Site-specificN-glycosylationofchickenserumIgGNorikoSuzuki1andYuanC.LeeDepartment of Biology, Johns Hopkins University, Baltimore, MD 21218ReceivedonSeptember27,2003;revisedonNovember7,2003;acceptedonNovember10,2003Avianserumimmunoglobulin(IgGor IgY) is functionallyequivalent to mammalian IgG but has one additional constantregion domain (CH2) in its heavy (H) chain. In chicken IgG,each H-chain contains two potential N-glycosylationsiteslocated on CH2 and CH3 domains. To clarify characteristicsof N-glycosylation on avian IgG, we analyze N-glycans fromchickenserumIgGbyderivatizationwith2-aminopyridine(PA) and identified by HPLC and MALDI-TOF-MS.Thereweretwotypesof N-glycans: (1) high-mannose-typeoligosaccharides (monoglucosylated 26.8%, others 10.5%)and(2)biantennarycomplex-typeoligosaccharides(neutral,29.9%; monosialyl, 29.3%; disialyl, 3.7%) on molar basis oftotal N-glycans. Toinvestigatethesite-specificlocalizationof different N-glycans, chicken serum IgG was digested withpapainandseparatedintoFab[containingvariableregions(VHVL) CH1 CL] andFc (containingCH3 CH4)fragments. Con A stained only Fc (CH3 CH4) and RCA-Istained only Fabfractions, suggesting that high-mannose-type oligosaccharides were located on Fc (CH3 CH4)fragments, andvariable regions of Fabcontains complex-typeN-glycans. MSanalysisof chickenIgG-glycopeptidesrevealed that chicken CH3 domain (structurally equivalent tomammalian CH2 domain) contained only high-mannose-typeoligosaccharides, whereas chicken CH2 domain containedonlycomplex-typeN-glycans. TheN-glycosylationpatternonavianIgGismoreanalogoustothatinmammalianIgEthanIgG, presumablyreflectingthestructural similaritytomammalian IgE.Keywords:Asn297/chickenserumIgG/IgG-Fc/monoglucosylatedhigh-mannose-type/N-glycan processingIntroductionAvian IgG, the predominant serumimmunoglobulin inbirds, iscloselyrelatedtobothmammalianIgGandIgE,basedontheirfunctional andstructural properties(Warret al., 1995). Incontrast tomammalianIgG, avianIgGcontainsoneadditional domainintheconstantregionofits heavy(H) chains (designatedupsilon, u), but it lacksfunctional hinge regions found in mammalian IgG(Figure1A)(Magoretal.,1992;Parvarietal., 1988;Warret al., 1995). In short, the CH3 and CH4 domains ofchicken/duckIgGresembletheCH2andCH3domainsofmammalianIgGinstructure,respectively,andtheequiva-lent of the CH2 domain in avian IgG is absent in mamma-lianIgG.BecauseofitsdistinctstructuraldifferencefrommammalianIgG, avianIgGisalsocalledIgY(LeslieandClem,1969;Warretal.,1995).TheIgY-likemoleculesarealsofoundinreptilesandamphibians(Fellahetal.,1993;Warr et al., 1995). Structural properties of IgY in birds andamphibians are rather close to mammalian IgE with respectto the number of CH domains as well as the organization ofintradomainandinterchaindisulfide bonds (Figure 1A)(Fellahet al.,1993; Parvariet al.,1988; Warret al.,1995).Itishypothesizedthat g andegeneshavebeengeneratedfromarelativelyrecent gene-duplicationevent andthatIgY-likemoleculewas theimmediateprogenitor bothofIgG and IgE (Parvari et al., 1988; Warr et al., 1995).Basedontheir aminoacidsequences, chicken(Parvarietal.,1988)andduck(Magoretal.,1992)IgGssharetwopotential N-glycosylationsites, predictablefromthecon-sensus sequence(sequon) inconstant regions (Figure1Aand 1B). One of them is located in the CH2 (Cu2) domain,which is absent in mammalian IgG. The other is located inthe CH3 (Cu3) domain, whichcorresponds tothe CH2(Cg2) domain of mammalian IgG (Asn297, Eu-numbering).Figure1Bshowsthesequencealignment forH-chainsofavian (chicken, duck) IgG (Cu-chains), mammalian(human) IgG1 (Cg1-chain), mammalian(human, mouse,rat) IgE(Ce-chains), and amphibian (Xenopus, axolotl)IgY(Cu-chains) aroundthe twosequons onavianIgGH-chains. The sequence alignment indicates that thelocationof bothpotential N-glycosylationsites of avianIgGsarealsoconservedinmammalianIgE(except CH2inhumanIgE). Incontrast, sequons of amphibianIgYare located at different positions fromavian IgGandmammalianIgG/IgE, probablyreflectingthe evolutionaldistances between mammals/avians and amphibians.We recentlyfoundthat pigeonserumIgGhas uniqueN-glycanfeatures,suchasthepresenceofalargequantityof highlygalactosylatedtriantennaryoligosaccharides aswellasmonoglucosylatedhigh-mannose-typeoligosaccha-rides (monoGlc-high-Man) (Suzukiet al., 2003), which arenot foundinmammaliannormal serumIgG. MonoGlc-high-Man is probably a characteristic in avian IgG becauseithadbeenalsofoundinchicken(Ohtaetal., 1991)andquail (Matsuuraet al., 1993) eggyolkIgGs andchickenserum IgG (Raju et al., 2000). Except avian IgGs, however,themonoGlc-high-Manisrarelyfoundinsecretedmatureglycoproteins,andonlytransientlyexistsonglycoproteinsduringfoldingprocessintheendoplasmicreticulum(ER).The currently proposedmechanismis that after correctfoldingofglycoproteins, themonoGlcresidueisremoved1Towhomcorrespondenceshouldbeaddressed;e-mail:nrsuzuki@jhu.eduGlycobiology vol.14 no.3#OxfordUniversityPress2004;allrightsreserved. 275Glycobiologyvol.14no.3pp.275292,2004DOI:10.1093/glycob/cwh031AdvanceAccesspublicationDecember23,2003 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from by a-glucosidase II (GII) (Helenius and Aebi, 2001; Parodi,2000). The retained monoGlc-high-Man of avian IgG mighthave resultedfromthe steric hindrance imposedby theuniqueconformational structuresofavianIgG. However,the relationship between the protein structure of avian IgGandits N-glycosylationpatternhas not beenexamined.Because chicken IgGis a goodsource of glycoproteinsthat contain monoGlc-high-Man and its peptide sequencesareknown,webelieveditisworthwhileinvestigatinghowsuch unique oligosaccharides exist in chicken IgG.Fig. 1. Structures of Igs and their N-glycosylation sites. (A) Structures of avian IgG, mammalian IgG, and mammalian IgE molecules. Avian(chicken, duck) IgGhas five domains in the heavy chain, termed VH (in variable region), CH1, CH2, CH3, and CH4 (in the constant region). The domainsof the light chains are termed VL (in the variable region) and CL (in constant region). The potential N-glycosylation sites on avian IgG are shown ashexagons. N-glycosylation on variable regions (gray hexagon) could occur sometimes, depending on the peptide structures. Location of disulfide bondslinking the chains were predicted from analogy to human IgE (Parvari et al., 1988; Wan et al., 2002), indicated with dotted bold lines. Numbering forchicken IgG H-chain (u-chain) is based on the deduced amino acid sequences from cDNA, starting from the first methionine in the leader region (Parvariet al., 1988; Reynaud et al., 1989). Position of actual N-glycosylated sites on human IgG1 and IgE were shown in hexagons. Interchain disulfide bonds arebased on human IgG1 and IgE structures, respectively (Paul, 1999; Wan et al., 2002), and indicated as bold lines. Numbering for mammalian Ig g-chain isbased on Eu numbering (Edelman et al., 1969). Numbering for mammalian Ig e-chain is modified from a reference (Dorrington and Bennich, 1978) tocomply with an human immunoglobulin e-chain of 547 amino acids. (B) Comparison of amino acid sequences around potential N-glycosylation sites onchicken IgG H-chain and others. Potential N-glycosylation sites were indicated as bold. Residue numbers on Asn were given for chicken IgG H-chain.Homologies of these sequences are not always high, but Trp and Cys residues (indicated with arrows), which are hallmarks of Ig domains, are wellconserved each other. Primary accession numbers for Entrez database are; chicken upsilon chain, CAA30161; duck upsilon chain, CAA46322; humangamma chain, CAC20454; human epsilon chain, AAB59424; mouse epsilon chain, EPC_MOUSE; rat epsilon chain, AAA41364; Xenopus upsilon chain,S04845; axolotl upsilon chain, CAA49247.N.SuzukiandY.C.Lee276 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from Inthis study, we first determine the detailedN-glycanstructuresofchickenserumIgGforthecomparisonwiththoseofpigeonIgGandthendemonstratethesite-specificN-glycosylationonchickenIgG. Ourdataindicatedthathigh-mannose-type N-glycans are exclusively located onCH3 domains (as we found in pigeon IgG), whereascomplex-typeN-glycansarepresent inCH2domainsandFab regions(most likelyvariable regions).N-glycosylationpatterns, including the oligosaccharide structures, onchicken IgGare more similar to mammalian IgEthanto IgG, which is probably due to the structural simi-larityinheritedinmolecular evolutionfromIgYtoIgElineage. Byanalogyofthe3DstructureofhumanIgE-Fc(containing Ce2-4 domains) (Wan et al., 2002), we speculatethat protein folding and assembly mechanisms of avian IgGenables retaining monoGlc-high-Man on the secretedproteins.ResultsStructuralanalysisofPA-derivatizedoligosaccharidesfromchickenIgGToinvestigate structural profile of N-glycans inchickenserum IgG, we utilized a 3D mapping technique (Takahashietal., 1995b), inwhich2-aminopyridine(PA)derivatizedN-glycans were chromatographed on high-performanceliquidchromatography(HPLC) using(1)anionexchangewithaDEAEcolumn, (2) reversed-phase withanocta-decylsilica(ODS)column,andthen(3)normalphasewithan Amide-80 column. The elution positions of eachPA-oligosaccharide were recordedas glucoseunits (GU)(TableI), andtheir structures weredeterminedbasedontheelutionpositionsandmatrix-assistedlaserdesorption/ionization time-of flight mass spectrometry (MALDI-TOFMS) data (Table I) by comparing with reference PA-derivatized oligosaccharides (Figure 2).TotalPA-oligosaccharidesfromchickenIgGweresepa-ratedintoneutral,mono-,anddisialyloligosaccharidesonaDEAEcolumn(Figure3A). MSanalysis revealedthatneutralfractions eluted between 5 and 15minon theODScolumn(Figure3B) wereHex711HexNAc2-PA(datanotshown), suggesting that these are high-mannose-type oligo-saccharides. The three major peaks (n-4, n-5, n-6) wereisolated, digestedwith a-mannosidase, andfurtherexam-ined by 2D HPLC mapping (Tomiya et al., 1988). Fractionn-4 showed the same elution position as Man9GlcNAc2-PAfrombovineRNaseB(Figure2), andbothmovedtothepositionofMan1GlcNAc2-PAafter a-mannosidasediges-tion (Figure 4A), suggesting that n-4 has the same structureas Man9GlcNAc2-PA. This was supported by the MS dataof n-4before(m/z1984.86; Hex9HexNAc2-PA) andafter(m/z687.97;Hex1HexNAc2-PA) a-mannosidasedigestion.Onthe other hand, neither n-5(m/z 1984.81; Hex9Hex-NAc2-PA) nor n-6 (m/z 2147.06; Hex10HexNAc2-PA)could be digested to Hex1HexNAc2-PA under the same con-ditions used for n-4 (Figure 4A). Mild a-mannosidase diges-tion(50mU/100pmolePA-N-glycans,37

