A novel pentaglycosylceramide in ostrich liver, IV4-ß-Gal-nLc4Cer, with terminal Gal(ß1–4)Gal, a xenoepitope recognized by human natural antibodies

Danièle Bouhours1, Jérôme Liaigre, Jeanne Naulet, Nicolaï V.Bovin2 and Jean-François Bouhours

INSERM U.437, F-44093 Nantes Cedex 1, France, and 2Shemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow GSP-7, 117871, Russia

Received on December 9, 1999; revised on March 28, 2000; accepted on April 10, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Thin layer chromatograms of ostrich liver neutral glycosphingolipids were immunostained with human sera. In addition to the expected staining of the Forssman pentaglycosylceramide by some sera, more polar and less abundant unknown glycolipids could be stained. Among them, the shortest carbohydrate chain glycolipid was purified and structurally characterized by mass spectrometry, proton NMR and methylation analysis. It was a novel pentaglycosylceramide of the neolactoseries terminated with the Gal(ß1–4)Gal determinant which is not expressed in mammalian species. Human antibodies affinity-purified on a synthetic Gal(ß1–4)Gal(ß1–4)Glc-Sepharose column recognized the newly characterized Gal(ß1–4)Gal-terminated pentaglycosylceramide, and, in addition, longer chain glycolipids. Occurrence of antibodies directed at the Gal(ß1–4)Gal epitope was studied by ELISA on 108 human sera. Anti-Gal(ß1–4)Gal antibodies were predominantly IgM, and their distribution was similar to that of anti-Gal({alpha}1–3)Gal and anti-Forssman IgMs. It was concluded that anti-Gal(ß1–4)Gal are natural antibodies, not previously identified in man. They can be considered as xenoantibodies directed at species which express Gal(ß1–4)Gal-terminated carbohydrate chains.

Key words: Galß4Gal/glycosphingolipid/xenoepitope/ human natural antibodies/electrospray-ion trap mass spectrometry/1H-NMR


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Interest for heterophile antigens (xenoantigens), has been revived by the prospect of grafting porcine organs to man in order to alleviate the shortage of human organs (Auchincloss and Sachs, 1998Go). Xenoantigens are the target of natural antibodies present in human serum. The major porcine xenoantigen recognized by human natural antibodies is the Gal{alpha}3Gal epitope (Good et al.,1992; Galili, 1993Go; Sandrin et al., 1993Go), which is expressed in two types of structures, the afucoB epitope Gal{alpha}3Galß4GlcNAc (Jalali-Araghi and Macher, 1994Go; Samuelsson et al., 1994Go) and the Gal{alpha}3Lex epitope (Bouhours et al., 1997Go, 1998b).

N-Glycolylneuraminic acid (HD antigen) (Hanganatziu, 1924; Deicher, 1926Go; Higashi et al., 1977Go) is the second heterophile antigen expressed in pig. Although the HD antigen is not expressed in man, the occurrence of preformed anti-HD antibodies is rare, but HD antigen is known to trigger a potent immune reaction.

Ostrich was interesting to study as an alternative to pig for experimental xenotransplantation, because, as a bird, it does not express the Gal{alpha}3Gal epitope or the HD antigen. However, it has been found that ostrich tissues express the Forssman pentaglycosylceramide (Bouhours et al., 1999Go), which is the third known heterophile antigen (Forssman, 1911Go; Siddiqui and Hakomori, 1971Go; Stellner et al., 1973Go; Kano et al., 1984Go).

As the occurrence of natural anti-Forssman antibodies in human serum (Young et al., 1979Go; Strokan et al., 1998Go) is not as well documented as that of anti-Gal{alpha}3Gal antibodies (Galili, 1993Go; Sandrin et al., 1993Go; Parker et al., 1994Go; McKane et al., 1998Go), the study of the anti-Forssman reactivity of human serum was reinvestigated by ELISA using purified Forssman antigen, and immunostaining of chromatograms of neutral glycolipids of ostrich liver. Surprisingly, it was found upon chromatogram-immunostaining that some sera were reactive at high dilution with glycolipids more polar and less abundant than the Forssman pentaglycosylceramide. One of these glycolipids was isolated from ostrich liver and characterized as Galß4Galß4GlcNAcß3Galß4GlcßCer. This is a novel structure related to blood group-active glycolipids and presenting at the non-reducing end a determinant which is unknown in man and in mammals. Glycolipids with more than five sugar residues were also recognized by affinity-purified human anti-Galß4Gal antibodies, but were not characterized in the present work. As the Galß4Gal disaccharide appeared as a potential xenoantigen, the occurrence of human antibodies directed at this epitope was investigated by ELISA. It was found that anti-Galß4Gal antibodies are mainly IgM and occur in a large population according to a distribution similar to that of natural anti-Gal{alpha}3Gal and anti-Forssman antibodies.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Chromatogram-immunostaining of ostrich liver neutral glycolipids with human serum IgM
It has been shown that ostrich liver chiefly expresses three neutral glycosphingolipids, the mono- and dihexosylceramides and the Forssman pentaglycosylceramide F-5 (Figure 1A, lane 2) (Bouhours et al., 1999Go). Random human sera were used for immunostaining of thin layer chromatograms of ostrich liver glycolipids in order to evaluate their IgM anti-Forssman reactivity.



