The prospect of grafting animal organs to man in order to alleviate the shortage of human organs (Auchincloss and Sachs, 1998; Tilney, 1998) has revived the interest for heterophilic antigens, now termed xenoantigens. It has been found that the main obstacle to xenotransplantation of pig organs to primates is the expression by porcine endothelial cells of the Gal[alpha]1->3Gal[beta]1->4GlcNAc[beta]1-> determinant (Good et al., 1992), which has been termed afucoB because it is structurally a non-fucosylated blood group B determinant (Bouhours et al., 1997). The afucoB epitope is not expressed by man and Old World primates which, in turn, have preformed antibodies against it (Galili et al., 1987, 1988). Another heterophile glycosidic antigen is the hydroxylated form of sialic acid, N-glycolylneuraminic acid, termed Hanganutziu-Deicher (HD) antigen (Hanganutziu, 1924; Deicher, 1926; Higashi et al., 1977). Absent in man and birds, the afucoB and HD epitopes are widely distributed among vertebrates. Although the occurrence of preformed antibodies against HD antigens is rare in man, exposure to the antigen triggers a strong immune reaction. In order to avoid early or delayed organ rejection via an antibody-dependent complement activation of the endothelium, it was proposed to look for birds which express neither the afucoB nor the HD xenoepitopes. The only bird species commensurate with man is ostrich (Taniguchi et al., 1996). Here, the glycosphingolipid composition of ostrich kidney and liver was investigated. It was found that a major glycolipid of both organs is the Forssman pentaglycosylceramide (Forssman, 1911; Siddiqui and Hakomori, 1971; Stellner et al., 1973), which is the third known heterophile oligosaccharide antigen (Kano et al., 1984). Mass spectrometry and 1H NMR spectroscopy analyses demonstrated a high percentage of hydroxylation of both components of ceramide, sphingoid base and fatty acid. The present study established that in addition to the Forssman pentaglycosylceramide found in many species, ostrich also expresses a Forssman tetraglycosylceramide not previously found in living animals.
Thin-layer chromatography
Thin-layer chromatography of ostrich kidney neutral glycolipids displayed a very simple pattern compared to porcine kidney neutral glycolipids, with only three glycolipids in sufficient quantity to be quantified by sphingosine assay (Figure
Figure 1. HPTLC of neutral glycosphingolipids. (A) Neutral glycosphingolipids of pig kidney cortex (lane 1), ostrich kidney (lane 2), and ostrich liver (lane 3) after chemical visualization with phenol/sulfuric acid. (B) Immunostaining with anti-Forssman monoclonal antibody of neutral glycolipids of ostrich liver (lane 4) and kidney (lane 5), and purified GL-4 from ostrich kidney (lane 6). Chromatography was developed in chloroform/methanol/water (60:35:8). Numbers in the right and left margins indicate the number of carbohydrate residues. GL, Glycosphingolipid; F, Forssman glycolipid. Mass spectrometry analysis
Ions obtained by electrospray-ion trap MS analysis of native glycolipids in the positive ion mode are sodium adducts of molecular ions [M + Na]+. The MS spectrum of kidney GL-5 displayed a series of molecular ions consistent with theoretical values for an oligosaccharide chain containing two HexNAc and three Hex (Figure
Figure 2. Electrospray-ion trap MS spectra of native Forssman-5 purified from ostrich kidney and liver. The masses indicated are the experimental values of monoisotopic masses [M+Na+]. d, d18:1 (sphingosine); t, t18:0 (phytosphingosine).
Molecular ions were submitted to collision-induced dissociation that gave rise to ions resulting from the cleavage of the glycosidic bonds. According to the nomenclature established by Domon and Costello (1996), fragments containing the nonreducing end of the oligosaccharide chain were labeled Ai, Bi, Ci, and the complementary fragments containing the aglycone Xj, Yj, Zj (Figures
Figure 3. Collision-induced dissociation of the molecular ion of Forssman-5 with d18:1/n16:0 ceramide. b2 and c2 in the MS3 spectrum (lower panel) indicate the dihexosyl fragments B and C of lactosylceramide Y2
Figure 4. Electrospray-ion trap MS of native Forssman-4 purified from ostrich kidney (upper panel) and collision-induced dissociation of the molecular ion m/z 1418.7 (d18:1/h24:0 ceramide) (lower panel). The masses indicated are the experimental values of monoisotopic masses [M+Na+]. d, d18:1 (sphingosine); t, t18:0 (phytosphingosine). The arrow in the MS spectrum (upper panel) marks the peak of the molecular ion m/z 1361.8 of globoside (d18:1/n24:0 ceramide).
