A prokaryote system optimization for rMEPLox expression: A promising non-toxic antigen for Loxosceles antivenom production.

Loxoscelism is the most dangerous araneism form in Brazil and antivenom therapy is the recommended treatment. Antivenom is produced by horse immunization with Loxosceles spider venom, which is toxic for the producer animal. Moreover, due to the high amount of venom required for horse hyperimmunization, new strategies for antigens obtention have been proposed. In this sense, our research group has previously produced a non-toxic recombinant multiepitopic protein derived from Loxosceles toxins (rMEPLox). rMEPLox was a successful immunogen, being able to induce the production of neutralizing antibodies, which could be used in the Loxoscelism treatment. However, rMEPLox obtention procedure requires optimization, as its production needs to be scaled up to suit antivenom manufacture. Therefore, an effective protocol development for rMEPlox production would be advantageous. To achieve this objective, we evaluated the influence of different cultivation conditions for rMEPLox optimum expression. The optimum conditions to obtain large amounts of rMEPlox were defined as the use of C43(DE3)pLysS as a host strain, 2xTY medium, 0.6 mM IPTG, biomass pre induction of OD600nm = 0.4 and incubation at 30 °C for 16 h. Following the optimized protocol, 39.84 mg/L of soluble rMEPLox was obtained and tested as immunogen. The results show that the obtained rMEPLox preserved the previously described immunogenicity, and it was able to generate antibodies that recognize different epitopes of the main Loxosceles venom toxins, which makes it a promising candidate for the antivenom production for loxoscelism treatment.

Unraveling the structure and function of CdcPDE: A novel phosphodiesterase from Crotalus durissus collilineatus snake venom.

This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (CdcPDE). CdcPDE was successfully isolated from whole venom using three chromatographic steps and represented 0.7% of total protein content. CdcPDE was inhibited by EDTA and reducing agents, demonstrating that metal ions and disulfide bonds are necessary for its enzymatic activity. The highest enzymatic activity was observed at pH 8-8.5 and 37 °C. Kinetic parameters indicated a higher affinity for the substrate bis(p-nitrophenyl) phosphate compared to others snake venom PDEs. Its structural characterization was done by the determination of the protein primary sequence by Edman degradation and mass spectrometry, and completed by the building of molecular and docking-based models. Functional in vitro assays showed that CdcPDE is capable of inhibiting platelet aggregation induced by adenosine diphosphate in a dose-dependent manner and demonstrated that CdcPDE is cytotoxic to human keratinocytes. CdcPDE was recognized by the crotalid antivenom produced by the Instituto Butantan. These findings demonstrate that the study of snake venom toxins can reveal new molecules that may be relevant in cases of snakebite envenoming, and that can be used as molecular tools to study pathophysiological processes due to their specific biological activities.

Improving the Pharmacodynamics and In Vivo Activity of ENPP1-Fc Through Protein and Glycosylation Engineering.

Enzyme replacement with ectonucleotide pyrophosphatase phospodiesterase-1 (ENPP1) eliminates mortality in a murine model of the lethal calcification disorder generalized arterial calcification of infancy. We used protein engineering, glycan optimization, and a novel biomanufacturing platform to enhance potency by using a three-prong strategy. First, we added new N-glycans to ENPP1; second, we optimized pH-dependent cellular recycling by protein engineering of the Fc neonatal receptor; finally, we used a two-step process to improve sialylation by first producing ENPP1-Fc in cells stably transfected with human α-2,6-sialyltransferase (ST6) and further enhanced terminal sialylation by supplementing production with 1,3,4-O-Bu3 ManNAc. These steps sequentially increased the half-life of the parent compound in rodents from 37 hours to ~ 67 hours with an added N-glycan, to ~ 96 hours with optimized pH-dependent Fc recycling, to ~ 204 hours when the therapeutic was produced in ST6-overexpressing cells with 1,3,4-O-Bu3 ManNAc supplementation. The alterations were demonstrated to increase drug potency by maintaining efficacious levels of plasma phosphoanhydride pyrophosphate in ENPP1-deficient mice when the optimized biologic was administered at a 10-fold lower mass dose less frequently than the parent compound-once every 10 days vs. 3 times a week. We believe these improvements represent a general strategy to rationally optimize protein therapeutics.

