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Glycosphingolipid patterns of the epithelial and non-epithelial compartments of rat large intestine
281
Biochimica et Biophysics Acta, 192 (1984) 281-292
Elsevier
BBA 51581
GLYCOSPHINGOLIPID PATTERNS OF THE EPITHELIAL COMPAR~ENTS OF RAT LARGE INTESTINE GUNNAR
C. HANSSON,
Department
of Medical
(Received
KARL-ANDERS
Biochemistry,
KARLSSON
AND NON-EPITHELIAL
and JAN THURIN
University of Giiteborg, I? 0. Box 330 31, S - 400 33 Gijteborg (Sweden)
September 28th, 1983)
Key words: Glycosphingolipid pattern; Compartmentation;
(Rat large intestine)
The epithelial cells and the non-epithelial residue from large intestine of two inbred rat strains were separated and the glycosphingolipids characterized in comparison with earlier detailed data from small intestine of the same strains. Total acid and non-acid glycolipids were prepared and the non-acid glycolipids were further fractionated into subgroups as acetylated derivatives on silicic acid. The fractions obtained were characterized mainly by #in-layer c~omat~phy, including binding of monoclonal anti-A and anti-B antibody to the chromatogram, and by direct-inlet mass spectrometry after derivatization. This combined technology allowed an overall conclusion from a small number of animals concerning relative amounts of glycolipids, microheterogeneity of blood group glycolipids and carbohydrate sequence and lipophilic components of major species of each subf~ction. As for the small intestine, the two separated ~omp~ments differed distinctly in composition, with blood group fucolipids being confined to the epithelial cells, and a series of glycolipids with probably internal Gala being restricted to the non-epithelial part. The main difference between large and small intestine concerned fucolipids of the epithelium. Three blood group B active glycolipids with four, six and seven sugars were detected which were absent from the small intestine. The four-sugar glycolipid was a major ~ycolipid with the structure Gala1 -+ 3Ga1(2 + laFuc)/31-+ 4Glc/31 + leer, as reported before. The six-sugar glycolipid was shown by mass spectrometry and NMR spectros4Glc/31 --f copy to have the probable structure Gala1 --+3Ga1(2 + laFuc)/31+ 3GlcNAc/31 --f 3GalBl+ 1Cer. The seven-sugar glycolipid had an additional fucose linked to N-acetylhexosamine, as shown by mass speetrometry. Three blood group A active glycolipids with four, six and seven sugars were found in both rat strains, with sequences analogous to the B glycolipids but with a terminal GalNAc instead of Gal. The fourand six-sugar blood group A compounds, but not the seven-sugar glycolipid, have been found before in the small intestine of one of the rat strains. In the small intestine, on the other hand, a branched-chain twelve-sugar blood group A active glycolipid has been found which was absent from the large intestine. Therefore large intestine of both rat strains expressed glycolipid-base blood group A and B activity, while small intestine lacked B activity and showed A activity only in one of the strains. Quantitatively the major glycolipids of the epitbelial cells of large intestine were monoglycosylceramides (glucosylceramides, and smaller amounts of galactosylceramides which were absent from small intestinal epithelhun) and tetraglycosyleeramides (including the A and B active species and a te~ahexosylceramide). The major li~philic components of the epithelial cell glycolipids were phytosphingosine and long-chain hydroxy fatty acids.
The glycohpid nomenclature used follows an IUPAC/IUB r~omm~dation (Eur. J. B&hem. (1977) 79, 11-21). Hex means hexose, HexN means N-acetyihexosamine and Cer means ceramide. Trihydroxy base is abbreviated t, and 2-hydroxy 0005-2760/84/$03&O
0 1984 Elsevier Science Publishers B.V.
fatty acid h. A figure before a colon specifies chain length, and after a coton, number of double bonds; t18 : 0 therefore means phytosphingosine or hydroxysphinganine.
282
Introduction The large intestine is of biomedical interest because of frequent diseases of man, including tumor formation from epithelial cells [l] and infections where bacteria are supposed to adhere to the epithelial cell surface [2,3]. In both these phenomena glycosphingolipids have been shown to be involved. A ganglioside of diagnostic interest [4] was recently identified in human colon cancer cells [5,6], and glycolipids of the globe series have been proposed as adhesion receptors for Escherichia co/i, causing urinary tract infection in man [7,8]. The present work is part of a collaboration with a group studying rat colon cancer [9,10] and of a systematic study on glycolipids as receptors for microorganisms [ 111. In earlier studies the small intestine of two inbred rat strains was subjected to a detailed structural analysis of glycolipids [12-151, including the isolation of epithelial cells of different maturity [16]. In the present case the large intestines of the same rat strains were used for preparation of epithelial and non-epithelial compartments to study the glycolipids. The approach was mainly a comparison with well-characterized glycolipids of the small intestine using thin-layer chromatography and mass spectrometry of subfractions after column chromatography of acetylated glycolipids. Monoclonal anti-blood-group A and B antibodies were applied directly on the thin-layer chromatogram [17,18], allowing a conclusion on microheterogeneity of blood group fucolipids. Direct-inlet mass spectrometry and a fractional evaporation. of derivatized glycolipid subfractions presented saccharide sequences permitting comparison with thin-layer chromatogram data [19]. Materials and Methods Animals Two inbred strains of rats were used, SpragueDawley strain (white rat) kept separate for more than 35 years, and a hooded strain (black-white), kept separate for 30 years at the Department of Pharmacology in Goteborg. Only male rats were used. Non-fasting animals, about 6 months old, were killed by decapitation under ether anaesthesia. The caecum-colon or colon was immediately removed and prepared for cell removal.
Remooal of epithelial cells Two preparations were used, one with colon cut right distal to caecum and one including caecum cut 1 cm proximal to the ileo-caecal valve. Both preparations were cut as far distal as possible. The caecum-colon preparation was ligated at the ileocaecal valve in a way that did not allow ileal mucosa to be in contact with the lumen of caecum. It was then cut open in the part of the preparation farthest away from the distal opening to avoid a blind end. The preparations were washed, tied onto plastic nipples, filled with solution A (96 mM NaCI/l.S mM KC1/27 mM sodium citrate/g mM KH,PO,/5.6 mM Na,HPO,, pH 7.3) and incubated for 15 min at 37°C as described [16], a procedure which was adapted from the method of Weiser [20]. This fraction together with one washing volume did not contain any cells and was discarded. The preparations were then sequentially incubated with aliquots of solution B (137 mM NaCl/3 mM KC1/1.5 mM KH,PO,/6.5 mM Na,HP0,/1.5 mM EDTA/O.S mM dithiothreitol, pH 7.3). During each incubation the intestine was elevated gently three times every 20 s and afterwards washed three times with one intestinal volume of solution B (the intestinal volume increased approx. 2-3% for every incubation): The incubation times were 6, 6, 5, 5, 5, 5, 5, 5, 5, and 10 min, respectively, and the fractions were collected in centrifuge tubes and centrifuged at 250 x g for 5 min. The supernatants including floating mucus were sucked off and the cells washed twice in phosphate-buffered saline, pH 7.3, by centrifugation at 250 x g for 5 min and suspended in phosphate-buffered saline. Cells were then taken for protein determination [21] and microscopy. For phase-contrast microscopy segments of intestine were taken after various incubation periods, fixed in formalin, embedded in wax, sectioned and stained with Van Gieson’s stain [22]. After centrifugation as above and removal of the buffer, 3 ml methanol were added to each fraction. The fractions were then pooled and stored at -20°C. The non-epithelial residues were cut off by the nipples and stored at -20°C. Preparation and fractionation of glycosphingolipids The pooled epithelial cells were extracted, subjected to mild alkaline methanolysis, dialysed and
283
separated into non-acid and crude acid glycolipid fractions. The total non-acid fractions and crude acid fractions from both epithelial cells and non-epithelial residues were prepared essentially as described in Ref. 16. The crude acid fractions from the same origins were acetylated and run on a silicic acid column as in Ref. 16, and then deacetylated with mild alkaline methanolysis [16] to yield a purified total acid fraction. 4 mg each of non-acid glycolipids of the epithelial cells and non-epithelial residue from the caecum-colon preparations of the white rat were acetylated twice overnight in darkness with chloroform/pyridine/acetic acid anhydride (1 : 1 : 1, by vol.). A fractionation of the acetylated mixture was done on a silicic acid column, using 10 g of Iatrobeads 6RS-8060 (Iatron Laboratories Inc., Tokyo) and eluting with 200 ml of a continuous gradient, O-10% (by vol.), of methanol in chloroform. Approx. 70 fractions, each of 3 ml, were collected, analysed by thin-layer chromatography and pooled together into six fractions. Mass spectrometry Direct-inlet mass spectrometry was done on total and on subfractionated non-acid glycolipids of the different origins. Permethylated [23] and LiAlH,-reduced permethylated [24] total glycolipid fractions were analysed on an AEI MS 902 instrument (Kratos) in the electron ionization mode using an Instem (Kratos) data system [19,25] and the ‘in beam’ technique [26,27]. The ion source temperature was raised linearly and mass spectra were recorded at fixed intervals and stored in the data memory. The selected ion curves showing intensity of different ions versus temperature were reproduced by a Tectronix hard copy unit. Mass spectra of permethylated, non-reduced glycolipid subfractions were analysed ‘in beam’ on a ZAB-HF instrument (VG Analytical). More details about technical conditions and interpretation of spectra have been given elsewhere [19,25-271. NMR analysis NMR spectroscopy was performed on the permethylated-reduced blood group B active hexaglycosylceramide. The spectrum was recorded on a Bruker 270 MHz instrument using 1 mg sample in 0.5 ml of C’HCl, at 313 K and 6200 pulses.
