Production of a Fungal Punicalagin-Degrading Enzyme by Solid-State Fermentation: Studies of Purification and Characterization.

The present work describes the purification of an enzyme capable of degrading punicalagin. The enzyme was produced by Aspergillus niger GH1 by solid-state fermentation, and the enzyme production was induced by using ellagitannins as the sole carbon source. The purification steps included the concentration by lyophilization, desalting, anionic exchange, and gel filtration chromatography. The enzyme kinetic constants were calculated by using punicalagin, methyl gallate, and sugar beet arabinans. The molecular mass of the protein was estimated by SDS-PAGE. The identified bands were excised and digested using trypsin, and the peptides were submitted to HPLC-MS/MS analysis. The docking analysis was conducted, and a 3D model was created. The purification fold increases 75 times compared with the cell-free extract. The obtained Km values were 0.053 mM, 0.53% and 6.66 mM for punicalagin, sugar beet arabinans and methyl gallate, respectively. The optimal pH and temperature for the reaction were 5 and 40 °C, respectively. The SDS-PAGE and native PAGE analysis revealed the presence of two bands identified as α-l-arabinofuranosidase. Both enzymes were capable of degrading punicalagin and releasing ellagic acid.

Influence of culture conditions on ellagitannase expression and fungal ellagitannin degradation.

The role and mechanism of elagitannase is misunderstood because it exhibited different activities due to the low purity or complexity of substrates, and there is no available information about the biochemical, physicochemical and molecular characteristics of the enzyme. This study was aimed to obtain enzymatic extracts by Aspergillus niger GH1 in solid-state fermentation, using dextrose and ellagitannins as inducers of ellagitannase. Protein and bioinformatic analysis were performed to identify the protein sequence expressed in terms of culture conditions. The presence of ellagitannins increased ellagitannase activity 1143-fold compared to dextrose. The higher ellagitannase activity was found at 18 h of culture (1143.30 U g-1PE). Three groups of proteins were identified in both cultures: ß-glucosidase, phospholipase C, and triacylglycerol lipase. However, only phospholipase C was overexpressed with ellagitannins as inducers, showing the most spontaneous reaction with punicalagin (ΔG -8.56). These results suggest that phospholipase could be involved in ellagitannins biosynthesis.

An Effective Protocol for Proteome Analysis of Medaka (Oryzias latipes) after Acute Exposure to Ionizing Radiation.

All terrestrial organisms are subject to evolutionary pressures associated with natural sources of ionizing radiation (IR). The legacy of human-induced IR associated with energy, weapons production, medicine, and research has changed the distribution and magnitude of these evolutionary pressures. To date, no study has systematically examined the effects of environmentally relevant doses of radiation exposure across an organismal proteome. This void in knowledge has been due, in part, to technological deficiencies that have hampered quantifiable environmentally relevant IR doses and sensitive detection of proteomic responses. Here, we describe a protocol that addresses both needs, combining quantifiable IR delivery with a reliable method to yield proteomic comparisons of control and irradiated Medaka fish. Exposures were conducted at the Savannah River Ecology Laboratory (SREL, in Aiken, SC), where fish were subsequently dissected into three tissue sets (carcasses, organs and intestines) and frozen until analysis. Tissue proteins were extracted, resolved by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and each sample lane was divided into ten equal portions. Following in-gel tryptic digestion, peptides released from each gel portion were identified and quantified by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to obtain the most complete, comparative study to date of proteomic responses to environmentally relevant doses of IR. This method provides a simple approach for use in ongoing epidemiologic studies of chronic exposure to environmentally relevant levels of IR and should also serve well in physiological, developmental, and toxicological studies.

Combination of the low anticoagulant heparin CX-01 with chemotherapy for the treatment of acute myeloid leukemia.

Relapses in acute myelogenous leukemia (AML) are a result of quiescent leukemic stem cells (LSCs) in marrow stromal niches, where they resist chemotherapy. LSCs employ CXCL12/CXCR4 to home toward protective marrow niches. Heparin disrupts CXCL12-mediated sequestration of cells in the marrow. CX-01 is a low-anticoagulant heparin derivative. In this pilot study, we combined CX-01 with chemotherapy for the treatment of AML. Induction consisted of cytarabine and idarubicin (7 + 3) with CX-01. Twelve patients were enrolled (median age, 56 years; 3 women). Three, 5, and 4 patients had good-, intermediate-, and poor-risk disease, respectively. Day 14 bone marrows were available on 11 patients and were aplastic in all without detectable leukemia. Eleven patients (92%) had morphologic complete remission after 1 induction (CR1). Eight patients were alive at a median follow-up of 24 months (4 patients in CR1). Three patients received an allogeneic stem cell transplant in CR1. Median disease-free survival was 14.8 months. Median overall survival was not attained at the maximum follow-up time of 29.4 months. No CX-01-associated serious adverse events occurred. Median day to an untransfused platelet count of at least 20 × 109/L was 21. CX-01 is well tolerated when combined with intensive therapy for AML and appears associated with enhanced count recovery and treatment efficacy.

