Poster Presentation 26th Annual Lorne Proteomics Symposium 2021

Hexosaminidase disruption impedes the biosynthesis of paucimannosidic proteins in HL-60 cells (#88)

Julian Ugonotti 1 , Ian Loke 2 , Yuqi Zhu 3 , Sayantani Chatterjee 1 , Harry C Tjondro 1 , Zeynep Sumer-Bayraktar 1 , Meral Topçu 4 , 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
  4. Department of Child Neurology, Hacettepe University, Ankara, Turkey

We have previously documented that human neutrophils are actively expressing immune-related glycoproteins carrying paucimannosidic glycans (Man1-3GlcNAc2Fuc0-1), a functional yet under-explored class of N-glycosylation in the human glycobiology. Whilst inference from the paucimannose-rich invertebrates and plants points to a β-N-acetyl-hexosaminidase (Hex)-driven production of paucimannosidic proteins from immature β1,2-linked N-acetylglucosamine-terminating glycoprotein intermediates in the mammalian glycosylation machinery, this hypothesis remains experimentally unsupported in human cells including neutrophils. We therefore sought to obtain evidence for the involvement of the Hex isoenzymes, which in humans exist in homodimeric (αα and ββ) and heterodimeric (αβ) variants arising from two coding genes, HEXA (α) and HEXB (β), in the biosynthesis of paucimannosidic proteins in human neutrophils. Our approach was to perform quantitative PGC-LC-MS/MS-based N-glycomics and C18-LC-MS/MS-based glycoproteomics of several CRISPR-Cas9-edited Hex-deficient neutrophil-like HL-60 mutant cell lines (two HEXA-/- and two HEXB-/- mutant lines) relative to an unedited HL-60 control line. Accurate disruption of the target genes and absence of off-target genetic mutations were validated using next-generation sequencing and established Hex activity assays. Both HEXA (24.0%) and HEXB (22.8%) disruption led to a relatively weak yet still significant reduction of paucimannosidic N-glycans relative to unedited HL-60 (33.3%). In particular, Man2-3GlcNAc2Fuc0-1 was reduced in the Hex-deficient mutants relative to unedited cells. Interestingly, an N-glycomics follow-up analysis of mature blood neutrophils from a patient diagnosed with early onset Sandhoff disease (HEXB-/-) recapitulated a reduced expression level of paucimannosidic N-glycans (20.7%) relative to levels found in mature blood neutrophils from a healthy age-matched donor (40.5%). This study provides evidence to support that several Hex isoenzyme variants encoded by HEXA and HEXB are directly responsible for the biosynthesis of paucimannosidic proteins in human neutrophils. These well-characterised Hex-deficient mutant cell lines displaying supressed levels of protein paucimannosylation will be useful to further our understanding of the functional roles of paucimannosidic proteins in neutrophil glycoimmunology.