Surface-exposed Glycoproteins of Hyperthermophilic <i>Sulfolobus solfataricus</i> P2 Show a Common <i>N-</i>Glycosylation Profile

Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the <i>N-</i>glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of <i>S. solfataricus</i>, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex₄(GlcNAc)₂ plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of <i>S. acidocaldarius</i>, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of <i>S. solfataricus</i> are <i>N-</i>glycosylated by the same sulfated oligosaccharide and we identified 56 <i>N-</i>glycosylation sites in this subproteome

Surface-exposed Glycoproteins of Hyperthermophilic <i>Sulfolobus solfataricus</i> P2 Show a Common <i>N-</i>Glycosylation Profile

Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the <i>N-</i>glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of <i>S. solfataricus</i>, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex₄(GlcNAc)₂ plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of <i>S. acidocaldarius</i>, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of <i>S. solfataricus</i> are <i>N-</i>glycosylated by the same sulfated oligosaccharide and we identified 56 <i>N-</i>glycosylation sites in this subproteome

Surface-exposed Glycoproteins of Hyperthermophilic <i>Sulfolobus solfataricus</i> P2 Show a Common <i>N-</i>Glycosylation Profile

Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the <i>N-</i>glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of <i>S. solfataricus</i>, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex₄(GlcNAc)₂ plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of <i>S. acidocaldarius</i>, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of <i>S. solfataricus</i> are <i>N-</i>glycosylated by the same sulfated oligosaccharide and we identified 56 <i>N-</i>glycosylation sites in this subproteome

Surface-exposed Glycoproteins of Hyperthermophilic <i>Sulfolobus solfataricus</i> P2 Show a Common <i>N-</i>Glycosylation Profile

Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the <i>N-</i>glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of <i>S. solfataricus</i>, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex₄(GlcNAc)₂ plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of <i>S. acidocaldarius</i>, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of <i>S. solfataricus</i> are <i>N-</i>glycosylated by the same sulfated oligosaccharide and we identified 56 <i>N-</i>glycosylation sites in this subproteome

Surface-exposed Glycoproteins of Hyperthermophilic <i>Sulfolobus solfataricus</i> P2 Show a Common <i>N-</i>Glycosylation Profile

Cell surface proteins of hyperthermophilic Archaea actively participate in intercellular communication, cellular uptake, and energy conversion to sustain survival strategies in extreme habitats. Surface (S)-layer glycoproteins, the major component of the S-layers in many archaeal species and the best-characterized prokaryotic glycoproteins, were shown to have a large structural diversity in their glycan compositions. In spite of this, knowledge on glycosylation of proteins other than S-layer proteins in Archaea is quite limited. Here, the <i>N-</i>glycosylation pattern of cell-surface-exposed proteins of Sulfolobus solfataricus P2 were analyzed by lectin affinity purification, HPAEC-PAD, and multiple mass spectrometry-based techniques. Detailed analysis of SSO1273, one of the most abundant ABC transporters present in the cell surface fraction of <i>S. solfataricus</i>, revealed a novel glycan structure composed of a branched sulfated heptasaccharide, Hex₄(GlcNAc)₂ plus sulfoquinovose where Hex is d-mannose and d-glucose. Having one monosaccharide unit more than the glycan of the S-layer glycoprotein of <i>S. acidocaldarius</i>, this is the most complex archaeal glycan structure known today. SSO1273 protein is heavily glycosylated and all 20 theoretical N-X-S/T (where X is any amino acid except proline) consensus sequence sites were confirmed. Remarkably, we show that several other proteins in the surface fraction of <i>S. solfataricus</i> are <i>N-</i>glycosylated by the same sulfated oligosaccharide and we identified 56 <i>N-</i>glycosylation sites in this subproteome

Progress in Detection and Structural Characterization of Glycosphingolipids in Crude Lipid Extracts by Enzymatic Phospholipid Disintegration Combined with Thin-Layer Chromatography Immunodetection and IR-MALDI Mass Spectrometry

In order to proceed in detection and structural analysis of glycosphingolipids (GSLs) in crude lipid extracts, which still remains a challenge in glycosphingolipidomics, we developed a strategy to structurally characterize neutral GSLs in total lipid extracts prepared from in vitro propagated human monocytic THP-1 cells, which were used as a model cell line. The procedure divides into (1) extraction of total lipids from cellular material, (2) enzymatical disintegration of phospholipids by treatment of the crude lipid extract with phospholipase C, (3) subsequent multiple thin-layer chromatography (TLC) overlay detection of individual GSLs with a mixture of various anti-GSL antibodies, and (4) structural analysis of immunostained GSLs directly on the TLC plate using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF MS) in combination with collision-induced dissociation (CID). Whereas GSLs were mostly undetectable in untreated crude lipid extracts, pretreatment with phospholipase C resulted in clear-cut mass spectra. MS¹ and MS² analysis gave similar results when compared to those obtained with a highly purified neutral GSL preparation of THP-1 cells, which served as a control. We could demonstrate in this study the feasibility of simultaneous multiple immunodetection of individual neutral GSLs in one and the same TLC run and their structural characterization in crude lipid extracts after phospholipase C treatment, thereby avoiding laborious and long-lasting sample purification. This powerful combinatorial technique allows for efficient structural characterization of GSLs in small tissue samples and takes a step forward in the emerging field of glycosphingolipidomics

Expression of glycosylated Pls increases biofilm formation of <i>S</i>. <i>aureus</i> Newman.

