Institute of Medical Biochemistry, Göteborg University, Box 440, SE 405 30 Göteborg, Sweden
Received on April 12, 2000; revised on July 7, 2000; accepted on July 12, 2000.
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Abstract |
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where R could be H, Fuc or NeuAc. Also observed were structures as
which indicated linear extension along both branches. Observed at higher masses were fully branched structures obtained by stepwise extension with
where R could be H, Fuc or NeuAc. Most probably further branching may occur along both the (13)- and the (1
6)-linked branches to give a partly dendritic structure. Structures with more than one sialic acid substituted could not be observed in the MALDI spectrum. Complementary information of the terminal sequences was obtained by FAB-MS analysis of permethylated undegraded PGCs. High-temperature gas chromatography/mass spectrometry of reduced and permethylated products from enzyme hydrolysis documented that Fuc was present in a blood group O sequence, Fuc-Hex-HexN-. Fucose may be placed on short (monolactosamine) or longer branches, while sialic acid seems to be restricted to monolactosamine branches. The conclusion is that human erythrocyte PGCs display microheterogeneity within terminal and internal parts of the poly-N-acetyllactosamine chains. The first branch from the ceramide end may be located at the second or third Gal and possibly also on the first Gal. Other branches may occur on every N-acetyllactosamine unit in fully branched domains, or there may be linear extensions between branches resulting in incompletely branched structures. The extended linear sequences may be present in both 3- and 6-linked antennae. Terminal structures are based on one, two or maybe higher number of N-acetyllactosamine units.
Key words: human erythrocytes/MALDI-TOF MS/FAB-MS/mass spectrometry/polyglycosylceramides/receptor activity/Helicobacter pylori/endo-ß-galactosidase
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Introduction |
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Results |
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where the first branch is on the first Gal. In the MALDI mass spectrum the higher mass ions could be assigned to structures extended from HexN-Hex-Hex-Cer by units of (2,2,y), y = 0,1. The major ions which belonged to the series (x + 1,x,y)Cer were accompanied by components 162 u lower in mass, which corresponded to lack of one hexose unit. This resulted in ions following series of (x,x,y)Cer, which indicated cleavage along both the (13)- and the (1
6)-linked branch. This important observation indicated that the PGC molecules could have linear extensions along both the (1
3)- and the (1
6)-linked branch. Further branching might result into a partly dendritic structure of the PGC molecules.
Ions that could be assigned to a series of branched neutral glycolipids continued with (4,3,y)Cer, y = 0,1 (24 and 12%, respectively)
where R = H or Fuc. Here and later on the structures are written as terminated on the (16)-linked branch but could as well be on the (1
3)-linked branch since it is not possible from mass spectrometry results to determine whether the linear extension occurs along the (1
3)- or the (1
6)-linked branches.
This structure was extended each time by
(2,2,y), y = 0,1, and R = H or Fuc, and observed up to (10,9,4)Cer. These structures could be rationalized as
where R = H or Fuc and j = 1 (4,3,y)Cer, y = 0,1 (24% and 12%, respectively); j = 2
(6,5,y)Cer, y = 0,1,2 (around 2%); j = 3
(8,7,y)Cer, y = 0,1,2,3 (below 1%); and j = 4
(10,9,y)Cer, y = 0,1,2,3,4 (below 0.3%). y is the number of fucoses substituted ranging from 0 up to a maximum of (x / 2 1) rounded off upwards. The mass difference between components with the same number of substituents (y) was 730 u (C28H46N2O20 = 730.26 [12C], 730.68 [average]).
Ions that could be assigned to structures which indicated linear extensions also at the (16)-linked branch were observed located 162 u lower in mass than the major series (x + 1,x,y)Cer. The series (x,x,y)Cer started with (3,3,0)Cer (4%),
and was extended by
(2,2,y), y = 0,1, and R = H or Fuc, each time up to (5,5,y)Cer and (7,7,y)Cer, where the number of fucoses y = 0 up to a maximum of (x / 2 2) rounded off upwards. The structure (5,5,y)Cer has two branching points either with (1) linear extension at the (16)-linked first branch and at the (1
3)-linked second branch,
(5,5,y)Cer or (2) linear extension at the (16)-linked second branch and at the (1
3)-linked second branch,
(5,5,y)Cer, where R = H or Fuc.
The next higher component in this series, the (7,7,y)Cer, y = 0,1,2,3, has three branching points (see also Table BI)
The possible linear extensions could be at a) the (16)- linked first branch and the (1
3)- linked third branch (R1 = H), b) the (1
6)- linked second branch and the (1
3)- linked third branch (R2 = H) or c) the (1
6)- and the (1
3)- linked third branch (R3 = H).
Ions that could be assigned to structures of neutral free oligosaccharides originating from internal parts of the polylactosamine core chain and cleaved at two positions either along the (13)- or the (1
6)-linked branches, were observed as a series (x,x,y)i where x was ranging from 3 up to 7, starting from (3,3,y)i, y = 0,1
and extended by
(2,2,y), y = 0,1 and R = H or Fuc, (730.26 u, 12C), each time up to (7,7,3)i. The internal neutral oligosaccharides in this series could be written as
where j = 1(3,3,y)i, y = 0,1 (18% and 9%, respectively); j = 2
(5,5,y)i, y = 0,1,2 (around 2%); j = 3
(7,7,y)i,y = 0,1,2,3 (around 0.5%). The number of substitutions (y) were from 0 up to (x / 2 1) rounded off upwards and R = H or Fuc.