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Fig. 1. HPTLC of neutral glycosphingolipids of ostrich liver. (A) Chemical visualization with phenol/sulfuric acid; (B and C) immunostaining with human sera reactive with Galß4Gal upon ELISA ((B) serum 97 at 1:500 dilution, and (C) serum 81 at 1:1000 dilution); (D) immunostaining with affinity-purified anti-Galß4Gal antibodies. Immunostainings were visualized with HPR-conjugated anti-human IgM. Lane 1, neutral glycolipids of pig kidney used as standard (14 nmol); lane 2, neutral glycolipids of ostrich liver (5 nmol); lanes 3, 5, and 8, neutral glycolipids of ostrich liver (2.5 nmol); lanes 4 and 7, purified GL-5 of ostrich liver (0.1 nmol); lane 6, purified Forssman pentaglycosylceramide (F-5) of ostrich liver (0.4 nmol). Chromatography was developed in chloroform/methanol/water (60:35:8). GL-1, monohexosylceramide; GL-2, dihexosylceramide; afucoB-5, IV3-{alpha}-Gal-nLc4Cer; F-5, IV3-{alpha}-GalNAc-Gb4Cer; GL-5, IV4-ß-Gal-nLc4Cer characterized in this paper. Nomenclature of glycolipids follows the IUPAC-IUB recommendations (1997).

 
Some sera immunostained F-5, whereas other did not. Surprisingly, glycolipids more polar than F-5 were occasionally stained, with concomitant staining of F-5 (Figure 1B, lane 3) or without it (Figure 1C, lanes 5–6). These glycolipids were hardly detected by chemical visualization compared to F-5 (Figure 1A, lane 2). One of them, termed GL-5 before structural characterization, migrated at the same level as the Gal{alpha}3Gal-terminated pentaglycosylceramide (afucoB-5) of porcine kidney (Figure 1A). However, it was not reactive with hen anti-Gal{alpha}3Gal antibodies (Bouhours et al., 1998aGo), thus confirming the lack of expression of this epitope in birds. GL-5 accounted for 3% of the neutral glycolipid content of ostrich liver (0.5 µmol per liver), that is about one tenth of the F-5 contribution (Bouhours et al., 1999Go).

Structural characterization of GL-5 in ostrich liver
Although GL-5 was present in very low amount in the liver glycolipid extract, it was purified by preparative thin layer chromatography with a sufficient yield and quality for structural characterization.

