Andrew J. Weston

Proteomic characterisation of toxins isolated from nematocysts of the South Atlantic jellyfish Olindias sambaquiensis

Surprisingly little is known of the toxic arsenal of cnidarian nematocysts compared to other venomous animals. Here we investigate the toxins of nematocysts isolated from the jellyfish Olindias sambaquiensis. A total of 29 unique ms/ms events were annotated as potential toxins homologous to the toxic proteins from diverse animal phyla, including cone-snails, snakes, spiders, scorpions, wasp, bee, parasitic worm and other Cnidaria. Biological activities of these potential toxins include cytolysins, neurotoxins, phospholipases and toxic peptidases. The presence of several toxic enzymes is intriguing, such as sphingomyelin phosphodiesterase B (SMase B) that has only been described in certain spider venoms, and a prepro-haystatin P-IIId snake venom metalloproteinase (SVMP) that activates coagulation factor X, which is very rare even in snake venoms. Our annotation reveals sequence orthologs to many representatives of the most important superfamilies of peptide venoms suggesting that their origins in higher organisms arise from deep eumetazoan innovations. Accordingly, cnidarian venoms may possess unique biological properties that might generate new leads in the discovery of novel pharmacologically active drugs.

A profile of an endosymbiont-enriched fraction of the coral Stylophora pistillata reveals proteins relevant to microbial-host interactions

This study examines the response of Symbiodinium sp. endosymbionts from the coral Stylophora pistillata to moderate levels of thermal “bleaching” stress, with and without trace metal limitation. Using quantitative high throughput proteomics, we identified 8098 MS/MS events relating to individual peptides from the endosymbiont-enriched fraction, including 109 peptides meeting stringent criteria for quantification, of which only 26 showed significant change in our experimental treatments; 12 of 26 increased expression in response to thermal stress with little difference affected by iron limitation. Surprisingly, there were no significant increases in antioxidant or heat stress proteins; those induced to higher expression were generally involved in protein biosynthesis. An outstanding exception was a massive 114-fold increase of a viral replication protein indicating that thermal stress may substantially increase viral load and thereby contribute to the etiology of coral bleaching and disease. In the absence of a sequenced genome for Symbiodinium or other photosymbiotic dinoflagellate, this proteome reveals a plethora of proteins potentially involved in microbial-host interactions. This includes photosystem proteins, DNA repair enzymes, antioxidant enzymes, metabolic redox enzymes, heat shock proteins, globin hemoproteins, proteins of nitrogen metabolism, and a wide range of viral proteins associated with these endosymbiont-enriched samples. Also present were 21 unusual peptide/protein toxins thought to originate from either microbial consorts or from contamination by coral nematocysts. Of particular interest are the proteins of apoptosis, vesicular transport, and endo/exocytosis, which are discussed in context of the cellular processes of coral bleaching. Notably, the protein complement provides evidence that, rather than being expelled by the host, stressed endosymbionts may mediate their own departure.

Proteomics Links the Redox State to Calcium Signaling During Bleaching of the Scleractinian Coral Acropora microphthalma on Exposure to High Solar Irradiance and Thermal Stress

Shipboard experiments were each performed over a 2 day period to examine the proteomic response of the symbiotic coral Acropora microphthalma exposed to acute conditions of high temperature/low light or high light/low temperature stress. During these treatments, corals had noticeably bleached. The photosynthetic performance of residual algal endosymbionts was severely impaired but showed signs of recovery in both treatments by the end of the second day. Changes in the coral proteome were determined daily and, using recently available annotated genome sequences, the individual contributions of the coral host and algal endosymbionts could be extracted from these data. Quantitative changes in proteins relevant to redox state and calcium metabolism are presented. Notably, expression of common antioxidant proteins was not detected from the coral host but present in the algal endosymbiont proteome. Possible roles for elevated carbonic anhydrase in the coral host are considered: to restore intracellular pH diminished by loss of photosynthetic activity, to indirectly limit intracellular calcium influx linked with enhanced calmodulin expression to impede late-stage symbiont exocytosis, or to enhance inorganic carbon transport to improve the photosynthetic performance of algal symbionts that remain in hospite. Protein effectors of calcium-dependent exocytosis were present in both symbiotic partners. No caspase-family proteins associated with host cell apoptosis, with exception of the autophagy chaperone HSP70, were detected, suggesting that algal loss and photosynthetic dysfunction under these experimental conditions were not due to host-mediated phytosymbiont destruction. Instead, bleaching occurred by symbiont exocytosis and loss of light-harvesting pigments of algae that remain in hospite. These proteomic data are, therefore, consistent with our premise that coral endosymbionts can mediate their own retention or departure from the coral host, which may manifest as “symbiont shuffling” of Symbiodinium clades in response to environmental stress.

O-Methyltransferase Is Shared between the Pentose Phosphate and Shikimate Pathways and Is Essential for Mycosporine-Like Amino Acid Biosynthesis in Anabaena variabilis ATCC 29413

The parent core structure of mycosporine‐like amino acids (MAAs) is 4‐deoxygadusol, which, in cyanobacteria, is derived from conversion of the pentose phosphate pathway intermediate sedoheptulose 7‐phosphate by the enzymes 2‐epi‐5‐epivaliolone synthase (EVS) and O‐methyltransferase (OMT). Yet, deletion of the EVS gene from Anabaena variabilis ATCC 29413 was shown to have little effect on MAA production, thus suggesting that its biosynthesis is not exclusive to the pentose phosphate pathway. Herein, we report how, using pathway‐specific inhibitors, we demonstrated unequivocally that MAA biosynthesis occurs also via the shikimate pathway. In addition, complete in‐frame gene deletion of the OMT gene from A. variabilis ATCC 29413 reveals that, although biochemically distinct, the pentose phosphate and shikimate pathways are inextricably linked to MAA biosynthesis in this cyanobacterium. Furthermore, proteomic data reveal that the shikimate pathway is the predominate route for UV‐induced MAA biosynthesis.

