Unveiling the peptidome diversity of Lachesismuta snake venom: Discovery of novel fragments of metalloproteinase, l-amino acid oxidase, and bradykinin potentiating peptides.

Snake venoms are known to be major sources of peptides with different pharmacological properties. In this study, we comprehensively explored the venom peptidomes of three specimens of Lachesismuta, the largest venomous snake in South America, using mass spectrometry techniques. The analysis revealed 19 main chromatographic peaks common to all specimens. A total of 151 peptides were identified, including 69 from a metalloproteinase, 58 from the BPP-CNP precursor, and 24 from a l-amino acid oxidase. To our knowledge, 126 of these peptides were reported for the first time in this work, including a new SVMP-derived peptide fragment, Lm-10a. Our findings highlight the dynamic nature of toxin maturation in snake venoms, driven by proteolytic processing, post-translational modifications, and cryptide formation.

An interdisciplinary therapy for lifestyle change is effective in improving psychological and inflammatory parameters in women with grade I obesity.

Obesity and depression, disorders associated with inflammation, have high incidences in women. Understanding the derangements present in the initial phase of obesity may point to factors that could help avoiding disease aggravation. The present study aimed at investigating the effects of a 6-months interdisciplinary therapy for weight loss in women with grade I obesity. Before and after the therapy, 37 middle-aged women donated blood and responded to questionnaires for depression and anxiety symptoms. Inflammatory parameters were evaluated in serum and a preliminary screening of the plasma proteome was performed. The therapy decreased anthropometric, psychological scores, and serum levels of inflammatory parameters. Depression and anxiety scores correlated positively with some inflammatory parameters. The proteomic analysis showed changes in proteins related to cholesterol metabolism and inflammatory response. Interdisciplinary therapy improves anthropometric and inflammatory statuses and ameliorating psychological symptoms. The decrease of MCP-1 levels after interdisciplinary therapy has not been reported so far, at the best of our knowledge. The present demonstration of positive associations of inflammatory markers and psychological scores indicate that these mediators may be useful to monitor psychological status in obesity. The present proteome data, although preliminary, pointed to plasma alterations indicative of improvement of inflammation after interdisciplinary therapy.

Multiomics Profiling of Toxins in the Venom of the Amazonian Spider Acanthoscurria juruenicola.

Acanthoscurria juruenicola is an Amazonian spider described for the first time almost a century ago. However, little is known about their venom composition. Here, we present a multiomics characterization of A. juruenicola venom by a combination of transcriptomics, proteomics, and peptidomics approaches. Transcriptomics of female venom glands resulted in 93,979 unique assembled mRNA transcript encoding proteins. A total of 92 proteins were identified in the venom by mass spectrometry, including 14 mature cysteine-rich peptides (CRPs). Quantitative analysis showed that CRPs, cysteine-rich secretory proteins, metalloproteases, carbonic anhydrases, and hyaluronidase comprise >90% of the venom proteome. Relative quantification of venom toxins was performed by DIA and DDA, revealing converging profiles of female and male specimens by both methods. Biochemical assays confirmed the presence of active hyaluronidases, phospholipases, and proteases in the venom. Moreover, the venom promoted in vivo paralytic activities in crickets, consistent with the high concentration of CRPs. Overall, we report a comprehensive analysis of the arsenal of toxins of A. juruenicola and highlight their potential biotechnological and pharmacological applications. Mass spectrometry data were deposited to the ProteomeXchange Consortium via the PRIDE repository with the dataset identifier PXD013149 and via the MassIVE repository with the dataset identifier MSV000087777.

Iron-deficient diet induces distinct protein profile related to energy metabolism in the striatum and hippocampus of adult rats.