C,overnight)ofn-6 yielded a major peak on either ODS or Amide-80column (GUODS7.1; GUAmide6.9; m/z 1498.60,Hex6HexNAc2-PA), which was transformed to a peak(GUODS6.5; GUAmide5.9; m/z 1336.26, Hex5HexNAc2-PA) on exhaustive digestion with a-mannosidase (200 mU/100 pmole PA-N-glycans, 37

C, two overnight). This mightbe a reflection of difficulty of removal of the a1-6 mannoseresidue by jack bean a-mannosidase.Althoughthe final product of a-mannosidase-digestedn-6hadthesamemassvalueasMan5GlcNAc2-PAfrombovineRNaseB(GUODS7.3;GUAmide6.1),itelutedatadistinctly different position on an ODS column (Figure 4A).These results suggest that one of the a-mannosylatedbranchesonn-6areblockedatthenonreducingterminus,so that a-mannosidase could not trim it to Man1GlcNAc2-PA.Theblockingismostlikelybyaglucosylationonthenonreducing terminal of Mana1-2Mana1-2Mana- branch,becausetherelativeelutionpositionof n-6onbothODSandAmide-80 columns were coincidental with those ofGlc1Man9GlcNAc2-PA(Tomiya et al., 1988). Fractionn-5, smaller by one hexose than n-6, yielded the sameproduct asn-6aftera-mannosidasedigestion, suggestingthatthisalsohasamonoglucosylatedbranch, butshorterby onea-mannoside residue thann-6. Because n-5 waseluted earlier than n-6 on the ODS column, one a1-2-mannoside residue on the Mana1-3Mana1-6Manb1-4GlcNAc arm is absent in n-5 (Tomiya et al., 1991; Tomiyaand Takahashi,1998).Thusthestructuresofn-4,n-5,andn-6 were deduced as shown in Table I.Elution positions of the remaining eight fractions of neu-tral oligosaccharides fromchickenIgG, n-9, n-10, n-11,n-12, n-14, n-15, n-16, and n-17, were coincidental onODSandAmide-80 columns withthose of humanIgGN-glycans F, J, H, L, M, N, O, and P, respectively (Figure 3B,Figure4B,andTableI).MALDI-TOFMS(TableI)gavegoodagreement withthese results. The structure assign-ments were also supported by digestion with b-galactosidaseand/or a-fucosidase (Figure 4B). After b-galactosidasedigestion, the elution positions of the products on the ODSand Amide-80 columns were shifted as follows: (1) both n-9andn-11yieldedthe same GlcNAc-terminatedstructure(N-glycanEinFigure2),whichhascore a1-6fucoseresi-dues andnobisecting GlcNAc; (2) bothn-10 andn-12yieldedthesameproduct(N-glycanIinFigure2), whichhasbisectingGlcNAcandno a1-6corefucose; (3) n-15,n-16, and n-17 all yielded the same product (N-glycan M inFigure2), whichhasbothcore a1-6fucoseandbisectingGlcNAc. After a-fucosidase digestion, n-9, n-11, n-14, n-15,n-16, andn-17yieldedtheexpectedrespectivefucose-lessstructures (N-glycans B, D, I, J, K, and L, respectively, as inFigure 2). Before the exoglycosidase-digestion, n-9 (GUODS13.5; GUAmide6.3) andn-10(GUODS13.5; GUAmide6.2)exhibit very close GUvalues. However they are distin-guished by m/z values (Table I) as well as by the sensitivityto a-fucosidase. Fractions n-15 (GUODS 19.5; GUAmide 6.5)andn-16(GUODS19.6; GUAmide6.6) alsoexhibit similarGU values, but a-fucosidase-treated n-15 and n-16 (i.e., N-glycansJandK,respectively)wereclearlydistinguishablebytheir elutionpositions ontheODScolumn. Thus thestructures of n-9, n-10, n-11, n-12, n-14, n-15, n-16, andn-17 were firmly assigned (Table I).Three fractions of monosialylatedPA-oligosaccharides(ms-5, ms-7, andms-8) andone fractionof disialylatedGlc1Man9GlcNAc2-AsnonchickenIgG-CH3domains277 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from TableI. Assignment of the major PA-oligosaccharides from chicken IgG based on HPLC and MSN.SuzukiandY.C.Lee278 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from PA-oligosaccharides (ds-7) were isolated, and theirstructures were analyzed with MALDI-TOF MS andHPLC(Figure4C). Compositions deducedfromtheMSdata were ms-5, NeuAcHex5HexNAc4dHex-PA; ms-7,NeuAcHex4HexNAc5dHex-PA; ms-8, NeuAcHex5Hex-NAc5dHex-PA; and ds-7, NeuAc2Hex5HexNAc5dHex-PA. After a-sialidasedigestion, elutionpositionsofms-5,ms-7, ms-8, andds-7shiftedaspredictedandwereindis-tinguishablefromthoseofhumanIgGN-glycansH,O,P,andP, respectively(Figure4C). TheirGUAmidedecreasedabout 0.30.4 after desialylation, suggesting that theirsialylationis a2-6linkageandnot a2-3(Nakagawaetal.,1995; Takahashi et al., 1995a,b; TomiyaandTakahashi,1998). The monosialylated branching position on ms-5,ms-7, andms-8 were determinedas follows: whenms-5and ms-8 were digested witha-fucosidase, followed byb-galactosidase, andthena-sialidase, the products wereidentical withN-glycansCandK, respectively(Figures4and 5). b-Galactosidase-treated ms-8 was identical to ms-7.When ms-7 was digested with a-fucosidase and a-sialidase,theproducts wereidenticalwithN-glycanK.Theseresultssuggest that monosialylated site in ms-5, ms-7, and ms-8 areontheMana1-3Manarm. Accordingly, thestructuresofthese monosialylated oligosaccharides were deduced asshowninTableI. Desialylatedds-7was further digestedwitha-fucosidase toyieldaN-glycanwithGal onbothterminals, having bisecting GlcNAc and no core a1-6fucose (N-glycanLinFigure 2). Therefore, structure ofds-7 was elucidated (Table I).Our HPLCstudyclearlyindicatedthat chickenserumIgGhas high-mannose-type (37.2%) and complex-type(62.8%) oligosaccharides. This ratio is in a range of that ofpigeonserumIgG(high-mannose-type, 33.3%; complex-type, 66.7%) (Suzuki et al., 2003). Thepresenceof high-mannose-type set avian IgG apart from human (Figure 3B,Figure 2) and other mammalian IgGs that possess complex-type oligosaccharides exclusively. MonoGlc-high-Man(Glc1Man89GlcNAc2) was71.2%ofthetotalhigh-mannose-typeN-glycans.Thispercentisalsointherangeofthatofpigeon IgG (61.7% of the total high-mannose type).Isolationandlectin-blottingsofchickenIgG-FabandFcChicken serum IgG was known to be cleaved into Fab andFc fragments by papain digestion (Dreesman and Benedict,1965;KuboandBenedict,1969),althoughtheexactcleav-agesiteshavenotbeenelucidatedyet. ToisolatechickenIgG-FabandFcfragments, papain-digestedchickenIgGwas applied to a DEAE-Sepharose column and eluted withagradient of NaCl toyieldthreepeaksfr. 1, fr. 2, andfr. 3, asshowninFigure6A. Immunoblottingswithanti-chickenIgG-Fcantibodyrevealedthatfr.1andfr.3wereFab and Fc, respectively (Figure 6B). Fr. 2 is mostly Fab butwas slightly contaminated by Fc fragments, which could beremoved with an affinity column using anti-chicken IgG-Fcantibody as affinant. The pass-through fraction of the affin-itycolumnwas designatedas fr.20. Fabwas detectedasbroad bands on sodium dodecyl sulfatepolyacrylamide gelelectrophoresis (SDSPAGE), perhaps due to peptide hetero-geneity of the variable regions (Figure 6B). Fc appeared as asharp band under the reducing condition (data not shown),but under the nonreducing condition it separated into threeaRelative quantity was calculated based on CLC-ODS elution profiles for neutral (67.1%), monosialyl (29.3%), anddisialyl (3.65%) PA-oligosaccharides, including minor peaks ( 51%/each peak). The sumof PA-oligosaccharides on thistable is 85.7%. Structures of minor peaks were not assigned.bm/z of neutral and sialylated oligosaccharides were detected as [MNa]

