the structural basis of blood group a-related glycolipids

The structural basis of blood group A-related glycolipidsin an A3 red cell phenotype and a potential explanationto a serological phenomenon

Lola Svensson1,2, Laura Bindila3,4, Jonas ngstrm5,Bo E Samuelsson2, Michael E Breimer5,Lennart Rydberg2, and Stephen MHenry6

2Department of Clinical Chemistry and Transfusion Medicine, Institute ofBiomedicine, The Sahlgrenska Academy at University of Gothenburg,Sahlgrenska University Hospital, Sahlgrenska Campus, 413 45 Gothenburg,Sweden; 3Institute for Medical Physics and Biophysics, University ofMnster, 48149 Mnster, Germany; 4Luxembourg Clinical Proteomics,Centre de Recherche Public, Strassen L-1445, Luxembourg; 5Department ofSurgery Institute of Clinical Sciences, The Sahlgrenska Academy atUniversity of Gothenburg, Sahlgrenska University Hospital, stra Campus,416 85 Gothenburg, Sweden; and 6Biotechnology Research Institute, AUTUniversity and KODE Biotech Ltd, Auckland 1142, New Zealand

Received on July 23, 2010; revised on August 20, 2010; accepted onSeptember 6, 2010

Glycolipids from the red cells of a rare blood group A sub-group individual, expressing the blood group A3 phenotypewith the classical mixed-eld agglutination phenomenon,A2(539G>A)/O1 genotype, and an unusual blood group A gly-colipid prole, were submitted to a comprehensive bio-chemical and structural analysis. To determine the natureof blood group A glycolipids in this A3 phenotype, struc-tural determination was carried out with complementarytechniques including proton nuclear magnetic resonance(1D and 2D), mass spectrometry (MS) (nano-electrosprayionization/quadrupole time-of-ight and tandem massspectrometry) and thin layer chromatography with immu-nostaining detection. As expected, total blood group Astructures were of low abundance, but contrary toexpectations extended-A type 2 and A type 3 glycolipidswere more dominant than A hexaglycosylceramides basedon type 2 chain (A-6-2 glycolipids), which normally isthe major A glycolipid. Several para-Forssman(GalNAc3GbO4) structures, including extended forms,were identied but surmised not to contribute to the classicmixed-eld agglutination of the A3 phenotype. It is pro-posed that the low level of A antigen combined with anabsence of extended branched glycolipids may be thefactor determining the mixed-eld agglutination phenom-enon in this individual.

Keywords: blood group A / glycolipid / immunochemistry /mass spectrometry / para-Forssman

Introduction

Blood group ABO antigens are present both as glycolipidsand as glycoproteins on the red cell membrane. These anti-gens are quantitatively expressed in a serologically denedmanner, with 80% of blood group A Europeans having verystrong A antigen expression and 20% having moderate Aantigen expression, representative of the A1 and A2 red cellphenotypes, respectively (Economidou et al. 1967; Schachteret al. 1973; Schenkel-Brunner 2000). A series of much less-frequent weak A subgroup red cell phenotypes each withdecreasing blood group A antigen from Aint, A3, Ax, Aend,Am, Ann, Abantu, Alae, Ay to Ael are also recognized. TheN-acetylgalactosaminyl transferase encoded by the bloodgroup A gene was mapped in 1990 (Yamamoto et al. 1990),and thereafter studies have established an enormous hetero-geneity between and within the subgroups (Olsson andChester 1996; Olsson et al. 2001). In most cases, subgroupsare due to recognized genetic mutations (Olsson et al. 2001);however, not all subgroups can be adequately explained bymutations within the ABO gene. In particular, with the excep-tion of the A3 sample in this paper, all other A3 phenotypingindividuals reported to date do not have a recognized ABOgene mutation (Olsson et al. 2001).Unlike the relatively well-investigated qualitative and quan-

titative differences in A antigen expression between thecommon A1 and A2 red cell phenotypes (Schachter et al.1973; Clausen, Levery, Nudelman et al. 1985; Svensson et al.2009), the qualitative differences of the weak A subgroups arestill largely unexplored. Studies with electron microscopy ofimmunogold-labelled red cells and immunochemistry of gly-colipids indicate that the weak A subgroups differ from A1and A2 both in the number of antigens present and by theirchemical nature (Heier et al. 1994; Svensson et al. 2005). Theweak A subgroup known as the A3 phenotype is one of themore unusual A subgroups as it is dened by the serologicalreaction referred to as mixed-eld agglutination: smallagglutinates in a sea of free unaggutinated cells (Klein andAnstee 2005). One recent ow cytometry study also showedthat there appears to be two populations of A cells in the A3phenotype, although the A3 sample in this paper (albeit with

1To whom correspondence should be addressed: Fax: +46-31-417631; e-mail:[email protected]

Glycobiology vol. 21 no. 2 pp. 162174, 2011doi:10.1093/glycob/cwq143Advance Access publication on October 6, 2010

The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected] 162

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a different genetic basis) in the same study was an exceptionwith only a single population of A cells (Hult and Olsson2010). Further evidence to support dual populations in the A3phenotype comes from the observation that A glycosyltrans-ferase activity could only be found in the stroma of A3agglutinates (Nakamura et al. 1989). Interestingly, earlystudies of A3 mixed-eld reactions found that the unaggluti-nated cells were agglutinable when retested with anti-A(Cartron et al. 1977), and despite no genetic evidence for anaberrant glycosyltransferase, 13 A3 individuals were found tohave an A glycosyltransferase with a different pH optima thanthat of the A1 glycosyltransferase (Nakamura et al. 1989).Determining the structural basis of unusual phenotypes, or

the insight they may give of background structures notusually accessible for identication in the presence of normaland dominating antigens, is of importance to understandingthe biology of blood group antigens. Subgroups provide aunique insight into the biosynthesis of ABO antigens, or atleast alternative biosynthetic pathways which may operateunder certain biological pressures. This paper describes acomprehensive biochemical and structural analysis of glyco-lipids isolated from one individual with a well-dened A3phenotype. The structural analysis is based on complementarystructural proling involving nuclear magnetic resonance(NMR), mass spectrometry (MS) and thin layer chromato-graphy (TLC) techniques. The data corroborated a previousstudy of this individual (Aweak-II), which revealed an A3 phe-notype, with a new genetic mutation (A304 in Blood GroupAntigen Gene Mutation Database; Blumenfeld and Patnaik2004), and an unusual total neutral glycolipid prole(Svensson et al. 2005). To distinguish the sample of thispaper from the undened ABO genetic background A3samples, hereafter it is termed the A304 A3 phenotype.