Characterisation of Pomegranate-Husk Polyphenols and Semi-Preparative Fractionation of Punicalagin.

INTRODUCTION: Pomegranate-husk is the main by-product generated from the pomegranate industry. It is a potential source of compounds highly appreciated by different costumers. Punicalagin is the main compound present in pomegranate-husk. OBJECTIVE: To characterise the pomegranate-husk total polyphenols by HPLC-ESI-MS and to establish a method for the recovery of punicalagin using a medium pressure liquid chromatography (MPLC) system. MATERIALS AND METHODS: The characterisation of total pomegranate-husk polyphenols was carried out using liquid chromatography coupled to mass spectrometry. Thus, 200 mg of pomegranate-husk polyphenols were fractionated by MPLC. The isolated punicalagin was characterised by HPLC-MS and was tested as standard reagent for the measurement of its scavenging capacity reducing DPPH and ABTS radicals. RESULTS: Twenty peaks were identified by analytical HPLC-MS analysis from the pomegranate-husk polyphenols. The main compounds were the punicalagin anomers, punicalin and ellagic acid. The MPLC method allowed three fractions to be obtained. In fraction three 39.40 ± 8.06 mg of punicalagin anomers (purity > 97.9%) were recovered. The scavenging capacity of punicalagin showed an IC50 of 109.53 and 151.50 µg/mL for DPPH and ABTS radicals, respectively. CONCLUSION: The MPLC system was an excellent tool for the separation of the main ellagitannins from pomegranate husk and for the isolation of punicalagin anomers. Fraction three was rich in high purity punicalagin anomers. The IC50 was obtained for DPPH and ABTS radicals. Copyright © 2017 John Wiley & Sons, Ltd.

Secretomic Insight into Glucose Metabolism of Aspergillus brasiliensis in Solid-State Fermentation.

The genus Aspergillus is ubiquitous in nature and includes various species extensively exploited industrially due to their ability to produce and secrete a variety of enzymes and metabolites. Most processes are performed in submerged fermentation (SmF); however, solid-state fermentation (SSF) offers several advantages, including lower catabolite repression and substrate inhibition and higher productivity and stability of the enzymes produced. This study aimed to explain the improved metabolic behavior of A. brasiliensis ATCC9642 in SSF at high glucose concentrations through a proteomic approach. Online respirometric analysis provided reproducible samples for secretomic studies when the maximum CO2 production rate occurred, ensuring consistent physiological states. Extracellular extracts from SSF cultures were treated by SDS-PAGE, digested with trypsin, and analyzed by LC-MS/MS. Of 531 sequences identified, 207 proteins were analyzed. Twenty-five were identified as the most abundant unregulated proteins; 87 were found to be up-regulated and 95 were down-regulated with increasing glucose concentration. Of the regulated proteins, 120 were enzymes, most involved in the metabolism of carbohydrates (51), amino acids (23), and nucleotides (9). This study shows the high protein secretory activity of A. brasiliensis under SSF conditions. High glucose concentration favors catabolic activities, while some stress-related proteins and those involved in proteolysis are down-regulated.

Immune Response Induced by an Immunodominant 60 kDa Glycoprotein of the Cell Wall of Sporothrix schenckii in Two Mice Strains with Experimental Sporotrichosis.

Cell wall (CW) components of fungus Sporothrix schenckii are the major inductors antigens of immune responses. The immunodominant 60 kDa glycoprotein (gp60) has been shown to be associated with the virulence of this fungus but its role in experimental sporotrichosis is unknown. In this work, the immunological effects of CW-purified gp60 were investigated in a model of experimental subcutaneous sporotrichosis in normal and gp60-preimmunized C57BL/6 and BALB/c mice strains which were then infected with S. schenckii conidia. Results showed that both mice strains use different cytokine profiles in order to fight S. schenckii infection; C57BL/6 mice seem to use a Th17 response while BALB/c mice tend to depend on a Th1 profile. Preimmunization with gp60 showed a downregulatory effect on the immune response since cytokines levels were diminished in both strains. There were no significant differences in the magnitude of dorsoplantar inflammation between gp60-preimmunized and nonimmunized mice of both strains. However, skin lesions due to the infection in gp60-preimmunized mice were more severe in BALB/c than in C57BL/6 mice, suggesting that the antigen exerts a higher downregulatory effect on the Th1 response.

