Glycobiology, 2000, Vol. 10, No. 12 1291-1309
© 2000 Oxford University Press
Polyglycosylceramides recognized by Helicobacter pylori: analysis by matrix-assisted laser desorption/ionization mass spectrometry after degradation with endo-ß-galactosidase and by fast atom bombardment mass spectrometry of permethylated undegraded material
Institute of Medical Biochemistry, Göteborg University, Box 440, SE 405 30 Göteborg, Sweden
Received on April 12, 2000; revised on July 7, 2000; accepted on July 12, 2000.
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Abstract |
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Human erythrocyte polyglycosylceramides (PGCs) are recognized by the gastric pathogen Helicobacter pylori and are based on a successively extended and highly branched N-acetyllactosamine core linked to ceramide and substituted by fucose and sialic acid. As a step in the identification of the binding epitope we earlier characterized intact PGCs by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS (Karlsson,H., Johansson,L., Miller-Podraza,H., and Karlsson,K-A. [1999] Glycobiology, 9, 765–778). In the present work, PGCs from human blood group O erythrocytes were digested with endo-ß-galactosidase (Bacterioides fragilis), an enzyme which cleaves the bond 3Galß1–4GlcNAc in linear but not branched poly-N-acetyllactosamine chains. The enzymatic digestion resulted in a mixture of neutral and sialic acid–containing glycolipids together with terminal and internal sequences of mainly neutral oligosaccharides. The products were analyzed by MALDI-TOF MS in both positive and negative ion mode which gave spectra where the ions could be assigned to structures of the neutral and acidic components, respectively. Among glycolipids found were
where R could be H, Fuc or NeuAc. Also observed were structures as
which indicated linear extension along both branches. Observed at higher masses were fully branched structures obtained by stepwise extension with
where R could be H, Fuc or NeuAc. Most probably further branching may occur along both the (1
3)- and the (16)-linked branches to give a partly dendritic structure. Structures with more than one sialic acid substituted could not be observed in the MALDI spectrum. Complementary information of the terminal sequences was obtained by FAB-MS analysis of permethylated undegraded PGCs. High-temperature gas chromatography/mass spectrometry of reduced and permethylated products from enzyme hydrolysis documented that Fuc was present in a blood group O sequence, Fuc-Hex-HexN-. Fucose may be placed on short (monolactosamine) or longer branches, while sialic acid seems to be restricted to monolactosamine branches. The conclusion is that human erythrocyte PGCs display microheterogeneity within terminal and internal parts of the poly-N-acetyllactosamine chains. The first branch from the ceramide end may be located at the second or third Gal and possibly also on the first Gal. Other branches may occur on every N-acetyllactosamine unit in fully branched domains, or there may be linear extensions between branches resulting in incompletely branched structures. The extended linear sequences may be present in both 3- and 6-linked antennae. Terminal structures are based on one, two or maybe higher number of N-acetyllactosamine units.
