Fingerprinting of large oligosaccharides linked to ceramide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: highly heterogeneous polyglycosylceramides of human erythrocytes with receptor activity for Helicobacter pylori

Hasse Karlsson, Lena Johansson, Halina Miller-Podraza1 and Karl-Anders Karlsson

Institute of Medical Biochemistry, Göteborg University, P.O. Box 440, SE-405 30 Göteborg, Sweden

Received on October 16, 1998; revised on February 1, 1999; accepted on February 1, 1999

Highly microheterogeneous polyglycosylceramides (PGCs) of human erythrocytes with an average composition of about 25 monosaccharides linked to ceramide were analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). The human gastric pathogen Helicobacter pylori was earlier shown to bind this glycosphingolipid mixture by thin-layer chromatogram binding assay. The receptor activity was present along the whole nonresolved chromatographic interval. Mass spectra of intact PGCs were compared with corresponding spectra of oligosaccharides enzymatically released from the ceramides. Two subfractions of PGCs containing less than one and more than one sialic acid residue per molecule were used. MALDI-MS spectra were recorded in both linear and reflectron mode with the accuracies of [le]0.08% and [le]0.02%, respectively, which allowed determination of the constituent parts of the detected ions in form of ceramide and number of hexoses, N-acetylhexosamines, fucoses and sialic acids. Molecular species were found based on ceramide with mainly sphingosine and fatty acids 24:0 and 24:1 (with less amounts of 22:0), and with a total number of monosaccharides ranging from 11 (neutral, m/z = 2605 for [M+Na]+) to 41 (one sialic acid, m/z = 8057 for [M-H]-). The saccharide composition obtained supported a successively extended and branched N-acetyllactosamine core with substitutions of fucoses (0 up to 8) and sialic acid (0 to 1). The reliable molecular analysis of large oligosaccharides linked to ceramide using this approach will be of great help for the further structure analysis in order to define the epitope for the sialic acid-dependent binding by the bacterium.

Key words: erythrocytes/Helicobacter pylori/MALDI-TOF MS/mass spectrometry/polyglycosylceramides

Introduction

Large membrane-associated oligosaccharides linked to ceramide, named megaloglycolipids or macroglycolipids, and carrying various blood group activities were first proposed by Gardas and Koscielak (Gardas and Koscielak, 1973). Preparations of human erythrocytes contained 90% carbohydrate, 7% amino acids, and about 2% sphingosine. The glycoconjugates were concluded to be unusually complex glycoproteins or glycolipids containing 30-50 saccharide residues per ceramide. The amino acid contents could be reduced to about 0.3% by alkali treatment (Gardas and Koscielak, 1974) which indicated the presence of glycolipids. Koscielak et al. (Koscielak et al., 1976) introduced the name polyglycosylceramides (PGCs) for these polar glycosphingolipids. Structural studies of a component isolated from blood group O erythrocytes provided evidence for a 22-sugar glycosphingolipid composed of eight N-acetyllactosamines in a straight chain with two branches of N-acetyllactosamine carrying blood group O determinants (Gardas, 1976a). Partial degradation of this preparation with fucosidase gave a product highly active with anti-i antibodies (Gardas, 1976b). A glycosphingolipid isolated from blood group A erythrocytes was proposed to contain 23 sugar residues and ending with blood group O and blood group A determinants (Gardas, 1978). Subsequent studies provided evidence for a high microheterogeneity of such polar preparations of human erythrocytes (Dejter-Juszynski et al., 1978; Koscielak et al., 1979; Fukuda and Hakomori, 1982; Zdebska et al., 1983; Hanfland et al., 1984).

Corresponding rabbit erythrocyte preparations revealed a more distinct, stepwise glycosylation (Hanfland et al., 1981). Ceramide-linked saccharides containing 10 (Hanfland et al., 1981), 15 (Dabrowski et al., 1984; Egge et al., 1985), 20 and 25 (Hanfland et al., 1988), 30, 35, and 40 (Kordowicz et al. 1986) monosaccharides could be differentiated. A complete structure of the 40-sugar glycolipid was determined with the aid of 600 MHz two-dimensional 1H NMR spectroscopy (Dabrowski et al., 1988). This glycolipid was an octa-antennary, regularly branched N-acetyllactosamine sequence ending with Gal[alpha]3 residues. Analogous structures with up to 20 sugars terminating with NeuAc[alpha]3 have been characterized from human placenta (Levery et al., 1989). More recently linear, multifucosylated poly-N-acetyllactosamine glycolipids with up to 16 sugars containing NeuAc[alpha]3 have been isolated from human leukocytes (Müthing et al., 1996; Stroud et al., 1996a,b).

Our own present interest in PGCs was aroused by the finding that the human gastric pathogen Helicobacter pylori was binding with high affinity to PGCs of human erythrocytes (Miller-Podraza et al., 1996, 1997b). The binding vanished upon treatment with neuraminidase, weak acid or mild periodate oxidation indicating a dependence of binding on sialic acid. Apparently, the binding was specific for human PGCs since preparations from animal sources, although containing sialic acid, were not recognized by the bacterium (Miller-Podraza et al., 1997a,c). A large number of other sialic acid-containing glycolipids and glycoproteins of human and animal origins were not recognized. At present, the only object for structural studies of the binding epitope is therefore human PGCs. To plan laborious synthesis and modeling studies, an average structure based on simply composition analysis is not enough. Binding and modification studies (Miller-Podraza et al., 1996, 1997a,b) must be related to a precisely defined sequence. Therefore subfractionation of complex mixtures of PGCs or enzymatic synthesis of defined carbohydrate structures will be necessary for final determination of the binding epitope.

Matrix-assisted laser desorption/ionization and electrospray are ionization techniques available for analysis of larger glycoconjugates. Ii et al. have used electrospray mass spectrometry (Ii et al., 1993) for the analysis of a synthetic 25-sugar saccharide of the type present in rabbit erythrocytes (Hanfland et al., 1988). The measured average molecular masses were 4441 Da and 9416 Da for the nonderivatized and the acetylated saccharide, respectively. However, this interesting approach is not applicable in our case due to the complexity of the mixtures.

The present paper reports that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a good method for detection of molecular species of PGCs in complex mixtures. The sample used was PGCs of human blood group O erythrocytes, corresponding to the preparation initially described (Gardas and Koscielak, 1973, 1974). However, in our case a rationalized and simplified preparative approach was used based on peracetylation (Miller-Podraza et al., 1993). This resulted in a complex PGC fraction suitable for further studies. The MALDI mass spectra of both neutral and sialylated glycosphingolipids revealed complex series with increasing number of N-acetyllactosamines and degree of fucosylation. The number of sugar residues ranged from about 10 up to 41, with a ceramide composed of mainly sphingosine in combination with 24:0 or 24:1 nonhydroxy fatty acid. The data supported earlier proposals of a successively extended and branched poly-N-acetyllactosamine core structure with terminal substitutions of Fuc and NeuAc in different combinations (Koscielak et al., 1976).

Results

The present work was performed on PGC material isolated from human blood group O erythrocytes. Four subfractions were analyzed in parallel: fraction 1 and 2 containing polyglycosylceramides and fraction A and B containing oligosaccharides. The PGC fractions were obtained by separation of acetylated derivatives on silica gel columns. Oligosaccharides were enzymatically released by ceramide glycanase from a mixture of PGCs. The oligosaccharides were separated by high-pH anion-exchange chromatography (HPAEC) and two of the eluted fractions were further analyzed. The chemical composition of the four fractions are summarized in Table I. Thin-layer chromatograms of fraction 2 (Figure 1, lane 1) were visualized by chemical staining (left) or by autoradiography after overlay with radiolabeled H.pylori cells (right). The microheterogeneity was evident from the unresolved chromatogram (left). The bacterial cells (right) bound to the same unresolved interval, indicating that the binding epitope documented earlier was located on PGCs with a successively extended core saccharide. Brain gangliosides (lane 2) were not bound. The absence of shorter glycolipids in the PGC mixtures (lane 1) was explained by the preparative procedure; the bulk of these glycolipids were extracted by conventional means before the erythrocyte stroma residue was peracetylated and the remaining shorter glycolipids were removed by Sephadex LH chromatography.


