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• W3043669394 abstract "Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called “targeting by timing.” Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, and N- and O-glycans present in each compartment were analyzed by LC–MS. We found abundant paucimannosidic N-glycans and lack of O-glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of “glycosylation by timing” during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function. Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called “targeting by timing.” Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, and N- and O-glycans present in each compartment were analyzed by LC–MS. We found abundant paucimannosidic N-glycans and lack of O-glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of “glycosylation by timing” during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function. Neutrophils are central cells of innate immunity, primarily dedicated to the killing of invading microbes. They are nondividing, short-lived white blood cells that, in large numbers and with impressive specificity, can perform numerous functions. It is well-established that mature neutrophils in circulation contain different granule subsets that harbor distinct proteins and other biomolecules (1Borregaard N. Cowland J.B. Granules of the human neutrophilic polymorphonuclear leukocyte.Blood. 1997; 89 (9160655): 3503-352110.1182/blood.V89.10.3503.3503_3503_3521Crossref PubMed Google Scholar, 2Faurschou M. Borregaard N. Neutrophil granules and secretory vesicles in inflammation.Microbes Infect. 2003; 5 (14613775): 1317-132710.1016/j.micinf.2003.09.008Crossref PubMed Scopus (804) Google Scholar). Sequential mobilization of granules allows for the cells to rapidly change their surface receptor repertoire and release matrix-destroying proteases to facilitate transmigration from the blood, extravasation into infected and inflamed tissues, and phagocytosis of microbes and cellular debris. Therefore, a distinct separation of proteins between the different granules and vesicles is important for neutrophils to appropriately respond to inflammation and infection. The neutrophil granules are sequentially formed during granulopoiesis, the process of neutrophil maturation in the bone marrow (1Borregaard N. Cowland J.B. Granules of the human neutrophilic polymorphonuclear leukocyte.Blood. 1997; 89 (9160655): 3503-352110.1182/blood.V89.10.3503.3503_3503_3521Crossref PubMed Google Scholar). Early during the promyelocyte stage, the azurophil granules (AG) are formed, followed by the specific and gelatinase granules (SG and GG), which develop sequentially during the myelocyte/metamyelocyte and band cell stages, respectively. Finally, the so-called secretory vesicles (SV) are formed from the plasma membrane (PM) of mature neutrophils through endocytosis. Because no subsequent exchange of proteins between fully synthesized compartments are thought to occur, the neutrophil proteins that are synthesized during the different stages of granulopoiesis end up in the corresponding granule type, a process known as “targeting by timing.” This was elegantly exemplified in studies correlating transcriptomics and proteomic data of maturing neutrophils and their granules, SV, and PM (1Borregaard N. Cowland J.B. Granules of the human neutrophilic polymorphonuclear leukocyte.Blood. 1997; 89 (9160655): 3503-352110.1182/blood.V89.10.3503.3503_3503_3521Crossref PubMed Google Scholar, 3Rørvig S. Østergaard 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 (23650620): 711-72110.1189/jlb.1212619Crossref PubMed Scopus (170) Google Scholar, 4Le Cabec V. Cowland J.B. Calafat J. Borregaard N. Targeting of proteins to granule subsets is determined by timing and not by sorting: the specific granule protein NGAL is localized to azurophil granules when expressed in HL-60 cells.Proc. Natl. Acad. Sci. U.S.A. 1996; 93 (8692836): 6454-645710.1073/pnas.93.13.6454Crossref PubMed Scopus (103) Google Scholar). The majority of granule and cell surface proteins are glycosylated (1Borregaard N. Cowland J.B. Granules of the human neutrophilic polymorphonuclear leukocyte.Blood. 