ResultsConrmation of mixed-eld agglutination phenomenonThe red cells of the A304 A3 sample were subjected toextended differential serology to prove the nature of themixed-eld agglutination (Figure 1). As expected, the A304A3 sample (lane I) showed the classic mixed-eld reaction ofboth agglutinating and non-agglutinating cells. When theagglutinating (lane II) and non-agglutinating cells (lane III)were puried and then retested with new reagents, both againshowed the mixed-eld agglutination reaction.

Total glycolipid pattern by TLC and TLCenzymeimmunoassay analysisAnalysis of the total neutral glycolipids extracted from the sixblood unit donations (Figure 2, lanes T) revealed a TLCprole qualitatively identical to the original single donation(Aweak-II) extract (Figure 2, lane T*). In comparison with thecontrol A1 Le(ab) ABH-secretor total glycolipids (Figure 2,lane A1) both Aweak-II samples (T* and T) reacted similarlyagainst anti-A 222, albeit quantitatively reduced in reactivity,despite being loaded at a 2.5 times higher concentration.However, against anti-A type 2 reactive monoclonal anti-bodies (MAbs) 2-24 and HH4, staining was unambiguouslyreduced. Anti-AB 2-41 was unreactive in the six-sugar region

but showed signicant reactivity in the extended glycolipidregions in agreement with previously reported data (Svenssonet al. 2005). Reactivity against three anti-B reactive reagentsconrmed this reactivity was not due to B structures (resultsnot shown). The anti-A type 3 reactive MAb TH1 revealed nobands in the regions expected for A type 3 glycolipids.Reactivity against anti-H type 3 MAb (HH14) and anti-H

type 2 (BE2) showed a pattern similar to a blood group A2phenotype control (result not shown).

Chemical staining of the medium-pressure liquidchromatography fractionsThe medium-pressure liquid chromatography (MPLC) separ-ation of the total neutral glycolipids resulted in seven fractions,each containing mixtures of complex glycolipids of increasingsize, and persistent contamination with globoside (GbO4) themajor glycolipid component in red cells, yet were sufcientlypure for MS and NMR analyses. Anisaldehyde semi-quantitativechemical staining (Figure 3, plate I) showed that fraction 1included glycolipids with 46 sugar residues, whereas fractions2 and 3 mainly contained 712 residues. The dominant struc-tures in fractions 4 and 5 were glycolipids with 8 to approxi-mately 12 sugar residues. Fractions 6 and 7, although poorlyresolved, represented the more extended glycolipids with morethan 10 sugar residues. Table I shows a summary of the repre-sentative glycolipids and weight of each of the seven fractions.

TLCenzyme immunoassay of the MPLC fractionsThe immunochemical staining was repeated on the MPLCfractions at a loading concentration of 10 g per lane.

Fig. 1. Mixed-eld agglutination serology. A304 A3 red cells are shown tohave typical mixed-eld agglutination (a mixture of positive (+) agglutinatedand unagglutinated negative () cells). Retesting of the agglutinated cells(lane II) and the unagglutinated cells (lane III) from the original reaction (laneI) again produced mixed-eld agglutination for both populations of cells.

Glycolipid structures in an A3 red cell phenotype

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Fraction 1 (Figure 3, plate II, lane 1), containing the four tosix-sugar-residue glycolipids, stained in the six-sugar region(relative migration, rm 6) with two monoclonal A antigenreactive reagents and the lectin Helix pomatia. With due con-sideration to the semi-quantitative nature of TLCenzymeimmunoassay (EIA), there was signicant contrast in bandintensity between the strong six-sugar bands with Lorne/H. pomatia and the moderate band with anti-A 2-24 (Figure 3,II) and the absence of reactivity with anti-AB 241.Compared with the unfractionated total samples (Figure 2),

the concentrated glycolipids in the MPLC fractions (Figure 3,II and III) could be more condently immunochemicallydened, particularly the presence of A type 2 (Hakomori1981) and A type 3 glycotopes (Clausen, Levery, Nudelmanet al. 1985; Clausen et al. 1986) (Figure 3, III). As expected,MAb 2-22 was strongly reactive in all fractions (Figure 3, III)and reactivity could be seen indicative of blood group A with8 (rm 3) and 10/12 (rm 1 0.5) sugar residues and probablypara-Forssman (GalNAc3GbO4, rm 6 0.5) (Ando et al.1982). Identity of these structures as A type 2 glycolipids wasindicated by their reactivity with the anti-A type 2 reactiveMAb (224). In addition, very weak reactions seen withanti-A type 3 MAb TH1 suggest the presence of some A-9-3(rm 3.0) and A-11-3 (rm 0.9) glycolipids in fractions 4 and 5,respectively (Figure 3, III). Expected glycolipid reaction pat-terns (when compared with the A2 phenotype) were observedwith anti-H type 2 and H type 3 reactive MAbs (results notshown).