Iron deficiency is a public health problem that affects all age groups. Its main consequence is anemia, but it can also affect cognitive functions. Although the negative effects of iron deficiency on cognitive function have been extensively described, the underlying mechanism has not been fully investigated. Thus, to gain an unbiased insight into the effects of iron deficiency (ID) on discrete brain regions, we performed a proteomic analysis of the striatum and hippocampus of adult rats subjected to an iron restricted (IR) diets for 30 days. We found that an IR diet caused major alterations in proteins related to glycolysis and lipid catabolism in the striatum. In the hippocampus, a larger portion of proteins related to oxidative phosphorylation and neurodegenerative diseases were altered. These alterations in the striatum and hippocampus occurred without a reduction in local iron levels, although there was a drastic reduction in liver iron and ferritin. Moreover, the IR group showed higher fasting glycaemia than the control group. These results suggest that brain iron content is preserved during acute iron deficiency, but the alterations of other systemic metabolites such as glucose may trigger distinct metabolic adaptations in each brain region. Abnormal energy metabolism precedes and persists in many neurological disorders. Thus, altered energy metabolism can be one of the mechanisms by which iron deficiency affects cognitive functions.

High-fat but not normal-fat intake of extra virgin olive oil modulates the liver proteome of mice.

PURPOSE: The metabolic benefits of the Mediterranean diet have been largely attributed to its olive oil content. Whether the ingested fat amount is relevant to these effects is not clear. We thus compared the effects of high-fat and normal-fat intake of extra-virgin olive oil (EVOO) on the liver proteome. METHODS: Three groups of mice were fed for 12 weeks with either normal-fat diets containing either soybean oil (control, C) or EVOO (NO) or a high-fat EVOO diet (HO). Body weight and food intake were measured weekly and serum parameters were analyzed. The liver was processed for data-independent acquisition mass spectrometry-based proteomics. The differentially expressed proteins among the groups were submitted to pathway enrichment analysis. RESULTS: The consumption of HO diet reduced food intake and serum triglycerides, while it preserved body weight gain, adiposity, and glycemia. However, it increased serum cholesterol and liver mass. The proteomic analysis showed 98 altered proteins, which were allocated in 27 significantly enriched pathways. The pathway analysis suggested stimulation of mitochondrial and peroxissomal ß-oxidation, and inhibition of lipid synthesis and gluconeogenesis in the HO group. Although the NO group failed to show significant liver proteome alterations, it presented reduced body fat, body weight gain, and serum triglycerides and glucose levels. CONCLUSION: The data indicate that the intake of the HO diet induced hepatic adjustments, which were partially successful in counteracting the detrimental outcomes of a high-fat feeding. Contrastingly, the NO diet had beneficial effects which were not accompanied by significant modifications on hepatic proteome.

Deep Profiling of the Cleavage Specificity and Human Substrates of Snake Venom Metalloprotease HF3 by Proteomic Identification of Cleavage Site Specificity (PICS) Using Proteome Derived Peptide Libraries and Terminal Amine Isotopic Labeling of Substrates (TAILS) N-Terminomics.

Snakebite is a major medical concern in many parts of the world with metalloproteases playing important roles in the pathological effects of Viperidae venoms, including local tissue damage, hemorrhage, and coagulopathy. Hemorrhagic Factor 3 (HF3), a metalloprotease from Bothrops jararaca venom, induces local hemorrhage and targets extracellular matrix (ECM) components, including collagens and proteoglycans, and plasma proteins. However, the full substrate repertoire of this metalloprotease is unknown. We report positional proteomic studies identifying >2000 N-termini, including neo-N-termini of HF3 cleavage sites in mouse embryonic fibroblast secretome proteins. Terminal amine isotopic labeling of substrates (TAILS) analysis identified a preference for Leu at the P1' position among candidate HF3 substrates including proteins of the ECM and focal adhesions and the cysteine protease inhibitor cystatin-C. Interestingly, 190 unique peptides matched to annotated cleavage sites in the TopFIND N-termini database, suggesting that these cleavages occurred at a site prone to cleavage or might have been generated by other proteases activated upon incubation with HF3, including caspases-3 and -7, cathepsins D and E, granzyme B, and MMPs 2 and 9. Using Proteomic identification of cleavage site specificity (PICS), a tryptic library derived from THP-1 monocytic cells was used as HF3 substrates for identifying protease cleavage sites and sequence preferences in peptides. A total of 799 unique cleavage sites were detected and, in accordance with TAILS analysis using native secreted protein substrates of MEF cells, revealed a clear preference for Leu at P1'. Taken together, these results greatly expand the known substrate degradome of HF3 and reveal potential new targets, which may serve as a basis to better elucidate the complex pathophysiology of snake envenomation.