Key words: human erythrocytes/MALDI-TOF MS/FAB-MS/mass spectrometry/polyglycosylceramides/receptor activity/Helicobacter pylori/endo-ß-galactosidase
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Introduction |
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Polyglycosylceramides (PGCs) are complex glycosphingolipids with a polymeric structure based on Galß4GlcNAc (N-acetyllactosamine) units (Koscielak et al., 1976
; Dejter-Juszynski et al., 1978; Dabrowski et al., 1988). They have been found in several human (Koscielak et al., 1976; Dejter-Juszynski et al., 1978; Levery et al., 1989) and animal (Slomiany et al., 1980; Dabrowski et al., 1988; Miller-Podraza et al., 1997) tissues and described as blood group-active (Koscielak et al., 1976, 1979; Egge et al., 1985; Dabrowski et al., 1988) and microbe binding molecules (Loomes et al., 1984; Liukkonen et al., 1992; Miller-Podraza et al., 1996). In contrast to PGCs of rabbit erythrocytes, which form a series of components with a regular 5-sugar interval between species (Dabrowski et al., 1988), PGCs of human erythrocytes display structural microheterogeneity. In PGCs of blood group O different branches may be terminated by Galß4, Fuc
2, NeuAc3, or NeuAc6 which results in complex series of molecules. In PGCs of other blood groups this variety is expected to be even higher. The molecular microheterogeneity of PGCs from human erythrocytes of blood group O was demonstrated recently by us using matrix-assisted laser desorption/ionization mass spectrometry (Karlsson et al., 1999). The composition agreed with the general formula of Hexx+2HexNxFucyNeuAczCer, abbreviated as (x + 2,x,y,z)Cer where x varied between 4 and 15 for the neutral components and between 5 and 17 for components with one sialic acid. The maximum number of substituted fucoses (y) in the series observed was (x / 2) rounded off upwards for the neutral components and (x / 2 – 1) rounded off upwards for the ones with one sialic acid. The sequence and anomerity of branches of human PGCs as well as distribution of terminal sugars has not previously been analyzed in detail. According to chemical degradations, branches within the poly-N-acetyllactosamine chains of erythrocyte PGCs should be located on every second N-acetyllactosamine unit (Zdebska et al., 1983). Investigations using MALDI-TOF MS performed in our laboratory (Karlsson et al., 1999) indicated on the other hand the presence of fully branched carbohydrate chains. In the present paper we provide evidence that both variants are present in PGCs from human erythrocytes. The conclusion is based on analysis of PGCs hydrolyzed by endo-ß-galactosidase from Bacteroides fragilis (Scudder et al., 1983, 1984), which cleaves linear but not branched poly-N-acetyllactosamine chains (Scudder et al., 1984; see sites of cleavage in Figure 1). The present studies allow insight into the organization of the core chains of PGCs and of the distribution of terminal monosaccharides, which may be of importance for understanding of the molecular basis of binding by Helicobacter pylori (Miller-Podraza et al., 1996), a human gastric pathogen of high clinical importance (Dunn et al., 1997; Karlsson, 1998, 2000).
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MALDI-TOF MS analysis of the products after digestion with endo-ß-galactosidase
This approach allowed a general survey of all released products from digestion with endo-ß-galactosidase. Although, the results from MALDI-MS alone cannot provide any information about the structure and branching of the components, the ion masses observed could be assigned to specific structures due to the specificity of the cleavage of the endo-ß-galactosidase. The analysis of the products by MALDI-MS in positive (Figure 2, Table BI) and negative (Figure 3, Table BII) ion modes provided structural information on neutral and acidic molecules, respectively. Both neutral and sialic acid-containing glycolipids were observed together with mainly neutral terminal and internal oligosaccharides. The neutral components were observed as sodium adduct ions, [M+Na]+, in the positive ion mode while the sialic acid–containing components were detected as [M-H]– in the negative ion mode.
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is the standard deviation and n is the number of masses observed.
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Spectrum in the positive ion mode
The most abundant ion observed in the MALDI spectrum in the positive ion mode could be assigned to the neutral glycolipid HexN-Hex-Hex-Cer, (2,1,0)Cer (100%, relative abundance in the mass spectrum), followed by the ion corresponding to Hex-Cer (72%). The latter product was due to slower enzymatic cleavage of the bond 3Gal1-4Glc1 (Figure 1). Present in low amount was also an ion that may be assigned to
where the first branch is on the first Gal. In the MALDI mass spectrum the higher mass ions could be assigned to structures extended from HexN-Hex-Hex-Cer by units of (2,2,y), y = 0,1. The major ions which belonged to the series (x + 1,x,y)Cer were accompanied by components 162 u lower in mass, which corresponded to lack of one hexose unit. This resulted in ions following series of (x,x,y)Cer, which indicated cleavage along both the (13)- and the (16)-linked branch. This important observation indicated that the PGC molecules could have linear extensions along both the (13)- and the (16)-linked branch. Further branching might result into a partly dendritic structure of the PGC molecules.
Ions that could be assigned to a series of branched neutral glycolipids continued with (4,3,y)Cer, y = 0,1 (24 and 12%, respectively)
where R = H or Fuc. Here and later on the structures are written as terminated on the (16)-linked branch but could as well be on the (13)-linked branch since it is not possible from mass spectrometry results to determine whether the linear extension occurs along the (13)- or the (16)-linked branches.