Structural analysis of A glycolipids by nano-electrosprayionization-quadrupole time-of-ight tandem massspectrometryBy nano-electrospray ionization (ESI)-quadrupole time-of-ight (QTOF) MS screening, a number of blood group Aglycolipid-related ions were detected in fractions 1, 4 and 5 asillustrated in Figures 46 and Table II. In total, 19 blood groupA glycolipid-related ions based on compositional assignment

were detected, indicative of A-6, A-8, A-9, A-10 and A-11 gly-colipids. The evaluation of glycolipid molecular ion patternsprovided evidence that A-8 structures dominate in number andabundance. The applied ionization conditions prompted theformation of doubly charged ions with a minimal, if any,in-source decay of glycosidic bonds. The A-6 glycolipid(Table II) was exclusively detected in its singly charged form.All ion species detected in the MS1 prole and postulatedbased on the compositional assignment to correspond to A gly-colipids (cf. also Table II) were conrmed by collision-induceddissociation (CID) tandem mass spectrometry (MS/MS) frag-mentation analysis. Basically, A-8, A-9 and A-10 type glyco-lipids were found to dominate the mixture. Sensitivity of theanalysis was sufciently high to allow the detection of verylow abundant species, such as A-6 glycolipids.Nano-ESI-QTOF MS identication of A-6 was initially

based on compositional analysis of corresponding singlycharged ions at m/z 1711.11 assigned to FucHexNAc2Hex3Cer(d18:1/24:0). Accurate structural elucidation of theions at m/z 1711.11 was achieved by CID MS/MS exper-iments (Figure 4) in the second-stage analysis. The resultantfragmentation pattern is unambiguously consistent with theA-6 structure having d18:1/24:0 Cer (Figure 4, insetscheme). The presence of a terminal fucose (Fuc) residue iseasily deducible from the immediate loss of 146 (atomicmass units) from the molecular ions, represented at m/z1565.02. The fragmentation pathway of the ions at m/z1565.02 follows a stepwise glycosidic bond cleavage, start-ing with the monosaccharide residues from the nonreducingend up to the Cer residue, and dening this way thesequence of the main HexNAc-Hex-HexNAc-Hex-Hex-Cerglycosidic backbone. The presence of the ions at m/z1507.99 corresponding to a neutral loss of the HexNAcresidue from the precursor ions clearly indicates the locationof HexNAc at the nonreducing end terminus and, at thesame time, that there exists a branching point for Fuc attach-ment within the remaining glycan chain. Given the presenceof Y3 type ions at m/z 1199.83, which denes the

Fig. 2. TLCEIA of total neutral glycolipids from the Aweak-II A3 phenotype donor (A304 A3) immunostained with selected blood group A reactive MAbs. Thenumbers to the right denote the approximate number of carbohydrate residues of the glycolipids in the corresponding band. Lane A1, common blood group A1phenotype control, 20 g loading; lane T*, the original Aweak-II glycolipid preparation, 50 g loading; lane T, new Aweak-II glycolipid preparation from sixpooled donations, 50 g loading. Individual plates were overlaid with either MAb anti-A 2-22, 2-24, anti-A type 2 HH4, anti-AB 2-41 or anti-A type 3 TH1,respectively, and then compiled to create the gure.

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HexNAc-Hex-Hex-Cer(d18:1/24:0) structure, and its counter-part B2-type ions at m/z 534.17, the attachment of the Fuc atthe terminal Gal residue is clearly revealed. Additionally, theB3-type ions at m/z 737.27, consistent with the HexNAc-(Fuc)Hex-HexNAc structure, and B4-type ions at m/z 899.63,

along with their counterpart fragments, support further thestructural architecture of the glycan chain as consistent withan A-6 glycolipid. The identity of the Cer moiety is directlyrevealed by the Y0-type ions at m/z 672.62 consistent withd18:1/24:0 and the series of Y1-Y3 type fragment ions,

Fig. 3. TLC analysis of the MPLC-puried glycolipid fractions. Lane numbers on plates equate with fraction numbers. Plate I, anisaldehyde chemical staining.Plates IIIII, immunostaining of fractions at a 10 g per lane loading but with A1 and A2 phenotype controls loaded at 20 g per lane due to the lower relativeabundance of individual glycolipid species in total fractions. Plate IV, GalNAc3GbO4 activity mapping of MAbs 2-22, 2-24 and 2-41. Plate II shows acomparison of the common A1 and A2 phenotyping controls and fraction 1 (containing the hexaglycosylceramides) and their respective reactivity against MAbanti-A Lorne, anti-AB MAb 241, anti-A type 2 MAb 224 and the lectin H. pomatia. Plate III shows the presence of various A antigens in the MPLCfractions as detected with the broadly reactive anti-A MAb 2-22 and specic A type 2 MAb 2-24 and A type 3 (MAb TH1) reagents. The control lane (A1) tothe right is blood group A1 glycolipids stained with MAb anti-A Lorne. Plate IV is GalNAc3GbO4 immunostained with selected anti-A and anti-AB MAbs.Lane A1, common blood group A1 phenotype control at 20 g per lane; lane p-F, puried GalNAc3GbO4 loaded at 6 g per lane. Plates are immunostainedwith the broadly reactive MAb anti-A 2-22, anti-A type 2 reactive 2-24 and anti-AB 2-41.


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whereas the N-type ions at m/z 264.28 correlate with thed18:1 long-chain base of the Cer part. Under the fragmenta-tion conditions applied, a number of fragment ions arisingfrom internal double- or triple-glycosidic-bond cleavageswere detected, which underscore more of the glycan chainstructure: the core disaccharide unit is supported by the ions

at m/z 347.12 (B5/B3) and the Y2-type ions at m/z 996.77;the (Fuc)HexHexNAcHex is indicated by the ions at m/z696.33, which then undergoes a neutral loss of Fuc todeliver the fragment ions at m/z 550.18.All other blood group A glycolipid species (listed in

Table II) rendered similar rich fragment ion patterns. Anadditional example is presented in Figure 5, where the frag-mentation patterns of the doubly charged ions at m/z 1304.58and the co-isolated ions at m/z 1305.60, respectively, areassigned to Fuc2Hex5HexNAc4Cer(d18:1/24:0,1) based on thetheoretical calculation. The resulting fragmentation pattern isconsistent with an A-11 structure with (d18:1/24:0) (Figure 6,I). Given the very low abundance of this species in the orig-inal mixture of fraction 5, the MS/MS pattern is remarkablyrich and the fragment ions abundance is sufcient to identifythe structure. Similar to the A-6 structure, the neutral loss of aHexNAc residue from the precursor ions delivering the frag-ment ions at m/z 1202.82 (Y8) indicates the HexNAc as aterminal residue at the nonreducing end. More prominent isthe neutral loss of one and two Fuc residues, characterizingthe degree of fucosylation of the molecule. Following the lossof HexNAc residue from the precursor ions, the series ofY-type ions starting with the ions at m/z 1048.74 arising from

