Discovery of a Distinct Superfamily of Kunitz-Type Toxin (KTT) from Tarantulas

Abstract

BackgroundKuntiz-type toxins (KTTs) have been found in the venom of animals such as snake, cone snail and sea anemone. The main ancestral function of Kunitz-type proteins was the inhibition of a diverse array of serine proteases, while toxic activities (such as ion-channel blocking) were developed under a variety of Darwinian selection pressures. How new functions were grafted onto an old protein scaffold and what effect Darwinian selection pressures had on KTT evolution remains a puzzle.Principal FindingsHere we report the presence of a new superfamily of KTTs in spiders (Tarantulas: Ornithoctonus huwena and Ornithoctonus hainana), which share low sequence similarity to known KTTs and is clustered in a distinct clade in the phylogenetic tree of KTT evolution. The representative molecule of spider KTTs, HWTX-XI, purified from the venom of O. huwena, is a bi-functional protein which is a very potent trypsin inhibitor (about 30-fold more strong than BPTI) as well as a weak Kv1.1 potassium channel blocker. Structural analysis of HWTX-XI in 3-D by NMR together with comparative function analysis of 18 expressed mutants of this toxin revealed two separate sites, corresponding to these two activities, located on the two ends of the cone-shape molecule of HWTX-XI. Comparison of non-synonymous/synonymous mutation ratios (ω) for each site in spider and snake KTTs, as well as PBTI like body Kunitz proteins revealed high Darwinian selection pressure on the binding sites for Kv channels and serine proteases in snake, while only on the proteases in spider and none detected in body proteins, suggesting different rates and patterns of evolution among them. The results also revealed a series of key events in the history of spider KTT evolution, including the formation of a novel KTT family (named sub-Kuntiz-type toxins) derived from the ancestral native KTTs with the loss of the second disulfide bridge accompanied by several dramatic sequence modifications.Conclusions/SignificanceThese finding illustrate that the two activity sites of Kunitz-type toxins are functionally and evolutionally independent and provide new insights into effects of Darwinian selection pressures on KTT evolution, and mechanisms by which new functions can be grafted onto old protein scaffolds.

The Toxicogenomic Multiverse: Convergent Recruitment of Proteins Into Animal Venoms

“…This toxin, named Conkunitzin-S1, blocks Shaker potassium channels with an IC 50 of approximately 60 nM. The Kunitz-type toxins in mygalomorph spider venoms also have a dual activity, acting as both trypsin inhibitors and K V channel blockers (170) For example, huwentoxin-XI, a Kunitz-type toxin from the Chinese tarantula Ornithoctonus huwena, competitively inhibits trypsin with K i = 68 nM (87), and it is also a weak blocker of K V 1.1 channels; the epitopes responsible for protease inhibition and K V channel block are spatially distinct and located at opposite ends of the cone-shaped molecule (170).…”

Spider toxins comprising disulfide‐rich and linear amphipathic domains: a new class of molecules identified in the lynx spider Oxyopes takobius

“…This signature may be reduced to the more general principal structural motif CX 6 CX n CC (C 1 X 6 C 2 X 5 C 4 C 5 in our case), and extra structural motif CXCX n CXC (C 6 XC 7 X m C 8 XC 9 in our case) that are characteristic of spider neurotoxins [23]. The principal structural motif and the extra structural motif in turn conform to the more general ICK motif CX 2-7 CX 3-11 CX 0-7 CX 1-17 CX [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] …”

Discoveries of Serine Protease Inhibitors from Scorpions

“…To detect whether SdPI possesses a similar active site to other Kunitz-type venom peptides, four residues (Lys12, Gly13, Lys14, and Ala15) were mutated [13,22,40]. The circular dichroism (CD) spectrum was performed for each of the mutants and it was found that each of the mutants, compared with that of the wild-type peptide, indicated no significant change in secondary structure, suggesting that they all adopted the same structural topology.…”

“…Serine protease inhibitors (SPIs) are ubiquitous in animals, plants as well as microorganisms and they play important roles in physiological processes such as blood coagulation, tissue remodeling, and proteolysis regulation [7][8][9][10]. According to previous reports, SPIs have been found in a variety of animal venoms, including snakes, scorpions, spider, cnidarians, cone snails, platypus, and hymenopterans, as well as the salivary secretions of hematophagous insects and leeches [11][12][13]. Importantly, serine protease inhibitors are a class of proteins involved in the regulation of serine and other types of proteases.…”

“…Among all classes, the Kunitz-type inhibitors are well characterized of them, possibly due to their abundance in several creatures [13,15,16]. Many studies reported that animal venoms contain Kunitz-type serine protease inhibitors [12,[17][18][19].…”