Identification and Characterization of a Redox Sensor Phosphodiesterase from Ferrovum sp. PN-J185 Containing Bacterial Hemerythrin and HD-GYP Domains.

The second messenger bis(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates numerous important physiological functions in bacteria. In this study, we identified and characterized the first dimeric, full-length, non-heme iron-bound phosphodiesterase (PDE) containing bacterial hemerythrin and HD-GYP domains (Bhr-HD-GYP). We found that the amino acid sequence encoded by the FV185_09380 gene from Ferrovum sp. PN-J185 contains an N-terminal bacterial hemerythrin domain and a C-terminal HD-GYP domain, which is characteristic of proteins with PDE activity toward c-di-GMP. Inductively coupled plasma optical emission spectroscopy analyses showed that Bhr-HD-GYP contains 4 equiv of iron atoms per subunit, suggesting both hemerythrin and HD-GYP domains have non-heme di-iron sites. A redox-dependent spectral change expected for oxo-bridged non-heme iron with carboxylate ligands was observed, and this redox interconversion was reversible. However, unlike marine invertebrate hemerythrin, which functions as an oxygen-binding protein, Bhr-HD-GYP did not form an oxygen adduct because of rapid autoxidation. The reduced ferrous iron complex of the protein catalyzed the hydrolysis of c-di-GMP to its linearized product, 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), whereas the oxidized ferric iron complex had no significant activity. These results suggest that Bhr-HD-GYP is a redox and oxygen sensor enzyme that regulates c-di-GMP levels in response to changes in cellular redox status or oxygen concentration. Our study may lead to an improved understanding of the physiology of iron-oxidizing bacterium Ferrovum sp. PN-J185.

Physical methods for studying flavoprotein photoreceptors.

Molecular mechanisms of dark-to-light state transitions in flavoprotein photoreceptors have been the subject of intense investigation. Blue-light sensing flavoproteins fall into three general classes that share aspects of their activation processes: LOV domains, BLUF proteins, and cryptochromes. In all cases, light-induced changes in flavin redox, protonation, and bonding states result in hydrogen-bond and conformational rearrangements important for regulation of downstream targets. Physical characterization of these flavoprotein states can provide valuable insights into biological function, but clear conclusions are often challenging to draw owing to complexities of data collection and interpretation. In this chapter, we briefly review the three classes of flavoprotein photoreceptors and provide methods for their recombinant production, reconstitution with flavin cofactor, and characterization. We then relate best practices and special considerations for the application of several types of spectroscopies, redox potential measurements, and X-ray scattering experiments to photosensitive flavoproteins. The methods presented are generally accessible to most laboratories.

Venom Composition in a Phenotypically Variable Pit Viper ( Trimeresurus insularis) across the Lesser Sunda Archipelago.

The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P-III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2 (PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA2 inhibitor, phospholipase B, cobra venom factor, 5'-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularis venoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularis venoms exhibited overall similarity to the other Trimeresurus complex species examined, with the exception of P. flavoviridis venom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularis venom proteins were recognized by Green Pitviper ( T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurus species, but incomplete antivenom-venom recognition was observed against P. flavoviridis venom proteins. These results demonstrate significant conservation in the venom composition of T. insularis across the Lesser Sunda archipelago relative to the other Trimeresurus species examined.

Therapeutic plasma exchange as a novel treatment for severe intrahepatic cholestasis of pregnancy: Case series and mechanism of action.

