Isolation and partial characterization of blood group A and H active glycosphingolipids of rat small intestine.

Blood group A and H active glycosphingolipids have been isolated from rat small intestine. By mass spectrometry of the permethylated and LiAlH4-reduced permethylated glycolipid derivatives, the A glycolipids were shown to contain four (A-4), six (A-6), and 12 (A-12) sugar residues, respectively. The anomeric structure of the A-4 and A-6 glycolipids was established by proton NMR spectroscopy of the permethylated-reduced derivatives. Acid degradation and gas chromatography were used for analysis of binding positions. The structures of the A-4 and A-6 glycolipids were GalNAcp alpha 1 leads to 3Galp(2 comes from 1Fucp alpha) beta 1 leads to Glcp beta 1 leads to 1Cer and GalNAcp alpha 1 leads to 3Galp(2 comes from 1Fucp alpha) beta 1 leads to 3GlcNAcp beta 1 leads to 4Galp beta 1 leads to 4Glcp beta 1 leads to 1Cer. The third glycolipid (A-12) was a branched dodecaglycosylceramide with two blood group A determinants. The complete structure of this glycolipid has not yet been solved. The blood group A activity was the same for the A-6 and A-12 glycolipids based on an equal number of blood group A determinants, but the activity of the A-4 compound was only about half of the others. The A-6 glycolipid was based on a type 1 (Gal beta 1 leads to 3GlcNAc) carbohydrate chain, thus differing from the already known isomer based on a type 2 chain (Gal beta 1 leads to 4GlcNAc) present in human erythrocyte. The blood group A activity of these two glycolipids was found to be identical. The three rat intestinal blood group A active glycolipids were exclusively located to the mucosa epithelial cells. The blood group H active tri- and pentaglycosylceramides (H-3 and H-5), presumed to be the precursors of the A-4 and A-6 glycolipids, were also identified. A 10-sugar glycolipid (H-10), a possible precursor of A-12, was not detected.

thus differing from the already known isomer based on a type 2 chain (Galpl + 4GlcNAc) present in human erythrocyte. The blood group A activity of these two glycolipids was found to be identical. The three rat intestinal blood group A active glycolipids were exclusively located to the mucosa epithelial cells. The blood group H active tri-and pentaglycosylceramides (H-3 and H-ti), presumed to be the precursors of the A-4 and A-6 glycolipids, were also identified. A 10-sugar glycolipid (H-lo), a possible precursor of A-12, was not detected.
Glycosphingolipids with blood group A determinants have been isolated from both human and animal tissues. Five different glycolipid-based blood group A type carbohydrate determinants have so far been identified (1-9). Several of these are linked to glycosylceramide backbones of varying complexity as reviewed by McKibbin (7). The blood group A determinants found in human tissues are a l l built on type 1 (Gall 3GlcNAc) or type 2 (Gall 4 4GlcNAc) carbohydrate chains (7) but in animal tissues core saccharides lacking Nacetylhexosamine have also been identified (1,2). In this * Supported by grants from the Swedish Medical Research Council, (No. 3967) and from the Medical Faculty, University of Goteborg. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Medical Biochemistry, University of Goteborg, Box 33031, S-400 33 + To whom correspondence should be sent at the Department of Goteborg, Sweden. paper, we describe the isolation and structural characterization of three blood group A active glycolipids of rat small intestine. One of these is a novel four-sugar compound based on lactosylceramide. In addition, the structure of a blood group H type pentaglycosylceramide is described. DEAE-cellulose chromatograohr -The non-acid lipids weze separated from acid lipids on DEAE-cellulose columns. The DEI\E-cellulose adsorbent was treated as described 1101. The column was packed in CHCL~-CHJOH 2:l (by "01. 1 The sample was applled in the same Solvent and allowed to eqmlibrate over night. The non-acid lipids were eluted by CHCl CH OH 2:l (by vol.1 followed by pure CH30H (25 ml per g adsorbent). The acia-li;ids were eluted by 5 % lw/vl LiCl in CHgOH 110 ml per g adsorbent1 and the Salt was removed by dia-1~5 1 5 agalnrt running tap water for 4 days.

