1. Šárka Cepáková, Sandra Stachová Leader: Mgr. Markéta Foldynová 28. 07. 2011 Protein glycosylations in cyanobacterium Synechocystis Schola ludus

2. Introduction Subject of research - post-translational modification - glycosylation Glycosylation: attachment of glycans to proteins (N-linked glycans, O-linked glycans) Model organism – cyanobacterium Synechocystis sp. PCC 6803 Differences in cultivation: in the presence of glucose X autotrophic cultivation

3. Hypotheses and aims How does glucose affect cultivation of cyanobacteria Synechocystis? Effect on the biosynthesis of proteins and pigments Reason: alternative glycosylation of an enzyme or regulatory factor Expectation: different glycosylation pattern in some proteins after cultivation in presence of glucose

4. Methods I Isolation of glycoproteins from cell extract: Glycoprotein Isolation Kit, ConA Fig.1: Purification column Separation: SDS-PAGE electrophoresis

5. Methods II Gel staining: Coomassie Blue Staining Pierce Glycoprotein Staining Kit Photographs taken by sensitive CCD camera

6. Results

7. a) Comparison glucose cultivation X autotrophic cultivation Cell membrane fraction I Fig.2: Autotrophic cultivation (SDS-PAGE, coomassie) Fig.3: Glucose cultivation (SDS-PAGE, coomassie) kDa 250 130 95 72 55 36 28 17 10

8. b) Comparison glucose cultivation X autotrophic cultivation Cell membrane fraction II Fig.4: Autotrophic cultivation (SDS-PAGE, coomassie) Fig.5: Glucose cultivation (SDS-PAGE, coomassie)

9. c) Comparison glucose cultivation X autotrophic cultivation Soluble proteins Fig.6: Autotrophic cultivation (SDS-PAGE, coomassie) Fig.7: Glucose cultivation (SDS-PAGE, coomassie)

10. d) Comparison glucose cultivation X autotrophic cultivation Y. H. Kim et al. (2009) Proteomics

11. Conclusions Some of N-glycosylated proteins are unique in absence / presence of glucose and will be determined by MASS SPECTROMETRY. We detected a few proteins that are O- or N- glycosylated in Synechocystis.

12. Acknowledgements Thanks to Markéta Foldynová and MBÚ Třeboň Thanks to Schola ludus and European social fund and budget of the Czech republic (CZ.1.07/2.3.00/09.0203)