Default biosynthesis pathway for blood group–related glycolipids in human small intestine as defined by structural identification of linear and branched glycosylceramides in a group O Le(a-b-) nonsecretor

Jonas Ångström2, Thomas Larsson3, Gunnar C. Hansson2, Karl-Anders Karlsson2 and Stephen Henry1,4

2 Institute of Medical Biochemistry and 3 Swegene Proteomics Center, Göteborg University, Box 440, SE 405 30, Sweden; and 4 Glycoscience Research Centre, Auckland University of Technology, Private Bag 92006, Auckland 1020, New Zealand, and Kiwi Ingenuity Limited, P.O. Box 39373, Howick, New Zealand

Received on March 4, 2003; revised on August 31, 2003; accepted on September 3, 2003


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Glycoconjugates of the GI tract are important for microbial interactions. The expression of histo-blood group glycosyltransferases governs both the expression of blood group determinants and in part the structure and size of the glycoconjugates. Using neutral glycolipids isolated from the small intestine of a rare blood group O Le(a-b-) ABH secretor-negative (nonsecretor) individual we were able to map the "default" pathway of the individual lacking ABO, Lewis, and secretor glycosyltransferases. Structures were deduced with combined analysis of mass spectrometry (MALDI-TOF and ESI-MS/MS), and 1H NMR (500 and 600 MHz). All structures present at a level >5% were structurally resolved and included two extended structures: Galß4(Fuc{alpha}3)GlcNAcß3(Galß4[Fuc{alpha}3]GlcNAcß6)Galß4GlcNAcß3Galß4Glcß1Cer and Galß3GlcNAcß3(Galß4[Fuc{alpha}3]GlcNAcß6)Galß3GlcNAcß3Galß4Glcß1Cer. The first, a novel component, is based on a type 2 chain and bears the Lex glycotopes on both its branches. The second, a major component, is based on a type 1 chain, which bears a 3-linked type 1 precursor (Lec) glycotope and a 6-linked Lex glycotope on its branches. This latter structure is identical to that previously isolated from plasma and characterized by MS and GC-MS but not by NMR. Structural resolution of these structures was supported by reanalysis of the blood group H–active decaosylceramides previously isolated from rat small intestine. Other minor linear monofucosylated penta-, hepta-, and difucosylated octaosylceramides, some bearing blood group determinants, were also identified. The cumulative data were used to define a default biosynthesis pathway where it can be seen that carbohydrate chain extension, in the absence of blood group glycosyltransferases, is controlled and regulated by non–blood group fucosylation and branching with type 2 Galß4GlcNAc branches.

Key words: ABO / glycosyltransferase / histo-blood group antigen / Lewis / secretor


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
An understanding of the biosynthesis and structural basis of glycosylation in the human gastrointestinal (GI) tract is essential to resolving the interactions these glycoconjugates may have with microorganisms and toward the biological pressures that have evolved the blood group polymorphisms. Previously several studies have immunochemically, immunohistologically, and partially structurally mapped the GI tract with respect to blood group related glycoconjugate expression (Szulman and Marcus, 1973Go; Falk et al., 1979Go; Breimer et al., 1983Go; Breimer 1984Go; Bjork et al., 1987Go; Holgersson et al., 1988Go; Finne et al., 1989Go; Henry et al., 1994Go, 1997Go).

These studies clearly established the complex interactions of the secretor, Lewis, and A/B glycosyltransferases in determining the glycoconjugate profile of individuals. In particular they established that the amount and type of histo-blood group–related glycotopes was highly dependent on the relative expression of the specific glycosyltransferases. Of note was the observation that absence of some glycosyltransferase combinations resulted in increased quantities of elongated/branched glycolipids (Henry et al., 1994Go, 1997Go). Although these studies established the presence of extended molecules, at the time they were unable to be fully structurally resolved mainly due to limitations of the technology and the relatively low quantity of sample available.

The blood group systems that affect blood group expression in the human GI tract include ABO, Lewis (FUT3), and secretor (FUT2) (as reviewed in Oriol, 1995Go). The various combinations of these different glycosyltransferases determine the range of glycoconjugate profiles expressed (Oriol, 1995Go). The rarest profile (<1% of Caucasians) belongs to the group O Le(a-b-) nonsecretor who lacks functional glycosyltransferases for the ABO, Lewis, and secretor systems and thus represents a "null" blood group phenotype in the GI tract.

