Oral Presentation 25th Annual Lorne Proteomics Symposium 2020

Granule-Specific N-Glycosylation and Chlorination Activity of Neutrophil Myeloperoxidase (#67)

Harry C. Tjondro 1 , Julian Ugonotti 1 , Sayantani Chatterjee 1 , Rebeca Sakuma 1 , Ian Loke 1 , Vignesh Venkatakrishnan 2 , Siyun Chen 3 , Weston Struwe 3 , Benjamin L. Parker 4 , Johan Bylund 2 , Oliver C. Grant 5 , Robert J. Woods 5 , Anna Karlsson 2 , Morten Thaysen-Andersen 1
  1. Biomolecular Discovery Research Centre, Macquarie University, Sydney, NSW, Australia
  2. Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
  3. Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, United Kingdom
  4. School of Biomedical Sciences, University of Melbourne, Melbourne, VIC, Australia
  5. Complex Carbohydrate Research Center, University of Georgia, Athens, GA, United States

The heavily glycosylated myeloperoxidase (MPO), a key component of neutrophil-mediated innate immunity, produces anti-microbial hypochlorous acid to combat pathogens, but the functional importance of MPO glycosylation remains unexplored. Herein, we first performed deep structural characterisation of the N-glycosylation decorating human MPO extracted from whole neutrophils (nMPO) using mass spectrometry-driven glycomics, glycopeptide, and intact glycoprotein profiling. Unusual site-specific signatures were observed including paucimannosidic N-glycans of Asn323 (47%) and Asn483 (56%), oligomannosidic N-glycans of Asn355 (97%) and Asn391 (64%) while Asn729 was unoccupied (44%) or carried equally peculiar chitobiose core-type N-glycans (33%). Native mass spectrometry and a novel mass photometry technique revealed extreme glycoform heterogeneity and the expected stoichiometry of the common dimeric (141-148 kDa) and less-abundant monomeric (70-73 kDa) nMPO forms. Native gel electrophoresis and glycopeptide profiling demonstrated that monomeric and dimeric nMPO displayed glycan differences of Asn483 located in the dimerization interface. The solvent accessibility of the glycosylation sites of maturely folded monomeric and dimeric MPO correlated with the degree of early- (Golgi) and late-stage (granule) N-glycan processing providing support for an oligomerisation-dependent glycan processing at Asn483. Excitingly, glycoprofiling of subcellularly-fractionated MPO demonstrated distinct granule-specific Asn355- and Asn391-glycosylation across the neutrophil compartments. Notably, the N-glycans carried by specific/gelatinase granule-resident MPO (Sp/Ge-MPO) displayed extreme truncation to GlcNAc╬▓-Asn at Asn355 and Asn391 whereas the azurophilic granule-resident MPO (Az-MPO) displayed oligomannosidic glycans at those sites. Interestingly, the Sp/Ge-MPO exhibited a comparably higher chlorination activity than Az-MPO, an observation that could be recapitulated for endoglycosidase H-treated nMPO simulating the Sp/Ge-MPO glycoform. Molecular dynamics simulations of granule-relevant MPO glycoforms were used to support that Asn355- and Asn391-glycans are able to perturb the conformation of the heme-containing active site of human MPO. We are the first to report on the granule-specific N-glycosylation and chlorination activity of neutrophil MPO revealing a novel fascinating feature of neutrophil glycobiology.