and [MH], respectively.cPA-derivatized reference N-glycans whose elution positions on both CLC-ODS and Amide-80 columns and [MNa]

coincide with the chicken IgG PA-oligosaccharides were indicated on the table. Because coincident reference oligosac-charides for n-5, n-6, ms-5, ms-7, ms-8, and ds-7 were not available, their relative elution positions on the HPLCcolumns were compared with those of the related reference N-glycans (see text). Structures of the reference compoundswere shown in Figure 2.dMonosaccharides were denoted by F, fucose; M, mannose; Glc, glucose; GN, N-acetylglucosamine; G, galactose; NA,N-acetylneuraminic acid.eN/A, not available.Glc1Man9GlcNAc2-AsnonchickenIgG-CH3domains279 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from bands, all stained with anti-chicken IgG-Fc antibody(Figure 6B, Coomassie brilliant blue [CBB] andanti-Fcantibodystaining). ThedifferentmobilityofthethreeFcfragmentsonthegelisattributabletopartialreductionofdisulfide bonds and/or multiple or alternative cleavagesduring papain digestion, as was found in mammalian IgG-Fc (Coligan, 1991).Onemajor N-terminal aminoacidsequenceof theiso-latedFcfragmentwasshowntobeSCSPIQL---(startingfrom Ser346), located near the initial part of CH3 domain.MALDI-TOF MS revealed that the [MH]

values of thewhole chicken IgG, Fab, and Fc were 167,940.56, 44,771.61,and53,925.79, respectively. ThevalueforFcwasclosetothe theoretical molecular mass of dimerizedCH3 CH4regions, apparently lacking CH2 domains. Therefore itwas designatedas Fc (CH3 CH4). The mass value forthewholechickenIgGmoleculewerealmostthesameaspreviouslyreported, andthevaluesforFabwereclosetothose for Fab0preparedbypepsindigestion(Sunet al.,Fig. 2. Structures of reference oligosaccharides isolated from humanIgG and bovine RNase B. All the oligosaccharides were derivatized withAP for HPLC and MALDI-TOF MS analysis. Monosaccharides weredenoted by F, fucose; M, mannose; GN, N-acetylglucosamine;G, galactose.Fig. 3. HPLC separation of PA-oligosaccharides from chicken serumIgG. (A) Total PA-oligosaccharides from chicken IgG were separatedinto neutral, mono-, and disialyl oligosaccharides on a DEAEcolumn. (B) Elution profiles of the neutral, mono-, and disialyl PA-oligosaccharides from chicken serum IgG on an ODS column. Structuresof human IgG N-glycans (AP) were shown in Figure 2. HumanIgG N-glycans I, J, and K were prepared with a-fucosidase-digestion ofhuman IgG N-glycans, and their elution positions on an ODS columnwere indicated. Each peak was collected and analyzed with MS. Fractionnumbers indicated for chicken IgG N-glycans correspond to those inTable I.N.SuzukiandY.C.Lee280 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from 2001). The molecular mass value of Fab agrees withtheoretical values of {(VLCL) (VHCH1)}but not{(VLCL) (VHCH1 CH2)}, suggesting that theFabfragmentalsolacksCH2domains.TheCH2domainsmighthavebeenlostbythepapaindigestion, presumablyby excessive fragmentation.ConcanavalinA(ConA)andanti-chickenIgG-Fcanti-bodystainedonlyFc(CH3 CH4) fractions (fr. 3), butRCA-IstainedFabfractions(fr. 1andfr. 20exclusively;Figure 6B). These data strongly suggest the gross differenceof glycans between chicken IgG-Fc (CH3 CH4) and Fab.Based on the lectin specificities, it is most likely thathigh-mannose-typeoligosaccharidesarelocatedontheFc(CH3 CH4) region, whereas galactosylated glycans are inFab regions. Oligosaccharides containing b-galactosidesonchickenIgG-FabwereN-glycans (i.e., complex-type),becauseglycoamidaseF(GAF)-treatedchickenIgG-Fabcouldnolonger be stainedwithRCA-I lectin(datanotshown). The N-glycosylation on Fab fragments most likelyoccurredonvariableregions,thatis,VLand VHdomains(Figure 1),as seeninmammalianserumIgG.N-glycosyla-tions on VL and VH occur only when the peptide sequencespossesstheN-glycosylationsignals,andthelocationofN-glycosylation sites are varied among polyclonal serum IgG.Fig. 4. Structural analysis of N-glycans from chicken IgG by a 2Dmapping technique. (A) Elution position of high-mannose-type PA-oligosaccharides from chicken IgG (n-4, triangle; n-5, square; n-6, opencircle) and bovine RNase B (solid circle) on ODS and Amide-80 columns.Structures of bovine RNase B N-glycans (M9 and M5) are shown inFigure 2. Arrows with dot-dot-dashed lines indicate the changes of thecoordinates of N-glycans after digestion with a-mannosidase. (B) Elutionpositions of neutral complex-type PA-oligosaccharides from chicken(open circle) and human IgG (solid circle). Arrows with dashed lines andwith dotted lines indicate the changes of the coordinates of N-glycansafter digestion with b-galactosidase and a-fucosidase, respectively. Alsosee Figure 2 for structures. (C) Elution positions of mono- (ms-5, square;ms-7, triangle; ms-8, reversed triangle), and disialylated (ds-7, opencircle) PA-oligosaccharides from chicken IgG and human IgG (solidcircle). Arrows with solid lines, dashed lines, and dotted lines indicate thechanges of the coordinates of N-glycans after digestion with a-sialidase,b-galactosidase, and a-fucosidase, respectively.Fig. 5. Sequential exoglycosidase digestions for determination of themonosialylated branch of biantennary N-glycans from chicken serumIgG. Products of exoglycosidase-treated ms-5, ms-7, and ms-8 wereanalyzed with ODS and Amide-80 columns and compared with referenceN-glycans (Figure 2). Monosaccharides were denoted by F, fucose;M, mannose; GN, N-acetylglucosamine; G, galactose; NA,N-acetylneuraminic acid.Glc1Man9GlcNAc2-AsnonchickenIgG-CH3domains281 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from Therefore, N-glycosylation sites on Fab fragments were notdetermined by isolating glycopeptides originating from VLand VH.N-glycosylationonchickenIgG-CH3domainThe known amino acid sequences of chickenIg upsilon (u)chains (H-chains)indicate theexistenceof onlyonepoten-tial N-linkedglycosylationsitelocatedontheCH3-CH4domains(Figure1A)(Parvari etal., 1988). Thiswascon-firmed by GAF treatment and SDSPAGE (data notshown). Wealsoconfirmedthepresenceof N-glycansontheCH3domainwithMALDI-TOFMS. MSof trypticglycopeptides fromthe reducedformof chickenIgG-Fc(CH3 CH4)fragmentareasshowninFigure7,andthe[MH]