Figure 1. Thin-layer chromatograms (silica gel 60 TLC plates, Merck, Germany) of fraction 2 of polyglycosylceramides (lane 1) and brain gangliosides (lane 2) detected with anisaldehyde (left) or by autoradiography after overlay with radiolabeled Helicobacter pylori cells (right). Developing solvent was propanol/0.25% KCl in water/methanol/chloroform, 7:5:1:0.5 (by vol.).

Table I. Chemical composition of fractions used for MALDI-MSa
Fraction Hex/SFb SA/SF Hex/SA
Polyglycosylceramides
   Fraction 1 11.6 0.5 23.2
   Fraction 2 13.3 1.6 8.3
Oligosaccharides
   Fraction A     73.5
   Fraction B     12.7
aDetermined by colorimetric method.
bAbbreviations: Hex, hexose; SA, sialic acid; SF, sphingosine.


Glycosphingolipids from human sources are composed of a ceramide part and relatively few different monosaccharides with unique incremental masses (e.g., fucose, 146.1 Da; hexose, 162.1 Da; N-acetylhexosamine, 203.2 Da; N-acetylneuraminic acid, 291.3 Da; average masses). The molecular weights provided from the MALDI mass spectra recorded both in the linear and reflectron mode with an accuracy of [le]0.08% and [le]0.02%, respectively, allowed determination of the saccharide compositions. Higher masses were detected in the linear mode with a more symmetrical mass distribution envelope. These mass spectra probably reflected the true contents of the fractions better than the data obtained in reflectron mode. The detected ions in the reflectron mode were shifted towards lower mass ions, but the resolution was higher and the mass measurements were more accurate. Neutral components were detected as [M+Na]+ in the positive ion mode, and sialylated species were detected as [M-H]- in the negative ion mode.

Fraction 1 with less sialylated PGCs

The MALDI-TOF mass spectra in positive ion linear and reflectron mode of fraction 1 are shown in Figure 2, A and B, respectively. The observed series of pseudomolecular ions, [M+Na]+, were assigned to a model structure of Hex(x+2)HexNAc(x)Fuc(y)Cer, with x varying from 4 to 15 and y from 0 to 7. The major ceramides were a mixture of sphingosine (d18:1) and nonhydroxy fatty acids 24:0 and 24:1 of approximately equal abundance (Table II). The composition of the ceramide could be observed in the resolved reflectron spectra of ions of lower masses. Less abundant ions composed of the ceramides d18:1-22:0 and d18:1-22:1 were also observed (Table III). [M+Na]+ ions of PGCs were observed from (6,4,1)Cer up to (17,15,6)Cer in the linear mode and up to (15,13,6)Cer in the reflectron mode. The different oligosaccharide series observed started with no fucose up to a maximum of x+1/y=2 (see general formula), which corresponded to a fully branched core of N-acetyllactosamines with terminals of fucose. There was a mass difference of 365 amu (Hex + HexNAc) between series which had the same number of fucoses but with a difference of one N-acetyllactosamine. The molecular mass distribution was centered around m/z = 4500, corresponding to (10,8,3)Cer and (10,8,4)Cer in linear mode (Figure 2A), and around m/z = 4400, corresponding to (10,8,3)Cer in reflectron mode (Figure 2B).


Figure 2. MALDI-TOF mass spectrum of fraction 1 in (A) positive ion linear mode and (B) positive ion reflectron mode. The annotations are made as HexNAc.Fuc (Tables II and III). The * indicates the less abundant d18:1-22:0 ceramide.

Fraction 2 with more sialylated PGCs

MALDI-TOF mass spectra of the more sialylated second fraction were only observed in negative ion linear mode (Figure 3). The observed ions were [M-H]-, which is consistent with components containing one sialic acid (Sugiyama et al., 1997). Similar series as observed in fraction 1 were also detected in this fraction with the distinction that one fucose was exchanged for a sialic acid residue. The observed [M-H]- ions could be assigned to model structures composed of Hex(x+2)HexNAc(x)Fuc(y)NeuAc(1)Cer, with x varying from 5 to 17 and y from 0 to 6. The major ceramide was composed of d18:1 and fatty acid 24:0. Due to the lower resolution achieved in linear mode the presence of d18:1-24:1 could not be distinguished. Similar to fraction 1 d18:1-22:0 ceramide was observed 28 amu below the components containing the more abundant ceramide (Table V), but d18:1-22:1 was not detected (Figure 3). Series from (7,5,0,1)Cer up to (19,17,4,1)Cer were detected (Table IV and Figure 3). The molecular mass distribution was centered around m/z = 5000, that is (11,9,2,1)Cer and (11,9,3,1)Cer (Figure 3). The sodium adduct ions of the type [M+Na-2H]- were located as satellites 22 amu above the [M-H]- ions of the more abundant ceramide components.

