{"doi":"10.1093/glycob/cwab108","title":"<i>N</i>-acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical 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-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.","journal":"Glycobiology","year":2021,"id":168252,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":31,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9514,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":557105,"name":"Rebeca Kawahara","orcid":"0000-0002-0292-2637","position":1,"is_corresponding":false},{"id":696940,"name":"Ian Loke","orcid":"0000-0002-2558-0975","position":2,"is_corresponding":false},{"id":464911,"name":"Yuqi Zhu","orcid":"0000-0002-2553-8021","position":3,"is_corresponding":false},{"id":696941,"name":"Sayantani Chatterjee","orcid":"0000-0002-7007-0333","position":4,"is_corresponding":false},{"id":696942,"name":"Harry C. Tjondro","orcid":"0000-0001-5037-0091","position":5,"is_corresponding":false},{"id":696943,"name":"Zeynep Sumer‐Bayraktar","orcid":"0000-0001-7064-2728","position":6,"is_corresponding":false},{"id":241859,"name":"Sriram Neelamegham","orcid":"0000-0002-1371-8500","position":7,"is_corresponding":false},{"id":557107,"name":"Morten Thaysen‐Andersen","orcid":"0000-0001-8327-6843","position":8,"is_corresponding":false},{"id":696939,"name":"Julian Ugonotti","orcid":"0000-0002-3455-6766","position":0,"is_corresponding":true}],"reference_count":56,"raw_metadata":null,"created_at":"2026-07-18T23:46:02.603031Z","pmid":"34939086","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}