Mishandling of the therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils
Mishandling of the therapeutic peptide glucagon generates cytotoxic amyloidogenic fibrils
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DOI：
10.1023/b:pham.0000033016.36825.2c
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发表时间：
2004-07-01
影响因子：
3.7
通讯作者：
Yajima, T
中科院分区：
文献类型：
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作者：
Onoue, S;Ohshima, K;Yajima, T
目的.一些治疗性肽表现出淀粉样蛋白生成特性，其在体外引起对神经元细胞的不溶性和细胞毒性。在这里，我们表征了单体治疗肽的构象变化，其纤维状聚集体，以防止在临床应用过程中淀粉样蛋白的形成。将包括胰高血糖素、猪促胰液素和鲑鱼降钙素的治疗性肽以1 mg/ml至80 mg/ml的浓度溶解在酸性溶液中，然后在37 ℃下老化。通过圆二色谱（CD）、电子显微镜（EM）、β折叠特异性染料染色和尺寸排阻色谱（SEC）评估淀粉样蛋白生成特性。同时测定细胞毒性特征。通过在2.5 mg/ml或更高浓度下老化24 h，单体胰高血糖素转化为纤维状聚集体，其由富含β折叠的结构和胰高血糖素的多聚体状态组成。尽管在1 mg/ml临床浓度下老化1天未观察到聚集，但30天老化导致产生纤维状聚集体。抗胰高血糖素血清的添加显著抑制单体胰高血糖素的纤维转化。胰高血糖素原纤维诱导PC 12细胞和NIH-3 T3细胞显著的细胞死亡并激活凋亡酶caspase-3。半胱天冬酶抑制剂以剂量依赖性方式减弱这种毒性，表明细胞凋亡信号通路参与胰高血糖素的纤维形成。与胰高血糖素相反，鲑鱼降钙素在40 mg/ml的高得多的浓度下表现出聚集，而胰泌素在高达75 mg/ml的浓度下不显示聚集。这些结果表明，在浓缩条件下的老化过程中，胰高血糖素通过其富含β折叠的分子间结构自缔合，以诱导纤维状聚集体。胰高血糖素具有与病理相关肽如β-淀粉样蛋白（Abeta）（1-42）和朊病毒蛋白片段（PrP）（106-126）相同的淀粉样蛋白生成倾向，包括构象变化为富含β折叠的结构和通过激活半胱天冬酶的细胞毒性作用。这些结果表明，不适当的制备和应用治疗胰高血糖素可能会导致不良的不溶性产物和副作用，如淀粉样变性在临床应用。
Purpose. Some therapeutic peptides exhibit amyloidogenic properties that cause insolubility and cytotoxicity against neuronal cells in vitro. Here, we characterize the conformational change in monomeric therapeutic peptide to its fibrillar aggregate in order to prevent amyloidogenic formation during clinical application.Methods. Therapeutic peptides including glucagon, porcine secretin, and salmon calcitonin were dissolved in acidic solution at concentrations ranging from 1 mg/ml to 80 mg/ml and then aged at 37 degreesC. Amyloidogenic properties were assessed by circular dichroism ( CD), electron microscopy (EM), staining with beta-sheet-specific dyes, and size-exclusion chromatography ( SEC). Cytotoxic characteristics were determined concomitantly.Results. By aging at 2.5 mg/ml or higher for 24 h, monomeric glucagon was converted to fibrillar aggregates consisting of a beta-sheet-rich structure with multimeric states of glucagon. Although no aggregation was observed by aging at the clinical concentration of 1 mg/ml for 1 day, 30-day aging resulted in the generation of fibrillar aggregates. The addition of anti-glucagon serum significantly inhibited fibrillar conversion of monomeric glucagon. Glucagon fibrils induced significant cell death and activated an apoptotic enzyme, caspase-3, in PC12 cells and NIH-3T3 cells. Caspase inhibitors attenuated this toxicity in a dose-dependent manner, indicating the involvement of apoptotic signaling pathways in the fibrillar formation of glucagon. On the contrary to glucagon, salmon calcitonin exhibited aggregation at a much higher concentration of 40 mg/ml and secretin showed no aggregation at the concentration as high as 75 mg/ml.Conclusions. These results indicated that glucagon was self-associated by its beta-sheet-rich intermolecular structure during the aging process under concentrated conditions to induce fibrillar aggregates. Glucagon has the same amyloidogenic propensities as pathologically related peptides such as beta-amyloid (Abeta)(1-42) and prion protein fragment (PrP)(106-126) including conformational change to a beta-sheet-rich structure and cytotoxic effects by activating caspases. These findings suggest that inappropriate preparation and application of therapeutic glucagon may cause undesirable insoluble products and side effects such as amyloidosis in clinical application.