Thin-layer chromatography Thin-layer chromatograms were run on HPTLC Si 60 precoated nanoplates (Merck). Detection was done with the anisaldehyde reagent [28] and the resorcinol reagent [29]. Thin-layer chromatography on a borate-containing layer was done as described [ 301. Binding of monoclonal anti-blood-group antibody to a thin-layer plate (chromatogram binding assay) The chromatogram binding assay was done as described in Ref. 18, which is a method adapted from Ref 17. The anti-blood-group B antibody E, 83-52 was derived from cell culture supernatants of hybridomas that were the result of immunizing mice with Capanpancreas carcinoma cells, and its specificity has been reported [18]. The antiblood-group A antibody M/D C,K,C,, was obtained by immunizing mice with human erythrocytes of blood group A and was produced in ascites. It was a gift from Arne Lundblad, Department of Clinical Chemistry, University of Lund, Sweden. The antibody reacts with human erythrocytes of blood group A but not of 0 or B. It reacts with isolated glycolipids with monofucosyl or difucosyl blood group A determinants on a type 1 or .type 2 saccharide chain (unpublished results). Results Epithelial cell isolation Epithelial cells were removed essentially as described earlier [16]. This cell-releasing method with chelating agents but no enzyme gives a good yield of viable cells [31-341. Biopsies taken before and after cell removal showed a total release of epithelial cells without any apparent damage to the basal membrane (Fig. 1). For colon, in contrast to small intestine [16], a sequential removal of villus tip cells and crypt cells, as viewed with phase-contrast microscopy (not reproduced), was not found. Conglomerates of cells resembling whole crypt bottoms were seen in almost all of the eluted fractions, which indicated a more random removal from caecum and colon than from small intestine, which may be explained by the different histology of the two mucosae [35]. From the white rat strain two preparations were done, one from five animals
284
TABLE
I
COMPARISON BETWEEN ABSOLUTE WEIGHTS OF LYOPHILIZED TISSUE, TOTAL PROTEIN AND TOTAL ACID AND NON-ACID GLYCOSPHINGOLIPID FRACTIONS FROM EPITHELIAL AND NON-EPITHELIAL CELL COMPARTMENTS OF TWO INBRED RAT STRAINS Figures are given in mg/animal. black-white rat, two animals.
White
rat, ten animals
and
White rat caecum + colon
Black-white rat caecum + colon
Epithelial
Epithelial
Non-epithelial
cells
cells
_
230 100
cells Dry weight Protein 85 Acid glycolipids 0.28 Non-acid glycolipids 0.81
Non-epithelial cells 280 120
85
0.52
0.30
0.90
0.84
0.84
1.35
1
3 4 5 7
Fig. 1. Light microscopy of the colon of the white rat before (top) and after (bottom) epithelial cell removal. Staining as given in Materials and Methods. Magnification x 410.
IO
ABCDEFGH with colon only and one from ten animals with caecum-colon. A caecum-colon preparation from two animals of the black-white rat strain was done for comparison. Data from these preparations are shown in Table I. Epithelial cell total non-acid glycolipids The thin-layer chromatogram of Fig. 2 of the non-acid glycosphingolipids of the epithelial cells of large intestine (lanes B-D) showed striking differences compared to the same fraction of the
Fig. 2. Thin-layer chromatogram of total non-acid glycosphingolipids from the epithelial cells (A-D) and non-epithelial residues (E-H), from the small intestine of the white rate (A and E), from colon of the white rate (B and F), from coloncaecum of the white rat (C and G), and from colon-caecum of the black-white rat (D and H). The amount of glycolipids applied corresponded to 8 mg protein for the epithelial fractions B-D, 2 mg protein for A and 4 mg protein for the residue fractions E-H. The numbers to the left indicate numbers of sugars of the glycolipids. All bands were green with the anisaldehyde reagent, as for glycolipids, except for the bands marked x. Solvent was chloroform/methanol/water, 60 : 35 : 8 (by vol.).
285
small intestine (lane A). Major glycolipids of the large intestine appear in the intervals of one, three, four and about six sugars. Minor differences in the seven sugar interval can be seen between the two rat strains (lanes C and D) and between the two preparations from colon only and caecum-colon (lanes B and C). The amounts of cells estimated as protein from the epithelium of the two strains were almost identical as were also the total amounts of non-acid and acid fractions obtained from these cell preparations (Table I). The glycosphingolipid species in the epithelial cells of caecum-colon of the white rat as found by mass spectrometry are summarized in Fig. 3. The permethylated-reduced glycosphingolipid mixture was introduced by the ‘in beam’ technique. The source was heated linearly and mass spectra (scans in Fig. 3) were recorded at fixed intervals [19]. The change in relative intensity of selected ions for separate glycolipids was reproduced as curves along the temperature and scan scales. The ions selected in this case are usually relatively abundant [19] and contained the complete saccharide chain to-
m/z
25 240°C
For more detailed information 4 mg of total non-acid glycolipids of epithelial cells of caecumcolon from ten white rats were acetylated and
Base
R
t18:O h24:O t18:O h24:O
1066
HexHexHex-CH2
t18:O h24:O
1270
HexHexHexHex-CH2FHNMe-R
t18:O h24:O
Hex(Fuc)-Hex-CH2yHNMe-
894
0
non-acid glycolipids of the epithelial
cells
Hex-CHzcHNMe-R ~ex~ex-CH2FHNMe-R
862
16O'C
Subfractionated
F-Fragment
608
Scan
gether with one fatty acid species and part of the long-chain base. The fatty acid species selected for reproduction were chosen from complete paper spectra to allow a minimum of interference from other non-relevant fragments. Oligohexosylceramides with one to four sugars (m/z 608, 862, 1066 and 1270, respectively) were detected together with blood group type fucolipids with H-type terminal (m/z 894), B-type terminal (m/z 1098, 1675 and 1793) and A-type terminal (m/z 1267 and 1702). The sequences of the sugars as given in the simplified formulas were deduced from supplementing ions found in the complete spectra of permethylated and permethylated-reduced mixtures (not shown). Phytosphingosine. was the major longchain base, as shown by m/z 396 of spectra of permethylated mixtures.
R
tl8:O
16:0
16:0
1098
Hex-Hex(Fuc)-Hex-CH2
R
t18:O
1267
HexN-Hex(Fuc)-Hex-CH2FHNMe-
R
t18:O h24:O
R R
t18:O h24:O
1675
(X10)
1793 1702
(X10) (X10)
Hex-Hex(Fuc)-HexNHexHex-CH2FHNMeHex-H~x(Fuc)-H~xN(Fuc)H~xHw-CH~
R
t18:O h20:O t18:O h24:O
50 325OC
Fig. 3. Selected ion curves of the fatty acid-carbohydrate-containing peaks from mass spectrometry of the major non-acid glycosphingohpids of the epithelial cells in colon-caecum of the white rat. The curves reproduced correspond to relative abundances of saccharide plus fatty acid (R) ions (see simplified formulas) of glycosphingolipids as a function of evaporation temperature. 60 gg of permethylated-reduced mixture were evaporated ‘in beam’ by raising the ion source temperature linearly with 5.2 K/mm. Spectra were recorded every 38 s. The electron energy was 50 eV, accelerating voltage 4 kV and trap current 500 PA. The letters to the left mark the structures and refer to Fig. 4 and the text. For abbreviations, see footnote on page 281.
286
4
3
12
12
3
4
5
6
3
4
5
6
123456
7
8
1,2
3
123456
4
5
123456
6,7 8
Fig. 4. Thin-layer chromatograms of the fractions obtained after fractionation of the acetylated glycosphingolipids of the epithelial cells (A) and non-epithelial residue (B) of the white rat. Acetylated fractions (1) and deacetylated fractions (2) were visualized by the anisaldehyde reagent. Chromatograms 3 and 4 were autoradiographed (72 h) after labelling a plate identical to A-2 with monoclonal ascites diluted lt 100) and anti-B antibody (ES 83-52, culture supernatant diluted 1+4), anti-A antibody (M/D C,K,C,,, respectively. The amounts of sample used in A-3 and A-4 were one-third of the amount in A-2. For chromatogram A-2 about 1% was applied of fractions 1 and 2,4% of fraction 4, and 10% of fractions 3, 5 and 6. For B-l and B-2 about 5% of each fraction was applied. Solvent was chloroform/methanol (96 : 4 by vol.) for plate 1, and chloroform/methanol/water (60 : 35 : 8 by vol.) for plates 2, 3 and 4. The bands marked by letters refer to structures in Fig. 3 and in the text. The bands marked x refer to non-glycolipid contaminants.
eluted by a continuous gradient from a silicic acid column. 70 fractions were pooled into six major fractions, which were deacetylated and analysed by thin-layer chromatography using anisaldehyde or antibody for the detection, and by mass spectrometry after permethylation. As shown in Fig. 4 several glycolipids had a reversed order of migration after deacetylation (chromatogram A-2) compared to acetylated derivatives (chromatogram Al), which migrate in the same order as they elute from the column. For the identification of several glycolipid
species a comparison was done with glycolipids isolated from rat small intestine, both concerning chromatographic mobility, mass spectrometric fragmentation and immunological properties [12]. Blood group active glycolipids In chromatograms A-3 and A-4 of Fig. 4 blood group active glycolipids were detected with monoclonal anti-A and anti-B antibodies, respectively. For both antibodies there were bands stained with a chromatographic mobility corresponding to four, six and seven sugars.
