Poster Presentation 27th Annual Lorne Proteomics Symposium 2022

Immuno-active and antimicrobial paucimannosidic proteins form via a non-canonical truncation pathway in neutrophils (#181)

Julian Ugonotti 1 , Rebeca Kawahara 1 , Ian Loke 2 , Yuqi Zhu 3 , Sayantani Chatterjee 1 , Harry C Tjondro 1 , Zeynep Sumer-Bayraktar 1 , Sriram Neelamegham 3 , Morten Thaysen-Andersen 1
  1. Department of Molecular Sciences, Macquarie University, Sydney, NSW, Australia
  2. Cordlife Group Limited, Singapore
  3. Department of Chemical and Biological Engineering, University at Buffalo, Buffalo, New York, United States of America

We recently discovered that human neutrophils express immuno-active and microbicidal 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 formation, we here set out to test if homologous human Hex isoenzymes encoded by HEXA and HEXB drive a similar truncation pathway in human neutrophils [1]. To this end, we firstly 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, ELISA, proteomics and Hex enzyme 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 paucimannosidic protein formation via a hitherto unexplored truncation pathway in human 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 HL-60 mutants relative to unedited controls. Quantitative glycoproteomics recapitulated the truncation-to-elongation switch in all Hex-disrupted HL-60 mutants and showed a pronounced switch for N-glycoproteins co-trafficking with Hex to the azurophilic granules of neutrophils such as myeloperoxidase. To support the Hex-paucimannose link, we then documented that neutrophils isolated from an early-onset Sandhoff disease patient (HEXB-/-) display dramatically reduced paucimannosylation relative to neutrophils from an age-matched unaffected donor. We conclude that both human Hex α and β mediate the formation of immuno-active and antimicrobial paucimannosidic proteins via a putative non-canonical truncation pathway in neutrophils.

  1. [1] Ugonotti J, Kawahara R, Loke I, Zhu Y, Chatterjee S, Tjondro HC, Sumer-Bayraktar Z, Neelamegham S, Thaysen-Andersen M. N-acetyl-β-D-hexosaminidases mediate the generation of paucimannosidic proteins via a putative non-canonical truncation pathway in human neutrophils. Glycobiology, accepted Oct 12, 2021.