Table II. MALDI-MS dataa of fraction 1 with less sialylated polyglycosylceramides and d18:1-24:0/1 ceramides
Structuree In Fig.f Formula CMb [M+Na]+ OM (linear) [M+Na]+ [Delta] (linear) OM (reflectr.) [M+Na]+ [Delta] (reflectr.)
(6,4,1)Cer 4.1 C116H205(203)N5O57 2603.89 2605.3 +1.4 2604.85 +0.96
(6,4,2)Cer 4.2 C122H215(213)N5O61 2750.03 2749.8 -0.2 2750.65 +0.62
(7,5,0)Cer 5.0 C124H218(216)N6O63 2823.08 2824.9 +1.8 2823.62 +0.54
(7,5,1)Cer 5.1 C130H228(226)N6O67 2969.22 2969.6 +0.4 2969.90 +0.68
(7,5,2)Cer 5.2 C136H238(236)N6O71 3115.37 3115.5 +0.1 3115.67 +0.30
(8,6,0)Cer 6.0 C138H241(239)N7O73 3188.42 3189.4 +1.0 3189.14 +0.72
(8,6,1)Cer 6.1 C144H251(249)N7O77 3334.56 3335.1 +0.6 3335.18 +0.62
(8,6,2)Cer 6.2 C150H261(259)N7O81 3480.70 3481.0 +0.3 3481.15 +0.45
(8,6,3)Cer 6.3 C156H271(269)N7O85 3626.85 3626.5 -0.4 3627.24 +0.39
(9,7,0)Cer 7.0 C152H264(262)N8O83 3553.76 3553.3 -0.5 3554.11 +0.35
(9,7,1)Cer 7.1 C158H274(272)N8O87 3699.90 3700.8 +0.9 3700.67 +0.77
(9,7,2)Cer 7.2 C164H284(282)N8O91 3846.04 3846.7 +0.7 3846.36 +0.32
(9,7,3)Cer 7.3 C170H294(292)N8O95 3992.19 3992.6 +0.4 3992.74 +0.55
(9,7,4)Cer 7.4 C176H304(302)N8O99 4138.33 4137.9 -0.4 4138.85 +0.52
(10,8,0)Cer 8.0 C166H287(285)N9093 3919.09 - - 3919.87 +0.78
(10,8,1)Cer 8.1 C172H297(295)N9O97 4065.24 4065.2 0.0 4065.91 +0.67
(10,8,2)Cer 8.2 C178H307(305)N9O101 4211.38 4211.6 +0.2 4211.83 +0.45
(10,8,3)Cer 8.3 C184H317(315)N9O105 4357.52 4357.4 -0.1 4358.12 +0.60
(10,8,4)Cer 8.4 C190H327(325)N9O109 4503.67 4503.1 -0.6 4504.17 +0.50
(11,9,1)Cer 9.1 C186H320(318)N10O107 4430.57 4431.7 +1.1 4430.49 -0.08
(11,9,2)Cer 9.2 C192H330(328)N10O111 4576.72 4576.3 -0.4 4576.98 +0.26
(11,9,3)Cer 9.3 C198H340(338)N10O115 4722.86 4722.7 -0.2 4723.62 +0.76
(11,9,4)Cer 9.4 C204H350(348)N10O119 4869.00 4868.4 -0.6 4869.42 +0.42
(11,9,5)Cer 9.5 C210H360(358)N10O123 5015.15c 5013.9 -1.3 5015.20 +0.05
(12,10,0)Cer 10.0 C194H333(331)N11O113 4649.77 - - 4649.66 -0.11
(12,10,1)Cer 10.1 C200H343(341)N11O117 4795.91 - - 4796.63 +0.72
(12,10,2)Cer 10.2 C206H353(351)N11O121 4942.05 4941.7 -0.4 4942.22 +0.17
(12,10,3)Cer 10.3 C212H363(361)N11O125 5088.20 5086.5 -1.7 5088.57 +0.37
(12,10,4)Cer 10.4 C218H373(371)N11O129 5234.34 5233.6 -0.7 5235.32 +0.98
(12,10,5)Cer 10.5 C224H383(381)N11O133 5380.48c 5379.2 -1.3 5380.83 +0.35
(13,11,3)Cer 11.3 C226H386(384)N12O135 5453.53 5452.3 -1.2 - -
(13,11,4)Cer 11.4 C232H396(394)N12O139 5599.68 5598.8 -0.9 5599.31 -0.37
(13,11,5)Cer 11.5 C238H406(404)N12O143 5745.82c 5744.5 -1.3 5745.20 -0.62
(13,11,6)Cer 11.6 C244H416(414)N12O147 5891.96c 5889.9 -2.1 5892.00 +0.04
(14,12,4)Cer 12.4 C246H419(417)N13O149 5965.02 5961.1 -3.9 5964.81 -0.21
(14,12,5)Cer 12.5 C252H429(427)N13O153 6111.16c 6109.0 -2.2 - -
(14,12,6)Cer 12.6 C258H439(437)N13O157 6257.30c 6257.5 +0.2 6257.50 +0.20
(15,13,4)Cer 13.4 C260H442(440)N14O159 6330.35 6331.7 +1.4 - -
(15,13,5)Cer 13.5 C266H452(450)N14O163 6476.50c 6474.1 -2.4 6476.62 +0.12
(15,13,6)Cer 13.6 C272H462(460)N14O167 6622.64c 6620.1 -2.5 6623.43 +0.79
(16,14,6)Cer 14.6 C286H485(483)N15O177 6987.98c 6987.1 -0.9 - -
(16,14,7)Cer 14.7 C292H495(493)N15O181 7134.13c 7137.1 +3.0 - -
(17,15,6)Cer 15.6 C300H508(506)N16O187 7353.31c 7352.9 -0.4 - -
        n = 40d [sigma] = 1.3d n = 37d [sigma] = 0.36d
      Accuracy: 0.02-0.05%   0.006-0.014%
OM, Obs.mass; [Delta], obs-calc; CM, calc.mass.
aLinear and reflectron data in positive mode.
bCalculated average mass of mean value of the ceramides d18:1-24:0 and d18:1-24:1.
cIsobaric structures are possible due to 5 MFuc + 0.04 = 2 MHexHexNAc.
d[sigma] is the standard deviation and n is the number of masses observed.
e(Hex,HexNAc,Fuc)Cer.
fIn Fig. HexNAc.Fuc.

Table III. MALDI-MS dataa of fraction 1 with less sialylated polyglycosylceramides and d18:1-22:0 ceramide
Structuree Formula CMb [M+Na]+ OM (linear) [M+Na]+ [Delta] (linear) OM (reflectron) [M+Na]+ [Delta] (reflectron)
(6,4,1)Cer C114H201N5O57 2576.84 2577.8 +1.0 - -
(7,5,2)Cer C134H234N6O71 3088.32 3089.5 +1.2 - -
(8,6,0)Cer C136H237N7O73 3161.37 3161.8 +0.4 - -
(8,6,1)Cer C142H247N7O77 3307.51 - - - -
(8,6,2)Cer C148H257N7O81 3453.66 3454.5 +0.8 3453.67 +0.01
(8,6,3)Cer C154H267N7O85 3599.80 3599.4 -0.4 3600.89 +1.09
(9,7,1)Cer C156H270N8O87 3672.85 3671.3 -1.6 3672.65 -0.20
(9,7,2)Cer C162H280N8O91 3818.99 3820.6 +1.6 3818.51 -0.48
(9,7,3)Cer C168H290N8O95 3965.14 3966.0 +0.9 3965.43 +0.29
(10,8,2)Cer C176H303N9O101 4184.33 4183.5 -0.8 - -
(10,8,3)Cer C182H313N9O105 4330.47 4330.2 -0.3 4330.75 +0.28
(10,8,4)Cer C188H323N9O109 4476.62 4477.4 +0.8 4476.92 +0.30
(11,9,3)Cer C196H336N10O115 4695.81 4694.0 -1.8 - -
(11,9,4)Cer C202H346N10O119 4841.96 4841.6 -0.4 4842.24 +0.28
(12,10,3)Cer C210H359N11O125 5061.15 - - 5062.00 +0.85
(12,10,4)Cer C216H369N11O129 5207.29 5204.2 -3.1 5207.19 -0.10
(12,10,5)Cer C222H379N11O133 5353.44c - - 5353.49 +0.05
(13,11,4)Cer C230H392N12O139 5572.63 - - 5572.61 -0.02
(13,11,5)Cer C236H402N12O147 5718.77c - - 5718.05 -0.72
      n = 14d [sigma] = 1.3d n = 13d [sigma] = 0.49d
      Accuracy: 0.03-0.05%   0.009-0.014%
OM, Obs.mass; [Delta], obs-calc; CM, calc.mass.
aLinear and reflectron data in positive mode.
bCalculated average mass.
c Isobaric structures are possible due to 5MFuc + 0.04 = 2MHexHexNAc.
d[sigma] is the standard deviation and n is the number of masses observed.
eStructure (Hex,HexNAc,Fuc)Cer.


Figure 3. MALDI-TOF mass spectrum of fraction 2 in the negative ion linear mode showing the monosialylated species annotated as HexNAc.Fuc (Table IV and V). The less abundant ceramide d18:1-22:0 is indicated by *.