287
A blood group B active tetraglycosylceramide was detected in lane 2 of chromatogram A-4 and corresponds to a major band marked f in lane 2 in chromatogram A-2 and detected with anisaldehyde. Mass spectrometry of this fraction showed the curve of m/z 1098 of Fig. 3 and sequence ions in accordance with the structure Gal& -+ 3Gal(2 + IcwFuc)/Il + 4Glcfll + 1Cer reported before for a rat large intestinal glycolipid [36]. The four-sugar blood group A active glycolipid of lanes 4 and 5 of chromatogram A-3 of Fig. 4 showed up as the band marked g in lanes 4 and 5 of chromatogram A-2. By mass spectrometry of these two fractions it was likely that the glycolipid was identical to the species isolated from rat small intestine and shown to have the structure GalNAcal + 3Gal(2 t- 1cwFuc)pl-+ 4Glc/31 -+ 1Cer [12,13]. This species produced the curve of m/z 1267 of Fig. 3 from the total mixture. The blood group A active hexaglycosylceramide in lanes 4-6 of chromatograms A-2 and A-3 of Fig. 4 and marked j may be identical to the structure GalNAccJ --+ 3Gal(2 c- laFuc),& -+ 3GlcNAc/31 + 3GalPl -+ 4Glc/31 -+ 1Cer identified in the small intestine [12,13]. Evidence for this sequence was given by the curve of m/z 1702 of Fig. 3 and by sequence ions from the permethylated sub-fractions. The seven-sugar glycolipid marked k in lane 5 of chromatogram A-2 had a difucosyl blood group A determinant. Evidence for this was the appearance of m/z 1057 and 1025 (1057-32) in the mass spectrum of the permethylated subfraction corresponding to the terminal pentasaccharide HexN-Hex(Fuc)-HexN(Fuc)as shown before [37]. Concerning the two blood group B active glycolipids marked h and i in chromatogram A-2 of Fig. 4, mass spectrometry of total epitheliaI glycolipids (Fig. 3) as well as permethylated fraction 4 gave evidence for the two B-type sequences shown at the m/z 1675 and 1793 curves of Fig. 3. Sequence ions after permethylation for a B-type monofucosyl glycolipid were found at m/z 842 and 810 (842-32) for four sugars and 1046 for five sugars, as well as M - 1 at m/z 1931 for the dominating species with phytosphingosine and hydroxy 20 : 0 fatty acid. The B-type difucosyl glycolipid was evidenced by m/z 1016 and 984 (101632) for five sugars and 1220 for six sugars, as well as 2105 for
M - 1 of the species with phytosphingosine and hydroxy 20 : 0 fatty acid. After further fractionation on silicic acid the six-sugar glycolipid was obtained pure for NMR spectroscopy as the permethylated-reduced derivative. The data obtained are in line with the structure Galrvl --) 3Gal(2 + lfuFuc)@ -+ 3GlcNac/31 + 3Gal/31 --* 4Glc/31 --, lCer, which is analogous to the six-sugar blood group A glycolipid [13]. Work on the preparation and further analysis of these two blood group B glycolipids is in progress. Although it appears from the relative intensities of the bands after detection with anisaldehyde (lane 4 of chromatogram A-2) that the six-sugar B-glycolipid predominated over the seven-sugar species, the reverse order of intensity was shown by antibody staining (lane 4, chromatogram A-4). This is in agreement with earlier data which showed a preference of this antibody for a difucosyl B deter~nant [18]. Other non-acid giycolipids of the epithelial cells The major glycolipid in large intestinal epithelium was monoglycosylcerarnide, as shown in Fig. 2, Fig. 3 (curve a) and Fig. 4 (lanes 1 of chromatogram A-l and A-2). The fraction was shown by separation on a borate-impregnated layer [3OJ to contain mainly glucosylceramide with phytosphingo~ine and hydroxy fatty acid and a smaller amount of galactosylceramide with the same ceramide. A smaller amount of diglycosylceramide was shown as band b in lane 1 of chromatogram A-2 of Fig. 4, and this was supported by mass speetrometry (curve b of Fig. 3). In the three-sugar interval two glycolipid species were detected, marked e and c in lane 2 of chromatogram A-2 (Fig. 4). These were also present in the small intestinal epithelium (Fig. 2) and identified as Fuccul -+ 2Galpl --f 4GlcPl + 1Cer [12,15] and globotriaosylceramide [12,14]. This conclusion was supported by mass spectrometry (Fig. 3 and not reproduced). Lane 3 of chromatogram A of Fig. 4 was almost free of blood group A or B activity and this fraction was shown by mass spectrometry to contain a tetrahexosylceramide, marked d (see also curve d of Fig. 3), a hex~exosylcer~de and a hexaglycosylceramide with five hexoses and a terminal fucose. These sequences were found also
288
in the small intestine and the first one was identified as Gala1 + 3Galal - 4Gal/31 + 4GlcPl ---) 1Cer [12,14]. Non-acid glycolipids of the non-epithelial residue As evident from the chromatogram of total non-acid glycolipids in Fig. 2, the patterns were similar for small and large intestine except for quantitative differences. The small intestine (sample E) had more di- and triglycosylceramides and relatively more of the rapidly moving band of monoglycosylceramide than colon (sample F), which was shown on a borate-containing layer [30] to be composed of glucosylceramide only, with mainly sphingosine and nonhydroxy fatty acids. There were no detectable strain differences (samples G and H), except for a higher amount of total glycolipid from the black-white strain (Table I). The qualitative similarity between small and large intestines was further substantiated after fractionation of the non-acid glycolipids as acetylated derivatives, pooling into eight fractions and deacetylation. Thin-layer chromatography (Fig. 4, chromatogram B), mass spectrometry after permethylation and permethylation-reduction and analysis with the two monoclonal antibodies revealed only one glycolipid in addition to those already shown for the small intestine [12]. Reactivity with anti-blood-group A or B antibody was lacking, except for a weak staining of band 1 in lane 8 of chromatogram B-2. The mass spectrum after permethylation of this fraction showed fragments for a pentaglycosylceramide with a sequence as for the Forssman glycolipid, with m/z 260 for terminal hexosamine, 505 for two hexosamines, and 709, 913 and 1117 for three, four and five sugars, respectively. All five sugars, two hexosamines and three hexoses, were also present in the fragment at m/z 1190 including part of the longchain base [38]. The dominating ceramide contained sphingosine and 24 : 0 fatty acid, as shown by m/z 1792 for M - 1. The glycolipid migrated, however, more slowly than authentic Forssman glycolipid from dog intestine [39,403. As this anti-A antibody was shown to cross-react weakly with Forssman glycolipid (unpublished data) the terminal sugar of the new glycolipid probably was GalNAccu. Work on the structure of this glycolipid is in progress.
Acid glycolipids of both compartments us shown by thin-layer chromatography An overall analysis by thin-layer chromatography was performed on total acid glycolipids of epithelial cells and non-epithelial residue of colon-caecum of the two rat strains in comparison with the corresponding two compartments of small intestine (Fig. 5). The major ganglioside of all fractions of large intestine seemed to be hematoside with N-acetylneuraminic acid, in contrast to small intestine where the epithelial cells contained hematoside with N-glycoloylneuraminic acid [12,41]. Also, the large intestine epithelium contained sulfatide, cerebroside 3-sulphate (not
Fig. 5. Thin-layer chromatogram of the total acid glycolipids from the epithelial cells (l-3) and non-epithelial residues (4-6), from the small intestine of the white rat (I and 4), from the colon-caecum of the white rat (2 and S), and from the coloncaecum of the black-white rat (3 and 6). A, N-acetylneuraminosyllactosylceramide; B, N-glycoloylneuraminosyllactosylceramide. Solvent was methylacetate/2-propanol/72 mM CaCI/S M NH, (45: 35: 15: 10 by vol.) and the detection reagent resorcinol. Bands marked x were yellow.
289
shown), which was lacking in the small intestine. The two strains differed concerning the total amount of crude acid glycolipids (Table I) and possibly in their ganglioside patterns. Several bands remain unidentified. Discussion The preparation of epithelial cells seemed to be quantitative as judged by light microscopy. A contamination of the non-epithelial residue with epithelial cells was also practically ruled out by the immunological analysis, which did not detect blood group active bands in the residues except for a Forssman-like glycolipid. As the detection limit for this assay is at the nanogram level [17,18] and
large amounts of glycolipids were put on the plate (data not shown), the negative result means that the actual contamination of epithelial cells in the residue after preparation should be less than one part in 104. On the other hand, it may be more difficult to rule out a contamination of the epithelial cell fraction with ceils from the non-epithelial residue. However, preliminary results from staining with a monoclonal antibody directed against isoglobotetraosylceramide, which is a major glycolipid of the residue, showed the practical absence of this glycolipid in the epithelial cell fraction (work in progress). The sequential dissociation of epithelial cells from the basal membrane according to level of cell differentiation, as found for the small intestine
TABLE II SOME EXAMPLES
OF STRAIN AND COMPARTMENT
DIFFERENCES
OF EPITHELIAL
GLYCOSPHINGOLIPIDS
LacCer, Gal/31 + 4GIcj31+ 1Cer.
Blood group H Fucal + 2LacCer Blood group A GalNAcal + 3 LacCer Fucal + 2 GaINAcal+ Fucal + 2
3
GaINAwl+ Fucal + 2
3
Gal/31 + 3GlcNAc/31+
3LacCer
Gal-HexN(-Fuc)-Hex-Hex-Cer
GalNAccrl + 3 Fucoll+ 2 Gal/31 -+ 3/4GlcNAcPl+ Gal/31 + 3GlcNAc/31* GalNAcal + 3 Galbl + 3GlcNAcPl+ Fuccrl-+ 2
Gala1 + 3
Small intestine
Large intestine
Small intestine
Large intestine ’
+
+
+
+
_
+
-
+
+
+
_
+
-
+
-
_
+
-
-
+
_
+
-
+
-
+
+
_
+
+
6 3LacCer 3
Blood group B Galal* Fucal + ZLacCer Gala1 + 3 Fucal ~ 2Gal/31 + 3GIcNAcPl+
Black-white rat
White rat
Glycosphingolipid structure
LacCer
a Identified by mass spectrometry of total non-acid glycohpids of whole intestine and chromatogram binding assay.
