Abstract
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.
Highlights
Ribosome-inactivating proteins (RIPs), widely distributed in higher plant tissues, can inactivate eukaryotic ribosomes and and irreversibly inhibit protein synthesis by N-glycosidase activity which catalytically cleaves the N-glycoside bond at position A4324 of the rat liver 28S rRNA1–7
We proposed that mono-PEGylated α-Momordica charantia L. (MC) can be successfully produced by 20 kDa mPEG-ALD conjugation with α-MC in a weakly acidic pH environment
The final product contained some other PEGylated byproducts, we can control the whole purification process and obtain high homogenous mono-PEGylated α-MC by using our purification process described in this manuscript
Summary
Ribosome-inactivating proteins (RIPs), widely distributed in higher plant tissues, can inactivate eukaryotic ribosomes and and irreversibly inhibit protein synthesis by N-glycosidase activity which catalytically cleaves the N-glycoside bond at position A4324 of the rat liver 28S rRNA1–7. In our earlier studies for the non-specific PEGylation with the amino groups on the side chain of lysines and N-terminus, the modified α-MC had acceptable bioactivity including longer half-life time in the bloodstream and decreased immunogenicity in vivo and in vitro[13,22,23,24]. This method is convenient for the formation of the PEGylated conjugates, it often leads to the non-homogenous mixture consisted of other PEGylated byproducts. This was the first study concerning the mono-PEGylation of Alpha-momorcharin as a potential therapeutic agent
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