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Glycobiology Pages 533-545  

Rapid and sensitive GC/MS characterization of glycolipid released Gal[alpha]1,3Gal-terminated oligosaccharides from small organ specimens of a single pig
Introduction
Results
Discussion
Material and methods
Acknowledgments
Abbreviations
References

Rapid and sensitive GC/MS characterization of glycolipid released Gal[alpha]1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Rapid and sensitive GC/MS characterization of glycolipid released Gal[alpha]1,3Gal-terminated oligosaccharides from small organ specimens of a single pig

Annika E.Bäcker3, Jan Holgersson1, Bo E.Samuelsson, Hasse Karlsson2

Institute for Laboratory Medicine, Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg University, SE413 45 Göteborg, Sweden and 1Division of Clinical Immunology, Karolinska Institute, Huddinge University Hospital, SE141 86 Huddinge, Sweden, and 2Department of Medical Biochemistry, Göteborg University, Medicinaregatan 9A, SE413 90 Göteborg, Sweden

Received on September 2, 1997; revised on December 12, 1997; accepted on January 15, 1998

Pig to human xenotransplantation is considered a possible solution to the prevailing chronic lack of human donor organs for allotransplantation. The Gal[alpha]1,3Gal determinant is the major porcine xenogeneic epitope causing hyperacute rejection following human antibody binding and complement activation. In order to characterize the tissue distribution of Gal[alpha]1,3Gal-containing and blood group-type glycosphingolipids in pig, acid and nonacid glycosphingolipids were isolated from the kidney, small intestine, spleen, salivary gland, liver, and heart of a single pig obtained from a semi-inbred strain homozygous at the SLA locus. Glycolipids were analyzed by thin-layer immunostaining using monoclonal antibodies, and following ceramide glycanase cleavage as permethylated oligosaccharides by gas chromatography, gas chromatography-mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry. The kidney contained large amounts of Gal[alpha]1,3Gal-containing penta- and hexasaccharides having carbohydrate sequences consistent with the Gal[alpha]1,3nLc4 and Gal[alpha]1,3Lex structures, respectively. The former structure was tentatively identified in all organs by GC/MS. The presence of extended Gal[alpha]1,3Gal-terminated structures in the kidney and heart was suggested by antibody binding, and GC/MS indicated the presence of a Gal[alpha]1,3nLc6 structure in the heart. The kidney, spleen, and heart contained blood group H pentaglycosylceramides based on type 1 (H-5-1) and type 2 (H-5-2) chains, and H hexaglycosylceramides based on the type 4 chain (H-6-4). In the intestine H-5-1 and H-6-4 were expressed, in the salivary gland H-5-1 and H-5-2, whereas only the H-5-1 structure was identified in the liver. Blood group A structures were identified in the salivary gland and the heart by antibody binding and GC/MS, indicating an organ-specific expression of blood group AH antigens in the pig.

Key words: oligosaccharides/glycosphingolipids/gas chromatography-mass spectrometry/matrix-assisted laser desorption/ionization/pig/xenotransplantation

Introduction

Human allotransplantation is nowadays a generally accepted treatment of choice for several illnesses. The major hindrance toward widened indications for organ transplantation as the preferred treatment, is the chronic lack of donor organs. Xenotransplantation, i.e., transplantation of tissue between different species, is considered one promising possible solution to this problem. The pig is looked upon as the most suitable donor species for practical, ethical, and economical reasons. The human blood group system ABO (based on carbohydrate epitopes) is closely related to the pig blood group system AO. The main problem in xenografting between discordant species (i.e., pig to human) is the hyperacute rejection (HAR) which leads to cessation of the blood flow within minutes following transplantation (Shons and Najarian, 1974, 1975). Even though other mechanisms of rejection will ensue after HAR (e.g., delayed xenorejection, DXR), the general belief is that these may be controlled by available immunosuppressive drugs (Bach et al., 1996).

HAR is considered to be caused by preformed, natural antibodies in the recipient species reacting with antigens exposed on the endothelium in donor organ vessels, an interaction which leads to complement and endothelial cell activation, platelet aggregation (Bach et al., 1994; Samuelsson et al., 1994), extravasation of white blood cells (Lasky, 1992), a perturbed endothelial layer, and eventually rejection. The xenogenic pig antigens reacting with human naturally occurring antibodies have turned out to be carbohydrates, the major one being the Gal[alpha]1,3Gal epitope (Galili, 1994). The Gal[alpha]1,3Gal epitope is not expressed in Old World monkeys and humans due to an inactivation of the gene encoding the [alpha]1,3 galactosyltranferase ([alpha]1,3 GT) (Galili, 1991). Immunohistochemical studies with Griffonia simplicifolia isolectin B4 of pig heart, liver, and kidney sections indicate a heterogeneous expression of the Gal[alpha]1,3Gal epitope within the organs, with a preferential, nonhomogenous expression in small vessels (Galili et al., 1987).

The structural elucidation of the cell surface carbohydrates is a major obstacle due to the structural complexity, microheterogeneity, and low abundance of specific cell surface carbohydrates, which upon isolation are obtained in complex mixtures (Holgersson et al., 1992). It is nowadays clear that carbohydrates play a significant role in a range of important biological contexts, i.e., cell-cell recognition (Varki, 1993) and host-microbial interactions (Karlsson, 1991) as well as in the field of xenotransplantation (see above). This further emphasizes the urgent need of improved, more sensitive, and structurally informative analytical techniques. Mass spectrometry, 1H NMR spectroscopy, and degradative techniques comprise the standard battery of methods used for structural elucidation of isolated, pure oligosaccharides or glycolipids. To some extent structural information may be obtained also from complex mixtures using these techniques. However, gas chromatography-mass spectrometry has been used to resolve permethylated oligosaccharide mixtures containing compounds with up to seven sugar residues in the carbohydrate chain (Hansson et al., 1989).

