High Throughput Isolation and Glycosylation Analysis of IgG–Variability and Heritability of the IgG Glycome in Three Isolated Human Populations

• DOI: 10.1074/mcp.m111.010090
• Publication Date: 2011-06-09T04:32:13Z
• Authors: Maja Pučić; Ana Knežević; Jana Vidič; Barbara Adamczyk; Mislav Novokmet; Ozren Polašek; Olga Gornik; Sandra Šupraha-Go...; Mark R. Wormald; Irma Redžić; Harry Campbell; Alan Wright; Nicholas D. Hastie; James F. Wilson; Igor Rudan; Manfred Wuhrer; Pauline M. Rudd; Djuro Josić; Gordan Lauc
• Keywords: Glycome; Fucosylation; N-linked glycosylation

All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity therapeutic antibodies intravenous immunoglobulins. We have developed a novel 96-well protein monolithic plate used it to rapidly isolate from plasma 2298 individuals three isolated human populations. N-glycans were released by PNGase F, labeled 2-aminobenzamide analyzed hydrophilic interaction chromatography fluorescence detection. The majority structural features glycome consistent previous studies, but sialylation was somewhat higher than reported previously. Sialylation particularly prominent core fucosylated glycans containing two galactose residues bisecting GlcNAc where median level nearly 80%. Very high variability between observed, approximately times total glycome. For example, neutral without fucose varied 1.3 19%, difference that significantly affects effector functions natural antibodies, predisposing or protecting particular diseases. Heritability generally 30 50%. individual's age associated significant decrease increase GlcNAc, whereas other functional elements glycosylation did not change much age. Gender an important predictor for any glycan. An observation is competition glycosyltransferases, occurs vitro, appear be relevant vivo, indicating final glycan structures are simple result competing enzymatic activities, carefully regulated outcome designed meet prevailing physiological needs. Glycosylation widespread post-translational modification capable producing changes proteins. Contrary N-glycan structure, essential multicellular life (1Marek K.W. Vijay I.K. Marth J.D. A recessive deletion GlcNAc-1-phosphotransferase gene results peri-implantation embryonic lethality.Glycobiology. 1999; 9: 1263-1271Crossref PubMed Scopus (118) Google Scholar), mutations genes involved modifications antennae common cause large part individual phenotypic variations exist humans organisms. membrane receptors modulates adaptive properties cell communication its environment (2Dennis J.W. Lau K.S. Demetriou M. Nabi I.R. Adaptive Regulation at Cell Surface N-Glycosylation.Traffic. 2009; 10: 1569-1578Crossref (163) Scholar). Deregulation wide range diseases, including cancer, diabetes, cardiovascular, congenital, immunological infectious disorders (3Crocker P.R. Paulson J.C. Varki A. Siglecs roles immune system.Nat. Rev. Immunol. 2007; 7: 255-266Crossref (1416) Scholar, 4Marth Grewal P.K. Mammalian immunity.Nat. 2008; 8: 874-887Crossref (529) 5Ohtsubo K. cellular mechanisms health disease.Cell. 2006; 126: 855-867Abstract Full Text PDF (2092) Variations great significance because has been demonstrated structure function polypeptide parts glycoproteins (6Skropeta D. effect on enzyme activity.Bioorg. Med. Chem. 17: 2645-2653Crossref (142) example this type regulation (IgG). Each heavy chain carries single covalently attached bi-antennary highly conserved asparagine 297 residue each CH2 domains Fc region molecule. oligosaccharides structurally stability antibody (7Kobata N-linked sugar chains G: unique pattern, roles.Biochim. Biophys. Acta. 1780: 472-478Crossref (88) In addition, 15–20% normal also bear complex variable regions light chain, both (8Jefferis R. recombinant therapeutics.Biotechnol. Prog. 2005; 21: 11-16Crossref (445) 9Zhu Ottensmeier C.H. Du M.Q. McCarthy H. Stevenson F.K. Incidence potential sites distinguishes subsets Burkitt's lymphoma mucosa-associated lymphoid tissue lymphoma.Br. J. Haematol. 