This structure was extended each time by
(2,2,y), y = 0,1, and R = H or Fuc, and observed up to (10,9,4)Cer. These structures could be rationalized as
where R = H or Fuc and j = 1 (4,3,y)Cer, y = 0,1 (24% and 12%, respectively); j = 2 (6,5,y)Cer, y = 0,1,2 (around 2%); j = 3 (8,7,y)Cer, y = 0,1,2,3 (below 1%); and j = 4 (10,9,y)Cer, y = 0,1,2,3,4 (below 0.3%). y is the number of fucoses substituted ranging from 0 up to a maximum of (x / 2 – 1) rounded off upwards. The mass difference between components with the same number of substituents (y) was 730 u (C28H46N2O20 = 730.26 [12C], 730.68 [average]).
Ions that could be assigned to structures which indicated linear extensions also at the (16)-linked branch were observed located 162 u lower in mass than the major series (x + 1,x,y)Cer. The series (x,x,y)Cer started with (3,3,0)Cer (4%),
and was extended by
(2,2,y), y = 0,1, and R = H or Fuc, each time up to (5,5,y)Cer and (7,7,y)Cer, where the number of fucoses y = 0 up to a maximum of (x / 2 – 2) rounded off upwards. The structure (5,5,y)Cer has two branching points either with (1) linear extension at the (16)-linked first branch and at the (13)-linked second branch,
(5,5,y)Cer or (2) linear extension at the (16)-linked second branch and at the (13)-linked second branch,
(5,5,y)Cer, where R = H or Fuc.
The next higher component in this series, the (7,7,y)Cer, y = 0,1,2,3, has three branching points (see also Table BI)
The possible linear extensions could be at a) the (16)- linked first branch and the (13)- linked third branch (R1 = H), b) the (16)- linked second branch and the (13)- linked third branch (R2 = H) or c) the (16)- and the (13)- linked third branch (R3 = H).
Ions that could be assigned to structures of neutral free oligosaccharides originating from internal parts of the polylactosamine core chain and cleaved at two positions either along the (13)- or the (16)-linked branches, were observed as a series (x,x,y)i where x was ranging from 3 up to 7, starting from (3,3,y)i, y = 0,1
and extended by
(2,2,y), y = 0,1 and R = H or Fuc, (730.26 u, 12C), each time up to (7,7,3)i. The internal neutral oligosaccharides in this series could be written as
where j = 1(3,3,y)i, y = 0,1 (18% and 9%, respectively); j = 2(5,5,y)i, y = 0,1,2 (around 2%); j = 3(7,7,y)i,y = 0,1,2,3 (around 0.5%). The number of substitutions (y) were from 0 up to (x / 2 – 1) rounded off upwards and R = H or Fuc.
Ions due to internal oligosaccharides cleaved from PGC structures at three positions with linear extensions along both the (13)- and the (16)- linked branches were observed as series of the form (x – 1,x,y)i, where x could be 2, 4 and y = 0,1;
(2,3,0)i (9%) and
(4,5,y)i (around 2%), with R = H or Fuc.
Ions due to neutral terminal oligosaccharides, originating from PGCs cleaved at one position, were observed from Hex-HexN-Hex, (2,1,0)t (5%), and Fuc-Hex-HexN-Hex, (2,1,1)t (32%), to
(4,3,y)t, y = 0,1,2 (8%, 4%, and 4%, respectively) where R = H or Fuc. Even an extended structure of the last one was observed as
(6,5,1)t (2%) where R = H or Fuc.
The results obtained from MALDI-TOF MS in the positive ion mode of the neutral glycolipids and oligosaccharides are presented in Table BI. Examples of possible neutral products obtained after digestion with endo-ß-galactosidase are shown in Figure 4 for isomers of (8,6,y)Cer.
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Spectrum in the negative ion mode
The most abundant ion observed in the MALDI spectrum in negative ion mode could be assigned to the sialic acid containing glycolipid with the structure (4,3,0,1)Cer (base peak, 100% relative abundance in the mass spectrum).
