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Effects of Arg26 and Lys27 mutation on the bioactivity of HNTX-Ⅳ

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Frontiers in Biology 2007年第1期2卷 75-79页

作者： XIONG Xia XU Xia LI Dongling CHEN Ping liang songping College of Life Sciences Hunan Normal UniversityChangsha 410081China

Hainantoxin-Ⅳ(HNTX-Ⅳ)was isolated from the Chinese bird spider Ornithoctorcs hainana and identified as a novel antagonist of tetrodotoxin-sensitive(TTX-S)sodium *** revealed by the solution structure of HNTX-Ⅳ solved by two-dimensional nuclear magnetic resonance(2D-NMR),HNTX-Ⅳ adopts an inhibitor cystine knot *** check the role of basic residues during HNTX-Ⅳ’s interaction with TTX-S sodium channels,R26A and K27A mutants of HNTX-Ⅳ were constructed by solid-phase chemical *** synthesized peptides were purified and refolded under optimized oxidation *** synthesis and folding were confirmed by MALDI-TOF mass spectrometry and NMR spectroscopy,*** the whole-cell patch-clamp technique,Lys27 but not Arg26 was identified as a key residue for HNTX-Ⅳ’s bioactivity against TTX-S sodium channels,because R26A-HNTX-Ⅳ showed slightly reduced activity and K27A-HNTX-Ⅳ showed almost no inhibition.

关键词： Hainantoxin-Ⅳ mutant solid-phase synthesis bioactivity

A COMPARISON OF THE PRIMARY STRUCTURES OF LACTATE DEHYDROGENASE ISOZYMES M4 FROM GIANT PANDA, RED PANDA,BLACK BEAR AND DOG

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Science in China,Ser.B 1987年第3期30卷 270-282页

作者：梁宋平张龙翔 Department of Biology Peking University

Lactate dehydrogenase isozymes M4 have been isolated and purified from red panda （Ailurus fulgens）,black bear （Selenarctos thibetanus） and dog （Canis familiars） by affinity chromatography and comparedwith that from giant panda （Ailuropoda melanoleuca）. Experimental results have shown that the N-ter-mini, C-termini and the molecular weights of LDH-M subunits of red panda, black bear and dog arethe same as those of the LDH-M subunit of giant panda. Analysis and comparison of HPLC peptidemaps from the tryptic digests of the isozymes of red panda, black bear and dog have shown that mostof their peptide fragments had the same retention time and amino acid composition as the correspondingpeptide fragments from giant panda. Fragments with different retention times and/or amino acid compo-sitions were sequentced. Careful examination of those variant amino acid residues demonstrated clearlythat the primary structure of giant panda LDH-M subunit is unique and it appears that the giant pandamight be classified as

关键词： LDH RED PANDA,BLACK BEAR AND DOG A COMPARISON OF THE PRIMARY STRUCTURES OF LACTATE DEHYDROGENASE ISOZYMES M4 FROM GIANT PANDA

Evaluation of strategy for analyzing mouse liver plasma membrane proteome

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Science China(Life Sciences) 2007年第6期50卷 731-738页

作者： CHEN Ping ZHANG LiJun LI XuanWen WANG XiE CAO Rui LIU Zhen XIONG JiXian PENG Xia WEI YingJuan YING XingFeng WANG XianChun liang songping College of Life Sciences Hunan Normal UniversityChangsha 410081China

Plasma membrane(PM)proteome is one of the major subproteomes present in the cell,and is very important in liver *** the present work,C57 mouse liver PM was purified by density-gradient *** purified PM was verified by electron microscope analysis and Western *** results showed that the PM was enriched by more than 20-fold and the contamination of mitochondria was reduced by 2-fold compared with the homogenization *** were separated by 2DE and 1DE,trypsin-digested and submitted to ESI-Q-TOF and MALDI-TOF-TOF mass spectrometry or directly digested in solution and analyzed by LC-ESI ion trap mass *** all,547 non-redundant mouse liver PM proteins were identified,of which 34%contributed to plasma membrane or plasma membrane-related *** study optimized and evaluated the HLPP plasma membrane proteome analysis method and made a systematic analysis on PM proteome.