1997; 89 (9160655): 3503-352110.1182/blood.V89.10.3503.3503_3503_3521Crossref PubMed Google Scholar). Glycosylation adds structural and functional heterogeneity to proteins by the attachment of oligosaccharides to specific amino acid residues. The N-glycans are attached to amino acid consensus sequences Asn-Xaa-Ser/Thr (where Xaa ≠ Pro), whereas O-glycans (mucin-type, O-GalNAc-type) are attached to Ser and Thr. Specific glycan structures and/or epitopes are involved in multiple aspects of the immune response, including neutrophil function. The glycosylation of neutrophil proteins is diverse and functionally important for the inflammatory response, e.g. by binding to immune regulating lectins such as selectins, siglecs, galectins, C-type lectins, and microbial adhesins (5Phillips M.L. Nudelman E. Gaeta F.C. Perez M. Singhal A.K. Hakomori S. Paulson J.C. ELAM-1 mediates cell adhesion by recognition of a carbohydrate ligand, sialyl-Lex.Science. 1990; 250 (1701274): 1130-113210.1126/science.1701274Crossref PubMed Scopus (1301) Google Scholar, 6Walz G. Aruffo A. Kolanus W. Bevilacqua M. Seed B. Recognition by ELAM-1 of the sialyl-Lex determinant on myeloid and tumor cells.Science. 1990; 250 (1701275): 1132-113510.1126/science.1701275Crossref PubMed Scopus (887) Google Scholar, 7Yamaoka A. Kuwabara I. Frigeri L.G. Liu F.T. A human lectin, galectin-3 (epsilon bp/Mac-2), stimulates superoxide production by neutrophils.J. Immunol. 1995; 154 (7897228): 3479-3487PubMed Google Scholar, 8Carlin A.F. Uchiyama S. Chang Y.C. Lewis A.L. Nizet V. Varki A. Molecular mimicry of host sialylated glycans allows a bacterial pathogen to engage neutrophil Siglec-9 and dampen the innate immune response.Blood. 2009; 113 (19196661): 3333-333610.1182/blood-2008-11-187302Crossref PubMed Scopus (302) Google Scholar, 9Graham S.A. Antonopoulos A. Hitchen P.G. Haslam S.M. Dell A. Drickamer K. Taylor M.E. Identification of neutrophil granule glycoproteins as Lewisx-containing ligands cleared by the scavenger receptor C-type lectin.J. Biol. Chem. 2011; 286 (21561871): 24336-2434910.1074/jbc.M111.244772Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Earlier MS studies documenting protein glycosylation in whole intact neutrophils showed the presence of Galβ1–4GlcNAc, i.e. N-acetyllactosamine chains (LacNAc) linked to lipids or protein (10Babu P. North S.J. Jang-Lee J. Chalabi S. Mackerness K. Stowell S.R. Cummings R.D. Rankin S. Dell A. Haslam S.M. Structural characterisation of neutrophil glycans by ultra sensitive mass spectrometric glycomics methodology.Glycoconj. J. 2009; 26 (18587645): 975-98610.1007/s10719-008-9146-4Crossref PubMed Scopus (59) Google Scholar, 11Karlsson A. Miller-Podraza H. Johansson P. Karlsson K.A. Dahlgren C. Teneberg S. Different glycosphingolipid composition in human neutrophil subcellular compartments.Glycoconj. J. 2001; 18 (11602807): 231-24310.1023/A:1013183124004Crossref PubMed Scopus (12) Google Scholar) and that some of the LacNAc moieties were modified with fucose and/or terminal sialic acid residues, generating epitopes such as Lewis X (Lex; Galβ1–4(Fucα1–3)GlcNAcβ-R) and sialyl-Lewis X (sLex; Neu5Acα2–3Galβ1–4(Fucα1–3)GlcNAcβ-R). These epitopes are the main ligands of the endothelial selectins involved in neutrophil capture and rolling on the endothelium, enabling transmigration to the peripheral tissue (12Foxall C. Watson S.R. Dowbenko D. Fennie C. Lasky L.A. Kiso M. Hasegawa A. Asa D. Brandley B.K. The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewisx oligosaccharide.J. Cell Biol. 1992; 117 (1374413): 895-90210.1083/jcb.117.4.895Crossref PubMed Scopus (654) Google Scholar, 13Nimrichter L. Burdick M.M. Aoki K. Laroy W. Fierro M.A. Hudson S.A. Von Seggern C.E. Cotter R.J. Bochner B.S. Tiemeyer M. Konstantopoulos K. Schnaar R.L. E-selectin receptors on human leukocytes.Blood. 