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• W2674157678 abstract "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. 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. Neutrophils, the most abundant immune cell population in blood, are integral to the human innate immune system by forming a first line of defense against invading pathogens and by assisting in resolving inflammation. Chemically distinct granule compartments, which are characterized by their unique proteome content including an arsenal of antimicrobial proteins and peptides, are found within neutrophils, most notably, the primary (azurophilic), secondary (specific) and tertiary (gelatinase) granules (1.Borregaard N. Sorensen O.E. Theilgaard-Monch K. Neutrophil granules: a library of innate immunity proteins.Trends Immunol. 2007; 28: 340-345Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). Other neutrophilic compartments including the secretory vesicles and the ficolin granules have been reported (2.Rorvig S. Honore C. Larsson L.I. Ohlsson S. Pedersen C.C. Jacobsen L.C. Cowland J.B. Garred P. Borregaard N. Ficolin-1 is present in a highly mobilizable subset of human neutrophil granules and associates with the cell surface after stimulation with fMLP.J. Leukoc. Biol. 2009; 86: 1439-1449Crossref PubMed Scopus (74) Google Scholar). The large family of serine proteases, which includes human neutrophil elastase (HNE)1, proteinase 3, azurocidin, and neutrophil cathepsin G (nCG), are preferentially stored in the azurophilic granules, but also reside in the specific and gelatinase granules (3.Rorvig S. Ostergaard O. Heegaard N.H. Borregaard N. Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane: correlation with transcriptome profiling of neutrophil precursors.J. Leukoc. Biol. 2013; 94: 711-721Crossref PubMed Scopus (126) Google Scholar) and on the cell surfaces of activated neutrophils albeit at lower levels (4.Owen C.A. Campbell M.A. Sannes P.L. Boukedes S.S. Campbell E.J. Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases.J. Cell Biol. 1995; 131: 775-789Crossref PubMed Scopus (317) Google Scholar). These potent serine proteases are serving crucial immune functions central to the diapedesis, chemotaxis, and bactericidal activities of neutrophils including phagocytosis and degranulation (5.Nauseef W.M. Borregaard N. Neutrophils at work.Nat. Immunol. 2014; 15: 602-611Crossref PubMed Scopus (434) Google Scholar). Protective functions of serine proteases were demonstrated in mice by a higher frequency of Mycobacterium tuberculosis infection upon inhibition of the serine protease activity (6.Reece S.T. Loddenkemper C. Askew D.J. Zedler U. Schommer-Leitner S. Stein M. Mir F.A. Dorhoi A. Mollenkopf H.J. Silverman G.A. Kaufmann S.H. Serine protease activity contributes to control of Mycobacterium tuberculosis in hypoxic lung granulomas in mice.J. Clin. Invest. 2010; 120: 3365-3376Crossref PubMed Scopus (57) Google Scholar). This was supported by the observation that serine protease deficient human neutrophils lack bactericidal activity, leaving affected individuals severely immune compromised (7.Gombart A.F. Koeffler H.P. Neutrophil specific granule deficiency and mutations in the gene encoding transcription factor C/EBP(epsilon).Curr. Opin. Hematol. 2002; 9: 36-42Crossref PubMed Scopus (79) Google Scholar). Among the serine proteases, HNE is particularly important in chronic inflammatory lung diseases e.g. cystic fibrosis (CF) (8.Le Gars M. Descamps D. Roussel D. Saussereau E. Guillot L. Ruffin M. Tabary O. Hong S.S. Boulanger P. Paulais M. Malleret L. Belaaouaj A. Edelman A. Huerre M. Chignard M. Sallenave J.M. Neutrophil elastase degrades cystic fibrosis transmembrane conductance regulator via calpains and disables channel function in vitro and in vivo.Am. J. Respir. Crit. Care Med. 2013; 187: 170-179Crossref PubMed Scopus (0) Google Scholar) and in hematological disorders e.g. cyclic and severe congenital neutropenia (9.Tidwell T. Wechsler J. Nayak R.C. Trump L. Salipante S.J. Cheng J.C. Donadieu J. Glaubach T. Corey S.J. Grimes H.L. Lutzko C. Cancelas J.A. Horwitz M.S. Neutropenia-associated ELANE mutations disrupting translation initiation produce novel neutrophil elastase isoforms.Blood. 