Mapping the major proteome of reproductive fluids and sperm membranes of rams: From the cauda epididymis to ejaculation.

The present study evaluated the major proteome of ram seminal plasma and the main secretions that contribute to its formation, such as the cauda epididymal and accessory sex gland fluids. The study also investigated sperm membrane protein profiles before and after ejaculation. First, semen was collected from six rams (using artificial vagina) to obtain seminal plasma and ejaculated sperm. Then, rams were vasectomized for collection of accessory sex gland fluid (using artificial vagina). Next, rams were slaughtered and cauda epididymal fluid (CEF), seminal vesicle fluid, bulbourethral gland fluid and cauda epididymal sperm were properly collected. Proteins from reproductive fluids and sperm membranes were analyzed by 2-D SDS-PAGE, tandem mass spectrometry and bioinformatics. There we 386 proteins and 256 isoforms identified in all samples. The most abundant seminal plasma proteins were BSP1, BSP5 and spermadhesins (bodhesin-2 and spermadhesin Z13-like). These proteins were present in similar patterns in maps of accessory sexgland fluid, with very low quantities in the CEF and absent in the bulbourethral gland secretion. Thus, practically all BSPs and spermadhesins come from seminal vesicles. Bulbourethral gland fluid brought bactericidal/permeability-increasing protein-containing Family A member 1 isoforms, superoxide dismutase [Cu-Zn] and betamicroseminoprotein to seminal plasma. CEF was the major provider of clusterin, epididymal-specific lipocalin-5-like isoform, epididymal secretory gluthathione peroxidase, epididymal secretory protein E1 and prostaglandin-H2 D-isomerase to seminal plasma. Albumin came from all reproductive fluids. BSPs and spermadhesins were present in 2-D maps of ejaculated sperm but absent in cauda epididymal sperm. These proteins come from the seminal vesicles and bind to sperm at the moment of ejaculation. Other proteins of ejaculated and epididymal sperm membranes were mostly associated to energy production, cell adhesion and proteolytic activity (ATP synthases, disintegrin, metalloproteinase domain-containing protein 32, carboxypeptidase Q and cytosol aminopeptidase). In conclusion, there is a well-orchestrated sequence of events to form the major seminal plasma proteome, with specific contributions from cauda epididymis, seminal vesicles and bulbourethral glands. The present data contribute to a better understanding of male reproductive biology and how sperm functions are affected by the noncellularmicro environment of semen.

A thermostable chitinase from the antagonistic Chromobacterium violaceum that inhibits the development of phytopathogenic fungi.

The biocontrol activity of some soil strains of Chromobacterium sp. against pathogenic fungi has been attributed to secreted chitinases. The aim of this work was to characterize biochemically a recombinant chitinase (CvChi47) from C. violaceum ATCC 12472 and to investigate its effects on phytopathogenic fungi. CvChi47 is a modular enzyme with 450 amino acid residues, containing a type I signal peptide at the N-terminal region, followed by one catalytic domain belonging to family 18 of the glycoside hydrolases, and two type-3 chitin-binding domains at the C-terminal end. The recombinant enzyme was expressed in Escherichia coli as a His-tagged protein and purified to homogeneity. The native signal peptide of CvChi47 was used to direct its secretion into the culture medium, from where the recombinant product was purified by affinity chromatography on chitin and immobilized metal. The purified protein showed an apparent molecular mass of 46 kDa, as estimated by denaturing polyacrylamide gel electrophoresis, indicating the removal of the signal peptide. CvChi47 was a thermostable protein, retaining approximately 53.7% of its activity when heated at 100 °C for 1 h. The optimum hydrolytic activity was observed at 60 °C and pH 5. The recombinant chitinase inhibited the conidia germination of the phytopathogenic fungi Fusarium oxysporum and F. guttiforme, hence preventing mycelial growth. Furthermore, atomic force microscopy experiments revealed a pronounced morphological alteration of the cell surface of conidia incubated with CvChi47 in comparison to untreated cells. Taken together, these results show the potential of CvChi47 as a molecular tool to control plant diseases caused by these Fusarium species.