In this article, we have used high-temperature gas chromatography-mass spectrometry of ceramide glycanase released oligosaccharides in combination with thin-layer immunostaining of intact glycolipids to rapidly screen the expression of putative xenoreactive, glycolipid-borne carbohydrate epitopes in small specimens of different organs of a semi-inbred pig homozygous at the SLA locus. The method, where we used small amounts (about 13-145 g) of material from each single organ, is to be compared to the commonly used large-scale preparations using kilograms of material from different individuals. The information which can be interpreted from these small sample amounts is to be compared to the information obtained from conventional carbohydrate characterizations.

The permethylated samples were analyzed by matrix-assisted laser desorption/ionization mass spectrometry in order to get the dynamic mass range of the mixtures. This in turn reflects the possibility to analyze the components present by GC/MS.

Results

Thin-layer immunostaining

The TH-5 MAb, detecting the Gal[alpha]1,3Gal epitope, bound clearly to a five-sugar compound in the kidney, salivary gland and heart glycolipids (Figure 1a). There were also strong staining of six-sugar compounds in the kidney glycolipids, weak staining of six-sugar structures in the heart, and additional staining of eight- and 10-sugar components in the kidney and heart glycolipids. The Griffonia simplicifolia lectin IB4 showed a similar pattern with binding in the five sugar region in the kidney and heart glycolipids. Additional binding was seen in the six sugar region of the kidney and in the seven sugar region of the heart fraction (not shown). The second [alpha]-Gal MAb used was a [alpha]-human IgG MAb. The binding pattern was very similar to the TH-5 MAb but with an additional weak binding in the five sugar region of the spleen fraction (not shown). The anti-B antibody reacted with components of all organs tested that migrated in the six-sugar region on the TLC plate (Figure 1b), although the staining of small intestinal glycolipids in this region was very weak. In addition, a five-sugar component was strongly stained in the kidney using the anti-B antibody (Figure 1b). Surprisingly, the anti-A antibody bound distinctly to a six-sugar compound in the salivary gland, and to six- and seven-sugar compounds in the heart (Figure 1c). The anti-H type 2 chain-specific antibody bound to five-sugar components of all organs and to a seven/eight-sugar compound in the heart and salivary gland glycolipids (not shown). The antibody specific for blood group H type 4 structures reacted diffusely with six-sugar compounds of all organs (not shown). The anti-Ley antibody detected a six-sugar compound in the glycolipid mixtures obtained from the small intestine, kidney, salivary gland and heart, and an eight/nine-sugar compound in the small intestine, spleen, kidney, and heart glycolipids (not shown). No reactivity was seen with the anti-Lex antibody (not shown). The anti-Gb4Cer specific antibody stained in the four-sugar region of all organs tested (not shown).


Figure 1. CBA of the total non-acidic glsphl fractions and the polar fractions of a single blood group O pig. Thin-layer immunostaining of glycolipids isolated from the small intestine (si), spleen (s), kidney (k), salivary gland (sg), liver (l), and heart (h) of a single pig blood group O-typed on its red blood cells. The TH-5 (a), A582 (b), and A581 (c) antibodies were used to identify glycolipids carrying Gal[alpha]1,3Gal-, blood group B-, and blood group A-determinants, respectively. In (a), the polar fractions containing glycolipids with 4 and more sugar residues in the carbohydrate chain were also applied (lanes si4, s4, k4, sg4, l4, and h4). The reference compounds were Gal[alpha]1,3nLc4Cer (r1), B-6-1 (r2), and A-6-1 (r3).

Gas chromatography and gas chromatography-mass spectrometry

Thin-layer chromatograms of separated glycosphingolipids isolated from the kidney, small intestine, spleen, salivary gland, liver, and the heart are shown in Figure 2, together with the total ion chromatograms (TIC) from GC/MS and matrix-assisted laser desorption/ionization mass spectra obtained from the analyses of the corresponding ceramide glycanase released, permethylated oligosaccharides. The peak numbers in the TICs correlate to a specific glycosphingolipid whose structure is given in Table I. The characteristic sequence fragment ions (Bi and Zj) for all components are listed in Table II. The gas chromatograms of ceramide glycanase released oligosaccharides from the different organs were consistently reproduced in the GC/MS experiments as assessed by the conserved scan times and peak intensities (cf. Figure 2b, he, to Figure 3). Mass spectra of special interest for the discussion were selected from the GC/MS analysis and shown in Figure 4. The EI+ mass spectra of permethylated oligosaccharides contained sequence oxonium ions (Bi ions) including the nonreducing end of the saccharide chain and, in the presence of a hexoseamine, fragment ions (Zi ions) (Domon and Costello, 1988) including the reducing end of the saccharide chain and arising from an inductive cleavage of the glycosidic bond at the nonreducing end of the HexNAc residue. Together, these complementary ions gave the complete sequence of the oligosaccharide in most cases.

