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Discovery of a distinct superfamily of Kunitz-type toxin (KTT) from tarantulas.

Yuan CH, He QY, Peng K, Diao JB, Jiang LP, Tang X, Liang SP - PLoS ONE (2008)

Bottom Line: Kuntiz-type toxins (KTTs) have been found in the venom of animals such as snake, cone snail and sea anemone.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.These 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.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory for Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, PR China.

ABSTRACT

Background: Kuntiz-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 findings: Here 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 (omega) 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/significance: These 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.

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Effects of HWTX-XI on Kv channels.(A) 1 µM HWTX-XI evidently reduced the control potassium currents amplitude in rat DRG neurons by 41.7±1.8% (n = 5). (B) Concentration-response relationship for HWTX-XI inhibition of potassium currents expressed on rat DRG neurons. Each data point (mean±S.E.) arises from 5–7 cells. The solid line through the data is a fit of I/Imax = 1/[1+exp(C–IC50)/Κ]. (C) Effect of HWTX-XI on steady-state current-voltage relationship of potassium channels on rat DRG neurons. DRG cells were held at −80 mV and stepped to test potentials of −80 to +60 mV (mean±SD, n = 4). (D–F) 1 µM HWTX-XI evidently reduced the control potassium currents (Kv1.1, D; Kv1.2, E; Kv1.3, F) amplitude in rat DRG neurons by 41.7±1.8% (n = 5). (G) Concentration-response relationship for HWTX-XI inhibition of potassium currents expressed on rat DRG neurons. Each data point (mean±S.E.) arises from 5–7 cells. The solid line through the data is a fit of I/Imax = 1/[1+exp(C−IC50)/Κ]. (H) At a concentration of 10 µM, HWTX-XI produced a rapid (τ≈12±2 s for steady-state inhibition, n = 5) inhibition which is readily reversible with the time constant of 21±3 s upon removal of the toxin. (I–J) The current-voltage curves of Kv1.1 (I) and Kv1.3(J) currents activated by 3 µM and 5 µM HWTX-XI respectively.
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pone-0003414-g003: Effects of HWTX-XI on Kv channels.(A) 1 µM HWTX-XI evidently reduced the control potassium currents amplitude in rat DRG neurons by 41.7±1.8% (n = 5). (B) Concentration-response relationship for HWTX-XI inhibition of potassium currents expressed on rat DRG neurons. Each data point (mean±S.E.) arises from 5–7 cells. The solid line through the data is a fit of I/Imax = 1/[1+exp(C–IC50)/Κ]. (C) Effect of HWTX-XI on steady-state current-voltage relationship of potassium channels on rat DRG neurons. DRG cells were held at −80 mV and stepped to test potentials of −80 to +60 mV (mean±SD, n = 4). (D–F) 1 µM HWTX-XI evidently reduced the control potassium currents (Kv1.1, D; Kv1.2, E; Kv1.3, F) amplitude in rat DRG neurons by 41.7±1.8% (n = 5). (G) Concentration-response relationship for HWTX-XI inhibition of potassium currents expressed on rat DRG neurons. Each data point (mean±S.E.) arises from 5–7 cells. The solid line through the data is a fit of I/Imax = 1/[1+exp(C−IC50)/Κ]. (H) At a concentration of 10 µM, HWTX-XI produced a rapid (τ≈12±2 s for steady-state inhibition, n = 5) inhibition which is readily reversible with the time constant of 21±3 s upon removal of the toxin. (I–J) The current-voltage curves of Kv1.1 (I) and Kv1.3(J) currents activated by 3 µM and 5 µM HWTX-XI respectively.

Mentions: The toxin also can inhibit potassium channels expressed in rat dorsal root ganglion neurons (Fig. 3A–C). As shown in Fig. 3A, 1 µM HWTX-XI reduced the amplitude of control potassium currents maximally by 41.7±1.8% (n = 5). The inhibition of HWTX-XI was concentration-dependent with an IC50 value of 11.6 nM (Fig. 3B). Current-voltage curves showed that potassium currents were activated at around −30 mV. From the current-voltage relationship, it was determined that HWTX-XI produced a distinct inhibition of potassium currents at all test pulses, and that the inhibition was voltage-dependent (Fig. 3C).


