CN117903278A - 一种蝎毒多肽LmTx-1及其应用 - Google Patents
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Abstract
本发明提供了一种蝎毒多肽LmTx‑1及其应用,属于生物多肽技术领域。本发明的一种蝎毒多肽LmTx‑1来源于海南双针蝎,其氨基酸全序列为:KDGYPIYSTGKSKGCKIECVINNKYCDKECTLKGGSSGYCYFWKLACYCE GLPDSVAVWTYAENTC。经膜片钳实验验证多肽毒素LmTx‑1高效抑制电压门控钠通道Nav1.4。基于多肽毒素LmTx‑1在骨骼肌钠通道Nav1.4上的显著的效果,可以推断这类多肽可以提供非常强效的肌肉松弛作用。多肽毒素LmTx‑1对其他电压门控钠通道抑制效果较弱,在保持较强皮肤抗皱功能的同时,显著降低了对心肌钠通道,脑钠通道抑制的风险。
Description
技术领域
本发明属于多肽技术领域,特别涉及一种蝎毒多肽LmTx-1及其应用,蝎毒多肽LmTx-1具体来源于海南双针蝎的多肽毒素。
背景技术
电压门控钠(Voltage-gated sodium channel;Nav)通道是一种重要的跨膜结构蛋白,广泛表达于人体的兴奋性细胞,如神经元、心脏和骨骼肌肌细胞。目前已发现Nav通道的突变可导致多种人类疾病,如癫痫、疼痛障碍、肌肉麻痹、强直性肌痉挛症和心脏疾病等。电压门控钠通道在哺乳动物中有9个亚型(Nav1.1-Nav1.9),其中电压门控钠通道1.4(Nav1.4)由SCN4A基因编码,特异的在骨骼肌中表达。Nav1.4可以控制神经-肌肉接头的端板电位产生和T-tubules膜的去极化扩散,从而启动和调节骨骼肌的收缩。编码人类骨骼肌Nav1.4通道的SCN4A基因突变会导致至少五种不同的骨骼肌疾病:钾加重性肌强直、先天性副肌强直、高钾性周期性麻痹、低钾性周期性麻痹和先天性肌无力综合征,Nav1.4通道也是公认的治疗骨骼肌相关疾病药物的热点靶标之一。
蝎子毒液是极其复杂的化学混合物,其中最为丰富的是肽类。是极具潜力的开发新型药理学工具和新型药物的活性分子资源库。大部分蝎子毒素作用于细胞膜上的各种电压门控离子通道或配体门控通道,因此它们是神经生物学研究的重要分子探针,也是发展新型药物的先导分子。
海南双针蝎(Lychas mucronatus)是一种毒蝎,广泛分布于东南亚和中国南方。毒腺转录组分析显示Lychas mucronatus毒液中毒素样肽的多样性较高,并鉴定出一些新的肽毒素。
发明内容
有鉴于此,本发明要解决的技术问题是提供一种蝎毒多肽LmTx-1及其应用。多肽毒素LmTx-1就是从其粗毒中纯化到的一种新型肽类毒素。LmTx-1是一种特异性作用于骨骼肌钠通道Nav1.4的抑制剂。通过阻断电压门控钠通道的电流,进而抑制动作电位的产生。基于LmTx-1在骨骼肌钠通道Nav1.4上的显著的效果,可以推断这类多肽可以提供非常强效的肌肉松弛作用,提供迅速即时的抗皱效果。
本发明的一种来源于海南双针蝎(Lychas mucronatus)的蝎毒多肽LmTx-1,其氨基酸序列如下:
N-Lys-Asp-Gly-Tyr-Pro-Ile-Tyr-Ser-Thr-Gly-Lys-Ser-Lys-Gly-Cys-Lys-Ile-Glu-Cys-Val-Ile-Asn-Asn-Lys-Tyr-Cys-Asp-Lys-Glu-Cys-Thr-Leu-Lys-Gly-Gly-Ser-Ser-Gly-Tyr-Cys-Tyr-Phe-Trp-Lys-Leu-Ala-Cys-Tyr-Cys-Glu-Gly-Leu-Pro-Asp-Ser-Val-Ala-Val-Trp-Thr-Tyr-Ala-Glu-Asn-Thr-Cys-C。多肽毒素LmTx-1由66个氨基酸残基组成,含有8个半胱氨酸,形成4对二硫键C1-C8,C2-C5,C3-C6,和C4-C7(数字表示半胱氨酸残基在多肽序列中的相对位置),分子量7355.6Da,等电点为7.66。三字母氨基酸转换成单字母缩写的结果如下:
