Purification and characterization of two pathogenesis-related class 10 protein isoforms with ribonuclease activity from the fresh Angelica sinensis roots

• Published in: Plant physiology and biochemistry 2018-07, Vol.128, p.66-71
• Main Authors: Pan, Jianru, Wang, Xiangling, Li, Lingling, Li, Xian, Ye, Xiaoqiang, Lv, Di, Chen, Cuihuang, Liu, Shutao, He, Huocong
• Format: Article
• Language: English
• Subjects: Angelica sinensis; Glycoprotein; Isoform; Pathogenesis-related (PR) class 10 protein; Purification; Ribonuclease activity
• ISSN: 0981-9428; 1873-2690
• DOI: 10.1016/j.plaphy.2018.04.040

In this study, two pathogenesis-related (PR) class 10 protein isoforms, ASPR-1 and ASPR-2, were purified from fresh roots of the Chinese medicinal plant Angelica sinensis (A. sinensis) using 80% ammonium sulfate precipitation, Sephadex G50 gel filtration chromatography, and DEAE-Sepharose ion-exchange chromatography. The molecular masses of ASPR-1 and ASPR-2 were estimated to be 16.66 kDa and 16.46 kDa, respectively, using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isoforms are both glycoproteins containing glycosyl contents of 1.8% (ASPR-1) and 3.4% (ASPR-2). The two isoforms were predominantly present as monomers, but they partially dimerized in solution. The 15 N-terminal amino acids of ASPR-1 were determined to be GIQKTEVEAPSTVSA, with significant sequence homology to certain PR-10 proteins. ASPR-2 was also identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis to be a PR-10 protein. The isoforms both exhibited ribonuclease (RNase) activity, with ASPR-2 having higher specific activity (128.85 U mg−1) than ASPR-1 (68.67 U mg−1). The isoforms had the same optimal temperature of 50 °C but different optimal pH values of 5.0 (ASPR-1) and 6.0 (ASPR-2). The RNase activities of the isoforms were both stable for 30 min at 50 °C, rapidly decreasing at higher or lower processing temperatures. However, ASPR-1 retained higher residual activity (89.4%–80.9%) than ASPR-2 (74.3%–67.9%) at temperatures from 40 °C to 60 °C. These results provide additional information to enrich the current knowledge of poorly annotated A. sinensis proteins. •Two pathogenesis-related class 10 protein isoforms were first purified from fresh Angelica sinensis roots.•The isoforms are predominantly present as monomers but partially dimerized in solution.•They both have ribonuclease activity.•ASPR-2 has higher specific activity while ASPR-1 are more thermotolerant.•This is the first observation of PR-10 protein glycosylation in fresh plant materials.