Table IV. MALDI-MS dataa of fraction 2 with more sialylated polyglycosylceramides and d18:1-24:0/1 ceramides
Structuree In Fig.f Formula CMb[M-H]- OM [M-H]- [Delta]
(7,5,0,1)Cer 5.0 C135H235(233)N7O71 3090.34 3091.7 +1.4
(8,6,0,1)Cer 6.0 C149H258(256)N8O81 3455.67 3457.4 +1.7
(8,6,1,1)Cer 6.1 C155H268(266)N8O85 3601.82 3602.7 +0.9
(9,7,0,1)Cer 7.0 C163H281(279)N9O91 3821.01 3822.7 +1.7
(9,7,1,1)Cer 7.1 C169H291(289)N9O95 3967.16 3968.6 +1.4
(9,7,2,1)Cer 7.2 C175H301(299)N9O99 4113.30c 4112.9 -0.4
(9,7,3,1)Cer 7.3 C181H311(309)N9O103 4259.44c 4258.9 -0.5
(10,8,0,1)Cer 8.0 C177H304(302)N10O101 4186.35 4188.6 +2.3
(10,8,1,1)Cer 8.1 C183H314(312)N10O105 4332.49 4333.9 +1.4
(10,8,2,1)Cer 8.2 C189H324(322)N10O109 4478.64c 4478.6 0.0
(10,8,3,1)Cer 8.3 C195H334(332)N10O113 4624.79c 4624.5 -0.3
(11,9,0,1)Cer 9.0 C191H327(325)N11O111 4551.69 4553.7 +2.0
(11,9,1,1)Cer 9.1 C197H337(335)N11O115 4697.83 4699.5 +1.7
(11,9,2,1)Cer 9.2 C203H347(345)N11O119 4843.97c 4844.8 +0.8
(11,9,3,1)Cer 9.3 C209H357(355)N11O123 4990.12c 4990.9 +0.8
(11,9,4,1)Cer 9.4 C215H367(365)N11O127 5136.26c 5135.3 -1.0
(12,10,0,1)Cer 10.0 C205H350(348)N12O121 4917.02 4918.2 +1.2
(12,10,1,1)Cer 10.1 C211H360(358)N12O125 5063.17 5064.9 +1.7
(12,10,2,1)Cer 10.2 C217H370(368)N12O129 5209.31 5209.9 +0.6
(12,10,3,1)Cer 10.3 C223H380(378)N12O133 5355.45 5356.7 +1.3
(12,10,4,1)Cer 10.4 C229H390(388)N12O137 5501.60 5501.8 +0.2
(13,11,0,1)Cer 11.0 C219H373(371)N13O131 5282.36 5282.4 0.0
(13,11,1,1)Cer 11.1 C225H383(381)N13O135 5428.51 5429.7 +1.2
(13,11,2,1)Cer 11.2 C231H393(391)N13O139 5574.65c 5575.1 +0.5
(13,11,3,1)Cer 11.3 C237H403(401)N13O143 5720.79c 5721.3 +0.5
(13,11,4,1)Cer 11.4 C243H413(411)N13O147 5866.93c 5866.7 -0.2
(13,11,5,1)Cer 11.5 C249H423(421)N13O151 6013.08c 6012.6 -0.5
(14,12,0,1)Cer 12.0 C233H396(394)N14O141 5647.70 5647.2 -0.5
(14,12,1,1)Cer 12.1 C239H406(404)N14O145 5793.84 5792.9 -0.9
(14,12,2,1)Cer 12.2 C245H416(414)N14O149 5939.99c 5940.3 +0.3
(14,12,3,1)Cer 12.3 C251H426(424)N14O153 6086.13c 6086.7 +0.6
(14,12,4,1)Cer 12.4 C257H436(434)N14O157 6232.27c 6233.0 +0.7
(14,12,5,1)Cer 12.5 C263H446(444)N14O161 6378.41c 6378.8 +0.4
(15,13,1,1)Cer 13.1 C253H429(427)N15O155 6159.18 6157.6 -1.6
(15,13,2,1)Cer 13.2 C259H439(437)N15O159 6305.32c 6304.8 -0.5
(15,13,3,1)Cer 13.3 C265H449(447)N15O163 6451.47c 6451.6 +0.1
(15,13,4,1)Cer 13.4 C271H459(457)N15O167 6597.61c 6597.0 -0.6
(15,13,5,1)Cer 13.5 C277H469(467)N15O171 6743.75c 6744.3 +0.6
(15,13,6,1)Cer 13.6 C283H479(477)N15O175 6889.89c 6889.0 -0.9
(16,14,1,1)Cer 14.1 C267H452(450)N16O165 6524.52 6524.7 +0.2
(16,14,2,1)Cer 14.2 C273H462(460)N16O169 6670.66c 6670.1 -0.6
(16,14,3,1)Cer 14.3 C279H472(470)N16O173 6816.80c 6815.2 -1.6
(16,14,4,1)Cer 14.4 C285H482(480)N16O177 6962.95c 6961.8 -1.2
(16,14,5,1)Cer 14.5 C297H492(490)N16O181 7109.09c 7107.9 -1.2
(16,14,6,1)Cer 14.6 C297H502(500)N16O185 7255.23c 7255.9 +0.7
(17,15,2,1)Cer 15.2 C287H485(483)N17O179 7036.00c 7034.8 -1.2
(17,15,3,1)Cer 15.3 C293H495(493)N17O183 7182.14c 7181.6 -0.5
(17,15,4,1)Cer 15.4 C299H505(503)N17O187 7328.28c 7328.0 -0.3
(17,15,6,1)Cer 15.6 C311H525(523)N17O195 7620.57c 7618.6 -2.0
(18,16,2,1)Cer 16.2 C301H508(506)N18O189 7401.33c 7399.4 -1.9
(18,16,3,1)Cer 16.3 C307H518(516)N18O193 7547.48c 7545.8 -1.7
(18,16,4,1)Cer 16.4 C313H528(526)N18O197 7693.62c 7692.6 -1.0
(18,16,5,1)Cer 16.5 C319H538(536)N18O201 7839.76c 7838.0 -1.8
(19,17,3,1)Cer 17.3 C321H541(539)N19O203 7912.81c 7911.6 -1.2
(19,17,4,1)Cer 17.4 C327H551(549)N19O207 8058.96c 8057.4 -1.6
        n = 55d [sigma] = 1.1d
        Accuracy: 0.01-0.04%
OM, Obs.mass; [Delta], obs-calc; CM, calc.mass.
aLinear data in negative mode.
bMean value of the ceramides d18:1-24:0 and d18:1-24:1.
c Isobaric structures are possible due to 5 MFuc + 0.04 = 2 MHexHexNAc and 2 MFuc.= 1.02 + MNeuAc.
d[sigma] is the standard deviation and n is the number of observed masses.
eStructure (Hex,HexNAc,Fuc,NeuAc)Cer.
fIn Fig. HexNAc.Fuc.

Table V. MALDI-MS dataa of fraction 2 with more sialylated polyglycosylceramides and d18:1-22:0 ceramide
Structuree In Fig.f Formula CMb [M-H]- OM [M-H]- [Delta]
(8,6,0,1)Cer 6.0 C147H254N8O81 3428.63 3430.5 +1.9
(9,7,0,1)Cer 7.0 C161H287N9O95 3940.11 3942.2 +2.1
(10,8,0,1)Cer 8.0 C175H300N10O101 4159.31 4158.1 -1.2
(10,8,1,1)Cer 8.1 C181H310N10O105 4305.45 4308.4 +3.0
(10,8,2,1)Cer 8.2 C187H320N10O109 4451.59c 4451.3 -0.3
(10,8,3,1)Cer 8.3 C193H330N10O113 4597.73c 4596.5 -1.2
(11,9,0,1)Cer 9.0 C189H323N11O111 4524.64 4525.5 +0.9
(11,9,1,1)Cer 9.1 C195H333N11O115 4670.79 4672.0 +1.2
(11,9,2,1)Cer 9.2 C201H343N11O119 4816.93c 4818.5 +1.6
(11,9,3,1)Cer 9.3 C207H353N11O123 4963.07c 4962.4 -0.7
(11,9,4,1)Cer 9.4 C213H363N11O127 5109.22c 5107.9 -1.3
(12,10,0,1)Cer 10.0 C203H346N12O121 4889.98 4888.4 -1.6
(12,10,2,1)Cer 10.2 C215H366N12O129 5182.27c 5182.9 +0.6
(12,10,3,1)Cer 10.3 C221H376N12O133 5328.41c 5328.7 +0.3
(12,10,4,1)Cer 10.4 C227H386N12O137 5474.55c 5473.9 -0.7
(13,11,0,1)Cer 11.0 C217H369N13O131 5255.32 5252.7 -2.6
(13,11,2,1)Cer 11.2 C229H389N13O139 5547.60c 5546.8 -0.8
(13,11,3,1)Cer 11.3 C235H399N13O143 5693.75c 5691.9 -1.9
(13,11,4,1)Cer 11.4 C241H409N13O147 5839.89c 5840.9 +1.0
(14,12,3,1)Cer 12.3 C249H422N14O153 6059.08c 6057.0 -2.1
(14,12,4,1)Cer 12.4 C255H432N14O157 6205.23c 6205.7 +0.5
(15,13,4,1)Cer 13.4 C269H455N15O167 6570.56c 6573.0 +2.4
(15,13,6,1)Cer 13.6 C281H475N15O175 6862.85c 6861.6 -1.3
(16,14,3,1)Cer 14.3 C277H468N16O173 6789.76c 6790.1 +0.3
(16,14,6,1)Cer 14.6 C295H498N16O185 7228.19c 7226.3 -1.9
(17,15,1,1)Cer 15.1 C279H471N17O175 6862.81c 6861.6 -1.2
(17,15,6,1)Cer 15.6 C309H521N17O195 7593.53c 7594.2 +0.7
        n = 27d [sigma] = 1.5d
        Accuracy: 0.02-0.04%
OM, Obs.mass; [Delta], obs-calc; CM, calc.mass.
aLinear data in negative mode.
bCalculated average mass.
cIsobaric structures are possible due to 5 MFuc + 0.04 = 2 MHexHexNAc and 2 MFuc= 1.02 + MNeuAc.
d[sigma] is the standard deviation and n is the number of masses observed.
eStructure (Hex,HexNAc,Fuc,NeuAc)Cer.
fIn Fig. HexNAc.Fuc.

