Abstract
Mortality due to snakebite is a serious public health problem, and available therapeutics are known to induce debilitating side effects. Traditional medicine suggests that seeds of Mucuna pruriens can provide protection against the effects of snakebite. Our aim is to identify the protein(s) that may be important for snake venom neutralization and elucidate its mechanism of action. To this end, we have identified and purified a protein from M. pruriens, which we have named MP-4. The full-length polypeptide sequence of MP-4 was obtained through N-terminal sequencing of peptide fragments. Sequence analysis suggested that the protein may belong to the Kunitz-type protease inhibitor family and therefore may potentially neutralize the proteases present in snake venom. Using various structural and biochemical tools coupled with in vivo assays, we are able to show that MP-4 does not afford direct protection against snake venom because it is actually a poor inhibitor of serine proteases. Further experiments showed that antibodies generated against MP-4 cross-react with the whole venom and provide protection to mice against Echis carinatus snake venom. This study shows that the MP-4 contributes significantly to the snake venom neutralization activity of M. pruriens seeds through an indirect antibody-mediated mechanism.
Highlights
Death due to venomous snakes is an important public health problem in many tropical and subtropical countries
This study shows that the MP-4 contributes significantly to the snake venom neutralization activity of M. pruriens seeds through an indirect antibody-mediated mechanism
Our studies show that the MP-4 protein contributes substantially to protection against snake venom by M. pruriens seeds through an antibody-mediated mechanism and not through direct inhibition of venom proteases
Summary
The precipitated protein in each ammonium sulfate fraction was subjected to centrifugation at 12,000 ϫ g for 1 h at 4 °C. The major protein bands in the 40 and 60% ammonium sulfate fractions were transferred onto a polyvinylidene difluoride (PVDF) membrane using 10 mM CAPS buffer (pH 11.0). The final 25 residues at the C terminus were built based on homologous sequences available in the sequence database This endoproteinase Glu-C-digested peptide fragment was non-reproducible, and different combinations of amino acids of this peptide were used for homologous database search. 10 mg mlϪ1 of this protein was mixed thoroughly with EcV in 1:1 ratio (w/w) This mixture was incubated for 1 h at 4 °C, and 400 l of this solution was administered intraperitoneally to each mouse in the sample group. The regions corresponding to long loops in the structure, the first five N-terminal residues, and the last 25 C-terminal residues and
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