Detection of 2 immunoreactive antigens in the cell wall of Sporothrix brasiliensis and Sporothrix globosa.

The cell wall of members of the Sporothrix schenckii complex contains highly antigenic molecules which are potentially useful for the diagnosis and treatment of sporotrichosis. In this study, 2 immunoreactive antigens of 60 (Gp60) and 70 kDa (Gp70) were detected in the cell wall of the yeast morphotypes of Sporothrix brasiliensis and Sporothrix globosa.

Heparan sulfate deficiency disrupts developmental angiogenesis and causes congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a common birth malformation with a heterogeneous etiology. In this study, we report that ablation of the heparan sulfate biosynthetic enzyme NDST1 in murine endothelium (Ndst1ECKO mice) disrupted vascular development in the diaphragm, which led to hypoxia as well as subsequent diaphragm hypoplasia and CDH. Intriguingly, the phenotypes displayed in Ndst1ECKO mice resembled the developmental defects observed in slit homolog 3 (Slit3) knockout mice. Furthermore, introduction of a heterozygous mutation in roundabout homolog 4 (Robo4), the gene encoding the cognate receptor of SLIT3, aggravated the defect in vascular development in the diaphragm and CDH. NDST1 deficiency diminished SLIT3, but not ROBO4, binding to endothelial heparan sulfate and attenuated EC migration and in vivo neovascularization normally elicited by SLIT3-ROBO4 signaling. Together, these data suggest that heparan sulfate presentation of SLIT3 to ROBO4 facilitates initiation of this signaling cascade. Thus, our results demonstrate that loss of NDST1 causes defective diaphragm vascular development and CDH and that heparan sulfate facilitates angiogenic SLIT3-ROBO4 signaling during vascular development.

The secondary cell wall polysaccharide of Bacillus anthracis provides the specific binding ligand for the C-terminal cell wall-binding domain of two phage endolysins, PlyL and PlyG.

Endolysins are bacteriophage enzymes that lyse their bacterial host for phage progeny release. They commonly contain an N-terminal catalytic domain that hydrolyzes bacterial peptidoglycan (PG) and a C-terminal cell wall-binding domain (CBD) that confers enzyme localization to the PG substrate. Two endolysins, phage lysin L (PlyL) and phage lysin G (PlyG), are specific for Bacillus anthracis. To date, the cell wall ligands for their C-terminal CBD have not been identified. We recently described structures for a number of secondary cell wall polysaccharides (SCWPs) from B. anthracis and B. cereus strains. They are covalently bound to the PG and are comprised of a -ManNAc-GlcNAc-HexNAc- backbone with various galactosyl or glucosyl substitutions. Surface plasmon resonance (SPR) showed that the endolysins PlyL and PlyG bind to the SCWP from B. anthracis (SCWPBa) with high affinity (i.e. in the µM range with dissociation constants ranging from 0.81 × 10(-6) to 7.51 × 10(-6) M). In addition, the PlyL and PlyG SCWPBa binding sites reside with their C-terminal domains. The dissociation constants for the interactions of these endolysins and their derived C-terminal domains with the SCWPBa were in the range reported for other protein-carbohydrate interactions. Our findings show that the SCWPBa is the ligand that confers PlyL and PlyG lysin binding and localization to the PG. PlyL and PlyG also bound the SCWP from B. cereus G9241 with comparable affinities to SCWPBa. No detectable binding was found to the SCWPs from B. cereus ATCC (American Type Culture Collection) 10987 and ATCC 14579, thus demonstrating specificity of lysin binding to SCWPs.

SPR and differential proteolysis/MS provide further insight into the interaction between PGIP2 and EPGs.

By using surface plasmon resonance (SPR) technology, the kinetics of the interaction of various fungal endopolygalacturonases (EPGs) (13 EPGs) with Phaseolus vulgaris (bean) PGIP2 was carried out to determine whether or not there is any interaction between polygalacturonases-inhibiting protein (PGIP) and EPG. The effect of polygalacturonic acid (PGA) on these interactions was also evaluated. The results show that all EPGs evaluated bind to PGIP2, except for AnPGb and the strength of the interaction depends on the EPG/PGIP2 pairing. Further, the presence of PGA has a moderate to strong effect on the EPG/PGIP2 interaction and the strength of the effect is dependent on the exact EPG/PGIP2 pairing. The differences in affinity in the absence and presence of PGA, suggest a certain level of cooperativity. These results indicate a three-component complex similar to that observed for the heparin-ATIII-thrombin, the FGF-FGFR-heparin, or the hedgehog-interference hedgehog-heparan complexes. This data points to an architecture in which the inhibitor binds at a location distant from the substrate binding site. Furthermore, we applied differential proteolysis mass spectrometry (DPMS) to study the location of the binding site between EPG and PGIP2. DPMS studies indicate that PGIP2 does not bind AnPGII, AnPGa, and AnPGc directly over the active site but instead binds on the face opposite to the active site, creating an allosteric interaction.

