Mosquito-Derived Anophelin Sulfoproteins Are Potent Antithrombotics

Abstract

The anophelins are small protein thrombin inhibitors that are produced in the salivary glands of the <i>Anopheles</i> mosquito to fulfill a vital role in blood feeding. A bioinformatic analysis of anophelin sequences revealed the presence of conserved tyrosine residues in an acidic environment that were predicted to be post-translationally sulfated <i>in vivo</i>. To test this prediction, insect cell expression of two anophelin proteins, from <i>Anopheles albimanus</i> and <i>Anopheles gambiae</i>, was performed, followed by analysis by mass spectrometry, which showed heterogeneous sulfation at the predicted sites. Homogeneously sulfated variants of the two proteins were subsequently generated by chemical synthesis via a one-pot ligation-desulfurization strategy. Tyrosine sulfation of the anophelins was shown to significantly enhance the thrombin inhibitory activity, with a doubly sulfated variant of the anophelin from <i>A. albimanus</i> exhibiting a 100-fold increase in potency compared with the unmodified homologue. Sulfated anophelins were also shown to exhibit potent <i>in vivo</i> anticoagulant and antithrombotic activity.