Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-Cho, Syowa-Ku, 466-8560 Nagoya, Japan; Department of Pulmonary Medicine, National Hospital Organization Higashi Nagoya Hospital, 5-101 Umemorizaka, Meito-Ku, 465-8620 Nagoya, Japan.
Department of Quality Control, Matsuurayakugyo Co., Ltd., 24-21 Enjo-Chou, Syowa-Ku, 466-0054 Nagoya, Japan.
J Mycol Med. 2019 Dec;29(4):345-351. doi: 10.1016/j.mycmed.2019.05.001. Epub 2019 Sep 24.
Elastase, produced by Aspergillus fumigatus and A. flavus, is an important pathogenic factor in pulmonary aspergillosis. We investigated the possibility of using A. fumigatus-derived A. fumigatus elastase inhibitor (AFUEI) as a therapeutic agent. As native-AFUEI (N-AFUEI) has an extremely low yield, we generated a synthetic-AFUEI (S-AFUEI) and investigated whether S-AFUEI has a biological activity against A. fumigatus elastase (AFUE) and inhibits cytotoxicity.
A. fumigatus was cultured in Yeast Carbon Base (YCB) -elastin culture medium for 3-7 days, and AFUE was purified by chromatography using DE52 cellulose and Sephadex G-75 column. Elastolytic activity was examined using Glt-Ala-Ala-Pro-Leu-pNA (GAAPLNA) as the substrate. The hydrolytic activity of AFUE was determined using the characteristic substrates, fibrinogen and collagen (Type IV), and human cell cytotoxicity was measured colorimetrically. Furthermore, the inhibitory effect of S-AFUEI on these activities was examined.
We confirmed that S-AFUEI demonstrated elastase inhibitory activity and heat stability equivalent to that demonstrated by N-AFUEI, and inhibited human collagen hydrolytic activity and human fibrinogen hydrolytic activity. Further, S-AFUEI inhibited cytotoxicity in AFUE human pulmonary artery endothelial cells (HPAEC), human small airway epithelial cells (HSAEC), and human pulmonary alveolar epithelial cells (HPAEpiC).
As S-AFUEI strongly inhibited cytotoxicity induced by elastase in human-derived cells, it could prove beneficial for the treatment of pulmonary aspergillosis.
烟曲霉和黄曲霉产生的弹性蛋白酶是肺曲霉病的重要致病因素。我们研究了使用烟曲霉衍生的烟曲霉弹性蛋白酶抑制剂（AFUEI）作为治疗剂的可能性。由于天然-AFUEI（N-AFUEI）的产量极低，我们生成了一种合成-AFUEI（S-AFUEI），并研究了 S-AFUEI 是否对烟曲霉弹性蛋白酶（AFUE）具有生物学活性并抑制细胞毒性。
烟曲霉在酵母碳基础（YCB）-弹性蛋白培养基中培养 3-7 天，并用 DE52 纤维素和 Sephadex G-75 柱通过色谱法纯化 AFUE。使用 Glt-Ala-Ala-Pro-Leu-pNA（GAAPLNA）作为底物检查弹性酶活性。使用特征底物纤维蛋白原和胶原蛋白（IV 型）以及人细胞细胞毒性通过比色法测定 AFUE 的水解活性。此外，还检查了 S-AFUEI 对这些活性的抑制作用。
我们证实 S-AFUEI 表现出与 N-AFUEI 相当的弹性酶抑制活性和热稳定性，并抑制人胶原蛋白水解活性和人纤维蛋白原水解活性。此外，S-AFUEI 抑制 AFUE 人肺动脉内皮细胞（HPAEC）、人小气道上皮细胞（HSAEC）和人肺泡上皮细胞（HPAEpiC）中的细胞毒性。
由于 S-AFUEI 强烈抑制了人源细胞中弹性酶诱导的细胞毒性，因此它可能有益于肺曲霉病的治疗。