Collision-induced dissociation of the kidney GL-5 molecular ion m/z 1452.6 (d18:1/n16:0 ceramide) (Figure
The MS spectrum of native kidney GL-4, a minor glycolipid, displayed molecular ions for an oligosaccharide chain with two HexNAc and two Hex (Figure
The mass spectrometry analysis of kidney GL-4 indicated the oligosaccharide chain HexNAcOHexNAcOHexOHexO, which suggested that ostrich kidney GL-4 is a Forssman tetraglycosylceramide. Its reactivity with the anti-Forssman antibody confirmed this hypothesis (Figure
MS spectra were interpreted assuming that only the 18 carbon atoms species of sphingosine and phytosphingosine were present and that the major molecular weight heterogeneity represented the fatty acids distribution. This hypothesis was substantiated by mass analysis in the ion trap of the fragments generated from molecular ions containing [alpha]-hydroxylated fatty acids by two (MS3) or three (MS4) rounds of isolation and dissociation. Fragmentation of the monohexosylceramide ion (Figure
Figure 5. MS4 collision-induced dissociation of the molecular ion m/z 1418.7 (d18:1/h24:0 ceramide) of kidney F-4 (upper panel) and MS3 dissociation of the molecular ion m/z 1598.9 (t18:0/h24:0 ceramide) of kidney F-5 (lower panel).
The MS spectrum of liver GL-4 mainly displayed molecular ions for a globoside with a ceramide portion containing C18 sphingosine and n16:0 (m/z 1250), n18:0 (m/z 1278.1), n22:0 (m/z 1333.8), n24:0 (m/z 1361.8), and h24:0 (m/z 1377.8). Molecular ions assignable to the Forssman tetraglycosylceramide could be detected. Their contribution to the spectrum was minor, consistent with the lack of reactivity of liver GL-4 with the anti-Forssman antibody.
The molecular ions for kidney GL-2 were consistent with a dihexosyl carbohydrate chain. The prevalent ceramides contained C18 sphingosine linked to nonhydroxylated C16 fatty acid (m/z 884.5) and [alpha]-hydroxylated C22, 23, and 24 fatty acids (m/z 984.5, 998.4, and 1012.4, respectively). The molecular ions for kidney GL-1 indicated a monohexosyl carbohydrate chain. Ceramides contained mainly [alpha]-hydroxylated C22, 23, and 24 fatty acids either linked to C18 sphingosine (m/z 822.6, 836.8, 850.8, respectively), or C18 phytosphingosine (m/z 840.7, 854.8, 868.7, respectively). Methylation analysis
Characterization of individual sugars and their linkages was obtained by methylation analysis (Figure
The GC profile of GL-2 yielded peaks for 2,3,6-tri-O-Me-Gal, 2,3,6-tri-O-Me-Glc, and 2,3,4,6-tetra-O-Me-Gal. Their relative intensities were consistent with an equal contribution of galabiose and lactosylceramide in kidney, and a 1:4 ratio in liver. The GC profile of GL-1 yielded peaks for 2,3,4,6-tetra-O-Me-Gal and 2,3,4,6-tetra-O-Me-Glc, indicating the presence of galactosyl and glucosylceramide in a 1:2 ratio in kidney, and the reverse in liver. 1H NMR spectroscopy
The 400 MHz 1H NMR spectrum of F-5 displayed two [alpha]-anomeric and three [beta]-anomeric proton signals (Figure
Table I.
Figure 6. Methylation analysis of Forssman-4 and Forssman-5 purified from ostrich kidney. Arrows in the GC profile of F-4 indicate impurities.
Figure 7. Proton NMR spectra of ostrich kidney and liver and sheep erythrocyte Forssman-5, and ostrich kidney Forssman-4, obtained at 400 MHz and 55°C in DMSO referenced to internal Me4Si. The spectra shown correspond to the downfield region, olefinic methine (R-5, R-4, cis-vinyl) and anomeric oligosaccharide protons, and the upfield region for the [alpha]-carbonyl (nFA-2), allylic, alkyl (R-6) and acetamido methyl (NAc) protons. In the spectrum of ostrich kidney F-4, arrows indicate anomeric protons of globoside oligosaccharide chain at 4.231 p.p.m. ([beta]-Glc), 4.297 p.p.m. ([beta]-Gal), 4.568 p.p.m. ([beta]-GalNAc) and 4.848 p.p.m. ([alpha]-Gal).