In the healthy GI tract, as with all other tissues of epithelial origin, type 1 (Galß3GlcNAc-R)–based blood group glycotopes dominate. There is significant data reporting the presence of type 1–based glycolipids with short chains bearing blood group glycotopes, in particular; type 1 precursor (Lec), type 1 H (Led), Lea, Leb, type 1 A, type 1 B, ALeb, and BLeb. In contrast, data on type 1–based extended glycosphingolipids of intestinal origin is scarce. To date only six type 1–based branched glycosphingolipid structures, all from rat small intestine, have been characterized by 1H nuclear magnetic resonance (NMR) (Bouhours et al., 1992Go; Breimer et al., 1982Go; Hansson, 1983Go).

In the absence of definitive NMR data of extended intestinal blood group–related glycolipids, reanalysis of the previously determined rat structures with type 1 inner core structures was necessary for unambiguous structural resolution. With the exception of the fucose residues, the two rat blood group H–active decaglycosylceramides (Breimer et al., 1982Go) most closely resembled the potential branched structures present in our human sample. They were thus reanalyzed in identical solvents and temperatures for comparative purposes.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Rat intestinal H-10 sample: NMR structural analysis
The lack of dimethyl sulfoxide (DMSO)/D2O NMR data for branched glycolipids, especially with regard to type 1 core structures, prompted us to reanalyze the rat H-10 sample. This was previously structurally characterized as Fuc{alpha}2Galß3GlcNAcß3(Fuc{alpha}2Galß3/4GlcNAcß6)Galß3GlcNAcß3Galß4Glcß1Cer with a 60/40 type 1/type 2 mixture on the 6-linked branch (Breimer et al., 1982Go). The 1D NMR and corresponding 2D correlation spectroscopy (COSY) spectra were determined (not shown), and the relevant chemical shifts summarized in Table I. All interpretations were consistent with the literature (Dabrowski et al., 1981Go; Breimer et al., 1982Go; Hanfland et al., 1984Go; Clausen et al., 1985Go; Levery et al., 1986Go, 1988Go; Holmes and Levery, 1989Go).


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Table I. Chemical shifts (30°C) of saccharide proton resonances of two branched blood group H decaglycosylceramides isolated from rat small intestine (1 and 2) and two branched deca- and nonaglycosylceramides carrying Lex epitopes isolated from human small intestine of a blood group O Le(a-b-) nonsecretor individual (3 and 4)

 
Human Le(a-b-) nonsecretor intestinal samples
Fractionation and samples E24-5 and E24-6
The open column fractionation resulted in two fractions suitable for glycolipid analysis and representative of the total nonacid glycolipids of a group O Le(a-b-) nonsecretor individual. Fraction E24-5 contained the minor glycolipids (those with eight or fewer sugars), whereas sample E24-6 contained predominantly larger glycolipids as well as small amounts of the glycolipids present in sample E24-5.

Structural analysis of sample E24-5
Mass spectrometry (MS) analysis indicated that fraction E24-5 contained glycolipid components having four to eight sugars (results not shown), and exhibiting a profile similar to the lower m/z components (i.e., m/z 1100–2000) shown in Figure 1 for the later eluting fraction E24-6. These short glycolipids are also identified completely or inferred from partial assignments of the anomeric region in the NMR spectrum. The dominating structure in E24-5 is represented by Lec-4 (Table II, 1), which is estimated to be approximately 75% of the E24-5 sample. Also clearly evident are Fuc{alpha}3 and Fuc{alpha}4 signals, representing Lex and Lea glycotopes, respectively. The structures carrying these glycotopes are mainly present as Lea-5 and Lex-5 (Table II, 5 and 6 respectively). However, the presence of a small amount of Lex-7 (internal glycotope; Levery et al., 1986Go), repetitive Lex-8 (Hakomori et al., 1984Go) and repetitive Lea-8 (Stroud et al., 1991Go) may be inferred from the shifts of the Galß3/4 residues of the internally located glycotopes. Additionally, a small amount of the type 1 H-5 glycolipid (Table II, 3) is seen, as well as a barely visible Fuc{alpha}2 signal attributable to Leb-6 (Blaszczyk-Thurin et al., 1987Go). The presence of Lea-5 and Leb-6 is compatible with earlier immunochemical findings of this Le(a-b-) nonsecretor individual (Henry et al., 1994Go, 1997Go).



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Fig. 1. MALDI-TOF spectrum of sample E24-6. The m/z ranges, due to ceramide differences, attributable to the various structures (insets) are shown with their peaks numbered and further defined in Table III.

