Solange M.T. Serrano

Insights into and speculations about snake venom metalloproteinase (SVMP) synthesis, folding and disulfide bond formation and their contribution to venom complexity

As more data are generated from proteome and transcriptome analyses of snake venoms, we are gaining an appreciation of the complexity of the venoms and, to some degree, the various sources of such complexity. However, our knowledge is still far from complete. The translation of genetic information from the snake genome to the transcriptome and ultimately the proteome is only beginning to be appreciated, and will require significantly more investigation of the snake venom genomic structure prior to a complete understanding of the genesis of venom composition. Venom complexity, however, is derived not only from the venom genomic structure but also from transcriptome generation and translation and, perhaps most importantly, post‐translation modification of the nascent venom proteome. In this review, we examine the snake venom metalloproteinases, some of the predominant components in viperid venoms, with regard to possible synthesis and post‐translational mechanisms that contribute to venom complexity. The aim of this review is to highlight the state of our knowledge on snake venom metalloproteinase post‐translational processing and to suggest testable hypotheses regarding the cellular mechanisms associated with snake venom metalloproteinase complexity in venoms.

Exploring snake venom proteomes: multifaceted analyses for complex toxin mixtures

Snake venom proteomes are complex mixtures of a large number of distinct proteins. In a sense, the field of snake venom proteomics has been under investigation since the very earliest biochemical studies on venoms where peptides and proteins were isolated and structurally and biologically characterized. With the recent developments in mass spectrometry for the identification of proteins, coupled with venom gland transcriptomes, has the field of snake venom proteomics began to flourish. These developments have led to exciting insights into the protein composition of venoms and subsequently their pathological activities. In this review, we will discuss the state of art of snake venom proteomics. Although we have not reached the ultimate goal of characterizing and quantifying all unique proteins in a venom proteome, current technologies have opened many opportunities for high‐throughput proteomic studies that have gone beyond simple protein identification to analyzing various functional aspects, such as post‐translational modifications, proteolytic processing and toxin‐target interactions. In this review, we will discuss the technological approaches used in the study of venom proteomics highlighting the advances made and future directions.

Approaching the Golden Age of Natural Product Pharmaceuticals from Venom Libraries: An Overview of Toxins and Toxin-Derivatives Currently Involved in Therapeutic or Diagnostic Applications

Poisons and the toxins found in venomous and poisonous organisms have been the focus of much research over the past 70 years, most of which has been directed at understanding the biochemical and physiological mechanisms by which they elicit their dramatic pathological consequences. Much knowledge has been gained in terms of how poisons and venoms and their composite toxins give rise to the syndromes associated with envenoming and poisoning and in some isolated cases there have been a few such agents promoted for therapeutic use. However, it has only been in the past decade that an explosion of interest has occurred in mining these natural, highly evolved libraries of bioactive toxins and poisons for use in pharmacotherapeutics as drugs or drug leads as well as in diagnostic applications. We ascribe this recent phenomenon to advances in toxinology which have provided investigators with a relatively thorough understanding of the nature of venoms and their biologically active toxins: particularly with regard to the peptidomes and proteomes of venoms. This is in conjunction with our greatly improved understanding of the etiology of many human diseases and the identification of sites of potential therapeutic intervention. In this review we provide an overview of some of the toxins, toxin derivatives or poisons from animal venoms and secretions which are in various stages of development for use as pharmaceuticals or diagnostics in human diseases. As one will recognize, developments in this field suggest that toxinology is now entering a golden age in terms of the identification and use of toxins as potent novel pharmaceuticals.

Timeline of key events in snake venom metalloproteinase research.

It is reasonable to state that snake venom toxinology has been actively pursued for at least the past 400 to 500 years. Early on it was appreciated that the venoms of the Viperidae produced profound local effects, notably hemorrhage. For the past 100 years, with the advent of modern chemistry and biochemistry significant progress has been gained regarding the function, structure and role of the snake venom metalloproteinases (SVMPs) in viperid venom pathogenesis. In this review we provide a concise, chronological presentation of the key significant studies that have led to our current understanding of these intriguing toxins.

