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J. Biol. Chem., Vol. 277, Issue 8, 6230-6239, February 22, 2002

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The Surface Layer (S-layer) Glycoprotein of Geobacillus stearothermophilus NRS 2004/3a
ANALYSIS OF ITS GLYCOSYLATION^*

Christina Schäffer^§, Thomas Wugeditsch, Hanspeter Kählig^¶, Andrea Scheberl, Sonja Zayni, and Paul Messner

From the  Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, A-1180 Wien, Austria and ^¶ the Institut für Organische Chemie, Universität Wien, A-1090 Wien, Austria

Geobacillus stearothermophilus NRS 2004/3a possesses an oblique surface layer (S-layer) composed of glycoprotein subunits as the outermost component of its cell wall. In addition to the elucidation of the complete S-layer glycan primary structure and the determination of the glycosylation sites, the structural gene sgsE encoding the S-layer protein was isolated by polymerase chain reaction-based techniques. The open reading frame codes for a protein of 903 amino acids, including a leader sequence of 30 amino acids. The mature S-layer protein has a calculated molecular mass of 93,684 Da and an isoelectric point of 6.1. Glycosylation of SgsE was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [2)--L-Rhap-(13)--L-Rhap-(12)--L-Rhap-(1]_n = 13-18, with a 2-O-methyl group capping the terminal trisaccharide repeating unit at the non-reducing end of the glycan chains. The glycan chains are bound via the disaccharide core 3)--L-Rhap-(13)--L-Rhap-(1 and the linkage glycose -D-Galp in O-glycosidic linkages to the S-layer protein SgsE at positions threonine 620 and serine 794. This S-layer glycoprotein contains novel linkage regions and is the first one among eubacteria whose glycosylation sites have been characterized.

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