Table I. Oligosaccharide structures in different organs from a single blood group O pig
TIC Peak Name Structure M (Da)
Kidney
2 Gb4 HexN-O-Hex-O-Hex-O-Hex 903.5
3 nLc4 Hex-O-HexN-O-Hex-O-Hex 903.5
6 H-5-2 dHex-O-Hex-O-HexN-O-Hex-O-Hex 1077.6
7 H-5-1 dHex-O-Hex-O-HexN-O-Hex-O-Hex 1077.6
9 Gal[alpha]1,3nLc4 Hex-O-Hex-O-HexN-O-Hex-O-Hex 1107.6
12 H-6-4 dHex-O-Hex-O-HexN-O-Hex-O-Hex-O-Hex 1281.7
13 Gal[alpha]1,3Lex Hex-O-Hex-O-HexN(dHex-O-)-O-Hex-O-Hex 1281.7
16 HexN Gal[alpha]a HexN-O-Hex-O-Hex-O-HexN-O-Hex-O-Hex 1352.7
Intestine
2 Gb4 HexN-O-Hex-O-Hex-O-Hex 903.5
7 H-5-1 dHex-O-Hex-O-HexN-O-Hex-O-Hex 1077.6
9 Gal[alpha]1,3nLc4 Hex-O-Hex-O-HexN-O-Hex-O-Hex 1107.6
12 H-6-4 dHex-O-Hex-O-HexN-O-Hex-O-Hex-O-Hex 1281.7
Spleen
2 Gb4 HexN-O-Hex-O-Hex-O-Hex 903.5
3 nLc4 Hex-O-HexN-O-Hex-O-Hex 903.5
6 H-5-2 dHex-O-Hex-O-HexN-O-Hex-O-Hex 1077.6
7 H-5-1 dHex-O-Hex-O-HexN-O-Hex-O-Hex 1077.6
9 Gal[alpha]1,3nLc4 Hex-O-Hex-O-HexN-O-Hex-O-Hex 1107.6
12 H-6-4 dHex-O-Hex-O-HexN-O-Hex-O-Hex-O-Hex 1281.7
Salivary gland
2 Gb4 HexN-O-Hex-O-Hex-O-Hex 903.5
3 nLc4 Hex-O-HexN-O-Hex-O-Hex 903.5
6 H-5-2 dHex-O-Hex-O-HexN-O-Hex-O-Hex 1077.6
7 H-5-1 dHex-O-Hex-O-HexN-O-Hex-O-Hex 1077.6
9 Gal[alpha]1,3nLc4 Hex-O-Hex-O-HexN-O-Hex-O-Hex 1107.6
15 A-6-1a HexN-O-Hex(dHex-O)-O-HexN-O-Hex-O-Hexa 1322.7
Liver
2 Gb4 HexN-O-Hex-O-Hex-O-Hex 903.5
7 H-5-1 dHex-O-Hex-O-HexN-O-Hex-O-Hex 1077.6
9 Gal[alpha]1,3nLc4 Hex-O-Hex-O-HexN-O-Hex-O-Hex 1107.6
Heart
1 GM3 NeuAc-O-Hex-O-Hex 815.4
2 Gb4 HexN-O-Hex-O-Hex-O-Hex 903.5
3 nLc4 Hex-O-HexN-O-Hex-O-Hex 903.5
4 GM2 oligos HexN-O-Hex(NeuAc-O)-O-Hex 1060.5
5 FucGg4 dHex-O-Hex-O-HexN-O-Hex-O-Hex 1077.6
6 H-5-2 dHex-O-Hex-O-HexN-O-Hex-O-Hex 1077.6
7 H-5-1 dHex-O-Hex-O-HexN-O-Hex-O-Hex 1077.6
8 GD3 NeuAc-O-NeuAc-O-Hex-O-Hex 1176.6
9 Gal[alpha]1,3nLc4 Hex-O-Hex-O-HexN-O-Hex-O-Hex 1107.6
10 GM1 Hex-O-HexN-O-Hex(NeuAc-O)-O-Hex 1254.6
11 Ley dHex-O-Hex-O-HexN(dHex-O)-O-Hex-O-Hex 1251.7
12 H-6-4 dHex-O-Hex-O-HexN-O-Hex-O-Hex-O-Hex 1281.7
14 Fuc GM1 dHex-O-Hex-O-HexN-O-Hex(NeuAc-O)-O-Hex 1438.7
15 A-6-1 HexN-O-Hex(dHex-O)-O-HexN-O-Hex-O-Hex 1322.7
16 HexN Gal[alpha] HexN-O-Hex-O-Hex-O-HexN-O-Hex-O-Hex 1352.7
17 A Ley HexN-O-Hex(dHex-O-)-O-HexN(dHex-O)-O-Hex-O-Hex 1496.8
18 Ext. H-5-2 dHex-O-Hex-O-HexN-O-Hex-O-HexN-O-Hex-O-Hex 1526.8
19 Ext. Gal[alpha] Hex-O-Hex-O-HexN-O-Hex-O-HexN-O-Hex-O-Hex 1556.8
aTrace amounts indicated by GC (not shown), thin-layer immunostaining and detected by GC/MS (mass spectrum not shown).

Table II. Characteristic sequence fragment ions in EI+ mass spectra of methylated oligosaccharides
Sequence fragment ions
(Bi, Bi-MeOH and Zj) m/z Present in structure
dHex- (B1) 189 FucGg4, H-5, Ley, H-6-4, FucGM1, Ext.H-5-2
Hex- (B1) 219, 187 nLc4, Gal[alpha]1,3nLc4, Gal[alpha]1,3Lex, GM1, Ext.Gal[alpha]
HexN- (B1) 260, 228 Gb4, Gg4, GM2, A-6-1, HexNGal[alpha], ALey
dHex-Hex- (B2) 393, 361 FucGg4, H-5, Ley, H-6-4, FucGM1, Ext.H-5-2
Hex-Hex- (B2) 423 Gal[alpha]1,3nLc4, Gal[alpha]1,3Lex
Hex-HexN- (B2) 464, 432 nLc4, GM1
HexN-Hex- (B2) 464, 432 Gb4
HexN-Hex(dHex)- (B2) 638 A-6-1, ALey
dHex-Hex-HexN- (B3) 638 FucGg4, H-5, H-6-4, FucGM1, Ext.H-5-2
Hex-Hex-HexN- (B3) 668 Gal[alpha]1,3nLc4, Ext.Gal[alpha]
dHex-Hex-HexN(dHex)- (B3) 812 Ley
Hex-Hex-HexN(dHex)- (B3) 842 Gal[alpha]1,3Lex
dHex-Hex-HexN-Hex- (B4) 842 Ext.H-5-2
Hex-Hex-HexN-Hex- (B4) 872 Ext.Gal[alpha]
HexN-Hex-(dHex)-HexN- (B3) 883 A-6-1
HexN-Hex-Hex-HexN- (B4) 913 HexNGal[alpha]
HexN-Hex(dHex)-HexN(dHex)- (B3) 1057 ALey
dHex-Hex-HexN-Hex-HexN- (B5) 1087 Ext.H-5-2
Hex-Hex-HexN-Hex-HexN- (B5) 1117 Ext.Gal[alpha]
-HexN-Hex-Hex (Z3) 668 nLc4, FucGg4, H-5, Gal[alpha]1,3nLc4, HexNGal[alpha],
Ext.H-5-2,Ext.Gal[alpha]
-HexN(dHex)-Hex-Hex (Z3) 842 Ley, Gal[alpha]1,3Lex, ALey
-HexN-Hex-Hex-Hex (Z4) 872 H-6-4
-HexN-Hex-HexN-Hex-Hex (Z5) 1117 Ext.H-5-2, Ext.Gal[alpha]
NeuAc- (B1) 376, 344 GM3, GM2, GM1, FucGM1
NeuAc-NeuAc- (B2) 737 GD3
-HexN-Hex(NeuAc)-Hex (Z3) 1029 FucGM1

