N-acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical truncation pathway in human neutrophils. (18th October 2021)

• Record Type: Journal Article
• Authors: Ugonotti, Julian; Kawahara, Rebeca; Loke, Ian; Zhu, Yuqi; Chatterjee, Sayantani; Tjondro, Harry C; Sumer-Bayraktar, Zeynep; Neelamegham, Sriram; Thaysen-Andersen, Morten
• Is Part Of: Glycobiology. Volume 32:Number 3(2022)
• Page Start: 218
• Page End: 229
• Publication Date: 2021-10-18
• Subjects: truncation pathway -- paucimannose -- N-glycosylation -- neutrophil -- N-acetyl-β-D-hexosaminidase; Glycoproteins -- Periodicals; Glycolipids -- Periodicals; Glycoconjugates -- Periodicals; 572.567
• DOI: 10.1093/glycob/cwab108
• Languages: English
• ISSNs: 0959-6658

Abstract

Abstract: We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N -glycans (Man1-3 GlcNAc2 Fuc0–1 ), 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 Man2-3 GlcNAc2 Fuc1, 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.