Fig. 4. Nano-ESI-QTOF fragmentation spectrum of singly charged ions at m/z 1711.11 detected in fraction 5 consistent with FucHexNAc2Hex3Cer(d18:1/24:0)and assigned to the A-6 glycolipid. Co-isolated are the ions at m/z 1709.10 which correspond to A-6 Cer(d18:1/24:1), giving rise to Y-type fragment ionsdownshifted by 2 amu (due to the monounsaturated C24 fatty acid) compared with the ions derived from precursor at m/z 1711.11. Inset: Fragmentation schemeof the A-6 glycolipid with Cer(d18:1/24:0).

Table I. The weight and dominant range of glycolipids in the MPLC fractions

MPLCfractions

Weight(mg)

Carbohydrateperglycolipida

1 15 462 2 793 1 8124 0.5 8125 0.7 10126

the subsequent neutral loss of FucHex, and ending withY1-type ions, HexCer, essentially concludes the carbohydratechain structure of an A-11 glycolipid. The HexNAc-Hex-HexNAc-Hex-Hex-Cer structure represented at m/z 1565.18 isextended by two HexNAc-(Fuc)Hex units, which is typicalfor an A-11-3 structure. The fragment ions at m/z 1873.30along with the fragmentation of ions at m/z 1202.78 to1048.74, respectively, clearly point to the attachment of twoFuc residues at the rst two Hex residues in the glycan chainat the nonreducing end. These structural elements along withthe B-type ions, B2 (HexNAc-(Fuc)Hex) and B3(HexNAc-(Fuc)Hex-HexNAc) and a considerable number ofinternal double- and triple-glycosidic-bond cleavages(Figure 6, I) represent a reliable set of ngerprint ions fordiagnosing an A-11-3 structure.An interesting feature revealed by data interpretation from

Figure 5 is the existence of structural elements correspondingto an overlapping, isobaric structure, other than A-11. Thefragment ions at m/z 1150.33 related to a neutral loss of aFucHex residue from the precursor ions along with the ionsderived by the subsequent neutral loss of the second Fucresidue at m/z 1077.25, and the ions at m/z 1768.09 support-ing the HexNAc-HexNAc-Hex-HexNAc-Hex-Hex-Cer

fragment reveal the existence of another species, as depictedin Scheme II (Figure 6). Even though at very low abun-dance, these peaks depict a reliable isotopomer pattern fortheir identication and assignment. In this conguration(Scheme II in Figure 6), a repeating (Fuc)Hex-HexNAcmotif at the nonreducing end terminus extends the HexNAc-Hex-HexNAc-Hex-Hex-Cer core structure at m/z 1565.18,indicating a seemingly extended X2-type glycolipid. Thepresence of X2 glycolipid was also detected by NMR(Table III), while structural elements suggestive for othermembers of extended X2 glycolipid series were also foundto interfere with other blood group A glycolipid fragmenta-tion patterns. A more detailed study of such an extended X2glycolipid as well as of extended GalNAc3GbO4 series(data not shown) will be undertaken in the near future tocomprehensively identify the number and type of the struc-tures in the series.

Structural analysis of A glycolipids by proton NMRThe proton NMR (1H-NMR) spectrum of fraction 1 is shownin Figure 7 and identied structures and references (Clausen,Levery, McKibbin et al. 1985; Holgersson et al. 1990; Thorn

Fig. 5. Nano-ESI-QTOF fragmentation spectrum of doubly charged ions at m/z 1304.58 detected in fraction 5 consistent with Fuc2HexNAc4Hex5Cer(d18:1/24:1)and assigned to the A-11 glycolipid. Co-isolated are the ions at m/z 1305.60 which corresponds to a glycolipid with 11 sugar residues and Cer(d18:1/24:1),giving rise to Y-type fragment ions up shifted by 2 amu compared with the ions derived from precursor at m/z 1304.58. Fragmentation patterns are alsoconsistent with an isobaric structure, possibly an extended X2 glycolipid.


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et al. 1992; Teneberg et al. 1994; Angstrom et al. 2004;Miller-Podraza et al. 2005) are summarized in Table III. Thedominating structures were as expected GbO4, H-5-2 andLea-5 in the descending order. Smaller contributions fromGalNAc3GbO4, X2, FucGgO4 and FucGbO5 are also evidentas are small contributions from Lex-5 and A-6-2. A closerexamination of the Fuc2 and GalNAc3 signals of the

A-6-2 revealed, however, an overlapping component whichmost likely stems from the corresponding signals of the A-4-6glycolipid (GalNAc3(Fuc2)Gal4Glc1Cer) (Breimer et al.1982; Bjork et al. 1987). An intermediate contributionattributed to neolactotetraosylceramide and/or neolactohexao-sylceramide (nLc-4 and/or nLc-6) is also deduced from theintensity of the GlcNAc3 resonance at 4.65 ppm which

Fig. 6. Fragmentation scheme of the ions at m/z 1304.58 for (I) the A-11 glycolipid with Cer(d18:1/24:01), the major species and (II) an isobaric structure,possibly extended X2 identied as low abundant species.