A proteomics-metabolomics approach indicates changes in hypothalamic glutamate-GABA metabolism of adult female rats submitted to intrauterine growth restriction.

PURPOSE: Intrauterine growth restriction (IUGR) has been shown to induce the programming of metabolic disturbances and obesity, associated with hypothalamic derangements. The present study aimed at investigating the effects of IUGR on the protein and metabolite profiles of the hypothalamus of adult female rats. METHODS: Wistar rats were mated and either had ad libitum access to food (control group) or received only 50% of the control intake (restricted group) during the whole pregnancy. Both groups ate ad libitum throughout lactation. At 4 months of age, the control and restricted female offspring was euthanized for blood and tissues collection. The hypothalami were processed for data independent acquisition mass spectrometry-based proteomics or targeted mass spectrometry-based metabolomics. RESULTS: The adult females submitted to IUGR showed increased glycemia and body adiposity, with normal body weight and food intake. IUGR modulated significantly 28 hypothalamic proteins and 7 hypothalamic metabolites. The effects of IUGR on hypothalamic proteins and metabolites included downregulation of glutamine synthetase, glutamate decarboxylase, glutamate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate, and up-regulation of NADH dehydrogenase and phosphoenolpyruvate. Integrated pathway analysis indicated that IUGR affected GABAergic synapse, glutamate metabolism, and TCA cycle, highly interconnected pathways whose derangement has potentially multiple consequences. CONCLUSION: The present findings suggested that the effects of IUGR on GABA/glutamate-glutamine cycle may be involved in the programming of obesity and hyperglycemia in female rats.

Identification and characterization of RSIY-11, a novel seminal peptide derived from semenogelin-1, which acts as a neutral endopeptidase inhibitor modulating sperm motility.

PURPOSE: Based on prior reports demonstrating that neutral endopeptidase (NEP) inhibitors increase sperm motility, the goal of our studies was to identify endogenous seminal peptides that inhibit NEP and investigate their potential effect on sperm motility. METHODS: Peptidomic analysis was performed on human seminal fluid, identifying 22 novel peptides. One peptide, named RSIY-11, derived from semenogelin-1, was predicted through sequence analysis to be a substrate and/or potential inhibitor of NEP. Enzymatic analysis was conducted to determine the inhibitory constant (Ki) of RSIY-11 as an inhibitor of NEP. Total and progressive sperm motility was determined at baseline and 30 and 60 min following addition of RSIY-11 to seminal fluid in 59 patients undergoing an infertility workup at an urban medical center. Additionally, the effects of RSIY-11 on sperm motility were evaluated in 15 of the 59 patients that met criteria for asthenospermia. RESULTS: RSIY-11 was shown to act as a competitive inhibitor of NEP with a Ki of 18.4 ± 1.6 µM. Addition of RSIY-11 at concentrations of 0.75 µM, 7.5 µM, and 75 µM significantly increased sperm motility at all time points investigated, with increases of 6.1%, 6.9%, and 9.2% at 60 min, respectively. Additionally, within the subgroup of patients with asthenospermia, RSIY-11 at concentrations of 0.75 µM, 7.5 µM, and 75 µM significantly increased sperm motility at all time points investigated, with increases of 7.6%, 8.8%, and 10.6% at 60 min, respectively. CONCLUSIONS: RSIY-11 is a newly identified semenogelin-1-derived peptide present in seminal fluid. RSIY-11 acts as a potent competitive inhibitor of NEP, which when added to seminal fluid significantly increases sperm motility. RSIY-11 could play a potential role in the treatment for male factor infertility related to asthenospermia and improve intrauterine insemination outcomes.

Intrauterine Growth Restriction Programs the Hypothalamus of Adult Male Rats: Integrated Analysis of Proteomic and Metabolomic Data.

