A new type of antimicrobial protein with multiple histidines from the hard tick, Amblyomma hebraeum.

A novel 11 kDa antimicrobial protein, named as hebraein, and having a unique amino acid sequence, was purified from the hemolymph of fed female Amblyomma hebraeum ticks. A full-length cDNA clone encoding hebraein was isolated from a cDNA library made from tick synganglia. Hebraein consists of 102 amino acids, including 6 cysteine residues; has 9 histidines in its C-terminal domain that are mainly present as HX repeats; and has no significant similarity to any known protein. The secondary structure prediction is very clearly all alpha-helical (4-6 helices) except for a very short extension at the C terminus. Such high alpha-helical content is quite different from known antimicrobial proteins. Recombinant hebraein and a mutant lacking the histidine residues in the C-terminal domain were constructed and expressed. Assayed at the slightly acidic pH equivalent of fed female tick hemolymph, the wild-type and the histidine-rich recombinant hebraein had stronger antimicrobial activities than the histidine-deficient mutant. The pH-dependent properties of histidine-rich antimicrobial proteins may allow the design of agents that would function selectively in specific pH environments. The results from protein profiling of hemolymph, analyzed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry combined with ProteinChip technology and RT-PCR analysis suggested that this antimicrobial protein was up-regulated by blood feeding. Our findings describe a new type of antimicrobial protein with multiple cysteine and histidine residues, and with unique secondary structure.