND METHODS: Articular chondrocytes from equine metacarpophalangeal joints were separated into hydrophobic and hydrophilic fractions; trypsin-digested proteins were analysed by nanoLC-MS/MS. RESULTS: A total of 315 proteins were identified. The phase extraction method yielded a high proportion of membrane proteins (56%) including CD276, S100-A6 and three VDAC isoforms. DISCUSSION: Defining the chondrocyte membranome is likely to reveal new biomarker targets for conventional and biological drug discovery.

Ras GTPase-like protein MglA, a controller of bacterial social-motility in Myxobacteria, has evolved to control bacterial predation by Bdellovibrio.

Bdellovibrio bacteriovorus invade Gram-negative bacteria in a predatory process requiring Type IV pili (T4P) at a single invasive pole, and also glide on surfaces to locate prey. Ras-like G-protein MglA, working with MglB and RomR in the deltaproteobacterium Myxococcus xanthus, regulates adventurous gliding and T4P-mediated social motility at both M. xanthus cell poles. Our bioinformatic analyses suggested that the GTPase activating protein (GAP)-encoding gene mglB was lost in Bdellovibrio, but critical residues for MglA(Bd) GTP-binding are conserved. Deletion of mglA(Bd) abolished prey-invasion, but not gliding, and reduced T4P formation. MglA(Bd) interacted with a previously uncharacterised tetratricopeptide repeat (TPR) domain protein Bd2492, which we show localises at the single invasive pole and is required for predation. Bd2492 and RomR also interacted with cyclic-di-GMP-binding receptor CdgA, required for rapid prey-invasion. Bd2492, RomR(Bd) and CdgA localize to the invasive pole and may facilitate MglA-docking. Bd2492 was encoded from an operon encoding a TamAB-like secretion system. The TamA protein and RomR were found, by gene deletion tests, to be essential for viability in both predatory and non-predatory modes. Control proteins, which regulate bipolar T4P-mediated social motility in swarming groups of deltaproteobacteria, have adapted in evolution to regulate the anti-social process of unipolar prey-invasion in the "lone-hunter" Bdellovibrio. Thus GTP-binding proteins and cyclic-di-GMP inputs combine at a regulatory hub, turning on prey-invasion and allowing invasion and killing of bacterial pathogens and consequent predatory growth of Bdellovibrio.

Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1ß.

INTRODUCTION: Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1ß (IL-1ß). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyclooxygenase-2 specific nonsteroidal pro-inflammatory drug carprofen. METHODS: Cartilage explant cultures were established by using metacarpophalangeal joints from horses euthanized for purposes other than research. Samples were treated as follows: no treatment (control), IL-1ß (10 ng/ml), carprofen (100 µg/ml), and carprofen (100 µg/ml) + IL-1ß (10 ng/ml). Explants were incubated (37°C, 5% CO2) over twelve day time courses. High-throughput nano liquid chromatography/mass spectrometry/mass spectrometry uncovered candidate proteins for quantitative western blot analysis. Proteoglycan loss was assessed by using the dimethylmethylene blue (DMMB) assay, which measures the release of sulfated glycosaminoglycans (GAGs). RESULTS: Mass spectrometry identified MMP-1, -3, -13, and the ECM constituents thrombospondin-1 (TSP-1) and fibronectin-1 (FN1). IL-1ß stimulation increased the release of all three MMPs. IL-1ß also stimulated the fragmentation of FN1 and increased chondrocyte cell death (as assessed by ß-actin release). Addition of carprofen significantly decreased MMP release and the appearance of a 60 kDa fragment of FN1 without causing any detectable cytotoxicity to chondrocytes. DMMB assays suggested that carprofen initially inhibited IL-1ß-induced GAG release, but this effect was transient. Overall, during the two time courses, GAG release was 58.67% ± 10.91% (SD) for IL-1ß versus 52.91% ± 9.35% (SD) with carprofen + IL-1ß. CONCLUSIONS: Carprofen exhibits beneficial anti-inflammatory and anti-catabolic effects in vitro without causing any detectable cytotoxicity. Combining proteomics with this explant model provides a sensitive screening system for anti-inflammatory compounds.