 

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Table II. Chemical shifts at 30°C of anomeric saccharide proton resonances of reference compounds relevant to the present work

 

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Table III. Tentative structures assigned to MALDI m/z peaks (and major peaks confirmed by ESI-MS/MS)

 
MS analysis of sample E24-6
Tetra-pentaosylceramides
Sample E24-6 was analyzed by matrix-assisted laser desorption/ionization (MALDI) (Figure 1, Table III). Evidence for expected sodiated tetrapentaosylceramides with a range of ceramides was obtained over the range of m/z 1266–1542. The structures that the major peaks represent have all been resolved by electrospray ionization (ESI) tandem MS (MS/MS) and/or NMR (results not shown) and were predominant in sample E24-5 (as before).

Heptaosylceramides
MALDI m/z peaks correlating with monofucosylated heptaosylceramides were found over the range of m/z 1777 to m/z 1907, representing a variety of ceramides (Figure 1, Table III). Major ions were analyzed by ESI-MS/MS. The collision-induced dissociation (CID) spectrum of the m/z 900.0 doubly charged molecular sodium adduct [M + 2Na]2+ is shown in Figure 2. The spectrum correlates with two different Hex4HexNAc2Fuc1 structures, both having d18:1,h16:0 ceramide tails.



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Fig. 2. ESI-MS/MS spectrum of two monofucosylated heptaosylceramides correlating with Hex4HexNAc2Fuc1 structures (inset schematic drawings) having d18:1,h16:0 ceramide tails present in sample E24-6. CID spectrum of the m/z 900.0 doubly charged molecular sodium adduct [M + 2Na]2+ correlates with these structures, as shown in the schematic theoretical fragmentation drawings. Monoisotopic peaks are annotated.

 
This spectrum shows expected B- and Y-ions as indicated in the inset schematic diagrams (Figure 2). Because the m/z 900.0 [M + 2Na]2+ parent ion represents two different structures, one subterminally fucosylated and the other internally fucosylated, they share some features but are distinct for others. Distinguishing B-ions, corresponding with the expected saccharide fragments for structure I, were found at m/z 534.2 (I:B3) and m/z 696.2 (I:B4). B-ions found at m/z 388.1 and m/z 550.2, although attributable to II:B2 and II:B3, respectively, could also arise from structure I, which had lost fucose (I:B3-F and II:B4-F). Distinguishing Y-ions, corresponding with the expected saccharide-ceramide fragments for structure II, were found at 1249.8 (II:Y4) and m/z 1411.8 (II:Y5). Y-ions found at m/z 1103.7 and m/z 1265.8, although attributable to I:Y3 and I:Y4, respectively, could also arise from structure II (II:Y4-F and II:Y5-F), which had lost fucose. Overall the ESI-MS/MS fragmentation patterns were strongly supportive of the Hex4HexNAc2Fuc1 structures shown in the insets of Figure 2.

Octaosylceramides
MALDI m/z peaks, suggestive of a difucosylated octaosylceramide with a d18:1,h16:0 ceramide, were found at m/z 1923.3 (Figure 1, Table III). Other evidence for this structure with different ceramides was inconclusive. This structure was analyzed by ESI-MS/MS (Figure 3). The CID spectrum of the m/z 973.0 doubly charged molecular sodium adduct [M + 2Na]2+ correlates with a Hex4HexNAc2Fuc2 structure having a d18:1,h16:0 ceramide tail. This spectrum shows expected B- and Y-ions as indicated in the inset schematic diagram (Figure 3). CID of the m/z 973.0 [M + 2Na]2+ parent ion produced peaks at m/z 1777.0 [M - F]+, m/z 1630.9 [M - 2F]+, m/z 900.0 [M - F]2+, and m/z 826.9 [M - 2F]2+, showing the presence of two fucoses. Overall the ESI-MS/MS fragmentation patterns were strongly supportive of the Hex4HexNAc2Fuc2 structure shown in the inset of Figure 3.



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Fig. 3. ESI-MS/MS spectrum of a difucosylated octaosylceramide correlating with a Hex4HexNAc2Fuc2 structure (inset schematic drawing) having d18:1,h16:0 ceramide tail present in sample E24-6. CID spectrum of the m/z 973.0 doubly charged molecular sodium adduct [M + 2Na]2+ correlates with this structure, as shown in the schematic theoretical fragmentation drawing. Monoisotopic peaks are annotated.

 
Nonaosylceramides
MALDI m/z peaks correlating with monofucosylated nonaglycosylceramides were found over the range of m/z 2142 to m/z 2272, representing a variety of ceramides (Figure 1, Table III). Major ions were analyzed by ESI-MS/MS. The CID spectrum of the m/z 1082.6 doubly charged molecular sodium adduct [M + 2Na]2+ is shown in Figure 4. The spectrum correlates with a Hex5HexNAc3Fuc1 structure having a d18:1,h16:0 ceramide tail and shows expected B- and Y-ions as indicated in the inset schematic diagram (Figure 4). CID of the m/z 1082.6 [M + 2Na]2+ parent ion produced peaks at m/z 1996.1 [M - F]+ and m/z 1009.5 [M - F]2+ (and absence of [M-2F] singly or doubly charged ions), showing the presence of a single fucose. Overall the ESI-MS/MS fragmentation patterns were strongly supportive of the Hex5HexNAc3Fuc1 structure shown in the inset of Figure 4.