(4,3,0,1)Cer. It is written as terminated by NeuAc on the (16)- linked branch but this could as well be on the (13)-linked branch as discussed above. This indicated that the sialic acid may be preferentially substituted at the first branching point of the PGC molecules. In conformity with the neutral components, ions could be assigned to structures extended by units of (2,2,y) each time (730.26 u (12C), 730.68 u (average mass), creating series of ions of the form (x + 1,x,y,1)Cer.
Series that started with (4,3,0,1)Cer,
was extended by
(2,2,y), y = 0,1, and R = H or Fuc, each time and was observed up to (12,11,4,1)Cer with a mass difference of 730 u between series with the same number of fucoses substituted (y).
These structures could be presented as
where R = H or Fuc and j = 1(6,5,y,1)Cer, y= 0,1 (15% and 13%, respectively); j = 2(8,7,y,1)Cer, y = 0,1,2 (below 10%); j = 3(10,9,y,1)Cer, y = 0,1,2,3 (below 4%) and j = 4(12,11,y,1)Cer, y = 0,1,2,3,4 (below 1.5%). The number of fucoses (y) was ranging from 0 up to a maximum of (x / 2 – 2) rounded off upwards, where x is the number of HexN’s present.
Ions which could be assigned to structures with linear extensions along both the (13)- and the (16)- linked branches were observed among the sialic acid-containing components in similarity with the neutral ones. These ions were observed as series of the type (x,x,y,1)Cer, located 162 u lower in mass than the main series of (x + 1,x,y,1)Cer. The (6,5,0,1)Cer structure was accompanied by the (5,5,0,1)Cer (7%),
(5,5,0,1)Cer, with two branching points and linear extension at both the (13)- and the (16)-linked second branch. Here it is assumed that NeuAc was substituted at the first branch and that the main elongation was along the (13)-linked branch. Ions from the extended structure (7,7,y,1)Cer, y = 0,1 (4% and 5%, respectively) was observed accompanying the (8,7,y,1)Cer structures. This structure will have three branching points with linear extension at either the (16)-linked second or third branch,
(7,7,y,1)Cer, linear extension at the (16)- linked second and at the (13)- linked third branch (R1 = HexN, R2 = R-Hex-HexN) or only at the third branch (R1 = R-Hex-HexN, R2 = HexN) with R = H or Fuc.
Similarly the (9,9,y,1)Cer, y = 0,1,2 (1%, 1%, and 2%, respectively) structures with four branching points could have linear extensions at the second, third, or fourth branch.
Also ions that could be assigned to structures of fully branched PGCs, unaffected by the enzyme, were observed as (9,7,3,1)Cer (2%), (11,9,4,1)Cer (2.5%) and (13,11,5,1)Cer (1%).
The structure of the components in this series could be presented as
where for j = 1(9,7,3,1)Cer; j = 2(11,9,4,1)Cer and j = 3(13,11,5,1)Cer. The extension each time in this series was
(2,2,1), corresponding to a calculated mass difference between the components of 876.82 u (average mass).
Only two negative ions in the MALDI spectrum could be assigned to internal acidic oligosaccharides cleaved at two positions, the structure (3,3,0,1)i and its extension (5,5,0,1)i:
(3,3,0,1)i (12%) and
(5,5,0,1)i (3%). This observation indicated that the sialic acid could be substituted also within the poly-N-acetyllactosamine chain.
Two negative ions could also be assigned to structures from terminal sequences of sialic acid-containing fragments, namely the structures (4,3,0,1)t and (4,3,1,1)t, respectively:
(4,3,0,1)t (4%), and
(4,3,1,1)t (2%), which indicated that substitution by sialic acid was possible in the nonreducing end of the PGCs. The products from internal and terminal sequences above could also be a result of the slower enzymatic cleavage of the bond 3Gal-4Glc1 close to the ceramide.
The results from MALDI-TOF MS in the negative ion mode of the sialylated components are presented in Table BII. Examples of possible sialic acid-containing products formed after cleavage with endo-ß-galactosidase are shown in Figure 5 for isomers of (10,8,y,1)Cer and (11,9,y,1)Cer.