INTRODUCTION: Intrahepatic cholestasis of pregnancy is characterised by pruritus and elevated serum bile acids. The pruritus can be severe, and pharmacological options achieve inconsistent symptomatic improvement. Raised bile acids are linearly associated with adverse fetal outcomes, with existing management of limited benefit. We hypothesised that therapeutic plasma exchange removes pruritogens and lowers total bile acid concentrations, and improves symptoms and biochemical abnormalities in severe cases that have not responded to other treatments. METHODS: Four women with severe pruritus and hypercholanemia were managed with therapeutic plasma exchange. Serial blood biochemistry and visual analogue scores of itch severity were obtained. Blood and waste plasma samples were collected before and after exchange; individual bile acids and sulfated progesterone metabolites were measured with HPLC-MS, autotaxin activity and cytokine profiles with enzymatic methods. Results were analysed using segmental linear regression to describe longitudinal trends, and ratio t tests. RESULTS: Total bile acids and visual analogue itch scores demonstrated trends to transiently improve following plasma exchange, with temporary symptomatic benefit reported. Individual bile acids (excluding the drug ursodeoxycholic acid), and the sulfated metabolites of progesterone reduced following exchange (P = .03 and P = .04, respectively), whilst analysis of waste plasma demonstrated removal of autotaxin and cytokines. CONCLUSIONS: Therapeutic plasma exchange can lower potentially harmful bile acids and improve itch, likely secondary to the demonstrated removal of pruritogens. However, the limited current experience and potential complications, along with minimal sustained symptomatic benefit, restrict its current use to women with the most severe disease for whom other treatment options have been exhausted.

Comprehensive Snake Venomics of the Okinawa Habu Pit Viper, Protobothrops flavoviridis, by Complementary Mass Spectrometry-Guided Approaches.

The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA2). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA2s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5'-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.

Isolation, purification and characterization of 5'-phosphodiesterase from Aspergillus fumigatus.

5'-Phosphodiesterase (5'-PDE) catalyzes the hydrolysis of ribonucleic acid to obtain a mixture of ribonucleotides, such as 5'-guanosine monophosphate and 5'-adenosine monophosphate. In this study, a 5'-PDE was newly isolated and purified from Aspergillus fumigatus. Following purification, this enzyme exhibited a specific activity of 1036.76 U/mg protein, a molecular weight of 9.5 kDa, and an optimal temperature and pH for enzyme activity of 60°C and 5.0, respectively. However, its activity was partially inhibited by Fe3+, Cu2+, and Zn2+, but slightly improved by the presence of K+ and Na+. Additionally, chemical-modification experiments were also applied to investigate the structural information of 5'-PDE, in which the residues containing carboxyl and imidazole groups were essential for enzyme activity based on their localization in the 5'-PDE active site. Furthermore, purified 5'-PDE could specifically catalyze the synthesis of ribonucleotides with a Vmax 0.71 mmol/mg·min and a KM of 13.60 mg/mL.

Purification and Characterization of RhoPDE, a Retinylidene/Phosphodiesterase Fusion Protein and Potential Optogenetic Tool from the Choanoflagellate Salpingoeca rosetta.

RhoPDE is a type I rhodopsin/phosphodiesterase gene fusion product from the choanoflagellate Salpingoeca rosetta. The gene was discovered around the time that a similar type I rhodopsin/guanylyl cyclase fusion protein, RhoGC, was shown to control phototaxis of an aquatic fungus through a cGMP signaling pathway. RhoPDE has potential as an optogenetic tool catalyzing the hydrolysis of cyclic nucleotides. Here we provide an expression and purification system for RhoPDE, as well as a crystal structure of the C-terminal phosphodiesterase catalytic domain. We show that RhoPDE contains an even number of transmembrane segments, with N- and C-termini both located on the cytoplasmic surface of the cell membrane. The purified protein exhibits an absorption maximum at 490 nm in the dark state, which shifts to 380 nm upon exposure to light. The protein acts as a cGMP-selective phosphodiesterase. However, the activity does not appear to be modulated by light. The protein is also active with cAMP as a substrate, but with a roughly 5-7-fold lower kcat. A truncation consisting solely of the phosphodiesterase domain is also active with a kcat for cGMP roughly 6-9-fold lower than that of the full-length protein. The isolated PDE domain was crystallized, and the X-ray structure showed the protein to be a dimer similar to human PDE9. We anticipate that the purification system introduced here will enable further structural and biochemical experiments to improve our understanding of the function and mechanism of this unique fusion protein.

The single cyclic nucleotide-specific phosphodiesterase of the intestinal parasite Giardia lamblia represents a potential drug target.

