Unlike plants, invertebrates and other “lower” organisms, mammals are considered to lack proteins displaying asparagine (N)-linked paucimannosylation (mannose1-3fucose0-1N-acetylglucosamine2Asn). Enabled by technology advancements in system-wide characterization of glycans and glycopeptides, we provide data documenting that protein paucimannosylation is a significant host-derived biomolecular signature of neutrophil-rich sputum from pathogen-infected human lungs. These signatures appear negligible in pathogen-free sputum undergoing inflammation. Five paucimannosidic N-glycan structures were carried by compartment-specific and inflammation-associated proteins localizing to the specialized azurophilic granules of human neutrophils including myeloperoxidase (MPO), azurocidin and neutrophil elastase. The timely expressed human azurophilic granule-resident b-hexosaminidase A displayed the capacity to generate paucimannosidic N-glycans by trimming hybrid/complex type N-glycan intermediates with relative broad substrate-specificity. Immunocytochemistry showed that paucimannosidic N-glycoepitopes significantly colocalized with b-hexosaminidase A and the azurophilic marker MPO in human neutrophils. Furthermore, maturation stage-specific expression of genes coding for paucimannosidic proteins and biosynthetic enzymes indicated a novel spatio-temporal biosynthetic route in early neutrophil maturation in the promyelocytic stage. Importantly, absence of bacterial exoglycosidase activities and paucimannosidic N-glycans excluded an exogenous origin of paucimannosylation. Paucimannosidic proteins from isolated and sputum neutrophils were preferentially secreted upon inoculation with virulent P. aeruginosa. Finally, paucimannosidic proteins displayed affinities to mannose-binding lectin and bacteriostatic activities towards virulent P. aeruginosa suggesting immune-related functions of paucimannosylation in activated human neutrophils. In conclusion, we here document the structure, function and biosynthesis of human paucimannosylation; we are the first to show that human neutrophils produce, store and, upon activation, selectively secrete bioactive paucimannosidic proteins into sputum of lungs undergoing pathogen-based inflammation.
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|Published - Jul 2015
|Congress of the Federation of European Biochemical Societies (FEBS) (40th : 2015): the Biochemical Basis of Life - Berlin, Germany
Duration: 4 Jul 2015 → 9 Jul 2015