AGRIMONY Yao Meng,1,2 Shuangfeng Liu,1 Juan Li,3 Yanfa Meng,3 Xiaojun Zhao2,41School of Medical Laboratory Science, Chengdu Medical College, Chengdu, China; 2West China Hospital Laboratory of Nanomedicine and Institute for Nanobiomedical Technology and Membrane Biology, Sichuan University, Chengdu, China; 3Key Laboratory of Bio-resources and Eco-environment Ministry of Education/Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu, China; 4Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USABackground: Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) derived from Momordica charantia L.have been confirmed to possess antitumor and antivirus activities due to their RNA-N-glycosidase activity.However, strong immunogenicity and short plasma half-life limit their clinical application.
To solve this problem, the two proteins were modified with (mPEG)2-Lys-NHS (20 kDa).Methodology/principal findings: In this article, a novel purification strategy for the two main type I ribosome-inactivating proteins (RIPs), α-MMC and MAP30, was successfully developed for laboratory-scale preparation.Using this dramatic method, 200 mg of α-MMC and about 120 mg of MAP30 was obtained in only one purification process from 200 g of Momordica charantia seeds.
The homogeneity and some other properties of the two proteins were assessed by gradient SDS-PAGE, electrospray ionization quadruple mass spectrometry, and N-terminal sequence analysis as well as Western blot.Two polyethylene glycol (PEG)ylated proteins were synthesized and purified.Homogeneous mono-, di-, or midi-dresses tri-PEGylated proteins were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry.
The analysis of antitumor and antivirus activities indicated that the serial PEGylated RIPs preserved moderate activities on JAR choriocarcinoma cells and herpes simplex virus-1.Furthermore, both PEGylated proteins showed about 60%–70% antitumor and antivirus activities, and at the same time decreased 50%–70% immunogenicity when compared with their unmodified counterparts.Conclusion/significance: α-MMC and MAP30 obtained from this novel purification strategy can meet the requirement of a large amount of samples for research.
Their chemical modification can solve the problem of strong immunogenicity and meanwhile preserve moderate activities.All these findings suggest the potential application of PEGylated α-MMC and PEGylated MAP30 as antitumor and antivirus agents.According to these results, PEGylated RIPs can be constructed with nanomaterials to be a targeting drug that can further decrease immunogenicity and side effects.
Through nanotechnology we can make them low-release drugs, which can further prolong their half-life period in the human body.Keywords: ribosome-inactivating proteins, alpha-momorcharin, momordica anti-HIV protein, antitumor, antivirus, (mPEG)2-Lys-NHS (20 kDa), immunogenicity.