Figure 2. TLC analysis of total and polar ([ge]4 sugars) nonacid glycolipid fractions isolated from the kidney (k, k4), small intestine (si, si4), spleen (s, s4), salivary gland (sg, sg4), liver (l, l4), and heart (h, h4) of a single blood group O pig, together with the gas chromatograms and matrix-assisted laser desorption/ionization mass spectra of the corresponding permethylated, ceramide glycanase-released oligosaccharides. About 4 µg of each fraction was applied for HPTLC, chromatographed with chloroform/methanol/H2O, 60/35/8 (by volume), and treated with the anisaldehyde reagent for visualization. The different structures identified in each oligosaccharide mixture by GC, MALDI-MS, and GC/MS were numbered for identification, and listed according to that numbering in Table I. The MALDI-mass spectra show the lithium adduct molecular ions [M+Li]+ of oligosaccharides identified in the different mixtures.

Kidney

The TIC from the GC/MS analysis together with the MALDI mass spectrum of permethylated oligosaccharides released from glsphl of pig kidney (k) are shown in Figure 2a. Eight components were identified by these methods (see Table I). In the mass spectrum of component 9 (not shown) a Hex-O-Hex-O-HexNAc-O-Hex- sequence was deduced from the oxonium ions at m/z 219 (B1), 423 (B2), 668 (B3), and 872 (B4). The structure from the reducing end (-HexN-O-Hex-O-Hex) was given by the fragment ion at m/z 668 (Z3) due to inductive cleavage at the non-reducing end of the HexNAc residue. The sequence of the pentasaccharide (M = 1107.6) is consistent with the Gal[alpha]1,3nLc4 structure, whose presence in the kidney fraction is inferred from the reactivity of the anti-Gal and anti-B antibodies with a five-sugar component in the TLC-immunostaining experiments (Figure 1a and b, lanes k and k4). The mass spectrum of component 13 in the chromatogram revealed a Hex-O-Hex-O-(dHex-O-)HexNAc-O-Hex-O-Hex- sequence based on the presence of oxonium ions at m/z 189 (B1[beta]), 219 (B1[alpha]), 423 (B2[alpha]), 842 (B3), and 1046 (B4), a sequence-specific fragment at m/z 636 (B3-(dHex-OH)), and the inductive ions at m/z 842 (Z3[alpha]) and at m/z 1077 (Z3[beta]) (Figure 4A). Together with immunostaining data, which suggest a Gal[alpha]1,3Gal-terminated structure among the six-sugar compounds (Figure 1a,b), the sequence supports the presence of a Gal[alpha]1,3Lex gsphl in the kidney-a structure that was recently identified by Bouhours and coworkers in pig kidney cortex (Bouhours et al., 1997).

Small intestine

Four components were identified by GC/MS and MALDI-MS of small intestinal oligosaccharides (Figure 2a, si) and their structures, as revealed by GC/MS and thin-layer immunostaining, are listed in Table I. Characteristic sequence fragment ions for the components are listed in Table II.

Spleen

At least six components were identified in the spleen (Figure 2a, s). The mass spectrum of component 12 (not shown) revealed a structure with a molecular mass of 1281.7 Da. The structure was found in most of the organs examined, and parts of its sequence dHex-O-Hex-O-HexNAc-O-Hex-O-Hex was deduced from oxonium ions observed at m/z 189 (B1), 393 (B2), 638 (B3), 842 (B4), and 1046 (B5). The sequence of the reducing end, -HexNAc-O-Hex-O-Hex-O-Hex, was completed by the fragment ion at m/z 872 (Z4). This sequence may correspond to the type 4 chain blood group H hexaglycosylceramide, whose presence was suggested by thin-layer immunostaining using anti-H type 4 specific antibodies. The remaining structures and characteristic sequence ions in the spleen are given in Table II and 2.

Table III. MAbs and lectins used in the thin layer immunostaining experiments
Antibody/lectin Specificity Code no. Reference
Anti A Terminal trisaccharide Dakopatts A 581, Denmark Holgersson et al., 1988
Anti B Terminal trisaccharide/weak
Gal[alpha]1,3Gal		 Dakopatts A 582, Denmark Holgersson et al., 1988
Anti H Type 1 and 2 chains Dakopatts A 583, Denmark Holgersson et al., 1988
Anti H H-5-2, H-3 Lot 9BH R0001,
Chembiomed, Canada		  
Anti Lex Lex terminal, type 2 chain SH-1 *
Anti Ley Ley terminal, type 2 chain AH-6 Abe et al., 1983
Anti Lea Lea terminal, type 1 chain XALA, Chembiomed, Canada Holgersson et al., 1990a
Anti Leb Leb terminal, type 1 chain/
H-5-1		 9ALB, Chembiomed, Canada Holgersson, et al., 1990a
Anti Gal[alpha]1,3Gal[beta] Gal[alpha]1,3Gal[beta] terminal,
type 2 chain		 TH-5 **
Anti Gal[alpha]1,3Gal Gal[alpha]1,3Gal[beta] terminal,
type 2 chain		 P3393 ***
IB-4 isolectin Gal[alpha] terminal Griffonia simplicifolia,
Vector Labs., USA		 Peters and Goldstein,
1979
< tblfn>*A.Singhal, S.Nance, and S.Hakomori, unpublished observations.
**J.Thorn, S.Hakomori, and H.Clausen, unpublished observations.
***V.Strokan, J.Mölne, C.Svalander, and M.E.Breimer, unpublished observations.