Proteomic identification and purification of seed proteins from native Amazonian species displaying antifungal activity.

MAIN CONCLUSION: Seeds of native species from the rain forest (Amazon) are source of chitinases and their protein extracts exhibited strong and broad antifungal activity. Numerous plant species native to the Amazon have not yet been chemically studied. Studies of seeds are scarcer, since adversities in accessing study areas and seasonality pose constant hurdles to systematic research. In this study, proteins were extracted from seeds belonging to endemic Amazon species and were investigated for the first time. Proteolytic activity, peptidase inhibitors, and chitinases were identified, but chitinolytic activity predominated. Four proteins were purified through chromatography and identified as lectin and chitinases by MS/MS analyses. The proteins were examined for inhibition of a phytopathogen (Fusarium oxysporum). Analyses by fluorescence microscopy suggested binding of propidium iodide to DNA of fungal spores, revealing that spore integrity was lost when accessed by the proteins. Further structural and functional analyses of defensive proteins belonging to species facing highly complex ecosystems such as Amazonia should be conducted, since these could provide new insights into specificity and synergism involving defense proteins of plants submitted to a very complex ecosystem.

Gut peptidases from a specialist herbivore of latex plants are capable of milk protein hydrolysis: Inputs for hypoallergenic milk formulas.

Transitory allergies to cow milk proteins in infants or adults have become a public health problem. Although extensively or partially hydrolyzed cow milk protein formulas are available, these products are costly. Therefore, studies into innovative enzymes to digest cow milk proteins are needed. Danaus plexippus gut peptidases were purified and examined with regard to cow milk protein hydrolysis. The peptidases hydrolyzed caseins and whey proteins. However, after heat treatment, there was a significant improvement in ß-lactoglobulin hydrolysis. The hydrolyzed cow milk proteins were not recognized by anti-casein antibodies and only reacted slightly with antibodies against whey proteins. This performance was better than that of partially hydrolyzed formulas and similar to that of an extensively hydrolyzed formula. These results suggest that D. plexippus gut peptidases are suitable and innovative enzymes to produce hypoallergenic cow milk protein formulas.

Proteomic analysis of follicular fluid from tropically-adapted goats.

The present study was conducted to characterize the major proteome of ovarian follicular fluid from locally-adapted, "Canindé" goats in the northeast of Brazil. Eight estrous cycling goats received a hormonal treatment consisting of medroxyprogesterone acetate, D-cloprostenol and FSH. Fluid was collected by laparoscopy from small (<3mm), medium (3-4mm) and large (>4mm) follicles and then, proteins were analyzed by 2-D SDS-PAGE and tandem mass spectrometry. Thirty-six proteins were identified in the goat follicular fluid, including albumin, immunoglobulins, ceruloplasmin, complement factor B, alpha-1B-glycoprotein precursor, serotransferrin, complement C3 and serpins, among others. Albumin and immunoglobulins were the most abundant proteins. Protein concentrations were similar in the fluid from small (45.3±3.1mg/mL), medium (44.2±3.3mg/mL) and large follicles (45.1±2.3mg/mL). The intensities of spots identified in 2-D gels as serotransferrin, zinc-alpha-2-glycoprotein-like, complement factor B and complement protein C3 differed (P<0.05) among follicle categories. The amount of serotransferrin was greater in the medium than small follicles (P<0.05). Content of zinc-alpha-2-glycoprotein-like, complement factor B and complement C3 was greater (P<0.05) in the fluid of large follicles than in medium follicles. Based on gene ontology, the major molecular functions associated with goat follicular fluid proteins were binding and catalytic activity, while the main biological processes were related to regulation, cellular processing, location and the immune system. In conclusion, the major proteome of the follicular fluid from goats subjected to hormonal stimulation was elucidated in the present study. Also, molecules associated with follicle development are potential biomarkers of oocyte competence were prevalent.

