Obtention of bioactive peptives (cryprides) by the action of trypsin and serine proteases from the venom of Bothrops jararaca on endogenous substrates

Abstract

 Serineproteases and metalloproteases are the main Bothrops jararaca venom enzymes acting   on the victim’s tissues and proteins. As a result of their direct actions on tissue proteins, these   proteases could generate peptides with specific actions in cells or other mechanisms. The   most common sources for bioactive peptides are natural precursor proteins. Recent studies   have shown that a new class of proteins not named as precursors, the crypteins, may, in some   conditions, originate bioactive peptides, or cryptides. New cryptides generated by the action of   the venom serinoproteases and by commercial trypsin on endogenous substrates, were isolated,   then biochemically and biologically characterized. Serineproteases from B. jararaca venom   were separated from the whole venom using an HPLC molecular exclusion column, verifying   the activity of the fractions on the chosen substrates (myoglobin, hemoglobin, immunoglobin   G and collagen). These substrates were incubated with the venom serineproteases as well   as with trypsin. The resulting peptides were separated by fractionation by HPLC and the   fractions were tested on cell cultures for proliferative or cytotoxic effects. Active fractions   were rechromatographed in order to obtain the pure bioactive peptides. After the activity   was confirmed, the peptides were sequenced and synthesized. Trypsin activity on myoglobin   generated peptides (ALELFR, TGHPETLEK, GLSDGEWQQVLNVWGK) presenting   proliferative activity on fibroblasts and endothelial cells. 3D modeling of myoglobin, using   Cn3D software, showed that the three peptides are located on the surface of the protein.   Bioactive fractions were also found after digestion of the other substrates mentioned above   with trypsin, but they were not yet isolated and sequenced. Digestion of myoglobin with the   venom serineproteases generated an HPLC profile similar to the one obtained with trypsin.   This suggests that the cryptides here described may indeed be generated at the the snake bite   site, causing secondary effects, not neutralized by serumtherapy. This study suggests that the   venom serineproteases may generate cryptides with relevant effects through their actions on   highly available protein substrates at the bite site.