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FAB-MS analysis of intact permethylated PGCs
To investigate further terminal substitutions in PGC molecules, the undegraded material was permethylated and analyzed by fast atom bombardment mass spectrometry (FAB-MS). The analysis was not sensitive enough to give information in the molecular region due to the extensive fragmentation that followed the ionization. However, abundant oxonium ions (Bi) were formed adjacent to each HexN residue which gave valuable sequence information concerning terminal fragments with up to 15 monosaccharides per mol (Figure 6, Table III). The fragment ions could often be followed in series with one N-acetyllactosamine unit mass difference (449.3 u). Bi ions from neutral components of the PGCs could be followed from linear structures such as Hex-HexN+, (1,1,0)+, at m/z 464; Fuc-Hex-HexN+, (1,1,1)+, at m/z 638; Hex-HexN-Hex-HexN+, (2,1,0)+, at m/z 913; Fuc-Hex-HexN-Hex-HexN+, (2,2,1)+, at m/z 1088; and Fuc-Hex-HexN-Hex-HexN-Hex-HexN+, (3,3,1)+ at m/z 1537.
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Bi ions of sialic acid-containing components were detected as NeuAc+ at m/z 376, NeuAc-Hex+ at m/z 580 and NeuAc-Hex-HexN+, (1,1,0,1)+, at m/z 825. Fragment ions from a component with two sialic acids were found at m/z 2086, (3,3,0,2)+. Components containing two sialic acids which were found in FAB-MS (the Bi ion at m/z 2086) could not be detected as the corresponding terminal oligosaccharide, (4,3,0,2)t, in the MALDI spectrum. The results from FAB-MS on permethylated PGCs are summarized in Table III.
Supporting evidence by EI-MS analysis of degraded permethylated PGCs
To confirm the conclusions from MALDI-TOF MS and FAB-MS concerning terminal sequences of potential interest for bacterial binding two approaches by EI-MS were applied. One was high-temperature GC/MS of reduced and permethylated hydrolysis products (Figure 7), the other was direct inlet EI-MS of two permethylated subfractions after separation of hydrolysis products by ion exchange chromatography (not reproduced). As was possible to interpret from Figure 7, major components from the TIC (total ion chromatogram) provided evidence for linear internal and terminal extensions. Component A, HexN-Hex, (1,1,0)iol corresponding to (1,1,0)i not detected in MALDI-MS, a nonsubstituted trisaccharide, B, (2,1,0)tol, corresponding to (2,1,0)t, and a fucosylated tetrasaccharide, C, (2,1,1)tol, corresponding to (2,1,1)t were found. Furthermore, the C saccharide was evidence for the blood group O determinant, Fuc2Galß4GlcNAc, and no ions were found corresponding to the Lewis x, Galß4(Fuc3)GlcNAc, or Lewis y, Fuc2Galß4(Fuc3)GlcNAc, determinant. Component D, (2,1,1)iol, corresponding to (2,2,1)i may have contained an internal Fuc linked to Gal, but this is inconclusive due to low amounts. No component was found corresponding to a tetrasaccharide with terminal NeuAc, indicating the existence of only nonextended NeuAcGalßGlcNAc, in contrast to extended fucosylated branches (component C, and Table III from FAB-MS). Component E (corresponding to (2,3,0)t) provided evidence for linear extensions on both (13)- and (16)-linked branches. Component F corresponded to (3,3,0)i from MALDI-MS. No other components from the enzymatic hydrolysis could be chromatographed due to their high molecular weights.
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The EI-MS spectrum (not shown) of the permethylated neutral subfraction from enzyme hydrolysis contained relatively abundant sequence ions identical with those from FAB-MS (Table III), at m/z 464, 638, 913, and 1088. In addition, fragment ions at m/z 1159 and 1333 were evidence for nonfucosylated (six sugars) and fucosylated (seven sugars) sequences with a HexN branch in the middle of the core chain. The EI-MS spectrum (not shown) of the permethylated acidic subfraction from enzyme hydrolysis contained the relatively abundant fragment ions at m/z 376, 580, and 825, also detected by FAB-MS (Table III). Co-presence of ions at m/z 464 and 638 proved that nonsubstituted and fucosylated terminals existed in the acidic fraction.