290
[16], was not possible using very similar conditions for the large intestine. Instead, conglomerates of cells apparently also from crypt bottoms were continuously eluted. A gentle mechanical scraping of the mucosa may possibly give access to more differentiated cells for separate study (cf. Ref. 42). Furthermore, the less differentiated cells can be prepared from a single-cell suspension by velocity sedimentation as shown in Ref. 43. The glycolipids of the two compartments prepared were worked up and compared with results of a detailed investigation done before on the two fractions from small intestine [12]. Some distinct differences were found, primarily restricted to the epithelial cells. Although all data were not presented, the non-epithelial residues of small and large intestine were very similar except for quantitative differences. A glycolipid similar to but apparently not identical to the Forssman glycolipid was found in large but not small intestine. A weak reactivity with the anti-A antibody indicated a terminal GalNAc in a-linkage. A blood group B-type hexaglycosylceramide with GalNAc instead of GlcNAc earlier identified in small intestine [12] was also concluded to exist in large intestine, based on immunology and mass spectrometry. This glycolipid did not bind to the anti-B monoclonal antibody used here [18], but was shown to react in a haemagglutination-inhibition assay [44] with conventional polyclonal anti-B antiserum, similar to the case for the glycolipid from small intestine [12]. The analysis was done on fraction 3 of chromatogram B-2 of Fig. 4, which also produced mass spectra in accordance with the existence of this sequence (band marked m in the chromatogram). The situation for the epithelial cells differed distinctly for large and small intestines and also between the two inbred strains of rat, as summarized for some glycolipids in Table II. Blood group B active glycolipids with four, six and seven sugars were identified in large intestine of both strains but were absent from small intestine. Concerning blood group A active glycolipids, the small intestine of the black-white rat, but not of the white rat, contained species with four, six and twelve sugars [12,45]. In the large intestine, however, both strains expressed blood group A activity of glycolipids with four, six and seven sugars,
probably with structures analogous to the B active glycolipids. The 12-sugar branched-chain species was apparently lacking in the large intestine, as shown by the absence of blood group A activity in this chromatographic interval. As the rat strains were inbred these differences are not due to heterogeneity within the limited populations used. One may also note that conventional ABH blood group typing on erythrocytes as a reference is not adequate in the rat, as these cells apparently do not reflect the blood group activities found in the epithelial cells [46], in contrast to the human [47,48]. The dominating glycolipids of the epithelial cells of large intestine were glucosylceramides and tetraglycosylceramides, including A and B active glycolipids and a tetrahexosylceramide. As apparent from mass spectrometry, the major lipophilic components were phytosphingosine and long-chain 2-hydroxy fatty acids, a situation similar to that in the epithelial glycolipids of small intestine [12]. The methodological approach of the present work has several advantages. A prefractionation of a total fraction into groups of glycolipids as acetylated derivatives before analysis by thin-layer chromatography including immunolabelling and by mass spectrometry may give advanced structural information without the need for pure glycolipid species. The reversed order of chromatographic mobility in some cases for acetylated compared with non-derivatized glycolipid may be diagnostic. Consider, for example, the B active glycolipids which eluted about one fraction ahead of the A active glycolipids with the same number of sugars (Fig. 4). Evidently, a terminal hexosamine may give a retardation compared to hexose in acetylated glycolipids. The reversed order of migration was true for the non-derivatized glycolipids, as shown by bands f and g in chromatogram of Fig. 4, which are B and A active tetraglycosylceramides, respectively. A second example was fraction 3 of chromatogram A-2 (Fig. 4) which contained glycolipids with four and six sugars and lacking hexosamine, which were eluted ahead of corresponding structures with terminal hexosamine present in the non-epithelial residue in fractions 5-7 (chromatogram B-2 of Fig. 4). Without this pre-separation into groups the com-
291
parison of mass spectral data with immunological detection in the case of minor components would have been overshadowed by information from major components. On the other hand, a further separation into pure glycolipid species would have needed both more material and laborious steps. The present approach may be limited in amount to 100 pg of each subfraction. The importance of the new binding assay for monoclonal antibodies [4,17,18] is obvious in the present case in the analysis of glycolipid mixtures. The location of a particular band is rather strictly related to the number of sugars in the glycolipid, allowing a direct comparison with mass spectral data. Analysis with conventional radioimmunoassay in microwells [17] of the different fractions from column chromatography may tell the total activity of the mixture in this fraction but not that the activity may be restricted to particular molecular species. In the actual case the chromatogram binding assay of the mixtures clearly showed the presence of A and B active glycolipids with four, six and seven sugars in the epithelial cell fractions and the absence of these in the non-epithelial residue. The two intestinal compartments analysed have different germ layer origins [49], the epithelial compartment deriving mainly from the endoderm (except for mesodermal lymphocytes), and the residue mainly from the mesoderm (except for ectodermal neuronal elements). Apparently, the glycolipids of large and small intestine differ in their endodermal but not in their mesodermal parts. This difference may in part be due to different functions, including a larger number of mucus-producing goblet cells in the large intestine [35]. The localization of classical blood group determinants exclusively to the epithelial cells, and GalNAc-containing members of the globoseries of glycolipids exclusively to the non-epithelial residue has been shown before for small intestine of several animal species [12,50]. Small and large intestine evidently express different glycosyltransferases for the synthesis of blood group active glycolipids. The B transferase is restricted to large intestine and apparently works on the same substrates as the A transferase of the same tissue, producing four-, six- and seven-sugar glycolipids (compare Table II). In the small in-
testine the enzyme for adding fucose to GlcNAc, for synthesis of the seven-sugar products, is missing. Instead small intestine may synthesize branched-chain glycolipids, which are lacking in large intestine. Interestingly, the two inbred rat strains differed in small but not in large intestine, with a probable lack of A transferase in small intestine of the white rat. Acknowledgements The authors are grateful to I. Pascher for help with derivatizations of glycolipids, and to W. Pimlott for mass spectrometry. Antibody E, 83-52 was a kind gift from H. Koprowski, Wistar Institute, Philadelphia, and antibody M/D C,K,C,, from A. Lundblad, Department of Clinical Chemistry, University of Lund. The work was supported by grants from the Swedish Medical Research Council, from the Medical Faculty, University of Goteborg, and from the Swedish Society of Medical Research, Karolinska Institute, Stockholm. References Robbins, S.L. (1974) Pathologic Basis of Disease, Saunders, Philadelphia Davis, B.D., Dulbecco, R., Eisen, H.N. and Ginsberg, H.S. (1980) Microbiology, 3rd Edn., Harper & Row, Philadelphia Beachey, E.H. (1980) Bacterial Adherence, in Receptors and Recognition, Series B, Vol. 6 (Cuatrecasas, P. and Greaves, M.F., eds.), Champman and Hall, London Magnani, J.L., Brockhaus, M., Smith, D.F., Ginsburg, V., Blaszczyk, M., Mitchell, K.F., Steplewski, Z. and Koprowski, H. (1981) Science 212, 55-56 Magnani, J.L., Nilsson, B., Brockhaus, M., Zopf, D., Steplewski, Z., Koprowski, H. and Ginsburg, V. (1982) J. Biol. Chem. 257, 14365-14369 Falk, K.-E., Karlsson, K.-A., Larson, G., Thurin, J., Blaszczyk, M., Steplewski, Z. and Koprowski, H. (1983) Biochem. Biophys. Res. Commun. 110, 383-391 Kallenius, G., Molby, R., Svensson, S.B., Winberg, J., Lundblad, A., Svensson, S. and Cedergren, B. (1980) FEMS Lett. I, 279-302 Leffler, H. and Svanborg-Ed&n, C.S. (1980) FEMS Lett. 8, 127-134 9 Sjogren, H.O. (1980) Cancer 45, 1229-1233 10 Hansson, G.C., Karlsson, K.-A., Larson, G., Stromberg, N., Teneberg, S., Thurin, J., Brodin, T., Sjogren, H.O., Hellstrom, I. and Hellstrom, K.-E. (1983) in Glycoconjugates (Chester, A.M., Heineg%rd, D., Lundblad, A. and Svensson, S., eds.), Proceedings of the 7th International Symposium on glycoconjugates, pp. 854-855, Lund, Sweden
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11 Hansson, G.C., Karlsson, K.-A., Larson, G., Lindberg, A., Stromberg, N. and Thurin, J. (1983) in Glycoconjugates (Chester, A.M., Heinegard, D., Lundblad, A. and Svensson,
12 13 14
15 16 17
18
19
S., eds.), Proceedings of the 7th International Symposium on Glycoconjugates, pp. 631-632, Lund, Sweden Breimer, M.E., Hansson, G.C., Karlsson, K.-A. and Leffler, H. (1982) J. Biol. Chem. 257, 557-568 Breimer, M.E., Hansson, G.C., Karlsson, K.-A. and Leffler, H. (1982) J. Biol. Chem. 257, 906-912 Angstrom, J.. Breimer. M.E.. Falk, K.-E., Hansson, G.C., Karlsson, K.-A., Leffler, H. and Pascher. 1. (1982) Arch. Biochem. Biophys. 213. 7088725 Breimer, M.E., Hansson, G.C., Karlsson, K.-A. and Leffler. H. (1980) B&him. Biophys. Acta 617, 85-96 Breimer, M.E., Hansson, G.C., Karlsson, K.-A. and Leffler, H. (1981) Exp. Cell Res. 135, 1-13 Brockhaus, M., Steplewski, 2.. Koprowski, H., Karlsson. K.-A., Larson, G. and Ginsburg, V. (1981) J. Biol. Chem. 256, 13223-12225 Hansson, G.C., Karlsson, K.-A., Larson, G., McKibbin, J.M., Blaszczyk, M.. Herlyn, M., Steplewski. Z. and Koprowski, H. (1983) J. Biol. Chem. 258, 4091-4097 Breimer, M.E., Hansson, G.C., Karlsson, K.-A., Leffler, H.. Pimlott, W. and Samuelsson, B.E. (1979) Biomed. Mass
Spectrom. 6. 231-241 20 Weiser, M.M. (1973) J. Biol. Chem. 248, 7, 2536-2541 N.J., Farr, A.L. and Randall. 21 Lowry, O.H., Rosebrough, R.J. (1951) J. Biol. Chem. 193, 265-275 B. (1948) Mikroskopische Technik, 5th Edn., 22 Romeis, Oldenburg, Munich 23 Hakomori, S.-I. (1964) J. Biochem. (Tokyo) 55, 205-208 13, 3643-3647 24 Karlsson, K.-A. (1974) Biochemistry 25 Karlsson, K.-A. (1976) in Glycolipid Methodology (Witting, L.A.. ed.), pp. 97-122, American Oil Chemists’ Society, Champaign, IL 26 Breimer, M.E., Hansson, G.C., Karlsson, K.-A., Leffler, H., Pimlott, W. and Samuelsson, B.E. (1981) FEBS Lett. 124, 289-303 27 Burlingame, A.L., Dell, A. and Russell, D.H. (1982) Anal. Chem. 54, R363-R409 Springer28 Stahl, E. (1962) Dtinnschicht-Chromatographie. Verlag, Berlin