Comparative proteomics reveals recruitment patterns of some protein families in the venoms of Cnidaria

ABSTRACT Cnidarians are probably the oldest group of animals to be venomous, yet our current picture of cnidarian venom evolution is highly imbalanced due to limited taxon sampling. High‐throughput tandem mass spectrometry was used to determine venom composition of the scyphozoan Chrysaora lactea and two cubozoans Tamoya haplonema and Chiropsalmus quadrumanus. Protein recruitment patterns were then compared against 5 other cnidarian venom proteomes taken from the literature. A total of 28 putative toxin protein families were identified, many for the first time in Cnidaria. Character mapping analysis revealed that 17 toxin protein families with predominantly cytolytic biological activities were likely recruited into the cnidarian venom proteome before the lineage split between Anthozoa and Medusozoa. Thereafter, venoms of Medusozoa and Anthozoa differed during subsequent divergence of cnidarian classes. Recruitment and loss of toxin protein families did not correlate with accepted phylogenetic patterns of Cnidaria. Selective pressures that drive toxin diversification independent of taxonomic positioning have yet to be identified in Cnidaria and now warrant experimental consideration. HIGHLIGHTSEarly diverging metazoans offer a phylogenetic anchor to study evolution of the venom trait.Venom proteomes of the medusozoans Chrysaoralactea, Tamoya haplonema and Chiropsalmus quadrumanus are presented.Toxin recruitment and retention patterns do not always correlate with accepted phylogeny.Factors that drive toxin diversification independent of phylogeny merit closer scrutiny.

THU0184 Identification of biomarkers in SLE pregnancies using urinary proteomics

Background Pre-eclampsia affects 2-7% of all pregnancies (1), is a leading cause of maternal morbidity and mortality and increases perinatal mortality five-fold (2). Women with Systemic Lupus Erythematosus (SLE) have a particularly high risk of developing pre-eclampsia (3) and patients with specific organ involvement (i.e. lupus nephritis) are at higher risk of developing pre-eclampsia earlier in their pregnancy (4). Objectives This study aims to identify human pregnancy biomarkers in the urinary proteome to improve the diagnosis and/or prediction of pre-eclampsia in patients with SLE. Methods Second trimester urine samples were collected from 3 women with SLE and lupus nephritis who subsequently developed pre-eclampsia, 5 SLE gestation-matched patients who did not develop pre-eclampsia and 6 healthy controls. Samples were prepared following our optimized workflow using in-gel trypsin digestion followed by LC-MS/MS. Spectral abundance of differentially expressed proteins was analysed using minimal stringency settings in Scaffold software. Results Our preliminary data suggest that pre-eclampsia results in a distinctive urinary proteome in women with lupus nephritis, but also in controls compared to women without pre-eclampsia (Figure 1a and b). One hundred and sixteen proteins were identified; of these, 6 proteins were found to be present only in the SLE pre-eclampsia urine samples (arrow). Furthermore, two proteins were found to be present in SLE pre-eclampsia (arrow), but showed a difference in SLE non-pre-eclampsia urinary samples and were absent in the samples from women with SLE without pre-eclampsia (circled in black) (Figure 1b). Conclusions Previous work in this group involving pre-eclamptic samples has used targeted mass spectrometry for relative quantitation in a validation sample set. Our study shows differentially expressed proteins, representing potential biomarker candidates of pre-eclampsia in patients with SLE/APS and hence warrant further investigation. References Sibai B et al., Lancet 2005;365:785-99. Roberts J.M.; Placenta 2002;23:359-72. Salmon JE et al., PLoS Med. 2011;8:e1001013. Bramham K et al., J Rheumatol 2011 Jun 1. Disclosure of Interest K. Schreiber Grant/Research support from: ESF - Biolupus, K. Bramham: None Declared, H. Mistry: None Declared, O. Barrutia-Ateka: None Declared, S. Lynham: None Declared, A. Weston: None Declared, M. Ward: None Declared, L. M. Bertolaccini: None Declared, L. C. Chappell: None Declared, M. Khamashta: None Declared

Identification of a novel urinary proteomic signature at time-of-disease in women with pre-eclampsia

Background Early detection/diagnosis of pre-eclampsia allows appropriate monitoring and targeting of therapeutic strategies. Urinary proteomics is a rapidly advancing technique which gives functional insight into gene expression in living organisms. Urine is an ideal medium for study as it is readily available, easily obtained, less complex than other bodily fluids and potentially a rich source of biomarkers. Hypothesis A distinctive pre-eclampsia urinary proteome profile compared to gestation matched controls can be identified using this methodology. Methods A proteome profile for time-of-disease urine samples from six preeclamptic and six gestation-matched controls was established using a validated workflow including selective immunodepletion, 1D SDS-PAGE gel fractionation, in-gel digestion of gel sections, LC-MS/MS analysis, spectral analysis and selection of candidate proteins for selective reaction monitoring (SRM) verification. Results 327 proteins were identified using minimal stringency in Scaffold. SRM peaks of proteotypic peptides showed six proteins were present only in pre-eclamptic urine samples and an additional single protein was present in controls and absent from preeclamptic samples. For protection of intellectual property protein identities are intentionally omitted. Conclusions A urinary proteomic signature can be identified in time-of-disease pre-eclampsia samples. Validation of this proteomic profile and identification of a similar mid-trimester signature for prediction of pre-eclampsia will facilitate stratification of care and surveillance.