RESULTS
The total non-acid glycolipid fraction isolated from blackwhite rat small intestine is shown in Fig. 1, lane T. All bands stained for carbohydrate (green) with the anisaldehyde reagent (10). Several glycolipid bands were present having a thin layer mobility as for compounds from one up to 12 sugar residues. The total amount of non-acid glycolipids obtained from 120 animals was 1.2 g which corresponded to 6.8 mg/g of tissue (dry weight) or about 9 mg/animal.
Isolation of Pure Glycolipids-The A-4 glycolipid was purified by repeated column chromatography as acetylated derivative. The native glycolipid fraction, eluted with 0-15 volume % CH:,OH in CHCln from the silicic acid column described above, was acetylated. Thin layer chromatography revealed that the distance between the acetylated tri-and tetraglyco-
sylceramides was widened compared to the native derivatives. This change in relative thin layer mobility was probably due to the amino sugar present in the tetraglycosylceramide fraction. In between these two major compounds, a weak glycolipid band was detected. The acetylated mono-to tetraglycosylceramide fraction was applied on a LiChroprep column eluted by 0, 0,0.5, 1, 1.5,2, and 75 volume W CH,OH in CHC1:I. The fraction eluted with 1.5 volume 5% contained A-4 and part of the ordinary tri-and tetraglycosylceramides. This component was further purified in a similar way on several Li-Chroprep columns until a pure single band was seen on the thin layer plate. After deacetylation, a major band was seen migrating as a triglycosylceramide (Fig. 1, lane A-4). Two faint bands were also present migrating just in front of and behind the major compound. The weight of this fraction obtained from 80 animals was 4.6 mg. In addition, a 3.8-mg fraction was obtained which was contaminated by the triglycosylceramide.
The A-6 glycolipid was isolated from 1.2 g of total non-acid glycolipids altogether (120 animals). The batch of glycolipids having five to eight carbohydrate residues, isolated with 15-30 volume B CH:jOH in CHCh by repeated silicic acid column chromatography as described above, was further fractionated on several silicic acid columns. Two fractions containing the six-sugar components were obtained partly pure. These were acetylated and the major components seen on thin layer chromatography were purified by repeated LiChroprep column chromatography. These glycolipids were eluted from the columns with 0-0.5 volume B CHsOH in CHCh due to a higher ethanol contamination of the CHCln as noted above (compare the A-4 glycolipid). In this way, three acetylated glycolipid fractions were obtained which were pure or nearly pure as seen on the thin layer plate. After deacetylation, only one fraction was homogeneous (Fig. 1, lane A-6). The other two were contaminated with more slow moving components. The weight of the pure glycolipid fraction was 11.3 mg but the total weight of the A-6 component in the total glycolipid fraction was estimated to about 40 mg.
The A-12 glycolipid was isolated from the native fraction eluted with 30-100 volume 5% CH:IOH in CHCLI from the initial silicic acid column described above. This fraction was shown by thin layer chromatography to consist of a major component and several minor ones. The major glycolipid was purified by repeated silicic acid chromatography. The partly pure fraction obtained was acetylated and further fractionated on Li-Chroprep columns. A pure fraction eluted with 1.75 to 2 volume B CH:,OH in CHCl,, was deacetylated, and this fraction was finally purified on a silicic acid column. 8.0 mg was obtained pure (Fig. 1, lane A-12) from 120 animals and, in addition, about 7 mg was present in nonpure fractions.
During the preparation of the A-6 compound, a glycolipid fraction was eluted as acetylated derivative just in front of the A-6 glycolipid. After deacetylation, this fraction showed a major band migrating as a five-sugar compound. It was further purified on a silicic acid column and a homogeneous fraction was eluted with 15 volume $6 CH,,OH in CHCh The weight of this fraction was 3.7 mg, isolated from 120 animals.
Structural Characterization of the A-4 Glycolipid-The mass spectra of the permethylated and permethylated-reduced A-4 glycolipid are shown in Figs. 2 and 3, respectively. A series of intense ions, formed by a loss of part of the long chain base, was found at m/e 1125 to 1237 (Fig. 3). These ions are evidence for four-sugar residues (1 Fuc, 1 hexosamine, and In addition to the A-4 glycolipid, small amounts of a contaminating trihexosylceramide were seen in the mass spectra. This was best evident in the reduced spectrum where the peaks at m/e 924 (16:O fatty acid), 980 (200), lo08 (220), and 1036 (24:O) were due to three hexoses and the fatty acid which is similar to the series at m / e 1125 to 1237 for the A-4 glycolipid. Terminal hexose was shown by m / e 219 and 187 (219 -32). In the permethylated spectrum (Fig. 2), trihexosylceramide fragments were found at m / e 219, and 187 and at m / e 1038, 1066, and 1094. This contamination explained the weak band seen in front of the major A-4 band on the thin layer plate (Fig. 1, lane A-4). The major A-4 band consisted of the longer fatty acid (20:O to 24:O) species and the weak band seen behind was due to the shorter fatty acid (16:O) species.

