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[Synthesis, refolding and identification of pharmacological activities of neurotoxin JZTX-XI and R3A-JZTX-XI].
Sheng Wu Gong Cheng Xue Bao. 2011 Jun; 27(6):900-8.SW

Abstract

Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.

Authors+Show Affiliations

Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, Hunan Normal University, Changsha 410081, China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

English Abstract
Journal Article
Research Support, Non-U.S. Gov't

Language

chi

PubMed ID

22034819

Citation

Chi, Yupeng, et al. "[Synthesis, Refolding and Identification of Pharmacological Activities of Neurotoxin JZTX-XI and R3A-JZTX-XI]." Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology, vol. 27, no. 6, 2011, pp. 900-8.
Chi Y, Deng M, Wu Y, et al. [Synthesis, refolding and identification of pharmacological activities of neurotoxin JZTX-XI and R3A-JZTX-XI]. Sheng Wu Gong Cheng Xue Bao. 2011;27(6):900-8.
Chi, Y., Deng, M., Wu, Y., Luo, J., Rong, M., Zhang, Y., Zhang, D., Zeng, X., & Liang, S. (2011). [Synthesis, refolding and identification of pharmacological activities of neurotoxin JZTX-XI and R3A-JZTX-XI]. Sheng Wu Gong Cheng Xue Bao = Chinese Journal of Biotechnology, 27(6), 900-8.
Chi Y, et al. [Synthesis, Refolding and Identification of Pharmacological Activities of Neurotoxin JZTX-XI and R3A-JZTX-XI]. Sheng Wu Gong Cheng Xue Bao. 2011;27(6):900-8. PubMed PMID: 22034819.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Synthesis, refolding and identification of pharmacological activities of neurotoxin JZTX-XI and R3A-JZTX-XI]. AU - Chi,Yupeng, AU - Deng,Meichun, AU - Wu,Yuanyuan, AU - Luo,Ji, AU - Rong,Minqiang, AU - Zhang,Yiya, AU - Zhang,Dongyi, AU - Zeng,Xiongzhi, AU - Liang,Songping, PY - 2011/11/1/entrez PY - 2011/11/1/pubmed PY - 2013/1/11/medline SP - 900 EP - 8 JF - Sheng wu gong cheng xue bao = Chinese journal of biotechnology JO - Sheng Wu Gong Cheng Xue Bao VL - 27 IS - 6 N2 - Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas. SN - 1000-3061 UR - https://www.unboundmedicine.com/medline/citation/22034819/[Synthesis_refolding_and_identification_of_pharmacological_activities_of_neurotoxin_JZTX_XI_and_R3A_JZTX_XI]_ L2 - http://www.xenbase.org/literature/article.do?method=display&articleId=44119 DB - PRIME DP - Unbound Medicine ER -