The C-terminal fragment of axon guidance molecule Slit3 binds heparin and neutralizes heparin's anticoagulant activity.

Slit3 is a large molecule with multiple domains and belongs to axon guidance families. To date, the biological functions of Slit3 are still largely unknown. Our recent study demonstrated that the N-terminal fragment of Slit3 is a novel angiogenic factor. In this study, we examined the biological function of the C-terminal fragment of human Slit3 (HSCF). The HSCF showed a high-affinity binding to heparin. The binding appeared to be heparin/heparan sulfate-specific and depends on the size, the degree of sulfation, the presence of N- and 6-O-sulfates and carboxyl moiety of the polysaccharide. Functional studies observed that HSCF inhibited antithrombin binding to heparin and neutralized the antifactor IIa and Xa activities of heparin and the antifactor IIa activity of low-molecular-weight heparin (LMWH). Thromboelastography analysis observed that HSCF reversed heparin's anticoagulation in global plasma coagulation. Taken together, these observations demonstrate that HSCF is a novel heparin-binding protein that potently neutralizes heparin's anticoagulation activity. This study reveals a potential for HSCF to be developed as a new antidote to treat overdosing of both heparin and LMWH in clinical applications.

Initial proteome analysis of caffeine-induced proteins in Aspergillus tamarii using two-dimensional fluorescence difference gel electrophoresis.

Caffeine is toxic to most microorganisms. However, some filamentous fungi, such as Aspergillus tamarii, are able to metabolize this alkaloid when fed caffeine as the sole nitrogen source. The aim of the present work was to identify intracellular A. tamarii proteins, regulated by caffeine, using fluorescence difference two-dimensional gel electrophoresis. Specific proteins from two culture media of A. tamarii grown either on ammonium sulfate or caffeine as the sole nitrogen source were analysed by mass spectrometry. Thirteen out of a total of 85 differentially expressed spots were identified after database search. Identified up-regulated proteins include phosphoglycerate kinase, malate dehydrogenase, dyp-type peroxidase family protein, heat shock protein, Cu, Zn superoxidase dismutase and xanthine dehydrogenase. Some of the proteins identified in this study are involved in the caffeine degradation pathway as well as in stress response, suggesting that stress proteins could be involved in caffeine metabolism in filamentous fungi.

Proteomic analysis of ripening tomato fruit infected by Botrytis cinerea.

Botrytis cinerea, a model necrotrophic fungal pathogen that causes gray mold as it infects different organs on more than 200 plant species, is a significant contributor to postharvest rot in fresh fruit and vegetables, including tomatoes. By describing host and pathogen proteomes simultaneously in infected tissues, the plant proteins that provide resistance and allow susceptibility and the pathogen proteins that promote colonization and facilitate quiescence can be identified. This study characterizes fruit and fungal proteins solubilized in the B. cinerea-tomato interaction using shotgun proteomics. Mature green, red ripe wild type and ripening inhibited (rin) mutant tomato fruit were infected with B. cinerea B05.10, and the fruit and fungal proteomes were identified concurrently 3 days postinfection. One hundred eighty-six tomato proteins were identified in common among red ripe and red ripe-equivalent ripening inhibited (rin) mutant tomato fruit infected by B. cinerea. However, the limited infections by B. cinerea of mature green wild type fruit resulted in 25 and 33% fewer defense-related tomato proteins than in red and rin fruit, respectively. In contrast, the ripening stage of genotype of the fruit infected did not affect the secreted proteomes of B. cinerea. The composition of the collected proteins populations and the putative functions of the identified proteins argue for their role in plant-pathogen interactions.

Heparin-induced leukocytosis requires 6-O-sulfation and is caused by blockade of selectin- and CXCL12 protein-mediated leukocyte trafficking in mice.

