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Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity

Nature Chemical Biology volume 10, pages 816–822 (2014)Cite this article

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Abstract

The Campylobacter jejuni protein glycosylation locus (pgl) encodes machinery for asparagine-linked (N-linked) glycosylation and serves as the archetype for bacterial N-linked glycosylation. This machinery has been functionally transferred into Escherichia coli, enabling convenient mechanistic dissection of the N-linked glycosylation process in this genetically tractable host. Here we sought to identify sequence determinants in the oligosaccharyltransferase PglB that restrict its specificity to only those glycan acceptor sites containing a negatively charged residue at the −2 position relative to asparagine. This involved creation of a genetic assay, glycosylation of secreted N-linked acceptor proteins (glycoSNAP), that facilitates high-throughput screening of glycophenotypes in E. coli. Using this assay, we isolated several C. jejuni PglB variants that could glycosylate an array of noncanonical acceptor sequences, including one in a eukaryotic N-glycoprotein. These results underscore the utility of glycoSNAP for shedding light on poorly understood aspects of N-linked glycosylation and for engineering designer N-linked glycosylation biocatalysts.

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Figure 1: Specific detection of glycosylated proteins using the glycoSNAP assay.
Figure 2: Structure- and sequence-guided mutagenesis of PglB.
Figure 3: PglB mutants exhibit relaxed substrate specificity.
Figure 4: Glycosylation of a native eukaryotic protein by PglB variants.

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Acknowledgements

We thank C. Guarino (Cornell University) for plasmid pSF-ClPglB and pBS-scFv13-R4DQNAT, J. Merritt (Glycobia, Inc.) for plasmid pMW07-pglΔB, B. Clemons (California Institute of Technology) for plasmid pET33b-ClStt3, M. Aebi (ETH Zürich) for providing antiserum used in this work, and R. Sherwood for his technical assistance acquiring the LC-MS/MS raw data files. This material is based on work supported by the US National Science Foundation grant CBET 1159581 (to M.P.D.) and National Institutes of Health (NIH) grant R44 GM088905-01 (to A.C.F. and M.P.D.) and NIH Shared Instrumentation Grant grant 1S10RR025449-01 (to S.Z.).

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Authors and Affiliations

  1. School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA

    Anne A Ollis & Matthew P DeLisa

  2. Proteomics and Mass Spectrometry Core Facility, Cornell University, Ithaca, New York, USA

    Sheng Zhang

  3. Glycobia Inc., Ithaca, New York, USA

    Adam C Fisher

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  1. Anne A Ollis
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Contributions

A.A.O. designed research, performed research, analyzed data and wrote the paper. S.Z. performed MS analysis, analyzed MS data and wrote the paper. A.C.F. conceptualized project, designed research and analyzed data. M.P.D. conceptualized project, designed research, analyzed data and wrote the paper.

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Correspondence to Matthew P DeLisa.

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Competing interests

A.C.F. is an employee of Glycobia, Inc. A.C.F. and M.P.D. have a financial interest in Glycobia, Inc.

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Ollis, A., Zhang, S., Fisher, A. et al. Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity. Nat Chem Biol 10, 816–822 (2014). https://doi.org/10.1038/nchembio.1609

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