N-Linked Glycosylation of Folded Proteins by the Bacterial Oligosaccharyltransferase

Kowarik, Michael; Numao, Shin; Feldman, Mario F.; Schulz, Benjamin L.; Callewaert, Nico; Kiermaier, Eva; Catrein, Ina; Aebi, Markus

doi:10.1126/science.1134351

Cited by 213 publications

(238 citation statements)

References 21 publications

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“…Fitting with the characteristics of this peptide-binding groove, Ost6p bound peptides rich in hydrophobic amino acids and with acidic residues. Consistent with the peptides described here (Supporting Information Table 1 and above), the peptide previously shown to bind to the peptide-binding groove of Ost6L (Cwp1p [21][22][23][24][25][26][27][28][29][30][31] ; DSEEFGLVSIR) also shares these characteristics. 23 We did not detect significant binding of peptides to wild type Ost3L.…”

Section: Discussionsupporting

confidence: 80%

“…23 The transient noncovalent binding of hydrophobic stretches of polypeptide by Ost3/ 6p that we observe here would also efficiently slow protein folding of nascent polypeptide in vivo, as these hydrophobic stretches will tend to be positioned internal to folded domains. N-glycosylation by OTase requires unfolded polypeptide substrate, 28,29 and so inhibition of substrate protein folding by Ost3/6p binding sequences internal to mature protein structures is an efficient strategy to enable optimal presentation of protein substrate to the active site of OTase.…”

Section: Discussionmentioning

confidence: 99%

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Polypeptide binding specificities of Saccharomyces cerevisiae oligosaccharyltransferase accessory proteins Ost3p and Ost6p

et al. 2011

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Asparagine-linked glycosylation is a common and vital co-and post-translocational modification of diverse secretory and membrane proteins in eukaryotes that is catalyzed by the multiprotein complex oligosaccharyltransferase (OTase). Two isoforms of OTase are present in Saccharomyces cerevisiae, defined by the presence of either of the homologous proteins Ost3p or Ost6p, which possess different protein substrate specificities at the level of individual glycosylation sites. Here we present in vitro characterization of the polypeptide binding activity of these two subunits of the yeast enzyme, and show that the peptide-binding grooves in these proteins can transiently bind stretches of polypeptide with amino acid characteristics complementary to the characteristics of the grooves. We show that Ost6p, which has a peptide-binding groove with a strongly hydrophobic base lined by neutral and basic residues, binds peptides enriched in hydrophobic and acidic amino acids. Further, by introducing basic residues in place of the wild type neutral residues lining the peptide-binding groove of Ost3p, we engineer binding of a hydrophobic and acidic peptide. Our data supports a model of Ost3/6p function in which they transiently bind stretches of nascent polypeptide substrate to inhibit protein folding, thereby increasing glycosylation efficiency at nearby asparagine residues.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Polypeptide binding specificities of Saccharomyces cerevisiae oligosaccharyltransferase accessory proteins Ost3p and Ost6p

et al. 2011

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“…[21] As a positive control, to confirm that the glycosylation machinery of C. jejuni functions under the chosen conditions, the plasmid pEC(AcrA-per), expressing periplasmic soluble AcrA of C. jejuni with the PelB signal peptide and a Cterminal hexahistidine tag, was transferred to electrocompetent E. coli BL21 Star (DE3) cells containing pACYCpgl. [29] Analysis of the expression product by Western immunoblotting, using a heptasaccharide specific antibody (anti-pgl antibody), confirmed glycosylation of AcrA ( Figure 3, lane 8).…”

Section: Design Of An Sgse Neoglycoprotein Carrying a C Jejuni Heptamentioning

confidence: 85%

“…In another neoglycoprotein production experiment, ssPelB and ssTorA were successfully used for the transfer of the N-glycosylation target protein AcrA of C. jejuni to the periplasm of E. coli, demonstrating that bacterial N-glycosylation can occur independently of the protein translocation machinery. [21,29] Concerning periplasmic targeting of S-layer proteins in general, MBP has been reported to export the S-layer protein SbsA of G. stearothermophilus PV/72 to the periplasm of E. coli. [32] The different S-layer fusion proteins were expressed in E. coli BL21 Star (DE3) carrying the respective expression plasmids.…”