Composition of the ceramides of the PGCs
Detailed information of the ceramide composition of the PGCs was achieved from the MALDI-MS spectra in the positive ion mode. From the pseudomolecular ions [M+Na]+ of Hex-Cer and Hex-HexN-Hex-Hex-Cer the most abundant ceramides were determined to be (d18:1–24:0), (d18:1–24:1), (d18:1–22:0), (d18:1–22:1) and smaller amounts of (d18:1-h24:0), (d18:1-h24:1), (d18:1–26:0), and (d18:1–26:1). The results are presented in Table IV.
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The analysis by FAB-MS also provided information concerning the ceramide composition of the investigated PGC molecules, where Zo fragment ions (Domon and Costello, 1988
) at m/z 660.7, 658.6, 632.6, and 630.5 indicated ceramides with sphingosine d18:1 and nonhydroxy fatty acids 24:0, 24:1, 22:0, and 22:1 with the ratio of about 4:4:2:1. The conclusion on ceramide composition is in line with earlier analyses of the PGCs (Koscielak et al., 1976; Dejter-Juszynski et al., 1978; Miller-Podraza et al., 1993). |
Discussion |
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Combining the results from MALDI-MS of all products generated by the enzymatic cleavage by endo-ß-galactosidase and the terminal sequence ions obtained from FAB-MS of permethylated PGCs together with EI-MS and the earlier obtained structural results from MALDI-TOF MS on undegraded PGCs (Karlsson et al., 1999
) allow a rather good characterization of the structural organization of the carbohydrate chains of human erythrocyte PGCs. There are strong indications, that PGCs contain both incompletely and fully branched poly-N-acetyllactosamine domains. All internal and terminal oligosaccharides released by the enzyme together with all glycolipids left unaffected by the enzyme had compositions supporting branched structures where the sum of N-acetyllactosamine units and terminal HexNAcs in a molecule is an odd number. Undegraded PGCs contain molecules based on both even and odd numbers of N-acetyllactosamine units (odd x in the PGC formula, Hexx+2HexNxFucyNeuAczCer). The products remaining after degradation with endo-ß-galactosidase of the PGC molecules would be branched glycolipids up to the linear extension where the cleavage occurred, and fully branched glycolipids left unaffected by the enzyme, together with cleavage products as terminal and internal oligosaccharides. The MALDI mass spectra, both in positive and negative ion mode, were dominated by ions which could be assigned to glycolipid structures that could be followed in series. The structures assigned within each series started with no fucose substituted up to fully fucosylated molecules. The assignment of these highly branched structures gave a detailed picture of the overall structural organization of the PGC molecules. The neutral components of the PGCs seemed to have more frequently linear extension of the first N-acetyllactosamine unit since the MALDI spectrum in positive mode was dominated by the ion corresponding to HexN-Hex-Hex-Cer, (2,1,0)Cer. A lower abundance of Hex-Cer supported a relatively slow hydrolysis at lactose. Some evidence existed for a branching at the first Gal.
In negative mode the most abundant ion in the MALDI spectrum could be assigned to the structure (4,3,0,1)Cer with the branching at the second Gal. However, positive ions with an abundance of 24% and 12%, respectively, could be assigned to structures of the neutral glycolipids (4,3,y)Cer, y = 0,1. Here the branching occurred at the second Gal from the ceramide.
For neutral PGCs the first branching point may more frequently be located on the third Gal than on the second Gal from the ceramide. Linear extensions between branches seemed to be frequent in both neutral and sialic acid-containing components. Ions which could be assigned to series where the branching started at the second Gal from the ceramide were observed for both neutral (as series [x + 1,x,y]Cer) and sialic acid–containing PGCs (as series [x + 1,x,y,1]Cer). Linear extensions took place after the first, second, third, fourth, and fifth branching point, respectively, for the sialic acid–containing glycolipids belonging to the series (x + 1,x,y,1)Cer. Ions from fully branched structures, unaffected by the enzyme, were not detected in the MALDI spectrum for the neutral components but were observed in the case of the sialic acid–containing PGCs. These structures followed the series of the form (x + 2,x,y,1)Cer for normal PGCs. One important result in this investigation was the observation of ions which could be assigned to structures with linear extensions at both the (13)- and the (16)-linked branches. Further branching may occur along the extended (16)-linked branch which may result in a partly dendritic structure of the PGCs.