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Fig. 4. ESI-MS/MS spectrum of a branched monofucosylated nonaosylceramide correlating with a Hex5HexNAc3Fuc1 structure (inset schematic drawing) having d18:1,h16:0 ceramide tail present in sample E24-6. CID spectrum of the m/z 1082.6 doubly charged molecular sodium adduct [M + 2Na]2+ correlates with this structure, as shown in the schematic theoretical fragmentation drawing. Monoisotopic peaks are annotated.

 
Decaosylceramides
MALDI m/z peaks correlating with a difucosylated decaosylceramide were found over the range of m/z 2272 to m/z 2418 and are representative of a variety of ceramides (Figure 1, Table III). Major ions were analyzed by ESI-MS/MS. CID spectrum of the m/z 1155.6 doubly charged molecular sodium adduct [M + 2Na]2+ is shown (Figure 5). The spectrum correlates with a Hex5HexNAc3Fuc2 structure having a d18:1,h16:0 ceramide tail and shows expected B- and Y-ions as indicated in the inset schematic diagram (Figure 5). CID of the m/z 1155.6 [M + 2Na]2+ parent ion produced peaks at m/z 2142.1 [M - F]+, m/z 1996.0 [M - 2F]+, m/z 1082.5 [M - F]2+, and m/z 1009.5 [M - F]2+, showing the presence of two fucoses. Overall the ESI-MS/MS fragmentation patterns were strongly supportive of the branched Hex5HexNAc3Fuc2 structure shown in the inset of Figure 5.



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Fig. 5. ESI-MS/MS spectrum of a branched difucosylated decaosylceramide correlating with a Hex5HexNAc3Fuc2 structure (inset schematic drawing) having d18:1,h16:0 ceramide tail present in sample E24-6. CID spectrum of the m/z 1155.6 doubly charged molecular sodium adduct [M + 2Na]2+ correlates with this structure as shown in the schematic theoretical fragmentation drawing. Monoisotopic peaks are annotated.

 
Proton NMR analysis of sample E24-6
MS of fraction E24-6 revealed the presence of two main components: a dominating nine-sugar glycolipid having a Hex5HexNAc3Fuc1 composition and a ten-sugar glycolipid having a Hex5HexNAc3Fuc2 composition as described. The NMR spectrum of the anomeric region shown in Figure 6 reveals that only these two components significantly contribute to the spectral features. The minor species (decaosylceramide) is estimated to be approximately 30% of the major species (nonaosylceramide) as judged by the relative intensity of the Fuc{alpha} signals seen at 4.82 ppm and 4.84 ppm.



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Fig. 6. Anomeric region of the 1D proton NMR spectrum of the human E24-6 sample. This spectrum was acquired at 500 MHz and 30°C with the sample dissolved in DMSO/D2O (98/2, by volume). Two fucosylated glycosylceramides are identified. Molecule 3, a difucosylated decaosylceramide based on a type 2 core, bears Lex glycotopes on both its branches. Molecule 4, the dominating structure, is a monofucosylated nonaosylceramide based on a type 1 core and bears unsubstituted Lec on its 3-linked branch and Lex on its 6-linked type 2 branch. The different structures are labeled with Arabic numerals, whereas the corresponding sugar residues are labeled with Roman numerals as seen between the structural formulae (inset). Resonances are recorded as molecules 3 and 4 in Table I, and a 2D COSY spectrum of this sample can be seen in Figure 7.

 


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Fig. 7. COSY spectrum of the human E24-6 sample. The spectrum was acquired at 600 MHz and 30°C with the sample dissolved in DMSO/D2O (98/2, by volume). Only H1/H2 cross-peaks have been labeled using the same numbering scheme as in Figure 6.

