Structural Diversity of Human Gastric Mucin Glycans
2017; Elsevier BV; Volume: 16; Issue: 5 Linguagem: Inglês
10.1074/mcp.m117.067983
ISSN1535-9484
AutoresChunsheng Jin, Diarmuid T. Kenny, Emma C. Skoog, Médea Padra, B Adamczyk, Varvara Vitizeva, Anders Thorell, Vignesh Venkatakrishnan, Sara K. Lindén, Niclas G. Karlsson,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoThe mucin O-glycosylation of 10 individuals with and without gastric disease was examined in depth in order to generate a structural map of human gastric glycosylation. In the stomach, these mucins and their O-glycosylation protect the epithelial surface from the acidic gastric juice and provide the first point of interaction for pathogens such as Helicobacter pylori, reported to cause gastritis, gastric and duodenal ulcers and gastric cancer. The rational of the present study was to map the O-glycosylation that the pathogen may come in contact with. An enormous diversity in glycosylation was found, which varied both between individuals and within mucins from a single individual: mucin glycan chain length ranged from 2–13 residues, each individual carried 34–103 O-glycan structures and in total over 258 structures were identified. The majority of gastric O-glycans were neutral and fucosylated. Blood group I antigens, as well as terminal α1,4-GlcNAc-like and GalNAcβ1–4GlcNAc-like (LacdiNAc-like), were common modifications of human gastric O-glycans. Furthemore, each individual carried 1–14 glycan structures that were unique for that individual. The diversity and alterations in gastric O-glycosylation broaden our understanding of the human gastric O-glycome and its implications for gastric cancer research and emphasize that the high individual variation makes it difficult to identify gastric cancer specific structures. However, despite the low number of individuals, we could verify a higher level of sialylation and sulfation on gastric O-glycans from cancerous tissue than from healthy stomachs. The mucin O-glycosylation of 10 individuals with and without gastric disease was examined in depth in order to generate a structural map of human gastric glycosylation. In the stomach, these mucins and their O-glycosylation protect the epithelial surface from the acidic gastric juice and provide the first point of interaction for pathogens such as Helicobacter pylori, reported to cause gastritis, gastric and duodenal ulcers and gastric cancer. The rational of the present study was to map the O-glycosylation that the pathogen may come in contact with. An enormous diversity in glycosylation was found, which varied both between individuals and within mucins from a single individual: mucin glycan chain length ranged from 2–13 residues, each individual carried 34–103 O-glycan structures and in total over 258 structures were identified. The majority of gastric O-glycans were neutral and fucosylated. Blood group I antigens, as well as terminal α1,4-GlcNAc-like and GalNAcβ1–4GlcNAc-like (LacdiNAc-like), were common modifications of human gastric O-glycans. Furthemore, each individual carried 1–14 glycan structures that were unique for that individual. The diversity and alterations in gastric O-glycosylation broaden our understanding of the human gastric O-glycome and its implications for gastric cancer research and emphasize that the high individual variation makes it difficult to identify gastric cancer specific structures. However, despite the low number of individuals, we could verify a higher level of sialylation and sulfation on gastric O-glycans from cancerous tissue than from healthy stomachs. Gastric cancer is the second most common cause of cancer-associated death and fourth most commonly diagnosed cancer worldwide (1.Ferlay J. Shin H.R. Bray F. Forman D. Mathers C. Parkin D.M. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008.Int. J. Cancer. 2010; 127: 2893-2917Crossref PubMed Scopus (13610) Google Scholar). Annually, 0.7 million patients with gastric cancer die globally (2.Carter D. New global survey shows an increasing cancer burden.Am. J. Nurs. 2014; 114: 17Google Scholar). The cancer is associated with glycosylation changes, but how alteration of gastric mucins relates to gastric cancer pathogenesis remains unknown. Despite the protection by mucins and the acidic gastric juice and proteolytic enzymes, the bacterium Helicobacter pylori manage to thrive in the gastric lining, infecting about half of the world's population (3.Cover T.L. Blaser M.J. Helicobacter pylori in health and disease.Gastroenterology. 2009; 136: 1863-1873Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). There is a direct correlation between infection and gastric cancer, where 0.1–3% of infected individuals develop gastric adenocarcinoma or mucosa-associated lymphoid tissue lymphoma and another 10–15% develop symptomatic gastritis or gastric and duodenal ulcers, whereas the majority show no symptoms (4.Suerbaum S. Michetti P. Helicobacter pylori infection.N. Engl. J. Med. 2002; 347: 1175-1186Crossref PubMed Scopus (2170) Google Scholar). In the stomach, MUC5AC and MUC6 are the major secreted mucins, whereas MUC1 is the dominant membrane-associated mucin. MUC5AC is produced by the surface epithelium, whereas MUC6 is secreted from the deep glands of the gastric mucosa (5.De Bolos C. Garrido M. Real F.X. MUC6 apomucin shows a distinct normal tissue distribution that correlates with Lewis antigen expression in the human stomach.Gastroenterology. 1995; 109: 723-734Abstract Full Text PDF PubMed Scopus (253) Google Scholar, 6.Teixeira A. David L. Reis C.A. Costa J. Sobrinho-Simoes M. Expression of mucins (MUC1, MUC2, MUC5AC, and MUC6) and type 1 Lewis antigens in cases with and without Helicobacter pylori colonization in metaplastic glands of the human stomach.J. Pathol. 2002; 197: 37-43Crossref PubMed Scopus (42) Google Scholar). Both MUC5AC and MUC6 are large oligomeric mucins that occur as distinct glycoforms (7.Linden S. Mahdavi J. Hedenbro J. Boren T. Carlstedt I. Effects of pH on Helicobacter pylori binding to human gastric mucins: identification of binding to non-MUC5AC mucins.Biochem. J. 2004; 384: 263-270Crossref PubMed Scopus (83) Google Scholar). In gastric precancerous lesions and cancer, altered expression of MUC5AC, MUC6, MUC2, and MUC5B has been described, with MUC2 being a marker for intestinal metaplasia (8.Buisine M.P. Devisme L. Maunoury V. Deschodt E. Gosselin B. Copin M.C. Aubert J.P. Porchet N. Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. I. Stomach. A relationship to gastric carcinoma.J. Histochem. Cytochem. 2000; 48: 1657-1666Crossref PubMed Scopus (85) Google Scholar, 9.Ilhan O. Han U. Onal B. Celik S.Y. Prognostic significance of MUC1, MUC2 and MUC5AC expressions in gastric carcinoma.Turk. J. Gastroenterol. 2010; 21: 345-352Crossref PubMed Scopus (40) Google Scholar). The gastric surface and foveolar epithelium are formed by a single layer of tall columnar mucin-producing cells that have a basal nucleus below an apical cup of mucin. These cells have a turnover rate of 3–6 days, but the mucus layer produced in these cells have an even shorter life span: the production rate from start of glycosylation until release at the apical side is about 6 h (10.Navabi N. Johansson M.E. Raghavan S. Linden S.K. Helicobacter pylori infection impairs the mucin production rate and turnover in the murine gastric mucosa.Infect. Immun. 