Pattern of protein expression in the epididymis of Oligoryzomys nigripes (Cricetidae, Sigmodontinae).

In the epididymis, epithelial cells work in a concerted manner to create a luminal environment for sperm maturation, transport, and storage. However, the cell functions may be affected by anthropogenic factors, causing negative impacts on male fertility. In our study, we describe the pattern of protein expression in the epithelium and luminal fluid from epididymis of Oligoryzomys nigripes, a South American sigmodontine rodent whose reproductive biology has been little studied. Nine animals were captured from a preserved area of Atlantic Forest, where the exposure to anthropogenic influences is minimal. Epididymides were processed for histological analysis under light and epifluorescence microscopy, in which we used cell-specific markers aquaporin 9 (AQP9), vacuolar H+-ATPase (V-ATPase), and cytokeratin 5 (KRT5). Other samples were assessed for protein expression using shotgun proteomics. Similar to laboratory rodents, principal cells expressed AQP9 in their stereocilia. Basal cells, identified by KRT5 labeling, presented lateral body projections and a few axiopodia going toward the lumen. Clear cells expressed V-ATPase in their sub-apical vesicles and microplicae, and showed different shapes along the duct. Shotgun proteomics detected 51 proteins from epididymal supernatant. Most of them have been previously described in other species, indicating that they are well conserved. Twenty-three proteins detected in O. nigripes have not been described in epididymis from other South American sigmodontine rodents, confirming that the secretion pattern is species-specific. Our findings in O. nigripes from a protected area may help to create a baseline for studies investigating the effects of anthropogenic factors on functionality of the epididymal epithelium.

A Chitin-binding Protein Purified from Moringa oleifera Seeds Presents Anticandidal Activity by Increasing Cell Membrane Permeability and Reactive Oxygen Species Production.

Candida species are opportunistic pathogens that infect immunocompromised and/or immunosuppressed patients, particularly in hospital facilities, that besides representing a significant threat to health increase the risk of mortality. Apart from echinocandins and triazoles, which are well tolerated, most of the antifungal drugs used for candidiasis treatment can cause side effects and lead to the development of resistant strains. A promising alternative to the conventional treatments is the use of plant proteins. M. oleifera Lam. is a plant with valuable medicinal properties, including antimicrobial activity. This work aimed to purify a chitin-binding protein from M. oleifera seeds and to evaluate its antifungal properties against Candida species. The purified protein, named Mo-CBP2, represented about 0.2% of the total seed protein and appeared as a single band on native PAGE. By mass spectrometry, Mo-CBP2 presented 13,309 Da. However, by SDS-PAGE, Mo-CBP2 migrated as a single band with an apparent molecular mass of 23,400 Da. Tricine-SDS-PAGE of Mo-CBP2 under reduced conditions revealed two protein bands with apparent molecular masses of 7,900 and 4,600 Da. Altogether, these results suggest that Mo-CBP2 exists in different oligomeric forms. Moreover, Mo-CBP2 is a basic glycoprotein (pI 10.9) with 4.1% (m/m) sugar and it did not display hemagglutinating and hemolytic activities upon rabbit and human erythrocytes. A comparative analysis of the sequence of triptic peptides from Mo-CBP2 in solution, after LC-ESI-MS/MS, revealed similarity with other M. oleifera proteins, as the 2S albumin Mo-CBP3 and flocculating proteins, and 2S albumins from different species. Mo-CBP2 possesses in vitro antifungal activity against Candida albicans, C. parapsilosis, C. krusei, and C. tropicalis, with MIC50 and MIC90 values ranging between 9.45-37.90 and 155.84-260.29 µM, respectively. In addition, Mo-CBP2 (18.90 µM) increased the cell membrane permeabilization and reactive oxygen species production in C. albicans and promoted degradation of circular plasmid DNA (pUC18) from Escherichia coli. The data presented in this study highlight the potential use of Mo-CBP2 as an anticandidal agent, based on its ability to inhibit Candida spp. growth with apparently low toxicity on mammalian cells.