29 Svennerholm,
L. (1963) J. Neurochem.
10, 613-623
30 Karlsson, K.-A., Samuelsson, B.E. and Steen, G.O. (1973) Biochim. Biophys. Acta 306, 317-328 31 Stern, B.K. and Jensen, W.E. (1966) Nature 209, 789-790 32 Culling, C.F.A., Ried, Ph.E., Trueman, L.S. and Dunn, W.L. (1973) Proc. Sot. Exp. Biol. Med. 142, 434-438 33 Perret, V., Lev, R. and Pigman, W. (1977) Gut 18, 382-385 34 Roediger, W.E.W. and Truelove, SC. (1979) Gut 20, 484-488 35 Bloom, W. and Fawcett, D.W. (1975) A Textbook of Histology, 10th Edn., Saunders, Philadelphia 36 Hansson, G.C., Karlsson, K.-A. and Thurin, J. (1980) Biochim. Biophys. Acta 620, 270-280 37 Smith, E.L., McKibbin. J.M., Karlsson, K.-A., Pascher, I. and Samuelsson. B.E. (1975) Biochemistry 14, 2120-2124 38 Karlsson, K.-A., Leffler, H. and Samuelsson. B.E. (1974) J. Biol. Chem. 249, 4819-4823 39 Sung. S.-S. and Sweeley. C.C. (1979) Biochim. Biophys. Acta 475, 295-298 40 Hansson, G.C., Karlsson, K.-A., Larson, G., McKibbin, J.M., Stromberg, N. and Thurin, J. (1983) Biochim. Biophys. Acta 750, 214-216 41 Angstrom, J., Breimer, M.E., Falk, K.-E., Griph. I., Hansson, G.C., Karlsson, K.-A. and Leffler, H. (1981) J. Bio;hem. 90, 909-921 42 Lipkin, H. (1973) Physiol. Rev. 53, 891-915 43 Brasitus, T.A. (1982) Anal. Biochem. 123, 364-372 44 Dahlgren, K.-E., Emilsson, P.M., Hansson, G.C. and Samuelsson, B.E. (1981) J. Immunol. Methods 44, 223-234 45 Breimer, M.E., Hansson, G.C., Karlsson, K.-A. and Leffler. H. (1980) FEBS Lett. 114, 51-56 46 Breimer, M.E., Hansson, G.C., Karlsson, K.-A. and Leffler, H. (1980) in ACS Symposium Series No. 128, Cell Surface Glycolipids (Sweeley, CC., ed), pp. 79-104, American Chemical Society, Washington DC 47 Race, R.R. and Sanger, R. (1975) Blood Groups in Man, 6th Edn., Blackwell Scientific Publications, Oxford 48 Breimer, M.E. (1984) Arch. Biochem. Biophys., in the press 49 Arey, L.B. (1974) Developmental Anatomy, 7th Edn., Saunders, Philadelphia 50 Breimer, M.E., Hansson, G.C., Karlsson, K.-A. and Leffler, H. (1981) J. B&hem. 90, 589-609
Biochimica et Biophysics Acta, 192 (1984) 281-292
Elsevier
BBA 51581
GLYCOSPHINGOLIPID PATTERNS OF THE EPITHELIAL COMPAR~ENTS OF RAT LARGE INTESTINE GUNNAR
C. HANSSON,
Department
of Medical
(Received
KARL-ANDERS
Biochemistry,
KARLSSON
AND NON-EPITHELIAL
and JAN THURIN
University of Giiteborg, I? 0. Box 330 31, S - 400 33 Gijteborg (Sweden)
September 28th, 1983)
Key words: Glycosphingolipid pattern; Compartmentation;
(Rat large intestine)
The epithelial cells and the non-epithelial residue from large intestine of two inbred rat strains were separated and the glycosphingolipids characterized in comparison with earlier detailed data from small intestine of the same strains. Total acid and non-acid glycolipids were prepared and the non-acid glycolipids were further fractionated into subgroups as acetylated derivatives on silicic acid. The fractions obtained were characterized mainly by #in-layer c~omat~phy, including binding of monoclonal anti-A and anti-B antibody to the chromatogram, and by direct-inlet mass spectrometry after derivatization. This combined technology allowed an overall conclusion from a small number of animals concerning relative amounts of glycolipids, microheterogeneity of blood group glycolipids and carbohydrate sequence and lipophilic components of major species of each subf~ction. As for the small intestine, the two separated ~omp~ments differed distinctly in composition, with blood group fucolipids being confined to the epithelial cells, and a series of glycolipids with probably internal Gala being restricted to the non-epithelial part. The main difference between large and small intestine concerned fucolipids of the epithelium. Three blood group B active glycolipids with four, six and seven sugars were detected which were absent from the small intestine. The four-sugar glycolipid was a major ~ycolipid with the structure Gala1 -+ 3Ga1(2 + laFuc)/31-+ 4Glc/31 + leer, as reported before. The six-sugar glycolipid was shown by mass spectrometry and NMR spectros4Glc/31 --f copy to have the probable structure Gala1 --+3Ga1(2 + laFuc)/31+ 3GlcNAc/31 --f 3GalBl+ 1Cer. The seven-sugar glycolipid had an additional fucose linked to N-acetylhexosamine, as shown by mass speetrometry. Three blood group A active glycolipids with four, six and seven sugars were found in both rat strains, with sequences analogous to the B glycolipids but with a terminal GalNAc instead of Gal. The fourand six-sugar blood group A compounds, but not the seven-sugar glycolipid, have been found before in the small intestine of one of the rat strains. In the small intestine, on the other hand, a branched-chain twelve-sugar blood group A active glycolipid has been found which was absent from the large intestine. Therefore large intestine of both rat strains expressed glycolipid-base blood group A and B activity, while small intestine lacked B activity and showed A activity only in one of the strains. Quantitatively the major glycolipids of the epitbelial cells of large intestine were monoglycosylceramides (glucosylceramides, and smaller amounts of galactosylceramides which were absent from small intestinal epithelhun) and tetraglycosyleeramides (including the A and B active species and a te~ahexosylceramide). The major li~philic components of the epithelial cell glycolipids were phytosphingosine and long-chain hydroxy fatty acids.
The glycohpid nomenclature used follows an IUPAC/IUB r~omm~dation (Eur. J. B&hem. (1977) 79, 11-21). Hex means hexose, HexN means N-acetyihexosamine and Cer means ceramide. Trihydroxy base is abbreviated t, and 2-hydroxy 0005-2760/84/$03&O
0 1984 Elsevier Science Publishers B.V.
fatty acid h. A figure before a colon specifies chain length, and after a coton, number of double bonds; t18 : 0 therefore means phytosphingosine or hydroxysphinganine.
282
Introduction The large intestine is of biomedical interest because of frequent diseases of man, including tumor formation from epithelial cells [l] and infections where bacteria are supposed to adhere to the epithelial cell surface [2,3]. In both these phenomena glycosphingolipids have been shown to be involved. A ganglioside of diagnostic interest [4] was recently identified in human colon cancer cells [5,6], and glycolipids of the globe series have been proposed as adhesion receptors for Escherichia co/i, causing urinary tract infection in man [7,8]. The present work is part of a collaboration with a group studying rat colon cancer [9,10] and of a systematic study on glycolipids as receptors for microorganisms [ 111. In earlier studies the small intestine of two inbred rat strains was subjected to a detailed structural analysis of glycolipids [12-151, including the isolation of epithelial cells of different maturity [16]. In the present case the large intestines of the same rat strains were used for preparation of epithelial and non-epithelial compartments to study the glycolipids. The approach was mainly a comparison with well-characterized glycolipids of the small intestine using thin-layer chromatography and mass spectrometry of subfractions after column chromatography of acetylated glycolipids. Monoclonal anti-blood-group A and B antibodies were applied directly on the thin-layer chromatogram [17,18], allowing a conclusion on microheterogeneity of blood group fucolipids. Direct-inlet mass spectrometry and a fractional evaporation. of derivatized glycolipid subfractions presented saccharide sequences permitting comparison with thin-layer chromatogram data [19]. Materials and Methods Animals Two inbred strains of rats were used, SpragueDawley strain (white rat) kept separate for more than 35 years, and a hooded strain (black-white), kept separate for 30 years at the Department of Pharmacology in Goteborg. Only male rats were used. Non-fasting animals, about 6 months old, were killed by decapitation under ether anaesthesia. The caecum-colon or colon was immediately removed and prepared for cell removal.