Salivary gland

The salivary gland contained five oligosaccharide components. Components 6 and 7 in the TIC (Figure 2b, sg) were shown by mass spectrometry (Figure 4, B and C, respectively) to be pentasaccharide isomers with a molecular mass of M = 1077.6 Da. In both cases the sequence dHex-O-Hex-O-HexN- was given by the fragment ions at m/z 189 (B1), m/z 393 (B2), and m/z 638 (B3). The structure from the reducing end (-HexN-O-Hex-O-Hex) was determined by the fragment ion at m/z 668 (Z3). The isomers could be differentiated on basis of differences in retention times and in intensity ratio between the fragment ions at m/z 182 and m/z 228 (Egge, 1978; Karlsson et al., 1989). In the presence of an internal carbon 4-substituted GlcNAc the ratio is very high, and the ion at m/z 182 is often base peak. If a 3-substituted GlcNAc is present, the fragment ion at m/z 228 is very intense, resulting in a very low intensity ratio between the two ions. The isomers were not completely chromatographically resolved, but it has been found that components containing an internal 4-substituted GlcNAc always elute in front of the isomer with a 3-substituted GlcNAc (Karlsson et al., 1989).


Figure 3. Gas chromatogram of permethylated oligosaccharides from glsphl of pig heart as detected by the flame ionization detector. Compare this gas chromatogram to the TIC from GC/MS (Figure 2b, he) of the same oligosaccharide mixture. The inset is a gas chromatogram which shows the partly resolved oligosaccharides corresponding to H-5-2/H-5-1 (labeled 6 and 7). Chromatographic conditions: fused silica column, 10 m × 0.25 mm i.d. coated with 0.03 µm P5264; H2 as carrier gas with a linear gas velocity of 125 cm/s at 70°C. One microliter of sample was injected on the column at 70°C (1 min), programmed to 200°C at 50°C/min followed by 10°C/min up to 400°C. Gas chromatograph used was a Hewlett-Packard 5890 Series II. Table I lists the labeled structures.


Figure 4. Mass spectra of kidney component number 13 (A), salivary gland components number 6 (B) and number 7 (C), and heart components number 14 (D) and number 19 (E).

Liver

Three oligosaccharide components were identified from the liver glycolipid mixture using GC, GC/MS, and MALDI-MS (Figure 2b, li). The structures and their characteristic sequence fragment ions are given in Table II and 2.

Heart

The most complex organ was the pig heart with 18 identified components. Acidic glycolipids are usually removed in a standard non-acid glycosphingolipid isolation procedure (see Materials and methods). However, in the case of the heart some acidic glycolipids were still present when the fraction was analyzed by GC/MS (Figure 5) and MALDI-MS thereby increasing the structural complexity of the oligosaccharide mixture. The spectrum of component 14 is shown in Figure 4D and indicates a hexasaccharide structure (M = 1438.7 Da). Oxonium ions at m/z 189 (B1[alpha]), 393 (B2[alpha]), and 638 (B3[alpha]) gave the sequence dHex-O-Hex-O-HexNAc- from the nonreducing end of the saccharide, and the fragment ion at m/z 1029 (Z3) indicated a reducing end sequence containing a NeuNAc substituted hexose, -HexNAc-O-(NeuNAc-O)-Hex-O-Hex-, thereby completing the sequence as given in Figure 4D. The NeuNAc oxonium ion was observed at m/z 376 (B1[beta]), and a characteristic oxonium ion derived from the sialic acid containing oligosaccharide was identified at m/z 1379 [M-COOCH3]+. The sequence could correspond to, and be derived from, the FucGM1 ganglioside.

Revealed from its spectrum (not shown), peak no. 16 (Figure 2b, he) was identified as a HexNAc-terminated hexasaccharide (M = 1352.7 Da). The sequence, HexNAc-O-Hex-O-Hex-O-HexNAc-, was determined from the oxonium ions at m/z 260 (B1), 668 (B3), and 913 (B4). Most of the intensity of m/z 668 is assumed to be contributed by the fragment ion (Z3) derived from the reducing end sequence, -HexNAc-O-Hex-O-Hex.

Two heptasaccharide oligosaccharides with molecular masses [M+Li]+ of 1533.8 (not shown) and 1563.8 Da (Figure 4E), were identified as components 18 and 19, respectively (Figure 2b, he). The difference of 30 mass units represents the difference in mass between a Hex and a dHex. The reducing end sequence, -HexNAc-O-Hex-O-HexNAc-O-Hex-O-Hex, was common to both structures and suggested by the inductive ions at m/z 668 (Z3) and 1117 (Z5). The nonreducing end sequence, dHex-O-Hex-O-HexNAc-O-Hex-O-HexNAc-, of component 18 was deduced from the oxonium ions at m/z 361 (B2-MeOH), 638 (B3), 842 (B4), and 1087 (B5) (spectrum not shown; see Table II for characteristic sequence fragment ions). The fragment ion at m/z 1381 (M-145) has empirically been found to be present in spectra of structures containing a dHex-O-Hex- terminal (Karlsson et al., 1987). The non-reducing end sequence, Hex-O-Hex-O-HexNAc-O-Hex-O-HexNAc- of component 19, was confirmed by the oxonium ions at m/z 187 (B1-MeOH), 668 (B3), 872 (B4), 1117 (B5), and 1321 (B6) (Figure 4E). Supported by the TH-5 binding of a 7/8-sugar compound in the heart glycolipids (Figure 1a, lane h4), this sequence may correspond to an extended Gal[alpha]1,3Gal-terminated glycolipid based on the nLc6Cer structure. This glycolipid was just recently identified in blood group O pig kidneys (Bouhours et al., 1997) but have not been found earlier in the pig heart.


Figure 5. TIC (broken line) and mass chromatogram (solid line) of the NeuNAc oxonium ion m/z 376 in the GC/MS analysis of pig heart oligosaccharides. The sialylated components are observed in the mass chromatogram (i.e., ganglioside oligosaccharides) and their structures are listed in Table I.

Discussion

The rapid GC/MS technique using small sample specimens is to be compared to the conventional large-scale preparation and characterization of glycospingolipids. The advantage of the technique is the possibility to analyze different organs in one single individual, and also different parts of one single organ (i.e., the medulla and the cortex of the kidney can easily be compared). In the large-scale preparation the possibility for an individual characterization is lost in favor of more starting material, e.g., kilograms of organs, but larger amounts of starting material do not always give the advantage of more structural information. More complex structures are often not possible to purify into single component fractions even in large-scale preparations, and the structural information of the mixture is therefore often difficult to interpret in mass- and NMR spectra. The GC/MS method allows picomoles of different components in a mixture to be analyzed and each carbohydrate sequence determined.