Fraction A with free oligosaccharides of low sialic acid content

Fraction A with a low sialic acid content (Table VI) and ions of the neutral species were observed in both positive ion linear and reflectron mode (Figure 4A,B). Series of [M+Na]+ ions were observed in analogy with fraction 1 of intact PGCs following the similar general formula Hex(x+2)HexNAc(x)Fuc(y), with x varying from 4 to 16 and y from 0 to 8. However, the MALDI-TOF mass spectra of oligosaccharides were better resolved than spectra of PGCs. Series starting from (6,4,0), to (16,14,7), were observed in reflectron mode, and up to (18,16,6) in linear mode (Table VI and Figure 4A,B). The molecular mass distribution was centered around m/z = 4200 in linear mode, corresponding to (11,9,3) and (11,9,4) (Figure 4A), and around m/z = 3700 in reflectron mode, corresponding to (10,8,3) (Figure 4B). As no ceramide was attached, the different series of fucose substitution were more obvious. The series of fucosylation started with none up to full substitution with more than one Fuc per two N-acetyllactosamine units, supporting the hypothesis of a successively extended branched core with terminal fucoses.

Fraction B with free oligosaccharides of higher sialic acid content

Fraction B contained sialic acid corresponding to monosialylated oligosaccharides (Table VII). The [M-H]- ions produced by MALDI were detected in negative ion linear and reflectron mode. The observed series of the general formula Hex(x+2)HexNAc(x)Fuc(y)NeuAc(1)Cer, with x varying from 6 to 16 and y from 0 to 7, were in agreement with those of fraction 2 of intact polyglycosylceramides. Series from (8,6,0,1) up to (18,16,6,1) and up to (17,15,6,1) were observed in linear and reflectron mode, respectively (Table VII and Figure 5A,B). The molecular mass distribution was centered around m/z = 4300, corresponding to (11,9,2,1) and (11,9,3,1) in linear mode (Figure 5A), and around m/z = 3800, corresponding to (10,8,2,1) in reflectron mode (Figure 5B). The sialylated oligosaccharides were detected in the reflectron mode in contrast to the sialylated PGCs which resulted in higher resolution. The [M-H]- ions in linear mode were accompanied by sodium adduct ions, [M+Na-2H]-, in similarity to fraction 2 of PGCs.

Table VI. MALDI-MS dataa of fraction A with free oligosaccharides of low sialic acid content
Structuree In Fig.f Formula CMb [M+Na]+ OM (linear) [M+Na]+ [Delta] (linear) OM (reflectron) [M+Na]+ [Delta] (reflectron)
(6,4,0) 4.0 C68H114N4O51 1826.64 1826.4 -0.2 1826.31 -0.33
(6,4,1) 4.1 C74H124N4O55 1972.78 1972.2 -0.6 1972.27 -0.51
(6,4,2) 4.2 C80H134N4O59 2118.93 2117.6 -1.3 2118.96 +0.03
(7,5,0) 5.0 C82H137N5O61 2191.98 2191.2 -0.8 2191.25 -0.73
(7,5,1) 5.1 C88H147N5O65 2338.12 2338.6 +0.5 2337.63 -0.49
(7,5,2) 5.2 C94H157N5O69 2484.26 - - 2483.79 -0.47
(8,6,0) 6.0 C96H160N6O71 2557.31 - - 2557.12 -0.19
(8,6,1) 6.1 C102H170N6O75 2703.46 2703.3 -0.2 2703.27 -0.19
(8,6,2) 6.2 C108H180N6O79 2849.60 2849.5 -0.1 2849.45 -0.15
(8,6,3) 6.3 C114H190N6O83 2995.74 2995.5 -0.2 2995.61 -0.13
(9,7,0) 7.0 C110H183N7O81 2922.65 2922.1 -0.6 2922.60 -0.05
(9,7,1) 7.1 C116H193N7O85 3068.79 3069.1 +0.3 3068.74 -0.05
(9,7,2) 7.2 C122H203N7O89 3214.94 3214.8 -0.1 3214.81 -0.13
(9,7,3) 7.3 C128H213N7O93 3361.08 3361.2 +0.1 3361.25 +0.17
(9,7,4) 7.4 C134H223N7O97 3507.22 3507.5 +0.3 3506.55 -0.67
(10,8,0) 8.0 C124H206N8O91 3287.99 3288.4 +0.4 3288.24 +0.25
(10,8,1) 8.1 C130H216N8O95 3434.13 3434.0 -0.1 3434.32 +0.19
(10,8,2) 8.2 C136H226N8O99 3580.27 3580.9 +0.6 3580.51 +0.24
(10,8,3) 8.3 C142H236N8O103 3726.42 3727.4 +1.0 3726.29 -0.13
(10,8,4) 8.4 C148H246N8O107 3872.56 3873.1 +0.5 3872.89 +0.33
(11,9,0) 9.0 C138H229N9O101 3653.33 3653.3 0.0 3653.38 +0.06
(11,9,1) 9.1 C144H239N9O105 3799.47 3800.3 +0.8 3799.79 +0.32
(11,9,2) 9.2 C150H249N9O109 3945.61 3945.6 0.0 3945.65 +0.04
(11,9,3) 9.3 C156H259N9O113 4091.76 4091.7 -0.1 4091.97 +0.21
(11,9,4) 9.4 C162H269N9O117 4237.90 4238.6 +0.7 4238.09 +0.19
(11,9,5) 9.5 C168H279N9O121 4384.04c 4384.1 +0.1 4384.31 +0.27
(12,10,0) 10.0 C152H252N10O111 4018.66 4019.7 +1.0 4018.97 +0.31
(12,10,1) 10.1 C158H262N10O115 4164.81 4165.3 +0.5 4164.48 -0.33
(12,10,2) 10.2 C164H272N10O119 4310.95 4311.5 +0.6 4310.97 +0.02
(12,10,3) 10.3 C170H282N10O123 4457.09 4457.3 +0.2 4457.35 +0.26
(12,10,4) 10.4 C176H292N10O127 4603.24 4603.6 +0.4 4603.44 +0.20
(12,10,5) 10.5 C182H302N10O131 4749.38c 4749.2 -0.2 4749.69 +0.31
(13,11,1) 11.1 C172H285N11O125 4530.14 4531.2 +1.1 4530.22 +0.08
(13,11,2) 11.2 C178H295N11O129 4676.29 4676.6 +0.3 4676.51 +0.22
(13,11,3) 11.3 C184H305N11O133 4822.43 4822.3 -0.1 4822.97 +0.54
(13,11,4) 11.4 C190H315N11O137 4968.57 4969.2 +0.6 4969.07 +0.50
(13,11,5) 11.5 C196H325N11O141 5114.72c 5115.2 +0.5 5115.25 +0.53
(13,11,6) 11.6 C202H335N11O145 5260.86c 5261.1 +0.2 5261.27 +0.41
(14,12,1) 12.1 C186H308N12O135 4895.48 4895.0 -0.5 4895.95 +0.47
(14,12,2) 12.2 C192H318N12O139 5041.62 5041.9 +0.3 5041.82 +0.20
(14,12,3) 12.3 C198H328N12O143 5187.77 5186.8 -1.0 5188.13 +0.36
(14,12,4) 12.4 C204H338N12O147 5333.91 5333.8 -0.1 5334.09 +0.18
(14,12,5) 12.5 C210H348N12O151 5480.05c 5479.5 -0.6 5480.02 -0.03
(14,12,6) 12.6 C216H358N12O155 5626.20c 5625.0 -1.2 5626.15 -0.05
(15,13,2) 13.2 C206H341N13O149 5406.96 5405.8 -1.2 5407.10 +0.14
(15,13,3) 13.3 C212H351N13O153 5553.10 5551.5 -1.6 5552.99 -0.11
(15,13,4) 13.4 C218H361N13O157 5699.25 5697.9 -1.4 5699.16 -0.09
(15,13,5) 13.5 C224H371N13O157 5845.39c 5844.0 -1.4 5845.65 +0.26
(15,13,6) 13.6 C230H381N13O165 5991.53c 5989.7 -1.8 5991.67 +0.14
(15,13,7) 13.7 C236H391N13O169 6137.68c 6134.9 -2.8 6138.88 +1.20
(16,14,2) 14.2 C220H364N14O159 5772.30 5773.5 +1.2 5770.63 -1.67
(16,14,3) 14.3 C226H374N14O163 5918.44 5916.1 -2.3 5917.99 -0.45
(16,14,4) 14.4 C232H384N14O167 6064.58 6063.8 -0.8 6063.58 -1.00
(16,14,5) 14.5 C238H394N14O171 6210.73c 6208.7 -2.0 - -
(16,14,6) 14.6 C244H404N14O175 6356.87c 6355.3 -1.6 - -
(16,14,7) 14.7 C250H414N14O179 6503.01c 6501.3 -1.7 - -
(17,15,3) 15.3 C240H397N15O173 6283.78 6279.5 -4.3 - -
(17,15,4) 15.4 C246H407N15O177 6429.92 6428.1 -1.8 - -
(17,15,5) 15.5 C252H417N15O181 6576.07c 6573.7 -2.4 - -
(17,15,6) 15.6 C258H427N15O185 6722.21c 6718.5 -3.7 - -
(17,15,8) 15.8 C270H447N15O193 7014.49c 7010.5 -3.9 - -
(18,16,3) 16.3 C254H420N16O183 6649.12 6646.5 -2.6 - -
(18,16,5) 16.5 C266H440N16O191 6941.40c 6939.3 -2.1 - -
(18,16,6) 16.6 C272H450N16O195 7087.55c 7081.9 -5.7 - -
        n = 62d [sigma] = 1.4d n = 53d [sigma] = 0.44d
        Accuracy: 0.02-0.08%   0.007-0.02%

