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Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis

Nature Chemical Biology volume 9, pages 367–373 (2013)Cite this article

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Abstract

Asparagine-linked glycosylation is a complex protein modification conserved among all three domains of life. Herein we report the in vitro analysis of N-linked glycosylation from the methanogenic archaeon Methanococcus voltae. Using a suite of synthetic and semisynthetic substrates, we show that AglK initiates N-linked glycosylation in M. voltae through the formation of α-linked dolichyl monophosphate N-acetylglucosamine, which contrasts with the polyprenyl diphosphate intermediates that feature in both eukaryotes and bacteria. Notably, AglK has high sequence homology to dolichyl phosphate β-glucosyltransferases, including Alg5 in eukaryotes, suggesting a common evolutionary origin. The combined action of the first two enzymes, AglK and AglC, afforded an α-linked dolichyl monophosphate glycan that serves as a competent substrate for the archaeal oligosaccharyl transferase AglB. These studies provide what is to our knowledge the first biochemical evidence revealing that, despite the apparent similarity of the overall pathways, there are actually two general strategies to achieve N-linked glycoproteins across the domains of life.

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Figure 1: N-linked glycosylation across the three domains of life.
Figure 2: N-linked glycosylation in M. voltae.
Figure 3: AglK is a Dol-P-GlcNAc synthase.
Figure 4: AglC is a UDP-Glc-2,3-diNAcA glycosyltransferase.
Figure 5: The OTase AglB uses Dol-P disaccharide to generate an N-linked glycopeptide.

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Acknowledgements

This work was supported by a grant from the US National Institutes of Health (GM039334 to B.I.) The authors are grateful to M. Jaffee for help with initial expression studies, J. Simpson (Department of Chemistry Instrumentation Facility, Massachusetts Institute of Technology) for assistance with NMR acquisition, W. Whitman (University of Georgia) for the gift of both M. voltae genomic DNA and M. maripaludis cell pellets and E. Swiezewska (Polish Academy of Sciences) for providing a sample of racemic short dolichols for preliminary studies.

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  1. Angelyn Larkin

    Present address: Present address: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.,

  2. Angelyn Larkin and Michelle M Chang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Angelyn Larkin, Michelle M Chang, Garrett E Whitworth & Barbara Imperiali

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Michelle M Chang, Garrett E Whitworth & Barbara Imperiali

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Contributions

B.I., A.L., M.M.C. and G.E.W. designed the experiments. A.L., M.M.C. and G.E.W. performed the experiments. The manuscript was written through the contributions of all of the authors.

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Correspondence to Barbara Imperiali.

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Larkin, A., Chang, M., Whitworth, G. et al. Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis. Nat Chem Biol 9, 367–373 (2013). https://doi.org/10.1038/nchembio.1249

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