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

被引:15
作者
Ugonotti, Julian [1 ]
Kawahara, Rebeca [1 ]
Loke, Ian [2 ]
Zhu, Yuqi [3 ]
Chatterjee, Sayantani [1 ]
Tjondro, Harry C. [1 ]
Sumer-Bayraktar, Zeynep [1 ]
Neelamegham, Sriram [3 ]
Thaysen-Andersen, Morten [1 ,4 ]
机构
[1] Macquarie Univ, Dept Mol Sci, Balaclava Rd, Sydney, NSW 2109, Australia
[2] Cordlife Grp Ltd, 1 Yishun Ind St, Singapore 768160, Singapore
[3] Univ Buffalo State Univ New York, Dept Chem & Biol Engn, 906 Furnas Hall, Buffalo, NY 14260 USA
[4] Macquarie Univ, Biomol Discovery Res Ctr, Balaclava Rd, Sydney, NSW 2109, Australia
基金
美国国家卫生研究院;
关键词
truncation pathway; paucimannose; N-glycosylation; neutrophil; N-acetyl-beta-D hexosaminidase; GLYCAN; GRANULES; BINDING; GLYCOPROTEOMICS; GANGLIOSIDOSIS; GLYCOSYLATION; BIOSYNTHESIS; GLYCOPEPTIDE; SUBUNITS; CELLS;
D O I
10.1093/glycob/cwab108
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N-glycans (Man(1-3)GlcNAc(2)Fuc(0-1)), but their biosynthesis remains elusive. Guided by the well-characterized truncation pathway in invertebrates and plants in which the N-acetyl-beta-D-hexosaminidase (Hex) isoenzymes catalyze paucimannosidic protein (PMP) formation, we here set out to test if the homologous human Hex alpha and beta 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 Man(2-3) GlcNAc(2)Fuc(1), 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 alpha and beta mediate PMP formation via a putative noncanonical truncation pathway in neutrophils.
引用
收藏
页码:218 / 229
页数:12
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