2014; 123: 562-569Crossref PubMed Scopus (28) Google Scholar, 10.Horwitz M.S. Duan Z. Korkmaz B. Lee H.H. Mealiffe M.E. Salipante S.J. Neutrophil elastase in cyclic and severe congenital neutropenia.Blood. 2007; 109: 1817-1824Crossref PubMed Scopus (174) Google Scholar, 11.Nayak R.C. Trump L.R. Aronow B.J. Myers K. Mehta P. Kalfa T. Wellendorf A.M. Valencia C.A. Paddison P.J. Horwitz M.S. Grimes H.L. Lutzko C. Cancelas J.A. Pathogenesis of ELANE-mutant severe neutropenia revealed by induced pluripotent stem cells.J. Clin. Invest. 2015; 125: 3103-3116Crossref PubMed Scopus (45) Google Scholar). The ELANE gene encoding the monomeric and heavily glycosylated HNE is highly expressed in the promyelocytic stage of cells committed to undergo myeloid lineage differentiation into neutrophils within the bone marrow (12.Kettritz R. Neutral serine proteases of neutrophils.Immunol. Rev. 2016; 273: 232-248Crossref PubMed Scopus (29) Google Scholar). The directly encoded gene product is an inactive preproprotein precursor that is converted to the mature serine protease upon extensive proteolytic processing and post-translational modification (13.Lindmark A. Persson A.M. Olsson I. Biosynthesis and processing of cathepsin G and neutrophil elastase in the leukemic myeloid cell line U-937.Blood. 1990; 76: 2374-2380Crossref PubMed Google Scholar). The 27 amino acid-long signal peptide of HNE is quickly removed by signal peptidases, followed by the addition and processing of N-glycosylation and the removal of the N- and C-terminal propeptides by other peptidases (14.Gullberg U. Lindmark A. Lindgren G. Persson A.M. Nilsson E. Olsson I. Carboxyl-terminal prodomain-deleted human leukocyte elastase and cathepsin G Are efficiently targeted to granules and enzymatically activated in the rat basophilic/mast cell Line RBL.J. Biol. Chem. 1995; 270: 12912-12918Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). It has been proposed that adaptor protein 3 (15.Benson K.F. Li F.Q. Person R.E. Albani D. Duan Z. Wechsler J. Meade-White K. Williams K. Acland G.M. Niemeyer G. Lothrop C.D. Horwitz M. Mutations associated with neutropenia in dogs and humans disrupt intracellular transport of neutrophil elastase.Nat. Genet. 2003; 35: 90-96Crossref PubMed Scopus (132) Google Scholar) and serglycin (16.Niemann C.U. Abrink M. Pejler G. Fischer R.L. Christensen E.I. Knight S.D. Borregaard N. Neutrophil elastase depends on serglycin proteoglycan for localization in granules.Blood. 2007; 109: 4478-4486Crossref PubMed Scopus (70) Google Scholar) direct HNE and other proteins expressed in promyelocytes to the azurophilic granules by timing rather than by specific protein signals/motifs (17.Cowland J.B. Borregaard N. Granulopoiesis and granules of human neutrophils.Immunol. Rev. 2016; 273: 11-28Crossref PubMed Scopus (124) Google Scholar). Mature HNE is predicted to consist of 218 amino acid residues, and harbors four disulfide bonds and four putative N-glycosylation sites (Asn88, Asn124, Asn173, Asn185, numbering based on the preproprotein sequence) (supplemental Fig. S1), leaving the mature protein product with an apparent molecular mass of 25–27 kDa (18.Sinha S. Watorek W. Karr S. Giles J. Bode W. Travis J. Primary structure of human neutrophil elastase.Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 2228-2232Crossref PubMed Scopus (170) Google Scholar). The catalytic activity of HNE relies on its His-Asp-Ser triad, a conserved structural feature mirrored in other serine proteases, in which Ser202 confers the proteolytical activity (19.Korkmaz B. Horwitz M.S. Jenne D.E. Gauthier F. Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases.Pharmacol. Rev. 2010; 62: 726-759Crossref PubMed Scopus (420) Google Scholar). The potent HNE can be inhibited at the site of inflammation by endogenous serine protease inhibitors (serpins) including α1-antitrypsin (A1AT), an abundant plasma N-glycoprotein produced by hepatocytes (20.Lussier B. Wilson A.A. Alpha-1 Antitrypsin:.The Protein. pp. 17–30, Humana Press, New York. 2016; : 17-30Google Scholar). HNE may be released from neutrophils upon stimuli-induced activation by lipopolysaccharide (LPS) or N-formylmethionine-leucyl-phenylalanine (fMLP) (21.Grigorieva D.V. Gorudko I.V. Sokolov A.V. Kostevich V.A. Vasilyev V.B. Cherenkevich S.N. Panasenko O.M. Myeloperoxidase Stimulates Neutrophil Degranulation.Bull. Exp. Biol. Med. 2016; 161: 495-500Crossref PubMed Scopus (18) Google Scholar, 22.Clemmensen S.N. Jacobsen L.C. Rorvig S. Askaa B. Christenson K. Iversen M. Jorgensen M.H. Larsen M.T. van Deurs B. Ostergaard O. Heegaard N.H. Cowland J.B. Borregaard N. Alpha-1-antitrypsin is produced by human neutrophil granulocytes and their precursors and liberated during granule exocytosis.Eur. J. Haematol. 2011; 86: 517-530Crossref PubMed Scopus (0) Google Scholar). In the extracellular environment, HNE serves multiple protective and regulatory roles within the innate immune system. For example, HNE inhibits the growth of Gram-negative bacteria such as Pseudomonas aeruginosa in the airways (23.Hirche T.O. Benabid R. Deslee G. Gangloff S. Achilefu S. Guenounou M. Lebargy F. Hancock R.E. Belaaouaj A. Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa.J. Immunol. 2008; 181: 4945-4954Crossref PubMed Google Scholar) and facilitates the release of anti-inflammatory cortisol from corticosteroid binding globulin (CBG) at inflammatory sites (24.Sumer-Bayraktar Z. Grant O.C. Venkatakrishnan V. Woods R.J. Packer N.H. Thaysen-Andersen M. Asn347 glycosylation of corticosteroid-binding globulin fine-tunes the host Immune response by modulating proteolysis by pseudomonas aeruginosa and neutrophil elastase.J. Biol. Chem. 2016; 291: 17727-17742Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). However, because of its high enzymatic activity, excessive secretion of HNE can damage the connective tissues of the lungs by causing proteolytic degradation of elastin, fibronectin and collagen, as commonly observed in chronic obstructive pulmonary disease and in chronic inflammation (25.Sinden N.J. Baker M.J. Smith D.J. Kreft J.U. Dafforn T.R. Stockley R.A. alpha-1-antitrypsin variants and the proteinase/antiproteinase imbalance in chronic obstructive pulmonary disease.Am. J. Physiol. Lung Cell Mol. Physiol. 2015; 308: L179-190Crossref PubMed Scopus (37) Google Scholar). Imbalanced A1AT-based inhibition may contribute to this uncontrolled proteolysis by HNE in such conditions (26.Hoenderdos K. Condliffe A. The neutrophil in chronic obstructive pulmonary disease.Am. J. Respir. Cell Mol. Biol. 2013; 48: 531-539Crossref PubMed Scopus (192) Google Scholar). HNE is also implicated in the formation of neutrophil extracellular traps (NETs) (27.Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191: 677-691Crossref PubMed Scopus (882) Google Scholar, 28.Metzler K.D. Goosmann C. Lubojemska A. Zychlinsky A. Papayannopoulos V. A myeloperoxidase-containing complex regulates neutrophil elastase release and actin dynamics during NETosis.Cell Reports. 2014; 8: 883-896Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar), but the role(s) of NETs in innate immune defense are not fully elucidated (29.Sorensen O.E. Borregaard N. Neutrophil extracellular traps - the dark side of neutrophils.J. Clin. Invest. 2016; 126: 1612-1620Crossref PubMed Scopus (173) Google Scholar). Despite the established biological significance of HNE, the site-specific structure and function of the HNE N-glycosylation remains undocumented. To the best of our knowledge, only studies using nuclear magnetic resonance and X-ray crystallography have provided qualitative and still incomplete structural insights into the N-glycosylation of HNE. Short mannose-terminating N-glycans were primarily reported on Asn124 and Asn173, whereas Asn88 was generally reported as being unoccupied on HNE (30.Watorek W. Halbeek H. Travis J. The isoforms of human neutrophil elastase and cathepsin G differ in their carbohydrate side chain structures.Bio Chem Hoppe Seyler. 