The full branching of human PGCs was suggested earlier on the basis of analysis by MALDI-TOF MS of intact PGCs, where high degree of fucosylation was shown (Karlsson et al., 1999). Fucose in PGCs of blood group O represents primarily the H-epitope (Fuc2Galß4GlcNAc) and is located terminally. High-temperature GC/MS documented this and no evidence was obtained for the presence of Lewis determinants.
Another important observation was, that fucose in human PGCs may be located on di- or monolactosamine terminal extensions while sialic acid is present only on monolactosamine branches. Worth noticing is that both Fuc and NeuAc may be present in the same molecules. The present work was based on the assumption that endo-ß-galactosidase is cleaving linear but not branched poly-N-acetyllactosamine structures (Figure 1). According to Scudder et al. (1983, 1984) branching Gal in the neolacto chains remain resistant to hydrolysis even at very high enzyme concentrations (up to 2.5 U/ml). Our own results confirm that the endo-ß-galactosidase specificity is very restricted.
In summary, human erythrocyte PGCs are microheterogenous within terminal and internal parts of the poly-N-acetyllactosamine chain. The first branch may be located on the first, second or third Gal and linear extensions may occur between branches. The linear extensions may occur along both the (13)- and the (16)-linked branch probably followed by further branching resulting in a partly dendritic structure. There may be fully branched regions within the poly-N-acetyllactosamine chain and there are examples of structures which are fully branched. A summarizing formula is shown in Figure 8.
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The structure of the minimum binding epitope for H. pylori (Miller-Podraza et al., 1996
) is still unknown. It was made likely that the bacterial binding to human PGCs is based on a second sialic acid–dependent binding which is separate from the binding to NeuAc3Gal present in many glycoconjugates, since this later was not expressed after bacterial growth in broth, in contrast to the PGC binding (Miller-Podraza et al., 1996). The binding was restricted to human PGCs and dependent on sialic acid, since removal of sialic acid by mild acid or neuraminidase completely abolished the binding. The linkage is probably NeuAc3Gal (Johansson et al., 1999). A clue for the epitope may be the removal of binding after mild periodate oxidation, which only cleaves off C9, or C9 and C8, from the glycerol tail of NeuAc. A question may be how this small change may affect recognition of a probably rather complex epitope of PGCs. Molecular dynamics simulations based on the information from the present work (J.Ångström, unpublished observations) revealed a bisected hydrogen bond between C9 of NeuAc and the (13)- and the (16)-linked GlcNAcs of the branch (see Figure in Karlsson, 1998). However, the product after oxidation gave a very different dominating conformation. Therefore, the sequence 
may hypothetically provide the binding epitope for H.pylori, including part of NeuAc and branching characteristics.
This structure is probably not common to all sialic acid-containing PGCs in the mixture since digestion with endo-ß-galactosidase revealed components belonging to the series (x + 1,x,y,1)Cer observed in the MALDI mass spectrum (see Figure 3). These components have consecutive branching with the second branch at the third Gal and not at the fourth Gal as the structure above. However, as the component (4,3,0,1)Cer was dominating in the MALDI mass spectrum in negative mode and the internal oligosaccharide (3,3,0,1)i was present, some of the human PGC components in the series (x + 2,x,y,1)Cer may have the structure above as an internal part of the molecules (see for example the isomers of [10,8,y,1]Cer and [11,9,y,1]Cer in Figure 5).
