Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Unusual Site- and Granule-Specific N-Glycosylation of Human Myeloperoxidase from Resting Neutrophils (#884)

Harry Tjondro 1 , Julian Ugonotti 1 , Sayantani Chatterjee 1 , Ian Loke 2 , Si-Yun Chen 3 , Hannes Hinneburg 1 , Benjamin L Parker 4 , Vignesh Venkatakrishnan 5 , Regis Dieckmann 5 , Weston Struwe 3 , Anna Karlsson 5 , Morten Thaysen-Andersen 1
  1. Department of Molecular Sciences, Macquarie University, Sydney, NSW, Australia
  2. Department of Biological Science, National University of Singapore, Singapore
  3. Department of Chemistrty, University of Oxford, Oxford, South East England, United Kingdom
  4. Department of Physiology, University of Melbourne, Melbourne, VIC, Australia
  5. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

Myeloperoxidase (MPO), which predominantly resides in the primary granules of resting neutrophils but reportedly also in other neutrophil compartments at lower levels, is an important glycoprotein in neutrophil-mediated innate immunity. Most structural features of human neutrophil MPO and its capacity to produce anti-microbial hypochlorous acid are well-documented, but the exact site- and granule-specific glycosylation of MPO remains undocumented, despite the recognised importance of N-glycosylation for its structure-function. Herein, we performed a deep structural characterisation of the N-glycosylation of human MPO from healthy donors using LC-MS/MS-based glycomics, intact glycopeptide and glycoprotein profiling. Quantitative glycomics and glycopeptide analyses of MPO from neutrophil lysates revealed that all five N-glycosylation sites of the heavy chain MPO monomer are conjugated with heterogeneous N-glycans albeit with strong site-specific differences. Paucimannosidic N-glycans were predominantly found to be linked to Asn323 (47%) and Asn483 (56%), oligomannosidic N-glycans were mainly carried by Asn355 (97%) and Asn391 (64%) while Asn729 was largely unoccupied (44%) or modified with chitobiose core N-glycans (33%). Importantly, the glycoprofiles of the intact MPO monomer and dimer obtained using high-resolution top-down mass spectrometry recapitulated the heterogeneity observed at the bottom-up level as supported by matching experimental and theoretical intact glycoprotein masses. Further, glycoprofiling of MPO from isolated neutrophil granules intriguingly demonstrated that the N-glycosylation of Asn355, Asn391 and Asn729 varied dramatically whereas Asn323 and Asn483 were similarly glycosylated on MPO across the studied neutrophil compartments. Particularly the abundant primary granule-resident MPO displayed unique glycosylation signatures comprising a higher proportion of oligomannosidic N-glycans on the three variable glycosylation sites. In conclusion, this study represents the most detailed structural characterisation of MPO N-glycosylation to date. The complex site- and granule-specific N-glycosylation of MPO, shown here for the first time, adds fundamental knowledge that aid our understanding of the fascinating glycobiology underpinning neutrophil-mediated immunity.