 
It can be concluded that both of these compounds are branched structures having identical 6-linked branches, because only a single ß-anomeric resonance at 4.50 ppm ascribable to GlcNAcß6 is present. The 0.09 ppm downfield shift of this resonance, relative to an unsubstituted lactosamine branch (Levery et al., 1988Go), indicates attachment of an {alpha}-fucose on this residue in the 3-position, resulting in a Lex glycotope (Galß4[Fuc{alpha}3]GlcNAcß6). In fact, the chemical shifts of the Fuc{alpha}3 signal at 4.82 ppm and the GlcNAcß6 signal at 4.50 ppm are in perfect agreement with the shifts observed for a Lex-substituted globopentaosylceramide structure isolated from mouse kidney (Lanne et al., 1995Go), as listed in Table II (molecule 7). Focusing on the minor component, it can also be surmised that its 3-linked branch must consist of a Lex glycotope, as judged by the Fuc{alpha}3 signal at 4.84 ppm and the GlcNAcß3 signal at 4.69 ppm. This coincides with the corresponding values observed for Lex-5 (Table II, 6). Furthermore, from the connectivity of the overlapping Fuc{alpha}3 H6 methyl resonances at 1.00 ppm, the likewise overlapping H5 signals could be located at 4.66 ppm. The 3-linked branch of the major component is also easily identifiable as Galß3GlcNAcß3 from a comparison with the corresponding segment of lactotetraosylceramide (Table II, 1), where the terminal Galß3 is found at 4.13 ppm and the penultimate GlcNAcß3 at 4.79 ppm (Holmes and Levery, 1989Go). For both components, the H1 protons of the Galß4 residue of the Lex branches are assigned to overlapping resonances at 4.28 ppm, since the corresponding H2 resonances are found at 3.28 ppm values typical of terminal galactose in Lex glycotopes (Levery et al., 1986Go).

Anomeric configurations remaining to be established are those of the two different core tetrasaccharides, that is, whether they belong to the lacto (type 1) or neolacto series (type 2). Starting with the minor component (decaosylceramide), the doublet of the core GlcNAcß3 is poorly visible in the 1D spectrum and is only seen as a shoulder on the high-field side of the Fuc{alpha}3 H5 resonance at 4.66 ppm. However, in the COSY spectrum (Figure 7, 3:III) the positions of the H1 and H2 resonances at 4.64 ppm and 3.35 ppm, respectively, are clearly revealed and identifies this residue as a type 2 GlcNAcß3. Both the H1 and H2 resonances are shifted somewhat to a higher field due to the influence of the Fuc{alpha}3 residues of the Lex branches. The H1 resonance of the core GlcNAcß3 of the major component (nonaosylceramide), is found at 4.76 ppm (Table I, 4:III), a value that is consistent with a type 1 GlcNAcß3 residue equivalent to the rat H-10 type 1 structures described (Table I, 1:III and 4:III). This is also corroborated later by the identification of a core Galß3 residue.

The H1 resonance originating from the branching Galß4 of the minor component (decaosylceramide) should be expected around 4.29 ppm, and the Galß4 residue closest to the glucose of either component is expected in the neighborhood of 4.26 ppm (e.g., Hanfland et al., 1984Go). From the COSY spectrum (Figure 7, 3:IV), H1 and H2 resonances at 4.27 ppm and 3.43 ppm (Table I, 3:IV) respectively, are found. These values correspond closely to those expected except for the H1 resonance of the branching Galß4, which is somewhat shifted to a higher field due to the Fuc{alpha}3 residues of the Lex branches. Two of the three remaining H1 resonances at 4.20 ppm (H2 at 3.02 ppm) and 4.21 ppm (H2 at 3.00 ppm) are assigned to Glcß1 bound to different ceramides (Levery et al., 1986Go), whereas the third H1 resonance, also at 4.21 ppm, in analogy with rat H-10 is assigned to the branching Galß3 of the major component (nonaosylceramide) because its H2 resonance was found at 3.41 ppm.

To summarize, the following two structures can be deduced from the combined analysis of the MS and NMR data presented: Galß4(Fuc{alpha}3)GlcNAcß3(Galß4[Fuc{alpha}3]GlcNAcß6)Galß4GlcNAcß3Galß4Glcß1Cer and Galß3GlcNAcß3(Galß4[Fuc{alpha}3]GlcNAcß6)Galß3GlcNAcß3Galß4Glcß1Cer.

The decaosylceramide represents a novel structure, whereas the nonaosylceramide is identical to the structure isolated from blood group O Le(a-b-) plasma and characterized by MS and gas chromatography–MS but not by NMR (Hanfland et al., 1986Go).

