Thirty new molecules are discovered in snake venom

Researchers at the Butantan Institute map a set of venom peptides from three species of genus Bothrops, including the jararaca, and find molecules with potential pharmacological activity.

By Karina Toledo

Agência FAPESP – Thirty new molecules – some with potential pharmacological activity – have been discovered at the Butantan Institute during a study that mapped a set of peptides found in the venom of three species of snakes of the Bothrops genus, including the jararaca.

 

“The objective of the work was to describe the complexity of the peptidome, or set of peptides, found in the venom of Bothrops jararaca, Bothrops cotiara (BC) and Bothrops fonsecai (BF),” explained Solange Maria de Toledo Serrano, coordinator of the study.

 

The results of the study, considered to be the most in-depth study conducted on a peptidome, were published in an article in the November edition of Molecular & Cellular Proteomics.  

 

The scientists sequenced 44 peptides, 30 of which were previously unknown. Among the other molecules identified, at least four of those tested presented bradykinin potentiation activity and angiotensin-converting enzyme inhibition (ACE inhibition), which are processes involved in controlling arterial pressure.

 

The first bradykinin peptide potentiator isolated in jararaca venom, back in the 1960s, paved the way for an entire class of antihypertensive medicines, including captopril.

 

For the researchers at the Butantan Institute, who have studied proteolytic venom for some time, employing mass spectrometry and bioinformatics was important for mapping the enzymatic cleavage points on toxins, particularly those where metalloproteinases act when the venom proteins are broken down during processing for analysis.

 

The analysis was conducted at the Applied Toxinology Center (CAT), one of FAPESP’s Research, Innovation and Dissemination Centers (CEPIDs), during the post-doctoral studies of Alexandre Keiji Tashima, who is currently a professor at the Department of Exact and Earth Sciences at Universidade Federal de São Paulo (Unifesp), Diadema campus.

 

To identify the peptides found in the samples of venom from the three Bothrops species, the first step was to separate the peptides from the proteins (which are larger molecules), explained Tashima.

 

“We separated the proteic fraction from the peptidic fraction, which together correspond to the majority of the substances found in the secretion, through a process called solid-phase extraction,” he commented.

  

Afterward, the peptidic fraction was analyzed using a mass spectrometer, a device that measures the mass to charge ratio of ionized molecules, to obtain information on the intact molecules and their fragments.

 

“The major difficulty in the case of the venom peptidome is the lack of a database that allows automated identification of amino acids. In the majority of cases, sequencing must be performed manually,” explained Tashima.

 

According to Serrano, this is the reason that knowledge about venom proteomes advances much quicker than knowledge about peptidomes. The researcher’s group has already investigated a set of proteins produced by these three species in previous studies.

 

“Serpent venoms are rich sources of biologically active peptides; nevertheless, because of the small number of sequences deposited in databases, advances in the discovery of new molecules have occurred slowly. But such advances are even more critical for rare species, such as B. cotiara and B. fonsecai, both of which are considered to be at risk of extinction and for which there are few published articles in the literature,” commented Serrano.

 

Unexpected results

 

After sequencing the polypeptide chains, the researchers were surprised to find that the peptidomes of the fresh venom samples were much less complex than those of lyophilized venom. 

 

“When venom is subjected to laboratory conditions, the proteolytic enzymes naturally found in the secretion operate and begin to degrade proteins, creating more peptides,” explained Tashima.

 

The scientists compared three types of samples: fresh venom collected in the presence of proteolytic enzymes, diluted lyophilized venom in solution with proteolytic enzymes and lyophilized venom diluted in acidic solution. The last presented the greatest number of protein fragments or, rather, underwent the most degradation.

 

“We did not expect to observe such strong degradation of the proteins. Now, we will need to study the impact of this, for example, on the production of antivenin, which is normally produced using lyophilized venom,” affirms Tashima.

 

Snakes of the Bothrops genus are responsible for approximately 90% of the snake-related accidents that occur in Brazil, explained Tashima. The majority of cases involve the jararaca, which is found throughout the country. In contrast, B. cotiara is found only in araucaria forests and B. fonsecai in the Atlantic Rainforest.

 

For Hugo Aguirre Armelin, coordinator of CAT-CEPID, the study reveals the advantages of a proteomic approach for studying venom. “FAPESP’s funding [for this initiative] is ending this year but has created a laboratory equipped with a state of the art spectrometer that will allow us to conduct detailed analyses of complex structures, such as serpent venom. Furthermore, the funding allowed us to train qualified researchers,” he said.

 

The article “Peptidomics of Three Bothrops Snake Venoms: Insights into the Molecular Diversification of Proteomes and Peptidomes” (doi: 10.1074/mcp.M112.019331) can be read at www.mcponline.org/content/11/11/1245.abstract.