OM, Obs.mass; [Delta], obs-calc; CM, calc.mass.
aLinear and reflectron data in positive mode. In linear mode the mass of the ions were determined using [M+H]+ = 2466.7 of ACTH clip 18-39 and [M+H]+ = 5734.5 of bovine insulin as internal standards.
bCalculated average mass.
cIsobaric structures are possible due to 5 MFuc + 0.04 = 2 MHexHexNAc.
d [sigma] and n represent the standard deviation and the number of masses observed.
eStructure (Hex,HexNAc,Fuc).
fIn Fig. HexNAc.Fuc.

The ceramide portion

The molecular masses of the ceramides (Mcer) were determined from the mass difference between intact PGCs (MPGC) and the released oligosaccharides (Moligos), Mcer = MPGC - Moligos + H, (Table VIII). The determined value of the most abundant ceramide of fraction 1, 632.1±1.6 Da in linear mode and 632.5 ± 0.6 Da in reflectron mode, was close to the calculated mean value 632.11 Da for d18:1-24:0 and d18:1-24:1. The mass of the less abundant ceramide was determined to 604.6 ± 1.6 Da and 605.0 ± 0.6 Da in linear and reflectron mode, respectively. The calculated value for the ceramide d18:1-22:0 was 605.06 Da.

The most abundant ceramide of fraction 2 was determined to 631.8 ± 1.2 Da in linear mode, which was consistent with the calculated mean value of 632.11 Da for d18:1-24:0 and d18:1-24:1. The less abundant ceramide was determined to 604.1 ± 1.7 Da and the calculated value for d18:1-22:0 was 605.06 Da (Table VIII).

Discussion

MALDI-TOF MS fingerprinting of complex mixtures of PGCs was shown in this paper to be superior to other MS techniques. Fast atom bombardment (FAB) MS has earlier been used on permethylated high-mass glycosphingolipids (>5000 Da), and molecular ions were recorded (Hanfland et al., 1981; Hanfland et al., 1984; Egge et al., 1985; Levery et al., 1989). However, FAB MS suffers from lack of sensitivity for high-mass molecular ions due to the extensive fragmentation at the hexosamine residues of permethylated glycolipids with repeating N-acetyllactosamine units. As mentioned above electrospray MS has been used for the analysis of a synthetic 25-sugar saccharide but the method is insufficient for complex mixtures as those handled here. In the present work, PGCs were isolated from human erythrocytes and separated into two fractions, 1 and 2, by silica gel chromatography. Two oligosaccharide fractions, A and B, were obtained by ceramide glycanase hydrolysis of the total mixture of PGCs followed by HPAEC. Fraction 1 and A contained mainly neutral components while fraction 2 and B were composed of sialylated species. The MALDI mass spectrum of fraction 1 showed great similarity to the spectrum of the released oligosaccharides of fraction A. In both cases [M+Na]+ ions and oligosaccharide series with increasing degree of fucosylation were detected. In positive ion linear mode, series from (6,4,1)Cer up to (17,15,6)Cer were observed for fraction 1, whereas from (6,4,0) up to (18,16,6) were found for fraction A. The molecular mass distribution envelope was centered around (10,8,3)Cer and (10,8,4)Cer in fraction 1, whereas fraction A was centered around (11,9,3) and (11,9,4). A conformity was observed between the MALDI spectra of sialylated species in fraction 2 and the released oligosaccharides in fraction B. In the negative ion linear mode, series from (7,5,0,1)Cer up to (19,17,4,1)Cer and from (8,6,0,1) up to (18,16,6,1) were detected for [M-H]- ions of fraction 2 and B, respectively. The molecular mass distribution envelope was in both cases centered around the saccharide composition (11,9,2) and (11,9,3). The mass difference between the intact PGCs and the released oligosaccharides with identical sugar composition fits exactly with the ceramide mass. This is conclusive evidence for the existence of such large glycolipids in human erythrocytes. In addition, MALDI-TOF MS revealed a microheterogeneity of these large glycolipids which is far beyond what could be detected by earlier used methods.