As can be seen in the 1D spectrum of E24-6 (Figure 6) there are also three very minor contributions (2–3% each) stemming from compound(s) having a type 1 H determinant located on a 3-linked branch (Fuc{alpha}2 H1 resonance at 4.98 ppm) and/or a 6-linked branch (Fuc{alpha}2 H1 resonance at 4.96 ppm). The presence of a Galß3 H1 resonance at 4.43 ppm, typical of type 1 H determinants, is also evident (see rat H-10 structures in Table I). The identity of the third component seen at 4.92 ppm is unclear.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The presence, absence, and combinations of the various polymorphic histo-blood group glycosyltransferases significantly affect glycosylation of the human GI tract (Oriol, 1995Go). The possible combinations of the common alleles of the ABO, secretor, and Lewis systems result in 14 markedly different glycoconjugate profiles (Henry, 2001Go). These range from individuals expressing all the glycosyltransferases (group AB, Lewis-positive, secretor-positive) through to those individuals lacking all the glycosyltransferases (group O, Lewis-negative, secretor-negative). This rare latter individual, lacking the ABO, Lewis, and secretor glycosyltransferases and the subject of this article, is important in the study of glycoconjugate biosynthesis as it defines the "default" pathway.

Published data on the structures present in Lewis-negative, secretor-negative individuals is rare. Two previous reports, one studying pooled plasma (Hanfland et al., 1986Go) and the other small intestine (Henry et al., 1997Go), have concluded that lactotetraosylceramide is the dominating blood group–related glycolipid together with fucosylated extended branched structures. Other fucosylated structures are also present but at relatively low levels. Using more advanced techniques, we were able to revisit the structural analysis of small intestinal glycolipids from a group O Le(a-b-) nonsecretor individual (Henry et al., 1997Go). In addition to the human intestinal glycolipids, type 1 inner-core branched glycolipids previously isolated from rat small intestine were reanalyzed. Although the rat structures were fucosylated, their NMR resonances were important to establish unambiguously the nature of the inner core structure of the human sample. Although neither of the rat molecules were present or expected in this human group O Le(a-b-) secretor negative sample, similar or identical structures could be predicted in group O Le(a-b-) secretor human intestine.

In the human sample we were able to show the presence of two extended branched structures, one a decaosylceramide bearing Lex on both branches and based on a type 2 internal chain. The other was a nonaosylceramide representing an extension of the dominating type 1 precursor (Lec-4) with a further unsubstituted 3-linked type 1 unit (Galß3GlcNAc) and a 6-linked fucosylated type 2 branch in the form of Lex. This molecule is identical to that previously isolated from pooled plasma of individuals of the same phenotype, although not unambiguously structurally resolved at that time (Hanfland et al., 1986Go).

Again and as previously noted, all the dominant structures larger than lactotetraosylceramide were fucosylated. Although some trace immunochemical evidence exists for Lea, type 1 H and Leb fucosylation (Henry et al., 1994Go; 1997Go), almost all fucosylation observed was on type 2 chains – and almost all type 2 chains were fucosylated. The virtual absence of type 1 fucosylation, as evidenced by lactotetraosylceramide and unsubstituted type 1 branches, clearly shows that the fucosyltransferase activity present during biosynthesis is restricted to type 2 chains. It would be expected that if these type 1 branches exist in Lewis/secretor positive individuals they would be appropriately fucosylated into H, Lea and Leb glycotopes.

It was clear from the studies on this sample that two types of glycolipid dominate; one being lactotetraosylceramide and the other extended structures. Subject to consideration and reservations about variables in the isolation procedures, which may be selective, we estimate that the relative ratios of lactotetraosylceramide, nonaosylceramide, decaosylceramide, and all other remaining glycolipids with more than four saccharides is approximately 50:30:10:10 by weight. It would be expected that losses that occur in the isolation procedure will predominantly affect the extended structures (Henry et al., 1997Go), and therefore they may be underestimated. Using polyglycosylceramide isolation procedures (Miller-Podraza et al., 1997Go), there is no evidence that polyglycosylceramides are present in human small intestine, as they are in red cell membranes (Miller-Podraza, personal communication). Furthermore, it should be noted that this analysis relates to glycolipids; glycoprotein glycosylation was not studied and may show significant variance to glycolipids.

Using structural data generated from glycolipids isolated from this rare group O Le(a-b-) nonsecretor phenotype, we are now able to speculate on the biosynthesis of glycolipids in the human GI tract in the absence of ABO, Lewis, and secretor glycosyltransferases (Figure 8). Essentially two pathways operate, one for the type 1–based molecules (Galß3GlcNAc-R) and the other for the type 2–based (Galß4GlcNAc-R) molecules. In the type 2 pathway, ceramide dihexose (Galß4Glcß-cer) is converted into neolactotetraosylceramide, with a small amount converted into a range of linear structures bearing Lex internal and terminal glycotopes. However, most of the neolactotetraosylceramide appears to be then elongated with a type 2 (Galß4GlcNAc) unit, then branched, and both branches fucosylated. Although the order of fucosylation of the 3-linked branch is not known for certain (i.e., before or after branching), it is expected to occur after branching, as it is known that terminal and internal fucosylation, at least in rats, prevents branch formation (Leppänen et al., 1997Go).