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PGC material
Total PGC fraction of human erythrocyte membranes, blood group O, was prepared by the peracetylation method as described earlier (Miller-Podraza et al., 1993
). Endo-ß-galactosidase digestion was performed essentially according to Scudder et al. (1984). In a typical experiment the incubation mixture contained 100 µg of PGCs, 135 µl of water, 15 µl of 0.5 M acetate buffer (pH 5.8, containing 2 mg/ml bovine serum albumin and 1 mg/ml sodium taurodeoxycholate) and 30 µl of endo-ß-galactosidase (15 mU, Bacteroides fragilis, Boehringer Mannheim, GmbH, Germany). The incubation was performed overnight at 37°C. The mixture after digestion was desalted using Sephadex G-15 column (1 x 15 cm), which was packed and run in water. The sugar fraction (eluting before the salt fraction) was collected and used for MALDI-TOF MS analysis. For FAB-MS the undegraded PGCs were permethylated as described (Ciucanu and Kerek, 1984). Ion exchange separation of the endo-ß-galactosidase products into a neutral and an acidic fraction was done using a DEAE cellulose column (1 x 3 cm, acetate form, packed in 5% aqueous ethanol). After application of the desalted material the column was washed with 10 column volumes of 5% aqueous ethanol and 2 column volumes of 0.5 M CH3COONH4 in the same solvent, resulting in a neutral and an acidic fraction, respectively. The acidic fraction was desalted by freeze drying and Sephadex G-15 chromatography. The two fractions were permethylated and analyzed by direct inlet EI-MS. One portion of the total hydrolysis mixture was reduced in an excess of NaBH4 in water at room temperature over night and permethylated for high-temperature GC/MS.
MALDI-TOF MS
MALDI mass spectra were acquired on a TofSpec-E time-of-flight mass spectrometer (Micromass, Manchester, England) equipped with delayed extraction and a nitrogen laser (337 nm, 4 ns pulse, LSI, Boston, MA) operated in the reflectron mode at ±20 kV acceleration voltage. Matrix and calibrations used as described previously (Karlsson et al., 1999).
FAB-MS
FAB-MS was performed on a JEOL SX-102A spectrometer (JEOL, Tokyo, Japan) in positive ion mode and using 6 keV Xenon atoms. 1 µl of the sample dissolved in chloroform/methanol (1:1) was added to the matrix that consisted of a mixture of glycerol/3-nitrobenzyl alcohol (1:1). Mass spectrometric conditions: a) acceleration voltage +10 kV; mass range scanned, m/z 100–2400; linear magnet scan with scan time, 25 s; resolution, 1200 (m/
m, 10% valley definition); b) acceleration voltage +8 kV; mass range scanned, m/z 2000–3200; linear magnet scan with scan time, 10.5 s; resolution, 1200 (m/m, 10% valley definition).
EI-MS
High-temperature GC/MS was performed on a Hewlett-Packard 5890-II gas chromatograph coupled to a JEOL SX-102A spectrometer (JEOL, Tokyo, Japan). Other conditions were as described previously (Karlsson et al., 1994). Direct inlet EI-MS was performed on a JEOL SX-102A mass spectrometer (JEOL, Tokyo, Japan) using the in-beam technique (Breimer et al., 1980). The electron energy was 70 eV, the trap current 300 µA and the acceleration voltage +10 kV. The temperature in the ion source was programmed from 150°C to 410°C at a rate of 15°C/min.
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This work was supported by grants from the Swedish Medical Research Council (No. 3967, 10435 and 13395–01), the Swedish Research Council for Engineering Sciences, the Foundations of the National Board of Health and Welfare, the Swedish Foundation for Strategic Research, the Knut and Alice Wallenberg Foundation, and the IngaBritt and Arne Lundberg Foundation.
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; FAB-MS, fast atom bombardment mass spectrometry; EI-MS, electron ionization mass spectrometry; GC/MS, gas chromatography/mass spectrometry; PGCs, polyglycosylceramides; Hex, hexose; HexN, N-acetylhexosamine; Fuc, fucose; NeuAc, N-acetylneuraminic acid; Cer, ceramide where d18:1 means sphingosine (1,3-dihydroxy-2-amino-trans-4-octadecene) and 24:1 means monounsaturated tetracosanoic acid. The PGCs are described by the general formula Hexx+2HexNxFucyNeuAczCer and abbreviated according to the number of monosaccharides present as (x + 2,x,y,z)Cer.
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1 To whom correspondence should be addressed
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