1993; 374: 385-393Crossref PubMed Google Scholar, 31.Hansen G. Gielen-Haertwig H. Reinemer P. Schomburg D. Harrenga A. Niefind K. Unexpected active-site flexibility in the structure of human neutrophil elastase in complex with a new dihydropyrimidone inhibitor.J. Mol. Biol. 2011; 409: 681-691Crossref PubMed Scopus (31) Google Scholar). These otherwise powerful analytical techniques for structural elucidation are, however, typically not suitable for accurate profiling of N-glycoproteins. The distribution of HNE glycoforms, glycosylation site occupancy and the presence of other post-translational modifications on HNE were also not described in these past studies. Functionally, genetic mutations were induced near, but not directly at, the putative N-glycosylation sites of HNE in a myeloid cell line to assess how the local structures around the N-glycosylation sites affect HNE folding, stability and activity (32.Kollner I. Sodeik B. Schreek S. Heyn H. von Neuhoff N. Germeshausen M. Zeidler C. Kruger M. Schlegelberger B. Welte K. Beger C. Mutations in neutrophil elastase causing congenital neutropenia lead to cytoplasmic protein accumulation and induction of the unfolded protein response.Blood. 2006; 108: 493-500Crossref PubMed Scopus (0) Google Scholar). Mutations near Asn88 and Asn124 altered the N-glycan processing, secretion and the proteolytic activity of HNE, but the N-glycosylation structure and function relationship of HNE was not investigated. In fact, given the importance of HNE in mediating a functional host immune response (33.Kawata J. Yamaguchi R. Yamamoto T. Ishimaru Y. Sakamoto A. Aoki M. Kitano M. Umehashi M. Hirose E. Yamaguchi Y. Human neutrophil elastase induce interleukin-10 expression in peripheral blood mononuclear cells through protein kinase C theta/delta and phospholipase pathways.Cell J. 2016; 17: 692-700PubMed Google Scholar, 34.Chawla A. Alatrash G. Philips A.V. Qiao N. Sukhumalchandra P. Kerros C. Diaconu I. Gall V. Neal S. Peters H.L. Clise-Dwyer K. Molldrem J.J. Mittendorf E.A. Neutrophil elastase enhances antigen presentation by upregulating human leukocyte antigen class I expression on tumor cells.Cancer Immunol. Immunother. 2016; 65: 741-751Crossref PubMed Google Scholar), and the increasing appreciation that protein N-glycosylation significantly modulates the function of proteins (35.Salazar V.A. Rubin J. Moussaoui M. Pulido D. Nogues M.V. Venge P. Boix E. Protein post-translational modification in host defense: the antimicrobial mechanism of action of human eosinophil cationic protein native forms.FEBS J. 2014; 281: 5432-5446Crossref PubMed Scopus (12) Google Scholar, 36.Hulsmeier A.J. Tobler M. Burda P. Hennet T. Glycosylation site occupancy in health, congenital disorder of glycosylation and fatty liver disease.Sci. Rep. 2016; 6: 33927Crossref PubMed Scopus (8) Google Scholar), the structure/function relationship of the N-glycosylation of this vital serine protease remains understudied. Herein, we present the first detailed site-specific structural and functional characterization of HNE N-glycosylation from resting and activated human neutrophils from healthy individuals. Structural analyses revealed unusual N-glycans occupying three utilized HNE N-glycosylation sites i.e. Asn88, Asn124, and Asn173. The putative Asn185 site was not glycosylated. It was found that the N-glycosylation of HNE is more likely to contribute to immunomodulatory functions during innate immune processes than interfering directly with the HNE enzymatic activity. These observations are important to increase our understanding of the structure, functional importance and spatiotemporal regulation of central neutrophil proteins carrying unusual N-glycosylation. Human resting neutrophils were isolated from the peripheral blood of a single healthy male donor after informed consent was obtained. The collection, handling and biomolecular analysis of healthy human neutrophils were approved by the Human Research Ethics Committee at Macquarie University, Sydney, Australia (Reference no. 5201500409). Neutrophils were isolated over multiple separate experiments from 40 ml freshly drawn blood collected in EDTA tubes (BD Biosciences, Australia). The donor and the collection volume, time and day were kept constant to reduce unnecessary donor variation. The neutrophils were isolated with polymorphprep density centrifugation (Axis Shield, Norway) followed by hypotonic lysis of the remaining erythrocytes with cold filtered high purity (MilliQ) water. Cell counts (> 1 × 107 cells/ml) and viabilities (>90%) of isolated neutrophils were determined using a Muse cell analyzer (Merck-Millipore, Australia). The purity of the neutrophil cell population (>95%) was estimated based on Wright-Giemsa stained cells prepared using a cytospin centrifuge (Thermo Scientific, Australia). Low abundance (∼2–3%) of other interfering cell types including erythrocytes, basophils and eosinophils were identified. The isolated neutrophils were used immediately for the structural and functional experiments. Enzymatically active human HNE (UniProtKB accession number: P08426) was purified from blood-isolated resting neutrophils from a pool of healthy individuals (Lee BioSolutions, MI, product number 342–40-1) using active site-based affinity and ion exchange liquid chromatography. Upon arrival, HNE was highly pure (>95%) and displayed full structural and functional integrity (i.e. showed high enzyme activity on various substrates) as assessed by SDS-PAGE (Invitrogen, Australia) and immunoblotting with rabbit anti-HNE (1:500, kind gift from Prof Niels Borregaard, University of Copenhagen, Denmark) and by its ability to rapidly cleave CBG (24.Sumer-Bayraktar Z. Grant O.C. Venkatakrishnan V. Woods R.J. Packer N.H. Thaysen-Andersen M. Asn347 glycosylation of corticosteroid-binding globulin fine-tunes the host Immune response by modulating proteolysis by pseudomonas aeruginosa and neutrophil elastase.J. Biol. Chem. 2016; 291: 17727-17742Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). HNE was stored in a soluble form at 1.9 μg/μl in 50 mm sodium acetate and 600 mm sodium chloride buffer, pH 5.5 at 4 °C until use. Protein concentrations were determined at 280 nm absorbance with a percent extinction coefficient (ε1%) of 10 using a Nanodrop spectrophotometer (Thermo Scientific). HNE (20 μg) was proteolytically inactivated with 1.5 mm PMSF, 90 min, 22 °C. Subsequently, the cysteine residues of HNE were reduced using 10 mm dithiothreitol (DTT) in 100 mm ammonium bicarbonate (pH 8.4), 45 min, 56 °C and alkylated using 25 mm iodoacetamide in 100 mm ammonium bicarbonate (pH 8.4) (both final concentrations), 30 min in the dark, 22 °C. The protein was then blotted on a primed 0.45 μm PVDF membrane (Merck-Millipore) and stained with Direct Blue (Sigma-Aldrich, Australia). The stained protein spots were excised, transferred to separate wells in a flat bottom polypropylene 96-well plate (Corning Life Sciences, Australia), blocked with 1% (w/v) polyvinylpyrrolidone in 50% (v/v) methanol and washed with MilliQ water. N-glycans were then released and handled as previously described (37.Jensen P.H. Karlsson N.G. Kolarich D. Packer N.H. Structural analysis of N- and O-glycans released from glycoproteins.Nat. Protoc. 2012; 7: 1299-1310Crossref PubMed Scopus (219) Google Scholar). In brief, enzymatic release was performed using 3.5 U Flavobacterium meningosepticum N-glycosidase F (Roche, Australia) per 20 μg HNE in 10 μl water/well, 16 h, 37 °C. The unstable amino groups (-NH2) of the reducing end GlcNAc residues of N-glycosidase F-released N-glycans were allowed to spontaneously convert to hydroxyl groups (-OH) in weak acid using 100 mm ammonium acetate, pH 5, 1 h, 22 °C to facilitate for subsequent quantitative reduction to glycan alditols. Reduction was carried out using 1 m sodium borohydride in 50 mm potassium hydroxide, 3 h, 50 °C. The reaction was stopped by glacial acetic acid quenching. Dual desalting was then performed in micro-solid phase extraction (SPE) formats using strong cation exchange/C18 (where N-glycans are not retained) and porous graphitised carbon (PGC) (where N-glycans are retained) as stationary phases, respectively. The desalted N-glycans were eluted from the PGC-SPE columns using 40% (v/v) ACN containing 0.1% (v/v) aqueous TFA, dried, and dissolved in 10 μl MilliQ water for N-glycan analysis. Bovine fetuin carrying sialo-N-glycans (Sigma-Aldrich) and the paucimannose-rich nCG (38.Loke I. Packer N.H. Thaysen-Andersen M. Complementary LC-MS/MS-based N-Glycan, N-Glycopeptide, and intact N-glycoprotein profiling reveals unconventional Asn71-glycosylation of human neutrophil cathepsin G.Biomolecules. 