Remooal of epithelial cells Two preparations were used, one with colon cut right distal to caecum and one including caecum cut 1 cm proximal to the ileo-caecal valve. Both preparations were cut as far distal as possible. The caecum-colon preparation was ligated at the ileocaecal valve in a way that did not allow ileal mucosa to be in contact with the lumen of caecum. It was then cut open in the part of the preparation farthest away from the distal opening to avoid a blind end. The preparations were washed, tied onto plastic nipples, filled with solution A (96 mM NaCI/l.S mM KC1/27 mM sodium citrate/g mM KH,PO,/5.6 mM Na,HPO,, pH 7.3) and incubated for 15 min at 37°C as described [16], a procedure which was adapted from the method of Weiser [20]. This fraction together with one washing volume did not contain any cells and was discarded. The preparations were then sequentially incubated with aliquots of solution B (137 mM NaCl/3 mM KC1/1.5 mM KH,PO,/6.5 mM Na,HP0,/1.5 mM EDTA/O.S mM dithiothreitol, pH 7.3). During each incubation the intestine was elevated gently three times every 20 s and afterwards washed three times with one intestinal volume of solution B (the intestinal volume increased approx. 2-3% for every incubation): The incubation times were 6, 6, 5, 5, 5, 5, 5, 5, 5, and 10 min, respectively, and the fractions were collected in centrifuge tubes and centrifuged at 250 x g for 5 min. The supernatants including floating mucus were sucked off and the cells washed twice in phosphate-buffered saline, pH 7.3, by centrifugation at 250 x g for 5 min and suspended in phosphate-buffered saline. Cells were then taken for protein determination [21] and microscopy. For phase-contrast microscopy segments of intestine were taken after various incubation periods, fixed in formalin, embedded in wax, sectioned and stained with Van Gieson’s stain [22]. After centrifugation as above and removal of the buffer, 3 ml methanol were added to each fraction. The fractions were then pooled and stored at -20°C. The non-epithelial residues were cut off by the nipples and stored at -20°C. Preparation and fractionation of glycosphingolipids The pooled epithelial cells were extracted, subjected to mild alkaline methanolysis, dialysed and
283
separated into non-acid and crude acid glycolipid fractions. The total non-acid fractions and crude acid fractions from both epithelial cells and non-epithelial residues were prepared essentially as described in Ref. 16. The crude acid fractions from the same origins were acetylated and run on a silicic acid column as in Ref. 16, and then deacetylated with mild alkaline methanolysis [16] to yield a purified total acid fraction. 4 mg each of non-acid glycolipids of the epithelial cells and non-epithelial residue from the caecum-colon preparations of the white rat were acetylated twice overnight in darkness with chloroform/pyridine/acetic acid anhydride (1 : 1 : 1, by vol.). A fractionation of the acetylated mixture was done on a silicic acid column, using 10 g of Iatrobeads 6RS-8060 (Iatron Laboratories Inc., Tokyo) and eluting with 200 ml of a continuous gradient, O-10% (by vol.), of methanol in chloroform. Approx. 70 fractions, each of 3 ml, were collected, analysed by thin-layer chromatography and pooled together into six fractions. Mass spectrometry Direct-inlet mass spectrometry was done on total and on subfractionated non-acid glycolipids of the different origins. Permethylated [23] and LiAlH,-reduced permethylated [24] total glycolipid fractions were analysed on an AEI MS 902 instrument (Kratos) in the electron ionization mode using an Instem (Kratos) data system [19,25] and the ‘in beam’ technique [26,27]. The ion source temperature was raised linearly and mass spectra were recorded at fixed intervals and stored in the data memory. The selected ion curves showing intensity of different ions versus temperature were reproduced by a Tectronix hard copy unit. Mass spectra of permethylated, non-reduced glycolipid subfractions were analysed ‘in beam’ on a ZAB-HF instrument (VG Analytical). More details about technical conditions and interpretation of spectra have been given elsewhere [19,25-271. NMR analysis NMR spectroscopy was performed on the permethylated-reduced blood group B active hexaglycosylceramide. The spectrum was recorded on a Bruker 270 MHz instrument using 1 mg sample in 0.5 ml of C’HCl, at 313 K and 6200 pulses.
Thin-layer chromatography Thin-layer chromatograms were run on HPTLC Si 60 precoated nanoplates (Merck). Detection was done with the anisaldehyde reagent [28] and the resorcinol reagent [29]. Thin-layer chromatography on a borate-containing layer was done as described [ 301. Binding of monoclonal anti-blood-group antibody to a thin-layer plate (chromatogram binding assay) The chromatogram binding assay was done as described in Ref. 18, which is a method adapted from Ref 17. The anti-blood-group B antibody E, 83-52 was derived from cell culture supernatants of hybridomas that were the result of immunizing mice with Capanpancreas carcinoma cells, and its specificity has been reported [18]. The antiblood-group A antibody M/D C,K,C,, was obtained by immunizing mice with human erythrocytes of blood group A and was produced in ascites. It was a gift from Arne Lundblad, Department of Clinical Chemistry, University of Lund, Sweden. The antibody reacts with human erythrocytes of blood group A but not of 0 or B. It reacts with isolated glycolipids with monofucosyl or difucosyl blood group A determinants on a type 1 or .type 2 saccharide chain (unpublished results). Results Epithelial cell isolation Epithelial cells were removed essentially as described earlier [16]. This cell-releasing method with chelating agents but no enzyme gives a good yield of viable cells [31-341. Biopsies taken before and after cell removal showed a total release of epithelial cells without any apparent damage to the basal membrane (Fig. 1). For colon, in contrast to small intestine [16], a sequential removal of villus tip cells and crypt cells, as viewed with phase-contrast microscopy (not reproduced), was not found. Conglomerates of cells resembling whole crypt bottoms were seen in almost all of the eluted fractions, which indicated a more random removal from caecum and colon than from small intestine, which may be explained by the different histology of the two mucosae [35]. From the white rat strain two preparations were done, one from five animals
284
TABLE
I
COMPARISON BETWEEN ABSOLUTE WEIGHTS OF LYOPHILIZED TISSUE, TOTAL PROTEIN AND TOTAL ACID AND NON-ACID GLYCOSPHINGOLIPID FRACTIONS FROM EPITHELIAL AND NON-EPITHELIAL CELL COMPARTMENTS OF TWO INBRED RAT STRAINS Figures are given in mg/animal. black-white rat, two animals.
White
rat, ten animals
and
White rat caecum + colon
Black-white rat caecum + colon
Epithelial
Epithelial
Non-epithelial
cells
cells
_
230 100
cells Dry weight Protein 85 Acid glycolipids 0.28 Non-acid glycolipids 0.81
Non-epithelial cells 280 120
85
0.52
0.30
0.90
0.84
0.84
1.35
1
3 4 5 7
Fig. 1. Light microscopy of the colon of the white rat before (top) and after (bottom) epithelial cell removal. Staining as given in Materials and Methods. Magnification x 410.
IO
ABCDEFGH with colon only and one from ten animals with caecum-colon. A caecum-colon preparation from two animals of the black-white rat strain was done for comparison. Data from these preparations are shown in Table I. Epithelial cell total non-acid glycolipids The thin-layer chromatogram of Fig. 2 of the non-acid glycosphingolipids of the epithelial cells of large intestine (lanes B-D) showed striking differences compared to the same fraction of the
Fig. 2. Thin-layer chromatogram of total non-acid glycosphingolipids from the epithelial cells (A-D) and non-epithelial residues (E-H), from the small intestine of the white rate (A and E), from colon of the white rate (B and F), from coloncaecum of the white rat (C and G), and from colon-caecum of the black-white rat (D and H). The amount of glycolipids applied corresponded to 8 mg protein for the epithelial fractions B-D, 2 mg protein for A and 4 mg protein for the residue fractions E-H. The numbers to the left indicate numbers of sugars of the glycolipids. All bands were green with the anisaldehyde reagent, as for glycolipids, except for the bands marked x. Solvent was chloroform/methanol/water, 60 : 35 : 8 (by vol.).
285
small intestine (lane A). Major glycolipids of the large intestine appear in the intervals of one, three, four and about six sugars. Minor differences in the seven sugar interval can be seen between the two rat strains (lanes C and D) and between the two preparations from colon only and caecum-colon (lanes B and C). The amounts of cells estimated as protein from the epithelium of the two strains were almost identical as were also the total amounts of non-acid and acid fractions obtained from these cell preparations (Table I). The glycosphingolipid species in the epithelial cells of caecum-colon of the white rat as found by mass spectrometry are summarized in Fig. 3. The permethylated-reduced glycosphingolipid mixture was introduced by the ‘in beam’ technique. The source was heated linearly and mass spectra (scans in Fig. 3) were recorded at fixed intervals [19]. The change in relative intensity of selected ions for separate glycolipids was reproduced as curves along the temperature and scan scales. The ions selected in this case are usually relatively abundant [19] and contained the complete saccharide chain to-
m/z
25 240°C
For more detailed information 4 mg of total non-acid glycolipids of epithelial cells of caecumcolon from ten white rats were acetylated and
Base
R
t18:O h24:O t18:O h24:O
1066
HexHexHex-CH2
t18:O h24:O
1270
HexHexHexHex-CH2FHNMe-R
t18:O h24:O
Hex(Fuc)-Hex-CH2yHNMe-
894
0
non-acid glycolipids of the epithelial
cells
Hex-CHzcHNMe-R ~ex~ex-CH2FHNMe-R
862
16O'C
Subfractionated
F-Fragment
608
Scan
gether with one fatty acid species and part of the long-chain base. The fatty acid species selected for reproduction were chosen from complete paper spectra to allow a minimum of interference from other non-relevant fragments. Oligohexosylceramides with one to four sugars (m/z 608, 862, 1066 and 1270, respectively) were detected together with blood group type fucolipids with H-type terminal (m/z 894), B-type terminal (m/z 1098, 1675 and 1793) and A-type terminal (m/z 1267 and 1702). The sequences of the sugars as given in the simplified formulas were deduced from supplementing ions found in the complete spectra of permethylated and permethylated-reduced mixtures (not shown). Phytosphingosine. was the major longchain base, as shown by m/z 396 of spectra of permethylated mixtures.
R
tl8:O
16:0
16:0
1098
Hex-Hex(Fuc)-Hex-CH2
R
t18:O
1267
HexN-Hex(Fuc)-Hex-CH2FHNMe-
R
t18:O h24:O
R R
t18:O h24:O
1675
(X10)
1793 1702
(X10) (X10)
Hex-Hex(Fuc)-HexNHexHex-CH2FHNMeHex-H~x(Fuc)-H~xN(Fuc)H~xHw-CH~
R
t18:O h20:O t18:O h24:O
50 325OC
Fig. 3. Selected ion curves of the fatty acid-carbohydrate-containing peaks from mass spectrometry of the major non-acid glycosphingohpids of the epithelial cells in colon-caecum of the white rat. The curves reproduced correspond to relative abundances of saccharide plus fatty acid (R) ions (see simplified formulas) of glycosphingolipids as a function of evaporation temperature. 60 gg of permethylated-reduced mixture were evaporated ‘in beam’ by raising the ion source temperature linearly with 5.2 K/mm. Spectra were recorded every 38 s. The electron energy was 50 eV, accelerating voltage 4 kV and trap current 500 PA. The letters to the left mark the structures and refer to Fig. 4 and the text. For abbreviations, see footnote on page 281.