Table II. List of ion species corresponding to A-glycolipids detected by nano-ESI-QTOF MS in three fractions and their structural assignment

Structural assignment Ceramide Name m/z

1 HexNAc(Fuc)HexHexNAcHexHex d18:1/24:1 A-6-2 1709.10+

HexNAc(Fuc)HexHexNAcHexHex d18:1/24:0 A-6-2 1711.11+

4 HexNAc(Fuc)HexHexNAcHexHexNAcHexHex d18:1/18:0 A-8-2 1006.832+

HexNAc(Fuc)HexHexNAcHexHexNAcHexHex d18:1/20:1 A-8-2 1019.822+






5 HexNAc(Fuc)HexHexNAcHexHexNAcHexHex d18:1/18:0 A-8-2 993.412+




HexNAc(Fuc)HexHexNAc(Fuc)HexHexNAcHexHex d18:1/24:1 A-9-3 1122.102+

HexNAc(Fuc)HexHexNAc(Fuc)HexHexNAcHexHex d18:1/24:0 A-9-3 1123.112+

HexNAc(Fuc)HexHexNAcHexHexNAcHexHexNAcHexHex d18:1/24:1 A-10-2 1231.522+

HexNAc(Fuc)HexHexNAcHexHexNAcHexHexNAcHexHex d18:1/24:0 A-10-2 1231.532+

HexNAc(Fuc)HexHexNAc(Fuc)HexHexNAcHexHexNAcHexHex d18:1/24:1 A-11-3 1304.582+

HexNAc(Fuc)HexHexNAc(Fuc)HexHexNAcHexHexNAcHexHex d18:1/24:0 A-11-3 1305.602+

N.B. All ion species were subjected to CID MS/MS experiments for structure elucidation.Bold indicates the blood group A epitope.

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cannot be due solely to the internal GlcNAc3 resonance ofthe X2 glycolipid (cf. the intensities of resonances EIII andEV). The P1 structure may also be present in small amountsbut is in that case masked primarily by GbO4 andGalNAc3GbO4 resonances.1D and 2D NMR evidence (not shown) for the presence of

different structures in fractions 25 containing extended gly-colipid chains is summarized in Table IV. Most noticeable isthe dominating presence of GbO4 and H-7-2/H-9-2 in all fourfractions whereas blood group A type 2 structures are evidentin very small amounts having 8 or 10 sugar residues. Allother structures are also present in very small amounts exceptfor an extended form of P1, which is present approximately inthe same amount as the H structures in fraction 4. Evidencefor a second extended structure stems from the observed per-turbations of the anomeric shift of the terminal GalNAc3residue of GalNAc3GbO4 in fraction 2. However, the natureof this extension from an NMR point of view is unclear atpresent. As expected, the extension of H-5-2 by one or twolactosamine fragments yielding H-7-2 and H-9-2 is present asare the structures H-8-3 and H-10-3 which are precursors forthe similarly found A-9-3 and A-11-3 structures. It should benoted that only the signature resonances stemming from theglycotope residues have been recognized for different A andH structures. No additional or informative spectra wereobtained from fractions 6 and 7. Novel glycolipid structuresnot related to blood group A, such as series of extendedGalNAc3GbO4, extended X2 and extended P1 glycolipidswere detected by both proton NMR and MS (Table IV).

Discussion

The A3 phenotype is rare occurring with a frequency of

for a structural analysis, particularly NMR spectroscopy, isdemanding. Unfortunately, confounding issues posed by thepoor solubility of puried extended glycolipids severely com-promises the ability to obtain optimal levels of purity, and

instead fractions containing mixtures of glycolipids have to beused. However, by using three independent techniques, comp-lementary advantages were exploited: TLC with immuno-chemical detection dened the approximate size and type ofthe glycotope(s) with a variety of monoclonal reagents; MSanalysis with high sensitivity determines the existence of low-abundance structures and provides the structural architectureof glycolipids; proton NMR including 2D methods allows theidentication of specic sugars and linkage signatures. Itshould be noted that, by tandem MS analysis of underivatizedA type glycolipids, the discrimination between Gal3 andGal4 linkages is not achievable, as ring cleavage or differen-tial fragment patterns are not readily obtainable.Derivatization methods such as permethylation (Domon et al.1990) were not explored in this study due to the increasedcomplexity this procedure would introduce to already highlyheterogeneous mixtures. Chain type as either 2 or 3 was there-fore determined by immunochemistry and NMR.Nevertheless, given the fact that A type 3 chains are doublyfucosylated, whereas A type 2 chains are monofucosylated(for species with the same number of monosaccharide resi-dues), and experimental conditions were applied to preventingin-source defucosylation, the type of the A chain could bepostulated from tandem MS as well. By combining the resultsof all methods, an accurate and condent identication of thered cell glycolipids of this A304 A3 phenotyping individualwas possible even for complex glycolipid molecular speciespresent in trace amounts.It was fortunate that this A304 A3 individual was an ABO

non-secretor, thereby eliminating the presence of type 1 Astructures acquired by red cells from plasma from theinterpretations. There are ve signicant observations relatedto the glycolipids of this individual. First, relative to the A1/A2 phenotype, there are very low levels of blood group A gly-colipids, which is expected as this is a requisite of a weak Aphenotype. Second is the below-detection limits of A-6-2 inthe total glycolipid fraction even when compared with the Axsubgroup (Svensson et al. 2005). Only in the puried fractionscould A-6-2 be conrmed by both MAb 2-24 (Figure 3, II)and structural analysis (nano-ESI-QTOF MS (CID MS/MS)and NMR). Third was the conrmed presence ofGalNAc3GbO4, which stained with certain anti-A reagents

Table IV. Summary of glycolipid structures identified in the seven MPLCfractions

Structure Fraction(s) TLCEIA MS NMR

nLc-4 1 2 4 7 + +nLc-6 1 2 + +nLc-8 4 5 + +H-5-2 1 + + +A-6-2 1 +a +b +b

H-7-2 2 3 4 5 + + +A-8-2 3 4 5 + + +H-9-2 4 5 6 + + +A-10-2 5 + + +H-8-3 3 4 5 + + +A-9-3 4 5 + + +H-10-3 4 5 6 + + +A-11-3 5 + +GbO4 1 2 3 4 5 6 7 + + +GalNAc3GbO4 1 + + +ext. GalNAc3GbO4 2 3 4 5 6 7 + + +Lea-5/Lex-5 1 + + +ext. Lea/Lex 4 5 + +P1 1 2 7 + +

b +b

ext. P1 4 + +X2 1 7 + +Gb3, nLc-3 1 7 +Galactosyl-Ab 4 5 6 +FucGgO4 1 + +FucGbO4 1 + +A-4-6 1 + +Hex5HexNAc4Fuc2 5 +Hex4HexNAc3Fuc 5 6 +Hex6HexNAc2Fuc 4 5 +Hex6HexNAc2 4 +H-10-2 4 5 6 +H-12-2 5 6 +>H-12 5 6 7 +

nLc-8, neolactooctaosylceramide; nLc-3, neolactotriaosylceramide. Blankspace, not tested or no known Mab reagent available for detection; ext.,extended.aVery weak to weak reactions.bVery low levels.