2015; 5: 1832-1854Crossref PubMed Scopus (30) Google Scholar) were included as control glycoproteins to ensure efficient N-glycan release, clean-up and analysis. Glycan standards were used to determine some structural aspects of HNE N-glycans. M2 N-glycan isoforms were generated from chicken ovalbumin (Sigma-Aldrich) by sequential exoglycosidase digestion using 1 U Streptococcus pneumoniae β-N-acetylhexosaminidase and 1 U Jack bean α1,3/6-linkage specific mannosidase (both from ProZyme, CA) using buffers and incubation times as recommended by the manufacturer. Synthetically generated and structurally validated M2 N-glycan (Product number MC0420) was obtained from Dextra Laboratories, Reading, UK. The N-glycan standards were reduced and desalted using PGC-SPE as described above prior to PGC-LC-MS/MS analysis. Purified N-glycans were separated and detected by PGC-LC-MS/MS performed on an Agilent 1260 Infinity HPLC connected to an ESI ion trap mass spectrometer (LC/MSD Trap XCT Plus Series 1100, Agilent Technologies, Australia). The N-glycans (3 μl injection volume, ∼200 pmol) were loaded on a PGC HPLC capillary column (Hypercarb KAPPA, 5 μm particle size, 200 Å pore size, 180 μm inner diameter x 100 mm length, Thermo Scientific) and separated using a linear gradient of 0–45% (v/v) ACN/10 mm ammonium bicarbonate over 85 min at a 2 μl/min flow rate. The acquisition range was m/z 300–2200 and detection was performed in negative ionisation polarity mode with data-dependent acquisition. The top-three most abundant precursors in each full scan spectrum were selected for MS/MS using resonance activation (ion trap) CID performed with smart fragmentation (start/end amplitude 30–200%) at 1.0 V and an isolation window of 4 Th with a maximum accumulation time of 200 ms. Smart ion charge control was enabled with a smart parameter setting target of m/z 900. ESI was performed using a capillary voltage of +3.2 kV, a nitrogen drying gas flow of 6 liters/min at 325 °C, and a nitrogen-based nebuliser pressure of 12 psi. Dynamic exclusion was inactivated over the entire LC-MS/MS acquisition period. The mass spectrometer was calibrated using a tune mix (Agilent Technologies). The mass accuracy of the precursor and product ions was typically better than 0.5 Da. The N-glycan mixtures were analyzed in technical triplicates (n = 3). Mass spectra were viewed and inspected using DataAnalysis v4.0 (Bruker Daltonics, Australia). GlycoMod (http://web.expasy.org/glycomod/) and GlycoWorkbench v2.1 assisted in the annotation and visualization of the N-glycans (39.Ceroni A. Maass K. Geyer H. Geyer R. Dell A. Haslam S.M. GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans.J. Proteome Res. 2008; 7: 1650-1659Crossref PubMed Scopus (599) Google Scholar, 40.Cooper C.A. Gasteiger E. Packer N.H. GlycoMod–a software tool for determining glycosylation compositions from mass spectrometric data.Proteomics. 2001; 1: 340-349Crossref PubMed Google Scholar). Approximately 10 μg HNE was reduced and alkylated as described above, but without PMSF-based inactivation to allow HNE to partially “self-digest” to generate appropriate (glyco)peptides for downstream LC-MS/MS analysis. Alkylation was quenched with 30 mm DTT (final concentration) before in-solution proteolytic digestion was carried out using porcine trypsin (sequencing grade, Promega, Australia) at a 1:20 enzyme/substrate ratio (w/w) in 50 mm ammonium bicarbonate, pH 8.4, 18 h, 37 °C. The majority (∼70%) of the resulting peptide mixture was enriched for glycopeptides as described previously, but with a minor modification (41.Mysling S. Palmisano G. Hojrup P. Thayse" @default.
• W2674157678 created "2017-06-30" @default.
• W2674157678 creator A5014554542 @default.
• W2674157678 creator A5033697331 @default.
• W2674157678 creator A5037819491 @default.
• W2674157678 creator A5043680660 @default.
• W2674157678 creator A5090521668 @default.
• W2674157678 date "2017-08-01" @default.
• W2674157678 modified "2023-10-06" @default.
• W2674157678 title "Paucimannose-Rich N-glycosylation of Spatiotemporally Regulated Human Neutrophil Elastase Modulates Its Immune Functions*." @default.
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