Dataset Information

N-Acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative non-canonical truncation pathway in human neutrophils

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-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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell

DISEASE(S): Acute Leukemia

SUBMITTER: Julian Ugonotti

LAB HEAD: Dr. Morten Thaysen-Andersen

PROVIDER: PXD027664 | Pride | 2023-03-11

REPOSITORIES: Pride

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Publications

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

Ugonotti Julian J Kawahara Rebeca R Loke Ian I Zhu Yuqi Y Chatterjee Sayantani S Tjondro Harry C HC Sumer-Bayraktar Zeynep Z Neelamegham Sriram S Thaysen-Andersen Morten M

Glycobiology 20220301 3

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 ...[more]

PMID: 34939086

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Dataset Information

N-Acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative non-canonical truncation pathway in human neutrophils

Publications

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

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