Characterization of Elastolytic Protease, Asnilase from Aspergillus nidulans and Its Cytotoxicity on Human Endothelial and Epithelial Cells
Yumiko Komori *
Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan
Yoshiyuki Okumura
Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan and Department of Quality Control, Matsuurayakugyo Co., Ltd., 24-21 Enjo-chou, Syowa-ku, Nagoya 466-0054, Japan
Kazuhito Kamiya
Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan
Kenji Ogawa
Department of Pulmonary Medicine, National Hospital Organization Higashi Nagoya Hospital, 5-101 Umemorizaka, Meito-ku, Nagoya 465-8620, Japan
Toshiaki Nikai
Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tenpaku-ku, Nagoya 468-8503, Japan
*Author to whom correspondence should be addressed.
Abstract
Aims: To elucidate the pathogenicity of Aspergillus nidulans, elastolytic protease was isolated from the culture supernatant of the isolate, and its biological activity and cytotoxicity were examined.
Methodology: A. nidulans (NBRC 4340) spores were cultured on YCB-elastin medium for three days and elastolytic protease was isolated from the culture supernatant by DEAE-Cellulose anion exchange chromatography. Elastolytic activity was measured by using GAAPLNA (Glutaryl-L-alanyl-L-alanyl-L-prolyl-L-leucine p-nitroanilide) as the substrate. Molecular mass and isoelectric point were determined by polyacrylamide gel electrophoresis. Substrate specificities were measured by using fibrinogen, collagen, and oxidized insulin B chain. Cytotoxicity of Asnilase on human pulmonary artery endothelial cells (HPAEC), human small airway epithelial cells (HSAEC), human bronchial/tracheal epithelial cells (HBTEC) and human pulmonary alveolar epithelial cells (HPAEpiC) was determined with colorimetric method.
Results: An elastolytic protease isolated from the culture supernatant of A. nidulans was found to be homogeneous as indicated by a single band after polyacrylamide gel electrophoresis (PAGE), and the final preparation named as Asnilase. Molecular mass of this enzyme was determined to be 33,800 Da and the isoelectric point was 4.2. Asnilase cleaved the Aα and Bβ chains of fibrinogen, collagen and hydrolyzed His(5)-Leu(6) and Glu(13)-Ala(14) bonds of oxidized insulin B chain. Cytotoxic effects for various human pulmonary cells were observed when Asnilase was added at concentration of 2.0-8.0 mg/mL.
Conclusion: It is shown in our current investigation that Asnilase is a newly isolated elastolytic protease from A. nidulans. It is responsible for pulmonary aspergillus infection.
Keywords: Aspergillus nidulans, elastolytic protease, proteolytic specificity, cytotoxicity