The different constituent monosaccharides and their linkages were concluded from methylation analysis of human erythrocyte PGCs (Koscielak et al., 1976; Miller-Podraza et al., 1993). NeuAc-, Fuc-, Gal-, -2Gal-, -3Gal-, -3,6Gal- and -4GlcNAc- were found, which suggested a branched N-acetyllactosamine core with terminals of Gal, sialic acid and fucose. The high degree of fucosylation of some PGC species revealed by the MALDI MS is in line with branched structures. Saccharides characterized by x/y < 2 (seen only in series with odd number of HexNAcs, e.g., in (11,9,5) or (13,11,6) of fraction A) may be branched at every N-acetyllactosamine unit since Fuc in the investigated PGCs occurs primarily as terminal sugar in H-blood group determinants. The PGCs also contain Lewis X and Y determinants with Fuc[alpha]3 linked to GlcNAc detectable by specific monoclonal antibodies; however, the 1,3,4-substituted GlcNAc occurs in mixtures of PGCs only in trace amounts as shown by analysis of partially methylated alditol acetates (not shown here). Less fucosylated structures could be explained by elongations without branching or by the presence of terminal Gal residues in a fully branched core. Thus, the series with x/y [ge] 2 could be explained by one or more positions lacking a branch anywhere in the core structure and having a repeated N-acetyllactosamine in linear sequence. In fact, we have evidence that human erythrocyte PGCs are heterogeneous both concerning substitutions and degree of branching. Degradation with endo-[beta]-galactosidase not expected to cleave between branches spaced by only one N-acetyllactosamine, produces series of ceramide-linked and free oligosaccharides, whose composition agrees with cleavage of internal parts of poly-N-acetyllactosamine core chains (Miller-Podraza et al., unpublished observations). This indicates that the enzyme adding GlcNAc in 6 position during the biosynthesis of human red cell PGCs is of insufficient activity to produce a fully branched core. This differs from rabbit red cells, where the PGCs were regularly branched and fully terminated with Gal[alpha]3 (Dabrowski et al., 1988). Interpretation of MALDI-TOF spectra of endo-[beta]-galactosidase degradation products of human erythrocyte PGCs will be discussed elsewhere. Recent studies on the enzymatic synthesis of branched polylactosaminoglycans using enzymes from human embryonal carcinoma cells presented evidence that 6-linked branches may be added midchain to linear 3-linked N-acetyllactosamine chains (Leppänen et al., 1997, 1998). The proposal was that branched structures like PGCs are synthesized from preformed linear sequences of N-acetyllactosamines coupled in 3-linkages by a [beta]1,6-N-acetylglucosaminyltransferase which is able to add GlcNAc on any Gal along the chain. If this enzyme is not active enough, the result would be insufficient branching and sensitivity to endo-[beta]-galactosidase as found for PGCs. This incomplete branching and the successive extension with N-acetyllactosamine combined with the varying substitution with fucose and sialic acid produces a high degree of microheterogeneity, which is reflected by the unresolved extended interval on the thin-layer chromatogram (Figure 1).

Table VII. MALDI-MS dataa of fraction B with free oligosaccharides of higher sialic acid content
Structured In Fig.e Formula CMb [M-H]- OM (linear) [M-H]- [Delta] (linear) OM (reflectron) [M-H]- [Delta] (reflectron)
(8,6,0,1) 6.0 C107H177N7O79 2824.57 2825.2 +0.6 2824.18 -0.39
(8,6,1,1) 6.1 C113H187N7O83 2970.72 2972.1 +1.4 2971.13 +0.42
(8,6,2,1) 6.2 C119H197N7O87 3116.86c 3117.8 +0.9 3115.80 -0.35
(9,7,0,1) 7.0 C121H200N8O89 3189.91 3190.3 +0.4 3189.50 -0.41
(9,7,1,1) 7.1 C127H210N8O93 3336.05 3336.0 0.0 3336.33 +0.28
(9,7,2,1) 7.2 C133H220N8O97 3482.20c 3482.5 +0.3 3482.47 +0.27
(9,7,3,1) 7.3 C139H230N8O101 3628.34c 3629.4 +1.1 3628.49 +0.15
(10,8,0,1) 8.0 C135H223N9O99 3555.25 3555.5 +0.3 3555.41 +0.16
(10,8,1,1) 8.1 C141H233N9O103 3701.39 3702.2 +0.8 3701.12 -0.27
(10,8,2,1) 8.2 C147H243N9O107 3847.53c 3848.2 +0.7 3847.53 0.00
(10,8,3,1) 8.3 C153H253N9O111 3993.68c 3994.2 +0.5 3993.59 -0.09
(11,9,0,1) 9.0 C149H246N10O109 3920.59 3920.4 -0.2 3920.56 -0.03
(11,9,1,1) 9.1 C155H256N10O113 4066.73 4067.8 +1.1 4066.54 -0.19
(11,9,2,1) 9.2 C161H266N10O117 4212.87c 4213.1 +0.2 4212.75 -0.12
(11,9,3,1) 9.3 C167H276N10O121 4359.02c 4359.0 0.0 4358.91 -0.11
(11,9,4,1) 9.4 C173H286N10O125 4505.16c 4505.5 +0.3 4504.60 -0.56
(12,10,0,1) 10.0 C163H269N11O119 4285.92 4286.7 +0.8 4285.67 -0.25
(12,10,1,1) 10.1 C169H279N11O123 4432.07 4432.0 -0.1 4432.26 +0.19
(12,10,2,1) 10.2 C175H289N11O127 4578.21c 4577.6 -0.6 4577.79 -0.42
(12,10,3,1) 10.3 C181H299N11O131 4724.35c 4724.7 +0.4 4724.04 -0.31
(12,10,4,1) 10.4 C187H309N11O135 4870.50c 4869.9 -0.6 4869.77 -0.72
(13,11,0,1) 11.0 C177H292N12O129 4651.26c 4652.4 +1.1 4651.17 -0.09
(13,11,1,1) 11.1 C183H302N12O133 4797.40 4798.6 +1.2 4796.70 -0.70
(13,11,2,1) 11.2 C189H312N12O137 4943.55c 4943.9 +0.4 4942.82 -0.73
(13,11,3,1) 11.3 C195H322N12O141 5089.69c 5089.3 -0.4 5088.69 -1.00
(13,11,4,1) 11.4 C201H332N12O145 5235.83c 5235.8 0.0 5235.31 -0.52
(13,11,5,1) 11.5 C207H342N12O149 5381.98c 5381.2 -0.8 5381.97 -0.01
(14,12,0,1) 12.0 C191H315N13O139 5016.60 5016.4 -0.2 5016.01 -0.59
(14,12,1,1) 12.1 C197H325N13O143 5162.74 5162.9 +0.2 5162.31 -0.43
(14,12,2,1) 12.2 C203H335N13O147 5308.88c 5309.3 +0.4 5307.59 -1.29
(14,12,3,1) 12.3 C209H345N13O151 5455.03c 5454.6 -0.4 5454.34 -0.69
(14,12,4,1) 12.4 C215H355N13O155 5601.17c 5600.7 -0.5 5601.36 +0.19
(14,12,5,1) 12.5 C221H365N13O159 5747.31c 5747.6 +0.3 5746.45 -0.86
(15,13,1,1) 13.1 C211H348N14O153 5528.08 5526.3 -1.8 5527.56 -0.52
(15,13,2,1) 13.2 C217H358N14O157 5674.22c 5674.5 +0.3 5673.21 -1.01
(15,13,3,1) 13.3 C223H368N14O161 5820.36c 5819.0 -1.4 5819.77 -0.59
(15,13,4,1) 13.4 C229H378N14O165 5966.51c 5964.2 -2.3 5967.24 +0.74
(15,13,5,1) 13.5 C235H388N14O169 6112.65c 6111.2 -1.5 6113.04 +0.39
(15,13,6,1) 13.6 C241H398N14O173 6258.79c 6256.5 -2.3 6258.51 -0.28
(16,14,1,1) 14.1 C225H371N15O163 5893.42 5895.3 +1.9 - -
(16,14,2,1) 14.2 C231H381N15O167 6039.56c 6038.1 -1.5 - -
(16,14,3,1) 14.3 C237H391N15O171 6185.70c 6187.1 +1.4 6184.28 -1.42
(16,14,4,1) 14.4 C243H401N15O175 6331.85c 6331.0 -0.9 6331.91 +0.06
(16,14,5,1) 14.5 C249H411N15O179 6477.99c 6476.5 -1.5 - -
(16,14,6,1) 14.6 C255H421N15O183 6624.13c 6623.7 -0.4 6625.48 +1.35
(17,15,2,1) 15.2 C245H404N16O177 6404.90c 6402.4 -2.5 - -
(17,15,4,1) 15.4 C257H424N16O185 6697.18c 6696.0 -1.2 - -
(17,15,5,1) 15.5 C263H434N16O189 6843.33c 6842.8 -0.5 6843.76 +0.43
(17,15,6,1) 15.6 C269H444N16O193 6989.47c 6989.1 -0.4 6991.69 +2.22
(17,15,7,1) 15.7 C275H454N16O197 7135.61c 7133.3 -2.3 - -
(18,16,2,1) 16.2 C259H427N17O187 6770.23c 6769.0 -1.2 - -
(18,16,3,1) 16.3 C265H437N17O191 6916.38c 6915.8 -0.6 - -
(18,16,4,1) 16.4 C271H447N17O195 7062.52c 7059.2 -3.3 - -
(18,16,5,1) 16.5 C277H457N17O199 7208.66c 7206.6 -2.1 - -
(18,16,6,1) 16.6 C283H467N17O203 7354.81c 7351.8 -3.0 - -
        n = 55n [sigma] = 1.2n n = 44n [sigma] = 0.65n
        Accuracy: 0.02-0.04%   0.009-0.02%
OM, Obs.mass; [Delta], obs-calc; CM, calc.mass.
[sigma] is the standard deviation, and n is the number of masses observed.
aLinear and reflectron data in negative mode.
bCalculated average mass.
cIsobaric structures are possible due to 5 MFuc + 0.04 = 2 MHexHexNAc and 2 MFuc= 1.02 + MNeuAc.
dStructure (Hex,HexNAc,Fuc,NeuAc).
eIn Fig. HexNAc.Fuc.