molecular ions areassignedinTableII. Peakswith m/z higher than 5000 were affected by GAF treatment,producing two new peaks of m/z 4036.66 and 4294.34. Thelargerofthesetwopeakshavingadditional EandKresi-dues on N-terminal side could have arisen from incompletetrypsin digestion (Table II). The N-glycans on the glycopep-tides were assignedtobe exclusively high-mannose-typeglycans, because the masses of the glycopeptides were com-pletelyshiftedaftertreatment withendo-b-N-acetylgluco-saminidase H(Endo H). The intact glycopeptides wereassigned as Hex610HexNAc2-peptide (Table II). Aftera-mannosidasedigestion,someoftheintactglycopeptideschangedtoHex1HexNAc2peptide,butalargeamountofHex56HexNAc2-peptide was also produced (Figure 7).Concomitant withthedecreaseinthepeaksof Hex6Hex-NAc2peptides after exhaustivea-mannosidasedigestion,peaksof Hex5HexNAc2peptidesincreased. NopeaksforHex24HexNAc2-peptideweredetected, evenwhenexcessa-mannosidase was used in the digestion. This suggests thepresenceof Glc-cappedoligomannosyl branch. ThereforeHex56HexNAc2peptides must have been derived fromlarger monoglucosylatedoligosaccharides. Hex1HexNAc2peptidecanbeassignedasManb1-4GlcNAcb1-4GlcNAc-peptide,whichcanbederivedfromglycopeptidesofhigh-mannose type without monoglucosylation. These resultssuggest that bothmonoglucosylatedandnonglucosylatedhigh-mannose-typeoligosaccharideswereonthesamesiteof CH3 domain.N-glycosylationonchickenIgGCH2domainOneofthetwopotential N-glycosylationsitesonchickenIgG CH domains is located on the CH2 domain. To demon-strate the actual glycosylation at this site, the correspondingglycopeptides were isolated. Tryptic peptides of wholechickenIgGwerepreparedasdescribedinMaterialsandmethods, andtheelutionprofilesonaC18columnbeforeandafter GAFtreatment were compared. Three of thepeaks, designatedfr. A, B, andC, shiftedtheirpositionsafter GAFtreatment (datanot shown). Theactual peptidesequence analysis of the pooled fr. A indicated its completeagreement withthe predictedpeptide sequence withtheN-glycosylationsite onthe CH2domain(Table II). Likewise,MS and peptide sequencing data indicated that fr. B and fr.Cwere shorter andlonger glycopeptides fromthe CH3domain, respectively. TheCH2glycopeptides beforeandafter GAF digestion or sequential exoglycosidase digestionswere analyzedwithMALDI-TOFMS(Figure 8). Peaksaroundm/z28003300(Figure8A) wereeliminatedafterGAF digestion, resulting in a peak of m/z 1035 (Figure 8B).The m/z value of the GAF-treated glycopeptide corre-sponded to the expected [MH]

molecular ion for de-N-glycosylated CH2 glycopeptides prepared with trypsindigestion(TableII). Thesignalsat about m/z17001800were not shifted by GAF digestion, suggesting that they arenot N-glycosylated peptides. When CH2 glycopeptidewasdigestedwith a-sialidase, onepeak, atm/z3298, dis-appeared, and theintensityofthem/z3005peakincreased(Figure 8C). The mass difference indicates that a singleNeuAcwasremovedbya-sialidase. Thisagreeswiththefact that predominant sialylatedN-glycans derivedfromFig. 6. Separation of chicken IgG Fab and Fc by DEAE-Sepharose.(A) Elution profile of papain-digested chicken IgG on DEAE-Sepharose.One milligramof papain-digested chicken IgGwas loaded onto a DEAE-Sepharose column (1 ml) and eluted by a linear gradient of NaCl in10 mM TrisHCl (pH 8.0). (B) Localization of N-glycans in chicken IgG.Lectin- and immunoblottings of chicken IgG Fab and Fc (CH3 CH4).Fractions from the DEAE-Sepharose (fr. 1 and 3) and affinity column(fr. 20) were heat-denatured with sample buffer containing 3% SDSwithout reducing, separated by SDSPAGE (12.5% gel, 1 mg/lane),transferred to polyvinyl difluoride membranes, and stained with CBB,Con A, RCA-I, or anti-chicken IgG-Fc antibodies.N.SuzukiandY.C.Lee282 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from chickenserumIgGis monosialylatedbiantennaryoligo-saccharidesasshowninTableI. a-Sialidase-treatedCH2glycopeptides were further digested sequentially withb-galactosidase (Figure 8D), b-N-acetylhexosaminidase(Figure 8E), and a-fucosidase (Figure 8F), and the [MH]

oftheproductateachstepwasrecorded.Newlyproducedpeaks of glycopeptides were unambiguously assignedasshown in Table II, and the results indicated that N-glycanson the CH2 domain are exclusively complex-type.HomologymodelingofchickenIgG-Fc(CH2CH4)Basedontheassumptionthatthe3DstructureofchickenIgGissimilartothat of humanIgE, the3DstructureofchickenIgG-Fc(CH2CH4)waspredictedbyhomologymodeling(Figure9).Thecrystalstructureofrecombinanthuman IgE-Fc (CH2 CH4) (PDB ID, 1o0v) was utilized asatemplate. Like the template structure, chickenIgG-Fc(CH2 CH4) was built to form an asymmetric homodimerwith highly bent CH2CH3 junctions. One of the N-glyco-sylation sites on chicken IgG u-chains, Asn407 on the CH3domain corresponded well to Asn394 on human IgEe-chains (Figure 1A, 1B), and the same orientation asfoundinthetemplates was adopted. High-mannose-typeoligosaccharidesatAsn407arelocatedinthecaveformedbytwoCH3 CH4regionchains,andpartiallyburiedbytheCH2domains,suggestingthatthisCH2domainsmayconfer steric hindrance to access oligosaccharides atAsn407. The model alsopredicts that without FabandCH2 regions, two CH3 CH4 region chains can onlyformaframewithacavebut cannot confer strict sterichindrancetoretainmonoglucosylatedhigh-mannose-typeoligosaccharidesatAsn407intheframe. IfentirechickenIgG molecule formed Y-shaped structure as seen inmammalian IgG(Harris et al., 1997), terminal glucoseresiduesonGlc1Man89GlcNAc2-oligosaccharides, whichoccupy wider space thanMan5GlcNAc2-, wouldreadilyprotruded fromthe (CH3 CH4) frame and would beaccessible toa-glucosidase II in the ER. On the otherhand, N-glycosylationsites at Asn308onCH2domainsare exposedonthe surface of the molecule tobe easilyaccessible by processing enzymes. Thus this model supportsour experimental results showing the site-specific N-glycanson chicken IgG and can give a rational explanation for theexpression mechanismof monoGlc-high-Man on avian IgG.DiscussionCarbohydrate chains on glycoproteins can have diverseroles, such as mediators of protein foldings, tags forintracellular and extracellular trafficking, protein stabilizer,conferringhydrophilicproperties, protectors against pro-teolyticdigestion, ligandsof carbohydratebindingrecep-tors, and so on (Varki, 1993). Glycans can assume differentstructuresdependingontheirbiological,biochemical,andstructural properties of the individual glycoproteins as wellastheirlocationincellsand/orinbodies. Suchstructuraland functional complexity of glycans often makes it difficultto understand their biological function immediately.However, comparison of oligosaccharide structures andglycosylation patterns among glycoproteins with similarFig. 7. MALDI-TOF MS analysis of the glycopeptides fromchicken IgG-Fc (CH3 CH4). Tryptic digest of chicken IgG-Fc(CH3 CH4) was analyzed with MALDI-TOF MS before andafter digestion with GAF, Endo H, or a-mannosidase. Assignment ofthe [MH]

