Identification and biochemical analysis of a homolog of a sulfate transporter from a vanadium-rich ascidian Ascidia sydneiensis samea

Background

Several species of ascidians accumulate extremely high levels of vanadium ions in the vacuoles of their blood cells (vanadocytes). The vacuoles of vanadocytes also contain many protons and sulfate ions. To maintain the concentration of sulfate ions, an active transporter must exist in the blood cells, but no such transporter has been reported in vanadium-accumulating ascidians.

Methods

We determined the concentration of vanadium and sulfate ions in the blood cells (except for the giant cells) of Ascidia sydneiensis samea. We cloned cDNA for an Slc13-type sulfate transporter, AsSUL1, expressed in the vanadocytes of A. sydneiensis samea. The synthetic mRNA of AsSUL1 was introduced into Xenopus oocytes, and its ability to transport sulfate ions was analyzed.

Results

The concentrations of vanadium and sulfate ions in the blood cells (except for the giant cells) were 38 mM and 86 mM, respectively. The concentration of sulfate ions in the blood plasma was 25 mM. The transport activity of AsSUL1 was dependent on sodium ions, and its maximum velocity and apparent affinity were 2500 pmol/oocyte/h and 1.75 mM, respectively.

General significance

This could account for active uptake of sulfate ions from blood plasma where sulfate concentration is 25 mM, as determined in this study.     

Anaerobic degradation of citrate under sulfate reducing and methanogenic conditions

Citrate is an important component of metal processing effluents such as chemical mechanical planarization wastewaters of the semiconductor industry. Citrate can serve as an electron donor for sulfate reduction applied to promote the removal of metals, and it can also potentially be used by methanogens that coexist in anaerobic biofilms. The objective of this study was to evaluate the degradation of citrate with sulfate-reducing and methanogenic biofilms. During batch bioassays, the citrate, acetate, methane and sulfide concentrations were monitored. The results indicate that independent of the biofilm or incubation conditions used, citrate was rapidly fermented with specific rates ranging from 566 to 720 mg chemical oxygen demand (COD) consumed per gram volatile suspended solids per day. Acetate was found to be the main fermentation product of citrate degradation, which was later degraded completely under either methanogenic or sulfate reducing conditions. However, if either sulfate reduction or methanogenesis was infeasible due to specific inhibitors (2-bromoethane sulfonate), absence of sulfate or lack of adequate microorganisms in the biofilm, acetate accumulated to levels accounting for 90–100% of the citrate-COD consumed. Based on carbon balances measured in phosphate buffered bioassays, acetate, CO₂ and hydrogen are the main products of citrate fermentation, with a molar ratio of 2:2:1 per mol of citrate, respectively. In bicarbonate buffered bioassays, acetogenesis of H₂ and CO₂ increased the yield of acetate. The results taken as a whole suggest that in anaerobic biofilm systems, citrate is metabolized via the formation of acetate as the main metabolic intermediate prior to methanogenesis or sulfate reduction. Sulfate reducing consortia must be enriched to utilize acetate as an electron donor in order to utilize the majority of the electron-equivalents in citrate.     

Are bioleaching rates determined by the available particle surface area concentration?

It is well known that pulp density and particle size determine the available surface area concentration and have an influence in the overall rate of bioleaching of minerals. As metal solubilization takes place through the surface area of the particles, it can be expected that different combinations of pulp densities and particle sizes giving the same surface area concentration would determine the same leaching rate. The objective of this work was to test this hypothesis on the effect of surface area concentration, pulp density and particle size of the biooxidation of a pyritic gold concentrate by the thermophilic Archaeon Sulfolobus metallicus in shake flasks. The gold concentrate was used at 2.5%, 5%, 10%, and 15% w/v pulp density and at four size fractions: 150–106, 106–75, 75–38 and –38 μm. Temperature was 68°C and the initial pH was 2.0. Results showed that the volumetric productivities of iron and sulfate depend not only on the surface area concentration but also on pulp density and particle size considered separately. These two variables not only determine surface area but also exert additional effects on the process, so the hypothesis was not confirmed. Maximum attained iron productivity was 1.042 g/l day with the 75–38 μm fraction at 5% pulp density. Maximum sulfate productivity was 4.279 g/l day with the 75–38 μm fraction at 10% pulp density.     

