Pharmacological investigation of ribosome inactivating protein (RIP) – like protein extracted from Annona squamosa L. seeds

Abstract

Ribosome inactivating proteins (RIPs), are plant proteins with N Glycosidase activity. Annona squamosa is traditionally used in medicine but RIP presence is less explored. The present study aimed to isolate and structurally analyse RIP- like protein from the seeds of Annona squamosa (ARIP) and investigate its in vitro pharmacological bioactivities.ARIP was isolated with step wise purification procedures – Cation exchange and Gel filtration column chromatography, Ultrafiltration and SDS PAGE were adopted to purify and separate ARIP. The protein bands were eluted and tested for the presence of ARIP by N Glycosidase activity. The trypsin digested peptides were characterized by LC-MS/ESI-MS and searched for match using MASCOT search engine. Pairwise sequence alignment of peptide sequences were attempted to predict the similarity with known RIP in Magnoliophyta. In vitro antimicrobial, antimutagenic and cytotoxic activity of the the purified fraction of the ARIP was also studied.Two proteins with band sizes 21 kDa and 28 KDa confirmed to have ARIP like activity. The LC-MS/ESI-MS mass spectra between 9 and 17 min yielded 19 peaks for 21 kDa protein and 18 peaks for 18 peaks for 28 kDa protein. Although database search revealed very low similarity with known RIPs, the closest similarity observed was with Populus trichocarpa which is known to have Type 2 Ricin B chain RIP. On sequence alignment with RIPs, the peptides revealed similarity with RIPs of type II category. Phamacological investigation also suggested strong in vitro antimicrobial, antimutagenic and cytotoxic activity, proving to be a promising candidate as drugs in future.Our study have observed that ARIP was novel protein with unique sequence but lectin binding properties. They have shown remarkable antimicrobial, antimutagenic and cytotoxic activity. Crystallization of the protein followed by X-ray characterization could help to study the structure of the protein, active sites which would in turn give clear focus on the usage of this RIP in treatment of various diseases.