2013; 81: 829-837Crossref PubMed Scopus (58) Google Scholar), demonstrating that both the mucin repertoire and glycosylation theoretically can change rapidly. The carbohydrate structures present on mucosal surfaces vary according to cell lineage, tissue location, and developmental stage (11.Linden S.K. Sutton P. Karlsson N.G. Korolik V. McGuckin M.A. Mucins in the mucosal barrier to infection.Mucosal Immunol. 2008; 1: 183-197Crossref PubMed Scopus (778) Google Scholar). The massive O-glycosylation of the mucins protects them from proteolytic enzymes and induces a relatively extended conformation. The dominating type of carbohydrate chains on mucins consist of extended oligosaccharides initiated with N-acetylgalactosamine (GalNAc) linked to a hydroxyl group on serine or threonine, elongated by the formation of the so-called core structures (core 1–8), and followed by the backbone region (type 1 or 2 chain). The chains are terminated by e.g. fucose (Fuc), galactose (Gal of type 1 or 2 chain), GalNAc or sialic acid (Neu5Ac) residues in the peripheral region, forming histo-blood group antigens such as A, B, and H, or Lewis type antigens such as Lewis a (Lea), Leb, Lex, and Ley, as well as sialyl-Lea (sLea) and sLex structures. Immunohistochemical analysis has demonstrated that the Lea and Leb blood group antigens (Le type 1 structures) mainly appear in the surface epithelium, whereas the Lex and Ley antigens (Le type 2 structures) are expressed in mucous, chief and parietal cells of the glands (12.Linden S. Semino-Mora C. Liu H. Rick J. Dubois A. Role of mucin Lewis status in resistance to Helicobacter pylori infection in pediatric patients.Helicobacter. 2010; 15: 251-258Crossref PubMed Scopus (35) Google Scholar). Thus, the Le type-1 structures co-localize with MUC5AC whereas Le type-2 structures co-localize with MUC6 (12.Linden S. Semino-Mora C. Liu H. Rick J. Dubois A. Role of mucin Lewis status in resistance to Helicobacter pylori infection in pediatric patients.Helicobacter. 2010; 15: 251-258Crossref PubMed Scopus (35) Google Scholar), although this distribution is not always distinct (12.Linden S. Semino-Mora C. Liu H. Rick J. Dubois A. Role of mucin Lewis status in resistance to Helicobacter pylori infection in pediatric patients.Helicobacter. 2010; 15: 251-258Crossref PubMed Scopus (35) Google Scholar, 13.Murata K. Egami H. Shibata Y. Sakamoto K. Misumi A. Ogawa M. Expression of blood group-related antigens, ABH, Lewis(a), Lewis(b), Lewis(x), Lewis(y), CA19–9, and CSLEX1 in early cancer, intestinal metaplasia, and uninvolved mucosa of the stomach.Am J Clin Pathol. 1992; 98: 67-75Crossref PubMed Scopus (71) Google Scholar, 14.Taylor D.E. Rasko D.A. Sherburne R. Ho C. Jewell L.D. Lack of correlation between Lewis antigen expression by Helicobacter pylori and gastric epithelial cells in infected patients.Gastroenterology. 1998; 115: 1113-1122Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The carbohydrate structures present depend on the glycosyltransferases expressed in the cells, i.e. by the genotype of the individual. The terminal structures of mucin oligosaccharides are heterogeneous and vary between/within species and even with tissue location within a single individual (12.Linden S. Semino-Mora C. Liu H. Rick J. Dubois A. Role of mucin Lewis status in resistance to Helicobacter pylori infection in pediatric patients.Helicobacter. 2010; 15: 251-258Crossref PubMed Scopus (35) Google Scholar, 15.Chik J.H. Zhou J. Moh E.S. Christopherson R. Clarke S.J. Molloy M.P. Packer N.H. Comprehensive glycomics comparison between colon cancer cell cultures and tumours: implications for biomarker studies.J. Proteomics. 