KDGYPIYSTGKSKGCKIECVINNKYCDKECTLKGGSSGYCYFWKLACYCEGLPDSVAVWTYAENTC(SEQ ID NO.1)。
通过实验证明多肽毒素LmTx-1对Nav1.4具有高亲和力的抑制作用,和目前已经发现的其他Nav1.4抑制剂功能存在差异,因此,这种蝎子多肽毒素的发现可以作为解析蝎子多肽毒素与Nav1.4相互作用机制的分子工具,关于多肽毒素LmTx-1的富集,主要通过天然毒液分离所得。
经膜片钳实验验证多肽毒素LmTx-1高效抑制电压门控钠通道Nav1.4,其抑制作用是其他钠通道的十倍以上,是一种选择性作用于肌肉钠通道Nav1.4的抑制剂。通过阻断电压门控钠通道的电流,进而抑制动作电位的产生。基于多肽毒素LmTx-1在骨骼肌钠通道Nav1.4上的显著的效果,可以推断这类多肽可以提供非常强效的肌肉松弛作用,即提供迅速即时的抗皱效果。多肽毒素LmTx-1对其他电压门控钠通道抑制效果较弱,其在保持较强皮肤抗皱功能的同时,显著降低了对心肌钠通道,脑钠通道抑制的风险。
鉴于Nav通道的突变可导致多种人类疾病,如癫痫、疼痛障碍、肌肉麻痹、强直性肌痉挛症和心脏疾病等。而电压门控钠通道1.4(Nav1.4))可以控制神经-肌肉接头的端板电位产生和T-tubules膜的去极化扩散,从而启动和调节骨骼肌的收缩。因此,用多肽毒素LmTx-1制备抗肌强直的药物也是一个重要的研究方向,其中,肌强直为钾加重性肌强直(PAM)、先天性副肌强直(PMC)、高钾性周期性麻痹(HyperPP)、低钾性周期性麻痹(HypoPP)和先天性肌无力综合征(CMS)中的至少一种。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍。
图1表示蝎子多肽毒素LmTx-1纯化与验证,其中:A为海南双针蝎,Lychasmucronatus;B为海南双针蝎的RP-HPLC粗分色谱峰图,其中*号所示的是含有LmTx-1的目的峰;C图为含有LmTx-1的目标峰的第二轮RP-HPLC细分结果,*号所示的为LmTx-1目的峰;D图为C图*号所示目的峰的质谱结果;
图2表示LmTx-1对电压门控钠离子通道不同亚型的活性分析,在-10mV去极化电压刺激下,等待5min后,依次加入终浓度为0.25μM或2.5μMLmTx-1处理前(Control)和处理后(0.25μM或2.5μM LmTx-1)的通道电流图。A-G表示Nav1.2、Nav1.3、Nav1.4、Nav1.5、Nav1.6、Nav1.7和Nav1.8、Nav1.9通道。数据为mean±SEM,每个点对应的细胞数n=3-5。
图3表示LmTx-1抑制Nav1.4及其他钠通道电流的浓度效应关系曲线图。LmTx-1抑制Nav1.4通道电流的IC50=250nM±0.89。数据为mean±SEM,每个点对应的细胞数n=5-6;
图4表示LmTx-1对L型电压门控钙离子通道亚型的活性分析,在+10mV强去极化电压刺激下,依次加入终浓度为2.5μM LmTx-1处理前(Control)和处理后(2.5μM LmTx-1)的通道电流图。A、B分别表示Cav1.2、Cav1.3通道。数据为mean±SEM,每个点对应的细胞数n=3-5。
