title: New insights into the glycosylation of the surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a.
abstract: The surface of Geobacillus stearothermophilus NRS 2004/3a cells is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. To this S-layer glycoprotein, elongated glycan chains are attached that are composed of [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-L-Rhap-(1-->] repeating units, with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain and a core saccharide as linker to the S-layer protein. On sodium dodecyl sulfate-polyacrylamide gels, four bands appear, of which three represent glycosylated S-layer proteins. In the present study, nanoelectrospray ionization time-of-flight mass spectrometry (MS) and infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry were adapted for analysis of this high-molecular-mass and water-insoluble S-layer glycoprotein to refine insights into its glycosylation pattern. This is a prerequisite for artificial fine-tuning of S-layer glycans for nanobiotechnological applications. Optimized MS techniques allowed (i) determination of the average masses of three glycoprotein species to be 101.66 kDa, 108.68 kDa, and 115.73 kDa, (ii) assignment of nanoheterogeneity to the S-layer glycans, with the most prevalent variation between 12 and 18 trisaccharide repeating units, and the possibility of extension of the already-known -->3)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1--> core by one additional rhamnose residue, and (iii) identification of a third glycosylation site on the S-layer protein, at position threonine-590, in addition to the known sites threonine-620 and serine-794. The current interpretation of the S-layer glycoprotein banding pattern is that in the 101.66-kDa glycoprotein species only one glycosylation site is occupied, in the 108.68-kDa glycoprotein species two glycosylation sites are occupied, and in the 115.73-kDa glycoprotein species three glycosylation sites are occupied, while the 94.46-kDa band represents nonglycosylated S-layer protein.
authors.affiliation: Zentrum für NanoBiotechnologie, Universität für Bodenkultur Wien, A-1180 Vienna, Austria.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Steiner
authors.forename: Gottfried
authors.initials: G
authors.lastname: Pohlentz
authors.forename: Klaus
authors.initials: K
authors.lastname: Dreisewerd
authors.forename: Stefan
authors.initials: S
authors.lastname: Berkenkamp
authors.forename: Paul
authors.initials: P
authors.lastname: Messner
authors.forename: Jasna
authors.initials: J
authors.lastname: Peter-Katalinić
authors.forename: Christina
authors.initials: C
authors.lastname: Schäffer
mesh_terms: D001406:Bacillaceae / Q000201:enzymology*; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C055764:S-layer proteins