<p><b>(A)</b> Quantitative assay of biofilm formation. Strains were grown in TSB in microtiter plates. <i>S</i>. <i>epidermidis</i> RP62A and <i>S</i>. <i>carnosus</i> TM300 served as positive and negative controls, respectively. Data are shown as the mean of four independent experiments. Statistical significance is marked by asterisks. <b>(B)</b> Initial attachment to a plastic surface. Attached bacterial cells were analysed by phase-contrast microscopy, photographed and counted. Each assay was performed in triplicates. Data are shown as the mean of three independent experiments. <b>(C)</b> Biofilm formation on a glass surface. <i>S</i>. <i>epidermidis</i> RP62A and <i>S</i>. <i>carnosus</i> TM300 served as positive and negative controls, respectively. <b>(D)</b> Proteinase K (0.1 mg/ml) or <b>(E)</b> NaIO₄ (40 mM) treatment (+) of preformed biofilms in microtiter plates and untreated controls (-). <i>S</i>. <i>epidermidis</i> RP62A served as a control. Data are shown as the mean of three independent experiments. Statistical significance is marked by asterisks. <b>(F)</b> Biofilm formation in the presence (+) or absence (-) of DNase I (0.1 mg/ml). <i>S</i>. <i>epidermidis</i> RP62A served as a control. Data are shown as the mean of three independent experiments. Statistical significance is marked by asterisks.</p

The SD-repeat region of Pls is glycosylated.

<p><b>(A)</b> Schematic map of Pls from strain 1061 and its truncated derivatives encoded by the indicated plasmids. SD; SD-repeat region, G5; G5 domains, LPXTG; C-terminal cell-wall anchor motif. <b>(B)</b> SDS-PAGE (7.5% separation gel) (upper panel) and corresponding PAS staining (lower panel) of surface proteins from the strains <i>S</i>. <i>aureus</i> 1061 (lane 2), 1061<i>pls</i> (lane 3), 1061<i>pls</i> (pPLS4) (lane 4), 1061<i>pls</i> (pPLS6) (lane 5), 1061<i>pls</i> (pPLSsub1) (lane 6), 1061<i>pls</i> (pPLSsub2) (lane 7), 1061<i>pls</i> (pPLSsub3) (lane 8). The sizes of the marker proteins (lane 1; kDa) are indicated on the left.</p

Functional characterization of Pls glycosylation.

<p><b>(A)</b> Pls reduces the adherence of <i>S</i>. <i>aureus</i> to Fg, Fn, and endothelial cells independently of its glycosylation status. The wells of microtiter plates were coated with Fg, Fn or endothelial cells, blocked, and incubated with the bacteria. After washing, binding was assessed as arbitrary units in ELISA adherence assays. Results are shown as the mean of three independent experiments. Statistical significance is marked by asterisks. <b>(B)</b> Pls reduces the internalization of <i>S</i>. <i>aureus</i> by endothelial EA.hy 926 cells independently of its glycosylation status. The internalization of FITC-labeled <i>S</i>. <i>aureus</i> strains by adherent EA.hy 926 cells was assessed by flow cytometry and computed in relation to <i>S</i>. <i>aureus</i> strain Cowan 1, which was set to 100% internalization. Data are shown as the mean of three independent experiments. Statistical significance is marked by asterisks. <b>(C, D)</b> Pls reduces the phagocytosis of <i>S</i>. <i>aureus</i> by PMNs independently of its glycosylation status. The phagocytosis of FITC-labelled <i>S</i>. <i>aureus</i> strains by PMNs was assessed by flow cytometry and computed in relation to <i>S</i>. <i>aureus</i> SA113 (pCU1), which was set to 100%. Data are shown as the mean of three independent experiments. Statistical significance is marked by asterisks.</p

Determination of the modifying carbohydrate moieties.

<p><b>(A)</b> NanoESI Q-Tof mass spectrum of a hydrolysate obtained from a Pls preparation derived from <i>S</i>. <i>aureus</i> strain 1061 by incubation with 12.5% (v/v) acetic acid for 2 h at 95°C. For reasons of clarity only a few signals originating from (glyco)peptides derived from the SD repeats are labeled. A summary of all detected corresponding species is given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006110#ppat.1006110.s001" target="_blank">S1 Table</a>. <b>(B)</b> NanoESI Q-Tof fragment ion spectrum obtained from a CID experiment on the singly charged precursor glycopeptide ions with <i>m/z</i> 626.25. The insert shows the corresponding fragmentation scheme.</p