Blood Group A and H Actiue Glycolipids
Degradation and gas chromatography of the native A-4 glycolipid showed the presence of alditol acetates of Fuc, Glc, Gal, and GalNAc with a molar ratio of about 1:1:1:1 (Table I) in agreement with the mass spectrometric data. The gas chromatogram of the degradation products obtained from the permethylated glycolipid fraction is shown in Fig. 4, chromatogram A. The peaks were identified as the acetates of 2,3,4trimethylfucitol (designated Fucl -+), 2,3,6-trimethylglucitol (-+ 4Glcl -+), 4,6-dimethylgalactitol (2 ;Gall +) , and 3,4,6-trimethyl-2-N-methylacetamido-2-deoxygalactitol (GalNAcl -). The peaks from the contaminating trihexosylceramide were very small, which made them impossible to identify among the by-products from the degradation. The results of the degradation of the permethylated-reduced derivative are shown in Fig. 5, chromatogram A. The reduction of the amino sugar makes the glycosidic bond nearest the amino sugar nitrogen-resistant against degradation at the conditions used for the permethylated derivative, and therefore di-and trisaccharides are obtained' (21,22). Compared with the chromatogram of the permethylated derivative (Fig.   4A), the peaks due to the Gal + 2Gall + and the GalNAc (GalNAcl -+) were lacking and, instead, a peak with a very long retention time had appeared GalNAcl + 3Ga12 . The Fuc and Glc peaks were unchanged. This peak has been identified by mass spectrometry to be the acetate of 3-(3,4,6trimethyl-2-Nmethylacetamido -2deoxygalactopyranosyl) -4,6-dimethylgalactitol. The presence of this disaccharide excluded the possibility that Gal instead of Glc was located nearest the ceramide and also conclusively established that Fuc was bound to C-2 of the Gal and not to C-3.
The structural data presented so far established the type and sequence of sugars as well as the ceramide composition. Information on the anomerity of the glycosidic bonds was given by proton NMR spectroscopy. The interpretation of NMR spectra of intact permethylated and permethylatedreduced glycolipids was based on the fact that the coupling constants for a and P protons differ and that the chemical shifts of separate glycosidic bonds may differ (6,23,24). In addition, the reduction introduces a change of the chemical shift for some signals (24). The anomeric region of the NMR spectrum of the permethylated-reduced A-4 glycolipid is reproduced in Fig. 6, spectrum A. This spectrum showed a sharp signal of Fuc at 5.32 ppm (J1,2 = 3.4 Hz) due to an a proton. The signal seen at 5.09 ppm (J,,z = 2.7 Hz) probably originated from H-1 of a-GalNAc (24). The doublet at 4.20 ppm (J1.2 = 7.4 Hz) was due to H-1 of P-Glc and the signal at 4.28 ppm came from H-1 of /&Gal. The peak at 5.15 ppm was due to a contamination of the C'HCb solvent.
The trihexosylceramide found as a contaminant by the mass spectra gave a signal at 4.94 ppm, due to H-1 of the terminal &-Gal (23). The two other signals of this glycolipid were buried in the / 3 region at 4.28 and 4.20 pprn, respectively.
In conclusion, the structural studies of the A-4 glycolipid showed it to be a tetraglycosylceramide with a blood group A determinant and the following stmcture GalNAcpcrl + 3Galp(2 c laFucp)~l"-$4Glcp~l+ 1Cer.

