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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: MoP-205
Session: MS Contribution to Immunology
Presentation date: Mon, Aug 28, 2006
Presentation time: 09:50 – 11:20

Characterization of Immunomodulatory Action of Holothurian Triterpene Glycosides (Cucumariosides) in Mice Spleen Cells by Proteome Analysis

Cornelia Koy1, Rene Bernitz1, Brian Arbogast2, Valery G. Voinov2, Pawel S. Dmitrenok3, Dmitri L. Aminin3, Brigitte Mueller-Hilke4, Max L. Deinzer2, Michael O. Glocker1

1 University of Rostock, Rostock, Germany
2 Oregon State University, Corvallis, United States
3 Pacific Institute of Bioorganic Chemistry, Vladivostock, Russian Federation
4 University of Rostock, Rostock, Germany

Correspondence address: Cornelia Koy, University of Rostock, Proteome Center Rostock, Schillingallee 69, Rostock, D-18057 Germany.

Web site: http://pzr.uni-rostock.de

Keywords: Glycosides; Immunology; Mass Spectrometry, QTOF; Proteomic.

Novel aspect: Determination of immune response mechanisms of cells/organisms by proteome analysis for the production of pharmacologically-effective formulations.


Triterpene glycosides such as Cucumarioside A2-2 from Cucumaria japonica and Frondoside A from Cucumaria frondosa are characteristic metabolites of sea cucumbers and possess a wide spectrum of biological activity, such as antifungal, antimytotic, and immunomodulatory activity caused mainly by their ability to modify cellular membranes.1,2

For the investigation of biological effects of cucumariosides on the protein level 3 primary spleen cell cultures from Balb/c mice were prepared and exposed to cucumariosides for three hours. All experiments were performed in triplicate. Afterwards, cell cultures were pooled, yielding in ca. 4x106 cells (controls), 7x106 cells (Cucumarioside A2-2), and 3x106 cells (Frondoside A), respectively. Spleen cell cultures consisted of ca. 3 % monocytes, granulocytes and NK cells. CD 8+ T cell amount was about 22%, the CD 4+ T cell amount ca. 40%, B cell amount was ca. 23 %. Subsequently, protein extracts from exposed cell cultures and from control samples were prepared and subjected to 2-DE. IEF was performed in a pH range of 3-10, with a total of 500 µg protein per gel. 2D Gels were stained by Colloidal Coomassie blue. Image analysis showed ca. 1000 spots per gel. From these 31 protein spots were two-fold and more up-regulated upon exposure of cells to Cucumarioside A2-2. 17 protein spots were down-regulated two-fold or more. These spots are currently being identified by mass spectrometry. As control, also intact spleen tissue was analyzed by global proteomics, resulting in yet another reference gel with ca. 600 spots upon Coomassie staining. To obtain an overview of the resulting protein pattern, 96 protein spots were randomly picked and subjected to mass spectrometric identification. Two sets of protein spots were analyzed using either MALDI ToF MS or nanoLC ESI Q ToF MS. Up to now, a total of 80 proteins has been identified by the combination of both methods that yielded complementary results and showed the presence of highly active immune cells. Among the identified proteins are coronin 1A and Rho GDI beta that are characteristic for immune cells, showing that immunomodulatory action of cucumariosides on spleen cells is in well observable by the selected analytical approach.

1. D. L. Aminin, I. G. Agafonova, E. V. Berdyshew, E. G. Isachenko, S. A. Avilov, V. A. Stonik, J. Med. Food 4, 127 (2001).
2. I. G. Agafonova, D. L. Aminin, S. A. Avilov, V. A. Stonik, J. Agric. Food Chem. 51, 6982 (2003).
3. P. Lorenz, M. Bantscheff, S. Ibrahim, H.-J. Thiesen, M. O. Glocker, Clin. Chem. Lab. Med. 41, 1622 (2003).