具体实施方式
为让本领域的技术人员更加清晰直观的了解本发明,下面将结合附图,对本发明作进一步的说明。
材料与方法
1、毒液收集和毒素纯化
电刺激法采集海南双针蝎(图1-A所示)的蝎子毒液,冻干后的粗毒储存在-20℃的条件下用于后续实验。多肽毒素LmTx-1的分离纯化分为两个步骤:(1)用XB-C18柱(10mm×250mm,Welch Materials Inc.,Shanghai,China)在岛津高效液相色谱系统上进行分离纯化,每次上样约1mg粗毒。其中溶液A是乙腈(加入0.1%的三氟乙酸),溶剂B是水(加入0.1%的三氟乙酸),以3ml/min的流速进行线性梯度洗脱:15%的A冲洗5min,然后A从15%到60%的梯度运行45min。在215nm下监测吸光值,收集色谱峰(如图1-B所示,*号所示的是含有LmTx-1目的峰),在-80℃冰冻后进行冷冻干燥。(2)将目标组分用XB-C18柱(4.6mm×250mm,WelchMaterials Inc.,Shanghai,China)进行第二轮高效液相色谱(Watersalliance2695HPLCsystem)分析,其中乙腈梯度从15%到55%(乙腈每分钟增加1%,流速为1ml/min),*号所示的是LmTx-1目的峰(如图1-C所示)。
2、质谱鉴定
将CCA粉末溶于50%的乙腈(含有0.1%TFA)中,形成饱和溶液。在点样板上将0.5μL的样品溶液和0.5μL CCA溶液混匀,待溶液自然结晶后,通过MALDI-TOF-TOFMS质谱(ABSCIEXTOF/TOFTM 5800system,Applied Biosystems,USA)测定多肽LmTx-1的分子量(如图1-D所示)。
3、细胞培养和转染
ND7/23和HEK293T细胞用高糖DMEM液体培养基进行培养,培养于37℃,含有5%CO2的恒温培养箱,培养基中含有10%FBS和1%PS。细胞经胰蛋白酶消化、培养液稀释,在35mm培养皿中培养。当细胞密度长至90%时进行转染,转染按照X-treme GENEHP DNATransfection Reagent(Roche,Basel,Switzerland)的产品说明书中的操作步骤进行,将通道质粒和eGFP共转。Nav1.6和Nav1.8转染ND7/23细胞,其他钠通道转染Hek293T细胞。转染4-6小时后,将细胞接种在新的小皿上,在5%CO2、37℃条件下培养24小时后用于全细胞膜片钳记录。绿色荧光用于识别阳性转染细胞。
4、电生理记录
使用的数据采集放大器为EPC 10USB Amplifier(HEKA,德国)。硼硅酸盐玻璃毛细管电极(电极入液电阻控制在1.8~2.5MΩ)由P-97电极拉制仪(PC-10,Narishige)用1.5mm毛细管玻璃制作而成,玻璃电极热抛光后电极尖端口径为1.5~3.0μm,拉制完成后在玻璃电极内灌细胞内液。采用80%串联电阻补偿,使电压误差降至最低。在EPC-10USB放大器中进行全细胞记录时,对液接电位进行校正。在建立全细胞模式后,使用patchmaster记录电压依赖性电流,在20kHz时采样,并以5kHz滤波。
5、数据处理
使用Patch Master v2x92、Igor Pro 9、Office Excel 2019和Graph Pad Prism9.5分析数据。所有数据点均显示为独立实验单元的平均值±标准误。单因素方差分析用于评估多组之间的差异。
实施例1
