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PXD027664

PXD027664 is an original dataset announced via ProteomeXchange.

Dataset Summary
TitleN-Acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative non-canonical truncation pathway in human neutrophils
DescriptionWe recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N-glycans (Man1-3GlcNAc2Fuc0-1), but their biosynthesis remains elusive. Guided by the well-characterised truncation pathway in invertebrates and plants in which the N-acetyl-β-D-hexosaminidase (Hex) isoenzymes catalyse paucimannosidic protein (PMP) formation, we here set out to test if the homologous human Hex α and β subunits encoded by HEXA and HEXB drive a similar truncation pathway in human neutrophils. To this end, we performed quantitative glycomics and glycoproteomics of several CRISPR-Cas9-edited Hex-disrupted neutrophil-like HL-60 mutants (HEXA-/- and HEXB-/-) and matching unedited cell lines. Hex disruption was validated using next-generation sequencing, ELISA, proteomics and Hex activity assays. Excitingly, all Hex-disrupted mutants displayed significantly reduced levels of paucimannosylation, particularly of Man2-3GlcNAc2Fuc1, relative to unedited HL-60 suggesting that both HEXA and HEXB contribute to PMP formation via a hitherto unexplored truncation pathway in neutrophils. Quantitative N-glycomics indeed demonstrated reduced utilisation of a putative non-canonical truncation pathway in favour of the canonical elongation pathway in all Hex-disrupted mutants relative to unedited controls. Quantitative glycoproteomics recapitulated the truncation-to-elongation switch in all Hex-deficient mutants and showed a pronounced switch for N-glycoproteins co-trafficking with Hex to the azurophilic granules of neutrophils such as myeloperoxidase. Finally, we supported the Hex-PMP relationship by documenting that primary neutrophils isolated from an early-onset Sandhoff disease patient (HEXB-/-) displayed dramatically reduced paucimannosylation relative to neutrophils from an age-matched unaffected donor. We conclude that both human Hex α and β mediate PMP formation via a putative non-canonical truncation pathway in neutrophils.
HostingRepositoryPRIDE
AnnounceDate2023-11-14
AnnouncementXMLSubmission_2023-11-14_08:40:50.733.xml
DigitalObjectIdentifier
ReviewLevelPeer-reviewed dataset
DatasetOriginOriginal dataset
RepositorySupportUnsupported dataset by repository
PrimarySubmitterJulian Ugonotti
SpeciesList scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationListTMT6plex-126 reporter+balance reagent acylated residue
InstrumentQ Exactive HF
Dataset History
RevisionDatetimeStatusChangeLog Entry
02021-07-30 03:44:49ID requested
12023-03-11 02:34:11announced
22023-11-14 08:40:52announced2023-11-14: Updated project metadata.
Publication List
Ugonotti J, Kawahara R, Loke I, Zhu Y, Chatterjee S, Tjondro HC, Sumer-Bayraktar Z, Neelamegham S, Thaysen-Andersen M, -D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation pathway in human neutrophils. Glycobiology, 32(3):218-229(2022) [pubmed]
Keyword List
ProteomeXchange project tag: Biology/Disease-Driven Human Proteome Project (B/D-HPP), Human Proteome Project, Glycoproteomics (B/D-HPP)
submitter keyword: N-Acetyl-β-D-hexosaminidase, biosynthesis, paucimannose, truncation, neutrophil, mass spectrometry, N-glycosylation
Contact List
Dr. Morten Thaysen-Andersen
contact affiliationDepartment of Molecular Sciences, Macquarie University, Sydney, NSW, Australia
contact emailmorten.andersen@mq.edu.au
lab head
Julian Ugonotti
contact affiliationMacquarie University
contact emailjulian.ugonotti@hdr.mq.edu.au
dataset submitter
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Dataset FTP location
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