New Insights into the Glycosylation of the Surface Layer Protein SgsE from Geobacillus stearothermophilus NRS 2004/3a

Abstract

Protein glycosylation is an emerging field in bacteriology and, particularly, the envisaged medical and biotechnological applications make prokaryotic glycoproteins an interesting system for further analysis and exploitation (for details, see references 19, 23, 30, 33, 36, 40, 41, and 43). This endeavor requires a thorough understanding of the protein glycosylation processes that occur in the prokaryotic cell.The glycosylated surface layer (S-layer) protein SgsE from Geobacillus stearothermophilus NRS 2004/3a is a promising model for studies on prokaryotic glycosylation because of the homopolymeric nature of the glycan, which is composed only of rhamnose residues (35). In sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis, the mature S-layer glycoprotein is separated into four bands. Three of them represent broad bands in the molecular mass range of approximately 119 to 170 kDa which also give a positive periodic acid-Schiff (PAS) staining reaction, indicating the presence of covalently linked glycan chains (7,14,35). The 93-kDa band is nonglycosylated, and the estimated molecular mass concurs with the calculated mass derived from the amino acid sequence of the mature structural protein, SgsE, after cleavage of the 30-amino-acid signal peptide from the precursor protein (GenBank accession number AF328862) (35). Dependent on the cultivation conditions of the organism (batch versus continuous culture), variations exist in the degree of glycosylation of the individual protein bands, all of which possess identical N termini indicative of identical protein portions (20,35). Previous nuclear magnetic resonance (NMR) experiments have demonstrated that only one type of glycan chain is present on the individual glycoprotein species, however, with considerable length variations. The S-layer glycans consist of trisaccharide repeats with the structure [32)-␣-L-Rhap-(133)-␤-L-Rhap-(132)-␣-L-Rhap-(13] n and of a short core saccharide consisting of ␣-1,3-linked L-Rhap residues, attached to carbon 3 of a ␤-D-galactose residue that serves as the linkage sugar to the S-layer polypeptide backbone (35). So far, two glycosylation sites for O-linked glycans have been determined after a papain degradation experiment of SgsE glycoprotein, namely, amino acids threonine-620 and serine-794 (with the numbers referring to the positions on the precursor protein) (35). These data, however, cannot satisfactorily explain the existence of three broad S-layer glycoprotein bands on SDS-PAGE gels.To better understand the glycosylation pattern of the S-layer glycoprotein of G. stearothermophilus NRS 2004/3a, we have investigated the intact, glycosylated S-layer protein SgsE and selected glycopeptides derived thereof using different mass spectrometric approaches. These data will allow interpretation of S-layer protein glycosylation in a more general way, because similar SDS-PAGE patterns of S-layer glycoproteins have also been observed with other organisms (24).

Recombinant Glycans on an S‐Layer Self‐Assembly Protein: A New Dimension for Nanopatterned Biomaterials

“…For the introduction of a distinctive protein N-glycosylation consensus sequence from the C. jejuni AcrA N-glycoprotein replacing the naturally O-glycosylation sites of the S-layer protein, [19,20] a modified sgsE gene containing an artificial KpnI site at position nt 1830 (corresponding to amino acid position 611), coding for a protein with a PelB signal peptide and lacking the amino acid sequence between L 610 and E 804 was constructed. N-and Cterminal parts of sgsE were amplified separately by PCR using the primer pairs A_sgsE_for(NcoI)/sgsEteil_rev(KpnI) and sgsE-teil_for(KpnI)/sgsE_rev(XhoI), respectively.…”

“…[18,19] SgsE was chosen for proof of concept because it is naturally O-glycosylated with long-chain poly-L-rhamnans linked via a β-D-galactose residue to the amino acids threonine 590 , threonine 620 , and serine 794 of the S-layer protein. [19,20] The protein-inherent glycosylation sites are predicted to form a loop structure that is spatially accessible to the glycosylation machinery of the bacterium. Thus, these sites offer ideal targets for engineering of specific glycosylation sequences that would be recognized by heterologous oligosaccharyl:protein transferases, as required for S-layer neoglycoprotein design.…”

Functional Characterization of the Initiation Enzyme of S-Layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

“…The core unit consists of two or three L-rhamnose residues [32)-␣-L-Rhap-(133)-␣-L-Rhap-(133)-␣-L-Rhap- (13], which are O-glycosidically linked via a ␤-D-galactose residue to threonine 590 , threonine 620 , and serine 794 of the mature S-layer protein SgsE (39,43). The glycan chain has, on average, 15 identical L-rhamnose trisaccharide repeating units with the structure 3[2)-␣-LRhap-(133)-␤-L-Rhap-(132)-␣-L-Rhap-(13.…”

Molecular Basis of S-layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus

“…Considering the results from TLC and ESI-QTOF MS analysis of WsaC products (Fig. S7), it is very likely that WsaC is also capable of transferring one more ␣1,3-linked rhamnose to product V, yielding compound VI, which implies that this enzyme is responsible for the core variability present in the S-layer glycan (17).…”

“…O-glycosidically linked to threonine 590, threonine 620, and serine 794 of the S-layer protein SgsE (16,17).…”