Posttranslational generation of C-alpha-formylglycin in eukaryotic sulfatases: development of the biochemical approach for the characterisation and purification of the modifying enzymee

Charakterisierung und Anreicherung der Enzyme, das Formylglycinreste in Sulfatasen bildet

by Ljudmila Borissenko
Doctoral thesis
Date of Examination:2003-01-30
Date of issue:2003-07-15
Advisor:Prof. Dr. Dr. h.c. Kurt von Figura
Referee:Prof. Dr. Hans-Ulrich Mösch
Referee:Prof. Dr. Dietrich Gradmann
Referee:Prof. Dr. Bernd Herrmann
Persistent Address: http://dx.doi.org/10.53846/goediss-1394

 

 

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Abstract

English

Sulfatases carry at their active site a C Ñ-formylglycine (FGly) residue that is essential for enzyme activity. The formyl group of this FGly is directly involved in sulfate ester cleavage. A defect in FGly formation in human is the cause of a recessively inherited disease called Multiple Sulfatase Deficiency, characterised by the synthesis of the catalytically inactive sulfatase polypeptides and by the accumulation of their unprocessed substrates in the lysosomes. FGly is generated by oxidation of a concerved cystein (pro- and eukaryotes) or serine residue (prokaryotes) comprised in the sequience motif C/S-x-P-x-R. In eukaryotes the modification is catalysed by lumenal components of the endoplasmic reticulum during or after protein translocation and prior to folding of sulfatases. Under in vitro conditions, using an enriched and soluble protein fraction, FGly formation could be observed under strictly posttranslational conditions and independent of a signal peptide. The modification reaction was characterised kinetically and with respect to cofactor requirement, albeit the acceptor of the reducing equivalents during cystein oxidation remains unknown.So far the enzymatic machinery involved in FGly modification could not be identified. We tried to purify it from the reticuloplasm of bovine pancreas microsomes using a number of chromatographic techniques with the following identification of proteins. We established several chromatographic protocols for separation of luminal proteins on different columns (ion exchanger, gel filtration, hydrophobic interaction, lectin chromatography, affinity chromatography etc.). FGly generating enzyme was characterised kinetically and biochemically. By combination of chromatographic protocols we purified the FGly generating activity and identified the bands visualised after separation of proteins by SDS PAGE. Several possible candidates on the role of FGly generating enzyme are identified.
Keywords: sulfatase; posttranslational modification; formylglycin
Schlagwörter: Sulfatasen; Enzyme Modification; Formylglycin
 
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