关键词： liver plasma membrane proteomics mass spectrometry 2DE shotgun 1DE

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间斑寇蛛毒素的蛋白质化学与蛋白质组学研究新进展

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生物工程学报 2014年第7期30卷 1036-1043页

作者：王贤纯梁宋平湖南师范大学生命科学学院蛋白质化学与发育生物学教育部重点实验室湖南长沙410081

间斑寇蛛Latrodectus tredecimguttatus俗称"黑寡妇"蜘蛛。其毒素不仅存在于毒腺中,而且存在于其身体的其他部分、卵粒甚至新生幼蛛体内。研究间斑寇蛛毒腺和毒腺外材料中的毒素成分,探明它们之间的异同、进化关系和生物学作用,具有重... 详细信息

间斑寇蛛Latrodectus tredecimguttatus俗称"黑寡妇"蜘蛛。其毒素不仅存在于毒腺中,而且存在于其身体的其他部分、卵粒甚至新生幼蛛体内。研究间斑寇蛛毒腺和毒腺外材料中的毒素成分,探明它们之间的异同、进化关系和生物学作用,具有重要的理论和实际意义。现代蛋白质化学和蛋白质组学技术的发展为间斑寇蛛蛋白质和多肽毒素的研究提供了有效手段,从而可以同时从单一纯化蛋白质和组学的层面探究毒素作用的分子基础和作用机制。到目前为止,间斑寇蛛毒素的蛋白质化学与蛋白质组学研究已取得一定的进展,但相关研究尤其是毒腺外材料来源的毒素研究还有待进一步深入。

关键词：毒素蛋白质多肽间斑寇蛛进展

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Three tarantula toxins acting on Nav1.5 reveals the diversity of toxin-channel interaction

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Three tarantula toxins acting on Nav1.5 reveals the diversit...

第12届生物毒素研究及医药应用学术大会

作者： Xi Zhou Cheng Tang Yunxiao Zhang Zhonghua Liu songping liang College of Life Sciences Hunan Normal University

Tarantula toxins potently target voltage-gated sodium channels(Navs) through binding to voltage sensor domains,and they are powerful tools for the investigation of *** the present study,we isolated and characterized three peptide toxins named JZTX-36,-38 and-39 from venom of Chilobrachys *** toxins could modulate human Na1.5 currents with different effects on Na1.5 channel activation and inactivation ***-36 and JZTX-39 interacted with site 4 to inhibit the activation of Nav1.5 and shift the steady-state activation voltage in a depolarization direction,whereas JZTX-38,which differs to JZTX-39 with only one amino acid located at the extreme C-terminal of the peptide,bound to the site 3 and site 4 simultaneously and barely affected steady-state *** results not only further confirmed that the S3-S4 linkers of domain Ⅱ and domain Ⅳ are hot spots for toxin interaction,but also revealed functional diversification of tarantula ***,many toxins can target a same binding site and one toxin can have two or more binding sites in *** addition,our results also suggest that spider toxins might be ideal template for designing ion channel’s modulators and useful probes for investigating the mechanism of toxin-channel interaction.

关键词： Navl.5 tarantula toxins toxin-channel interaction

A designed chimeric Nav1.8 channel which can be functionally expressed in HEK293T cells

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A designed chimeric Nav1.8 channel which can be functionally...

第12届生物毒素研究及医药应用学术大会

作者： Cheng Tang Yunxiao Zhang Xi Zhou Zhonghua Liu songping liang College of Life Sciences Hunan Normal University

Among the nine VGSC subtypes,Nav1.7 and Nav1.8 are attractive therapeutic targets for ***,the heterologous expression of Nav1.8 in mammalian cell lines in past years has achieved limited *** this study,we designed and constructed a chimeric channel by replacing all the transmembrane segments of human Nav1.5 with those of rat Nav1.8 *** resulting hybrid channel was named as DCNav1.8 and was abundantly expressed in Hek293 t as tested by patch-clamp ***1.8 exhibited normal voltage gating properties with a Va and Vh of15.2±1.5 mV and-17.3±1.8 mV *** differences were observed when compared with those of DRG TTX-R Nav1.8 channel or native Nav1.8 channel expressed in ND7/23 *** importantly,the pharmacological properties of DCNav1.8 resembled those of native Nav1.8 by testing several known Nav1.8 antagonists（Lidocaine,APETx-2 and MrVIB）.Single site mutation of amino acids located in the domain Ⅱ extracellular loops of DCNav1.8 to the corresponding ones in Navl.9 did not changed the affinity of MrVIB to *** molecular basis for the preference of MrVIB to Nav1.8 might reside in the pore loop of channel’s domain Ⅲ.In summary,our study provided a reliable and convenient platform for Nav1.8 expression which could be used in full-auto patch clamp system for high-throughput Nav1.8 modifiers screening and in intensive study of the molecular mechanism of drug-Nav1.8 interaction.

关键词： Nav1.8 chimeric channel heterologous expression patch clamp

Molecular cloning,expression and solution structure of HWTX-XVIIb1 from the spiderOrnithoctonus huwena

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Molecular cloning,expression and solution structure of HWTX-...