Temperature adaptation of soil bacterial communities along an Antarctic climate gradient: predicting responses to climate warming

Soil microorganisms, the central drivers of terrestrial Antarctic ecosystems, are being confronted with increasing temperatures as parts of the continent experience considerable warming. Here we determined short‐term temperature dependencies of Antarctic soil bacterial community growth rates, using the leucine incorporation technique, in order to predict future changes in temperature sensitivity of resident soil bacterial communities. Soil samples were collected along a climate gradient consisting of locations on the Antarctic Peninsula (Anchorage Island, 67 °34′S, 68 °08′W), Signy Island (60 °43′S, 45 °38′W) and the Falkland Islands (51 °76′S 59 °03′W). At each location, experimental plots were subjected to warming by open top chambers (OTCs) and paired with control plots on vegetated and fell‐field habitats. The bacterial communities were adapted to the mean annual temperature of their environment, as shown by a significant correlation between the mean annual soil temperature and the minimum temperature for bacterial growth (T_min). Every 1 °C rise in soil temperature was estimated to increase T_min by 0.24–0.38 °C. The optimum temperature for bacterial growth varied less and did not have as clear a relationship with soil temperature. Temperature sensitivity, indicated by Q₁₀ values, increased with mean annual soil temperature, suggesting that bacterial communities from colder regions were less temperature sensitive than those from the warmer regions. The OTC warming (generally <1 °C temperature increases) over 3 years had no effects on temperature relationship of the soil bacterial community. We estimate that the predicted temperature increase of 2.6 °C for the Antarctic Peninsula would increase T_min by 0.6–1 °C and Q₁₀ (0–10 °C) by 0.5 units.     

Sulfate Activation Enzymes: Phylogeny and Association with Pyrophosphatase

The enzymes catalyzing the first two reactions in the sulfate activation pathway, ATP-sulfurylase (S) and APS-kinase (K), are fused as ‘KS’ in animals but are fused as ‘SK’ in select bacteria and fungi. We have discovered a novel triple fusion protein of K, S, and pyrophosphatase (P) in several protozoan genomes within the Stramenopile lineage. These triple domain fusion proteins led us to hypothesize that pyrophosphatase enzymes and sulfate activation enzymes physically interact to impact the thermodynamics of the sulfate activation pathway. In support of this hypothesis, we demonstrate through biochemical assays that separately encoded KS and P proteins physically interact and that KS/P complexes activate more sulfate than KS alone. We also conclude on the basis of phylogenetic analyses that all known KS fusions originate from a single fusion event early in the eukaryotic lineage. Strikingly, our analyses support the same conclusion for all known SK fusions. These observations indicate that the patchwork of fused and nonfused S and K genes observed in modern-day eukaryotes and prokaryotes are the result of the two ancestral fusion genes evolving by an assortment of gene fissions, duplications, deletions, and horizontal transfers in different lineages. Our integrative use of genomics, phylogenetics, and biochemistry to characterize pyrophosphatase as a new member of the sulfate activation pathway should be effective at identifying new protein members and connections in other molecular pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported here are available in the GenBank/DDBJ/EMBL databases under accession number EU352210.     

Evidence for L-dopa incorporation into cell proteins in patients treated with levodopa

Levodopa (L-dopa) is the most widely used agent for the symptomatic relief of Parkinson's disease. There is concern that chronic L-dopa treatment may be detrimental, with some studies suggesting that L-dopa may be neurotoxic. A potentially important mechanism whereby L-dopa may exert neurotoxic effects has been overlooked: that of the incorporation of L-dopa into proteins by protein synthesis. L-Dopa competes with tyrosine as a substrate in protein synthesis in vitro. We provide evidence that L-dopa can also be incorporated into proteins in vivo. Blood from L-dopa-treated and -non-treated patients was separated into protein, erythrocyte and lymphocyte fractions and levels of protein-incorporated dopa quantified by HPLC. Levels of protein-incorporated dopa were significantly increased in lymphocyte cell proteins from L-dopa-treated patients. This has not arisen from oxidative pathways as there was no evidence of oxidative damage to proteins. In addition, there was no increase in protein-incorporated dopa in erythrocytes, which are not actively synthesizing proteins. We suggest that protein-incorporated dopa could also be generated in the CNS. The accumulation of protein-incorporated dopa in cells is associated with oxidative stress and impaired function and could contribute to some of the problems associated with long-term L-dopa treatment.     

Differential effects of murine and human factor X on adenovirus transduction via cell-surface heparan sulfate

Serum coagulation factor X (FX) is proposed to play a major role in adenovirus tropism, promoting transduction by bridging the virus to cell-surface heparan sulfate proteoglycans (HSPGs). Both murine FX and human FX increased transduction by Ad.CMVfLuc, an adenovirus vector, in murine hepatocyte-like cells and human hepatocarcinoma cells. In contrast, only hFX increased transduction of several non-hepatic cancer cell lines and Chinese hamster ovary (CHO) cells. Not only was mFX unable to promote transduction in these cells, it competitively blocked hFX-enhanced transduction. Competition and HSPG digestion experiments suggested mFX- and hFX-enhanced transduction in hepatocyte-derived cells, and hFX-enhanced transduction in epithelial cancer cells were dependent on HSPGs. Ad·hFX-mediated transduction of CHO mutants unable to produce HSPGs was also curtailed. Hepatocyte-derived cells expressed substantially more HSPGs than the cancer cell lines. Dose-response curves and heparin-Sepharose binding suggested Ad·hFX has greater affinity for HSPGs than does Ad·mFX. In coagulation factor-depleted mice hFX also had enhanced ability, compared with mFX, to reconstitute hepatic adenovirus transduction. The results suggest that differences in Ad·hFX and Ad·mFX affinity to HSPGs may result in differences in their ability to enhance adenovirus transduction of many cells. These findings may have implications for murine models of adenovirus vector targeting.     