Programming of hypothalamic functions regulating energy homeostasis may play a role in intrauterine growth restriction (IUGR)-induced adulthood obesity. The present study investigated the effects of IUGR on the hypothalamus proteome and metabolome of adult rats submitted to 50% protein-energy restriction throughout pregnancy. Proteomic and metabolomic analyzes were performed by data independent acquisition mass spectrometry and multiple reaction monitoring, respectively. At age 4 months, the restricted rats showed elevated adiposity, increased leptin and signs of insulin resistance. 1356 proteins were identified and 348 quantified while 127 metabolites were quantified. The restricted hypothalamus showed down-regulation of 36 proteins and 5 metabolites and up-regulation of 21 proteins and 9 metabolites. Integrated pathway analysis of the proteomics and metabolomics data indicated impairment of hypothalamic glucose metabolism, increased flux through the hexosamine pathway, deregulation of TCA cycle and the respiratory chain, and alterations in glutathione metabolism. The data suggest IUGR modulation of energy metabolism and redox homeostasis in the hypothalamus of male adult rats. The present results indicated deleterious consequences of IUGR on hypothalamic pathways involved in pivotal physiological functions. These results provide guidance for future mechanistic studies assessing the role of intrauterine malnutrition in the development of metabolic diseases later in life.

New proline-rich oligopeptides from the venom of African adders: Insights into the hypotensive effect of the venoms.

BACKGROUND: The snakes from the Bitis genus are some of the most medically important venomous snakes in sub Saharan Africa, however little is known about the composition and effects of these snake venom peptides. Considering that the victims with Bitis genus snakes have exacerbate hypotension and cardiovascular disorders, we investigated here the presence of angiotensin-converting enzyme modulators on four different species of venoms. METHODS: The peptide fractions from Bitis gabonica gabonica, Bitis nasicornis, Bitis gabonica rhinoceros and Bitis arietans which showed inhibitory activity on angiotensin-converting enzyme were subjected to mass spectrometry analysis. Eight proline-rich peptides were synthetized and their potencies were evaluated in vitro and in vivo. RESULTS: The MS analysis resulted in over 150 sequences, out of which 32 are new proline-rich oligopeptides, and eight were selected for syntheses. For some peptides, inhibition assays showed inhibitory potentials of cleavage of angiotensin I ten times greater when compared to bradykinin. In vivo tests showed that all peptides decreased mean arterial pressure, followed by tachycardia in 6 out of 8 of the tests. CONCLUSION: We describe here some new and already known proline-rich peptides, also known as bradykinin-potentiating peptides. Four synthetic peptides indicated a preferential inhibition of angiotensin-converting enzyme C-domain. In vivo studies show that the proline-rich oligopeptides are hypotensive molecules. GENERAL SIGNIFICANCE: Although proline-rich oligopeptides are known molecules, we present here 32 new sequences that are inhibitors of the angiotensin-converting enzyme and consistent with the symptoms of the victims of Bitis spp, who display severe hypotension.

Exploring potential virulence regulators in Paracoccidioides brasiliensis isolates of varying virulence through quantitative proteomics.

Few virulence factors have been identified for Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis. In this study, we quantitatively evaluated the protein composition of P. brasiliensis in the yeast phase using minimal and rich media to obtain a better understanding of its virulence and to gain new insights into pathogen adaptation strategies. This analysis was performed on two isolates of the Pb18 strain showing distinct infection profiles in B10.A mice. Using liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis, we identified and quantified 316 proteins in minimal medium, 29 of which were overexpressed in virulent Pb18. In rich medium, 29 out of 295 proteins were overexpressed in the virulent fungus. Three proteins were found to be up-regulated in both media, suggesting the potential roles of these proteins in virulence regulation in P. brasiliensis. Moreover, genes up-regulated in virulent Pb18 showed an increase in its expression after the recovery of virulence of attenuated Pb18. Proteins up-regulated in both isolates were grouped according to their functional categories. Virulent Pb18 undergoes metabolic reorganization and increased expression of proteins involved in fermentative respiration. This approach allowed us to identify potential virulence regulators and provided a foundation for achieving a molecular understanding of how Paracoccidioides modulates the host-pathogen interaction to its advantage.