The amount of work and money put into the preparation of small sample specimens compared to the conventional large-scale preparation is also to be considered. The structural information from the screening technique and the conventional large-scale preparation is to be compared with these considerations in mind.

The analysis of released, permethylated oligosaccharides was started by MALDI-MS in order to get the molecular mass range of the mixtures (Figure 2a,b). This was followed by capillary GC (Figure 3) which gave qualitative and semiquantitative estimates of the components found in the sample mixtures. The flame ionization detector of the gas chromatograph is less sensitive to siloxanes from the column bleed than the mass spectrometer (cf. Figure 2b, he, to Figure 3) (Karlsson et al., 1994). In the total ion chromatogram of the mass spectrometric analysis, the ion current from larger structures is much less than that derived from the siloxanes and will therefore not appear as peaks. However, if a mass chromatogram of a specific fragment ion is followed (e.g., Figure 5), structures otherwise buried in the TIC may appear. GC/MS becomes a very sensitive and specific analytical tool through the high resolving power of capillary gas chromatography using ultra-thin films, and the structural specificity of mass spectrometry. As the EI+ mass spectra of permethylated oligosaccharides are very simple to interpret, the sequence information given is straight forward. In combination with thin-layer immunostaining, where specific carbohydrate epitopes can be detected by monoclonal antibodies and associated to the approximate size of the carrier glycolipid through its TLC plate Rf-value, GC/MS becomes a very powerful tool in the structural elucidation of small amounts of complex oligosaccharide/glycolipid mixtures.

The high resolving power obtained in capillary GC using ultra thin films is demonstrated by the partly resolved isomers of the oligosaccharides corresponding to the H-5 type 2 and type 1 glycolipids (Figure 2a,b, components 6 and 7, respectively). Due to different relative intensities of the fragment ions at m/z 182 and m/z 228 the isomers could be differentiated on basis of their mass spectra provided they are chromatographically separated (Figure 4B,C) (Egge, 1978; Karlsson et al., 1989). This has been observed previously for the oligosaccharides corresponding to Lc4Cer (type 1 chain) and nLc4Cer (type 2 chain) (Teneberg and Karlsson, unpublished observations), and A-6-2 and A-6-1, respectively (Bouhours et al., 1990). Thus, limited linkage information can be achieved using high-temperature GC/MS in situations where conventional linkage analysis by degradation procedures and 1H NMR are excluded due to low sample amounts.

The strength of capillary gas chromatography was also illustrated by the analysis of pig heart oligosaccharides. Component 5 (Figure 2b, he) which is clearly distinct from the H-5-2 (component 6) and H-5-1 (component 7) has been interpreted by virtue of its mass spectrum to have a sequence consistent with the Fuc[alpha]1,2Gg4 structure. The mass spectrum showed striking similarities to the mass spectrum of H-5-1 (Figure 4C) except in the low mass region below m/z 200. However, in this region the relative intensities of different fragment ions are almost identical to the spectrum of component 14 (Figure 4D) which has a sequence consistent with Fuc[alpha]1,2GM1, another ganglio-series oligosaccharide.

The reactivity of the anti-A antibody with glycolipids isolated from the heart and salivary gland (Figure 1c, lanes sg and h) of a pig strain that was blood group-typed as blood group O, is surprising. Sequences consistent with a blood group A-6-1 structure was identified by GC/MS among the salivary gland and heart oligosaccharides (Table I), and a sequence corresponding to a difucosylated heptasaccharide with a blood group A determinant was found among the heart oligosaccharides (Table I). This intraindividual organ-specific expression of blood group glycolipids, which has been demonstrated before in other species (Hansson et al., 1980, 1983, 1984; Oriol et al., 1996) clearly illustrates the weakness of conventional blood group-typing using pig red blood cells (Breimer et al., 1996). It is important to consider this phenomenon in future pig to human xenotransplantations in order to avoid anti-blood group mediated rejections.

The Gal[alpha]1,3Gal epitope has been shown to be the major xenogeneic determinant responsible for hyperacute rejections of porcine organs perfused with human blood or transplanted into non-human primates. Its tissue distribution has been studied using the Gal[alpha]-specific lectin, Griffonia simplicifolia isolectin B4, and human AB serum. High expression has been found on porcine endothelium of all vascularized organs (Oriol et al., 1994). However, the specificity of this lectin is broader than just Gal[alpha]1,3Gal-terminated structures (Galili et al., 1987; Liu et al., 1994), which is why biochemical studies on the tissue distribution of these structures are justified. Porcine kidneys has been shown to contain the Gal[alpha]1,3nLc4Cer structure (Hendricks et al., 1990) and, recently, a Gal[alpha]1,3- terminated Lex structure was identified in pig kidneys by Bouhours (Bouhours et al., 1997). Removal of human anti-pig antibodies prior to transplantation is a proposed major strategy to prevent hyperacute rejection following pig to human xenotransplantation. This has been accomplished by pretransplant organ perfusion or plasmapheresis (Makowka et al., 1995; Breimer et al., 1996), and by extracorporally circulating the blood of the recipient through columns binding human immunoglobulins before transplantation (Gjörstrup and Watt, 1990; Leventhal et al., 1995). Protein A or immunoglobulin-binding antibodies are usually used as absorbers in such columns (Gjörstrup and Watt, 1990; Leventhal et al., 1995). More specific absorption of xenoreactive anti-pig antibodies has been achieved by perfusing the blood through columns derivatized with the major xenogeneic epitope, the Gal[alpha]1,3Gal disaccharide (Rieben et al., 1995). However, as shown in this article and by others (Bouhours et al., 1997), Gal[alpha]1,3-terminated structures other than the common Galili antigen, Gal[alpha]1,3nLc4, may be found in porcine tissues. It is therefore reasonable to believe that the porcine [alpha]1,3 GT is responsible for the biosynthesis of an array of Gal[alpha]1,3-containing immunodominant epitopes. Likewise, it is reasonable to believe that the human anti-pig antibody repertoire contains antibodies that can distinguish between these different epitopes, just as there are antibodies that can distinguish between blood group A determinants carried by different core saccharide chains (Rydberg et al., 1994). Efficient absorbers of human anti-pig xenoreactive antibodies should therefore contain a variety of Gal[alpha]1,3-terminated structures in order to absorb as much as possible of the human anti-pig, [alpha]1,3 GT-determined antibody repertoire (Liu et al., 1997).

