N-acetylmuramic acid as capping element of alpha-D-fucose-containing S-layer glycoprotein glycans from Geobacillus tepidamans GS5-97T.
Geobacillus tepidamans GS5-97(T) is a novel Gram-positive, moderately thermophilic bacterial species that is covered by a glycosylated surface layer (S-layer) protein. The isolated and purified S-layer glycoprotein SgtA was ultrastructurally and chemically investigated and showed several novel properties. By SDS-PAGE, SgtA was separated into four distinct bands in an apparent molecular mass range of 106-166 kDa. The three high molecular mass bands gave a positive periodic acid-Schiff staining reaction, whereas the 106-kDa band was nonglycosylated. Glycosylation of SgtA was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and electrospray ionization quadrupole time-of-fight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [-->2)-alpha-L-Rhap-(1-->3)-alpha-D-Fucp-(1-->](n=approximately 20), with D-fucopyranose having never been identified before as a constituent of S-layer glycans. The rhamnose residue at the nonreducing end of the terminal repeating unit of the glycan chain was di-substituted. For the first time, (R)-N-acetylmuramic acid, the key component of prokaryotic peptidoglycan, was found in an alpha-linkage to carbon 3 of the terminal rhamnose residue, serving as capping motif of an S-layer glycan. In addition, that rhamnose was substituted at position 2 with a beta-N-acetylglucosamine residue. The S-layer glycan chains were bound via the trisaccharide core -->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1--> to carbon 3 of beta-D-galactose, which was attached in O-glycosidic linkage to serine and threonine residues of SgtA of G. tepidamans GS5-97(T).
Kählig
Hanspeter
H
Department of Organic Chemistry, University of Vienna, Austria.
Kolarich
Daniel
D
Zayni
Sonja
S
Scheberl
Andrea
A
Kosma
Paul
P
Schäffer
Christina
C
Messner
Paul
P
D001406:Bacillaceae / Q000737:chemistry* / Q000648:ultrastructure; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005643:Fucose / Q000032:analysis* / Q000737:chemistry; D005690:Galactose / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009112:Muramic Acids / Q000032:analysis / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*; D011402:Pronase / Q000378:metabolism; D012210:Rhamnose / Q000737:chemistry; D017421:Sequence Analysis; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013912:Threonine / Q000737:chemistry
D006023:Glycoproteins; D008562:Membrane Glycoproteins; D009112:Muramic Acids; D011134:Polysaccharides; C055764:S-layer proteins; C031651:N-acetylmuramic acid; D005643:Fucose; D013912:Threonine; D012694:Serine; D011402:Pronase; D012210:Rhamnose; D005690:Galactose