Chemosynthesis and characterization of site-specific N-terminally PEGylated Alpha-momorcharin as apotential agent.

Alpha-momorcharin (α-MC), a type I ribosome-inactivating protein (RIP) isolated from Momordica charantia seeds, has been extensively studied for its antitumor, antiviral and antifungal activities. However, as an exogenous protein, problems associated with short half-life and strong immunogenicity have limited its clinical application. Poly (ethylene glycol) (PEG), as a polyether compound, is a well established and efficient modifier to develop it as a potential agent. Nevertheless, conventional PEGylation is not site-controlled and the conjugates are often not homogenous due to the generation of multi-PEGylated derivatives. To obtain a homogenous mono-PEGylated α-MC, the PEGylation was carried out by coupling a 20 kDa mPEG-butyraldehyde (mPEG-ALD) with α-MC. The product was separated and purified by MacroCap SP chromatography. Results from SDS-PAGE and MALDI-TOF MS revealed that the PEGylated α-MC consisted of one molecule mPEG and α-MC. Edman degradation confirmed that the N-terminal residue of α-MC was successfully coupled with mPEG-ALD. The mono-PEGylated α-MC possessed an extremely similar secondary structure to native α-MC through spectral analyses. In addition, it also showed low immunogenicity by double immunodiffusion and preserved moderate antitumor activity to three kinds of tumor cell lines in vitro. Finally, trypsin resistance was also considerably improved.

Α-MMC and MAP30, two ribosome-inactivating proteins extracted from Momordica charantia, induce cell cycle arrest and apoptosis in A549 human lung carcinoma cells.

α­Momorcharin (α­MMC) and momordica anti­human immunodeficiency virus protein (MAP30), produced by Momordica charantia, are ribosome­inactivating proteins, which have been reported to exert inhibitory effects on cultured tumor cells. In order to further elucidate the functions of these agents, the present study aimed to investigate the effects of α­MMC and MAP30 on cell viability, the induction of apoptosis, cell cycle arrest, DNA integrity and superoxide dismutase (SOD) activity. α­MMC and MAP30 were purified from bitter melon seeds using ammonium sulfate precipitation in combination with sulfopropyl (SP)­sepharose fast flow, sephacryl S­100 and macro­Cap­SP chromatography. MTT, flow cytometric and DNA fragmentation analyses were then used to determine the effects of α­MMC and MAP30 on human lung adenocarcinoma epithelial A549 cells. The results revealed that A549 cells were sensitive to α­MMC and MAP30 cytotoxicity assays in vitro. Cell proliferation was significantly suppressed following α­MMC and MAP30 treatment in a dose­ and time­dependent manner; in addition, the results indicated that MAP30 had a more potent anti­tumor activity compared with that of α­MMC. Cell cycle arrest in S phase and a significantly increased apoptotic rate were observed following treatment with α­MMC and MAP30. Furthermore, DNA integrity analysis revealed that the DNA of A549 cells was degraded following treatment with α­MMC and MAP30 for 48 h. The pyrogallol autoxidation method and nitrotetrazolium blue chloride staining were used to determine SOD activity, the results of which indicated that α­MMC and MAP30 did not possess SOD activity. In conclusion, the results of the present study indicated that α­MMC and MAP30 may have potential as novel therapeutic agents for the prophylaxis and treatment of cancer.

A novel method for simultaneous production of two ribosome-inactivating proteins, α-MMC and MAP30, from Momordica charantia L.

Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) from Momordica charantia L. have been confirmed to possess anti-tumor and anti-virus activities. Traditional purification methods of these two ribosome-inactivating proteins (RIPs) were separate which was time consuming and cost effective as well as low efficient. In order to obtain sufficient samples for researches, a strategy combining ion-exchange and gel filtration chromatography was developed and optimized in this study. Using this novel purification method, averagely 1162 mg of α-MMC and 535 mg of MAP30 were obtained from 400 g of Momordica charantia L seeds. The homogeneities of them were assessed by electrophoresis analysis. Determination of molecular weights of α-MMC and MAP30 were 28.585 kDa and 29.094 kDa by MALDI-TOF/TOF and pI were 9.02 and 9.12, respectively. The single glycoproteins were identified by Periodate-Schiff's base (PAS) and the saccharide content was tested to be 1.25% and 1.1% by anthrone-sulfuric acid method. Biological activities were evidenced by their ability to inhibit proliferation of lung adenocarcinoma A549 cell and to convert supercoiled plasmid pUC18 into relaxed forms. Finally, we also found that both two RIPs exhibited no superoxide dismutase (SOD) activity.