286
4
3
12
12
3
4
5
6
3
4
5
6
123456
7
8
1,2
3
123456
4
5
123456
6,7 8
Fig. 4. Thin-layer chromatograms of the fractions obtained after fractionation of the acetylated glycosphingolipids of the epithelial cells (A) and non-epithelial residue (B) of the white rat. Acetylated fractions (1) and deacetylated fractions (2) were visualized by the anisaldehyde reagent. Chromatograms 3 and 4 were autoradiographed (72 h) after labelling a plate identical to A-2 with monoclonal ascites diluted lt 100) and anti-B antibody (ES 83-52, culture supernatant diluted 1+4), anti-A antibody (M/D C,K,C,,, respectively. The amounts of sample used in A-3 and A-4 were one-third of the amount in A-2. For chromatogram A-2 about 1% was applied of fractions 1 and 2,4% of fraction 4, and 10% of fractions 3, 5 and 6. For B-l and B-2 about 5% of each fraction was applied. Solvent was chloroform/methanol (96 : 4 by vol.) for plate 1, and chloroform/methanol/water (60 : 35 : 8 by vol.) for plates 2, 3 and 4. The bands marked by letters refer to structures in Fig. 3 and in the text. The bands marked x refer to non-glycolipid contaminants.
eluted by a continuous gradient from a silicic acid column. 70 fractions were pooled into six major fractions, which were deacetylated and analysed by thin-layer chromatography using anisaldehyde or antibody for the detection, and by mass spectrometry after permethylation. As shown in Fig. 4 several glycolipids had a reversed order of migration after deacetylation (chromatogram A-2) compared to acetylated derivatives (chromatogram Al), which migrate in the same order as they elute from the column. For the identification of several glycolipid
species a comparison was done with glycolipids isolated from rat small intestine, both concerning chromatographic mobility, mass spectrometric fragmentation and immunological properties [12]. Blood group active glycolipids In chromatograms A-3 and A-4 of Fig. 4 blood group active glycolipids were detected with monoclonal anti-A and anti-B antibodies, respectively. For both antibodies there were bands stained with a chromatographic mobility corresponding to four, six and seven sugars.
287
A blood group B active tetraglycosylceramide was detected in lane 2 of chromatogram A-4 and corresponds to a major band marked f in lane 2 in chromatogram A-2 and detected with anisaldehyde. Mass spectrometry of this fraction showed the curve of m/z 1098 of Fig. 3 and sequence ions in accordance with the structure Gal& -+ 3Gal(2 + IcwFuc)/Il + 4Glcfll + 1Cer reported before for a rat large intestinal glycolipid [36]. The four-sugar blood group A active glycolipid of lanes 4 and 5 of chromatogram A-3 of Fig. 4 showed up as the band marked g in lanes 4 and 5 of chromatogram A-2. By mass spectrometry of these two fractions it was likely that the glycolipid was identical to the species isolated from rat small intestine and shown to have the structure GalNAcal + 3Gal(2 t- 1cwFuc)pl-+ 4Glc/31 -+ 1Cer [12,13]. This species produced the curve of m/z 1267 of Fig. 3 from the total mixture. The blood group A active hexaglycosylceramide in lanes 4-6 of chromatograms A-2 and A-3 of Fig. 4 and marked j may be identical to the structure GalNAccJ --+ 3Gal(2 c- laFuc),& -+ 3GlcNAc/31 + 3GalPl -+ 4Glc/31 -+ 1Cer identified in the small intestine [12,13]. Evidence for this sequence was given by the curve of m/z 1702 of Fig. 3 and by sequence ions from the permethylated sub-fractions. The seven-sugar glycolipid marked k in lane 5 of chromatogram A-2 had a difucosyl blood group A determinant. Evidence for this was the appearance of m/z 1057 and 1025 (1057-32) in the mass spectrum of the permethylated subfraction corresponding to the terminal pentasaccharide HexN-Hex(Fuc)-HexN(Fuc)as shown before [37]. Concerning the two blood group B active glycolipids marked h and i in chromatogram A-2 of Fig. 4, mass spectrometry of total epitheliaI glycolipids (Fig. 3) as well as permethylated fraction 4 gave evidence for the two B-type sequences shown at the m/z 1675 and 1793 curves of Fig. 3. Sequence ions after permethylation for a B-type monofucosyl glycolipid were found at m/z 842 and 810 (842-32) for four sugars and 1046 for five sugars, as well as M - 1 at m/z 1931 for the dominating species with phytosphingosine and hydroxy 20 : 0 fatty acid. The B-type difucosyl glycolipid was evidenced by m/z 1016 and 984 (101632) for five sugars and 1220 for six sugars, as well as 2105 for
M - 1 of the species with phytosphingosine and hydroxy 20 : 0 fatty acid. After further fractionation on silicic acid the six-sugar glycolipid was obtained pure for NMR spectroscopy as the permethylated-reduced derivative. The data obtained are in line with the structure Galrvl --) 3Gal(2 + lfuFuc)@ -+ 3GlcNac/31 + 3Gal/31 --* 4Glc/31 --, lCer, which is analogous to the six-sugar blood group A glycolipid [13]. Work on the preparation and further analysis of these two blood group B glycolipids is in progress. Although it appears from the relative intensities of the bands after detection with anisaldehyde (lane 4 of chromatogram A-2) that the six-sugar B-glycolipid predominated over the seven-sugar species, the reverse order of intensity was shown by antibody staining (lane 4, chromatogram A-4). This is in agreement with earlier data which showed a preference of this antibody for a difucosyl B deter~nant [18]. Other non-acid giycolipids of the epithelial cells The major glycolipid in large intestinal epithelium was monoglycosylcerarnide, as shown in Fig. 2, Fig. 3 (curve a) and Fig. 4 (lanes 1 of chromatogram A-l and A-2). The fraction was shown by separation on a borate-impregnated layer [3OJ to contain mainly glucosylceramide with phytosphingo~ine and hydroxy fatty acid and a smaller amount of galactosylceramide with the same ceramide. A smaller amount of diglycosylceramide was shown as band b in lane 1 of chromatogram A-2 of Fig. 4, and this was supported by mass speetrometry (curve b of Fig. 3). In the three-sugar interval two glycolipid species were detected, marked e and c in lane 2 of chromatogram A-2 (Fig. 4). These were also present in the small intestinal epithelium (Fig. 2) and identified as Fuccul -+ 2Galpl --f 4GlcPl + 1Cer [12,15] and globotriaosylceramide [12,14]. This conclusion was supported by mass spectrometry (Fig. 3 and not reproduced). Lane 3 of chromatogram A of Fig. 4 was almost free of blood group A or B activity and this fraction was shown by mass spectrometry to contain a tetrahexosylceramide, marked d (see also curve d of Fig. 3), a hex~exosylcer~de and a hexaglycosylceramide with five hexoses and a terminal fucose. These sequences were found also
288
in the small intestine and the first one was identified as Gala1 + 3Galal - 4Gal/31 + 4GlcPl ---) 1Cer [12,14]. Non-acid glycolipids of the non-epithelial residue As evident from the chromatogram of total non-acid glycolipids in Fig. 2, the patterns were similar for small and large intestine except for quantitative differences. The small intestine (sample E) had more di- and triglycosylceramides and relatively more of the rapidly moving band of monoglycosylceramide than colon (sample F), which was shown on a borate-containing layer [30] to be composed of glucosylceramide only, with mainly sphingosine and nonhydroxy fatty acids. There were no detectable strain differences (samples G and H), except for a higher amount of total glycolipid from the black-white strain (Table I). The qualitative similarity between small and large intestines was further substantiated after fractionation of the non-acid glycolipids as acetylated derivatives, pooling into eight fractions and deacetylation. Thin-layer chromatography (Fig. 4, chromatogram B), mass spectrometry after permethylation and permethylation-reduction and analysis with the two monoclonal antibodies revealed only one glycolipid in addition to those already shown for the small intestine [12]. Reactivity with anti-blood-group A or B antibody was lacking, except for a weak staining of band 1 in lane 8 of chromatogram B-2. The mass spectrum after permethylation of this fraction showed fragments for a pentaglycosylceramide with a sequence as for the Forssman glycolipid, with m/z 260 for terminal hexosamine, 505 for two hexosamines, and 709, 913 and 1117 for three, four and five sugars, respectively. All five sugars, two hexosamines and three hexoses, were also present in the fragment at m/z 1190 including part of the longchain base [38]. The dominating ceramide contained sphingosine and 24 : 0 fatty acid, as shown by m/z 1792 for M - 1. The glycolipid migrated, however, more slowly than authentic Forssman glycolipid from dog intestine [39,403. As this anti-A antibody was shown to cross-react weakly with Forssman glycolipid (unpublished data) the terminal sugar of the new glycolipid probably was GalNAccu. Work on the structure of this glycolipid is in progress.
Acid glycolipids of both compartments us shown by thin-layer chromatography An overall analysis by thin-layer chromatography was performed on total acid glycolipids of epithelial cells and non-epithelial residue of colon-caecum of the two rat strains in comparison with the corresponding two compartments of small intestine (Fig. 5). The major ganglioside of all fractions of large intestine seemed to be hematoside with N-acetylneuraminic acid, in contrast to small intestine where the epithelial cells contained hematoside with N-glycoloylneuraminic acid [12,41]. Also, the large intestine epithelium contained sulfatide, cerebroside 3-sulphate (not
Fig. 5. Thin-layer chromatogram of the total acid glycolipids from the epithelial cells (l-3) and non-epithelial residues (4-6), from the small intestine of the white rat (I and 4), from the colon-caecum of the white rat (2 and S), and from the coloncaecum of the black-white rat (3 and 6). A, N-acetylneuraminosyllactosylceramide; B, N-glycoloylneuraminosyllactosylceramide. Solvent was methylacetate/2-propanol/72 mM CaCI/S M NH, (45: 35: 15: 10 by vol.) and the detection reagent resorcinol. Bands marked x were yellow.