Of the organs analyzed, the kidney is clearly the organ that contains the highest amounts of Gal[alpha]1,3-terminated glycolipids. It is unlikely though that this level of expression in the kidney is explained by a higher level of expression solely on the kidney endothelium. It is more likely that other kidney structures, such as the proximal tubules, contribute to the overall expression level in the kidney (Oriol et al., 1993). Whether the proximal tubule, which is not in direct contact with the blood of the recipient, contributes to the humoral and cellular rejection of pig kidneys is not known. However, this possibility should be considered and kidneys may therefore be less suitable for initial trials of xenotransplantation because of the high xenoantigen load. In this respect, the cell-specific expression of the Gal[alpha]1,3Lex structure and its importance in hyperacute rejection of porcine xenografts warrants further study.

Material and methods

Preparation of glycosphingolipids

Total non-acid glycolipid fractions were isolated from different organ specimens of a blood group 0 pig using the method of Karlsson with slight modifications (Karlsson, 1987). Tissue samples were obtained from the kidney (13.8 g wet tissue weight, 6.2 g dry tissue weight), small intestine (57.3 g and 11.3 g, respectively), spleen (52.7 g wet tissue weight), salivary gland (56.7 g wet tissue weight), liver (93.6 g and 26.5 g, respectively), and heart (142.4 g wet tissue weight). Lipids were extracted from lyophilized tissues in a Soxhlet apparatus using chloroform/methanol mixtures. The different steps in the isolation procedure included alkaline methanolysis to remove alkali-labile phospholipids, silica columns to remove nonpolar lipids such as ceramides and methyl esters of fatty acids, DEAE-cellulose columns to separate the acidic glycolipids from the nonacidic glycolipid components, acetylation followed by additional silica columns to remove sphingomyelin which in its native state has the same polarity as some glsphl, and finally a second DEAE-cellulose column to remove residual acidic material (Karlsson, 1987).

Table IV. Reference glycolipids and their specificity used in the thin layer immunostaining experiments
References Origin Specificity Reference
H-5-2, Gb4Cer Total fraction of human
blood group O erythrocytes		 Blood group O
pentaglycosylceramide<		 Koscielak et al., 1973;
Stellner et al., 1973
A-6-1 Human meconium Blood group A type 1 chain
hexaglycosylceramide		 Karlsson and Larson,
1981
B-6-1 Human A1B Le(a-b-)
pancreas		 Blood group B type 1 chain
hexaglycosylceramide		 *
Lex Dog small intestine Blood group X
pentaglycosylceramide		 Hansson et al., 1983
Ley Dog small intestine Blood group Y
hexaglycosylceramide		 Hansson et al., 1983
Lea Human small intestine Blood group Lea
pentaglycosylceramide		 Smith et al., 1975
Leb Human small intestine Blood group Leb
hexaglycosylceramide		 Smith et al., 1975
Gal[alpha]1,3nLc4Cer Pig aorta Gal[alpha]1,3nLc4Cer **
Gal[alpha]1,3Gal-R Pig kidney Gal[alpha] terminating
glycosylceramides		 Rydberg et al., 1996
Gal[alpha]1,3Gal-R Blood group O pig kidney Gal[alpha] terminating
glycosylceramides		 Holgersson et al., 1990b
*A.E.Bäcker and J.Holgersson, unpublished observations.
**E.C.Hallberg et al., unpublished observations.

HPLC fractionation of glycosphingolipid mixtures

The non-acid glycosphingolipid mixtures were fractionated by silicic-acid column chromatography (Polygosil 60-5, Machery-Nagel, Düren, Germany) (Holgersson et al., 1991) on an HPLC system (LKB 2150 and 2152, Bromma, Sweden) using a linear gradient from 80/20/1 to 40/40/12 (by volume) of chloroform/methanol/water (Holgersson et al., 1991). A constant flow rate of 2 ml/min. was used over 280 min, and fractions of 4 ml were collected. Fractions containing glycolipids with [le]3 sugar residues (nonpolar) in the carbohydrate chain according to thin-layer mobility were pooled together, as were fractions containing glycolipids with [ge]4 sugars (polar) in their carbohydrate chains.

Ceramide glycanase-cleavage of glycosphingolipids

Two hundred micrograms of each polar glycolipid mixture were mixed with 30 µl (30 mg/ml) sodium cholate (Sigma) in chloroform/methanol, 2:1 (by volume). The samples were dried under a stream of nitrogen, resuspended in 200 µl 0.1 M sodium acetate buffer, pH 5.0, and incubated for 48 h at 37°C after adding 5 mU ceramide glycanase (Boehringer-Mannheim, Germany) (Li et al., 1986). The reaction mixtures were passed through prewashed C18 reversed phase SepPak cartridges (Waters Associates, USA) in order to remove ceramides and potential traces of noncleaved glycolipids (Hansson et al., 1989). To reach the optimal digestion conditions different amounts of ceramide glycanase and glycolipids were tested and evaluated. The effectiveness of the used digestion was determined with TLC and anisaldehyde staining of the ceramide fraction. The yield was estimated to be more than 90%. The oligosaccharide fractions eluted by water were lyophilized and permethylated (Larson et al., 1987) prior to GC, GC/MS, and MALDI-MS analysis.

