Structures of mammalian ER α-glucosidase II capture the binding modes of broad-spectrum iminosugar antivirals

Caputo, Alessandro T.; Alonzi, Dominic S.; Marti, Lucia; Reca, Ida-Barbara; Kiappes, J. L.; Struwe, Weston B.; Cross, Andreea-Ingrid; Basu, Souradeep; Lowe, E.D.; Darlot, Benoit; Santino, Angelo; Roversi, Pietro; Zitzmann, Nicole

doi:10.1073/pnas.1604463113

Cited by 68 publications

(82 citation statements)

References 71 publications

(72 reference statements)

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“…During peer review processes of our manuscript, crystal structure of the trypsinolyzed murine GII comprising GIIα and the G2B domain of β‐subunit has been reported . The murine GII structure was essentially identical with our fugal GII structure, indicating the evolutional conservation of the architecture of GII.…”

Section: Methodssupporting

confidence: 56%

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control

et al. 2016

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The glycoside hydrolase family 31 (GH31) α-glucosidases play vital roles in catabolic and regulated degradation, including the α-subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N-glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β-subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα-binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme.

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Section: Methodssupporting

confidence: 56%

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control

et al. 2016

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“…Development of selective ER α-glucosidase inhibitors would allow both confirmation that ER α-glucosidase inhibition (and not other enzymes) is responsible for the antiviral effect of iminosugars and avoidance of gastrointestinal side effects due to inhibition of intestinal glucosidases. Recently published structures of GluII [12], alone and in complex with MON-DNJ and castanospermine (see Chap. 19), provide the opportunity for rational drug design and greater understanding of the biochemical detail underlying the inhibition of this host enzyme.…”

Section: Discussionmentioning

confidence: 99%

Mechanisms of Antiviral Activity of Iminosugars Against Dengue Virus

Miller

Tyrrell

Zitzmann

2018

Advances in Experimental Medicine and Biology

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The antiviral mechanism of action of iminosugars against many enveloped viruses, including dengue virus (DENV), HIV, influenza and hepatitis C virus, is believed to be mediated by inducing misfolding of viral N-linked glycoproteins through inhibition of host endoplasmic reticulum-resident α-glucosidase enzymes. This leads to reduced secretion and/or infectivity of virions and hence lower viral titres, both in vitro and in vivo. Free oligosaccharide analysis from iminosugar-treated cells shows that antiviral activity correlates with production of mono- and tri-glucosylated sugars, indicative of inhibition of ER α-glucosidases. We demonstrate that glucose-mimicking iminosugars inhibit isolated glycoprotein and glycolipid processing enzymes and that this inhibition also occurs in primary cells treated with these drugs. Galactose-mimicking iminosugars that have been tested do not inhibit glycoprotein processing but do inhibit glycolipid processing, and are not antiviral against DENV. By comparison, the antiviral activity of glucose-mimetic iminosugars that inhibit endoplasmic reticulum-resident α-glucosidases, but not glycolipid processing, demonstrates that inhibition of α-glucosidases is responsible for iminosugar antiviral activity against DENV. This monograph will review the investigations of many researchers into the mechanisms of action of iminosugars and the contribution of our current understanding of these mechanisms for optimising clinical delivery of iminosugars. The effects of iminosugars on enzymes other than glucosidases, the induction of ER stress and viral receptors will be also put into context. Data suggest that inhibition of α-glucosidases results in inhibited release of virus and is the primary antiviral mechanism of action of iminosugars against DENV.

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“…N-linked glycans were purified and detected as described in ref. 53. The amount of reglucosylation was measured by determining the peak area of the PNGase F released 2AAlabeled species Man 9 GlcNAc 2 and Glc 1 Man 9 GlcNAc 2 using Waters Empower software.…”

Section: Methodsmentioning

confidence: 99%

Interdomain conformational flexibility underpins the activity of UGGT, the eukaryotic glycoprotein secretion checkpoint

Roversi

Marti

Caputo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170-kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively reglucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones, and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER retention and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, crystal structures of a full-length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc that terminates in two β-sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first β-sandwich and the fourth TRXL domain being encoded by nonconsecutive stretches of sequence. In addition to the crystal structures, a 15-Å cryo-EM reconstruction reveals interdomain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra interdomain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and reglucosylate its many different substrates and/or enable reglucosylation of N-linked glycans situated at variable distances from the site of misfold.

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Structures of mammalian ER α-glucosidase II capture the binding modes of broad-spectrum iminosugar antivirals

Cited by 68 publications

References 71 publications

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control

Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control

Mechanisms of Antiviral Activity of Iminosugars Against Dengue Virus

Interdomain conformational flexibility underpins the activity of UGGT, the eukaryotic glycoprotein secretion checkpoint

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