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Ugonotti J, Kawahara R, Loke I, et al. N-acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation pathway in human neutrophils. Glycobiology. 2021;doi: 10.1093/glycob/cwab108.
Ugonotti, J., Kawahara, R., Loke, I., Zhu, Y., Chatterjee, S., Tjondro, H. C., Sumer-Bayraktar, Z., Neelamegham, S., & Thaysen-Andersen, M. (2021). N-acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation pathway in human neutrophils. Glycobiology, . https://doi.org/10.1093/glycob/cwab108
Ugonotti, Julian, et al. "N-acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation pathway in human neutrophils." Glycobiology vol. (2021). doi: https://doi.org/10.1093/glycob/cwab108
Ugonotti J, Kawahara R, Loke I, Zhu Y, Chatterjee S, Tjondro HC, Sumer-Bayraktar Z, Neelamegham S, Thaysen-Andersen M. N-acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation pathway in human neutrophils. Glycobiology. 2021 Oct 18; doi: 10.1093/glycob/cwab108. Epub 2021 Oct 18. PMID: 34939086.
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Glycobiology. 2021 Oct 18; doi: 10.1093/glycob/cwab108. Epub 2021 Oct 18.

N-acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation pathway in human neutrophils.

Glycobiology

Julian Ugonotti, Rebeca Kawahara, Ian Loke, Yuqi Zhu, Sayantani Chatterjee, Harry C Tjondro, Zeynep Sumer-Bayraktar, Sriram Neelamegham, Morten Thaysen-Andersen

Affiliations

  1. Department of Molecular Sciences, Macquarie University, Balaclava Road, Macquarie Park, Sydney, NSW 2109, Australia.
  2. Cordlife Group Limited, 1 Yishun Industrial Street, Singapore 768160, Singapore.
  3. Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, 906 Furnas Hall, Buffalo, NY 14260, USA.
  4. Biomolecular Discovery Research Centre, Macquarie University, Balaclava Road, Macquarie Park, Sydney, NSW 2109, Australia.

PMID: 34939086 DOI: 10.1093/glycob/cwab108

Abstract

We 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-characterized truncation pathway in invertebrates and plants in which the N-acetyl-β-D-hexosaminidase (Hex) isoenzymes catalyze 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-KO and HEXB-KO) and matching unedited cell lines. Hex disruption was validated using next-generation sequencing, enzyme-linked immunosorbent assay (ELISA), quantitative proteomics and Hex activity assays. Excitingly, all Hex-disrupted mutants displayed significantly reduced levels of paucimannosylation, particularly 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 utilization of a putative noncanonical truncation pathway in favor 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-disrupted mutants and showed a greater switch for N-glycoproteins cotrafficking 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 noncanonical truncation pathway in neutrophils.

© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected].

Keywords: N-acetyl-β-D-hexosaminidase; N-glycosylation; neutrophil; paucimannose; truncation pathway

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