Production in Pichia pastoris, antifungal activity and crystal structure of a class I chitinase from cowpea (Vigna unguiculata): Insights into sugar binding mode and hydrolytic action.

A cowpea class I chitinase (VuChiI) was expressed in the methylotrophic yeast P. pastoris. The recombinant protein was secreted into the culture medium and purified by affinity chromatography on a chitin matrix. The purified chitinase migrated on SDS-polyacrylamide gel electrophoresis as two closely-related bands with apparent molecular masses of 34 and 37 kDa. The identity of these bands as VuChiI was demonstrated by mass spectrometry analysis of tryptic peptides and N-terminal amino acid sequencing. The recombinant chitinase was able to hydrolyze colloidal chitin but did not exhibit enzymatic activity toward synthetic substrates. The highest hydrolytic activity of the cowpea chitinase toward colloidal chitin was observed at pH 5.0. Furthermore, most VuChiI activity (approximately 92%) was retained after heating to 50 °C for 30 min, whereas treatment with 5 mM Cu2+ caused a reduction of 67% in the enzyme's chitinolytic activity. The recombinant protein had antifungal activity as revealed by its ability to inhibit the spore germination and mycelial growth of Penicillium herquei. The three-dimensional structure of VuChiI was resolved at a resolution of 1.55 Å by molecular replacement. The refined model had 245 amino acid residues and 381 water molecules, and the final R-factor and Rfree values were 14.78 and 17.22%, respectively. The catalytic domain of VuChiI adopts an α-helix-rich fold, stabilized by 3 disulfide bridges and possessing a wide catalytic cleft. Analysis of the crystallographic model and molecular docking calculations using chito-oligosaccharides provided evidences about the VuChiI residues involved in sugar binding and catalysis, and a possible mechanism of antifungal action is suggested.

Biochemical Profile, Biological Activities, and Toxic Effects of Proteins in the Rhinella schneideri Parotoid Gland Secretion.

Parotoid glands of amphibians are known for the production of several biologically active compounds having pharmacological and toxic effects in mammals. In the present work, a protein fraction obtained from Rhinella schneideri parotoid gland (RsPP) was characterized to study its biological and toxic effects. Rhinella schneideri parotoid secretion is composed of up to 30% (w/w) of soluble proteins. Tandem mass spectrometric analysis of the RsPP identified 104 proteins, including actin, beta-actin, ribosomal proteins, catalase, galectin, and uncharacterized proteins; however, no peptidases were found, and this result was reinforced by the absence of proteolytic activity. In addition, RsPP did not exhibit pro-coagulant or antibacterial effects. However, pretreatment of mice with different doses of RsPP intraperitoneally inhibited carrageenan-induced paw edema and increased tissue myeloperoxidase activity. RsPP also reduced interleukin 1ß levels in the peritoneal cavities and cell migration in the peritoneal cavities of an animal model of carrageenan-induced peritonitis. Subchronic treatment of animals with RsPP for 7 consecutive days did not alter the serum biochemical, renal, or liver parameters. However, a significant reduction in blood leukocyte count was observed. Our results showed that R. schneideri parotoid secretion contains proteins with anti-inflammatory and slight toxic effects.

First isolation and antinociceptive activity of a lipid transfer protein from noni (Morinda citrifolia) seeds.

