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Structure of Human Endo-Α-1,2-Mannosidase (MANEA), an Antiviral Host-Glycosylation Target

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Structure of Human Endo-Α-1,2-Mannosidase (MANEA), an Antiviral Host-Glycosylation Target Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target Łukasz F. Sobalaa,1, Pearl Z. Fernandesb,c, Zalihe Hakkib,c, Andrew J. Thompsona, Jonathon D. Howed, Michelle Hilld, Nicole Zitzmannd, Scott Daviese, Zania Stamatakie, Terry D. Buttersd, Dominic S. Alonzid, Spencer J. Williamsb,c,2, and Gideon J. Daviesa,2 aDepartment of Chemistry, University of York, York YO10 5DD, United Kingdom; bSchool of Chemistry, University of Melbourne, Parkville, VIC 3010, Australia; cBio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia; dOxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom; and eInstitute for Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, United Kingdom Edited by Gerald W. Hart, University of Georgia, Athens, GA, and accepted by Editorial Board Member Brenda A. Schulman September 28, 2020 (received for review July 1, 2020) Mammalian protein N-linked glycosylation is critical for glycopro- glucosylated mannose in the A branch, releasing Glc1–3Man and tein folding, quality control, trafficking, recognition, and function. Man8GlcNAc2; human MANEA acts faster on Glc1Man than on N-linked glycans are synthesized from Glc3Man9GlcNAc2 precur- Glc2,3Man glycans (9). The endomannosidase pathway allows sors that are trimmed and modified in the endoplasmic reticulum processing of ER-escaped, glucosylated high-mannose glycans on (ER) and Golgi apparatus by glycoside hydrolases and glycosyl- glycoproteins that fold independently of the calnexin/calreticulin transferases. Endo-α-1,2-mannosidase (MANEA) is the sole endo- folding cycle, allowing them to rejoin the downstream N-glycan acting glycoside hydrolase involved in N-glycan trimming and is maturation pathway. In murine BW6147 cells under normal con- located within the Golgi, where it allows ER-escaped glycoproteins ditions, flux through the endomannosidase pathway accounts for to bypass the classical N-glycosylation trimming pathway involv- approximately 15% of total flux through the secretory pathway ing ER glucosidases I and II. There is considerable interest in the (10); under conditions of glucosidase blockade through knockout use of small molecules that disrupt N-linked glycosylation as ther- or inhibition, MANEA can support as much as 50% of the normal apeutic agents for diseases such as cancer and viral infection. Here glycoprotein flux, depending on cellular expression levels (10). we report the structure of the catalytic domain of human MANEA MANEA activity accounts for the accumulation of Glc3Man tet- BIOCHEMISTRY and complexes with substrate-derived inhibitors, which provide rasaccharide in the urine of a neonate suffering from CDG-IIb, a insight into dynamic loop movements that occur on substrate rare genetic disease caused by a deficiency of ER glucosidase binding. We reveal structural features of the human enzyme that I (11). explain its substrate preference and the mechanistic basis for ca- Most enveloped viruses (e.g., coronaviruses, retroviruses, talysis. These structures have inspired the development of new ebolaviruses, hepatitis B virus [HBV], influenza viruses) contain inhibitors that disrupt host protein N-glycan processing of viral glycans and reduce the infectivity of bovine viral diarrhea and Significance dengue viruses in cellular models. These results may contribute CHEMISTRY to efforts aimed at developing broad-spectrum antiviral agents The glycosylation of proteins is a major protein modification and help provide a more in-depth understanding of the biology that occurs extensively in eukaryotes. Glycosidases in the se- of mammalian glycosylation. cretory pathway that trim N-linked glycans play key roles in protein quality control and in the specific modifications leading glycosylation | structural biology | secretory pathway | enzyme | antiviral to mature glycoproteins. Inhibition of glucosidases in the se- cretory pathway is a proven therapeutic strategy, that holds sparagine (N)-linked glycosylation is a protein modification great promise in the treatment of viral disease. The enzyme Athat is widespread in eukaryotes and is essential for protein endo-α-1,2-mannosidase (MANEA) provides an alternative quality control and trafficking (1). N-linked glycans contribute to processing pathway to evade glucosidase inhibitors. We report protein structure and stability, receptor targeting, development, the three-dimensional structure of human MANEA and com- and immune responses (2). N-linked glycosylation occurs in the plexes with enzyme inhibitors that we show act as antivirals secretory pathway and is initiated in