molecular ions detected were listed in Table II.Asterisks on the m/z values indicate the peaks of glycopeptidescontaining two additional amino acid residues (Glu-Lys, 257.29 Da)produced by alternative trypsin digestion. Because all trypticpeptide fragments are 54000 Da, they are not shown in the selectedwindows.Glc1Man9GlcNAc2-AsnonchickenIgG-CH3domains283 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from structures andfunctions canprovide useful informationaboutthestructuralrelationshipbetweentheoligosaccha-ride chains and the core proteins. Mammalian IgG is one ofthe best studied glycoproteins with regard to their carbohy-drate structures andfunctions, as well as the entire 3Dstructures(Deisenhofer,1981;Harrisetal.,1997;Kobata,1990; Matsudaet al., 1990; Mimuraet al., 2000; RadaevandSun, 2001). Indeed, it has beenusedas astructuralmodel forotherclassesofmammalianIgs(Burton, 1987;RuddandDwek, 1997). DetailedstudiesofcarbohydratechainsonIgsare, however, mostlylimitedtothosefrommammals,andlessattentionhasbeenpaidtootherverte-brate immunoglobulins.Our previous work for structural analysis of pigeon serumIgGN-glycans by HPLC, MS, and tandemMS haverevealed that the prominent N-glycans are triantennaryTableII. Assignment of the [MH]

molecular ion signals afforded by glycopeptides from chicken IgGaaData were compiled from the result of MALDI-TOF MS (Figures 7 and 8) analysis. All [MH]

values given were the averaged masses.bMonosaccharide composition indicated were deduced from the results of exoglycosidase digestions.cThese exoglycosidase digestions were sequentially performed as described in Materials and methods.dAfter deglycosylation with GAF, the glycosylated Asn residues were converted into Asp.eIncomplete trypsin digestion produced an alternative glycopeptide fragment containing Glu-Lys at the N-terminal site. [MH]

values given from thealternative were indicated in parentheses.N.SuzukiandY.C.Lee284 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from complextypeaswellashigh-mannose-type,bothofwhichare rarely found in mammalian normal serum IgGs(Hamako et al., 1993). When we compared the pigeon IgGN-glycanswiththosefromchickenIgGreportedbyothergroups (Ohta et al., 1991; Raju et al., 2000), we realized thatconfirming the N-glycan structures of chicken serum IgG isnecessary by our conventional methods (Suzuki et al., 2001;Takahashietal.,2001)forseveralreasons.First,althoughegg yolk IgG, which arises fromtransport of maternalantibodies by receptor-specific process (Pattersonet al.,1962),hasidenticalbiophysicalpropertieswithserumIgG(LoekenandRoth,1983),itisalsoreportedthateggyolkIgGdoes not mediateanaphylacticreactionwhichavianserumIgGdoes(FaithandClem, 1973). Inthisregard, apossibility that the transport of IgG to egg yolk is restrictedby certain carbohydrate structures have not been excluded.Second, Rajuet al. (2000) reportedchickenserumIgGN-glycans analyzed by MALDI-TOF MS, but the proposedstructuresareonlybasedonmassvaluesanddeducedbyanalogy to mammalian IgGs. Although simple MS is a con-venient tool for oligosaccharide analysis, it cannot distinguishisoforms, such as bisected biantennary and nonbisectedtriantennary oligosaccharides. We have demonstratedearlier that N-glycan structures and N-glycosylation patternof pigeon IgG are quite different from those of mammalianIgGs, so it was necessary to investigate the differencesbetween chicken and pigeon IgG N-glycans carefully.Thus we analyzed the N-glycan structures of chickenserumIgGbyHPLC, whichcandistinguishtriantennaryandbisectedbiantennarystructures(Tomiyaetal., 1988).Fig. 8. MALDI-TOF MS analysis for glycopeptides from chicken IgG CH2. The isolated chicken IgG CH2 glycopeptide (A) was treated withGAF (B) or sequentially digested with a-sialidase (C), b-galactosidase (D), b-N-acetyl-D-hexosaminidase (E), and a-fucosidase (F) and analyzed withMALDI-TOF MS. Assignment of the [MH]

