Mosquito-Derived Anophelin Sulfoproteins Are Potent Antithrombotics

The anophelins are
small protein thrombin inhibitors that are produced
in the salivary glands of the Anopheles 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 in vivo. To test
this prediction, insect cell expression of two anophelin proteins,
from Anopheles albimanus and Anopheles gambiae, 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 A. albimanus exhibiting
a 100-fold increase in potency compared with the unmodified homologue.
Sulfated anophelins were also shown to exhibit potent in vivo anticoagulant and antithrombotic activity.