The anomeric region of the 1H NMR spectrum of kidney F-4 displayed a series of four signals consistent with a Forssman-4 oligosaccharide chain, GalNAc[alpha]->GalNAc[beta]->Gal[alpha]->Gal[beta] (Figure
Forssman-5
GalNAc[alpha]1->3GalNAc[beta]1->3Gal[alpha]1->4Gal[beta]1->4Glc[beta]1
Reference
V
IV
III
II
I
4.74
4.56
4.82
4.27
4.17
Equine kidney, Dabrowski et al., 1980a
4.765
4.582
4.836
4.285
4.183
Sheep erythrocytes, this work
4.765
4.615
4.839
4.290
4.229
Kidney, this work
4.763
4.594
4.834
4.283
4.213
Liver, this work
GalNAc[alpha]1->3GalNAc[beta]1->3Gal[alpha]1->4Gal[beta]1
Forssman-4
IV
III
II
I
4.771
4.630
4.866
4.132
Kidney, this work
Tissue localization of the Forssman antigen
Positive immunofluorescent reactions of kidney and liver with the anti-Forssman antibody were observed on cryostat sections only, whereas conventional paraffin-embedded sections did not react with the antibody. Immunofluorescence was detected at the same location in ostrich as in guinea pig, in the connective tissue, vascular endothelial cells, the midsegment piece and collecting ducts of the ostrich nephron corresponding to the renal distal tubules and collecting ducts of guinea pig kidney, and the liver Kupffer cells.
The neutral glycolipid composition of ostrich kidney and liver lacked the major neutral glycolipids expressed in porcine kidney (Figure
Figure 8. Biosynthesis of Forssman antigens.
Mass spectrometry and 1H NMR spectroscopy analyses gave concordant data for a high degree of ceramide hydroxylation. [alpha]-hydroxylated fatty acids were prevalent in Forssman glycolipids of both kidney and liver. Fragmentation of the amide bond of [alpha]-hydroxylated fatty acid-containing mono- and dihexosylceramide, as molecular or daughter ions, yielded psychosine type fragments characteristic of the sphingoid base. Only C18 sphingoid bases were found. Liver F-5 only contained the dihydroxylated monounsaturated base sphingosine, whereas the trihydroxylated saturated base phytosphingosine was prominent in kidney F-5. The difference in base composition of the Forssman pentaglycosylceramides was consistent with their distinct tissue origins. Differential tissue expression of sphingoid bases in glycolipids makes ostrich similar to mammals. For example, the Forssman pentaglycosylceramide of the mouse small intestine, which only contains sphingosine (Gustavsson et al., 1996), has been localized in the nonepithelial tissue whereas the major epithelial glycolipid, asialo-GM1, contains phytosphingosine (Umezaki et al., 1989). The sphingoid base composition of kidney F-4 was different from that of kidney F-5, with sphingosine as the major base. This finding might indicate that kidney F-4 and F-5 are synthesized in different cell types. Phytosphingosine has been described in kidney glycolipids in man (Karlsson and Martensson, 1968), cow (Karlsson et al., 1973), horse (Karlsson et al., 1974), and pig (Holgersson et al., 1990). The present work indicates that expression of phytosphingosine in kidney glycolipids has been consistently maintained in species at least since the divergence of birds from the other vertebrates.
The minimum Forssman determinant GalNAc[alpha]1->3GalNAc[beta]1->R resembles the afucoB determinant Gal[alpha]1->3Gal[beta]1->R. It is synthesized from UDP-GalNAc and globoside by an [alpha]3-GalNAc transferase, the cDNA of which has been isolated from a canine kidney cell line (Haslam and Baenziger, 1996). The Forssman transferase has been found very similar to the murine [alpha]3-Gal transferase synthesizing the afucoB epitope (35% amino acid sequence similarity) and the human [alpha]3-GalNAc/Gal transferase synthesizing the A/B blood group determinants (42% amino acid sequence similarity). Therefore, from structural and genetic standpoints, the Forssman antigen is analogous to the histo-group A/B and afucoB determinants. However, A/B and afucoB determinants are built on lactoseries glycolipids and glycoproteins, whereas Forssman antigens are exclusively glycosphingolipids of the globoseries or galaseries, as demonstrated here.