2008; 112 (18579791): 3744-375210.1182/blood-2008-04-149641Crossref PubMed Scopus (108) Google Scholar). Deficiency in the sLex antigen presentation on the neutrophil cell surface causes leukocyte adhesion deficiency 2, associated with recurrent infection, persistent leukocytosis, and severe mental and growth retardation (14Etzioni A. Frydman M. Pollack S. Avidor I. Phillips M.L. Paulson J.C. Gershoni-Baruch R. Recurrent severe infections caused by a novel leukocyte adhesion deficiency.N. Engl. J. Med. 1992; 327 (1279426): 1789-179210.1056/NEJM199212173272505Crossref PubMed Scopus (444) Google Scholar, 15Etzioni A. Harlan J.M. Pollack S. Phillips L.M. Gershoni-Baruch R. Paulson J.C. Leukocyte adhesion deficiency (LAD) II: a new adhesion defect due to absence of sialyl Lewis X, the ligand for selectins.Immunodeficiency. 1993; 4 (7513226): 307-308PubMed Google Scholar). The interest in the roles played by Lex and sLex antigens in cell recruitment has resulted in more specific analyses of these neutrophil epitopes, on a subcellular level. Detailed analyses of glycan structures of neutrophil granule glycoproteins have been carried out (9Graham S.A. Antonopoulos A. Hitchen P.G. Haslam S.M. Dell A. Drickamer K. Taylor M.E. Identification of neutrophil granule glycoproteins as Lewisx-containing ligands cleared by the scavenger receptor C-type lectin.J. Biol. Chem. 2011; 286 (21561871): 24336-2434910.1074/jbc.M111.244772Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 16Lucka L. Fernando M. Grunow D. Kannicht C. Horst A.K. Nollau P. Wagener C. Identification of Lewis x structures of the cell adhesion molecule CEACAM1 from human granulocytes.Glycobiology. 2005; 15 (15317738): 87-10010.1093/glycob/cwh139Crossref PubMed Scopus (45) Google Scholar, 17Poland D.C. García Vallejo J.J. Niessen H.W. Nijmeyer R. Calafat J. Hack C.E. Van Het Hof B. Van Dijk W. Activated human PMN synthesize and release a strongly fucosylated glycoform of α1-acid glycoprotein, which is transiently deposited in human myocardial infarction.J. Leukoc. Biol. 2005; 78 (15647324): 453-46110.1189/jlb.1004566Crossref PubMed Scopus (29) Google Scholar, 18Theilgaard-Mönch K. Jacobsen L.C. Rasmussen T. Niemann C.U. Udby L. Borup R. Gharib M. Arkwright P.D. Gombart A.F. Calafat J. Porse B.T. Borregaard N. Highly glycosylated α1-acid glycoprotein is synthesized in myelocytes, stored in secondary granules, and released by activated neutrophils.J. Leukoc. Biol. 2005; 78 (15941779): 462-47010.1189/jlb.0105042Crossref PubMed Scopus (41) Google Scholar). Although glycosylation is known to be essential to neutrophil function (12Foxall C. Watson S.R. Dowbenko D. Fennie C. Lasky L.A. Kiso M. Hasegawa A. Asa D. Brandley B.K. The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewisx oligosaccharide.J. Cell Biol. 1992; 117 (1374413): 895-90210.1083/jcb.117.4.895Crossref PubMed Scopus (654) Google Scholar, 13Nimrichter L. Burdick M.M. Aoki K. Laroy W. Fierro M.A. Hudson S.A. Von Seggern C.E. Cotter R.J. Bochner B.S. Tiemeyer M. Konstantopoulos K. Schnaar R.L. E-selectin receptors on human leukocytes.Blood. 2008; 112 (18579791): 3744-375210.1182/blood-2008-04-149641Crossref PubMed Scopus (108) Google Scholar, 19Brazil J.C. Sumagin R. Cummings R.D. Louis N.A. Parkos C.A. Targeting of neutrophil Lewis X blocks transepithelial migration and increases phagocytosis and degranulation.Am. J. Pathol. 