Section: Periplasmic Targeting Of Sgsementioning

confidence: 99%

“…In Gram-negative bacteria, the oligosaccharyl:protein transferase is proposed to act in the periplasm [21] in a mechanism, which is, in contrast to eukaryotes, independent of the protein translocation machinery, and is able to glycosylate folded and unfolded proteins. [29] The aim of this study is the transfer of the C. jejuni heptasaccharide and the E. coli O7 polysaccharide onto the SgsE S-layer protein of G. stearothermophilus NRS 2004/3a, and the successful expression of the S-layer neoglycoproteins in E. coli. For proof of concept we specifically deal with: i) the establishment of a periplasmic targeting system for the S-layer protein as a prerequisite for S-layer neoglycoprotein production in E. coli, [30] ii) engineering of an N-glycosylation site in the naturally O-glycosylated S-layer protein to serve as a potential target for the PglB oligosaccharyl:protein transferase to act on, iii) the unequivocal proof of the neoglycoprotein nature of the engineered products, and, most importantly, iv) the demonstration of the self-assembly capability of the produced nanostructured composites.…”

Section: Introductionmentioning

confidence: 99%

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

Steiner

Hanreich

Kainz

et al. 2008

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Crucial biological phenomena are mediated through carbohydrates that are displayed in a defined manner and interact with molecular scale precision. We lay the groundwork for the integration of recombinant carbohydrates into a "biomolecular construction kit" for the design of new biomaterials, by utilizing the self-assembly system of the crystalline cell surface (S)-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a. SgsE is a naturally O-glycosylated protein, with intrinsic properties that allow it to function as a nanopatterned matrix for the periodic display of glycans. By using a combined carbohydrate/protein engineering approach, two types of S-layer neoglycoproteins are produced in Escherichia coli. Based on the identification of a suitable periplasmic targeting system for the SgsE self-assembly protein as a cellular prerequisite for Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts protein glycosylation, and on engineering of one of the natural protein O-glycosylation sites into a target for N-glycosylation, the heptasaccharide from the AcrA protein of Campylobacter jejuni and the O7 polysaccharide of E. coli are co-or post-translationally transferred to the S-layer protein by the action of the oligosaccharyltransferase PglB. The degree of glycosylation of the Slayer neoglycoproteins after purification from the periplasmic fraction reaches completeness. Electron microscopy reveals that recombinant glycosylation is fully compatible with the S-layer protein self-assembly system. Tailor-made ("functional") nanopatterned, self-assembling neoglycoproteins may open up new strategies for influencing and controlling complex biological systems with potential applications in the areas of biomimetics, drug targeting, vaccine design, or diagnostics.

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Glycosyltransferases, Chemistry of

Weadge¹,

Palcic²

2008

Wiley Encyclopedia of Chemical Biology

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Glycosyltransferases (GTs) form glycosidic bonds by catalyzing the transfer of saccharides from a donor to a wide variety of acceptors. The donors used by GTs are sugars conjugated to nucleotides, phosphates, or lipid phosphates; whereas acceptors consist of carbohydrates, proteins, lipids, DNA, and numerous small molecules such as antibiotics, flavonols, steroids, and so on. Together, the products of these reactions comprise the most diverse and abundant class of natural compounds found in nature. Numerous GTs are needed to synthesize these compounds because the formation of each distinct glycosidic linkage requires a different enzyme. The abundance of these enzymes is emphasized by the fact that GTs constitute 1% of the genes in all genomes sequenced. They are ubiquitous in every kingdom of life and in all compartments of the cell. Currently, over 32,000 GT ORFs have been classified into 90 families on the basis of amino acid sequence similarity. The structures for 64 GTs have been determined to date and generally reveal conserved architectures of a GT‐A or GT‐B fold, although other folds have been observed and are predicted. These crystal structures, together with biochemical data, have provided insight into the catalytic mechanism. GTs generally exhibit strict regio/stereospecificity and transfer with either retention or inversion of configuration at the anomeric carbon of the donor sugar. The importance of characterizing the precise activity of these enzymes is exemplified by the many genetic disorders that have been linked to aberrant glycosylation.

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N-Linked Glycosylation of Folded Proteins by the Bacterial Oligosaccharyltransferase

Cited by 213 publications

References 21 publications

Polypeptide binding specificities of Saccharomyces cerevisiae oligosaccharyltransferase accessory proteins Ost3p and Ost6p

Polypeptide binding specificities of Saccharomyces cerevisiae oligosaccharyltransferase accessory proteins Ost3p and Ost6p

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

Glycosyltransferases, Chemistry of

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