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Fig. 8. Proposed biosynthetic "default" pathway for the formation of blood group–related glycolipids in the small intestine of an adult group O, Lewis-negative, nonsecretor. The structures inside boxes are dominating (combined contribution estimated >90% by weight). All other structures are present only in low concentrations. Some molecules that are present at levels estimated at less than 2% are not included. The order of fucosylation on the linear molecules is speculative and remains to be proven by enzymatic studies.

 
In contrast to the type 2 pathway, branching in the type 1 pathway (in humans) is different in that the branched structure has mixed branches; the 6-linked branch was predominantly a type 2 unit and fucosylated, while the 3-linked branch was type 1 and almost always unfucosylated. This is in distinct contrast to the rat small intestine where the 6 linked branch could be either type 1 or type 2 (Breimer et al., 1982Go). However there was a small contribution at 4.96 p.p.m. (Fuc{alpha}2) and possibly 4.43 p.p.m. (Galß3) (2–3%) of the NMR spectrum (Figure 6) which indicates the presence of some 6-linked type 1 H branches in humans.

Essentially in the type 1 pathway, ceramide dihexose (Galß4Glcß1Cer) is converted into lactotetraosylceramide, and again a small amount is converted into a range of linear structures, this time bearing Lea internal and terminal glycotopes. However, in contrast to the type 2 pathway, most of the tetraosylceramide appears to remain unmodified. All the same, a significant amount is elongated with a type 1 (Galß3GlcNAc) unit into a lactohexaosylceramide (Henry et al., 1997Go). There was no evidence for 3-linked type 2 extension of the inner core, which is in agreement with the extended molecules found in the rat. After extension into type 1 lactohexaosylceramide, the molecule is then branched (6-linked) with a type 2 unit, and this type 2 branch is always fucosylated. This fucosylation appears to be complete, as there was no evidence for unsubstituted 6-linked type 2 branches. For the extended linear structures, we have suggested that the pathway involves chain extension of Lea or Lex, rather than extension and then fucosylation. This is speculative and remains to be proven by enzymatic studies.

No core structures larger than the molecules reported here have been found in Lewis-positive human small intestine (unpublished data). Only substitutions of these cores by secretor, Lewis, and ABO glycosyltransferases to form a limited range of structures have been found. Therefore, unlike red cells, in which chain extension and branching dominate, glycolipids of the small intestine are restricted in size and structure. This process of extension appears to be restricted by both branching and fucosylation. The reason for this restriction in chain extension is unknown, but as each different glycoconjugate may represent a potential receptor to which microorganisms can specifically attach (Karlsson, 1986Go; Hooper and Gordon, 2001Go), it is possible this limited range has developed as a result of biological tension. Only further understanding of the biosynthetic pathway, resulting in the formation of the various blood group glycoconjugates, will lead us to an understanding of their biological significance and the evolutionary pressures that drive carbohydrate blood group polymorphism.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Glycolipid samples
Two samples with extended intestinal glycolipids were obtained from previous studies. Sample H-10 was obtained from rat small intestine (Breimer et al., 1982Go), and human extended glycolipids were reisolated from a group O Le(a-b-) small intestinal sample (Henry et al., 1997Go). Glycolipids were prepared as described by Karlsson (1987)Go but also using an additional solvent of chloroform:methanol:water (40:40:12 by volume) to ensure recovery of the extended glycolipids from the silica columns (Henry et al., 1997Go). However, initial MALDI-time-of-flight (TOF) MS indicated incomplete O-deacetylation of internal HexNAcs by the original methanolysis method. Consequently all samples were redeacetylated by a more aggressive approach, which included the addition of water to allow for hydrolysis because this had proved successful for the preparation of polyglycosylceramides (Miller-Podraza et al., 1997Go).

The new deacetylation step uses a methanol:water: chloroform mix (65:35:16 by volume) containing 0.2 M KOH, (100 ml/g lipid) instead of simply 0.2 M KOH in methanol, and is allowed to proceed overnight instead of 3 h as previously described (Karlsson, 1987Go). The reactants are then dialysed as a two-phase system against water. This new step successfully removed almost all unwanted O-acetylated residues (as determined by MS) of native glycolipids. Overall and with respect to the changes indicated, the entire glycolipid isolation procedure involved solvent extraction of total lipids with methanol and chloroform:methanol (2:1 by volume) at 70°C; mild alkaline degradation with KOH followed by dialysis to remove lipids with alkali labile ester linkages; silica chromatography fractionation with chloroform:methanol (99:1 by volume) to remove unwanted nonpolar lipids and chloroform:methanol (1:3 by volume), methanol and chloroform:methanol:water (40:40:12 by volume) to recover a crude glycolipid containing sample. This sample was then loaded onto a DEAE cellulose ion exchange column and unbound material including the nonacid glycolipids were removed with chloroform:methanol (2:1 by volume) and methanol.