Table VIII. Ceramides found in fraction 1 and 2 of polyglycosylceramidesa
Fraction and mode Mcer (Da) measured Mcer calc. mass (Da) Ceramide Formula
Fraction 1, linear 632.1±1.6, n = 36b      
Fraction 1, reflectron 632.5±0.6, n = 35b 632.11 d18:1-24:0/1 C42H82(80)NO2
Fraction 2, linear 631.8±1.2, n = 36b      
Fraction 1, linear 604.6±1.6, n = 12b      
Fraction 1, reflectron 605.0±0.6, n = 13b 605.06 d18:1-22:0 C40H78NO2
Fraction 2 linear 604.1±1.7, n = 23b      
aFrom the mass difference between polyglycosylceramides and the corresponding released oligosaccharides, Mcer = MPGC-Moligos + H.bn is the number of masses used.


Figure 4. MALDI-TOF mass spectrum of fraction A in (A) positive ion linear mode and (B) positive ion reflectron mode. Annotations as HexNAc.Fuc (Table VI).


Figure 5. MALDI-TOF mass spectrum of fraction B in (A) negative ion linear mode and (B) negative ion reflectron mode. Annotations as HexNAc.Fuc (Table VII).

From these results, nothing can be concluded on the binding epitope for H.pylori. However, MALDI-TOF MS technique will be helpful in characterizing the structure of partial degradation products, which may retain binding activity. The binding of the bacterium is sialic acid dependent; removal of sialic acid by acid or by neuraminidase completely abolishes binding, and the same result is obtained after mild periodate oxidation, which eliminates only C-9, or C-8 and C-9, of the glycerol tail of sialic acid (Miller-Podraza et al., 1996). However, animal PGCs, which also contain sialic acid, are inactive (Miller-Podraza, et al., 1997c). The specificity for human PGCs may reside in an epitope conformation where sialic acid interacts with hydrogen bonds between C-9 of its glycerol tail and an incompletely branched N-acetyllactosamine core (Karlsson, 1998).

Materials and methods

Glycosphingolipid material

Bovine brain gangliosides (mixture of GM1, GD1a, GD1b, and GT1b) were purchased from Calbiochem (La Jolla, CA, USA). PGCs of human erythrocyte membranes, blood group O, were prepared in our laboratory according to the peracetylation procedure as described (Miller-Podraza et al., 1993). The main fraction obtained after Sephadex LH 60 chromatography was purified by silica gel chromatography (silica gel suspended in chloroform) using chloroform/methanol 3:1 as elution solvent. The separation resulted in two polyglycosylceramide fractions differing in sialic acid content (see Table 1).

Colorimetric assays

Hexose, sialic acid and sphingosine were assayed according to references given earlier (Miller-Podraza et al., 1993).

Binding assay

H.pylori, CCGU 17874 strain, was obtained from Culture Collection of Göteborg University (CCGU). Cultivation in Ham's F12 liquid medium and overlay of TLC plates with 35S-labeled cells were performed as described earlier (Karlsson and Strömberg, 1987; Miller-Podraza et al., 1996).

Hydrolysis by ceramide glycanase and separation of oligosaccharides

Digestion of PGCs by ceramide glycanase (Rhodococcus) was performed as described (Miller-Podraza et al., 1997c). The products were separated by phase partition in chloroform/methanol/water, 10:5:3 (Li and Li, 1989) and the carbohydrates contained in the upper phase were purified by Sephadex G-15 chromatography. The saccharide mixture was separated by HPAEC as described (Miller-Podraza et al., 1993).

MALDI-TOF MS

MALDI mass spectra were obtained on a TofSpec-E time-of-flight mass spectrometer (Micromass, Manchester, England) equipped with delayed extraction (DE) and a nitrogen laser (337 nm, 4 ns pulse, LSI, Boston, MA) operated in either the reflectron or linear mode. The accelerating voltage used was 20 kV in reflectron mode and 25 kV in linear mode. For delayed extraction a 2 kV potential difference between the probe and the extraction lens was applied with a time delay of 600 ns after each laser pulse. The molecular ions, [M+H]+, of the ACTH clip 18-39 at m/z = 2466.7 (average mass) and bovine insulin at m/z = 5734.5 (average mass) were used in positive ion mode for external mass calibration. The corresponding [M-H]- ions were used for calibration in the negative ion mode. As matrix 2,5-dihydroxybenzoic acid (about 15 mg/ml) in water was used for all samples and calibration compounds. Equal volumes (5 µl) of the sample and matrix solutions were mixed from which 1 µl was applied on the stainless steel target. The mixture was allowed to dry at ambient temperature before introduction into the mass spectrometer. The neutral glycolipids and oligosaccharides produced sodium adduct ions, [M+Na]+, and were detected in positive ion mode. The sialic acid containing components produced [M-H]- ions, which were detected in negative ion mode. Approximately 200-300 shots were accumulated per spectrum. The pressure in the TOF analyzer was 1 × 10-7 mbar. Although the delayed extraction data obtained in reflectron mode was isotopically resolved in the lower mass range (below 3000 amu) all spectra were smoothed to receive an average mass in order to simplify the data treatment.

Abbreviations

MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Hex, hexose; HexNAc, N-acetylhexosamine; Fuc, fucose; NeuAc, N-acetylneuraminic acid; PGCs, polyglycosylceramides. Polysaccharides are denoted in the text according to number of Hex, HexNAc, Fuc, and NeuAc units, respectively; e.g., (8,6,0,1) means that the molecule contains totally 15 monosaccharides. For ceramide abbreviations, d18:1 means sphingosine (1,3-dihydroxy-2-amino-4-trans-octadecane) and, e.g., 24:1 means a nonhydroxy fatty acid with 24 carbon atoms and one double bond.

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