Identification of rapeseed oleosins, a family of emulsifying proteins, and optimization of their extraction from seeds and defatted meals using organic solvents

摘要

In oilseeds, lipids are stored in oil bodies, also called oleosomes. They are composed of a core of triacylglycerols surrounded by a monolayer of phospholipids in which different proteins are inserted. Eight proteins were previously identified in Arabidopsis thaliana oleosomes, the most abundant ones belonging to the oleosin family. These seed oleosins, named S1, S2, S3, S4 and S5,are amphiphatic proteins containing a long central hydrophobic domain highly conserved and two terminal hydrophilic domains,and therefore display emulsifying properties. In addition, oleosins are expected to be key factors for the stability of oleosomes and their study may contribute, in particular for rapeseed, to improve oil extraction using mild technologies while preserving proteins quality. Here, we describe the oleosin content of Brassica napus oleosomes. These organelles have been purified by 6 flotation centrifugation steps in order to remove proteins non-specifically associated. Protein identification was carried out after SDS-PAGE separation, nano-LC-MS/MS analysis of trypsin peptides and comparison with A. thaliana protein database and B.napus EST databank. Major proteins were characterized as 15 different oleosins, representing up to 84% of oleosome proteins,and highly homologous to A. thaliana oleosins. Specific antibodies raised against the N-terminal domain of A. thaliana oleosins, cross-reacted with rapeseed oleosins.Immunoblot analysis corroborated proteomic results, showing the presence of B. napus oleosins orthologous to A. thaliana S1, S2,S3, S4 and S5. These antibodies were used to set up a semi-quantitative assay of rapeseed oleosins by dot-blot. This assay allowed the study of oleosin extraction from seeds using organic solvents. One mixture of chloroform/methanol gave optimal extraction of all the oleosins. This protocol was also used on seed defatted meals and allowed the specific extraction of oleosins, identified by immunoblot and proteomic analyses. Surprisingly, oleosins extracted from seed defatted meals displayed the same apparent molecular mass than in seeds, showing that they are not degraded during industrial process. Together with oleosins, napins and cruciferins were also identified in this extract. This is the first report showing the presence of oleosins in rapeseed defatted meals. A method for their specific extraction is described. The functional properties of this extract need to be further investigated to determine potential biotechnological applications.