BACKGROUND: Giardiasis is an intestinal infection correlated with poverty and poor drinking water quality, and treatment options are limited. According to the Center for Disease Control and Prevention, Giardia infections afflict nearly 33% of people in developing countries, and 2% of the adult population in the developed world. This study describes the single cyclic nucleotide-specific phosphodiesterase (PDE) of G. lamblia and assesses PDE inhibitors as a new generation of anti-giardial drugs. METHODS: An extensive search of the Giardia genome database identified a single gene coding for a class I PDE, GlPDE. The predicted protein sequence was analyzed in-silico to characterize its domain structure and catalytic domain. Enzymatic activity of GlPDE was established by complementation of a PDE-deficient Saccharomyces cerevisiae strain, and enzyme kinetics were characterized in soluble yeast lysates. The potency of known PDE inhibitors was tested against the activity of recombinant GlPDE expressed in yeast and against proliferating Giardia trophozoites. Finally, the localization of epitope-tagged and ectopically expressed GlPDE in Giardia cells was investigated. RESULTS: Giardia encodes a class I PDE. Catalytically important residues are fully conserved between GlPDE and human PDEs, but sequence differences between their catalytic domains suggest that designing Giardia-specific inhibitors is feasible. Recombinant GlPDE hydrolyzes cAMP with a Km of 408 µM, and cGMP is not accepted as a substrate. A number of drugs exhibit a high degree of correlation between their potency against the recombinant enzyme and their inhibition of trophozoite proliferation in culture. Epitope-tagged GlPDE localizes as dots in a pattern reminiscent of mitosomes and to the perinuclear region in Giardia. CONCLUSIONS: Our data strongly suggest that inhibition of G. lamblia PDE activity leads to a profound inhibition of parasite proliferation and that GlPDE is a promising target for developing novel anti-giardial drugs.

Basophil activation test: Implementation and standardization between systems and between instruments.

The basophil activation test (BAT) is a good ex vivo alternative for measuring hypersensitivity to an allergen in sensitized patients but still lacks standardization. In this present study, we have implemented one of the systems and proposed inter-systems, inter-instrument standardization. Our method for basophil activation and labeling on whole blood: EDTA in one step using BasoflowEx® and FlowCast® . Setup on Navios and fluorescence targets converted to set up FACSCanto™ instrument. Our results: 1) A CD203c/CD63 (BasoflowEx) method was adapted for EDTA samples and simplified. 2) Final washing and concentration and use of time parameter help acquiring as many basophils as possible, spare acquisition time and noise. 3) The modified method was validated according to ISO15189 with a precision at 5.1% RCV, linearity between 1 and 1/8 of anti-IgE stimulation. Results were very close with CCR3/CD63 system (FlowCast). 4) Standardization, between systems and even between instruments. Mean Fluorescence Intensity targets are proposed using standard beads (Cytocal® ) middle peak: FITC = 19.4; PE = 28.8 on Navios® corresponding to FITC = 4,966; PE = 7,373 for FACSCanto. Data analyzed on common software (Kaluza® ) were very closely correlated. 5) Co-labeling of B cells (CD20+) gives the possibility to monitor a significant drop of basophils under stimulation that could explain some underestimation in case of strong hypersensitivity. In conclusion, BAT would strongly benefit from easy implementation [EDTA, one step stimulation/labeling, wash, full sample analysis over time parameter, B cell relative basophil count] and standardization of instrument settings on MFI targets whatever system or instrument is used. © 2017 International Society for Advancement of Cytometry.

Comparison of total protein and enzyme levels in successive regenerations of venom from individual coralsnakes.

Coralsnakes produce highly potent neurotoxic venoms, but little is known about variations in specific enzyme components within a species or from one replenishment of venom to the next within the same animal. Since published studies are often conducted using venom pools from multiple snakes, individual differences are masked and variations among individual snakes and between subsequent venom regenerations from the same snake have rarely been documented. This study involves the analysis and comparison of four successive venom collections from each of nine individual coralsnakes in order to detect these differences. Significant variation was found within the successive re-synthesis of venom components. Even greater differences were observed between the venoms from similar individual snakes. Since studies of variation in enzymatic activity would be significant only if they were above these normal variations, it is important to be aware of these differences. These results suggest the importance of understanding the variations present within and between individuals of the same species when interpreting the potential significance of differences found as the result of genetic, environmental or ecological factors.

Identification and characterization of a novel phosphodiesterase from the metagenome of an Indian coalbed.

