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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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Relative activities of 18 mutants of HWTX-XI.The selection of substituted sites was based on a comparison of the primary structures of HWTX-XI, BPTI, and DTX-K. Values are normalized to the respective activities of wild-type HWTX-XI (100%). The detailed values can be found in supplemental materials Table S1.
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pone-0003414-g005: Relative activities of 18 mutants of HWTX-XI.The selection of substituted sites was based on a comparison of the primary structures of HWTX-XI, BPTI, and DTX-K. Values are normalized to the respective activities of wild-type HWTX-XI (100%). The detailed values can be found in supplemental materials Table S1.

Mentions: To determine the key residues of HWTX-XI for its both functions, 18 mutants were designed according to its solution 3D structure. All the mutant cDNAs of HWTX-XI were constructed through site-directed mutagenesis using HWTX-XI gene as a template and expressed in S. cerevisiae strain S-78. The activities of all the purified mutants for both trypsin inhibition and blockage of voltage-sensitive K+ channels were measured (Table 2). Fig. 5 is a bar chat illustration of the results according to the data of Table 2. The results show that K14 is mainly responsible for the inhibition function, because the mutation K14N led to a ≈105-fold reduction in inhibitory potency, while K14A showed no binding activity to trypsin at all, as measured by ITC. The surrounding residues, such as R12, S16, F17 and A15, make minor contributions to the binding activity. With regard to to K+ channel blocking, the residue Leu 6 seemed to be critical, as its mutation to Ala or Tyr led to a ≈200-fold reduction in inhibitory potency. The mutation R5I reduced this activity ≈14-fold indicating that it has a secondary role in the blocking function. The results shown in Fig. 5 also indicated that none of the mutation in the loop region of HWTX-XI influenced the inhibitory potency toward Kv1.1 channels and that the mutants of the key residues for Kv1.1 channel blockage have no effect on trypsin inhibition activities. These results revealed that there are two separate sites, corresponding to the two types of activities, located on the two ends of the cone-shape molecule HWTX-XI and that they are functionally independent.


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)

Relative activities of 18 mutants of HWTX-XI.The selection of substituted sites was based on a comparison of the primary structures of HWTX-XI, BPTI, and DTX-K. Values are normalized to the respective activities of wild-type HWTX-XI (100%). The detailed values can be found in supplemental materials Table S1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2561067&req=5

pone-0003414-g005: Relative activities of 18 mutants of HWTX-XI.The selection of substituted sites was based on a comparison of the primary structures of HWTX-XI, BPTI, and DTX-K. Values are normalized to the respective activities of wild-type HWTX-XI (100%). The detailed values can be found in supplemental materials Table S1.
Mentions: To determine the key residues of HWTX-XI for its both functions, 18 mutants were designed according to its solution 3D structure. All the mutant cDNAs of HWTX-XI were constructed through site-directed mutagenesis using HWTX-XI gene as a template and expressed in S. cerevisiae strain S-78. The activities of all the purified mutants for both trypsin inhibition and blockage of voltage-sensitive K+ channels were measured (Table 2). Fig. 5 is a bar chat illustration of the results according to the data of Table 2. The results show that K14 is mainly responsible for the inhibition function, because the mutation K14N led to a ≈105-fold reduction in inhibitory potency, while K14A showed no binding activity to trypsin at all, as measured by ITC. The surrounding residues, such as R12, S16, F17 and A15, make minor contributions to the binding activity. With regard to to K+ channel blocking, the residue Leu 6 seemed to be critical, as its mutation to Ala or Tyr led to a ≈200-fold reduction in inhibitory potency. The mutation R5I reduced this activity ≈14-fold indicating that it has a secondary role in the blocking function. The results shown in Fig. 5 also indicated that none of the mutation in the loop region of HWTX-XI influenced the inhibitory potency toward Kv1.1 channels and that the mutants of the key residues for Kv1.1 channel blockage have no effect on trypsin inhibition activities. These results revealed that there are two separate sites, corresponding to the two types of activities, located on the two ends of the cone-shape molecule HWTX-XI and that they are functionally independent.

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