A novel protein structure with kallikrein-like activity found in Thalassophryne nattereri fish venom

¹Magalhães G S, ¹Lopes-Ferreira  M, ¹Junqueira de Azevedo I M J, ²Spencer P J, ^3,4Valente R H, ⁵Juliano L, ⁴Fox, J W,  ¹Ho P L, ¹Moura-da-Silva A M.

1 Instituto Butantan-SP; 2 IPEN-SP; 3 FIOCRUZ-RJ; 4 University of Virginia-USA, 5 UNIFESP-SP

Thalassophrynenattereri fish envenoming is an important medical problem in North and Northeast of Brazil, causing in human victims intense pain and edema followed by necrosis. To elucidate the composition of the venom and establish structure-function relationships of the toxins, a representative cDNA library was obtained from T. nattereri venom gland. The clones obtained were sequenced and searched for homology in the DDBJ/GENBANK. Sequences from 51.6% of the clones were identified and related to cell activities or other functions and the remaining 48.4% were unknown. Those sequences were then translated and grouped into families. The major family, comprising 18% clones was named natterin. Additionally, T. nattereri venom major components were characterized by protein chemistry. The venom was submitted to FPLC/HPLC and the principal effects of the venom (nociception and edema) were mostly located in one peak (MS3). Previous experiments using whole venom showed that edema and nociception were related with tissue-kallikrein enzymes. Thus, MS3 fraction was tested for hydrolysis of human kininogen and kininogen-derived synthetic peptides, showing the release of kallidin (Lys-bradykinin). It was also observed that hydrolysis was inhibited by metal chelating agents but not by serino-, aspartyl- or cysteino-proteinase inhibitors. To elucidate the sequence of the protein(s) responsible for those effects, this fraction was submitted to N-terminal sequencing by Edman degradation and internal peptides by MS/MS spectrometry. Those sequences revealed to belong to the natterin family in the cDNA library, confirming a correlation between venom protein content and abundance in cDNA library. We could divide this family into 4 natterins, which cDNAs were completely sequenced and included internal and N-terminal peptides found in MS3 fraction. Natterin 4 was found due its homology (32%) with the others. Interestingly, despite the biological activity, natterin sequences did not present significant homology with tissue-kallikreins or any protein registered in databanks. Together with the lack of inhibition by conventional serino-proteinase inhibitors, this data suggest that the natterin family may represent a new structure of protein with kallikrein activity. The expression of natterin cDNAs may elucidate the requirements for the activity of this new category of enzyme.

Financial support: FAPESP

Correspondence to: anamoura@butantan.gov.br