Phosphoesterases are involved in the degradation of organophosphorus compounds. Although phosphomonoesterases and phosphotriesterases have been studied in detail, studies on phosphodiesterases are rather limited. In our search to find novel phosphodiesterases using metagenomic approach, we cloned a gene encoding a putative phosphodiesterase (PdeM) from the metagenome of the formation water collected from an Indian coal bed. Bioinformatic analysis showed that PdeM sequence possessed the characteristic signature motifs of the class III phosphodiesterases and phylogenetic study of PdeM enabled us to identify three distinct subclasses (A, B, and C) within class III phosphodiesterases, PdeM clustering in new subclass IIIB. Bioinformatic, biochemical and biophysical characterization of PdeM further revealed some of the characteristic features of the phosphodiesterases belonging to newly described subclass IIIB. PdeM is a monomer of 29.3 kDa, which exhibits optimum activity at 25°C and pH 8.5, but low affinity for bis(pNPP) as well as pNPPP. The recombinant PdeM possessed phosphodiesterase, phosphonate-ester hydrolase and nuclease activity. It lacked phosphomonoesterase, phosphotriesterase, and RNAse activities. Overexpression of PdeM in E.coli neither affected catabolite respression nor did the recombinant protein hydrolyzed cAMP in vitro, indicating its inability to hydrolyze cAMP. Although Mn2+ was required for the activity of PdeM, but addition of metals (Mn2+ or Fe3+) did not induce oligomerization. Further increase in concentration of Mn2+ upto 3 mM, increased α-helical content as well as the phosphodiesterase activity. Structural comparison of PdeM with its homologs showed that it lacked critical residues required for dimerization, cAMP hydrolysis, and for the high affinity binding of bis(pNPP). PdeM, thus, is a novel representative of new subclass of class III phosphodiesterases.

Phosphodiesterase from Daboia russelli russelli venom: purification, partial characterization and inhibition of platelet aggregation.

Phosphodiesterases (PDEs) belong to a super-family of enzymes that have multiple roles in the metabolism of extracellular nucleotides and regulation of nucleotide-based intercellular signalling. A PDE from Russell's viper (Daboia russelli russelli) venom (DR-PDE) was purified by gel filtration, ion exchange and affinity chromatographies. Homogeneity of the preparation was verified by SDS-PAGE, SE-HPLC and mass spectrometry. It was free from 5'-nucleotidase, alkaline phosphatase and protease activities. Identity of the enzyme was ensured from partial sequence homology with other PDEs. DR-PDE was inactivated by polyvalent anti-venom serum and metal chelators. The enzyme was partially inhibited by the root extracts of four medicinal plants but remained unaffected by inhibitors of intracellular PDEs. DR-PDE hydrolyses ADP and thus, strongly inhibits ADP-induced platelet aggregation in human platelet rich plasma. This study leads to better understanding of a component of Russell's viper venom that affects homoeostatic system of the victim.

A new Enpp1 allele, Enpp1(ttw-Ham), identified in an ICR closed colony.

We recently have reported on a novel ankylosis gene that is closely linked to the Enpp1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) gene on chromosome 10. Here, we have discovered novel mutant mice in a Jcl:ICR closed colony with ankylosis in the toes of the forelimbs at about 3 weeks of age. The mutant mice exhibited rigidity in almost all joints, including the vertebral column, which increased with age. These mice also showed hypogrowth with age after 16 weeks due to a loss of visceral fat, which may have been caused by poor nutrition. Histological examination and soft X-ray imaging demonstrated the ectopic ossification of various joints in the mutant mice. In particular, increased calcium deposits were observed in the joints of the toes, the carpal bones and the vertebral column. We sequenced all exons and exon/intron boundaries of Enpp1 in the normal and mutant mice, and identified a G-to-T substitution (c.259+1G>T) in the 5' splice donor site of intron 2 in the Enpp1 gene of the mutant mice. This substitution led to the skipping of exon 2 (73 bp), which generated a stop codon at position 354 bp (amino acid 62) of the cDNA (p.V63Xfs). Nucleotide pyrophosphohydrolase (NPPH) activity of ENPP1 in the mutant mice was also decreased, suggesting that Enpp1 gene function is disrupted in this novel mutant. The mutant mice reported in this study will be a valuable animal model for future studies of human osteochondral diseases and malnutrition.