Mass spectrometry.
The ESI-IT-MS spectrum of GL-5 displayed a prominent molecular ion (m/z 1411.9) (Figure 2, upper panel). It was consistent with an oligosaccharide chain containing one HexNAc and four Hex linked to a ceramide containing C18 sphingosine (d18:1) and C16 nonhydroxylated saturated fatty acid (n16:0). The m/z value of this ion was identical with that of the afucoB-5 (IV3-{alpha}-Gal-nLc4Cer) of porcine kidney with the same ceramide. A peak at m/z 1539.8 could be interpreted as the same oligosaccharide chain linked to a ceramide portion containing C24 hydroxylated fatty acid (h24:0). Molecular ion m/z 1411.9 was submitted to collision-induced dissociation (Figure 2, middle panel, MS2) in order to obtain the carbohydrate sequence. Loss of the terminal sugar generated the Y4 ion (m/z 1249.7), consistent with a terminal Hex. Loss of the penultimate carbohydrate residue generated the Y3 ion m/z 1087.8, consistent with a terminal disaccharide HexOHex. The major ion generated by the fragmentation was the Y2 dihexosylceramide ion m/z 884.9 which arose from the additional loss of a HexNAc, indicating that the terminal trisaccharide was HexOHexOHexNAc which appeared as the B3 ion (m/z 549.9). The Y1 monohexosylceramide ion was observed (m/z 722.8) with the corresponding tetrasaccharide B4 ion (m/z 712.4) for HexOHexOHexNAcOHex. The entire pentasaccharide chain HexOHexOHexNAcOHexOHex was manifested by B5 (m/z 874.5) and C5 (m/z 892.5) ions. Further fragmentation of the Y2 ion (Figure 2, lower panel, MS3) yielded the disaccharide ions HexOHex at the reducing end of the chain termed b2 (m/z 347.5) and c2 (m/z 365.8) by analogy with the B2 and C2 disaccharide ions at the nonreducing end of the oligosaccharide chain. Appearance of the Z0 ceramide ion (m/z 542.6) indicated the presence of a nonhydroxylated fatty acid (Bouhours et al., 1999Go). Although the chain length of the sphingoid base was not established by fragmentation of the Y2 ion, due to the lack of hydroxylation of the fatty acid (Bouhours et al., 1999Go), the only presence of sphingosine (d18:1) in other glycolipids of ostrich liver (Bouhours et al., 1999Go) was in favor of the same base in GL-5, consistent with the d18:1/n16:0 ceramide species.



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Fig. 2. Electrospray-ion trap MS of native IV4-ß-Gal-nLc4Cer purified from ostrich liver (upper panel) and collision-induced dissociation of the molecular ions m/z 1411.9 (middle panel, MS2) and m/z 884.9 (lower panel, MS3). The masses indicated in the upper panel are the experimental values of monoisotopic masses [M+Na]+. The scheme of dissociation corresponds to the nomenclature of Domon and Costello (1988)Go. The ions observed after dissociation in the ion trap are Yj = [Yj + H + Na]+, Bi = [Bi – H + Na]+, Ci = [Ci + H + Na]+ and Zj = [Zj – H + Na]+. d, d18:1 (dihydroxylated monounsaturated sphingosine); n, normal fatty acid; h, {alpha}-hydroxylated fatty acid.

 

Characterization of carbohydrate residues and linkages.
GC analysis of the partially methylated alditol acetates of GL-5 yielded peaks for 2,3,4,6-tetra-O-Me-Gal (terminal Gal ->1), 2,3,6-tri-O-Me-Gal (->4Gal1->), 2,3,6-tri-O-Me-Glc (->4Glc-1->), 2,4,6-tri-O-Me-Gal (->3Gal1->) and 3,6-di-O-Me-Glc-NAcMe (->4GlcNAc1->) (Figure 3, upper trace). Together with the sequence established by MS analysis, these data were consistent with a Gal(1–3/4)Gal(1–4)GlcNAc(1–3/4)Gal(1–4) Glc1 chain. The presence of a galactose substituted on C-4 made GL-5 different from afucoB-5 which does not contain -4Gal1-. However, methylation analysis could not determine which one of the two internal galactoses was substituted on C-4. The LiAlH4 reduction of the permethylated sugar chain is known to prevent cleavage of the internal GlcNAc-Gal linkage upon acid hydrolysis (Karlsson, 1974Go), and to give rise to a late eluting disaccharide peak (Karlsson and Larson, 1981Go). In the present analysis, the disaccharide GlcNAc-Gal peak was accompanied by the disappearance of the peak for -3Gal1- (Figure 3, lower trace). This result indicated that the GlcNAc was linked on C-3 of the previous Gal. Thus, the terminal disaccharide of GL-5 was Gal(1–4)Gal1-. It was essential to determine the anomerity of the linkage, in order to know whether the terminal disaccharide was Gal({alpha}1–4)Gal, the known blood group P1 determinant (Naiki et al., 1975Go), or Gal(ß1–4)Gal, a new epitope for glycolipids.



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Fig. 3. Gas chromatography of the partially methylated alditol acetates of methylated (upper trace), and methylated-reduced IV4-ß-Gal-nLc4Cer (lower trace).

