Venom Kunitz-type family
(BPTI-like protease inhibitor)

General Activity
Kunitz domains are the active domains of proteins that inhibit the function of serine proteases. They are relatively small with a length of about 50 to 60 amino acids. One example of one Kunitz-type protease inhibitor is aprotinin (also called bovine pancreatic trypsin inhibitor (BPTI)), which inhibits a spectrum of serine proteases including trypsin, chymotrypsin, plasmin and kallikrein.

This domain has also been found in venom proteins from most venomous taxa. These proteins are almost exclusively composed of this domain, thus grouping venom Kunitz-type proteins in a single family. Venom Kunitz-type proteins play important roles in envenomation by inhibiting serine proteases acting in living processes, such as the hemostatic system (Masci et al., 2000, Choo et al., 2012 ), or by blocking potassium channels (Harvey, 2001 ), or both (Yang et al., 2014, Schweitz et al., 1995, Chen et al., 2012 ).

Sequence characteristics
Venom Kunitz-type proteins are basic polypeptides with approximately 60 amino acids. They can be classified into two families based on different cysteine frameworks. One family retains the typical Kunitz-type architecture, with three highly conserved disulfide bridges (for example, see kappa-theraphotoxin-Hh1a from spider, dendrotoxin-K from snake, and kalicludines from sea anemone). The other family has only four or five cysteine residues, which results in the apparent “loss” of a conserved disulfide bridge (see conkunitzin-S1 from cone snail).

Evolution
It is suggested that the evolutionary process of Kunitz-type toxins probably occurred in three stages: old function molecules (with serine protease inhibition activity), bi-function toxins (with both serine protease and potassium channel inhibition activities) and new function toxins (with potassium channel inhibition activity). Thus, some Kunitz-type protease inhibitors have acquired the neurotoxin function, whereas others may have lost their protease inhibitory role and act only to block voltage-gated ion channels. Potent and specific neurotoxic potassium channel blockers with Kunitz-type motif are particularly well developed in snakes, while sea anemone, scorpion and spider toxins have developed only the dual-function Kunitz-type toxins, frequently with weak potassium channel blocking activity. One possible explanation is that selective pressure would have acted in order to keep the dual-function Kunitz-type toxins with weak potassium channel blocking activity in these latter animals, which already have other potent neurotoxins in their venoms/body extracts. By contrast, only potassium channel blockers presenting the Kunitz-type motif have been characterized from snake venoms so far (reviewed in Mourao and Schwartz, 2013 ).