2003; 120: 217-222Crossref (58) Decreased galactosylation rheumatoid arthritis over 25 years ago (10Parekh R.B. Dwek R.A. Sutton B.J. Fernandes D.L. Leung Stanworth Rademacher T.W. Mizuochi T. Taniguchi Matsuta Association primary osteoarthritis pattern serum IgG.Nature. 1985; 316: 452-457Crossref (989) Scholar) numerous subsequent studies revealed number consequences alterations glycans. addition sialic acids dramatically role IgGs converting them pro-inflammatory into anti-inflammatory agents (11Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory resulting sialylation.Science. 313: 670-673Crossref (1384) 12Anthony R.M. glycan: sialylated Fcs.J Clin Immunol Suppl. 2010; 1: S9-14Crossref (242) Another glycans, core, interferes binding FcγRIIIa dampens ability destroy target cells through dependent cell-mediated cytotoxicity (ADCC) 1The abbreviations are: (13Nimmerjahn Fcgamma as regulators responses.Nat Rev 34-47Crossref (2061) 14Ferrara C. Stuart Sondermann P. Brünker Umana carbohydrate FcgammaRIIIa Asn-162. element required affinity non-fucosylated glycoforms.J. Biol. 281: 5032-5036Abstract (296) Lack enhances clinical efficacy monoclonal exert ADCC mediated killing (15Shinkawa Nakamura Yamane N. Shoji-Hosaka E. Kanda Sakurada Uchida Anazawa Satoh Yamasaki Hanai Shitara absence presence N-acetylglucosamine IgG1 complex-type shows critical enhancing antibody-dependent cytotoxicity.J. 278: 3466-3473Abstract (1135) 16Iida S. Misaka Inoue Shibata Nakano Yamane-Ohnuki Wakitani Yano Nonfucosylated can evade inhibitory FcgammaRIIIa.Clin Cancer Res. 12: 2879-2887Crossref (179) 17Preithner Elm Lippold Locher Wolf da Silva A.J. Baeuerle P.A. Prang N.S. High concentrations needed compensate inhibition excess endogenous G.Mol. 43: 1183-1193Crossref (106) However, despite undisputed importance IgGs, scale study identifies heritability populations attempted. One major bottlenecks proteomics glycomics purification samples. Affinity liquid widely used, they versatile techniques purpose. combination supports exhibits many advantageous when compared conventional particulate (18Barut Podgornik Urbas L. Gabor B. Brne Vidic Plevcak Strancar Methacrylate-based short columns: enabling tools rapid efficient analyses biomolecules nanoparticles.J. Sep. Sci. 31: 1867-1880Crossref (47) 19Josić Buchacher Application monoliths fast conversion.J. Biochem. Methods. 2001; 49: 153-174Crossref (104) 20Jungbauer Hahn Polymethacrylate preparative industrial separation biomolecular assemblies.J. Chromatogr. 1184: 62-79Crossref (190) 21Roberts M.W. Ongkudon C.M. Forde G.M. Danquah M.K. Versatility polymethacrylate chromatographic biomolecules.J. 32: 2485-2494Crossref (48) 22Podgornik Convective Interaction Media (CIM)–short layer stationary phases.Biotechnol. Annu. 11: 281-333Crossref (57) Monoliths continuous phases cast piece very interconnected pores (23Barut media 28: 1876-1892Crossref (61) comparison transferred diffusion, porosity allows convective mass transport. This makes resolution dynamic capacity practically independent flow rate (24Brne Bencina Peterka Fast M using columns.J. 1144: 120-125Crossref (33) 25Iberer G. Jungbauer biopolymers: fourth generation sorbents.LC-GC. : 998-1005Google 26Urbas Barut Strlic Petric T.C. Depletion high-abundance proteins pseudo-affinity column.J. 1216: 2689-2694Crossref (32) 27Vlakh E.G. Tennikova T.B. Applications polymethacrylate-based high-performance chromatography.J. 2637-2650Crossref (120) rates low pressure drop enable processing volumes mixtures (28Tennikova Reusch Short beds: history introduction field.J. 1065: 13-17Crossref (53) monoliths, specifically poly(glycidyl methacrylate-co-ethylene dimethacrylate), possess all above mentioned characteristics supports. known good mechanical strength, pH resistance, surface area, porosity, attachment ligands epoxy groups (29Tetala K.K. van Beek T.A. Bioaffinity supports.J. 33: 422-438Crossref (92) most commonly bioaffinity isolation (30Berruex L.G. Freitag Comparison immobilized group specific performance monolith Pharm. Biomed. Anal. 2000; 24: 95-104Crossref (72) 31Gupalova T.V. Lojkina O.V. Pàlàgnuk