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

A.J. Thompson, R.J. Williams, Z. Hakki, D.S. Alonzi, T. Wennekes, T.M. Gloster, K. Songsrirote, E. Thomas-Oates, T.M. Wrodnigg, J. Spreitz, A.E. Stütz, T.D. Butters, S.J. Williams, G.J. Davies

Research output: Contribution to journalArticleAcademicpeer-review

52 Citations (Scopus)

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-a-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-a-mannosidase. Structures solved at resolutions 1.7–2.1 Å reveal a (ß/a)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 a-Glc-1,3-a-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-a-mannosidase inhibitor a-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, a-Glc-1,3-isofagomine, and with the reducing-end product a-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.
Original languageEnglish
Pages (from-to)781-786
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number3
DOIs
Publication statusPublished - 2012

Keywords

  • asparagine-linked oligosaccharides
  • glucosidase-ii-deficient
  • glycoprotein-biosynthesis
  • endomannosidase pathway
  • endoplasmic-reticulum
  • glycoside hydrolases
  • quality-control
  • inhibitors
  • cells
  • classification

Fingerprint Dive into the research topics of 'Structural and mechanistic insight into N-glycan processing by endo-a-mannosidase'. Together they form a unique fingerprint.

  • Cite this

    Thompson, A. J., Williams, R. J., Hakki, Z., Alonzi, D. S., Wennekes, T., Gloster, T. M., ... Davies, G. J. (2012). Structural and mechanistic insight into N-glycan processing by endo-a-mannosidase. Proceedings of the National Academy of Sciences of the United States of America, 109(3), 781-786. https://doi.org/10.1073/pnas.1111482109