2014; 108: 146-162Crossref PubMed Scopus (48) Google Scholar). Possibly, this structural diversity allows us to cope with diverse and rapidly changing pathogens, as reflected by the observation that susceptibility to specific pathogens differs between people with different histo-blood groups (16.Marionneau S. Cailleau-Thomas A. Rocher J. Le Moullac-Vaidye B. Ruvoen N. Clement M. Le Pendu J. ABH and Lewis histo-blood group antigens, a model for the meaning of oligosaccharide diversity in the face of a changing world.Biochimie. 2001; 83: 565-573Crossref PubMed Scopus (248) Google Scholar). Mucins appear to be the major carrier of aberrant glycosylation in carcinomas, and incomplete glycosylation, leading to expression of Tn and T antigens, and/or sialylation/sulfation are common (15.Chik J.H. Zhou J. Moh E.S. Christopherson R. Clarke S.J. Molloy M.P. Packer N.H. Comprehensive glycomics comparison between colon cancer cell cultures and tumours: implications for biomarker studies.J. Proteomics. 2014; 108: 146-162Crossref PubMed Scopus (48) Google Scholar, 17.Varki A. Kannagi R. Toole B.P. Glycosylation Changes in Cancer.in: Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Bertozzi C.R. Hart G.W. Etzler M.E. Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, The Consortium of Glycobiology Editors, La Jolla, California, Cold Spring Harbor (NY)2009Google Scholar). The sLex and sLea are frequently overexpressed in carcinomas, and expression of these antigens by epithelial carcinomas correlates with tumor progression, metastatic spread and poor prognosis (17.Varki A. Kannagi R. Toole B.P. Glycosylation Changes in Cancer.in: Varki A. Cummings R.D. Esko J.D. Freeze H.H. Stanley P. Bertozzi C.R. Hart G.W. Etzler M.E. Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, The Consortium of Glycobiology Editors, La Jolla, California, Cold Spring Harbor (NY)2009Google Scholar). Mucins from different individuals differ in their effect on H. pylori growth, adhesion and expression of virulence genes (18.Linden S. Mahdavi J. Semino-Mora C. Olsen C. Carlstedt I. Boren T. Dubois A. Role of ABO secretor status in mucosal innate immunity and H. pylori infection.PLoS Pathog. 2008; 4: e2Crossref PubMed Scopus (136) Google Scholar, 19.Skoog E.C. Sjoling A. Navabi N. Holgersson J. Lundin S.B. Linden S.K. Human gastric mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions with host cells.PLoS ONE. 2012; 7: e36378Crossref PubMed Scopus (71) Google Scholar, 20.Linden S. Nordman H. Hedenbro J. Hurtig M. Boren T. Carlstedt I. Strain- and blood group-dependent binding of Helicobacter pylori to human gastric MUC5AC glycoforms.Gastroenterology. 2002; 123: 1923-1930Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and the Leb and α1,4GlcNAc are two structural epitopes that have been shown to participate in regulation of H. pylori growth (21.Kawakubo M. Ito Y. Okimura Y. Kobayashi M. Sakura K. Kasama S. Fukuda M.N. Fukuda M. Katsuyama T. Nakayama J. Natural antibiotic function of a human gastric mucin against Helicobacter pylori infection.Science. 2004; 305: 1003-1006Crossref PubMed Scopus (287) Google Scholar, 22.Skoog E.C. Padra M. Aberg A. Gideonsson P. Obi I. Quintana-Hayashi M.P. Arnqvist A. Linden S.K. BabA dependent binding of Helicobacter pylori to human gastric mucins cause aggregation that inhibits proliferation and is regulated via ArsS.Sci. Rep. 2017; 7: 40656Crossref PubMed Scopus (36) Google Scholar). However, other, yet unknown, glycans may also affect H. pylori. The detailed characterization of O-glycosylation of a given tissue context is crucial for our understanding of its role during pathological and physiological conditions, such as H. pylori infection