Peptidomics of three Bothrops snake venoms: insights into the molecular diversification of proteomes and peptidomes.

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from l-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures.

Quantitative Peptidomics Using Reductive Methylation of Amines.

A number of different approaches have been used for quantitative peptidomics. In this protocol, we describe the method in which peptides are reacted with formaldehyde and sodium cyanoborohydride, which converts primary and secondary amines into tertiary amines. By using different combinations of regular reagents, deuterated reagents (2H), and reagents containing deuterium and 13C, it is possible to produce five isotopically distinct forms of the methylated peptides, which can be quantified by mass spectrometry. Peptides with free N-termini that are primary amines incorporate two methyl groups using this procedure, which differ by 2 Da for each of the five isotopic combinations. Peptides that contain unmodified lysine residues incorporate additional pairs of methyl groups, leading to larger mass differences between isotopic forms. The reagents are commercially available, relatively inexpensive, and chemically stable.

Identification of Peptides in Spider Venom Using Mass Spectrometry.

Spider venoms are composed of hundreds of proteins and peptides. Several of these venom toxins are cysteine-rich peptides in the mass range of 3-9 kDa. Small peptides (<3 kDa) can be fully characterized by mass spectrometry analysis, while proteins are generally identified by the bottom-up approach in which proteins are first digested with trypsin to generate shorter peptides for MS/MS characterization. In general, it is sufficient for protein identification to sequence two or more peptides, but for venom peptidomics it is desirable to completely elucidate peptide sequences and the number of disulfide bonds in the molecules. In this chapter, we describe a methodology to completely sequence and determine the number of disulfide bonds of spider venom peptides in the mass range of 3-9 kDa by multiple enzyme digestion, mass spectrometry of native and digested peptides, de novo analysis, and sequence overlap alignment.

Analysis of the Snake Venom Peptidome.

Snake venom peptidomes are known to be a large source of molecules with different pharmacological properties. The complexity and variability of snake venoms, the presence of proteinases, and the lack of complete species-specific genome sequences make snake venom peptidome profiling a challenging task that requires especial technical strategies for sample processing and mass spectrometric analysis. Here, we describe a method for assessing the content of snake venom peptides and highlight the importance of sampling procedures, as they substantially influence the peptidomic complexity of snake venoms.

Mice heterozygous for a null mutation of CPE show reduced expression of carboxypeptidase e mRNA and enzyme activity but normal physiology, behavior, and levels of neuropeptides.

Carboxypeptidase E (CPE) is an essential enzyme that contributes to the biosynthesis of the vast majority of neuropeptides and peptide hormones. There are several reports claiming that small decreases in CPE activity cause physiological changes in animals and/or cultured cells, but these studies did not provide evidence that neuropeptide levels were affected by decreased CPE activity. In the present study, we tested if CPE is a rate-limiting enzyme in neuropeptide production using CpeNeo mice, which contain a neomycin cassette within the Cpe gene that eliminates enzyme expression. Homozygous CpeNeo/Neo mice show defects found in Cpefat/fat and/or Cpe global knockout (KO) mice, including greatly decreased levels of most neuropeptides, severely impaired fertility, depressive-like behavior, adult-onset obesity, and anxiety-like behavior. Removal of the neomycin cassette with Flp recombinase under a germline promoter restored expression of CPE activity and resulted in normal behavioral and physiological properties, including levels of neuropeptides. Mice heterozygous for the CpeNeo allele have greatly reduced levels of Cpe mRNA and CPE-like enzymatic activity. Despite the decreased levels of Cpe expression, heterozygous CpeNeo mice are behaviorally and physiologically identical to wild-type mice, with normal levels of most neuropeptides. These results indicate that CPE is not a rate-limiting enzyme in the production of most neuropeptides, casting doubt upon studies claiming small decreases in CPE activity contribute to obesity or other physiological effects.