2016; 186 (26687991): 297-31110.1016/j.ajpath.2015.10.015Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), a comprehensive mapping of the granule and cell surface glycome is still lacking even of healthy resting (nonactivated) neutrophils. Recently, we have identified paucimannosidic glycans (Man1–3GlcNAc2Fuc0–1) carried by glycoproteins in the sputum of cystic fibrosis patients (20Venkatakrishnan V. Thaysen-Andersen M. Chen S.C. Nevalainen H. Packer N.H. Cystic fibrosis and bacterial colonization define the sputum N-glycosylation phenotype.Glycobiology. 2015; 25 (25190359): 88-10010.1093/glycob/cwu092Crossref PubMed Scopus (29) Google Scholar). These glycoproteins were traced back to the AG of human neutrophils (21Thaysen-Andersen M. Venkatakrishnan V. Loke I. Laurini C. Diestel S. Parker B.L. Packer N.H. Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum.J. Biol. Chem. 2015; 290 (25645918): 8789-880210.1074/jbc.M114.631622Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), for example, neutrophil elastase, where the unusually truncated glycans were shown to influence the binding of elastase to mannose-binding lectin and ɑ1-antitrypsin often present within inflamed tissues (22Loke I. Østergaard O. Heegaard N.H.H. Packer N.H. Thaysen-Andersen M. Paucimannose-rich N-glycosylation of spatiotemporally regulated human neutrophil elastase modulates its immune functions.Mol. Cell. Proteomics. 2017; 16 (28630087): 1507-152710.1074/mcp.M116.066746Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Herein, we provide a comprehensive characterization of the N- and O-glycans in neutrophil granules, isolated using subcellular fractionation and analyzed by porous graphitized carbon (PGC)–LC–MS/MS. We show vast differences in glycosylation between different granules. By building on the targeting-by-timing hypothesis, the granule-specific glycan differences therefore suggest that the glycosylation machinery undergoes alterations during granulopoiesis. The differences in glycosylation between granules could impact the immune modulating functions of neutrophils during inflammation. Subcellular organelles of healthy resting neutrophils were first separated by two-layer Percoll gradients into three fractions; AG, SG + GG, and SV + PM (Fig. 1a). Organelle lysis and extraction and solubilization of organelle proteins were followed by enzymatic release of N-glycans, and MS analyses enabled the unambiguous identification of 69 unique N-glycans. The obtained profiles between the three fractions differed vastly from each other (Fig. 1b). Paucimannosidic and complex N-glycans were predominantly identified in AG and SG + GG, respectively, whereas SV + PM contained oligomannose and complex glycans, at similar levels (40–50%). The analysis identified 36 N-glycans in AG, 60 in SG + GG, and 61 in SV + PM. Of the total 69 N-glycans, 27 were present in all organelles, and SG + GG and SV + PM were mostly similar, sharing 50 N-glycans (Fig. 1c). These qualitative differences were corroborated also on the quantitative level (Fig. 1d). Hence, the different organelles all had unique sets of glycan structures that were in some parts overlapping. The detailed list of N-glycans and their relative abundances in each fraction is found in Table S1. The hallmark of AG was the presence of paucimannosidic glycans, with a relative abundance of 49.4 ± 5.8% of all N-glycans. In contrast, only 18.7 ± 5.2% and 4.9 ± 0.9%, respectively, of all N-glycans were paucimannosidic in SG + GG and SV + PM (Fig. 1d). Six different paucimannosidic glycans were identified in AG, with the most abundant (65% of the paucimannose glycans) containing two mannoses and a core fucose (M2F; m/z 895.31−) (Fig. 2a and Fig. S1). In fact, within the class of paucimannosidic-type N-glycans, the M2F was clearly the most abundant paucimannosidic glycan species in all organelles (Fig. 2a), but