and gastric carcinogenesis. In addition, the alteration of O-glycosylation during gastric cancer progression, such as metastasis and cancer cell invasion, helps us to understand the control of O-glycosylation in gastric cancer. In this study, O-glycans from gastric adenocarcinoma tumors, normal mucosa of tumor-adjacent stomachs and normal mucosa are characterized. The diversity and alteration in gastric O-glycosylation broaden our understanding of the human gastric O-glycome and its implications for gastric cancer research. Gastric specimens were obtained after informed consent and approval of local ethics committees (Lund University Hospital, Lund, Sweden). Mucins were isolated from frozen gastric specimens as described previously (19.Skoog E.C. Sjoling A. Navabi N. Holgersson J. Lundin S.B. Linden S.K. Human gastric mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions with host cells.PLoS ONE. 2012; 7: e36378Crossref PubMed Scopus (71) Google Scholar). In brief, four of the specimens (P1T, P2T, P3T, and P4T) were from gastric adenocarcinoma tumors (intestinal type) and another three (P5TA, P6TA, and P7TA) were from macroscopically normal mucosa of tumor-adjacent stomachs (Table I). Two of the tumors contained both soluble (S) and insoluble mucins (I, e.g. P1TS and P1TI, in which the insoluble MUC2 mucin was later solubilized by reduction and alkylation) whereas the insoluble fractions from the other tumors did not contain MUC2, MUC5B, MUC6, or MUC5AC (i.e. were considered negative for mucins). The specimens (∼1.5 × 1.5 cm) of normal mucosa isolated from tissues adjacent to gastric tumors (tumor-adjacent, TA) were separated into fundus (F) versus pyloric antrum (A), surface (S) versus gland material (G) according to tissue location, e.g. P5TA-AS and P6TA-FG. In addition, three specimens (P8H, P9H, and P10H) were from the junction between antrum and corpus of patients who underwent elective surgery for morbid obesity. Mucins were isolated by isopycnic density gradient centrifugation from these materials as previously described (23.Nordman H. Davies J.R. Lindell G. de Bolos C. Real F. Carlstedt I. Gastric MUC5AC and MUC6 are large oligomeric mucins that differ in size, glycosylation and tissue distribution.Biochem. J. 2002; 364: 191-200Crossref PubMed Scopus (116) Google Scholar). Gradient fractions containing mucins were pooled together to obtain one sample for each gradient. The presence of MUC5AC, MUC6, MUC2, and MUC5B, as well as Leb, sLea, sLex, and α1,4-GlcNAc, were evaluated in previous study (19.Skoog E.C. Sjoling A. Navabi N. Holgersson J. Lundin S.B. Linden S.K. Human gastric mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions with host cells.PLoS ONE. 2012; 7: e36378Crossref PubMed Scopus (71) Google Scholar).Table IDistribution of O-glycan features on mucins from gastric adenocarcinoma tumor (Tumor), normal mucosa of tumor-adjacent tissue (Norm.tum.adj) and normal tissue (Normal). The relative amounts of the different glycan features are given in percentage (%) in the relation to the total sum of integrated peak areas in the LC-MS chromatogramsSamplesaTen Samples included four specimens (P1-P4) from gastric adenocarcinoma tumors (T), three (P5-P7) from normal mucosa of tumor-adjacent tissues (TA), and three (P8–10) from normal tissue (H). For mucins from tumors, they were divided into soluble (S) and insoluble mucins (I). Mucins from tumor-adjacent tissues were separated into fundus (F) versus pyloric antrum (A), surface (S) versus gland material (G) according to tissue location.Core 2bIdentification of structural epitopes was based on knowledge of gastric glycan biosynthesis and assumptions made on linkage configuration