V.G. Totolian A.A. Quantitative investigation different forms means 2002; 949: 185-193Crossref (59) 32Burnouf Goubran Radosevich technology extracorporeal plasmapheresis large-scale fractionation plasma.J. B Appl. 1998; 715: 65-80Crossref (23) four subclasses strongly bind fragments. Here we present development application Protein throughput first population Glycidyl methacrylate, ethylene dimethacrylate, cyclohexanol, 1-dodecanol purchased Sigma-Aldrich (St. Louis, MO). Photoinitiator CIBA (Basel, Switzerland) GE Healthcare (Uppsala, Sweden). Sodium acetate, sulfuric acid, hydrochloric acid (37%) obtained Merck (Darmstadt, Germany). buffers filtered 0.45 μm pore size filter composed Sartolon polyamide (Sartorius, Goettingen, plates frits, mean 36 microns, Chromacol (Welwyn Garden City, United Kingdom). Chemicals buffer preparations (phosphate buffered saline (PBS), Tris, HCl, NaOH, formic ammonium bicarbonate, propan-2-ol) Fisher Scientific (Pittsburgh, PA) Sigma-Aldrich. running SDS-PAGE Invitrogen (Carlsbad, CA). dl-dithiothreitol, iodoacetamide, persulfate, 2-aminobenzamide, sodium cyanoborohydride, acetic dimethyl sulfoxide ultra pure water (Purite Fusion 40 system, Purite Ltd., Thame, UK) throughout. based samples respondents who residents Croatian Adriatic islands Vis Korčula Northern Scottish Orkney Islands recruited within larger genetic epidemiology program sought investigate map influencing disease traits genetically (33Rudan I. Campbell Rudan Genetic epidemiological eastern Island isolates, Croatia: objective strategies.Coll. Antropol. 23: 531-546PubMed 34Rudan Biloglav Z. Vorko-Jović Kujundzic-Tiljak Stevanović Ropac Puntarić Cucević Salzer Effects inbreeding, endogamy, admixture, outbreeding health: (1001 Dalmatians) study.Croat. 47: 601-610PubMed genetic-epidemiology began 2002, continues today. sampling framework voting register Croatia, send postal invitations adult inhabitants (over 18 age); subjects volunteers Complex Disease Study, again aged years. sample consisted 906 island (39.4%), 915 (39.8%) 477 (20.8%) totaling individuals. entire 18–100 (median 56, interquartile 22 years). There 894 men (39.2%) 1384 women (60.8%), 20 people gender data missing. analysis performed only, more extensive familial links. genealogical information reconstructed Church Parish records provided subjects, then checked against allele sharing relatives quality control measure exclude errors. contained 809 relationships (including 205 parent-child, 123 sibling, 481 relationships). lower links standard errors arising rather shallow records, calculate estimates sample. members interviewed one trained surveyors, questionnaire (35Campbell Carothers A.D. Hayward Barac Pericic Janicijevic Smolej-Narancic Polasek O. Kolcic Weber J.L. Hastie N.D. Wright A.F. genome-wide heterozygosity biomedically quantitative traits.Hum. Mol. Genet. 16: 233-241Crossref (79) collected personal (name, date, place birth, gender, marital status, education occupation), selected health-related lifestyle variables (such diet smoking status), complaints, drug intake hospitalization records. Blood taken epruvetes anticoagulant immediately processed; separated centrifugation stored −70 °C. conformed ethical guidelines 1975 Declaration Helsinki. signed informed consent form before participating approved appropriate Ethics Board University Zagreb Medical School Research Committees Aberdeen. consisting (poly(glycidyl dimethacrylate)) phase coupled casted inside well custom prepared BIA Separations (Ljubljana, Slovenia). basic synthesized free-radical polymerization GMA cross-linking agent, EDMA, porogenic solvents, cyclohexanol dodecanol (60 vol.