title: Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum.
abstract: Unlike plants and invertebrates, mammals reportedly lack proteins displaying asparagine (N)-linked paucimannosylation (mannose(1-3)fucose(0-1)N-acetylglucosamine(2)Asn). Enabled by technology advancements in system-wide biomolecular characterization, we document that protein paucimannosylation is a significant host-derived molecular signature of neutrophil-rich sputum from pathogen-infected human lungs and is negligible in pathogen-free sputum. Five types of paucimannosidic N-glycans were carried by compartment-specific and inflammation-associated proteins of the azurophilic granules of human neutrophils including myeloperoxidase (MPO), azurocidin, and neutrophil elastase. The timely expressed human azurophilic granule-resident β-hexosaminidase A displayed the capacity to generate paucimannosidic N-glycans by trimming hybrid/complex type N-glycan intermediates with relative broad substrate specificity. Paucimannosidic N-glycoepitopes showed significant co-localization with β-hexosaminidase A and the azurophilic marker MPO in human neutrophils using immunocytochemistry. Furthermore, promyelocyte stage-specific expression of genes coding for paucimannosidic proteins and biosynthetic enzymes indicated a novel spatio-temporal biosynthetic route in early neutrophil maturation. The absence of bacterial exoglycosidase activities and paucimannosidic N-glycans excluded exogenous origins of paucimannosylation. Paucimannosidic proteins from isolated and sputum neutrophils were preferentially secreted upon inoculation with virulent Pseudomonas aeruginosa. Finally, paucimannosidic proteins displayed affinities to mannose-binding lectin, suggesting immune-related functions of paucimannosylation in activated human neutrophils. In conclusion, we are the first to document that human neutrophils produce, store and, upon activation, selectively secrete bioactive paucimannosidic proteins into sputum of lungs undergoing pathogen-based inflammation.
authors: From the Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales-2109, Australia, morten.andersen@mq.edu.au.
authors: Morten
authors: M
authors: Thaysen-Andersen
authors: From the Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales-2109, Australia.
authors: Vignesh
authors: V
authors: Venkatakrishnan
authors: From the Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales-2109, Australia.
authors: Ian
authors: I
authors: Loke
authors: the Institute of Nutrition and Food Sciences, University of Bonn, Bonn 53113, Germany, and.
authors: Christine
authors: C
authors: Laurini
authors: the Institute of Nutrition and Food Sciences, University of Bonn, Bonn 53113, Germany, and.
authors: Simone
authors: S
authors: Diestel
authors: the Diabetes and Obesity Program, Garvan Institute of Medical Research, Sydney, New South Wales-2010, Australia.
authors: Benjamin L
authors: BL
authors: Parker
authors: From the Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales-2109, Australia.
authors: Nicolle H
authors: NH
authors: Packer
mesh_terms: D001399:Azure Stains / Q000378:metabolism*; D015153:Blotting, Western; D002853:Chromatography, Liquid; D006031:Glycosylation; D018922:HL-60 Cells; D006801:Humans; D008362:Mannosides / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000302:isolation & purification; D013183:Sputum / Q000382:microbiology*; D053719:Tandem Mass Spectrometry
chemical_list: D001399:Azure Stains; D008362:Mannosides
keywords: Azurophilic Granule; Glycoprotein Biosynthesis; Glycoproteomics; Infection; Infectious Disease; Inflammation; N-linked Glycosylation; Neutrophil; Paucimannosylation; Sputum; β-Hexosaminidase A
references: J Biol Chem. 1995 Jul 21;270(29):17344-9
references: 7615537
references: Can Respir J. 2007 Mar;14(2):99-103
references: 17372637
references: Blood. 2005 Feb 15;105(4):1785-96
references: 15514007
references: Nat Rev Mol Cell Biol. 2012 Jul;13(7):448-62
references: 22722607
references: J Biol Chem. 2010 May 21;285(21):16351-9
references: 20332087
references: Biochim Biophys Acta. 2013 Nov;1833(11):2430-7
references: 23583305
references: Glycobiology. 2012 Nov;22(11):1440-52
references: 22798492
references: J Proteome Res. 2013 Dec 6;12(12):5791-800
references: 24090084
references: Methods Enzymol. 2006;417:359-89
references: 17132514
references: Trends Immunol. 2007 Aug;28(8):340-5
references: 17627888