 

1H NMR spectroscopy.
The main characteristic of the 400 MHz spectrum of GL-5 (Figure 4) was the lack of {alpha}-anomeric proton signal, and the presence of five ß-anomeric proton signals indicating that GL-5 was terminated by Gal(ß1–4)Gal. By comparing the GL-5 spectrum with the nLc4Cer and Gal{alpha}3-nLc4Cer spectra (Table I), signals at 4.178 and 4.706 p.p.m. were assigned to ß-Glc I-1 and ß-GlcNAc III-1, respectively. Signals for the three ß-Gal H-1 protons were more difficult to assign. The most deshielded signal at 4.290 p.p.m. probably originated in the internal ß-Gal II-1, as in IV3-{alpha}-Gal-nLc4Cer (Table I). The two other ß-Gal H-1 signals were almost superimposed. The ß-Gal IV-1 proton which resonates upfield when it is terminal (Table I; Levery et al., 1988Go) must be deshielded by the presence of ß-Gal V, although to a lesser extent than by {alpha}-Gal V (Table I; Bouhours et al., 1997Go). In addition, according to spectra obtained in D2O of terminal Galß4Gal oligosaccharide of Collocalia salivary mucin (Wieruszeski et al., 1987Go) and fish fertilized eggs glycoproteins (Taguchi et al., 1995Go), the terminal ß-Gal H-1 proton is more deshielded than the penultimate ß-Gal H-1 proton. Thus, the ß-Gal proton signals at 4.273 and 4.278 p.p.m. were tentatively assigned to ß-Gal IV-1 and V-1, respectively. The lactosamine core structure was supported by the ß-GlcNAc NAc signal in the acetamido-methyl region at 1.835 p.p.m.



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Fig. 4. Proton NMR spectrum of IV4-ß-Gal-nLc4Cer purified from ostrich liver. The spectrum was obtained at 400 MHz and 55°C in Me2SO referenced to internal Me4Si. Shown are the downfield region, olefinic methine (R-5, R-4) and anomeric oligosaccharide protons, and the upfield region for the {alpha}-carbonyl (nFA-2), alkyl (R-6) and acetamido methyl (NAc) protons. Arrows in the anomeric proton spectrum point to signals for traces of IV3-{alpha}-GalNAc-Gb4Cer (Forssman-5).

 

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Table I. Anomeric proton resonances (p.p.m.) of GL-5 in Me2SO at 55°C
 

The ceramide portion of GL-5 was clearly characterized by the nonhydroxylated fatty acid signal at 2.04 p.p.m., and signals at 5.39, 5.57, and 1.96 p.p.m. (R4, R5 and R6, respectively) characteristic of unsaturated dihydroxylated sphingosine, in agreement with the findings of MS analysis.

In conclusion, the analyses fully support the structure IV4-ß-Gal-nLc4Cer. It has the nLc4 core structure of blood group ABH- active and {alpha}3Gal-terminated glycolipids. As the epitope Galß4Gal has never been found in man or in mammals, the occurrence of antibodies against this determinant was investigated.

Determination of the reactivity of human sera with the xenoepitope Galß1–4Gal
108 human sera were tested by ELISA with Galß4Gal-PAA as solid phase antigen. It was found that anti-Galß4Gal antibodies do exist and that they are mainly of the IgM isotype. Anti-Galß4Gal IgM followed the same pattern of distribution as that of natural anti-Gal{alpha}3Gal and anti-Forssman IgM (Figure 5). High reactivity (absorbance over 1.0) was found in 6 sera for anti-Galß4Gal, 11 sera for anti-Gal{alpha}3Gal and 3 sera for anti-Forssman IgM (Table II). High anti-Gal{alpha}3Gal reactivity was associated with the anti-Galß4Gal reactivity in two sera, with additional anti-Forssman reactivity in one serum. It was concluded that anti-Galß4Gal IgMs represent a new type of human natural antibodies. The reactivity of human serum IgG with Galß4Gal-PAA was almost nonexistent (90% below absorbance 0.25) (Figure 6). However, four sera had high IgG reactivity (absorbance over 1.0), corresponding to high anti-Galß4Gal IgM reactivity for three of them.



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Fig. 5. Distribution of the IgM reactivities in 108 human sera. Sera were grouped according to the range of their O.D. responses in ELISA with Galß4Gal-PAA, Gal{alpha}3Gal-PAA and Forssman-5 glycolipid as solid phase antigens.

 

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Table II. IgM reactivity with Gal{alpha}3Gal and Forssman antigen of human sera with the highest IgM reactivity with Galß4Gal
 


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Fig. 6. Distribution of the IgG reactivities of 108 human sera in ELISA with Galß4Gal and Gal{alpha}3Gal. Same representation as in Figure 5.