% reaction mixture) described et al. (36Tennikova Belenkii B.G. Švec Liq. 1990; 13: 63-70Crossref (331) instead thermally initiated polymerization, UV used. preparation process mixture, consists monomers porogens, polymerized applying heat light. types property macroporous material distribution. photoinduced copolymerization 150 μl monomers, photoinitiator, solvents room temperature directly 96 plates. mixture irradiated constant intensity 5 × 8 W mercury lamp wavelength 312 nm (UVItec Ltd, Cambridge, exposure time up 180 min. Although instrument does active cooling, exceed °C thus effectively excluding thermal initiation. After completed, extensively washed ethanol wash out soluble compounds. average determined intrusive porosimetry (PASCAL 440 porosimeter, Thermoquest Italia, Rodano, Italy). distribution around 700 nm, comparable (37Monolithic Materials: Preparation, Properties Applications.in: Merhar Štrancar Deyl Journal Chromatography Library. vol. 67. Elsevier, Amsterdam2003: 51Google immobilization flushing solution acetate. Afterward flushed deionized deactivation remaining 0.5 acid. Before use, 10 column (CV) equilibrated CV (1X PBS, 7.4). Plasma (50 μl) diluted applied plate. filtration completed ∼5 five remove unbound elution solvent (0.1 2.5). Eluates 96-deep-well neutralized 7.0 neutralization (1 bicarbonate) maintain stability. application, regenerated following buffers: followed 0.1 afterward 1 PBS re-equilibrate monoliths. step procedure done under vacuum (cca. 60 mmHg reduction while samples, 500 during washing steps) manual set-up multichannel pipet, manifold (Beckman Coulter, Brea, CA) pump (Pall Life Sciences, Ann Arbor, MI). If period, 20% (v/v) 4 repeated use contaminants sometimes completely elute phase. cleaning protocol included NaOH precipitated 30% propan-2-ol bound hydrophobic lipids. removed precipitates diminish purity verified NuPAGE Novex 4–12% Bis-Tris gels Xcell SureLock Mini-Cell (Invitrogen) according manufacturer. Precision Plus Blue Standards (BioRad, Hercules, molecular weight marker. run V 45 min, stained GelCode (Pierce) visualized VersaDoc Imaging System (BioRad). Glycan release labeling previously (38Knežević Polašek Gornik Kolčić O'Donoghue Bones Rudd P.M. Lauc Variability, Environmental Determinants Human N-Glycome.J. Proteome 694-701Crossref block SDS-polyacrylamide gel digestion N-glycosidase F (ProZyme, microtiter achieve best preparation. extraction, fluorescently 2-aminobenzamide. enzymes, ProZyme (San Leandro, CA), digestions: Sialidase A™/NANase III (recombinant Arthrobacter ureafaciens, expressed Escherichia coli), mU; α(1–2,3,4,6)fucosidase (bovine kidney), 1.25 α(1–3,4)-fucosidase (almond meal), 1.6 β(1–3,4)-galactosidase testis), β(1–4)-galactosidase (Streptococcus pneumoniae), 2 β-N-acetylhexosaminidase/HEXase I Streptococcus pneumoniae, α(1–2,3,6)-mannosidase (jack bean), mU. Aliquots 2-AB pool dried down digested corresponding 1X concentrated manufacturers volume μl. overnight incubation 37 °C, enzymes AcroPrep Filter Plates, 10K Corporation, MI, USA). Digested HILIC-UPLC undigested equivalent. Fluorescently Waters Acquity UPLC quaternary manager, manager FLR detector set excitation emission wavelengths 330 420 respectively. Empower software, build 2145 (Waters, Milford, MA). Labeled BEH column, 100 2.1 mm i.d., 1.7 particles, mM formate, 4.4, acetonitrile Recently methods profiling (39Ahn Yu Y.Q. Gilar Separation columns packed microm sorbent.J Chromatogr Analyt Technol Biomed 878: 403-408Crossref (169) 40Bones Mittermayr Guttman Ultra identification cancer glycosylation.Anal 82: 10208-10215Crossref (155) starting point method linear gradient 75–62% 0.4 ml/min min analytical run. Samples maintained injection, system calibrated external hydrolyzed glucose oligomers retention converted units. Data automatic traditional integration algorithm after chromatogram manually corrected same intervals chromatograms manner 24 peaks amount peak % integrated area. MS peak, fractionated (HILIC) particles UltiMate Dual Gradient LC (Dionex, Sunnyvale, controlled Chromeleon software connected FP-2020 (Jasco, Easton, MD) To obtain adjusted 0.3 prolonged Collected fractions resuspended water. Nano-LC-ESI-MS/MS. Ultimate 3000 nano-LC (Dionex/LC Packings, Amsterdam, Netherlands) equipped reverse trap (C18 PepMap 100Å, μm, 300 mm; Dionex/LC Packings) nano 3 75 Packings). eluent (0.1% water) nL/min. disialylated extra 0.04% trifluoroacetic added injection