Table III. Glycolipid structures identified by NMR in the four to six-sugar region in rare blood group A erythrocytes from a single individual

Structure Trivial name VI V IV III IIa Ia NMR reference

A GbO4 GalNAc3 4.511 Gal4 4.796 Gal4 Glc1 Cer Holgersson et al. (1990)B H-5-2 Fuc2 5.034 Gal4 4.328 GlcNAc3 4.627 Gal4 Glc1 Cer Holgersson et al. (1990)C Lea-5 Gal3 4.32 (Fuc4) 4.767 GlcNAc3 4.737 Gal4 Glc1 Cer Angstrom et al. (2004)D GalNAc3GbO4 GalNAc3 4.525 GalNAc3 4.46 Gal4 4.80 Gal4 Glc1 Cer J. ngstrm, unpublishedE X2 GalNAc3 4.581 Gal4 4.296 GlcNAc3 4.65 Gal4 Glc1 Cer Thorn et al. (1992)F Lex-5 Gal4 4.306 (Fuc3) 4.836 GlcNAc3 4.705 Gal4 Glc1 Cer Angstrom et al. (2004)G FucGgO4 Fuc2 4.95 Gal3 4.34 GalNAc4 4.472 Gal4 Glc1 Cer Teneberg et al. (1994)H FucGbO5 Fuc2 4.95 Gal3 4.46 GalNAc3 4.46 Gal4 4.80 Gal4 Glc1 Cer Holgersson et al. (1990)I-1 A-6-2 GalNAc3 4.911 (Fuc2) 5.137 Gal4 4.383 GlcNAc3 4.605 Gal4 Glc1 Cer Clausen, Levery, McKibbin et al. (1985)I-2 A-4-6 GalNAc3 4.916 (Fuc2) 5.132 Gal4 Glc1 Cer Breimer et al. (1982)J nLc-4/nLc-6 Gal4a GlcNAc3 4.65 Gal4a GlcNAc3 4.65 Gal4 Glc1 Cer Miller-Podraza et al. (2005)

The identity of the anomeric proton resonances are given in the NMR spectrum shown in Figure 7.aChemical shift values are not given due to severe resonance overlap but can be found in the literature references given above.

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at the six-sugar interval, was present in extended forms andwas identied by structural MS analysis through specic frag-ment ions corresponding to HexNAcHexNAcHexHexHexCersequence and its extensions. Initially, it was considered thatthe GalNAc3GbO4 antigen could be contributing to the A3phenotype; however, since this structure has been found inpools of red cell membranes of all ABO types (Ando et al.1982; Angstrom et al. 1986), it is unlikely to be the cause ofthe A3 phenotype. Surprisingly, when we immunostained totalglycolipid fractions from blood group B and O individualswith the GalNAc3GbO4 reactive anti-A (MAb 222), wewere also able to clearly identify GalNAc3GbO4 bands in 6/6 B samples but in only 1/5 O samples (results not shown). Itis possible to speculate that this could be due to an alternativesubstrate/precursor activity of the B and A304 A3 glycosyl-transferases, and in the case of some O samples, a nontrun-cated enzyme incapable of utilizing H but still active againstglobotetraosylceramide, but this remains highly speculative.On the basis of these observations and the literature, it seemsevident that GalNAc3GbO4 antigen does not contribute tothe A3 phenotype but remains an important antigen to con-sider when using anti-A reagents by very sensitive techniquessuch as ow cytometry and TLCEIA. The fourth observationwas the presence of most of the expected extended structures,including A-8-2, A-10-2, A-9-3 and A-11-3. Why theseextended structures should still be synthesized, when theusually more dominant A-6-2 glycolipid is poorly represented,is an interesting questionand one which remains unresolved.The fth was both the absence of an extended structure andno evidence for any branched blood group A glycolipids inthe MS spectra, which raises the possibility that the absentextended structure may have been branched.After excluding recognized dual-cell population reasons for

mixed-eld agglutination, namely chimerism, transfusion,transplantation, fetal maternal bleeds or disease, the diagnosisof the A3 phenotype is solely on the basis of mixed-eldanti-A serological agglutination of red cells with reduced Aantigen expression. Intriguingly, ow cytometry has reason-ably suggested that dual populations of cells may be an expla-nation for the mixed-eld reactions observed (Hult and Olsson2010). This is also supported by ndings that the A glycosyl-transferase is found only on agglutinated cells (Nakamura et al.1989). Unfortunately, this dual population explanation formixed-eld agglutination does not hold for this A304 A3sample, as in the same ow cytometry study (Hult and Olsson2010), it did not have a dual population. Additional complexityis suggested by early observations that A3 cells initially notagglutinated in the mixed-eld reaction will agglutinate whensecondarily reacted with anti-A. We were also able to conrmthis unusual phenomenon of A3 red cells with this A304 A3sample and serological gel cards. Although the glycolipids ofthis A304 A3 individual may not be typical of all A3 pheno-types this individual unambiguously has A3 serology ofreduced A antigen and mixed-eld agglutination.Consequently, an alternative explanation for mixed-eldagglutination, not dependent on a dual-cell population, isneeded. This requires a population of cells to start with a lowantigen count close to the positive/negative agglutinationthreshold. If that population of cells is then also missing some