It has been proposed that the genes for the histo-group transferases arise by duplication and subsequent divergence of an ancestral gene (Yamamoto et al., 1991). In human, the afucoB determinant is not expressed, but two inactivated genes for the [alpha]3-galactosyltransferase are present, a pseudogen on chromosome 12 and the remnant of the ancestral gene in the vicinity of the A/B/O locus on chromosome 9 (Joziasse et al., 1991). Most mammals express the A/O histo group and the afucoB determinants. By contrast, the distribution of the Forssman antigen does not follow a clear evolutionary pathway. Among mammals, guinea pig, mouse, hamster, dog, cat, sheep, horse, and cow express Forssman antigens (Forssman, 1911; Tanaka and Leduc, 1956). Rabbit, pig and rat are nonexpressing. The same erratic expression is found in birds: goose and pigeon are negative whereas chicken is Forssman positive (Tanaka and Leduc, 1956; Kitamoto et al., 1980). Humans have been considered Forssman-negative, primarily because the Forssman antigen has not been found in erythrocytes, in concordance with the presence in human sera of natural antibodies against the widespread Forssman antigen (Young et al., 1979). However, all human sera are not equivalent as to their anti-Forssman reactivity which displays a great variability (Young et al., 1979). The Forssman antigen has been described in human gastric mucosa (Hakomori et al., 1977), lung (Yoda et al., 1980), and kidney (Breimer, 1985) in some individuals. Concordance between low levels of anti-Forssman antibodies and expression of the Forssman antigen has been noticed in the case of gastric tumors (Hakomori et al., 1977), but the relationship remains unclear for normal individuals.
In the present study, lack of Forssman reactivity of paraffin-embedded sections made unlikely the expression of the Forssman determinant in ostrich glycoproteins, whereas its expression in ostrich glycosphingolipids was supported by the Forssman reactivity of cryosections. The anti-Forssman antibody stained blood vessels in kidney and liver cryosections, demonstrating that in ostrich, as in chicken (Kitamoto et al., 1980) and in guinea pig, the Forssman antigens are present in the endothelium and connective tissue. Therefore, the death of cultured ostrich endothelial cells observed after incubation with native human serum (Taniguchi et al., 1996) could be explained by natural anti-Forssman-dependent complement mediated attack. In conclusion, besides physiologic barriers and anatomic differences which may prevent the transplantation of ostrich organs to man (Taniguchi et al., 1996), ostrich is not a better organ or cell donor than pig with regards to natural antibody-mediated hyperacute rejection, because of the endothelial expression of the common Forssman xenoepitope.
Purification of glycosphingolipids
Kidneys (150 g each) and liver (650 g) were collected after exsanguination, cut into small pieces and lyophilized. Lipids were extracted from lyophilized materials by successive incubations at 70°C first in methanol and then three times in the mixture chloroform/methanol (1:2; 4 ml/g) (Bouhours et al., 1992)). Glycosphingolipids were isolated from acetylated lipids by Florisil column chromatography (Saito and Hakomori, 1971). Neutral glycolipids were separated from acid glycolipids by DEAE-Sephadex A-25 (acetate form) column chromatography (Ueno et al., 1978).
Thin layer chromatography
Neutral glycolipids were chromatographed on HPTLC silica gel 60 aluminum plates (Merck) developed in chloroform/methanol/water (60:35:8). Immunostaining of chromatograms was done as described previously (Bouhours et al., 1987) with murine anti-A (NaM87-1F6, Centre Régional de Tranfusion Sanguine, Nantes, France), anti-type 2 H (MR3-517, Institut National de la Tranfusion Sanguine, Paris, France), anti-Leb/type 1 H (Wistar Institute), anti-B (Dako, Denmark) monoclonal antibodies, rat anti-Forssman monoclonal antibody (M1/22.25.8.HL, ATCC TIB 121), and chicken anti-Gal[alpha]1-3Gal polyclonal antibody (Bouhours et al., 1998). Murine monoclonal antibodies were detected with sheep biotinylated anti-mouse immunoglobulins, and rat monoclonal antibody with rabbit biotinylated anti-rat immunoglobulins, labeled with streptavidin-horseradish peroxidase (HRP) conjugate. Chicken polyclonal antibody was detected with HRP-labeled rabbit anti-chicken antibodies. Visualization was obtained by chemiluminescence with the ECL Western blotting system (Amersham). For structural analysis, glycolipids were separated by preparative HPTLC on Silica gel 60 glass plates (Merck) in chloroform/methanol/water (60:35:8). Visualization was done with ultraviolet light after spraying a 0.05% solution of primulin in aceton/water (4:1). Each glycolipid was scraped off the plate, and extracted from the gel in chloroform/methanol/water (30:60:8). Primulin was removed by chromatography of the purified glycolipid on DEAE Sephadex A-25 (acetate form).