Leukocytosis refers to an increase in leukocyte count above the normal range in the blood and is a common laboratory finding in patients. In many cases, the mechanisms underlying leukocytosis are not known. In this study, we examined the effects, the structural determinants, and the underlying mechanisms of heparin-induced leukocytosis, a side effect occurring in 0.44% of patients receiving heparin. We observed that heparin induced both lymphocytosis and neutrophilia, and the effects required heparin to be 6-O-sulfated but did not require its anticoagulant activity. Cell mobilization studies revealed that the lymphocytosis was attributable to a combination of blockage of lymphocyte homing and the release of thymocytes from the thymus, whereas the neutrophilia was caused primarily by neutrophil release from the bone marrow and demargination in the vasculature. Mechanistic studies revealed that heparin inhibits L- and P-selectin, as well as the chemokine CXCL12, leading to leukocytosis. Heparin is known to require 6-O-sulfate to inhibit L- and P-selectin function, and in this study we observed that 6-O-sulfate is required for its interaction with CXCL12. We conclude that heparin-induced leukocytosis requires glucosamine 6-O-sulfation and is caused by blockade of L-selectin-, P-selectin-, and CXCL12-mediated leukocyte trafficking.

Differential properties of Aspergillus niger tannase produced under solid-state and submerged fermentations.

Significant differences on structure, stability, and catalytic properties of tannase were found when this enzyme was produced under solid-state and submerged fermentations (SSF and SmF) by Aspergillus niger. The specific activity was 5.5 times higher on SSF than in SmF. Significant differences in isoelectric points of tannases were found. The pH optima for both types of enzyme was found at 6 and the pH stability of SSF and SmF tannase were at 6 and 5-8, respectively. The optimal temperature range was from 50 to 60 °C for SmF tannase and 60 °C for SSF tannase, and both enzyme types showed tolerance to high temperatures (60-70 °C). The SSF tannase showed a major specificity for methyl gallate substrate while SmF tannase for tannic acid. All metal ions tested, had an activity inhibition from 30-46% on SSF tannase. SDS-PAGE analysis as well as gel localization studies of both SSF and SmF purified tannases showed a single band with a molecular weight of 102 and 105 kDa, respectively. Different levels of glycosylation were found among SSF and SmF purified tannases. This is the first report about structural differences among tannase produced under SSF and SmF and this study provides basis for explanation of the stability and catalytic differences observed previously for this two tannase types.

The Botrytis cinerea early secretome.

The extracellular proteome, or secretome, of phytopathogenic fungi is presumed to be a key element of their infection strategy. Especially interesting constituents of this set are those proteins secreted at the beginning of the infection, during the germination of conidia on the plant surfaces or wounds, since they may play essential roles in the establishment of a successful infection. We have germinated Botrytis cinerea conidia in conditions that resemble the plant environment, a synthetic medium enriched with low molecular weight plant compounds, and we have collected the proteins secreted during the first 16 h by a double precipitation protocol. 2-D electrophoresis of the precipitated secretome showed a spot pattern similar for all conditions evaluated and for the control medium without plant extract. The proteins in 16 of these spots were identified by PMF and corresponded to 11 different polypeptides. Alternative determination of secretome composition by LC-MS/MS of tryptic fragments rendered a much larger number, 105 proteins, which included all previously identified by PMF. All proteins were functionally classified according to their putative function in the infection process. Key features of the early secretome include a large number of proteases, the abundance of proteins involved in the degradation of plant defensive barriers, and plenty of proteins with unknown function.

Site mapping and characterization of O-glycan structures on alpha-dystroglycan isolated from rabbit skeletal muscle.

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, alpha-dystroglycan (alpha-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown alpha-DG to be modified by both O-GalNAc- and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle alpha-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on alpha-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from alpha-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.

A proteomic study of pectin-degrading enzymes secreted by Botrytis cinerea grown in liquid culture.

Botrytis cinerea is a pathogenic filamentous fungus, which infects more than 200 plant species. The enzymes secreted by B. cinerea play an important role in the successful colonization of a host plant. Some of the secreted enzymes are involved in the degradation of pectin, a major component of the plant cell wall. A total of 126 proteins secreted by B. cinerea were identified by growing the fungus on highly or partially esterified pectin, or on sucrose in liquid culture. Sixty-seven common proteins were identified in each of the growth conditions, of which 50 proteins exhibited a SignalP motif. Thirteen B. cinerea proteins with functions related to pectin degradation were identified in both pectin growth conditions, while only four were identified in sucrose. Our results indicate it is unlikely that the activation of B. cinerea from the dormant state to active infection is solely dependent on changes in the degree of esterification of the pectin component of the plant cell wall. Further, these results suggest that future studies of the B. cinerea secretome in infections of ripe and unripe fruits will provide important information that will describe the mechanisms that the fungus employs to access nutrients and decompose tissues.