Deciphering the role of histidine 252 in mycobacterial adenosine 5'-phosphosulfate (APS) reductase catalysis

Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (APR) catalyzes the first committed step in sulfate reduction for the biosynthesis of cysteine and is essential for survival in the latent phase of tuberculosis infection. The reaction catalyzed by APR involves the nucleophilic attack by conserved Cys-249 on adenosine 5'-phosphosulfate, resulting in a covalent S-sulfocysteine intermediate that is reduced in subsequent steps by thioredoxin to yield the sulfite product. Cys-249 resides on a mobile active site lid at the C terminus, within a K(R/T)ECG(L/I)H motif. Owing to its strict conservation among sulfonucleotide reductases and its proximity to the active site cysteine, it has been suggested that His-252 plays a key role in APR catalysis, specifically as a general base to deprotonate Cys-249. Using site-directed mutagenesis, we have changed His-252 to an alanine residue and analyzed the effect of this mutation on the kinetic parameters, pH rate profile, and ionization of Cys-249 of APR. Interestingly, our data demonstrate that His-252 does not perturb the pK(a) of Cys-249 or play a direct role in rate-limiting chemical steps of the reaction. Rather, we show that His-252 enhances substrate affinity via interaction with the α-phosphate and the endocyclic ribose oxygen. These findings were further supported by isothermal titration calorimetry to provide a thermodynamic profile of ligand-protein interactions. From an applied standpoint, our study suggests that small-molecules targeting residues in the dynamic C-terminal segment, particularly His-252, may lead to inhibitors with improved binding affinity.     

Two distinct sites in sonic Hedgehog combine for heparan sulfate interactions and cell signaling functions

Hedgehog (Hh) proteins are morphogens that mediate many developmental processes. Hh signaling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hh proteins require heparan sulfate proteoglycans (HSPGs) for their normal distribution and signaling activity. Here, we have used molecular modeling to examine the heparin-binding domain of sonic hedgehog (Shh). In biochemical and cell biological assays, the importance of specific residues of the putative heparin-binding domain for signaling was assessed. It was determined that key residues in human (h) Shh involved in heparin and HSPG syndecan-4 binding and biological activity included the well known cationic Cardin-Weintraub motif (lysines 32-38) but also a previously unidentified major role for lysine 178. The activity of Shh mutated in these residues was tested by quantitation of alkaline phosphatase activity in C3H10T1/2 cells differentiating into osteoblasts and hShh-inducible gene expression in PANC1 human pancreatic ductal adenocarcinoma cells. Mutated hShhs such as K37S/K38S, K178S, and particularly K37S/K38S/K178S that could not interact with heparin efficiently had reduced signaling activity compared with wild type hShh or a control mutation (K74S). In addition, the mutant hShh proteins supported reduced proliferation and invasion of PANC1 cells compared with control hShh proteins, following endogenous hShh depletion by RNAi knockdown. The data correlated with reduced Shh multimerization where the Lys-37/38 and/or Lys-178 mutations were examined. These studies provide a new insight into the functional roles of hShh interactions with HSPGs, which may allow targeting this aspect of hShh biology in, for example, pancreatic ductal adenocarcinoma.     

Investigation of a sulfate transfer during autohydrolysis of a fucoidan from the brown alga Fucus evanescens by tandem ESIMS

A fucoidan from the brown alga Fucus evanescens was effectively depolymerized by autohydrolysis. Negative-ion electrospray ionization mass spectrometry (ESIMS) revealed that the mixture contained sulfated mono- and oligosaccharides with polymerization degree (DP) up to 6, having from 1 to 4 sulfate groups per molecule. The prevalence of oligosaccharides with even DP was observed. It could be explained by the tendency of the 3-linked α-l-fucopyranose residues to hydrolyze faster than 4-linked ones. The intermolecular sulfate transfer during autohydrolysis was detected by ESIMS, when equimolar quantities of d-Rib and d-Glc were added as acceptors. The products were singly-sulfated and hexose was about four times more effective as an acceptor, than pentose. It was impossible to record MS/MS spectra of the sulfate transfer products, since intensities of their ions were too low.