Purification, characterization, molecular cloning, and extracellular production of a novel bacterial glycerophosphocholine cholinephosphodiesterase from Streptomyces sanglieri.

A novel metal ion-independent glycerophosphocholine cholinephosphodiesterase (GPC-CP) of Streptomyces sanglieri was purified 53-fold from culture supernatant with 1.1% recovery (583 U/mg-protein). The enzyme functions as a monomer with a molecular mass of 66 kDa. The gene encoding the enzyme consists of a 1941-bp ORF that produces a signal peptide of 38 amino acids for secretion and a 646 amino acid mature protein with a calculated molecular mass of 70,447 Da. The maximum activity was found at pH 7.2 and 40°C. The enzyme hydrolyzed glycerol-3-phosphocholine (GPC) over a broad temperature range (37-60°C) and within a narrow pH range near pH 7. The enzyme was stable at 50°C for 30 min and between pH 5-10.5. The enzyme exhibited specificity toward GPC and glycerol-3-phosphoethanolamine and hydrolyzed glycerol-3-phosphate and lysophosphatidylcholine. However, the enzyme showed no activity toward any diacylglycerophospholipids and little activity toward other glycerol-3-phosphodiesters and lysophospholipids. The enzyme was not inhibited in the presence of 2 mM SDS and Mg(2+); however, Cu(2+), Zn(2+), and Co(2+) remarkably inhibited activity. Enzyme activity was also slightly enhanced by Ca(2+), Na(+), EDTA, DTT, and 2-mercaptoethanol. During the hydrolysis of GPC at 37°C and pH 7.2, apparent Vmax and turnover number (kcat) were determined to be 24.7 µmol min(-1) mg-protein(-1) and 29.0 s(-1), respectively. The apparent Km and kcat/Km values were 1.41 mM and 20.6 mM(-1) s(-1), respectively. GPC hydrolysis by GPC-CP might represent a new metabolic pathway for acquisition of a phosphorus source in actinomycetes.

Highly diverse protein library based on the ubiquitous (ß/α)8 enzyme fold yields well-structured proteins through in vitro folding selection.

Proper protein folding is a prerequisite for protein stability and enzymatic activity. Although directed evolution can be a powerful tool to investigate enzymatic function and to isolate novel activities, well-designed libraries of folded proteins are essential. In vitro selection methods are particularly capable of searching for enzymatic activities in libraries of trillions of protein variants, yet high-quality libraries of well-folded enzymes with such high diversity are lacking. We describe the construction and detailed characterization of a folding-enriched protein library based on the ubiquitous (ß/α)8 barrel fold, which is found in five of the six enzyme classes. We introduced seven randomized loops on the catalytic face of the monomeric, thermostable (ß/α)8 barrel of glycerophosphodiester phosphodiesterase (GDPD) from Thermotoga maritima. We employed in vitro folding selection based on protease digestion to enrich intermediate libraries containing three to four randomized loops for folded variants, and then combined them to assemble the final library (10¹4 DNA sequences). The resulting library was analyzed by using the in vitro protease assay and an in vivo GFP-folding assay; it contains ∼10¹² soluble monomeric protein variants. We isolated six library members and demonstrated that these proteins are soluble, monomeric and show (ß/α)8-barrel fold-like secondary and tertiary structure. The quality of the folding-enriched library improved up to 50-fold compared to a control library that was assembled without the folding selection. To the best of our knowledge, this work is the first example of combining the ultra-high throughput mRNA display method with selection for folding. The resulting (ß/α)8 barrel libraries provide a valuable starting point to study the unique catalytic capabilities of the (ß/α)8 fold, and to isolate novel enzymes.

Venom of the Brazilian spider Sicarius ornatus (Araneae, Sicariidae) contains active sphingomyelinase D: potential for toxicity after envenomation.

