Cloning, Function And Structure-function Relationship Of Three New Antimicrobial Peptides From The Venom Gland Of The Scorpion Heterometrus Spinifer

Scorpion venom peptides are powerful weapons of scorpions for prey and defense. It consists of disulfide-bridged peptides (DBPs) and non-disulfide-bridged peptides (NDBPs). Three new cysteine-free venom peptides, which are referred to as Heterin-1, Heterin-2and Spiniferin, respectively, were identified from the scorpion Heterometrus spinifer. Heterin-1, Heterin-2and Spiniferin are43,24and13residues long, respectively. Protein homology search showed that they belong to the3rd,4th and5th family of NDBPs from scorpions, respectively. They were classified as NDBP3.14, NDBP4.2and NDBP5.24, respectively. Secondary structure prediction showed that they are a-helical molecules. Helical-wheel projection of Heterin-1, Heterin-2and Spiniferin revealed that the molecules have distinct hydrophilic and hydrophobic faces, and thus are amphipathic molecules. Genomic analysis showed that all of them contain two exons disrupted by an intron located in the nucleotide sequence encoding the C-terminal region of the signal peptide. The genomic organizations of the three peptides consistent with those of the known Na+, K+or Cl--channel specific toxins from scorpions; this suggests that the genes of the cysteine-free and cysteine-rich peptides from scorpions were derived from a common ancestor. Antimicrobial assay demonstrated that Heterin-1possesses potent activities against both Gram-positive and Gram-negative bacteria. Among the tested bacterial species, Heterin-1is the most active against Bacillus megaterium and Micrococcus luteus with MICs of4.0μM and4.0μM, respectively. Heterin-2is able to potently inhibit the growth of Gram-positive bacteria with MICs from5.6μM to30.0μM; however, it has weaker activities against the tested Gram-negative bacteria. It is interesting to see that deletion of the C-terminal random coiled tail (KKD) in Heterin-2markedly changed the antimicrobial specificity and activity of the peptide. This finding suggests that peptide hydrophobicity is highly associated with the specificity of the peptide; decreasing hydrophilicity could be a good way to reduce toxicity of the antimicrobial peptides to mammaliancells. Spiniferin has very weak antimicrobial activities against both Gram-positive and Gram-negative bacteria. We found that introducing three net charges into the polar face of Spiniferin significantly increased its antimicrobial activity against the majority of the tested bacteria. This finding is consistent with the previous observation that net charge is important for both antimicrobial and hemolytic activities. However, in some instances, net charge on the polar face is not important for the antimicrobial activity of the peptide. These studies have expanded our understanding of the diversity, evolution and structure/function relationships of the cysteine-free peptides from scorpions.