第12届生物毒素研究及医药应用学术大会

作者： Liping Jiang Juan Lv Zhonghua Liu Meichun Deng Guangzhong Tu songping liang Department of Parasitology Xiangya School of MedicineCentral South University Beijing Institute of Mieroehemistry College of Life Sciences Hunan Normal University Department of Biochemistry School of Life SciencesCentralSouthUniversity

The venom of spider is an increasingly valuable library to discover novel componentsfortherapeutic applications and investigating the relationship of the structure-function of ion *** gene of HWTX-XV11b1 was directly cloned from thespider venom glands of Ornithoctonus *** order to elucidatethe structureand the biological activities,recombinant HWTX-XV11b1 was successfully expressed,furtherpurified and *** comprises 31 aminoacid residues including six cysteine *** MALDI-TOF MS,the measured monoisotopic mass of recombinant HWTX-XV11b1(3337.948 Da) agreed wellwith the calculated one(3344.35 Da)by forming four pairs of disulfide *** determine the structureofHWTX-XV11b1,a suiteof2 D *** allthebackbone protonsandtheside-chain *** calculationwas performed by *** was found that the main element of these secondary structure of HWTX-XV11b1 is a short triple-stranded antiparallel P-sheet and its threedimensionalstructure adopts an’inhibitor cystine-knot’(ICK) *** using whole-cell patch-clamp methods,HWTX-XV11b1 potently inhibit high-voltage-activated(HVA) CachanneIsinDRG *** findings provide a basis for the further Study of the structure-function relationships of HWTX-XV11b1.

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Najaatra venom peptide selectively blocks voltage-gated sodium channel Nav1.8 to abolish pain

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Najaatra venom peptide selectively blocks voltage-gated sodi...

第12届生物毒素研究及医药应用学术大会

作者： Fan Zhang Dongfan Tang Heng Zhang Xinzhou Wu Zhonghua Liu songping liang College of Life Sciences Hunan Normal University

The tetrodotoxin-resistant voltage-gated sodium channel(VGSC) Nav1.8 is expressed predominantly in small nociceptive primary afferent nerves and contributes to inflammatory and neuropathic *** we described the discovery of μ-EPTX-Na1 a,a unique 62-residue peptide from Najaatra venom,that potently blocked Nav1.8(IC50 of 167 nM) in rat dorsal root ganglion neurons,whereas micromolar concentration of u-EPTX-Nala showed no inhibition on Navl.9 and tetrodotoxin-sensitive VGSC currents in these *** the five VGSC subtypes(Nav1.3-1.5,Nav1.7 and Nav1.8) expressed heterologously,human Nav1.8 was the most sensitive to μ-EPTX-Na1(IC50 of 386 nM) with the selectivity at least 20-folds over other VGSC *** neuropathic and chronic inflammatory pain models,μ-EPTX-Nala displayed analgesic effect,even more potent than morphine in rodent models of acid-induced pain,formalin-induced secondary mechanical allodynia and intraplantar complete Freund’s adjuvant injection-induced pain,and similar to morphine in rodent models of thermal pain and partial nerve ligation-induced *** antinociceptive effect of this peptide was not associated with obvious side *** study establishes μ-EPTX-Nala as a promising lead molecule for the development of novel analgesics targeting Nav1.8,which might be suitable for treating a wide range of human pain pathologies.

关键词： Nav1.8 snake venom peptide antinociceptive

Identification of a novel Kv1.3 antagonist from the venom of the spider Macrothele raveni

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Identification of a novel Kv1.3 antagonist from the venom of...

第12届生物毒素研究及医药应用学术大会

作者： Bo Chen Minzhi Chen Xi Zhou songping liang Ping Chen Zhonghua Liu College of Life Sciences Hunan Normal University

The Kv1.3 voltage-gated potassium channel regulates membrane potential and calcium signaling in human effector memory T cells that are key mediators of autoimmune diseases such as multiple sclerosis,type 1 diabetes,and rheumatoid ***,subtype-specific Kv1.3 blockers have potential for treatment of autoimmune ***-Ⅷ,a novel potassium channel blocker,was purified from the venom of the spider Macrothele raveni and its amino acid sequence was determined by Edman degradation and mass spectrometry *** contains 35 amino acid residues with a molecular weight of 3965.2 Da,tightly folded by three disulfide *** peptide was shown to block preferentially the currents of *** did not affect several other ion channels tested at 10 M ***-response curves of Raventoxin-Ⅷ yielded an ICof 36.3nM for Kv1.3 ***-Ⅷ was expressed as a cleavable fusion to small ubiquitin-related modifier(SUMO) in the cytoplasm of the SHuffle T7 express Escherichia coli strain,which allows cytosolic disulfide bond *** by IMAC with selective elution of monomelic SUMO fusion followed by proteolytic cleavage and HPLC steps yielded pure homogeneous toxin.

关键词： Kvl.3 Raventoxin-Ⅷ antagonist expression