molecular ions detected were listed in Table II.Glc1Man9GlcNAc2-AsnonchickenIgG-CH3domains285 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from Moreover, we also determined the preferred galactosylationand sialylation branches in complex-type as well as isoformsof high-mannose-type oligosaccharides, which have notbeen reported for chicken serum IgG.Ourresultsconfirmedthatstructuralpropertiesofcom-plex-type N-glycans from chicken serum IgG is more similartothoseofhumanIgGthantothoseofpigeonserumIgG(Suzukietal.,2003),becausenotriantennaryorextendedbranches with b- and a-galactosylation were detected.Thisfactpointsoutthatstructuralpropertiesofcomplex-type N-glycan are somehow conserved between chicken andmammalianIgGs regardless of thedifferent N-glycosyla-tionsites but nolonger maintainedinpigeonIgG. Bothhuman andchicken IgGs possess biantennary complex-type oligosaccharides with and without core a1-6 Fucand/or bisecting GlcNAc. Both IgGs have a mono-galactosylatedbranchpredominantly onthe GlcNAcb1-2Mana1-6Manarm(N-glycansn-9, n-10, andn-15fromchickenIgG; showninTable I, N-glycans B, F, andNfrom human IgG; shown in Figures 2 and 3B). Monosialy-lation in chicken IgG(ms-5, ms-7, ms-8 in Table I)exclusively occurs on the Galb1-4GlcNAcb1-2Mana1-3-Manarm, whichis alsothe case innormal humanIgG(Takahashi et al., 1995b). There are, however, some notabledifference in structural patterns of complex-type N-glycansbetween human and chicken IgG. More than half ofthe complex-type N-glycans fromchicken IgGcontainbisecting GlcNAc and core a1-6 Fuc and are fully b-galac-tosylated (n-17, ms-8, and ds-7 in Table I). Althoughhuman IgGalso has the same structure, predominantoligosaccharides are core a1-6 fucosylated biantennaryN-glycans without bisecting GlcNAc (Figure 3B). Theobserved difference is consistent with an earlier report(Raju et al., 2000).We also confirmed that complex-type N-glycan structuresof chickenserumIgGwere mostlythe same as chickeneggyolkIgG(Ohtaet al., 1991) but different fromquailegg yolk IgG (Matsuura et al., 1993) or pigeon serum IgG.In contrast, high-mannose-type N-glycans includingmonoGlc-high-Manarewell conservedamongthem. Ourprevious data suggested that pigeon serum IgG-CH3domain also possess exclusively high-mannose-type (Suzukiet al., 2003). Because the same site-specific location of high-mannose-type was found eveninthe distantly removedavianorderssuchasGalliformes(chicken)andColumbi-formes (pigeon), this feature may be widely occurringamong avian IgGs.CH3domainofavianIgGisequivalenttoCH2domainofmammalianIgG. InmammalianIgG, theN-glycosyla-tion site is located at Asn297 on CH2 domains (Figure 1A),and is well conserved among mammals (Burton, 1987). TheN-glycosylation at Asn297 is essential because it influencesthermal stability of IgG, recognition by Fc receptors,associationwithcompliment component C1q, andinduc-tion of antigen-dependent cellular cytotoxicity (Kobata,1990; Mimura et al., 2000; Radaev and Sun, 2001).N-glycansatthissiteareexclusivelybiantennarycomplextype. ItisreportedthatrecombinantIgGpossessingonlyhigh-mannose-typeoligosaccharides is defectivefor com-plement activation(Wright andMorrison, 1994). CrystalstructuresofhumanandmouseIgGstudiedbyX-raydif-fraction revealed that their Fc region form a cavity betweenthetwog-chains, andN-glycans onAsn297canbeseenoccludingthecavityatthecenteroftheFc(Harrisetal.,1997).1Hnuclear magneticresonanceprovidedevidencethatconformationalchangesinthesugarchainscanaffectthe structure of the Fc (Matsuda et al., 1990). By analogy oftheN-glycansatAsn297onmammalianIgG,andbecauseof the structural similarity of the complex-type N-glycans ofchickenandmammalianIgG, one mayassume that theFig. 9. Ribbon representations of predicted 3D structures ofchicken IgG-Fc (CH2 CH4) by homology modeling. The two chains(A-chain, green and B-chain, orange) are indicated in two orthogonalviews. Backbones and side chains of Asn308, Asn407 (light blue), andconserved Cys residues (at 252, 264, 322, 340, 372, 431, 477, and 546,yellow) forming intradomain and interchain disulfide bridges areindicated. Man5GlcNAc2- linking to Asn407 are visualized bysuperimpose from the template structure (PDB ID: 1o0v). The atomcolors for oligosaccharide chains are carbon, white; oxygen, red;nitrogen, blue.N.SuzukiandY.C.Lee286 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from glycans at the corresponding position on chicken IgG(Asn407) are also complex type.However, we have demonstrated that N-glycosylationpatternofavianIgGisclosertothoseofmammalianIgEthantomammalianIgG. Althoughthenumberandposi-tions of N-glycosylationsites onavianIgG, mammalianIgG, and IgEare varied, one site (Asn297 on CH2 ofmammalianIgG,Asn394 on CH3ofIgE, Asn407 on CH3of avianIgG) iswell conservedamongthem(Figure1A,1B). The crystal structure of human IgE-Fc (including Ce2-4domains)(Wanetal., 2002)revealedthatN-glycansonAsn394(seeFigure1A)areburiedinacavitybetweenthetwo heavy chains, as seen in N-glycans on Asn297 of mam-malianIgG. UnlikemammalianIgG, however, N-glycansat Asn394 of IgE is exclusively high-mannose-typeoligosaccharides (Baenziger et al., 1974; DorringtonandBennich, 1978), although the precise structures of N-glycansatthispositionderivedfromtheentireIgEmolecule(i.e.,not truncated mutants) remain to be elucidated. OtherN-glycosylation sites on IgE are complex-type oligosacchar-ides. The presence of high-mannose-type N-glycans atAsn394 in IgE can be understood from the crystal structure,which shows that the gap between two Ce3 domains of thetwochainsarenarrowerthanthoseof twoCg2domainsand covered with Ce2 domains formed by highly bentstructureat Ce2Ce3junctions (Wanet al., 2002; Zhenget al., 1992). In contrast, mammalian IgG has a wider cavityin the Fc region, and its hinge is flexible enough to make theFccavitynot coveredbytheFabregions (Harris et al.,1997; Zheng et al., 1992). Enzymes for N-glycan processingapparentlycanaccess the N-glycans inCg2cavitymorereadily, leading to complex-type structure.Structural properties of chickenIgG based on the aminoacidsequencesareknowntobeclosetomammalianIgEinterms of the number of CHdomains as well as theorganization of intradomain and interchain disulfidebonds (Magor et al., 1992; Parvari et al., 1988; Warr et al.,1995)(Figure1A).Inaddition,bothofthemmediateana-phylactic reaction (Faith and Clem, 1973; Warr et al., 1995),anditisbelievedthattheyarecloserelativesinmolecularevolution (Warr et al., 1995). Moreover, as we have demon-strated in this study, the site-specific presence of high-mannose-typeN-glycanatAsn407onchickenIgGisalsosimilar tothat inhumanIgE(Asn394). Basedonthesesequencesandwithfunctional similarityinmind, wecon-structeda3Dstructuremodel ofchickenIgG-Fc(CH2CH4) (Figure 9). The model suggests that unless FabandCH2regions conferredsterichindranceagainst GII,oligosaccharides inthe cave of Fc (CH3 CH4) regionswere relatively exposed and could not retain monoglucosy-latedforms.AlthoughthebentstructureofentirechickenIgGwithFabregions shouldbe confirmedpreciselybyother biochemical and biophysical approaches, such a uniquestructureismost likelytoimposethesterichindrancetoN-glycan processing enzymes to retain Glc1Man89GlcNAc2structures at Asn407.GlycoproteinsbearingmonoGlc-high-Manoligosaccha-ridesareusuallyfoundintheERasanearlyintermediateinthebiosynthesis of N-glycans andaresupposedtoberecognizedbylectin-likechaperones,calnexin(CNX)and/orcalreticulin(CRT), toaidinthefoldingprocess. Afterthe correct folding, the terminal glucose is released by GII,followed bya-mannosidase action for further N-glycanprocessing (Helenius and Aebi, 2001). Although it isreportedthatCRTcanbindtochickenIgGinvitro(Patilet al., 2000; Saito et al., 1999), the role of CNX or CRT inprotein folding for IgG in ER is not clear. In contrast, it isreportedthat partiallyfoldedmammalianIgGH-chainsform complex with BiP (immunoglobulin heavy-chain bind-ingprotein) andsomeotherchaperones, includingUDP-Glc:glycoproteinglucosyltransferase, but not with CNXnor CRT (Meunier et al., 2002).Acurrentlyproposedmodel fortheproteinfoldingandassemblyof mammalianIgG(HaasandWabl, 1983; Leeetal.,1999)isthatVH,CH2,andCH3domainsonmam-malian IgG are folded first, then folding of the CH1 domainis accomplished by assembly with light (L) chains. Weexpected that this two-step protein folding process of mam-malian IgG and formation of the IgE-like highly bent struc-ture can account for the presence of monoGlc-high-Man onavianIgG-CH3domain. If thetwo-stepfoldingmodel isalso applicable to avian IgG, it follows that VH, CH3, andCH4 domains of the H-chains are folded first, thenassembledwithLchains toallowthe completionof theentireIgmolecule(Figure10). Afterthefoldingof avianCH3domain,deglucosylationonthisdomaincanonlybepartially carriedout, becauseavianIgG-CH3 domainpos-sesses bothmonoglucosylatedandnonglucosylatedhigh-mannose-type N-glycans. However, concurrent with butindependent of deglucosylation, the H-chains are assembledwith L-chains, and the fully folded molecule no longerconfersaccessibilitybyGII, sothattheIgG-CH3domaincan retain monoglucosylated N-glycans (Figure 10). On theotherhand,N-glycosylationsitesontheCH2domainandvariable regions are more exposed to allow advancedN-glycanprocessing,sothattheycanbepossessedtofullyb-galactosylated, bisected, andcore fucosylatedcomplextype N-glycans. CH3 domains of chicken IgGcontainsexclusively high-mannose-type oligosaccharides, whereasCH2 domains only contains complex-type, suggestingthat the presence of monoGlc-high-Man N-glycans ismainlyduetostrictsterichindrance,ratherthanrandom,incomplete cleavages by the processing enzymes.The reasonfor the N-glycosylationsite inacavityofFc region to be conserved among mammalian IgG, IgE, andavianIgGandyet withdifferent N-glycantypes remainsto be elucidated. Basu et al. (1993) had reported that humanIgElackingN-glycosylationattheAsn394bypointmuta-tions tended to self-aggregate, although the loss of N-glyco-sylation did not influence its binding to Fce receptors.Therefore the N-glycosylation is probably involved at leastin stabilization of the protein structures by conferringsuitablehydrophilicityinthecavityof IgE, aswell asinIgG. If the hypothesis that IgY evolved to mammalian IgGis correct, mammalian IgGmight have gained a hinge regionthat gives higher segmental flexibilitytoaccommodateamore diverse range of antigens, such as adjusting for cross-linkingof epitopesontwolargeantigens. Accompanyingwiththe changes inproteinstructures, N-glycans inthecavityof Fcregionof mammalianIgGbecamecomplex-typebutareconservedatthesamesite(Asn297)toconferthe protein stabilization.Glc1Man9GlcNAc2-AsnonchickenIgG-CH3domains287 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from MaterialsandmethodsMaterialsPapain (2crystallized), iodoacetamide, and alkaline phos-phataseconjugated ExtrAvidin were purchased fromSigma(St.Louis, MO). GAFisalso known asPNGaseF,glycopeptideN-glycosidaseF,orN-glycanase.OneunitofGAFactivity is definedas the amount of enzyme thatcatalyzes the release of N-linked oligosaccharides from1nmol denaturedribonuclease Bin1minat 37

