Total chemical synthesis of human psoriasin by native chemical ligation
Total chemical synthesis of human psoriasin by native chemical ligation
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DOI：
10.1021/bi051519g
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发表时间：
2005-11-08
期刊：
影响因子：
2.9
通讯作者：
Lu, WY
中科院分区：
文献类型：
--
作者：
Li, XQ;de Leeuw, E;Lu, WY
人银屑病素（S100 A7）是钙结合蛋白S100家族的成员，在银屑病患者的角质形成细胞中大量表达。迄今为止，在许多人类细胞类型中丰富的银屑病蛋白的确切生理功能仍不清楚。Schroder及其同事最近的一份报告表明，从人角质层提取物中纯化的银屑病蛋白通过螯合细菌生长所必需的Zn 2+离子选择性地杀死大肠杆菌，这表明在先天免疫防御微生物感染中发挥重要作用。我们使用固相肽合成结合天然化学连接法化学合成了100个氨基酸残基的N-末端乙酰化银屑病蛋白。从0.25 mmol规模的单次合成中获得了超过140 mg的高纯度和正确折叠的合成银肩病素。通过尺寸排阻色谱法分析合成的银肩病菌素显示蛋白质在溶液中形成同二聚体。圆二色性分析表明，银肩病菌素的α-螺旋度增加超过20%的CaCl 2或ZnCl 2的存在下，表明金属离子结合诱导的构象变化。基于圆二色性的滴定进一步确定了合成蛋白质结合两个Ca 2+和两个Zn 2+离子/二聚体，与已发表的结构发现一致。重要的是，这种合成蛋白杀死大肠杆菌的能力。并且ZnCl 2对杀死的抑制与从其天然来源分离的银屑病素相当。大量人类银屑病素的稳健合成途径应有助于对其生物学功能及其作用模式的研究。
Human psoriasin (S100A7), a member of the S100 family of calcium-binding proteins, is richly expressed in keratinocytes of patients suffering from psoriasis. To date, the exact physiological function of psoriasin abundant in many human cell types remains unclear. A recent report by Schroder and colleagues suggests that psoriasin, purified from human stratum corneum extracts, selectively kills Escherichia coli by sequestering Zn2+ ions essential for bacterial growth, indicative of an important role in innate immune defense against microbial infection. We chemically synthesized the N-terminally acetylated psoriasin of 100 amino acid residues using solid phase peptide synthesis in combination with native chemical ligation. More than 140 mg of highly pure and correctly folded synthetic psoriasin was obtained from a single synthesis on a 0.25 mmol scale. Analysis of synthetic psoriasin by size exclusion chromatography showed that the protein forms a homodimer in solution. Circular dichroism analysis indicated that the alpha-helicity of psoriasin increases by more than 20% in the presence of CaCl2 or ZnCl2, suggesting a metal ion binding induced conformational change. Circular dichroism based titration further established that the synthetic protein binds two Ca2+ and two Zn2+ ions per dimer, in agreement with the published structural findings. Importantly, the ability of the synthetic protein to kill E. coli and the inhibition of the killing by ZnCl2 is comparable to that of psoriasin isolated from its natural source. The robust synthetic access to large quantities of human psoriasin should facilitate studies of its biological functions as well as its mode of action.