or most of its branched structures, then the binding strength ofantibodies to them would be compromised (a phenomenonwell recognized in the serology of newborns; Clausen andHakomori 1989). When external shear forces are then appliedin serological reactions, it would be reasonable to expect thatmany of the cells less strongly bound in the agglutinates wouldshear away as free cells and result in a mixed-eld reaction.Such a postulate is also compatible with the observation thatthe free cells when recovered are agglutinable by antibody.Overall, we can condently state that the glycolipid

anomalies of this A304 A3 individual was a very low abun-dance of A structures, particularly A-6-2 and an absence ofbranched A glycolipids. The contribution of the glycoproteinsor polyglycosylceramides was not able to be considered. It isspeculated that the low level of A antigen combined with anabsence of extended branched glycolipids may be the factordetermining the mixed-eld agglutination phenomenon, atleast in this A304 A3 individual.

Materials and methodsBlood donorSix blood donations totaling approximately 1.0 kg of concen-trated red cells were collected over the period of 2 years fromthe original donor (Aweak-II) with the A3 (anti-A mixedeld), P1, Le(a+b) ABH non-secretor phenotype and theA2(539G>A)/O1 genotype as previously reported (Svensson et al.2005). Red cell units at the supplying Blood Centre wereleukocyte-depleted by ltration, washed free of plasma withbuffered isotonic saline and stored frozen at 20C until readyfor processing.

Mixed-eld agglutination serologyA304 A3 red cells were tested against Novaclone murinemonoclonal anti-A (Galileo; Dominion Biologicals Limited,Dartmouth, Nova Scotia, Canada) in a saline Diamed gel cardsystem (DiaMed, GmbH, Cressier FR, Switzerland) and separ-ated by centrifugation according to normal serological pro-cedures. Following centrifugal separation, the agglutinated (+)cells from the upper part of the gel were recovered from theunagglutinated () cells from the lower part of the gel. Cellswere recovered from several gel cards, washed, concentrationadjusted and then reacted again in the original system againstnew reagents. Blood group O and A2 cells were tested in par-allel as controls (not shown).

Neutral glycolipidsThe glycolipids from the thawed hemolyzed red cell poolwere extracted via a system of solvents and silica/ionexchange chromatography as previously described (Svenssonet al. 2005), resulting in 250 mg of total neutral glycolipids. Itshould be noted this method does not isolate polyglycosylcer-amides, i.e. those with >20 sugar residues.The total glycolipid mixture was subjected to TLC, chemi-

cal or immunochemical staining analyses, and 200 mg wasfurther rened by column chromatography (open column aswell as MPLC).


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The terminology used to describe glycolipids is as rec-ommended by IUPAC-IUB (Chester 1998). The abbreviation,for example, A-6-2 stands for blood group (group A), thenumber of carbohydrate units (six saccharides) and the typeof core structure (type 2) of the glycolipid (Holgersson et al.1992). With respect to Cer, for example, d18:1/24:0 means adihydroxy long chain base with 18 carbon atoms and onedouble bond combined with a saturated non-hydroxylated24-carbon atom fatty acid.

Medium-pressure liquid chromatographyTwo hundred milligrams of total neutral glycolipids were sep-arated into 20 fractions by the Bchi Sepacore Flash system(Bchi Labortechnik AG, Switzerland) using a silica S60(2050027, SDS, France) packed column. The ow rate wasset to 12.5 mL/min. The rst two fractions were eluted with asolvent mix of chloroform:methanol:water (CMW) 80:20:1v/v each after 15 min followed by four more fractions elutedwith CMW 70:25:4 v/v at 10 min intervals. Ten fractionswere then eluted with CMW 60:35:8 v/v at 5 min intervalsand two further fractions at 10 min intervals. Two nal frac-tions were eluted with CMW 40:40:12 v/v at 20 and 10 minintervals, respectively. Following TLC analysis (chemical andimmunochemical), the rst nine fractions were found tocontain no signicant blood group glycolipids or only shortchain compounds (less than ve residues) and were not ana-lyzed further. The remaining fractions were pooled into sevenfractions according to TLC mobility (Table I) and then sub-jected to extended immunochemical analysis and structuralcharacterization by MS and proton NMR spectroscopy. Noattempts to further purify these fractions were made due to thelow solubility in the solvent systems used and the risk of irre-versible loss of individual componentsa phenomenon pre-viously reported during the purication of glycolipidcomponents (Angstrom et al. 1986).

Thin layer chromatographyTLC plates (HPTLC Silica gel 60 aluminum sheets; Merck,Darmstadt, Germany) were loaded with 10, 20 or 50 g ofglycolipids per lane and separated in a solvent system ofCMW 60:35:8 v/v. The air-dried plates were either chemicallyvisualized with anisaldehyde (Karlsson 1987) or by immuno-chemistry (TLCEIA) (Schnaar and Needham 1994;Svensson et al. 2005).

TLCenzyme immunoassayTLCEIA involved staining TLC (CMW 60:35:8 v/v)-separated glycolipids with characterized primary murineMAbs, followed by alkaline phosphatase-conjugated anti-murine immunoglobulin (A0162; Sigma, St. Louis, MO) andBCIP/NBT chromogenic substrate (B5655; Sigma) (Schnaarand Needham 1994; Svensson et al. 2005). A similar protocolwas used for immunostaining with biotinylated H. pomatialectin, which detects GalNAc residues (L6512; Sigma)diluted at 1:1000 except the secondary detection was withalkaline phosphatase-conjugated streptavidin (S2890; Sigma).The blood group A, AB and H MAb reagents used for

TLCEIA and their reported specicities are listed in Table V

(Young et al. 1981; Clausen, Levery, Nudelman et al. 1985;Hirohashi et al. 1985; Le Pendu and Henry 2002; Svenssonet al. 2005). Blood group B MAb reagents used are anti-B(DAKO Corporation, Carpinteria), HEB-29 (GeneTex Inc.,Irvine) and MAb 035 (4th International Workshop onMonoclonal Antibodies Against Red Cells and RelatedAantigens).The specicity of the broadly reactive MAb 2-41 and the

anti-A type 2 MAb 2-24 was further investigated, and it wasfound that both were unreactive with pure NMR-veriedGalNAc3GbO4 pentaglycosylceramide (Ando et al. 1982),which migrates in the hexaglycosylceramide region on theTLC plate. In contrast, the other broadly reactive antibodiesMAbs 2-22 and MAb Lorne (result not shown) were bothstrongly reactive with this GalNAc3GbO4 structure(Figure 3, plate IV). On this basis, if a glycosylceramidepresent in hexaglycosylceramide region was reactive withMAbs 2-22 or Lorne but unreactive with MAb 2-41, it wasconsidered strongly indicative of the presence ofGalNAc3GbO4.

