Chemical and functional identification and characterization of novel sulfated α-conotoxins from the cone snail Conus anemone
Chemical and functional identification and characterization of novel sulfated α-conotoxins from the cone snail Conus anemone
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DOI：
10.1021/jm031010o
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发表时间：
2004-02-26
影响因子：
7.3
通讯作者：
Lewis, RJ
中科院分区：
文献类型：
--
作者：
Loughnan, ML;Nicke, A;Lewis, RJ
对翻译后修饰的多肽进行了LC/MS分析和诊断性筛选，结果表明在海葵粗毒中存在一种新的硫酸盐化多肽，其分子质量范围在-芋螺毒素范围内。对提取物的功能分析表明，该提取物对几种神经元烟碱型乙酰胆碱受体(NAChRs)具有活性。用LC/MS和分析定向分离鉴定了3种硫酸化α-芋螺毒素(Ania、Anib和ANIC)，并对其进行了纯化和测序。其中最活跃的α-Anib被进一步表征并用于研究翻译后修饰对亲和力的影响。人工合成的Anib在大鼠α3/β2 nAChR(IC50 0.3 nM)处显示出亚纳摩尔效力，而在大鼠α7 nAChR(IC50 76 NM)上的活性则降低了200倍。非磺化多肽[Tyr(16)]Anib在α3β2(IC50 0.6 nM)和α7(IC50 836 NAChR)处的活性分别降低2倍和10倍。同样，去除C-末端酰胺对α7(IC50 367 Nm)效价的影响大于α3β2 nAChR(IC50 0.5 nM)。对两个N-末端甘氨酸残基的逐级去除表明，这些残基影响了多肽的结合动力学。与类似的4/7-α-芋螺毒素序列的比较表明，残基11(丙氨酸或甘氨酸)和残基14(谷氨酰胺)是α3beta2选择性的重要决定因素，而C端的酰胺化和酪氨酸-16的硫化有利于α7的亲和力。
An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the -conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3/beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr(16)]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7(IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.