Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery

Vignesh Venkatakrishnan*, Regis Dieckmann, Ian Loke, Harry Tjondro, Sayantani Chatterjee, Johan Bylund, Morten Thaysen-Andersen, Niclas G. Karlsson, Anna Karlsson-Bengtsson

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called 'targeting-by-timing'. Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation and N- and O-glycans present in each compartment were analyzed by LC-MS. We found abundant paucimannosidic N-glycans and lack of O-glycans in the early-formed azurophil granules (AG), whereas the later-formed specific and gelatinase granules (SG and GG) and secretory vesicles contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis epitopes. Immuno-blotting and -histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils and their complexity increased progressively from AG to SG and then to GG, suggesting temporal changes in the glycosylation machinery indicative of 'glycosylation-by-timing' during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step towards a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.
Original languageEnglish
Pages (from-to)12648-12660
Number of pages13
JournalThe Journal of biological chemistry
Volume295
Issue number36
DOIs
Publication statusPublished - 4 Sep 2020

Bibliographical note

This research was originally published in the Journal of Biological Chemistry. Vignesh Venkatakrishnan, Régis Dieckmann, Ian Loke, Harry C. Tjondro, Sayantani Chatterjee, Johan Bylund, Morten Thaysen-Andersen, Niclas G. Karlsson, and Anna Karlsson-Bengtsson. Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery. J Biol Chem. 2020; Vol. 295, no. 36, pp. 12648–12660. © the Author(s).

Keywords

  • neutrophil
  • granule
  • targeting by timing
  • N-glycan
  • O-glycan
  • LacNAc
  • Lewis epitope
  • liquid chromatography
  • mass spectrometry (MS)
  • glycosylation
  • N-linked glycosylation
  • plasma membrane

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