and positions were summarized in material and methods.Core 1I antigenBlood groupH typeA typeB typeAB typeLea/xsLea/xLeb/yFucosylationα1,4-GlcNAcLacdiNAcsTnSialylationSulfationTumorP1TS81.117.91.1B89.2−0.2−0.90.60.190.64.06.30.78.90.8P1TI63.726.80.3B29.3−8.9−10.07.90.669.45.80.73.322.81.1P3TS62.735.12.1H31.2−−−0.7−−31.96.5−−21.37.1P2TS62.922.12.8A17.23.3−−2.72.0−24.38.30.910.659.815.7P4TS63.822.84.7A42.54.5−−4.11.7−47.018.51.211.324.721.1P4TI85.912.71.4A56.120.2−−0.70.4−83.67.35.8−4.2−Norm.tum.adjP6TA-FS84.012.35.4A11.11.3−−6.71.0−18.95.43.20.39.20.3P6TA-FG77.118.89.1A50.618.9−−5.7−0.273.716.51.30.54.70.3P6TA-AS80.016.71.3A69.516.4−−1.0−−86.05.52.4−6.81.3P6TA-AG85.314.722.5A33.36.4−−18.90.8−58.732.1−−14.1−P5TA-AS62.323.40.8AB19.10.94.20.326.023.70.545.51.60.212.567.71.4P5TA-AG68.820.95.5AB22.518.98.73.5−−−53.621.60.8−−−P7TA-AS68.813.2−AB71.7−−−−−−61.0−−−21.533.6P7TA-AG83.417.01.9AB34.950.92.31.314.8−6.193.39.63.60.31.00.4NormalP8H78.019.14.3A50.47.9−−6.00.30.962.23.62.10.99.50.3P9H93.25.44.5B64.8−15.8−7.00.60.588.88.08.01.37.1−P10H75.419.11.1AB46.90.81.20.615.513.11.468.53.92.52.237.61.1a Ten Samples included four specimens (P1-P4) from gastric adenocarcinoma tumors (T), three (P5-P7) from normal mucosa of tumor-adjacent tissues (TA), and three (P8–10) from normal tissue (H). For mucins from tumors, they were divided into soluble (S) and insoluble mucins (I). Mucins from tumor-adjacent tissues were separated into fundus (F) versus pyloric antrum (A), surface (S) versus gland material (G) according to tissue location.b Identification of structural epitopes was based on knowledge of gastric glycan biosynthesis and assumptions made on linkage configuration and positions were summarized in material and methods. Open table in a new tab In situ proximity ligation assay (PLA) was performed with paraffin-embedded sections from human gastric tissues for the detection of proximity of blood group antigens (ABH) and MUC5AC. These samples were obtained after written informed consent (Ersta Diaktioni, Sweden) in conjunction with obesity surgery and they had a normal histology. The Duolink II kit (Olink Bioscience, Uppsala, Sweden) was used according to the manufacturer's instructions. The paraffin-embedded sections were dewaxed and rehydrated. Heat induced antigen retrieval was performed using 10 mm Tris, 1 mm EDTA and 0.05% Tween 20, pH 9.0. The sections were incubated with blocking solution (Olink Bioscience) for 1 h at 37 °C. Primary antibodies against blood type H (monoclonal mouse anti-human blood group H antigen, clone A70-A/A9, at a concentration of 2.5 μg/ml, ThermoFisher Scientific, Waltham, MA), A (monoclonal mouse anti-human blood group A, clone HE-193, dilution 1:80, ThermoFisher Scientific), B (monoclonal mouse anti-human blood group B, clone HEB-29, dilution 1:40, Abcam, Cambridge, UK), and MUC5AC (polyclonal rabbit anti-oligomeric mucus/gel-forming MUC5Ac N-term aa552–567, at a concentration of 5 μg/ml, antibodies-online GmbH, Aachen, Germany) were used and incubated at 4 °C overnight. Antibodies conjugated with oligonucleotides were utilized to examine the proximity for 1 h at 37 °C (Olink Bioscience). Ligation and amplification were performed at 37 °C for 30 min and 90 min, respectively. The cell nuclei were visualized by DAPI. Sections were examined under a Zeiss Imager Z1 Axio fluorescence microscope (Zeiss, Welwyn Garden City, UK). The proximity ligation resulted in bright red fluorescent dots. Images were acquired using a Zeiss Axio cam MRm and the AxioVision Rel 4.8 software. Isolated mucins were dot-blotted onto PVDF membranes (Immobillin P, Millipore), stained with direct blue 71 (Sigma-Adrich) and destained with a solution of 10% acetic acid in 40% ethanol. The O-glycans were released from PVDF membranes as described previously (24.Karlsson N.G. Schulz B.L. Packer N.H. Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn.J. Am. Soc. Mass Spectrom. 2004; 15: 659-672Crossref PubMed Scopus (108) Google Scholar). Released O-glycans were analyzed by liquid-chromatography-mass spectrometry (LC-MS) using a 10 cm × 250 μm I.D. column, prepared in-house, containing 5 μm porous graphitized carbon (PGC) particles (Thermo Scientific). Glycans were eluted using a linear gradient from 0–40% acetonitrile in 10 mm ammonium bicarbonate over 40 min at a flow rate of ∼10 μl/min. The eluted O-glycans were detected using an LTQ mass spectrometer (Thermo Scientific) in negative-ion mode with an electrospray voltage of 3.5 kV, capillary voltage of −33.0 V and capillary temperature of 300 °C. Air was used as a sheath gas and mass ranges were defined depending on the specific structure to be analyzed. The data were processed using Xcalibur software (version 2.0.7, Thermo Scientific). Glycans were annotated from their MS/MS spectra manually and validated by available structures stored in UniCarb-DB database (2015–12 version) (25.Hayes C.A. Karlsson N.G. Struwe W.B. Lisacek F. Rudd P.M. Packer N.H. Campbell M.P. UniCarb-DB: a database resource for glycomic discovery.Bioinformatics. 2011; 27: 1343-1344Crossref PubMed Scopus (107) Google Scholar). The annotated structures were submitted to the UniCarb-DB database and they will be included in the next release at http://unicarb-db.org/references/339. For structural annotation, some assumptions were used in this study: monosaccharides in the reducing end were assumed as GalNAcol; GalNAc was used for HexNAc when identified in blood group A and LacdiNAc sequences, otherwise HexNAc was assumed to be GlcNAc; hexose was interpreted as Gal residues. The presence of core 1–4 has been reported in gastric tissue (15.Chik J.H. Zhou J. Moh E.S. Christopherson R. Clarke S.J. Molloy M.P. Packer N.H. Comprehensive glycomics comparison between colon cancer cell cultures and tumours: implications for biomarker studies.J. Proteomics. 2014; 108: 146-162Crossref PubMed Scopus (48) Google Scholar, 26.Hanisch F.G. Chai W. Rosankiewicz J.R. Lawson A.M. Stoll M.S. Feizi T. Core-typing of O-linked glycans from human gastric mucins. Lack of evidence for the occurrence of the core sequence Gal1–6GalNAc.Eur. J. Biochem. 1993; 217: 645-655Crossref PubMed Scopus (34) Google Scholar, 27.Brockhausen I. Pathways of O-glycan biosynthesis in cancer cells.Biochim. Biophys. Acta. 1999; 1473: 67-95Crossref PubMed Scopus (478) Google Scholar). In this study, reducing end with sequence of Hex-HexNAcol and retention time (RT) shorter than 8 min on PGC column was assumed be to core 1 disaccharide, Hex-(HexNAc-)HexNAcol as core 2 trisaccharide, HexNAc-HexNAcol as core 3 and 5 disaccharides with core 3 having shorter RT on PGC column (28.Jin C. Padra J.T. Sundell K. Sundh H. Karlsson N.G. Linden S.K. Atlantic Salmon Carries a Range of Novel O-Glycan Structures Differentially Localized on Skin and Intestinal Mucins.J. Proteome Res. 2015; 14: 3239-3251Crossref PubMed Scopus (46) Google Scholar), and HexNAc-(HexNAc-)HexNAcol as core 4 trisaccharide. The discovery of core 5 structures (isomeric to core 3) were assumed to be only present as di- and tri-saccharides, and they were validated with RT compared with standards obtained from our previous studies (29.Liu J. Jin C. Cherian R.M. Karlsson N.G. Holgersson J. O-glycan repertoires on a mucin-type reporter protein expressed in CHO cell pools transiently transfected with O-glycan core enzyme cDNAs.J. Biotechnol. 