This crude total nonacid lipid sample was acetylated and fractionated on an open silica column with the acetylated glycolipids recovered with the solvent chloroform:methanol (95:5 by volume). The acetylated glycolipids were deacetylated according to the original method but later redeacetylated by the method described here. The sample was then subjected to a DEAE cellulose ion-exchange column and a further silica column as described. The resultant total neutral glycolipids were then fractionated in an open silica column using the solvent system of chloroform:methanol: water (60:35:12 by volume) to recover the glycolipids as previously described (Henry et al., 1997Go). Two fractions for structural analysis were obtained; E24-5 containing predominantly shorter structures, and E24-6 containing extended structures.

MS
MALDI spectra were obtained on a TofSpec E (Micromass, Manchester, U.K.) equipped with a time lag focusing unit. All spectra were recorded in the reflectron mode, with an accelerating voltage of 20 kV. Half a microliter of matrix solution, 2,5-dihydroxybenzoic acid, 20 mg/ml (Aldrich, St. Louis, MO) dissolved in acetone, was deposited on the target and allowed to dry. One microliter of the sample solution was added to the matrix spot and the redissolved matrix–sample mixture was then allowed to dry in air. An external calibration sample (ethylated polyfructanes) was loaded in parallel. The monoisotopic masses obtained (sodium adducts) were compared to theoretical glycosphingolipid masses using an in-house program.

ESI-MS/MS data were acquired on a Q-Tof (Micromass) hybrid quadrupole time-of-flight instrument using the nanospray source. Samples were dissolved in methanol containing 1% H2O. Doubly charged ions (sodium adducts) were chosen and subjected to MS/MS using argon as the collision gas and with the collision energy varied between 30–80 V.

Spectra have been annotated with masses that correspond to monoisotopic peaks. Diagnostic fragments arise from fragmentation of the doubly charged parent ions. In this article we tried to make interpretation and annotation of the spectra as simple as possible for the nonspecialist to read. Because ESI-MS produces a lot of secondary ions by loss of branches, the nomenclature gets very complicated, especially with respect to annotation of figures. Consequently we use the Domon and Costello (1988)Go nomenclature only to indicate from which "end" of the molecule the primary ions originate.

The B series of ions, B-ions, represent the terminal saccharide fragments (less one hydrogen). The Y series correspond with those representing the ceramide containing fragment (plus one hydrogen). The ions are assigned a name according to the number of saccharides it contains, for instance, B3 are the three terminal saccharides. If a further bond is broken, for example loss of fucose, it is called B3-F, indicating the terminal trisaccharide which has lost fucose. Both the B and Y series also produce ions caused by the further loss of fucose. Singly and doubly charged molecular ions with one or two sodium adducts are also seen, [M + Na]+ and [M + 2Na]2+, respectively. Also, fragment ions can carry two charges and the state is designated 2+, for example, [M - F]2+. If not designated, it is singly charged. Other fragmentation ions (other than B and Y series) were often also present but have not been annotated or commented on.

Proton NMR spectroscopy
1H NMR spectra were acquired on Varian 500 and 600 MHz spectrometers at 30°C. Samples were dissolved in DMSO/D2O (98/2, by volume) after deuterium exchange. Two-dimensional double quantum-filtered COSY spectra were recorded by the standard pulse sequence (Marion and Wüthrich, 1983Go) using 32 scans per t1 increment in 2K* 256 point matrices.


    Acknowledgements
 
Professor Bo Samuelsson is gratefully acknowledged for support and technical advice. Use of the Varian 500 and 600 MHz spectrometers at the Swedish NMR Centre, Hasselblad Laboratory, Göteborg University, is gratefully acknowledged. This research was supported by grants from the Auckland University of Technology (grants 97/17 and 99/54), Auckland Medical Research Foundation (grant 81315), Kiwi Ingenuity Limited, the Swedish Research Council (grant 12628), the Lundberg Foundation, and the Wallenberg Foundation.


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: kiwi{at}aut.ac.nz Back


    Abbreviations
 
CID, collision-induced-dissociation; COSY, correlation spectroscopy; DMSO, dimethyl sulfoxide; ESI, electrospray ionization (MS-1 a quadrupole mass filter and MS-2 time-of-flight); GI, gastrointestinal; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NMR, nuclear magnetic resonance


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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