验证海南双针蝎多肽毒素LmTx-1对电压门控钠离子通道各亚型的作用,结果如图2和图3所示。实验前必须将细胞外液置于室温下平衡后再更换培养皿内的培养液,以防止溶液温度的剧烈变化。更换溶液时要防止细胞从培养皿底部脱落。倒置显微镜下选择细胞膜较为光滑、细胞质均匀的细胞,在室温20~25℃条件下进行膜片钳实验。形成全细胞记录模式后将细胞钳制在-90mV,钠离子通道给予-10mV的去极化电压进行单刺激记录电流;记录终浓度0.25μM或2.5μM的LmTx-1对钠通道各亚型的作用,Nav1.3、Nav1.5、Nav1.6、Nav1.7和Nav1.8通道电流部分抑制;终浓度250nM的LmTx-1可明显抑制Nav1.4通道电流,Nav1.2的抑制作用较弱,Nav1.9通道的电流大小无明显改变。多肽毒素LmTx-1数据为mean±SEM,每个点对应的细胞数n=3-5。如图3所示,发现多肽毒素LmTx-1选择性抑制Nav1.4的峰电流(IC50值为250nM±0.89),对Nav1.3、Nav1.5、Nav1.6、Nav1.7和Nav1.8通道电流部分抑制,对Nav1.2的抑制作用较弱,对Nav1.9通道的电流大小无明显改变。
实施例2
验证海南双针蝎多肽毒素LmTx-1对钙离子通道亚型的作用,钙通道活性鉴定时将细胞钳制为-80mV,在建立全细胞模式后,给予+10mV电压进行单刺激开始记录电流。记录终浓度2.5μM的多肽毒素LmTx-1对钙通道亚型的作用,如图4所示,在终浓度2.5μM的LmTx-1作用下Cav1.2、Cav1.3通道电流大小几乎不变,因此推测LmTx-1对大部分的L型钠钙道也无明显抑制作用。
总的来说,本发明涉及海南双针蝎多肽毒素LmTx-1的应用,通过实验证明多肽毒素LmTx-1对Nav1.4表现高效抑制作用,对其他钠通道作用较弱或无作用,而L型钙通道几乎无影响,是一种具有选择性的电压门控钠离子通道抑制剂,一定程度上可以规避成药后的心脏毒性。
上述对实施例的描述是为便于该技术领域的普通技术人员能理解和应用本发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于这里的实施例,本领域技术人员根据本发明的揭示,对于本发明做出的改进和修改都应该在本发明的保护范围之内。
Claims (6)
1.一种蝎毒多肽LmTx-1,其特征在于,其氨基酸序列如下:
N-Lys-Asp-Gly-Tyr-Pro-Ile-Tyr-Ser-Thr-Gly-Lys-Ser-Lys-Gly-Cys-Lys-Ile-Glu-Cys-Val-Ile-Asn-Asn-Lys-Tyr-Cys-Asp-Lys-Glu-Cys-Thr-Leu-Lys-Gly-Gly-Ser-Ser-Gly-Tyr-Cys-Tyr-Phe-Trp-Lys-Leu-Ala-Cys-Tyr-Cys-Glu-Gly-Leu-Pro-Asp-Se r-Val-Ala-Val-Trp-Thr-Tyr-Ala-Glu-Asn-Thr-Cys-C。
2.如权利要求1所述的蝎毒多肽LmTx-1在制备电压门控钠离子通道Nav1.4抑制剂中的应用。
3.如权利要求1所述的蝎毒多肽LmTx-1在制备抗肌强直的药物中的应用。
4.如权利要求3所述的应用,其特征在于,所述肌强直为钾加重性肌强直、先天性副肌强直、高钾性周期性麻痹、低钾性周期性麻痹和先天性肌无力综合征中的至少一种。
5.如权利要求1所述的蝎毒多肽LmTx-1在制备皮肤抗皱相关药品或者化妆品的药物中的应用。
6.一种电压门控钠离子通道Nav1.4的选择性抑制剂,其特征在于,包括权利要求1所述的蝎毒多肽LmTx-1。
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