Enolase, a plasminogen receptor isolated from salivary gland transcriptome of the ixodid tick Haemaphysalis flava

Abstract

Enolase, a multifunctional protein, is shown to act as a plasminogen receptor that contributes to fibrinolysis, which plays an important role in preventing the formation of blood clots during tick feeding. The study of enolase genes provides opportunities to develop a potential antigen target for tick control. So far, enolase has been identified in only a few species of ticks. Knowledge of the exact mechanisms of plasminogen activation and fibrinolysis by enolase as a plasminogen receptor is limited. Here, we cloned the enolase full-length complementary DNA (cDNA) from the salivary glands of Haemaphysalis flava, expressed it, and analyzed the function of the recombinant H. flava enolase. The enolase cDNA was 1988 bp in length and encoded 433 amino acid residues. It contained two domains and some highly conserved functional motifs including an assumed membrane re-association region "AAVPSGASTGI." The enolase exhibited 83.3 % amino acid similarity to that of the putative enolase of Ixodes ricinus, and 85 % to that of Ornithodoros moubata enolase. After eukaryotic expression in insect cells, Western blot analysis showed that the mouse antiserum against the hexahistidine-tagged recombinant enolase protein recognized a band of approximately 48 kDa. The recombinant enolase bound human plasminogen in a dose-dependent manner and enhanced plasminogen activation in the presence of host tissue plasminogen activator (t-PA), most probably to promote fibrinolysis and maintain blood flow at the host-tick interface. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that the expression level of enolase in salivary glands was significantly higher than in other tested tissues. Although the enolase was expressed in all developmental stages, it had the highest expression in the rapid blood feeding period of ticks. These findings indicate that the enolase might play an important role in blood feeding of H. flava.

Keywords: Enolase; Gene expression; Haemaphysalis flava; Plasminogen receptor.