Glycolipid analysis

Depending on the complexity of the mixtures, 5-10 µg of the nonacidic glsphl were applied in each lane on HPTLC plates (Si-60, Merck, Darmstadt, Germany; and HP-KF, Whatman, Maidstone, UK). The components were chromatographed in chloroform/methanol/H2O, 60/35/8 (by volume). Detection was accomplished by anisaldehyde (Karlsson et al., 1987) or by MAb immunostaining using a modification of the chromatogram binding assay by Magnani et al. (Magnani et al., 1980; Hansson et al., 1985) or the assay by Hynsjö et al. (Hynsjö et al., 1995). Bound primary anti-carbohydrate MAb were detected by 125I-labeled secondary antibodies or alkali phosphatase conjugated antibodies and visualized by autoradiography using a [gamma]-sensitive film or in the latter case by a visual color change on the TLC when adding the enzyme substrate. Antibodies, lectin and reference glsphl used for the experiments are listed in Table III and IV.

High-temperature capillary gas chromatography

Fused silica capillary columns (10 m × 0.25 mm i.d., Chrompack, Middelburg, The Netherlands) were D4-deactivated and statically coated with 0.03 µm of PS 264 (Fluka, Buchs, Switzerland) which was cross-linked using dicumyl peroxide (Blomberg et al., 1982). Capillary GC was performed using a Hewlett-Packard 5890-II gas chromatograph equipped with an on-column injector and a flame ionization detector (detector temperature, 400°C). Hydrogen was used as carrier gas with an oxygen trap (Oxypurge, Alltech) in the carrier gas line and a head pressure of 0.7 bar giving an average linear gas velocity of 125 cm/s at 70°C. The permethylated oligosaccharides were dissolved in 200 µl of ethyl acetate and 1 µl was injected on-column at 70°C (1 min) then programmed up to 200°C at 50°C/min followed by 10°C/min up to 400°C.

High-temperature gas chromatography-mass spectrometry

GC/MS was performed on a Hewlett-Packard 5890-II gas chromatograph interfaced to a JEOL SX-102A mass spectrometer (Jeol, Tokyo, Japan). The gas chromatograph was equipped with an on-column injector which was under electronic pressure control. Helium was used as carrier gas with an oxygen trap (Oxypurge, Alltech) in the carrier gas line and with a head pressure of 0.3 bar at the start giving an average linear gas velocity of 87 cm/s at 80°C. GC/MS was performed with the on-column injector in constant flow mode (1.9 ml/min) and vacuum compensation on with the electronic pressure control. The fused silica column tip was positioned in the ion source about 2 mm from the electron beam. Gas chromatographic conditions: fused silica column (10 m × 0.25 mm i.d.) coated with 0.03 µm of cross-linked PS 264 (Fluka, Buchs, Switzerland), 1 µl of sample dissolved in ethyl acetate was injected on-column at 80°C and the same two-step temperature program as described above for GC was used (Hansson and Karlsson, 1993).

Mass spectrometry conditions: acceleration voltage, +10 kV; electron energy, 70 eV; trap current, 300 µA; ion source temperature, 360 °C; GC/MS interface temperature, 380 °C; linear magnet scan and mass range scanned, m/z 100-1600; total cycle time, 1.4 s; resolution, 1400 (m/[Delta]m, 10% valley definition); pressure in the ion source region, 5·10-4 Pa.

Matrix-assisted laser desorption/ionization mass spectrometry

MALDI-MS was performed on a Micromass TofSpec-E spectrometer (Micromass, Manchester, England) in the positive reflectron mode at +22.5 kV acceleration voltage. A thin-film matrix surface was prepared using the fast evaporation technique from 2,5-dihydroxybensoic acidic (16.2 mg/ml in acetone containing 10 mM LiCl). The permethylated oligosaccharides were dissolved in 200-500 µl of ethyl acetate and 0.5 µl was applied on the matrix surface. The lithium adducts of the molecular ions [M+Li]+ were observed.

Acknowledgments

We thank Dr. Richard Binns, AFRC Babraham Institute, Cambridge, England, for the generous gift of pig organ tissues. We thank Drs. Henrik Clausen and Sen-itoroh Hakomori for the gifts of monoclonal antibodies. Prof. K.-A. Karlsson is acknowledged for use of the mass spectrometry equipment. This work was supported by the Swedish Medical Research Council no. 6521 (to B.S.), no. 13X-11574 (to J.H.), and nos. 3967, 10435 (to K.-A.K.), the IngaBritt and Arne Lundberg Foundation, The Swedish Dental Society, The Dental Society of Göteborg, The Royal Society of Arts and Sciences in Göteborg, and the Immunology Concerted Action (3026PL95004), Biotechnology Program Contract BI04-CT95-004 of the European Union Biotechnology Program and the Shared Cost Biotechnology Programme PL962242. Additional support to J.H. was obtained from the Swedish Cancer Society (no. 3645-B95), the Vårdal Foundation (no. A95 045), the King Gustaf V 80-Year Foundation, the Swedish Medical Society, the Magn. Bergvall Foundation, the Åke Wiberg Foundation, the Sigurd and Elsa Golje Foundation, the Tobias Foundation, the Pharmacia Research Foundation, and the Karolinska Institute.

Abbreviations

HAR, hyperacute rejection; DXR, delayed xenorejection; BSA, bovine serum albumin; FFA, free fatty acids; glsphl, glycosphingolipids; Lc4Cer, lactotetraosylceramide (Type 1 chain); nLc4Cer, neolactotetraosylceramide (Type 2 chain); FucGg4Cer, Fucosylated gangliotetraosylceramide; Gal, d-galactose; GalNAc, d-N-acetylgalactosamine; Glc, d-glucose; Hex, hexose; HexNAc, N-acetylhexosamine; MALDI-MS, matrix-assisted laser desorption/ionization mass spectrometry; MS, mass spectrometry; GC, gas chromatography; GC/MS, gas chromatography-mass spectrometry; TIC, total ion chromatogram; CBA, chromatogram binding assay; Gal[alpha]1,3nLc4Cer, Gal[alpha]3Gal[beta]4GlcNAc[beta]3Gal[beta]4Glc[beta]1Cer. Glycosphingolipids are denoted by the epitope name, the number of monosaccharides in the chain, and the core saccharide chain type. For example, A-6-1 denotes a six-sugar blood group A structure based on a type 1 chain. The nomenclature used for describing fragmentation and ions is in accordance with Domon and Costello (Domon and Costello, 1988). Enzymes: ceramide glycanase from leech (EC 3.2.1.4.5; Boehringer Mannheim.

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