C, pH7.5)wasfromProzyme(SanLeandro, CA), andEndoHfrom Streptomyces plicatus was a gift from Dr. C. E. Ballou(Berkeley, CA). L-(Tosylamido-2-phenyl) ethyl chloro-methyl ketone (TPCK)-treated trypsin (3 crystallized)was from Worthington Biochemical (Lakewood, NJ). Alka-line phosphataseconjugated Con A and RCA-I lectin werepurchased from EY Labs (San Mateo, CA). Biotin-conjugated anti-chicken IgG-Fc was from Biotrend Chemi-cals (Destin, FL). 5-Bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium kit for use with alkaline phosphatasewaspurchasedfromZymedLaboratories(SanFrancisco,CA). DEAE-Sepharose Fast Flow column (HiTrap, 1 mL)was from Amersham Pharmacia Biotech (Piscataway, NJ).TSKgel DEAE-5PWcolumn(7.5 75mm) andTSKgelAmido-80 column(4.6 250 mm) were fromTosoHaas(Montgomeryville, PA). Shim-Pack CLC-ODS column(6.0 150mm)wasfromShimadzu(Kyoto,Japan).Poly-vinylidene difluoride membranes for blotting and CentriconYM 10 were from Millipore (Bedford, MA). Chicken serumIgGwas fromPel-Freez Biologicals (Rogers, AR). BCAProtein Assay reagents and immobilized avidin (on 6%cross-linkedbeadedagarose)werefromPierce(Rockford,IL). Neuraminidase fromArthrobacter ureafaciens was agenerousgift fromDr. TsukadaandDr. Ohtaof KyotoResearch Institute (Uji, Japan). Other exoglycosidases usedwere b-galactosidase (from jack beans SeikagakuAmerica),b-N-acetyl-D-hexosaminidase(fromjackbean, Sigma), a-mannosidase (fromjackbean, Glyko), anda-fucosidase(frombeef kidney, Roche). The matrices for MALDI-TOFMS, 3,5-dimethoxy-4-hydroxycinnamicacid(sinapi-nic acid, SA), a-cyano-4-hydroxycinnamic acid (ACH),2,5-dihydroxybenzoicacid(DHB),and20,40,60-trihydroxy-acetophenonemonohydrate(THAP)werepurchasedfromAldrich (Milwaukee, WI).BuffersandstandardproceduresTris-buffered saline (TBS) contains 50 mMTrisHCl(pH7.4)and150mMNaCl. TBSTcontains0.1%Tween20 in TBS. Digestion buffer for papain treatment wasFig. 10. Diagram of hypothesis of folding and assembly of avian IgG. In the ER, nascent H-chains of avian IgG possesses Glc3Man9GlcNAc2 onboth CH2 and CH3 domains. Partially folded H-chains with Glc1Man9GlcNAc2 can be produced by concerted actions of a-glucosidase I, II (GI, GII),UDP-Glc:glycoprotein glucosyltransferase (GT) (Parodi, 2000), and some ER chaperones. When folding of VH, CH3, and CH4 domains anddimerization of H-chains are proceeded in analogy to mammalian g-chains (Lee et al., 1999), N-glycan processing enzymes such as GII and a-mannosidase I might be able to partially process Glc1Man9GlcNAc2 on CH3 domain. However, concurrent with but independent on deglucosylation,L-chains are assembled with the H-chains mediated by BiP and other ER chaperones, then the CH3 domains became sterically unaccessible to theprocessing enzymes after full folding and assembly of the avian IgG molecules. Although timing of the folding of CH2 domain is unknown, folded CH2domains might confer highly bent structure between Fab and Fc regions in analogy to mammalian IgE (Wan et al., 2002). N-glycans on CH2 domains,however, are amenable to processing enzymes and can become complex-type eventually.N.SuzukiandY.C.Lee288 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from 50 mMsodiumphosphate (pH7.0) containing 1 mMEDTAand10mMcysteine. ProceduresforSDSPAGE,lectin- and immunoblottings, and N-terminal sequenceanalyseswereasdescribedpreviously (Suzuki etal., 2001).ProteinconcentrationsweremeasuredbytheBCAassay(Smith et al., 1985) using bovine serumalbumin as astandard.PapaindigestionofchickenserumIgGPapainsuspension(28 mg/mL) was dilutedindigestionbuffertobe1mg/mLandincubatedat 37

Cfor10minfor activation. ChickenserumIgGdissolvedindigestionbuffer(2mg/ml)wasincubatedwiththeactivatedpapain(enzyme:substrate ratio of 1:100) at 37

Cfor 4 h. Thereactionwas terminatedby adding iodoacetamide (finalconcentration 30 mM) and incubation at room temperaturefor30mininthedark. Themixturewasdialyzedagainst10 mM TrisHCl (pH 8.0) for the following anion-exchangechromatography.IsolationofFabandFcfragmentsofchickenserumIgGAcolumnofDEAE-SepharoseFastFlow(HiTrap,1mL)waswashedwith1MNaClin10mMTrisHCl(pH8.0)andequilibratedwith10 mMTrisHCl (pH8.0). Afterpapain-digested chicken IgG (1 mg) was loaded, the columnwas washed with 10 mM TrisHCl (pH 8.0) for 20 min (flowrate 1mL/min), andthenconcentrationof NaCl intheelutionwaslinearlyincreasedupto0.3Mwithin120min(flow rate 0.5 mL/min). The major peaks detected by A280nmwere collected and concentrated with a Centricon YM-10 at4

C. One of the chicken IgG Fab fractions was incompletelyseparatedfromtheFcfraction,anditwasfurtherisolatedwith an affinity column. Avidin-agarose (250 mL) in a 1-mLsyringecolumnwas washedwith5ml water andequili-bratedwith5mLbindingbuffer (20mMsodiumphos-phate, pH7.4, with 500 mMNaCl). Biotin-conjugatedanti-chickenIgGFcantibodies(100 mg)wereloadedontothe columnandallowed tostand for 30 min, thenthecolumn was washed with 5 mL binding buffer. The samplewas loaded into the column and incubated for 30 min; thenthe column was washed with 2 mL binding buffer. The pass-throughfractionwas collectedandconcentratedwithaCentriconYM-10. All theaffinitypurificationproceduresdescribed were performed at 4

C.GAFandEndoHtreatmentofglycoproteinsForGAFdigestion, glycoproteins(15mg) weredissolvedwith30mL50 mMNaHPO4(pH7.5) containing 0.1%SDSand100mM2-mercaptoethanol andheatedat90

Cfor3minfordenaturation. Afterthesolutionwascooledtoroomtemperature, 1%(v/v) NP-40was addedtotheheat-denaturedglycoproteins. Themixturewasincubatedwith GAF(40 U/mg substrates) at 37

Cfor 16 h forcomplete de-N-glycosylation, and heated at 100

C for10mintoinactivateGAF.Forpartialde-N-glycosylation,glycoproteins were incubated with GAF(1 U/mg sub-strates)for10min, 40min, and3handheatedat100

Cfor 10 min.EndoHdigestionforglycoproteinswasperformedsimi-larly,but50mMsodiumacetate(pH5.5)wasusedasthereaction buffer. The glycoproteins before and after thedigestions were analyzed with SDSPAGE.ReductionandalkylationofglycoproteinsThe formations of intramolecular disulfide bonds on glyco-proteinswerereducedandblockedasfollows. Glycopro-teins(0.2mg)weredissolvedwith120 mL8Mguanidine-HCl in0.2MTrisHCl (pH8.0)andreducedwith60 mL0.18 M dithiothreitol in the 8 M guanidine solution at roomtemperaturefor1h. Forthiol alkylation, 240mL0.18Miodoacetamideintheguanidinesolutionwasaddedtothemixtureandincubatedatroomtemperaturefor30mininthedark. ThereactionmixturewasdialyzedagainstH2Oand lyophilized.PreparationandisolationofglycopeptidesOne-third of the reduced and alkylated glycoproteinsweresuspendedin50 mL50mMNH4HCO3,pH7.8,andincubatedwithTPCK-treatedtrypsinat 37

Covernight.Trypsinwas inactivatedbyheatingat 100

Cfor 5min.Aportion of the reaction mixture was further treatedwithGAF(1U/10mL) at 37

C, overnight. The peptidefragmentsbeforeandaftertreatmentwithGAFwereana-lyzedwithreversed-phase HPLConaShim-packCLC-ODScolumn(6.0 150mm). Themobilephasewas (A)0.05%trifluoroacetic acid (TFA) and (B) 90%CH3CNwith0.05%TFA. Elution(1ml/min)wasconductedbyalinear gradient of 050%of (B) in (A) developed over100min. EachpeakdetectedbyA210nmwascollectedandkept at 4