the overall abundance of M2F in SV + PM and SG + GG was <5% of total N-glycans as compared with 40% in the AG. Only the α1,6-isomer of the M2F glycan was observed in agreement with our previous reports (21Thaysen-Andersen M. Venkatakrishnan V. Loke I. Laurini C. Diestel S. Parker B.L. Packer N.H. Human neutrophils secrete bioactive paucimannosidic proteins from azurophilic granules into pathogen-infected sputum.J. Biol. Chem. 2015; 290 (25645918): 8789-880210.1074/jbc.M114.631622Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 22Loke I. Østergaard O. Heegaard N.H.H. Packer N.H. Thaysen-Andersen M. Paucimannose-rich N-glycosylation of spatiotemporally regulated human neutrophil elastase modulates its immune functions.Mol. Cell. Proteomics. 2017; 16 (28630087): 1507-152710.1074/mcp.M116.066746Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Another striking feature of AG is the high abundance of core fucosylation (80.4%) as compared with antenna fucosylation (19.6%). Oligomannose and complex type N-glycans predominate in the SV + PM fraction, with a relative abundance of 46.1 ± 3.7% and 49.1 ± 3.2%, respectively (Fig. 1, b and d). Oligomannose N-glycans in the SV + PM fraction showed low abundance of Man4 glycan and a varied distribution of Man5 to Man9 glycans (Fig. 2b). Oligomannose glycans were present also in AG and SG + GG, however at much lower levels (9.7 ± 0.7% and 19.9 ± 4.2%, respectively). The distribution between Man4 to Man9 glycans was similar in these organelles as in SV + PM (Fig. 2b). Complex N-glycans were observed in all fractions, with a higher abundance in the SG + GG (61.4 ± 9.5% of all N-glycans) as compared with the AG (40.7 ± 6.5%) and SV + PM (49.1 ± 3.2%) (Fig. 1d). Conventional glycans carrying single LacNAcs on both antennae were observed in AG, albeit in lower abundance (5.8 ± 1.8%) as compared with the other fractions. In contrast, one of the key glycosylation features in SG + GG and SV + PM was the presence of unusual, very-high-molecular-mass complex glycans extended with repeating LacNAc units (Fig. 3a). Because these glycans were characterized by broad peaks of extracted precursor ions caused by unresolved multiple isomers, their abundances were not considered during our initial analysis. Apart from oligomannose structures, the glycan profile of SV + PM was similar to that of the SG + GG with elongated complex N-glycans (Fig. S2). In SG + GG, complex-type N-glycans were predominantly biantennary fucosylated glycans with or without terminal sialic acid. The most abundant glycan (m/z 1111.92−) identified in SG + GG was a biantennary monosialylated and difucosylated glycan with both core and a single antenna fucosylation (Fig. 3a). The identified sialylated glycans were found to be mainly α2,6 linked to galactose on the 3′ arm and α2,3 linked forming sLex on the 6′ arm. In conclusion, complex N-glycans with LacNAc repeats were abundant in SG + GG and SV + PM but absent from AG. The structures of the extended LacNAcs were confirmed based on their composition related to their molecular mass, along with supporting PGC–LC retention time data and MS/MS spectral data that concertedly were able to confirm the sequence and branch points of the extended structures. Spectra were manually annotated for B-/Y- and C-/Z- ion series as well as diagnostic ions for various glycoepitopes (23Everest-Dass A.V. Abrahams J.L. Kolarich D. Packer N.H. Campbell M.P. Structural feature ions for distinguishing N- and O-linked glycan isomers by LC–ESI–IT MS/MS.J. Am. Soc. Mass Spectrom. 