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芋螺(Cone snail)是一种肉食性海洋软体动物[1],其分泌的芋螺毒液包含许多不同化合物的混合物,包括小分子、肽和酶。其中,含有很多种不同成分的神经活性毒素肽−芋螺毒素(Conotoxin, CTx)具有广泛的多样性,这些具有高活性和靶点选择性的神经小肽大多由6~50个氨基酸残基构成,富含二硫键,以特定的离子通道和受体为靶标,作为药理学工具和探针,具有较好的开发前景[2-4]。α−芋螺毒素 (α-conotoxin, α-CTx) 是目前研究最广泛和深入的CTxs。α-CTx分子质量较小,由12~20个氨基酸残基连接构成,是各种乙酰胆碱受体(nicotinic acetylcholine receptor,nAChR)亚型的选择性拮抗剂[5-6]。α-CTx一般含有4个半胱氨酸(Cys),根据二硫键的不同连接方式,可组成3种异构体,其中,大部分α-CTxs的活性形式为球型。另外,根据α-CTx半胱氨酸间氨基酸数量的不同将α-CTx分为:α-3/5、α-4/4、α-4/6、α-4/7等多种亚家族。α-CTx不仅作为分子探针,用以鉴别不同亚型的nAChR,在治疗与nAChR相关疾病时有着至关重要的作用[7-9],并且在成瘾、镇痛等神经生理、药理研究方面也扮演重要角色[10]。
罗素兰研究团队从疣缟芋螺中分离得到了一种 α-4/7型CTx LvIA,它由15个氨基酸残基组成,能够作用于nAChRs,对α3β2 nAChR具有较好的的活性(IC50为8.7 nmol·L−1),但是LvIA对α6/α3β2β3,α3β4和α6/α3β4 3种nAChRs亚型保持一定的高亲和力[11-13]。为了提高LvIA的选择专一性,前期实验室基于LvIA进行了设计和改造,获得了对应的共晶结构,同时也利用受体突变技术解析了它与α3β2 nAChR相互作用的分子机制[14-15]。研究发现,当第11位天冬氨酸(Asp, D)突变成丙氨酸(Ala, A)后对α3β2 nAChR的活性保持不变,共晶实验表明,第11位氨基酸影响着LvIA与α3β2 nAChR的结合作用[16-19]。第11位是否是LvIA的关键氨基酸位点,今后能否基于该位点对LvIA进行改造,有待深入研究。
本研究基于LvIA前期研究发现的关键氨基酸信息[20-22],主要是针对第11位氨基酸,对LvIA进行进一步设计改造,分别用精氨酸(Arg, R)和组氨酸(His, H)对Asp进行取代,设计了2种新型突变体,考察第11位氨基酸电性和侧链基团对LvIA活性的影响,旨在为LvIA的设计和开发提供依据。
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纯化线性肽,使其纯度高于95%,用于后续的氧化折叠反应,连接2对二硫键,将所得产物通过HPLC和MS鉴定,具体结果见图1-A、B。[D11H]LvIA在乙腈浓度23%时洗脱,洗脱时间为14.8 min;而[D11R]LvIA在乙腈浓度为25%时洗脱,洗脱时间是14.9 min。通过质谱确定了[D11H]LvIA和[D11R]LvIA的相对分子质量,分别为1 702.95 和1 721.99 Du,与理论值一致,且均比其线性肽的相对分子量少了4 Du,说明成功连接了2对二硫键并且末端酰胺化,见图1-C、图1-D。
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提取包含鼠源α3、β2这2种亚基的质粒,使用0.8%的琼脂糖凝胶电泳进行纯度检测。从图2-A可知,质粒条带清晰,未发生拖尾情况,超螺旋结构大小在2 500 bp左右,符合理论值大小。从图2-B可知,条带清晰可见,无弥散现象,超螺旋结构的大小在5 000~7 500 bp范围内。使用紫外分光光度计测定线性质粒的质量浓度,质量浓度皆大于0.2 g·L−1,可以保证后续试验的浓度要求。制备α3、β2这2种亚基的cRNA,所得RNA的凝胶电泳结果如图2-C,图中有1条明显的亮带,大小在750~1 000 bp之间,符合理论值,α3亚基的浓度为0.4 g·L−1,β2亚基的浓度为0.49 g·L−1。将2种亚基按照1∶1的比例混合,显微注射到酶解好的爪蟾卵母细胞中(图2-D),表达2~3 d,使用激动剂ACh刺激,可以在细胞膜上检测到内向电流,电流大小在200~2 000 nA范围内,如图2-E。
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分别将LvIA和2种11位的LvIA突变体在表达α3β2 nAChR的非洲爪蟾卵母细胞上进行电生理活性检测。在终浓度为10 μmol·L−1的孵育条件下,LvIA、 [D11R]LvIA和[D11H]LvIA对α3β2nAChR的阻断率分别为(95.8±2.8)%(n=6)、(9.8±2.5)%(n=7)和(12.4±3.1)%(n=5)(图3),与野生型LvIA相比,2种11位突变体对α3β2 nAChR活性几乎完全丧失。
为了进一步确定2种LvIA 的11位突变体对于α3β2 nAChR的阻断活性,笔者做了剂量反应曲线(图4),分别测定了[D11R]LvIA和[D11H]LvIA对α3β2 nAChR的IC50值,2种突变体对于α3β2 nAChR的IC50值分别为8 976 nmol·L−1和6 390 nmol·L−1(表1),活性与比本体的活性相比,分别降低了574.38%和408.62%。证明当第11位的Asp变化后,LvIA的活性大大降低。
Synthesis of new analogs of α-conotoxin LvIA mutated at the position 11 residue and their target activity