BACKGROUND: The spider family Sicariidae includes two genera, Sicarius and Loxosceles. Bites by Sicarius are uncommon in humans and, in Brazil, a single report is known of a 17-year old man bitten by a Sicarius species that developed a necrotic lesion similar to that caused by Loxosceles. Envenomation by Loxosceles spiders can result in dermonecrosis and severe ulceration. Sicarius and Loxosceles spider venoms share a common characteristic, i.e., the presence of Sphingomyelinases D (SMase D). We have previously shown that Loxosceles SMase D is the enzyme responsible for the main pathological effects of the venom. Recently, it was demonstrated that Sicarius species from Africa, like Loxosceles spiders from the Americas, present high venom SMase D activity. However, despite the presence of SMase D like proteins in venoms of several New World Sicarius species, they had reduced or no detectable SMase D activity. In order to contribute to a better understanding about the toxicity of New World Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from the Brazilian Sicarius ornatus spider and compare these with venoms from Loxosceles species of medical importance in Brazil. METHODOLOGY/PRINCIPAL FINDINGS: SDS-PAGE analysis showed variations in the composition of Loxosceles spp. and Sicarius ornatus venoms. Differences in the electrophoretic profiles of male and female venoms were also observed, indicating a possible intraspecific variation in the composition of the venom of Sicarius spider. The major component in all tested venoms had a Mr of 32-35 kDa, which was recognized by antiserum raised against Loxosceles SMases D. Moreover, male and female Sicarius ornatus spiders' venoms were able to hydrolyze sphingomyelin, thus showing an enzymatic activity similar to that determined for Loxosceles venoms. Sicarius ornatus venoms, as well as Loxosceles venoms, were able to render erythrocytes susceptible to lysis by autologous serum and to induce a significant loss of human keratinocyte cell viability; the female Sicarius ornatus venom was more efficient than male. CONCLUSION: We show here, for the first time, that the Brazilian Sicarius ornatus spider contains active Sphingomyelinase D and is able to cause haemolysis and keratinocyte cell death similar to the South American Loxosceles species, harmful effects that are associated with the presence of active SMases D. These results may suggest that envenomation by this Sicarius spider has the potential to cause similar pathological events as that caused by Loxosceles envenomation. Our results also suggest that, in addition to the interspecific differences, intraspecific variations in the venoms composition may play a role in the toxic potential of the New World Sicarius venoms species.

PdeB, a cyclic Di-GMP-specific phosphodiesterase that regulates Shewanella oneidensis MR-1 motility and biofilm formation.

Shewanella oneidensis MR-1, a gammaproteobacterium with respiratory versatility, forms biofilms on mineral surfaces through a process controlled by the cyclic dinucleotide messenger c-di-GMP. Cellular concentrations of c-di-GMP are maintained by proteins containing GGDEF and EAL domains, which encode diguanylate cyclases for c-di-GMP synthesis and phosphodiesterases for c-di-GMP hydrolysis, respectively. The S. oneidensis MR-1 genome encodes several GGDEF and EAL domain proteins (50 and 31, respectively), with a significant fraction (∼10) predicted to be multidomain (e.g., GGDEF-EAL) enzymes containing an additional Per-Arnt-Sim (PAS) sensor domain. However, the biochemical activities and physiological functions of these multidomain enzymes remain largely unknown. Here, we present genetic and biochemical analyses of a predicted PAS-GGDEF-EAL domain-containing protein, SO0437, here named PdeB. A pdeB deletion mutant exhibited decreased swimming motility and increased biofilm formation under rich growth medium conditions, which was consistent with an increase in intracellular c-di-GMP. A mutation inactivating the EAL domain also produced similar swimming and biofilm phenotypes, indicating that the increase in c-di-GMP was likely due to a loss in phosphodiesterase activity. Therefore, we also examined the enzymatic activity of purified PdeB and found that the protein exhibited phosphodiesterase activity via the EAL domain. No diguanylate cyclase activity was observed. In addition to the motility and biofilm phenotypes, transcriptional profiling by DNA microarray analysis of biofilms of pdeB (in-frame deletion and EAL) mutant cells revealed that expression of genes involved in sulfate uptake and assimilation were repressed. Addition of sulfate to the growth medium resulted in significantly less motile pdeB mutants. Together, these results indicate a link between c-di-GMP metabolism, S. oneidensis MR-1 biofilm development, and sulfate uptake/assimilation.