 
Human sera with high anti-Galß4Gal activity upon ELISA recognized several glycolipids in addition to IV4-ß-Gal-nLc4Cer. In order to know whether these glycolipids had Galß4Gal-terminated carbohydrate chains, anti-Galß4Gal antibodies were affinity-purified on a column made of the trisaccharide Galß4Galß4Glc- covalently bound by a spacer arm to Sepharose. Upon ELISA, the affinity-purified anti-Galß4Gal IgM reacted as the total serum with the synthetic disaccharide Galß4Gal-PAA, and also with IV4-ß-Gal-nLc4Cer. Furthermore, the purified anti-Galß4Gal IgM immunostained the same glycolipids (Figure 1D) as the whole serum (Figure 1C). Such an experiment (affinity-purification of antibodies, ELISA and TLC-immunostaining with the purified antibodies) was performed with several sera with high anti-Galß4Gal antibodies titer upon ELISA with Galß4Gal-PAA, with the same result. From these findings, it was concluded that, among the glycolipids occasionally stained by human sera (Figure 1B-C), a set of glycolipids shared the Galß4Gal epitope recognized by human natural antibodies (Figure 1D).


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The novel glycolipid characterized in ostrich liver is the fourth galactose-terminated pentaglycosylceramide of the neolactoseries (Table III). Glycolipid 1 (IV3-{alpha}-Gal-nLc4Cer) was first characterized in rabbit erythrocyte membrane (Stellner et al., 1973Go). Subsequently, it appeared that the Gal{alpha}3Gal determinant is expressed in all mammals except Old World monkeys, Apes, and Man, species in which anti-Gal{alpha}3Gal antibodies are found (Galili et al., 1987Go, 1988). More recently, it was shown that hyperacute rejection of pig organs transplanted in primates originates in the binding of these antibodies to pig endothelium (Good et al., 1992Go). Pig kidney and endothelial cells were found to express glycolipid 1 among other Gal{alpha}3Gal- terminated glycolipids (Bouhours et al., 1996Go, 1997, 1998b). Glycolipid 2 (IV3-ß-Gal-nLc4Cer) was characterized in human erythrocytes (Stellner and Hakomori, 1974Go). It has not been reported in other human tissues. However, the disaccharide Galß3Gal was characterized in the urine of a child with early myoclonic epileptic encephalopathy (Michalski et al., 1984Go). Furthermore, synthesis in vitro of the determinant Gal-ß3Galß4GlcNAc by human kidney microsomes indicated that the terminal ß3-galactosyltransferase is present in man (Bailly et al., 1988Go). Therefore, it is unlikely that human individuals express natural antibodies against IV3-ß-Gal-nLc4Cer, unless there is an expression polymorphism. Glycolipid 3 (IV4-{alpha}-Gal-nLc4Cer) was identified as the antigen conferring blood group P1 specificity (Naiki et al., 1975Go) which is expressed by about 75% of human beings. Individuals lacking the P1 antigen on their erythrocytes belong to blood group P2 (Bailly and Bouhours, 1995Go). In man, the Gal{alpha}4Galß4GlcNAc determinant is only carried by this unique pentaglycosylceramide (Naiki et al., 1975Go), and not by glycoproteins (Yang et al., 1994Go), whereas in pigeons and doves, it is present in mucin glycans (François-Gerard et al., 1980Go). Anti-P1 natural antibodies found in P2 individuals are usually of low strength. However, in case of immunological stimulation such as in pigeon breeders, their titer and strength rise considerably (Radermecker et al., 1975Go).

The discovery of glycolipid 4 (IV4-ß-Gal-nLc4Cer) in the present work and the lack of expression of the Galß4Gal determinant in man led to investigating the presence of natural antibodies in human serum. The human serum reactivity with Galß4Gal was significant only for IgM, and its distribution in a random population was similar to that of anti-Gal{alpha}3Gal and anti-Forssman IgM. No class switch was observed even in sera with high IgM reactivity, which is the fate of antibodies raised by oligosaccharides in a T cell independent manner. These findings were consistent with a natural antibody response. The study of the reactivity of affinity-purified anti-Galß4Gal natural antibodies with ostrich liver glycolipids indicated that IV4-ß-Gal-nLc4Cer shared the xenoepitope Galß4Gal with several longer carbohydrate chain glycolipids.