Carbohydrate composition (in per cent) of rat small intestine tetraglycosylceramide (A-4), hexaglycosylceramide (A-6), and dodecaglycosylceramide (A-12)
Chromatographic peak areas were measured without correction for individual sunars. The A-4 glycolipid inhibited hemagglutination of human blood group A red cells by anti-A antisera (Table 11).
Structural Characterization of the A-6 Glycolipid-The complete structure of the A-6 glycolipid was established by the same methods as for the A-4 compound. The mass spectra (Figs. 7 and 8) were very similar to those obtained from a hexaglycosylceramide of human small intestine (5). The degradation studies (Table I, Figs. 4B and 5B) revealed the addition of a + 3GlcNAcPl-3Gal@1+ saccharide residue compared to the A-4 compound. The anomeric region of the proton NMR spectrum of the permethylated-reduced A-6 glycolipid (Fig. 6B) was similar to that of the erythrocyte A-6 glycolipid (24). The only difference was the signal due to the H-1 of the P-Gal bound to the GlcNAc. In this case, this signal was present at 5.22 ppm = 7.4 Hz) due to a 1 + 3 bond (type 1 saccharide chain) compared to the erythrocyte compound (type 2 saccharide chain) where the corresponding signal was buried in the @-hexose region at 4.3 ppm (24).
Thus, the data presented established the A-6 glycolipid from rat small intestine as a hexaglycosylceramide with a blood group A determinant and based on a type 1 carbohydrate chain. The complete structure was GalNAcpcul 3Galp(2 c lFucpa)Pl+ 3GlcNAcpPl+ 3 G a l p p l j 4GlcpPl + 1Cer. The ceramide part was made up of almost exclusively trihydroxy 18:O long chain base in combination with both nonhydroxy and hydroxy 16:O-24:0 fatty acids.
This glycolipid inhibited the hemagglutination of human

Blood Group
A and H Active Glycolipids Fuc 01-2 -4Glc)l"ICER blood group A red blood cells by anti-A antisera ( Table 11). The immunological activity of the A-6 glycolipid was identical with that of the type 2 chain isomer of human erythrocytes and higher than that of the A-4 compound.
Structural Characterization of the A-12 Glycolipid-The total non-acid glycolipid fraction (Fig. 1, lane T) contained a major slow moving compound which was isolated pure (lane A-12). Mass spectrometry of the intact glycolipid showed it to be a branched dodecaglycosylceramide with two blood group A determinants (26). The branching point was located on the fourth sugar residue from ceramide and type 1 (Gall + 3GlcNAc) carbohydrate core saccharides were dominating. Acid degradation and gas chromatography of the native A-12 glycolipid revealed Fuc, Glc, Gal, GlcNAc, and GalNAc as constituent sugars (Table I). Due to the volume of data, the complete structure wiU be presented el~ewhere.~ The A-12 glycolipid showed a strong blood group A activity (Table 11) and the capacity to inhibit hemagglutination by anti-A antisera was similar to the A-6 glycolipid based on an equal number of single blood group A saccharide determinants.
Structural Characterization of the Blood Group H Type Precursors of the Blood Group A Glycolipids-The blood group A glycolipids presented here are probably synthesized by enzymatic addition of the terminal GalNAc to the corresponding blood group H precursors (27,28). In a recent paper, we described the presence of a blood group H active triglycosylceramide (H-3) isolated from the black-white rat small intestine (29). The structure was shown to be Fucpal --$ M. E. Breimer, G. C. Hansson, K.-A. Karlsson, and Leffler, H., manuscript in preparation. 2GalpPl+ 4Glcpbl+ 1Cer.
A glycolipid fraction, having a thin layer mobility as a fivesugar compound, isolated during the preparation of the A-6 glycolipid (see above) was structurally characterized by mass spectrometry of the permethylated and permethylated-reduced derivatives, by NMR spectroscopy of the reduced derivative and by degradation-gas chromatography of the native and permethylated glycolipid. The structural data (not shown) established this fraction to be a mixture of two components. The major one was a blood group H type pentaglycosylceramide with the probable structure Fucpal + 2GalpPl + 3GlcNAcpfil+ 3Galppl+ 4GlcpPl-1Cer. component was Gal-GalNAc-Gal-Glc-Cer. The NMR spectrum indicated that this glycolipid may be gangliotetraosylceramide. It was not possible to separate these two species due to lack of material and a very close chromatographic behavior.
A branched 10-sugar blood group H glycolipid (H-10) expected to be the precursor to the A-12 compound and identified in a white rat strain (30)3 was not present in the blackwhite rat small intestine.
Tissue Localization of Blood Group A and HGlycolipids-We have modified the technique of Weiser (31) for isolation of mucosa epithelial cells from rat small intestine (30,32). 3 The epithelial cells were completely removed from the intestinal stroma (32). 3 The total non-acid glycolipid fraction of the epithelial cells is shown in Fig. 1, lune C. Some glycolipids present in the total intestine were lacking in the epithelial cells. A detailed analysis of the epithelial cells and the residual stroma showed that blood group A and H glycolipids were exclusively present in the epithelial cells (30).3

