Definition of the bacterial N-glycosylation site consensus sequence

Kowarik, Michael; Young, N. Martin; Numao, Shin; Schulz, Benjamin L.; Hug, Isabelle; Callewaert, Nico; Mills, Dominic C.; Watson, David; Hernández, María Helena Ramírez; Kelly, John F.; Wacker, Michael; Aebi, Markus

doi:10.1038/sj.emboj.7601087

Cited by 324 publications

(347 citation statements)

References 44 publications

(78 reference statements)

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“…Inspection of the S-layer protein sequence revealed the presence of one putative Nglycosylation site at position N 879 (DVNQT; the glycosylated asparagine residue is underlined), conforming with the amino acid sequence requirement of the oligosaccharyl:protein transferase PglB of C. jejuni, which is the key enzyme for protein Nglycosylation. [22,28,35] Thus, in a first glycosylation approach, the potential of PglB to glycosylate native SgsE with its endogenous heptasaccharide Glc(GalNAc) 5 Bac substrate in E. coli was investigated by expression of ssPelB-A_SgsE from plasmid pET22b-A_SgsE in E. coli BL21 Star (DE3) harboring pACYCpgl. [21] However, no glycosylated protein was visible on a Western immunoblot, implying that the putative N-glycosylation site of SgsE at position N 879 was not recognized by PglB (not shown).…”

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

confidence: 99%

“…Plasmids pACYC184pgl, [21] pJHCV32, [24] pEC(AcrA-per), [28] and pMAF10 [22] have been described previously. All strains and plasmids are listed in Table 1.…”

Section: Europe Pmc Funders Author Manuscriptsmentioning

confidence: 99%

“…As a control, the soluble periplasmic C. jejuni protein AcrA expressed from pEC(AcrA_per) [28] was used in combination with pACYCpgl. pEC(AcrA_per) was kindly provided by Prof. Markus Aebi; pACYCpgl [21] was obtained form Prof. Brendan Wren.…”

Section: Engineering Of N-glycosylation Sites On Sgsementioning

confidence: 99%

“…[26] The heptasaccharide is transferred by the PglB oligosaccharyl:protein transferase to asparagine residues present in the target protein consensus sequence D-X-N-Z-S/T (where X and Z are any amino acid except proline). [27,28] Using E. coli as a host, it was shown that PglB has relaxed substrate specificity, transferring several O-antigen polysaccharides, carrying a 2-acetamido modification at the reducing-end sugar of the glycan, to distinct protein glycosylation sites. [22] Among the glycans tested in the course of this previous study was also the E. coli O7 antigen with the repeating unit…”

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

confidence: 99%

“…Plasmids pACYC184pgl, [21] pJHCV32, [24] pEC(AcrA-per), [28] and pMAF10 [22] have been described previously. All strains and plasmids are listed in Table 1.…”

Section: Europe Pmc Funders Author Manuscriptsmentioning

confidence: 99%

Section: Engineering Of N-glycosylation Sites On Sgsementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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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“…The structural conservation between Stt3 and prokaryotic enzymes is remarkable, considering their ~20% sequence identity. The bacterial enzymes have an extended acceptor sequon of DXNXS/T 30 . The −2 position D is stabilized by R331 in the PglB crystal structure 13 .…”

Section: Introductionmentioning

confidence: 99%

The atomic structure of a eukaryotic oligosaccharyltransferase complex

Bai

Wang

Zhao

et al. 2018

Nature

102

131

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SUMMARY N-glycosylation is a ubiquitous modification of eukaryotic secretory and membrane-bound proteins; about 90% of glycoproteins are N-glycosylated. The reaction is catalyzed by an eight-protein oligosaccharyltransferase complex, OST, embedded in the ER membrane. Our understanding of eukaryotic protein N-glycosylation has been limited due to the lack of high-resolution structures. Here we report a 3.5-Å resolution cryo-EM structure of the Saccharomyces cerevisiae OST, revealing the structures of Ost1–5, Stt3, Wbp1, and Swp1. We found that seven phospholipids mediate many of the inter-subunit interactions, and an Stt3 N-glycan mediates interaction with Wbp1 and Swp1 in the lumen. Ost3 was found to mediate the OST-Sec61 translocon interface, funneling the acceptor peptide towards the OST catalytic site as the nascent peptide emerges from the translocon. The structure provides novel insights into co-translational protein N-glycosylation and may facilitate the development of small-molecule inhibitors targeting this process.

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Glycosylation and Disease

Dwek

Markiv

2018

Encyclopedia of Life Sciences

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Glycosylation is the process of attachment of carbohydrates (glycans) to proteins and lipids to form the glycoproteins and glycolipids found in eukaryotic organisms. Glycosylation reactions are amongst the most common posttranslational modifications that have been reported on proteins, and they function to alter protein size, stability, charge and antigenicity. Glycosylation plays an important role in cancer, inherited diseases, pathogen–host interactions and immune recognition. Key Concepts The analysis of glycosylation is more complex than DNA and RNA or protein analysis as glycosylation is a non‐template‐driven event. N‐ linked glycosylation commences in the endoplasmic reticulum and is completed in the Golgi apparatus. O‐ linked glycosylation commences and is completed in the Golgi apparatus. N‐ linked glycosylation occurs on asparagine residues in the consensus sequon Asn‐X‐Ser/Thr. O‐ linked glycosylation occurs on serine and threonine residues. Many enzymes are involved in both N‐ and O‐ linked glycosylation. Glycans play a role in sperm–egg interactions, the implantation process and embryonic development. Congenital disorders in glycosylation result from autosomally dominant mutations in enzymes involved in glycosylation pathways. Glycosylation of proteins influences their half‐life in the serum. N‐ and O‐ linked glycosylation have been reported to be altered in cancer and implicated in all of the steps in the metastatic cascade.

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Definition of the bacterial N-glycosylation site consensus sequence

Cited by 324 publications

References 44 publications

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

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

The atomic structure of a eukaryotic oligosaccharyltransferase complex

Glycosylation and Disease

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