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摘要: 为了进一步探究第11位氨基酸的性质对LvIA靶点结合活性的影响,设计了LvIA的2个新型突变体[D11R]LvIA和[D11H]LvIA,即用2个碱性氨基酸−精氨酸(R)和组氨酸(H)分别替换原来的酸性氨基酸D。先人工合成了这2个新突变体的线性肽,然后采用2步氧化法进行折叠,以获得在第1位和第3位半胱氨酸(Cys 1~3)、第2位和第4位半胱氨酸(Cys 2~4)之间定点连接形成二硫键。经高效液相色谱分离纯化和质谱鉴定,合成了含有Cys(1~3, 2~4)二硫键连接方式的多肽,其分子质量正确,纯度在95%以上。利用双电极电压钳电生理学技术对这2种突变体与α3β2 nAChR的结合活性进行了检测。结果发现,当该位点的氨基酸性质由酸性转换为碱性后,对LvIA的活性影响巨大,直接导致对α3β2 nAChR的阻断活性丧失。[D11R]LvIA和[D11H]LvIA的活性与野生型LvIA相比分别降低了574.38%和408.62%。由此表明,第11位氨基酸的酸碱性对LvIA的活性至关重要。
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关键词:
- α−芋螺毒素LvIA /
- 突变体设计与合成 /
- α3β2烟碱型乙酰胆碱受体 /
- 靶点结合活性 /
- 电生理学技术
Abstract: α-snail toxin LvIA is a small peptide targeting α3β2 acetylcholine receptor (nAChR), which was found in the South China Sea snail. The structure and function of LvIA were previously analyzed in our laboratory. After alanine scanning mutation it was found that the mutant [D11A] LvIA, which was replaced by alanine (A) with aspartic acid at the site 11 of LvIA, maintained its activity against α3β2 nAChR. In order to further explore the effect of the properties of amino acid at the position 11 on the binding activity of LvIA target, two new mutants of LvIA [D11R] LvIA and [D11H] LvIA were designed, namely, two basic amino acids - arginine (R) and histidine (H) were used to replace the original acid amino acid D, respectively. The linear peptides of the two new mutants were synthesized and then folded by a two-step oxidation method to obtain a disulfide bond between the first and third cysteines (Cys 1-3) and the second and fourth cysteines (Cys 2-4). The peptides containing Cys (1-3, 2-4) disulfide bond were successfully synthesized by high performance liquid chromatography (HPLC) and mass spectrometry. The synthesized peptides were correct in molecular weight and their purity was above 95%. The binding activity of these two mutants to α3β2 nAChR was detected by two-electrode voltage clamp electrophysiology. The results showed that when the amino acid properties of the site were changed from acidic to alkaline, LvIA activity was greatly affected, resulting in direct loss of α3β2 nAChR blocking activity. The activity of [D11R] LvIA and [D11H] LvIA was 574.38% and 408.62% lower than that of wild-type LvIA, respectively. This suggests that the acidity and basicity of the 11th amino acid is crucial to the activity of LvIA. These results provide some reference for the optimization design and modification of LvIA in the future, based on which it is expected to obtain more specific peptide tools targeting α3β2 nAChR, which can provide a better pharmacological molecular probe for the study of the structure, function and distribution of an α3β2 nAChR receptor. -
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