DISCUSSION
The glycolipid composition of rat small intestine is very complex and we have isolated about 30 different carbohydrate structures of which several were earlier unknown (30,33). Three blood group A glycolipids with 4, 6, and 12 sugar residues and two blood group H compounds with 3 and 5 sugar residues were isolated from the small intestine of an inbred rat strain (black-white). In addition, a blood group H type compound with terminal Fuc linked to four hexoses was found in small amounts (33).3 The A-4 glycolipid was shown to have F u c d + 2 and GalNAcd -+ 3 linked to the Gal of lactosylceramide. The six-sugar compound (A-6) was based on a type 1 carbohydrate core structure which means Fucal + 2 and GalNAcd + 3 linked to the terminal galactose of lactotetraosylceramide. The third blood group A glycolipid (A-12) was a branched dodecaglycosylceramide with two blood group A determinants.
The exact location of the terminal Fuc and GalNAc on the subterminal Gal of the A-4 and A-6 glycolipids was established by degradation and gas chromatography of the permethylated-reduced derivative. The reduction of the amide group of N-acetylamino sugars makes the glycosidic bond on C-1 of the amino sugar resistant against acid degradation at the experimental conditions used for permethylated glycolipids (21). 3 The disaccharide obtained from the A-4 glycolipid showed that GalNAc was linked to C-3 of the subterminal Gal and therefore Fuc was located at the C-2 of the Gal. This information was also obtained for the A-6 compound by one of the two disaccharides obtained due to the presence of both GalNAc and GlcNAc. In addition, important information about the sequence of the sugars was given by these disaccharides.
The chemical identification of blood group A glycolipids was supported by their ability to inhibit hemagglutination of human red cells by anti-A antisera. The immunological activity was identical for the rat A-6 compound and the corresponding type 2 chain isomer of human erythrocyte. The reactivity of the A-6 and A-12 compounds was the same when corrected for the fact that each A-12 glycolipid contained two A saccharides. The A-4 glycolipid had a weaker blood group A activity. The identical reactivity of type 1 and 2 chain A-6 compounds indicated that the GlcNAc is not specifically involved in the binding of the antibodies to the antigen. However, the addition of Fucal + 4 by the Lewis enzyme to GlcNAc of the A-6 glycolipid or the corresponding blood group B-6 glycolipid completely abolishes the blood group activity (34) showing that some part of the antibody extends over the GlcNAc residue. The weak A activity of the A-4 glycolipid may be explained by the short carbohydrate chain (only one sugar) onto which the antigenic determinant is linked.
The A-4 glycolipid has not been described before and is so far the smallest blood group A active glycolipid isolated. Recently, a free tetrasaccharide was isolated from human urine and shown to have the same structure as that of the rat A-4 glycolipid (35). A corresponding blood group B active tetraglycosylceramide with terminal Gal instead of GalNAc was present in the large intestine of the black-white rat but has not been found in the small intestine (36).
The blood group A glycolipids present in the black-white rat small intestine are probably synthesized by addition of a terminal GalNAc to the corresponding blood group H precursors (7, 27, 28). Blood group H glycolipids having three (H-3) and five (H-5) sugars were present in the intestine but the branched 10-sugar compound (H-10) was lacking. The H-10 glycolipid was, however, present in another rat strain (white) together with the H-3 and H-5 glycolipids (30,37). In this strain, blood group A glycolipids were completely lacking. This finding indicates that there may be different enzymes for the synthesis of the short and long carbohydrate chains or that the A-12 glycolipid is synthesized by a multiglycosyltransferase system which does not release the precursor. Other explanations may be that the short and long carbohydrate chain glycolipids are synthesized in different cell compartments or that the enzyme is more effective on longer carbohydrate chains and completely converts the blood group H determinant to A.
In relation to this, one may note that the ceramide composition of the A-4 and H-3 glycolipids differed. The A-4 glycolipid contained non-hydroxy fatty acids having a bimodal chain length distribution with predominantly 16:O and 24:O acids while the H-3 glycolipid had mainly non-hydroxy 200 and 22:O acids (29). 3 This may indicate that the H-3 is not the precursor for the A-4 glycolipid or that certain molecular species are selected by the glycosyltransferase.
In human erythrocyte blood group A type glycolipids with

Blood Group
A and H Active Glycolipids 6,8,9, and 12 sugar residues have been proposed (4, 16,38). The 8-sugar compound, which was a nonbranched compound, was completely lacking in the rat small intestine.
All the blood group A and H active glycolipids of the rat small intestine were located in the mucosa epithelial cells.

This was shown by a detailed glycolipid analysis
of both isolated epithelial cells and the residual intestinal stroma (30,32). 3 The biological significance of the cell, tissue, and individual specificity of the distribution of glycolipids remain to be shown.