In this study a novel heat-stable lipid transfer protein, designated McLTP1, was purified from noni (Morinda citrifolia L.) seeds, using four purification steps which resulted in a high-purified protein yield (72 mg McLTP1 from 100g of noni seeds). McLTP1 exhibited molecular masses of 9.450 and 9.466 kDa, determined by electrospray ionisation mass spectrometry. The N-terminal sequence of McLTP1 (AVPCGQVSSALSPCMSYLTGGGDDPEARCCAGV), as analysed by NCBI-BLAST database, revealed a high degree of identity with other reported plant lipid transfer proteins. In addition, this protein proved to be resistant to pepsin, trypsin and chymotrypsin digestion. McLTP1 given intraperitoneally (1, 2, 4 and 8 mg/kg) and orally (8 mg/kg) caused an inhibition of the writhing response induced by acetic acid in mice. This protein displayed thermostability, retaining 100% of its antinociceptive activity after 30 min incubation at 80 °C. Pretreatment of mice with McLTP1 (8 mg/kg, i.p. and p.o.) also decreased neurogenic and inflammatory phases of nociception in the formalin test. Naloxone (2 mg/kg, i.p.) antagonised the antinociceptive effect of McLTP1 suggesting that the opioid mechanisms mediate the analgesic properties of this protein.

Crystal structure of an antifungal osmotin-like protein from Calotropis procera and its effects on Fusarium solani spores, as revealed by atomic force microscopy: Insights into the mechanism of action.

CpOsm is an antifungal osmotin/thaumatin-like protein purified from the latex of Calotropis procera. The protein is relatively thermostable and retains its antifungal activity over a wide pH range; therefore, it may be useful in the development of new antifungal drugs or transgenic crops with enhanced resistance to phytopathogenic fungi. To gain further insight into the mechanism of action of CpOsm, its three-dimensional structure was determined, and the effects of the protein on Fusarium solani spores were investigated by atomic force microscopy (AFM). The atomic structure of CpOsm was solved at a resolution of 1.61Å, and it contained 205 amino acid residues and 192 water molecules, with a final R-factor of 18.12% and an Rfree of 21.59%. The CpOsm structure belongs to the thaumatin superfamily fold and is characterized by three domains stabilized by eight disulfide bonds and a prominent charged cleft, which runs the length of the front side of the molecule. Similarly to other antifungal thaumatin-like proteins, the cleft of CpOsm is predominantly acidic. AFM images of F. solani spores treated with CpOsm resulted in striking morphological changes being induced by the protein. Spores treated with CpOsm were wrinkled, and the volume of these cells was reduced by approximately 80%. Treated cells were covered by a shell of CpOsm molecules, and the leakage of cytoplasmic content from these cells was also observed. Based on the structural features of CpOsm and the effects that the protein produces on F. solani spores, a possible mechanism of action is suggested and discussed.

Mo-CBP3, an antifungal chitin-binding protein from Moringa oleifera seeds, is a member of the 2S albumin family.

Mo-CBP3 is a chitin-binding protein from M. oleifera seeds that inhibits the germination and mycelial growth of phytopathogenic fungi. This protein is highly thermostable and resistant to pH changes, and therefore may be useful in the development of new antifungal drugs. However, the relationship of MoCBP3 with the known families of carbohydrate-binding domains has not been established. In the present study, full-length cDNAs encoding 4 isoforms of Mo-CBP3 (Mo-CBP3-1, Mo-CBP3-2, Mo-CBP3-3 and Mo-CBP3-4) were cloned from developing seeds. The polypeptides encoded by the Mo-CBP3 cDNAs were predicted to contain 160 (Mo-CBP3-3) and 163 amino acid residues (Mo-CBP3-1, Mo-CBP3-2 and Mo-CBP3-4) with a signal peptide of 20-residues at the N-terminal region. A comparative analysis of the deduced amino acid sequences revealed that Mo-CBP3 is a typical member of the 2S albumin family, as shown by the presence of an eight-cysteine motif, which is a characteristic feature of the prolamin superfamily. Furthermore, mass spectrometry analysis demonstrated that Mo-CBP3 is a mixture of isoforms that correspond to different mRNA products. The identification of Mo-CBP3 as a genuine member of the 2S albumin family reinforces the hypothesis that these seed storage proteins are involved in plant defense. Moreover, the chitin-binding ability of Mo-CBP3 reveals a novel functionality for a typical 2S albumin.

