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Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase

  1. Gideon J. Daviesa,1
  1. aDepartment of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom;
  2. bSchool of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia;
  3. cOxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom;
  4. dLaboratory of Organic Chemistry, Wageningen University, 6703 HB, Wageningen, The Netherlands;
  5. eCentre of Excellence in Mass Spectrometry, University of York, Heslington, York YO10 5DD, United Kingdom; and
  6. fInstitute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, A-8010 Graz, Austria

  1. Edited by Chi-Huey Wong, Academia Sinica, Taipei, Taiwan, and approved November 28, 2011 (received for review August 9, 2011)

Abstract

N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-α-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-α-mannosidase. Structures solved at resolutions 1.7–2.1 Å reveal a (β/α)8 barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc1/3Man9GlcNAc2 yields Glc1/3-Man. Using the bespoke substrate α-Glc-1,3-α-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-α-mannosidase inhibitor α-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, α-Glc-1,3-isofagomine, and with the reducing-end product α-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.

Footnotes

  • ↵1To whom correspondence may be addressed. E-mail: davies{at}ysbl.york.ac.uk or sjwill{at}unimelb.edu.au.

  • Author contributions: A.J.T., R.J.W., T.D.B., S.J.W., and G.J.D. designed research; A.J.T., R.J.W., Z.H., D.S.A., T.W., T.M.G., and K.S., performed research; R.J.W., Z.H., T.M.W., J.S., A.E.S., and T.D.B. contributed new reagents/analytic tools; A.J.T., R.J.W., J.E.T.-O., T.D.B., S.J.W., and G.J.D. analyzed data; and A.J.T., S.J.W., and G.J.D. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4acy, 4acz, 4ad0, 4ad1, 4ad2, 4ad3, 4ad4, and 4ad5).

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1111482109/-/DCSupplemental.

Freely available online through the PNAS open access option.

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