Bothrops jararaca venom proteome rearrangement upon neonate to adult transition.

The pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Similarly, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. In this study we used large and representative newborn and adult venom samples consisting of pools from 694 and 110 specimens, respectively, and demonstrate a significant ontogenetic shift in the venom proteome complexity of B. jararaca. 2-DE coupled to MS protein identification showed a clear rearrangement of the toxin arsenal both in terms of the total proteome, as of the glycoproteome. N-glycosylation seems to play a key role in venom protein variability between newborn and adult specimens. Upon the snake development, the subproteome of metalloproteinases undergoes a shift from a P-III-rich to a P-I-rich profile while the serine proteinase profile does not vary significantly. We also used isobaric tag labeling (iTRAQ) of venom tryptic peptides for the first time to examine the quantitative changes in the venom toxins of B. jararaca upon neonate to adult transition. The iTRAQ analysis showed changes in various toxin classes, especially the proteinases. Our study expands the in-depth understanding of venom complexity variation particularly with regard to toxin families that have been associated with envenomation pathogenesis.

Neuropeptidomic Analysis of a Genetically Defined Cell Type in Mouse Brain and Pituitary.

Neuropeptides and peptide hormones are important cell-cell signaling molecules that mediate many physiological processes. Unlike classic neurotransmitters, peptides undergo cell-type-specific post-translational modifications that affect their biological activity. To enable the identification of the peptide repertoire of a genetically defined cell type, we generated mice with a conditional disruption of the gene for carboxypeptidase E (Cpe), an essential neuropeptide-processing enzyme. The loss of Cpe leads to accumulation of neuropeptide precursors containing C-terminal basic residues, which serve as tags for affinity purification. The purified peptides are subsequently identified using quantitative peptidomics, thereby revealing the specific forms of neuropeptides in cells with the disrupted Cpe gene. To validate the method, we used mice expressing Cre recombinase under the proopiomelanocortin (Pomc) promoter and analyzed hypothalamic and pituitary extracts, detecting peptides derived from proopiomelanocortin (as expected) and also proSAAS in POMC neurons. This technique enables the analyses of specific forms of peptides in any Cre-expressing cell type.

Systemic Effects of Hemorrhagic Snake Venom Metalloproteinases: Untargeted Peptidomics to Explore the Pathodegradome of Plasma Proteins.

Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon that involves capillary disruption and blood extravasation. HF3 (hemorrhagic factor 3) is an extremely hemorrhagic SVMP of Bothrops jararaca venom. Studies using proteomic approaches revealed targets of HF3 among intracellular and extracellular proteins. However, the role of the cleavage of plasma proteins in the context of the hemorrhage remains not fully understood. The main goal of this study was to analyze the degradome of HF3 in human plasma. For this purpose, approaches for the depletion of the most abundant proteins, and for the enrichment of low abundant proteins of human plasma, were used to minimize the dynamic range of protein concentration, in order to assess the proteolytic activity of HF3 on a wide spectrum of proteins, and to detect the degradation products using mass spectrometry-based untargeted peptidomics. The results revealed the hydrolysis products generated by HF3 and allowed the identification of cleavage sites. A total of 61 plasma proteins were identified as cleaved by HF3. Some of these proteins corroborate previous studies, and others are new HF3 targets, including proteins of the coagulation cascade, of the complement system, proteins acting on the modulation of inflammation, and plasma proteinase inhibitors. Overall, the data indicate that HF3 escapes inhibition and sculpts the plasma proteome by degrading key proteins and generating peptides that may act synergistically in the hemorrhagic process.

Analysis of the ontogenetic variation in the venom proteome/peptidome of Bothrops jararaca reveals different strategies to deal with prey.

Previous studies have demonstrated that the pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Variation in the venom proteome is a well-documented phenomenon; however, variation in the venom peptidome is poorly understood. We report a comparative proteomic and peptidomic analysis of venoms from newborn and adult specimens of B. jararaca and correlate it with the evaluation of important venom features. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. The coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of SDS-PAGE, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and concanavalin A-binding proteins. Furthermore, we report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles and were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species.