Paucimannose-Rich N-glycosylation of Spatiotemporally Regulated Human Neutrophil Elastase Modulates Its Immune Functions
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Paucimannose-Rich N-glycosylation of Spatiotemporally Regulated Human Neutrophil Elastase Modulates Its Immune Functions. / Loke, Ian; Østergaard, Ole; Heegaard, Niels H H; Packer, Nicolle H; Thaysen-Andersen, Morten.
In: Molecular and Cellular Proteomics, Vol. 16, No. 8, 08.2017, p. 1507-1527.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Paucimannose-Rich N-glycosylation of Spatiotemporally Regulated Human Neutrophil Elastase Modulates Its Immune Functions
AU - Loke, Ian
AU - Østergaard, Ole
AU - Heegaard, Niels H H
AU - Packer, Nicolle H
AU - Thaysen-Andersen, Morten
N1 - © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2017/8
Y1 - 2017/8
N2 - Human neutrophil elastase (HNE) is an important N-glycosylated serine protease in the innate immune system, but the structure and immune-modulating functions of HNE N-glycosylation remain undescribed. Herein, LC-MS/MS-based glycan, glycopeptide and glycoprotein profiling were utilized to first determine the heterogeneous N-glycosylation of HNE purified from neutrophil lysates and then from isolated neutrophil granules of healthy individuals. The spatiotemporal expression of HNE during neutrophil activation and the biological importance of its N-glycosylation were also investigated using immunoblotting, cell surface capture, native MS, receptor interaction, protease inhibition, and bacteria growth assays. Site-specific HNE glycoprofiling demonstrated that unusual paucimannosidic N-glycans, particularly Manα1,6Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAcβ, predominantly occupied Asn124 and Asn173. The equally unusual core fucosylated monoantenna complex-type N-sialoglycans also decorated these two fully occupied sites. In contrast, the mostly unoccupied Asn88 carried nonfucosylated paucimannosidic N-glycans probably resulting from low glycosylation site solvent accessibility. Asn185 was not glycosylated. Subcellular- and site-specific glycoprofiling showed highly uniform N-glycosylation of HNE residing in distinct neutrophil compartments. Stimulation-induced cell surface mobilization demonstrated a spatiotemporal regulation, but not cell surface-specific glycosylation signatures, of HNE in activated human neutrophils. The three glycosylation sites of HNE were located distal to the active site indicating glycan functions other than interference with HNE enzyme activity. Functionally, the paucimannosidic HNE glycoforms displayed preferential binding to human mannose binding lectin compared with the HNE sialoglycoforms, suggesting a glycoform-dependent involvement of HNE in complement activation. The heavily N-glycosylated HNE protease inhibitor, α1-antitrypsin, displayed concentration-dependent complex formation and preferred glycoform-glycoform interactions with HNE. Finally, both enzymatically active HNE and isolated HNE N-glycans demonstrated low micromolar concentration-dependent growth inhibition of clinically-relevant Pseudomonas aeruginosa, suggesting some bacteriostatic activity is conferred by the HNE N-glycans. Taken together, these observations support that the unusual HNE N-glycosylation, here reported for the first time, is involved in modulating multiple immune functions central to inflammation and infection.
AB - Human neutrophil elastase (HNE) is an important N-glycosylated serine protease in the innate immune system, but the structure and immune-modulating functions of HNE N-glycosylation remain undescribed. Herein, LC-MS/MS-based glycan, glycopeptide and glycoprotein profiling were utilized to first determine the heterogeneous N-glycosylation of HNE purified from neutrophil lysates and then from isolated neutrophil granules of healthy individuals. The spatiotemporal expression of HNE during neutrophil activation and the biological importance of its N-glycosylation were also investigated using immunoblotting, cell surface capture, native MS, receptor interaction, protease inhibition, and bacteria growth assays. Site-specific HNE glycoprofiling demonstrated that unusual paucimannosidic N-glycans, particularly Manα1,6Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAcβ, predominantly occupied Asn124 and Asn173. The equally unusual core fucosylated monoantenna complex-type N-sialoglycans also decorated these two fully occupied sites. In contrast, the mostly unoccupied Asn88 carried nonfucosylated paucimannosidic N-glycans probably resulting from low glycosylation site solvent accessibility. Asn185 was not glycosylated. Subcellular- and site-specific glycoprofiling showed highly uniform N-glycosylation of HNE residing in distinct neutrophil compartments. Stimulation-induced cell surface mobilization demonstrated a spatiotemporal regulation, but not cell surface-specific glycosylation signatures, of HNE in activated human neutrophils. The three glycosylation sites of HNE were located distal to the active site indicating glycan functions other than interference with HNE enzyme activity. Functionally, the paucimannosidic HNE glycoforms displayed preferential binding to human mannose binding lectin compared with the HNE sialoglycoforms, suggesting a glycoform-dependent involvement of HNE in complement activation. The heavily N-glycosylated HNE protease inhibitor, α1-antitrypsin, displayed concentration-dependent complex formation and preferred glycoform-glycoform interactions with HNE. Finally, both enzymatically active HNE and isolated HNE N-glycans demonstrated low micromolar concentration-dependent growth inhibition of clinically-relevant Pseudomonas aeruginosa, suggesting some bacteriostatic activity is conferred by the HNE N-glycans. Taken together, these observations support that the unusual HNE N-glycosylation, here reported for the first time, is involved in modulating multiple immune functions central to inflammation and infection.
KW - Bacterial Infections/immunology
KW - Catalytic Domain
KW - Complement Activation
KW - Glycopeptides/metabolism
KW - Glycoproteins/metabolism
KW - Glycosylation
KW - Humans
KW - Immunity, Innate
KW - Inflammation/immunology
KW - Lectins/metabolism
KW - Leukocyte Elastase/antagonists & inhibitors
KW - Male
KW - Mannose/metabolism
KW - Neutrophils/enzymology
KW - Polysaccharides/metabolism
KW - Pseudomonas Infections/immunology
KW - Pseudomonas aeruginosa/immunology
KW - alpha 1-Antitrypsin/pharmacology
U2 - 10.1074/mcp.M116.066746
DO - 10.1074/mcp.M116.066746
M3 - Journal article
C2 - 28630087
VL - 16
SP - 1507
EP - 1527
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
SN - 1535-9476
IS - 8
ER -
ID: 210473645