Some aspects of the venom proteome of the Colubridae snake Philodryas olfersii revealed from a Duvernoy’s (venom) gland transcriptome

We investigated the putative toxins of Philodryas olfersii (Colubridae), a representative of a family of snakes neglected in venom studies despite their growing medical importance. Transcriptomic data of the venom gland complemented by proteomic analysis of the gland secretion revealed the presence of major toxin classes from the Viperidae family, including serine proteases, metalloproteases, C‐type lectins, Crisps, and a C‐type natriuretic peptide (CNP). Interestingly, the phylogenetic analysis of the CNP precursor showed it as a linker between two related precursors found in Viperidae and Elapidae snakes. We suggest that these precursors constitute a monophyletic group derived from the vertebrate CNPs.

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.

Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.

Bradykinin-potentiating peptides: Beyond captopril

The identification of novel endogenous and exogenous molecules acting in the complex mechanism of regulating the vascular tonus has always been of great interest. The discovery of bradykinin (1949) and the bradykinin-potentiating peptides (1965) had a pivotal influence in the field, respectively, in understanding cardiovascular pathophysiology and in the development of captopril, the first active-site directed inhibitor of angiotensin-converting enzyme, and used worldwide to treat human hypertension. Both discoveries originated from studies of envenoming by the snake Bothrops jararaca. The aim of the present article is to reveal that the snake proline-rich oligopeptides, known as bradykinin-potentiating peptides, are still a source of surprising scientific discoveries, some of them useful not only to reveal potential new targets but also to introduce prospective lead molecules for drug development. In particular, we emphasize argininosuccinate synthetase as a new functional target for one of bradykinin-potentiating peptides found in B. jararaca, Bj-BPP-10c. This decapeptide leads to argininosuccinate synthetase activation, consequently sustaining increased nitric oxide production, a critical endogenous molecule to reduce the arterial blood pressure.

The Cysteine-rich Domain of Snake Venom Metalloproteinases Is a Ligand for von Willebrand Factor A Domains ROLE IN SUBSTRATE TARGETING*

Snake venom metalloproteinases (SVMPs) are members of the Reprolysin family of metalloproteinases to which the ADAM (a disintegrin and metalloproteinase) proteins also belong. The disintegrin-like/cysteine-rich domains of the ADAMs have been implicated in their function. In the case of the SVMPs, we hypothesized that these domains could function to target the metalloproteinases to key extracellular matrix proteins or cell surface proteins. Initially we detected interaction of collagen XIV, a fibril-associated collagen with interrupted triple helices containing von Willebrand factor A (VWA) domains, with the PIII SVMP catrocollastatin. Next we investigated whether other VWA domain-containing matrix proteins could support the binding of PIII SVMPs. Using surface plasmon resonance, the PIII SVMP jararhagin and a recombinant cysteine-rich domain from a PIII SVMP were demonstrated to bind to collagen XIV, collagen XII, and matrilins 1, 3, and 4. Jararhagin was shown to cleave these proteins predominantly at sites localized at or near the VWA domains suggesting that it is the VWA domains to which the PIII SVMPs are binding via their cysteine-rich domain. In light of the fact that these extracellular matrix proteins function to stabilize matrix, targeting the SVMPs to these proteins followed by their specific cleavage could promote the destabilization of extracellular matrix and cell-matrix interactions and in the case of capillaries could contribute to their disruption and hemorrhage. Although there is only limited structural homology shared by the cysteine-rich domains of the PIII SVMPs and the ADAMs our results suggest an analogous function for the cysteine-rich domains in certain members of the expanded ADAM family of proteins to target them to VWA domain-containing proteins.

The long road of research on snake venom serine proteinases

It has long been recognized that snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, blood pressure and platelet aggregation. Therefore, SVSPs have been used as refined tools to study molecular mechanisms involved in the activation of key factors that control hemostasis and as therapeutic agents in various thrombotic and hemostatic conditions. The aim of this review is to highlight the state of our knowledge on the advances made in SVSP research since the 18th century. It includes the personal accounts of some distinguished scientists that addressed specific problems and contributed to advance the field.