the lumen of the endo- for bovine viral diarrhea and human dengue viruses. The plasmic reticulum (ER) through cotranslational transfer of the structure of MANEA will support inhibitor optimization and the triantennary Glc3Man9GlcNAc2 glycan from a lipid linked pre- development of more potent antivirals. cursor (2–5) (Fig. 1). The formation of mature glycoproteins re- quires the removal of the glucose residues, typically through the Author contributions: D.S.A., S.J.W., and G.J.D. designed research; Ł.F.S., P.Z.F., Z.H., A.J.T., α J.D.H., M.H., S.D., Z.S., D.S.A., and G.J.D. performed research; P.Z.F., Z.H., T.D.B., and S.J.W. action of -glucosidases I and II, which are localized within the contributed new reagents/analytic tools; Ł.F.S., Z.H., N.Z., D.S.A., S.J.W., and G.J.D. ana- rough ER. However, in most cell lines and in human patients, lyzed data; and Ł.F.S., D.S.A., S.J.W., and G.J.D. wrote the paper. genetic disruption of α-glucosidase I or II or inhibition of their The authors declare no competing interest. activities does not prevent the formation of mature glycoproteins This article is a PNAS Direct Submission.G.W.H.isaguesteditorinvitedbythe because of the action of Golgi endo-α-1,2-mannosidase (MANEA; Editorial Board. mannosidase, endo-alpha) (6), which provides a glucosidase- This open access article is distributed under Creative Commons Attribution-NonCommercial- independent pathway for glycoprotein maturation termed the NoDerivatives License 4.0 (CC BY-NC-ND). endomannosidase pathway (7). 1Present address: Department of Immunochemistry, Laboratory of Glycobiology, Hirszfeld cis medial Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 MANEA is present chiefly in / Golgi (84%) and the Wrocław, Poland. ER-Golgi intermediate compartment (15%) (8) and allows trim- 2To whom correspondence may be addressed. Email: [email protected] or gideon. ming of glucosylated mannose residues from Glc1–3Man9GlcNAc2 [email protected]. structures and B and C branch mannose-trimmed variants (see This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Fig. 1 for a definition of N-glycan branches). The action of doi:10.1073/pnas.2013620117/-/DCSupplemental. MANEA on Glc1–3Man9GlcNAc2 results in cleavage of the First published November 5, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2013620117 PNAS | November 24, 2020 | vol. 117 | no. 47 | 29595–29601 Downloaded by guest on September 30, 2021 Fig. 1. Simplified pathway for biosynthesis of N-linked glycans through the classical and endomannosidase pathways. En bloc transfer of the preformed Glc3Man9GlcNAc2 tetradecasaccharide (branches labeled A/B/C) from the dolichol precursor occurs cotranslationally to Asn residues within the consensus sequence Asn-Xxx-Ser/Thr. Trimming of glucose residues can be achieved through the classical pathway involving sequential action of α-glucosidases I and II. Alternatively, MANEA provides a glucosidase-independent pathway for glycoprotein maturation, through cleaving the glucose-substituted mannose residues. ER mannosidase I resides in quality control vesicles (QCV) (43). ER-associated degradation of terminally misfolded glycoproteins is omitted. glycoproteins within their envelope (12). Glycosylation is achieved and Bacteroides thetaiotaomicron (BtGH99) that act on related by coopting host cell glycosylation machinery during replication. structures within yeast mannans and that share ∼40% identity with Substantial efforts have been deployed in the use of inhibitors of MANEA (29–31). MANEA encodes a 462-aa protein that consists α-glucosidases I and II as antivirals (e.g., N-butyldeoxynojirimycin, of a single-pass type II membrane protein with a transmembrane 6-O-butanoylcastanospermine) for the treatment of HIV/AIDS helix followed by a stem region, followed by the catalytic domain (13), dengue (14, 15), and HBV (16, 17) (reviewed in refs. 18–20). (32). Here we reveal the structure of the catalytic domain of Inhibition of host glycosylation pathways, in particular ER glu- human MANEA. Through complexes with substrate, we show cosidases I and II and Golgi mannosidase I, interferes with the the architecture of the binding groove and identify key catalytic viral lifecycle by impairing protein folding and quality control, residues. We report structures with MANEA inhibitors that inducing the unfolded protein response or mistrafficking of viral were designed based on the structure of the glucosylated high- glycoproteins. These changes can lead to impairment of secretion mannose N-glycan. Finally, we show that inhibitors of MANEA (17, 21–23) or fusion (24–26) or evasion of host immunity (27). In act as antiviral agents against bovine viral diarrheal virus the case of HBV, when glucosidases are inhibited, mature HBV (BVDV) and dengue virus (DENV),
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