2013; 24 (23605685): 895-90610.1007/s13361-013-0610-4Crossref PubMed Scopus (101) Google Scholar). A representative MS/MS fragmentation of a four-LacNAc-containing glycan at m/z 15502− is shown in Fig. 3b. The presence of various fragment ions, including m/z 1039.52−, 1213.22−, 1294.82−, 1469.02−, 1713.41−, and 1917.41− and D-ions of 1692.41− and 1710.41−, suggests exclusive elongation of 6′ arm rather than 3′ arm elongation (24Harvey D.J. Fragmentation of negative ions from carbohydrates: part 3. Fragmentation of hybrid and complex N-linked glycans.J. Am. Soc. Mass Spectrom. 2005; 16 (15862766): 647-65910.1016/j.jasms.2005.01.006Crossref PubMed Scopus (209) Google Scholar, 25Harvey D.J. Royle L. Radcliffe C.M. Rudd P.M. Dwek R.A. Structural and quantitative analysis of N-linked glycans by matrix-assisted laser desorption ionization and negative ion nanospray mass spectrometry.Anal. Biochem. 2008; 376 (18294950): 44-6010.1016/j.ab.2008.01.025Crossref PubMed Scopus (166) Google Scholar) and as opposed to branched tri- or tetra-antennary glycans. These fragment ions were observed in the MS/MS fragmentation spectra of other glycans as well (Fig. S3), which support and confirm our assessment. Furthermore, the monosaccharide composition of glycans with additional LacNAc repeats contains either one or two terminal sialic acids, supporting the presence of elongated biantennary glycans instead of higher branched glycans. Broad signal peaks of higher m/z values suggest that tri- and tetra-antennary glycans elongated with LacNAc units may also be present but at a lower abundance. Information about the degree of LacNAc extensions could be obtained from the combined intensities of the broad LC peaks, which corresponds to structures potentially carrying three to ten LacNAc units (Fig. S4, top panel). It could be seen that structures containing six to eight LacNAc units were the most abundant in both SG + GG and SV + PM. Simple extrapolation of the data suggests that structures even longer than 10 LacNAc units could be present in both of these fractions, but they were due to structural ambiguity not included here. Fucosylation of the GlcNAc moiety in the LacNAc chains, building Lewis type epitopes, was observed in the SG + GG and SV + PM fractions. It should be noted that not all LacNAcs in an antenna were fucosylated. For the high-molecular-mass glycans, we calculated the number of Lewis epitopes per structure and their corresponding relative intensities (Fig. S4, bottom panel). Simple structures containing one to three Lewis epitopes were more abundant than structures containing higher numbers of fucose. Structures were found to be decorated with a maximum of seven fucose residues. The data also show that the amount of fucosylation per LacNAc was decreasing with the increasing number of Lewis epitopes. When analyzing the fractions for Le epitopes by immunoblotting analyses, the Lex antigens were mainly found in the SG + GG fraction, whereas sLex epitopes were detected predominantly in the SV + PM fraction (Fig. 3c). Using confocal microscopy, the presence of Lex was detected in intracellular granules, whereas sLex was mainly localized to vesicles close to the cytoplasmic membrane, suggesting that sLex is carried by glycans that are associated with the SV + PM (Fig. 3d). Overall, these data show that neutrophils extensively express complex N-glycans with elongated LacNAc repeats that are decorated with fucose (Lex; in SG + GG) and capped with sialic acid (sLex; in SV + PM). Using crude granule separation, the above have demonstrated that the neutrophil granules, in addition to their difference in size, density, and protein composition, also differs in their glycan composition (Fig. 1, b and d) (3Rørvig S. Østergaard 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 (23650620): 711-72110.1189/jlb.1212619Crossref PubMed Scopus (170) Google Scholar, 26Lominadze G. Ward R.A. Klein J.B. McLeish K.R. Proteomic analysis of human neutrophils.Methods Mol. Biol. 2006; 332 (16878704): 343-35610.1385/1-59745-048-0:343PubMed Google Scholar) and indicated some