Although the Galß4Gal determinant has not been shown previously in glycolipids, it has been already described in a bird sialylated salivary mucin (Wieruszeski et al., 1987Go), and in N-linked penta-antennary glycan chains of a sialoglycopeptide of fish fertilized eggs (Taguchi et al., 1995Go).

The enzyme responsible for the synthesis of the Galß4Gal determinant is a ß4-galactosyltransferase (ß4Gal-T) which must operate downstream from the known ß4Gal-T. It is provisionally termed Gal:ß4Gal-T, in order to distinguish it from the "mammalian" ß4-galactosyltransferases which catalyze the synthesis of lactose (Galß4Glc) and/or N-acetyllactosamine (Galß4GlcNAc), and should be termed Glc(NAc):ß4Gal-T. Seven members of the human ß4Gal-T gene family have been characterized (Almeida et al., 1999Go). As galactose and glucose are epimeric at C-4, Gal:ß4Gal-T and Glc(NAc):ß4Gal-T might be as different as Glc(NAc):ß4Gal-T and ß3Gal-T which are encoded by unrelated gene families (Almeida et al., 1998Go).

Expression of Galß4Gal in glycolipids of different birds such as chicken (not shown) and ratites (ostrich and emu), and in glycoproteins of birds (Wieruszeski et al., 1987Go) and fishes (Taguchi et al., 1995Go) indicates that the Gal:ß4Gal-T has a large species distribution. It might be encoded by a gene which has been inactivated in Mammalia at an unknown time during evolution, in a similar way as the inactivation of the {alpha}3Gal-T gene in catarrhines (Galili and Swanson, 1991Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Human sera
The human sera used in the present work for chromatography immunostaining and ELISA screening were random and anonymous sera taken at the central biochemistry laboratory of the hospital. They were heated at 56°C for 30 min for complement inactivation.

Purification of glycosphingolipids
Ostrich organs were collected after exsanguination, cut into small pieces and lyophilized. Lipids were extracted from lyophilized tissues by successive incubations at 70°C in methanol and chloroform/methanol (1:2). Glycolipids were purified from lipid extracts and separated into neutral and acid glycolipids as already described (Bouhours et al., 1992Go).

Thin layer chromatography
The glycosphingolipid composition of ostrich organs was analyzed by thin layer chromatography. The glycolipid solutions were chromatographed on silica gel 60 aluminum backed plates (Merck) developed in chloroform/methanol/water (60:35:8). For chemical visualization, chromatograms were treated with the phenol/sulfuric acid reagent. For immunostaining, they were treated as described previously (Bouhours et al., 1987Go) with human sera instead of monoclonal antibodies. Sera were diluted in PBS, pH 7.4, containing 1% BSA. Antibodies bound to glycolipids were detected with HRP-conjugated rabbit anti-human IgM diluted 1:1000 in PBS (Dako, Denmark). Polyclonal anti-Gal{alpha}3Gal antibodies raised in hens were also used (Bouhours et al., 1998aGo) with rabbit HPR-conjugated anti-chicken antibodies (Sigma). Visualization was obtained by chemiluminescence with the ECL Western blotting system (Amersham) and exposure to a blue light-sensitive autoradiography film (Hyperfilm ECL Western, 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 acetone/water (4:1). Individualized glycolipids were scraped off the plate, extracted in chloroform/methanol/water (30:60:8), and taken up in chloroform/methanol (2:1).

Quantitative measurements
Quantities of glycolipids were determined by measurement of the sphingosine content of the solutions of glycolipids purified from the tissues, according to a described procedure, taking into consideration that there is 1 mol of sphingosine per mol of glycolipid (Bouhours and Glickman, 1976Go). The percentage distribution of the glycolipids of a tissue was determined by sphingosine measurement in the 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.

Electrospray/ionization-ion trap mass spectrometry
Purified GL-5 was analyzed by mass spectrometry on a HP-Bruker-ESQUIRE-LC mass spectrometer (Bruker Daltonik, Bremen, Germany), following a described procedure (Bouhours et al., 1999Go). Ions obtained by electrospray-ion trap MS analysis of native glycolipids in the positive ion mode are sodium adducts of molecular ions [M + Na]+. Collision-induced dissociation of molecular ions gives rise to ions resulting from the cleavage of the glycosidic bonds. According to the nomenclature established by Domon and Costello (1988)Go, fragments containing the non-reducing end of the oligosaccharide chain are labeled Ai, Bi, Ci, and the complementary fragments containing the aglycone Xj, Yj, Zj (Figure 2, MS2). The major ions observed after dissociation of the molecular ion in the ion trap were Yj = [Yj+H+Na]+, Bi = [Bi-H+Na]+, Ci = [Ci+H+Na]+ and Zj = [Zj-H+Na]+.