Discovery of a distinct superfamily of Kunitz-type toxin (KTT) from tarantulas.

Yuan CH, He QY, Peng K, Diao JB, Jiang LP, Tang X, Liang SP - PLoS ONE (2008)

Effects of HWTX-XI on Kv channels.(A) 1 µM HWTX-XI evidently reduced the control potassium currents amplitude in rat DRG neurons by 41.7±1.8% (n = 5). (B) Concentration-response relationship for HWTX-XI inhibition of potassium currents expressed on rat DRG neurons. Each data point (mean±S.E.) arises from 5–7 cells. The solid line through the data is a fit of I/Imax = 1/[1+exp(C–IC50)/Κ]. (C) Effect of HWTX-XI on steady-state current-voltage relationship of potassium channels on rat DRG neurons. DRG cells were held at −80 mV and stepped to test potentials of −80 to +60 mV (mean±SD, n = 4). (D–F) 1 µM HWTX-XI evidently reduced the control potassium currents (Kv1.1, D; Kv1.2, E; Kv1.3, F) amplitude in rat DRG neurons by 41.7±1.8% (n = 5). (G) Concentration-response relationship for HWTX-XI inhibition of potassium currents expressed on rat DRG neurons. Each data point (mean±S.E.) arises from 5–7 cells. The solid line through the data is a fit of I/Imax = 1/[1+exp(C−IC50)/Κ]. (H) At a concentration of 10 µM, HWTX-XI produced a rapid (τ≈12±2 s for steady-state inhibition, n = 5) inhibition which is readily reversible with the time constant of 21±3 s upon removal of the toxin. (I–J) The current-voltage curves of Kv1.1 (I) and Kv1.3(J) currents activated by 3 µM and 5 µM HWTX-XI respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2561067&req=5

pone-0003414-g003: Effects of HWTX-XI on Kv channels.(A) 1 µM HWTX-XI evidently reduced the control potassium currents amplitude in rat DRG neurons by 41.7±1.8% (n = 5). (B) Concentration-response relationship for HWTX-XI inhibition of potassium currents expressed on rat DRG neurons. Each data point (mean±S.E.) arises from 5–7 cells. The solid line through the data is a fit of I/Imax = 1/[1+exp(C–IC50)/Κ]. (C) Effect of HWTX-XI on steady-state current-voltage relationship of potassium channels on rat DRG neurons. DRG cells were held at −80 mV and stepped to test potentials of −80 to +60 mV (mean±SD, n = 4). (D–F) 1 µM HWTX-XI evidently reduced the control potassium currents (Kv1.1, D; Kv1.2, E; Kv1.3, F) amplitude in rat DRG neurons by 41.7±1.8% (n = 5). (G) Concentration-response relationship for HWTX-XI inhibition of potassium currents expressed on rat DRG neurons. Each data point (mean±S.E.) arises from 5–7 cells. The solid line through the data is a fit of I/Imax = 1/[1+exp(C−IC50)/Κ]. (H) At a concentration of 10 µM, HWTX-XI produced a rapid (τ≈12±2 s for steady-state inhibition, n = 5) inhibition which is readily reversible with the time constant of 21±3 s upon removal of the toxin. (I–J) The current-voltage curves of Kv1.1 (I) and Kv1.3(J) currents activated by 3 µM and 5 µM HWTX-XI respectively.
Mentions: The toxin also can inhibit potassium channels expressed in rat dorsal root ganglion neurons (Fig. 3A–C). As shown in Fig. 3A, 1 µM HWTX-XI reduced the amplitude of control potassium currents maximally by 41.7±1.8% (n = 5). The inhibition of HWTX-XI was concentration-dependent with an IC50 value of 11.6 nM (Fig. 3B). Current-voltage curves showed that potassium currents were activated at around −30 mV. From the current-voltage relationship, it was determined that HWTX-XI produced a distinct inhibition of potassium currents at all test pulses, and that the inhibition was voltage-dependent (Fig. 3C).

Bottom Line: Kuntiz-type toxins (KTTs) have been found in the venom of animals such as snake, cone snail and sea anemone.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.These 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.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory for Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, PR China.

ABSTRACT

Background: Kuntiz-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 findings: Here 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 (omega) 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/significance: These 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.

Show MeSH
Related in: MedlinePlus