Quantitative measurements
Quantities of glycolipids were determined by measurement of sphingosine content according to a procedure described earlier (Bouhours and Glickman, 1976), either in glycolipid mixtures, or in suspensions of silica gel containing individual glycolipids scraped off the chromatogram of 40 nmol of glycolipid solution on a 2 cm streak, after visualization with primuline.
Methylation analysis
Purified glycolipids were permethylated by the method of Ciucanu and Kerek, (1984). The permethylated glycolipids were submitted to acetolysis, reduction and acetylation (Yang and Hakomori, 1971). Gas chromatography of the partially methylated alditol acetates was done on a 25 m × 0.32 mm fused silica capillary column wall-coated with 0.2 µm of OV-1. Analyses were performed on a Hewlett Packard 5890 gas chromatograph equipped with a flame ionization detector and operated in constant flow mode. Carrier gas was helium at a velocity of 40 cm s-1. Samples dissolved in hexane were injected (1 µl) on column at an oven temperature of 60°C. After 0.5 min, the oven temperature was raised to 125°C at a rate of 20°C min-1, and then up to 250°C at a rate of 5°C min-1.
Electrospray-ion trap mass spectrometry
Analyses were performed on a HP-Bruker ESQUIRE-LC mass spectrometer (Bruker Franzen, Bremen, Germany) equipped with an atmospheric pressure electrospray ionization source. Samples dissolved in ethyl acetate/methanol (1:1) were introduced in the electrospray source with a syringe pump at 2 µl/min. High voltages of the end plate and capillary were set at 3.5 and 4 kV, respectively. The nebulizer pressure was 1 psi. The drying nitrogen flow was 1 l/min and its temperature was 250°C. Capillary exit voltage was set at 350 V. Voltages of skimmer 1 and 2 were set at 80 V and 25 V, respectively. Spectra were acquired on the positive mode with the standard scanning range and a resolution of 0.6 m/z. For MS/MS operation, isolation windows were set in order to contain the three main isotopic molecular mass peaks, and fragmentation energy was set at the exact value that was necessary for the disappearance of the isolated ions. Spectra were averaged on 10-30 trap cycles and recorded as profile spectra. They were processed with the version 1.5c of the Bruker Data Analysis software.
1H-NMR spectroscopy
Each native glycolipid was equilibrated three times in deuterated methanol. The deuterated glycolipid was dissolved in 0.5 ml of Me2SO-d6 containing 2% D2O. Spectra were recorded at 400 MHz with 0.4 Hz digital resolution on a Bruker ARX-400 spectrometer. The probe temperature was 55°C. Chemical shifts are given relative to tetramethylsilane.
Histochemistry
Kidney and liver samples from an ostrich and a guinea pig (a known Forssman-positive mammal) were split in two parts: (1) one part was fixed in formalin 4%, paraffin-embedded, cut at 4 µm and the sections were rehydrated by routine histology techniques; (2) the second part was embedded in Tissue-Tek (Sakura Finetech, Bayer Diagnostic, France) and frozen in 2-methylbutane at liquid nitrogen temperature. Serial sections of 5 µm were obtained on a cryostat (Slee, London) and were fixed in acetone at -20°C for 4 min. Both types of tissue sections were incubated with rat anti-Forssman monoclonal antibody (diluted 1 to 2) for 60 min in a wet chamber. After washing (3 × 5 min), the sections were incubated for 60 min with fluorescein-labeled rabbit anti-rat Ig (diluted 1 to 20) (Dako, Denmark), washed again and mounted with 10 µl of Mowiol (Hoechst, Germany) under coverslides (Candelier et al., 1993). The fluorescence of stained sections was observed in an epifluorescence microscope (Aristopan, Leitz-Leika, Germany).
France Autruche (Chateaubriand, France) is gratefully acknowledged. This work was supported by the French National Institute for Medical Research (INSERM), by the Transvie Foundation (Nantes, France), and by the shared cost xenotransplantation contract IO4-CT97-2242 from the Immunology Biotechnology program DG XII from the European Union (EU).
GL, glycosphingolipid; F, Forssman glycolipid; MS, mass spectrometry; GC, gas chromatography; HPTLC, high performance thin layer chromatography; Me2SO-d6, hexadeuterated dimethyl sulfoxide; DMSO, dimethyl sulfoxide.
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