Methylation analysis
The carbohydrate composition and sugar linkages were determined by gas chromatography of the partially methylated alditol acetates. The purified glycolipid was permethylated by the method of Ciucanu and Kerek (1984)Go, then submitted to acetolysis, reduction with potassium borohydride and acetylation (Yang and Hakomori, 1971Go). Additional data on sugar sequence were obtained by reduction of the permethylated glycolipid before acetolysis, using LiAlH4 in diethylether according to Karlsson (1974)Go. Gas chromatography of the partially methylated alditol acetates was done on a Hewlett Packard 5890 gas chromatograph as already described (Bouhours et al., 1997Go).

1H-NMR spectroscopy
Data on the anomerity of the sugar linkages and on the degree of hydroxylation of the fatty acids and sphingoid base were obtained by 1H-NMR spectroscopy. The native glycolipid was equilibrated three times in deuterated methanol, and dried under nitrogen. The deuterated glycolipid was dissolved in 0.5 ml of Me2SO-d6/2%D2O. Spectra were recorded at 400 MHz with 0.4 Hz digital resolution on a Bruker ARX-400 spectrometer. Chemical shifts are given relative to tetramethylsilane.

Immunoreactivity of human serum
The screening of human sera for their level of xenoreactivity was performed by ELISA according to a published technique (Rieben et al., 1995Go) with some modifications. The antigens were polyacrylamide-linked disaccharides Gal(ß1–4)Gal-PAA and Gal({alpha}1–3)Gal-PAA (Syntesome, Munich, Germany) (Bovin, 1998Go). They were coated on Nunc Maxisorp microtiter plate (0.5 µg of disaccharide in 50 µl of PBS, pH 7.4). Purified Gal{alpha}3-, Galß4- and Forssman pentaglycosylceramides were coated on Dynex Immulon-1B plate (25–50 pmol in 50 µl of methanol/water (80:20) per well). Coating was performed at 4°C overnight for the PAA-disaccharides, and at room temperature for 1 h for the glycolipids. For blocking the non-specific interactions, the plates were washed three times and let to stand 30 min at room temperature with 50 µl of PBS pH 7.4 containing 1% BSA and 1% Tween 20 (PBS/BSA/Tween). Sera were diluted 1:40 in PBS/BSA/Tween, and the reaction was performed with 50 µl of diluted serum. A blank was done for each serum, using PBS or methanol/water instead of the coating solution. After 2 h at 37°C, plates were washed five times with 50 µl of PBS containing 0.05% Tween 20 (PBS/Tween), and then incubated 1 h at 37°C with HRP-conjugated anti-human IgM (Dako) diluted 1:1000 in PBS/Tween. Enzyme activity was determined with ABTS (Boehringer) as substrate. Color intensity was measured as O.D. at 405 nm in a Dynatech MRX microplate reader after incubation for 1 h at 37°C.

Affinity-purification of anti-Galß4Gal antibodies
Human serum (1 ml) was chromatographed on a column made with 2 ml of Galß4Galß4Glc-spacer-Sepharose (Syntesome) in PBS. The retained antibodies were eluted with 1% ammonium hydroxide. Eluates were immediately neutralized with 1 M KH2PO4. Affinity-purified immunoglobulins were used at the same dilution as the initial serum for TLC-immunostaining and ELISA.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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). We are thankful to Dr. Jerôme Lemoine (UMR 8576 CNRS/USTL, Université des Sciences et Technologies de Lille, France) for his contribution to MALDI analysis.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
GL, glycosphingolipids; F, Forssman glycolipid; ESI-IT-MS, electrospray/ionization-ion trap mass spectrometry; GC, gas chromatography; HPTLC, high performance thin layer chromatography; Me2SO-d6, hexadeuterated dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay.


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Table III. Galactose-terminated neolactoseries pentaglycosylceramides
 

    Footnotes
 
1 To whom correspondence should be addressed at: INSERM U.437, Centre Hospitalier Universitaire, 30 Boulevard Jean-Monnet, F-44093 Nantes Cedex 1, France Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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