289
shown), which was lacking in the small intestine. The two strains differed concerning the total amount of crude acid glycolipids (Table I) and possibly in their ganglioside patterns. Several bands remain unidentified. Discussion The preparation of epithelial cells seemed to be quantitative as judged by light microscopy. A contamination of the non-epithelial residue with epithelial cells was also practically ruled out by the immunological analysis, which did not detect blood group active bands in the residues except for a Forssman-like glycolipid. As the detection limit for this assay is at the nanogram level [17,18] and
large amounts of glycolipids were put on the plate (data not shown), the negative result means that the actual contamination of epithelial cells in the residue after preparation should be less than one part in 104. On the other hand, it may be more difficult to rule out a contamination of the epithelial cell fraction with ceils from the non-epithelial residue. However, preliminary results from staining with a monoclonal antibody directed against isoglobotetraosylceramide, which is a major glycolipid of the residue, showed the practical absence of this glycolipid in the epithelial cell fraction (work in progress). The sequential dissociation of epithelial cells from the basal membrane according to level of cell differentiation, as found for the small intestine
TABLE II SOME EXAMPLES
OF STRAIN AND COMPARTMENT
DIFFERENCES
OF EPITHELIAL
GLYCOSPHINGOLIPIDS
LacCer, Gal/31 + 4GIcj31+ 1Cer.
Blood group H Fucal + 2LacCer Blood group A GalNAcal + 3 LacCer Fucal + 2 GaINAcal+ Fucal + 2
3
GaINAwl+ Fucal + 2
3
Gal/31 + 3GlcNAc/31+
3LacCer
Gal-HexN(-Fuc)-Hex-Hex-Cer
GalNAccrl + 3 Fucoll+ 2 Gal/31 -+ 3/4GlcNAcPl+ Gal/31 + 3GlcNAc/31* GalNAcal + 3 Galbl + 3GlcNAcPl+ Fuccrl-+ 2
Gala1 + 3
Small intestine
Large intestine
Small intestine
Large intestine ’
+
+
+
+
_
+
-
+
+
+
_
+
-
+
-
_
+
-
-
+
_
+
-
+
-
+
+
_
+
+
6 3LacCer 3
Blood group B Galal* Fucal + ZLacCer Gala1 + 3 Fucal ~ 2Gal/31 + 3GIcNAcPl+
Black-white rat
White rat
Glycosphingolipid structure
LacCer
a Identified by mass spectrometry of total non-acid glycohpids of whole intestine and chromatogram binding assay.
290
[16], was not possible using very similar conditions for the large intestine. Instead, conglomerates of cells apparently also from crypt bottoms were continuously eluted. A gentle mechanical scraping of the mucosa may possibly give access to more differentiated cells for separate study (cf. Ref. 42). Furthermore, the less differentiated cells can be prepared from a single-cell suspension by velocity sedimentation as shown in Ref. 43. The glycolipids of the two compartments prepared were worked up and compared with results of a detailed investigation done before on the two fractions from small intestine [12]. Some distinct differences were found, primarily restricted to the epithelial cells. Although all data were not presented, the non-epithelial residues of small and large intestine were very similar except for quantitative differences. A glycolipid similar to but apparently not identical to the Forssman glycolipid was found in large but not small intestine. A weak reactivity with the anti-A antibody indicated a terminal GalNAc in a-linkage. A blood group B-type hexaglycosylceramide with GalNAc instead of GlcNAc earlier identified in small intestine [12] was also concluded to exist in large intestine, based on immunology and mass spectrometry. This glycolipid did not bind to the anti-B monoclonal antibody used here [18], but was shown to react in a haemagglutination-inhibition assay [44] with conventional polyclonal anti-B antiserum, similar to the case for the glycolipid from small intestine [12]. The analysis was done on fraction 3 of chromatogram B-2 of Fig. 4, which also produced mass spectra in accordance with the existence of this sequence (band marked m in the chromatogram). The situation for the epithelial cells differed distinctly for large and small intestines and also between the two inbred strains of rat, as summarized for some glycolipids in Table II. Blood group B active glycolipids with four, six and seven sugars were identified in large intestine of both strains but were absent from small intestine. Concerning blood group A active glycolipids, the small intestine of the black-white rat, but not of the white rat, contained species with four, six and twelve sugars [12,45]. In the large intestine, however, both strains expressed blood group A activity of glycolipids with four, six and seven sugars,
probably with structures analogous to the B active glycolipids. The 12-sugar branched-chain species was apparently lacking in the large intestine, as shown by the absence of blood group A activity in this chromatographic interval. As the rat strains were inbred these differences are not due to heterogeneity within the limited populations used. One may also note that conventional ABH blood group typing on erythrocytes as a reference is not adequate in the rat, as these cells apparently do not reflect the blood group activities found in the epithelial cells [46], in contrast to the human [47,48]. The dominating glycolipids of the epithelial cells of large intestine were glucosylceramides and tetraglycosylceramides, including A and B active glycolipids and a tetrahexosylceramide. As apparent from mass spectrometry, the major lipophilic components were phytosphingosine and long-chain 2-hydroxy fatty acids, a situation similar to that in the epithelial glycolipids of small intestine [12]. The methodological approach of the present work has several advantages. A prefractionation of a total fraction into groups of glycolipids as acetylated derivatives before analysis by thin-layer chromatography including immunolabelling and by mass spectrometry may give advanced structural information without the need for pure glycolipid species. The reversed order of chromatographic mobility in some cases for acetylated compared with non-derivatized glycolipid may be diagnostic. Consider, for example, the B active glycolipids which eluted about one fraction ahead of the A active glycolipids with the same number of sugars (Fig. 4). Evidently, a terminal hexosamine may give a retardation compared to hexose in acetylated glycolipids. The reversed order of migration was true for the non-derivatized glycolipids, as shown by bands f and g in chromatogram of Fig. 4, which are B and A active tetraglycosylceramides, respectively. A second example was fraction 3 of chromatogram A-2 (Fig. 4) which contained glycolipids with four and six sugars and lacking hexosamine, which were eluted ahead of corresponding structures with terminal hexosamine present in the non-epithelial residue in fractions 5-7 (chromatogram B-2 of Fig. 4). Without this pre-separation into groups the com-
291
parison of mass spectral data with immunological detection in the case of minor components would have been overshadowed by information from major components. On the other hand, a further separation into pure glycolipid species would have needed both more material and laborious steps. The present approach may be limited in amount to 100 pg of each subfraction. The importance of the new binding assay for monoclonal antibodies [4,17,18] is obvious in the present case in the analysis of glycolipid mixtures. The location of a particular band is rather strictly related to the number of sugars in the glycolipid, allowing a direct comparison with mass spectral data. Analysis with conventional radioimmunoassay in microwells [17] of the different fractions from column chromatography may tell the total activity of the mixture in this fraction but not that the activity may be restricted to particular molecular species. In the actual case the chromatogram binding assay of the mixtures clearly showed the presence of A and B active glycolipids with four, six and seven sugars in the epithelial cell fractions and the absence of these in the non-epithelial residue. The two intestinal compartments analysed have different germ layer origins [49], the epithelial compartment deriving mainly from the endoderm (except for mesodermal lymphocytes), and the residue mainly from the mesoderm (except for ectodermal neuronal elements). Apparently, the glycolipids of large and small intestine differ in their endodermal but not in their mesodermal parts. This difference may in part be due to different functions, including a larger number of mucus-producing goblet cells in the large intestine [35]. The localization of classical blood group determinants exclusively to the epithelial cells, and GalNAc-containing members of the globoseries of glycolipids exclusively to the non-epithelial residue has been shown before for small intestine of several animal species [12,50]. Small and large intestine evidently express different glycosyltransferases for the synthesis of blood group active glycolipids. The B transferase is restricted to large intestine and apparently works on the same substrates as the A transferase of the same tissue, producing four-, six- and seven-sugar glycolipids (compare Table II). In the small in-
testine the enzyme for adding fucose to GlcNAc, for synthesis of the seven-sugar products, is missing. Instead small intestine may synthesize branched-chain glycolipids, which are lacking in large intestine. Interestingly, the two inbred rat strains differed in small but not in large intestine, with a probable lack of A transferase in small intestine of the white rat. Acknowledgements The authors are grateful to I. Pascher for help with derivatizations of glycolipids, and to W. Pimlott for mass spectrometry. Antibody E, 83-52 was a kind gift from H. Koprowski, Wistar Institute, Philadelphia, and antibody M/D C,K,C,, from A. Lundblad, Department of Clinical Chemistry, University of Lund. The work was supported by grants from the Swedish Medical Research Council, from the Medical Faculty, University of Goteborg, and from the Swedish Society of Medical Research, Karolinska Institute, Stockholm. References Robbins, S.L. (1974) Pathologic Basis of Disease, Saunders, Philadelphia Davis, B.D., Dulbecco, R., Eisen, H.N. and Ginsberg, H.S. (1980) Microbiology, 3rd Edn., Harper & Row, Philadelphia Beachey, E.H. (1980) Bacterial Adherence, in Receptors and Recognition, Series B, Vol. 6 (Cuatrecasas, P. and Greaves, M.F., eds.), Champman and Hall, London Magnani, J.L., Brockhaus, M., Smith, D.F., Ginsburg, V., Blaszczyk, M., Mitchell, K.F., Steplewski, Z. and Koprowski, H. (1981) Science 212, 55-56 Magnani, J.L., Nilsson, B., Brockhaus, M., Zopf, D., Steplewski, Z., Koprowski, H. and Ginsburg, V. (1982) J. Biol. Chem. 257, 14365-14369 Falk, K.-E., Karlsson, K.-A., Larson, G., Thurin, J., Blaszczyk, M., Steplewski, Z. and Koprowski, H. (1983) Biochem. Biophys. Res. Commun. 110, 383-391 Kallenius, G., Molby, R., Svensson, S.B., Winberg, J., Lundblad, A., Svensson, S. and Cedergren, B. (1980) FEMS Lett. I, 279-302 Leffler, H. and Svanborg-Ed&n, C.S. (1980) FEMS Lett. 8, 127-134 9 Sjogren, H.O. (1980) Cancer 45, 1229-1233 10 Hansson, G.C., Karlsson, K.-A., Larson, G., Stromberg, N., Teneberg, S., Thurin, J., Brodin, T., Sjogren, H.O., Hellstrom, I. and Hellstrom, K.-E. (1983) in Glycoconjugates (Chester, A.M., Heineg%rd, D., Lundblad, A. and Svensson, S., eds.), Proceedings of the 7th International Symposium on glycoconjugates, pp. 854-855, Lund, Sweden
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