Rm scaleRm scales are included in all TLCEIA plates to indicate therelative position of various bands. The scale was set with 0 asthe origin and rm 6 for blood group A hexaglycosylceramide(Svensson et al. 2005).

Nano-ESI-QTOF MS and tandem MSMS experiments were conducted on an orthogonal time-of-ight mass spectrometer (Micromass, UK) equipped with anano-ESI ion source. For data acquisition and processing, theMassLynx software (Micromass) was used. The gas-phaseions were generated from solution using an in-house pulledglass capillary. The ESI voltage was applied onto a metal wireinserted in the glass capillary.All fractions were prepared to a stock concentration of

1 g/L in different solvent systems systematically

Table V. Monoclonal anti-A, anti-AB and anti-H reagents used for TLCEIA

Identity Clone Specificity Source

224a NaM200-16C5 A type 2 ETS, Pays de Loire,France

HH4 HH4 A type 2 H. Clausen, Copenhagen,Denmark

TH1 TH1 A type 3 H. Clausen, Copenhagen,Denmark

222a AY209 A +GalNAc3GbO4b

ETS, Bretagne, France

1401 11H5 A +GalNAc3GbO4b

Lorne laboratories,Reading, UK

241a BH517 A + B ETS, Bretagne, FranceHH14 HH14 H type 3 H. Clausen, Copenhagen,

DenmarkBE2 BE2 H type 2 H. Clausen, Copenhagen,

Denmark

aMAbs from the 4th International Workshop on Monoclonal AntibodiesAgainst Human Red Cells and Related Antigens.bGalNAc3GbO4 reactivity tested against pure GalNAc3GbO4 glycolipid(see Materials and methods).

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determined in accordance to the requirements for the solu-bility and ionization of individual fractions. Thus, fraction1 was prepared in methanol:water 10:1 v/v, fraction 5 inmethanol:water 4:1 v/v and fraction 4 in 100:5:2 methanol:chloroform:water. Fractions 1 and 5 were further diluted inmethanol and fraction 4 in 10:1 methanol:water v/v sol-utions to a nal concentration of approximately 5 pmol/L(calculated considering the average molecular mass of2500 Da for fractions 5 and 4 and 1500 Da for fraction 1).The mass spectrometer was operated in a positive-ionmode at electrospray potentials between 1000 and 1200 V.Sampling cone potential was ranged between 35 and 55 Vfor fractions 5 and 4 to enhance the ionization of largemolecules and prevent in-source glycosidic bond cleavages,particularly the labile Fuc linkages. For fraction 1, thesampling cone potential was varied between 65 and 120 Vgiven the presence of shorter glycolipids. For structuralelucidation, CID tandem MS experiments were performedemploying collision energy values between 45 and 160 eVdepending on the ion type, to obtain a comprehensive frag-ment ion pattern for an accurate structural identication.The collision gas pressure was set to 15 psi for all MS/MSexperiments. All mass spectra were externally calibratedusing a NaI solution.The tandem MS data assignment followed the nomenclature

established by Domon and Costello (1988).

Proton NMR spectroscopyIntact non-derivatized glycolipid samples were dissolved indimethylsulfoxide:D2O, 98:2 (v:v) after deuterium exchangeand spectra were acquired on a Varian 600 MHz spectrometerat 30C. Both 1D and 2D double quantum-ltered correlatedspectroscopy (Marion and Wuthrich 1983) proton NMR wereperformed. An extensive library of reference chemical shiftdata at the temperature of choice (30C) for previously iso-lated pure glycosphingolipids representing practically all res-onances of the structures in Table III was available forstructural identication.

Funding

This work was supported by Swedish Research Council/Medicine (grant no. 11612) and Claes Hgman SAGMANstipendium, Baxter Medical AB, Sweden. This project andL.S. was supported in part by the Biotechnology ResearchInstitute of AUT University.

Acknowledgements

The authors acknowledge the support and valuable adviceprovided throughout the study by Prof. Dr. JasnaPeter-Katalinic, University of Mnster, Germany. ProfessorsKarl-Anders Karlsson and Susann Teneberg at the Institute ofBiomedicine, Department of Medical Biochemistry and CellBiology, University of Gothenburg, Sweden, are thanked fortheir valued discussions.

Conict of interest statement

None declared.

Abbreviations

Cer, ceramide; CID, collision-induced dissociation; CMW,chloroform:methanol:water; ESI-QTOF MS, electrosprayionization-quadrupole time-of-ight mass spectrometry; Ext.,extended; Fuc, fucose; GalNAc3GbO4, para-Forssman;GbO4, globotetraosylceramide (globoside); Hex, hexose;HexNAc, hexosamine; 1H-NMR, proton NMR; MAb, mono-clonal antibody; MPLC, medium-pressure liquid chromato-graphy; MS, mass spectrometry; MS/MS, tandem massspectrometry; nLc-4, neolactotetraosylceramide; nLc-6, neo-lactohexaosylceramide; NMR, nuclear magnetic resonance;rm, relative migration; TLC, thin layer chromatography;TLCEIA, thin layer chromatographyenzyme immunoassay.

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the structural basis of blood group a-related glycolipids

Documents

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