Poster Presentation 23rd Annual Lorne Proteomics Symposium 2018

Glycosylation features of neutrophilic granules (#107)

Harry Tjondro 1 , Vignesh Venkatakrishnan 2 , Ian Loke 1 , Sayantani Chatterjee 1 , Regis Dieckmann 3 , Charlotte Horn 4 , Benjamin L Parker 5 , Nicolle H Packer 1 , Niels Borregaard 4 , Niclas G Karlsson 2 , Johan Bylund 6 , Anna Karlsson 3 , Morten Thaysen-Andersen 1
  1. Department of Molecular Sciences, Macquarie University, Sydney, NSW, Australia
  2. Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
  3. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
  4. The Granulocyte Research Laboratory, Department of Hematology, Rigshospitalet, University of Copenhagen, Copenhagen, Dernmark
  5. Charles Perkins Centre, School of Molecular Bioscience and School of Medicine, University of Sydney, Sydney, Australia
  6. Department of Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

Protein glycosylation is important in many processes of the innate immune system. Aberrant glycosylation can modulate the innate immune response including the anti-microbial function of neutrophils. Recently, we discovered a new type of protein N-glycosylation, paucimannosidic N-glycans (Man1-3GlcNAc2Fuc0-1) on intact bioactive proteins that appear to be significant immune-modulators within the primary granule of neutrophils (Loke et al., Mol Cell Proteomics, 2017). To improve our understanding of these glycoepitopes, we here provide a detailed map of the glycosylation of the various granules of human neutrophils. Resting neutrophils from healthy donors were isolated. After gentle plasma membrane disruption, granules were separated by density gradient centrifugation into four fractions containing primary, secondary and tertiary granules, and the secretory vesicles/plasma membrane. The identity of the isolated intact compartments was validated using ELISA of granule-specific markers. Disruption of the granules by sonication, followed by ultracentrifugation, allowed the separation of the luminal and membrane proteins. Released N­- and O-glycans from these proteins were analysed using PGC-LC-ESI-CID-MS/MS. Bottom-up and top-down glycoproteomics experiments were also performed using RP-LC-ESI-MS/MS. In total, 68 N-glycans and 9 O-glycans were characterised and quantified across the neutrophil granules. Interestingly, granule-specific N- and O-glycosylation was determined, as illustrated by the significant enrichment of paucimannosidic N-glycans and a complete absence of O-glycans in primary granules, and very large poly-LacNAc containing bi-antennary complex-type N-glycans within secondary granules. Additionally, distinct glycosylation differences were observed between the luminal and membrane proteins indicating differential glycan processing even within the individual granules. Bottom-up and top-down glycoproteomics confirmed that paucimannosylation is a significant glycoepitope within primary granules and that these unusual N-glycans are carried by intact proteins. This detailed glyco-map demonstrating peculiar granule-specific glycosylation features of healthy neutrophils is essential to further advance our understanding of granulocytic glycobiology in inflammation and infection.