references: Glycobiology. 2005 May;15(5):463-74
references: 15604090
references: Glycoconj J. 2009 Nov;26(8):975-86
references: 18587645
references: J Biol Chem. 2004 Oct 15;279(42):44123-32
references: 15302890
references: Biochem J. 2003 May 15;372(Pt 1):53-64
references: 12603202
references: J Biol Chem. 2013 Mar 8;288(10):6936-45
references: 23329837
references: J Proteome Res. 2013 Jul 5;12(7):3383-92
references: 23799247
references: Glycobiology. 2001 Jul;11(7):549-56
references: 11447134
references: Nat Med. 2008 Oct;14(10):1088-96
references: 18836458
references: Mol Cell Proteomics. 2005 Oct;4(10):1503-21
references: 15985654
references: Biochimie. 2001 Aug;83(8):703-12
references: 11530201
references: Biochem Biophys Res Commun. 2002 Apr 26;293(1):213-9
references: 12054586
references: J Biol Chem. 2012 Oct 19;287(43):35922-33
references: 22930755
references: Mol Cell Proteomics. 2015 Feb;14(2):277-88
references: 25452313
references: J Biol Chem. 2007 Sep 21;282(38):27825-40
references: 17636254
references: Blood. 2007 Mar 1;109(5):2066-77
references: 17105821
references: Nat Immunol. 2011 Nov;12(11):1035-44
references: 22012443
references: Mol Microbiol. 2006 Sep;61(6):1556-68
references: 16930149
references: J Biol Chem. 1991 Jan 25;266(3):1879-87
references: 1824846
references: Infect Immun. 1991 Oct;59(10):3760-7
references: 1894375
references: Glycobiology. 2005 Jun;15(6):1R-15R
references: 15647514
references: Glycobiology. 2012 Nov;22(11):1465-79
references: 22833316
references: J Proteome Res. 2014 Jan 3;13(1):277-88
references: 24295106
references: J Biol Chem. 2012 Jan 2;287(1):496-503
references: 22074924
references: Anal Chem. 2010 Jul 1;82(13):5598-609
references: 20536156
references: Biochem Soc Trans. 2011 Jan;39(1):349-54
references: 21265802
references: Blood. 2010 Jul 29;116(4):625-7
references: 20410504
references: Biochem Soc Symp. 2002;(69):117-34
references: 12655779
references: Sci Rep. 2013;3:1365
references: 23455567
references: Allergy Asthma Clin Immunol. 2006 Sep 15;2(3):98-108
references: 20525154
references: Nat Protoc. 2012 Jul;7(7):1299-310
references: 22678433
references: Fly (Austin). 2011 Jan-Mar;5(1):18-24
references: 21057214
references: Nat Med. 2014 May;20(5):511-7
references: 24784231
references: Lab Invest. 2000 May;80(5):617-53
references: 10830774
references: Blood. 2013 Jan 17;121(3):510-8
references: 23144171
references: J Biol Chem. 2012 Jul 13;287(29):24356-64
references: 22645129
references: Scand J Immunol. 1991 Dec;34(6):697-712
references: 1749920
references: Science. 2004 Mar 5;303(5663):1532-5
references: 15001782
references: J Biol Chem. 2006 Feb 24;281(8):4867-75
references: 16339150
references: J Proteomics. 2012 Feb 16;75(5):1472-85
references: 22138257
references: J Biol Chem. 2002 Jan 25;277(4):2562-72
references: 11707436
references: J Mol Biol. 2003 Apr 11;327(5):1093-109
references: 12662933
references: Front Immunol. 2014 Aug 25;5:404
references: 25202310
references: Glycobiology. 2006 May;16(5):440-61
references: 16449350
references: Genes Immun. 2005 Jun;6(4):319-31
references: 15789058
references: Biochim Biophys Acta. 2010 Oct;1804(10):2046-53
references: 20621206
references: Immunology. 2009 Mar;126(3):336-45
references: 18710403
references: Glycobiology. 2003 Mar;13(3):217-25
references: 12626421
references: Immunity. 2010 Nov 24;33(5):657-70
references: 21094463
references: Mol Cell Proteomics. 2012 Sep;11(9):571-85
references: 22573871
references: Mol Cell Proteomics. 2013 Oct;12(10):2935-51
references: 23764502
references: Carbohydr Res. 2009 Aug 17;344(12):1391-6
references: 19515361
references: J Leukoc Biol. 1998 Jul;64(1):72-7
references: 9665278
references: Blood. 1997 May 15;89(10):3503-21
references: 9160655
references: Biotechnol Prog. 2004 Jul-Aug;20(4):1273-9
references: 15296462
references: Glycoconj J. 1999 Feb;16(2):109-23
references: 10612411
references: Glycobiology. 2015 Jan;25(1):88-100
references: 25190359
references: J Mol Biol. 2006 Jun 16;359(4):913-29
references: 16698036
references: J Biol Chem. 2006 May 5;281(18):12776-85
references: 16522637
references: J Biol Chem. 2002 Dec 20;277(51):49143-57
references: 12361949
references: Nat Immunol. 2014 Jul;15(7):602-11
references: 24940954
references: Biol Chem. 2012 Aug;393(8):661-73
references: 22944671
references: J Leukoc Biol. 2013 Oct;94(4):711-21
references: 23650620