Šárka Cepáková, Sandra Stachová

Leader: Mgr. Markéta Foldynová

28. 07. 2011

Protein glycosylations in cyanobacterium Synechocystis

Schola ludus

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

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

Methods IIsolation of glycoproteins from cell extract: Glycoprotein Isolation Kit, ConA

Fig.1: Purification column

Separation: SDS-PAGE electrophoresis

Methods II

Gel staining:

• Coomassie Blue Staining• Pierce Glycoprotein Staining KitPhotographs taken by sensitive CCD camera

Results

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)

kDa250

130

95

72

55

36

2817

10

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)

c) Comparison glucose cultivation X autotrophic cultivation

Soluble proteins

Fig.6: Autotrophic cultivation (SDS-PAGE, coomassie) Fig.7: Glucose cultivation (SDS-PAGE, coomassie)

d) Comparison glucose cultivation X autotrophic cultivation

Y. H. Kim et al. (2009) Proteomics

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.

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)