C.PreparationandsepalationofPA-derivatizedoligosaccharidesforstructuralanalysesChicken serum IgG (reductive alkylated, 1 mg) weredigested with trypsin and chymotrypsin in 50 mMNH4HCO3, pH7.8, at 37

Covernight, andtheenzymeswereinactivatedbyheatingat100

Cfor5min.Oligosac-charides were released with GAFtreatment in 50 mMNH4HCO3, pH7.8, at37

Covernight. AfterinactivatingGAF by heating at 100

C for 5 min, the digest was lyophi-lized. Cations and peptides were removed with Dowex50W 2(H

form,50100mesh,250 mL,Sigma)packedin a 1-mL syringe. The column was washed with 1 mL H2O,andthecollectedeffluentwaslyophilized.Thesamplewasreconstituted with 100 mL H2O, loaded onto a Carbographtube(25mg,Alltech),andwashedwith400 mLH2O,thenelutedwith400mL50%CH3CNcontaining0.05%TFA.ForthePAderivatizationbyreductiveamination,lyophi-lizedoligosaccharidefractionsweredissolvedin40 mLPAsolution(1g/580mLinconcentratedHCl, pH6.8), andheatedat 90

Cfor 15 minwithheating block. FreshlypreparedNaCNBH3solution(7mg/4 mL)wasaddedintothe reaction mixture, then heated at 90

Cfor 1 h. PAoligosaccharides were fractionatedbygel filtrationonaSephadex G-15 column (1.040 cm, in 10 mM NH4- HCO3),Glc1Man9GlcNAc2-AsnonchickenIgG-CH3domains289 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from and the effluent was monitored with a fluorometer (excita-tion 300 nm, emission 360 nm) and lyophilized.The mixture of PA-oligosaccharides was separatedbyHPLC with three different columns as described previously(Nakagawa et al., 1995). In the first stage, the PA-oligosac-charideswereseparatedonaTSKgelDEAE-5PWcolumn(7.5 75 mm), and the neutral, monosialyl, and disialylfractions (monitoredbyfluorescence, excitation320nm,emission 400 nm) were collected separately and lyophilized.In the second stage, neutral, mono-, and disialylatedoligosaccharide fractions were individually dissolved inH2Oandseparatedona Shim-PackCLC-ODScolumn(6.0 150 mm), monitoring effluent with fluorescence(excitation 320 nm, emission 400 nm). Elution wasperformed at a flow rate of 1.0 mL/min at 55

C using eluentA (0.005% TFA) and eluent B (0.5 % 1-butanol in eluent A).The columnwas equilibratedwitha mixture of eluentsA:B90:10(v/v), andaftersampleinjection, theratiooftheeluentswaschangedlinearlytoA:B60:40in60min.Eachpeakwas collected, lyophilized, andanalyzedwithMALDI-TOF MS.Inthethirdstage, major peaks fromtheODScolumnwere dissolved with 50 mL eluent C (CH3CN:3%CH3COOH-trietylamine,pH7.3,65:35),andseparatedonaTSKgel Amide-80column(4.6 250mm), monitoringeffluent with fluorescence, excitation 300 nm, emission360nm. Elutionpositionof eachPA-oligosaccharidesonCLC-ODSandAmide-80columnswasexpressedinGUs,basedontheelutionpositionofisomaltoseseries.Fortheassignment of GUODS, analysis on a CLC-ODS column wasperformed using 10 mMsodium phosphate, pH 3.8(Tomiyaet al., 1988) insteadof 0.005%TFA. ReferencePA-derivatized oligosaccharides fromasialo-human IgGandbovineRNaseBwerepreparedbythesamemethod.Structures of the reference compounds were shown inFigure2. PA-derivatizedN-glycans A, B, C, D, I, J, K,and L (Figure 2) were obtained from a-fucosidase-digestedPA-oligosaccharides from human IgG.MALDI-TOFMSMALDI-TOFMS was performed on a Kompact SEQ(Kratos Analytical, Manchester, England), equippedwith a 337-nmnitrogen laser and set at 20 kVextrac-tionvoltage. Eachspectrumwas theaverageof 50lasershots. Glycoproteins, glycopeptides, and neutral oligo-saccharideswereanalyzedinthelinearpositive-ionmode,andsialylatedoligosaccharideswereanalyzedinthelinearnegative-ionmode. SAandACHwere usedas matricesin the analysis of glycoproteins and glycopeptides,respectively. Thesematrices weredissolvedtobe10mg/mLwith50%CH3CNwith0.05%TFA. Forneutral andsialylatedPA-derivatizedoligosaccharides, DHB(10 mg/mLin5 mMNaCl) andTHAP(2 mg/mLin25%CH3CNwith10mMdibasicammoniumcitrate)wereused,respec-tively (Papac et al., 1998). Samples (0.5 mL) were applied toatarget andmixedwithmatrix(0.5mL), andallowedtodryunderambientcondition(forSAorACH)orvacuum(forDHBorTHAP) at roomtemperaturepriortomassanalysis.Endo-andexoglycosidasedigestionforglycopeptidesandoligosaccharidesGlycopeptides(about90pmol/2.5mL)weredigestedwithGAF (1 U) in 50 mM NH4HCO3 (pH 7.8) or with Endo H(2.5mg)in20mMsodiumacetate(pH5.6)at37

Cover-night. For exoglycosidase digestion, glycopeptides were dis-solved with 20 mM sodium acetate (pH 4.5) to be 40 pmol/mL. Glycopeptides containing high-mannose-type oligosac-charides (90 pmol) were digested with a-mannosidase(250 mU/100 pmol glycopeptides) at 37

C overnight.Glycopeptides containing complex-type oligosaccharides(250 pmol) were sequentially digested with a-sialidase(0.63 mU/100 pmol of glycopeptides), b-galactosidase(0.38mU), b-N-acetyl-D-hexosaminidase (1.11mU), anda-fucosidase (0.5 mU). After the incubation at 37

Covernight andheat-inactivationat 90

Cfor5minat theeach step, a portion of the digestion products was analyzedwithMALDI-TOFMS. PA-derivatizedoligosaccharideswere digestedwith exoglycosidases in the same mannerand analyzed with HPLC as described.HomologymodelingAmodel of the3Dstructureof chickenIgG-Fc(CH2CH4)(fromSer250toGly567)wasconstructedusingtheSWISS-MODELprogram(Schwedeetal.,2003),whichismade public by the Swiss Institute of Bioinformatics(UniversityofBasel,Switzerland).ThecrystalstructureofhumanIgE-Fc(CH2CH4)(Wanetal.,2002)(PDBID:1o0v)wasusedasatemplateforthehomologymodelingbased on their 32% amino acid sequence identity as well astheiroverall structural similarity. Thesequencealignmentwas generated by T-Coffee (Notredame et al., 2000),which correctly aligned all Cys and Trp residues (hallmarksof Ig-like domains) conserved between the target andtemplate. The optimize mode for oligomer modeling in theSWISS-MODELserver was chosen for the appropriatemodeling. The predicted 3D structure was visualizedwithDeepViewprogram. Oligosaccharide chains (Man5-GlcNAc2-)atAsn407onchickenIgGwasimposedfromthe template.AcknowledgmentsTheauthorsaregratefulforDr.NoboruTomiyafortech-nicaladviceconcerningoligosaccharideanalysisbyHPLCand Dr. Hao-Chia Chen for peptide sequencing. This workwas supported by NIH Research Grant DK09970.AbbreviationsACH, a-cyano-4-hydroxycycinnamicacid;CBB,Coomas-siebrilliantblue;CNX,calnexin;ConA,concanavalinA;CRT, calreticulin; ER, endoplasmic reticulum; GII, a-glucosidaseII; GAF, glycoamidaseF; GU, glucoseunit;MALDI, matrix-assistedlaserdesorption/ionization; MS,mass spectrometry; ODS, octadecylsilica; PA, 2-aminopyr-idine;PBS,phosphatebufferedsaline;SA,3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid); SDSPAGE,sodium dodecyl sulfatepolyacrylamide gel electrophoresis;N.SuzukiandY.C.Lee290 by guest on September 5, 2012http://glycob.oxfordjournals.org/Downloaded from TBS, Tris-buffered saline; TFA, trifluoroacetic acid;THAP, 20,40,60-trihydroxyaetophenone monohydrate;TOF,timeofflightReferencesBaenziger, J., Kornfeld, S., and Kochwa, S. (1974) Structure of thecarbohydrateunitsof IgEimmunoglobulin. I. 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