Dataset Information

N-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.

SUBMITTER: Ugonotti J

PROVIDER: S-EPMC8966476 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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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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Project description: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.

Project description:β-N-acetyl-D-hexosaminidases have attracted significant attention due to their crucial role in diverse physiological functions including antibacterial synergists, pathogen defense, virus infection, lysosomal storage, and protein glycosylation. In particular, the GH3 β-N-acetyl-D-hexosaminidase of V. cholerae (VcNagZ), human GH20 β-N-acetyl-D-hexosaminidase B (HsHexB), and human GH84 β-N-acetyl-D-hexosaminidase (hOGA) are three important representative glycosidases. These have been found to be implicated in β-lactam resistance (VcNagZ), lysosomal storage disorders (HsHexB) and Alzheimer's disease (hOGA). Considering the profound effects of these three enzymes, many small molecule inhibitors with good potency and selectivity have been reported to regulate the corresponding physiological functions. In this paper, the best-known inhibitors PUGNAc and two of its derivatives (N-valeryl-PUGNAc and EtBuPUG) were selected as model compounds and docked into the active pockets of VcNagZ, HsHexB, and hOGA, respectively. Subsequently, molecular dynamics simulations of the nine systems were performed to systematically compare their binding modes from active pocket architecture and individual interactions. Furthermore, the binding free energy and free energy decomposition are calculated using the MM/GBSA methods to predict the binding affinities of enzyme-inhibitor systems and to quantitatively analyze the contribution of each residue. The results show that PUGNAc is deeply-buried in the active pockets of all three enzymes, which indicates its potency (but not selectivity) against VcNagZ, HsHexB, and hOGA. However, EtBuPUG, bearing branched 2-isobutamido, adopted strained conformations and was only located in the active pocket of VcNagZ. It has completely moved out of the pocket of HsHexB and lacks interactions with HsHexB. This indicates why the selectivity of EtBuPUG to VcNagZ/HsHexB is the largest, reaching 968-fold. In addition, the contributions of the catalytic residue Asp253 (VcNagZ), Asp254 (VcNagZ), Asp175 (hOGA), and Asp354 (HsHexB) are important to distinguish the activity and selectivity of these inhibitors. The results of this study provide a helpful structural guideline to promote the development of novel and selective inhibitors against specific β-N-acetyl-D-hexosaminidases.

Dataset Information

N-acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative noncanonical 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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