2015; 199: 77-89Crossref PubMed Scopus (27) Google Scholar, 30.Cherian R.M. Jin C. Liu J. Karlsson N.G. Holgersson J. A panel of recombinant mucins carrying a repertoire of sialylated O-glycans based on different core chains for studies of glycan binding proteins.Biomolecules. 2015; 5: 1810-1831Crossref PubMed Scopus (12) Google Scholar). O-glycans with linear cores (core 1, 3, and 5) were distinguished from branched cores (core 2 and 4) based on the presence of [M - H]− − 223 and [M - H]− - C3H8O4 (or 108) in MS/MS of structures with linear core (24.Karlsson N.G. Schulz B.L. Packer N.H. Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn.J. Am. Soc. Mass Spectrom. 2004; 15: 659-672Crossref PubMed Scopus (108) Google Scholar, 28.Jin C. Padra J.T. Sundell K. Sundh H. Karlsson N.G. Linden S.K. Atlantic Salmon Carries a Range of Novel O-Glycan Structures Differentially Localized on Skin and Intestinal Mucins.J. Proteome Res. 2015; 14: 3239-3251Crossref PubMed Scopus (46) Google Scholar, 31.Everest-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: 895-906Crossref PubMed Scopus (101) Google Scholar). Elongation was assumed to occur as N-acetyl-lactosamine units (Hex-HexNAc or Galβ1–4GlcNAcβ1–3). Terminal epitopes corresponding to blood group ABH, Lewis a/x, Lewis b/y and LacdiNAc were assumed based on the sequences detected in their MS/MS spectra (24.Karlsson N.G. Schulz B.L. Packer N.H. Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn.J. Am. Soc. Mass Spectrom. 2004; 15: 659-672Crossref PubMed Scopus (108) Google Scholar, 28.Jin C. Padra J.T. Sundell K. Sundh H. Karlsson N.G. Linden S.K. Atlantic Salmon Carries a Range of Novel O-Glycan Structures Differentially Localized on Skin and Intestinal Mucins.J. Proteome Res. 2015; 14: 3239-3251Crossref PubMed Scopus (46) Google Scholar, 31.Everest-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: 895-906Crossref PubMed Scopus (101) Google Scholar). Terminal HexNAc was assumed to be αGlcNAc, because distal β1,3GlcNAc residues were usually capped with Gal residues as result of highly active galactosyltransferases. Validation of smaller structures (<7 residues) was made by RT comparison with standards (29.Liu J. Jin C. Cherian R.M. Karlsson N.G. Holgersson J. O-glycan repertoires on a mucin-type reporter protein expressed in CHO cell pools transiently transfected with O-glycan core enzyme cDNAs.J. Biotechnol. 2015; 199: 77-89Crossref PubMed Scopus (27) Google Scholar, 30.Cherian R.M. Jin C. Liu J. Karlsson N.G. Holgersson J. A panel of recombinant mucins carrying a repertoire of sialylated O-glycans based on different core chains for studies of glycan binding proteins.Biomolecules. 2015; 5: 1810-1831Crossref PubMed Scopus (12) Google Scholar) and/or MS/MS spectral matching using Unicarb-DB database (25.Hayes C.A. Karlsson N.G. Struwe W.B. Lisacek F. Rudd P.M. Packer N.H. Campbell M.P. UniCarb-DB: a database resource for glycomic discovery.Bioinformatics. 2011; 27: 1343-1344Crossref PubMed Scopus (107) Google Scholar). Larger structures were identified by de novo sequencing of MS/MS spectra, epitope specific fragmentation and biosynthetic pathways (core type and blood group ABH). Proposed structures are depicted using the Symbol Nomenclature for Glycomics (SNFG) (32.Varki A. Cummings R.D. Aebi M. Packer N.H. Seeberger P.H. Esko J.D. Stanley P. Hart G. Darvill A. Kinoshita T. Prestegard J.J. Schnaar R.L. Freeze H.H. Marth J.D. Bertozzi C.R. Etzler M.E. Frank M. Vliegenthart J.F. Lutteke T. Perez S. Bolton E. Rudd P. Paulson J. Kanehisa M. Toukach P. Aoki-Kinoshita K.F. Dell A. Narimatsu H. York W. Taniguchi N. Kornfeld S. Symbol nomenclature for graphical representations of glycans.Glycobiology. 201
Referência(s)