Antifungal activity of naphthoquinoidal compounds in vitro against fluconazole-resistant strains of different Candida species: a special emphasis on mechanisms of action on Candida tropicalis.

In recent decades, the incidence of candidemia in tertiary hospitals worldwide has substantially increased. These infections are a major cause of morbidity and mortality; in addition, they prolong hospital stays and raise the costs associated with treatment. Studies have reported a significant increase in infections by non-albicans Candida species, especially C. tropicalis. The number of antifungal drugs on the market is small in comparison to the number of antibacterial agents available. The limited number of treatment options, coupled with the increasing frequency of cross-resistance, makes it necessary to develop new therapeutic strategies. The objective of this study was to evaluate and compare the antifungal activities of three semisynthetic naphthofuranquinone molecules against fluconazole-resistant Candida spp. strains. These results allowed to us to evaluate the antifungal effects of three naphthofuranquinones on fluconazole-resistant C. tropicalis. The toxicity of these compounds was manifested as increased intracellular ROS, which resulted in membrane damage and changes in cell size/granularity, mitochondrial membrane depolarization, and DNA damage (including oxidation and strand breakage). In conclusion, the tested naphthofuranquinones (compounds 1-3) exhibited in vitro cytotoxicity against fluconazole-resistant Candida spp. strains.

Proteomics changes during the incompatible interaction between cowpea and Colletotrichum gloeosporioides (Penz.) Penz and Sacc.

Anthracnose represents an important disease of cowpea [Vigna unguiculata L. (Walp.)] caused by the hemibiothrophic fungus Colletotrichum gloeosporioides that drastically reduces cowpea field production. In this study we investigated some biochemical aspects underlying the incompatible interaction between a resistant cowpea genotype and C. gloeosporioides using a proteomic approach. Analyses of two-dimensional gel electrophoresis patterns and protein identification indicate C. gloeosporioides infection-dependent cowpea leaf proteome changes associated with metabolism, photosynthesis, response to stress, oxidative burst and scavenging, defense signaling, and pathogenesis-related proteins. Moreover the C. gloeosporioides responsive proteins interaction network in cowpea revealed the interconnected modulation of key cellular processes involving particularly antioxidants proteins, photosynthetic apparatus forming proteins and proteins of the energetic metabolism that interact with each other suggesting that their expression changes are also important for resistance of cowpea to C. gloeosporioides.

Enhanced Synthesis of Antioxidant Enzymes, Defense Proteins and Leghemoglobin in Rhizobium-Free Cowpea Roots after Challenging with Meloydogine incognita.

The root knot nematodes (RKN), Meloydogine spp., particularly Meloidogyne incognita and Meloidogyne javanica species, parasitize several plant species and are responsible for large annual yield losses all over the world. Only a few available chemical nematicides are still authorized for RKN control owing to environmental and health reasons. Thus, plant resistance is currently considered the method of choice for controlling RKN, and research performed on the molecular interactions between plants and nematodes to identify genes of interest is of paramount importance. The present work aimed to identify the differential accumulation of root proteins of a resistant cowpea genotype (CE-31) inoculated with M. incognita (Race 3) in comparison with mock-inoculated control, using 2D electrophoresis assay, mass spectrometry identification and gene expression analyses by RT-PCR. The results showed that at least 22 proteins were differentially represented in response to RKN challenge of cowpea roots mainly within 4-6 days after inoculation. Amongst the up-represented proteins were SOD, APX, PR-1, ß-1,3-glucanase, chitinases, cysteine protease, secondary metabolism enzymes, key enzymes involved in ethylene biosynthesis, proteins involved in MAPK pathway signaling and, surprisingly, leghemoglobin in non-rhizobium-bacterized cowpea. These findings show that an important rearrangement in the resistant cowpea root proteome occurred following challenge with M. incognita.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves.

BACKGROUND: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). METHODS: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. RESULTS: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56­4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% ß-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx. GENERAL SIGNIFICANCE: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.