peculiar N-glycosylation features in the SG + GG fraction. We wanted to investigate the granule-specific glycosylation further using a higher resolution granule separation technique. The SG and GG fractions were separated using a three-layer Percoll gradient (Fig. 4a) and analyzed for high molecular mass N-glycans. Mass spectra of the N-glycans in the m/z 1200–2000 region revealed an enrichment of triply charged ions in the GG, whereas doubly charged ions were more abundant in the SG (Fig. 4b). It is important to note that the detection of more high-molecular-mass glycans in the isolated GG fraction (three-layer gradient) as compared with in the combined SG + GG fraction (two-layer gradient) is a consequence of absence of SG in the GG fraction. Consequently, the precursor ion (m/z 1933.73−) corresponding to eleven LacNAcs, four fucoses, and one sialic acid (Δmassof-0.016Da), the longest glycan detected, was identified only in the GG fraction of the three-layer gradient. The relative intensities of glycans, based on their predicted monosaccharide compositions, were grouped into different mass ranges. It is evident that the relative intensities of glycans containing five to eight LacNAcs per structure were more abundant in SG as compared with GG (Fig. 4c). In contrast, structures containing nine or more LacNAcs were more abundant in the GG fraction. Taken together, the data strongly suggest that the glycans in GG are further elongated and larger than those in the SG. The glycans identified in the granule fractions may originate either from the granule membrane or luminal (i.e. soluble) proteins. For SV, the lumen contains plasma proteins taken up during invagination of the plasma membrane during neutrophil terminal differentiation (27Borregaard N. Kjeldsen L. Rygaard K. Bastholm L. Nielsen M.H. Sengeløv H. Bjerrum O.W. Johnsen A.H. Stimulus-dependent secretion of plasma proteins from human neutrophils.J. Clin. Invest. 1992; 90 (1378856): 86-9610.1172/JCI115860Crossref PubMed Scopus (94) Google Scholar). To address which of the identified glycans are displayed on membrane-associated proteins, the membrane protein extracts were separated from the soluble protein extracts for each granule population (AG, SG, and GG) and the SV + PM obtained from the three-layered Percoll isolation method. Paucimannosidic glycans were enriched in the AG membrane protein fraction, whereas oligomannose glycans were enriched in the membrane fraction of SG and SV + PM (Fig. 5). In contrast, complex glycans were observed in high abundance on the soluble protein fraction in all granules. Apart from N-glycan analysis, proteins were also subjected to reductive β-elimination, after which the released O-glycans were analyzed. Notably, no O-glycans were detected in the AG, whereas SG + GG (Fig. 6a) and SV + PM (Fig. S5) demonstrated the presence of similar O-glycans, with a total of 17 O-glycans identified. The most abundant O-glycans were mono- and disialylated core 1 and core 2 glycans (Fig. 6a). Analogous to the LacNAc extension of the N-glycans, only one of the branches was elongated with LacNAcs on O-glycans. Sialic acid linked to galactose was found on the 3′ arm but not on the 6′ arm, whereas the fucose moiety was located on the 6′ arm together with LacNAc extensions as elucidated using molecular mass, PGC–LC retention time, and MS/MS fragmentation (Fig. 6b). Similar to N-glycans, O-glycans with Lex epitopes were identified, with fucosylation on 6′ arm and sialylation capping on the other arm (Fig. 6b). In conclusion, neutrophil granule proteins are modified with core 1 and core 2 O-glycans car" @default.
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• W3043669394 title "Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery" @default.
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