Ions due to internal oligosaccharides cleaved from PGC structures at three positions with linear extensions along both the (13)- and the (1
6)- linked branches were observed as series of the form (x 1,x,y)i, where x could be 2, 4 and y = 0,1;
(2,3,0)i (9%) and
(4,5,y)i (around 2%), with R = H or Fuc.
Ions due to neutral terminal oligosaccharides, originating from PGCs cleaved at one position, were observed from Hex-HexN-Hex, (2,1,0)t (5%), and Fuc-Hex-HexN-Hex, (2,1,1)t (32%), to
(4,3,y)t, y = 0,1,2 (8%, 4%, and 4%, respectively) where R = H or Fuc. Even an extended structure of the last one was observed as
(6,5,1)t (2%) where R = H or Fuc.
The results obtained from MALDI-TOF MS in the positive ion mode of the neutral glycolipids and oligosaccharides are presented in Table BI. Examples of possible neutral products obtained after digestion with endo-ß-galactosidase are shown in Figure 4 for isomers of (8,6,y)Cer.
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(4,3,0,1)Cer. It is written as terminated by NeuAc on the (16)- linked branch but this could as well be on the (1
3)-linked branch as discussed above. This indicated that the sialic acid may be preferentially substituted at the first branching point of the PGC molecules. In conformity with the neutral components, ions could be assigned to structures extended by units of (2,2,y) each time (730.26 u (12C), 730.68 u (average mass), creating series of ions of the form (x + 1,x,y,1)Cer.
Series that started with (4,3,0,1)Cer,
was extended by
(2,2,y), y = 0,1, and R = H or Fuc, each time and was observed up to (12,11,4,1)Cer with a mass difference of 730 u between series with the same number of fucoses substituted (y).
These structures could be presented as
where R = H or Fuc and j = 1(6,5,y,1)Cer, y= 0,1 (15% and 13%, respectively); j = 2
(8,7,y,1)Cer, y = 0,1,2 (below 10%); j = 3
(10,9,y,1)Cer, y = 0,1,2,3 (below 4%) and j = 4
(12,11,y,1)Cer, y = 0,1,2,3,4 (below 1.5%). The number of fucoses (y) was ranging from 0 up to a maximum of (x / 2 2) rounded off upwards, where x is the number of HexNs present.
Ions which could be assigned to structures with linear extensions along both the (13)- and the (1
6)- linked branches were observed among the sialic acid-containing components in similarity with the neutral ones. These ions were observed as series of the type (x,x,y,1)Cer, located 162 u lower in mass than the main series of (x + 1,x,y,1)Cer. The (6,5,0,1)Cer structure was accompanied by the (5,5,0,1)Cer (7%),
(5,5,0,1)Cer, with two branching points and linear extension at both the (13)- and the (1
6)-linked second branch. Here it is assumed that NeuAc was substituted at the first branch and that the main elongation was along the (1
3)-linked branch. Ions from the extended structure (7,7,y,1)Cer, y = 0,1 (4% and 5%, respectively) was observed accompanying the (8,7,y,1)Cer structures. This structure will have three branching points with linear extension at either the (1
6)-linked second or third branch,
(7,7,y,1)Cer, linear extension at the (16)- linked second and at the (1
3)- linked third branch (R1 = HexN, R2 = R-Hex-HexN) or only at the third branch (R1 = R-Hex-HexN, R2 = HexN) with R = H or Fuc.
Similarly the (9,9,y,1)Cer, y = 0,1,2 (1%, 1%, and 2%, respectively) structures with four branching points could have linear extensions at the second, third, or fourth branch.
Also ions that could be assigned to structures of fully branched PGCs, unaffected by the enzyme, were observed as (9,7,3,1)Cer (2%), (11,9,4,1)Cer (2.5%) and (13,11,5,1)Cer (1%).
The structure of the components in this series could be presented as
where for j = 1(9,7,3,1)Cer; j = 2
(11,9,4,1)Cer and j = 3
(13,11,5,1)Cer. The extension each time in this series was
(2,2,1), corresponding to a calculated mass difference between the components of 876.82 u (average mass).
Only two negative ions in the MALDI spectrum could be assigned to internal acidic oligosaccharides cleaved at two positions, the structure (3,3,0,1)i and its extension (5,5,0,1)i:
(3,3,0,1)i (12%) and
(5,5,0,1)i (3%). This observation indicated that the sialic acid could be substituted also within the poly-N-acetyllactosamine chain.
Two negative ions could also be assigned to structures from terminal sequences of sialic acid-containing fragments, namely the structures (4,3,0,1)t and (4,3,1,1)t, respectively:
(4,3,0,1)t (4%), and
(4,3,1,1)t (2%), which indicated that substitution by sialic acid was possible in the nonreducing end of the PGCs. The products from internal and terminal sequences above could also be a result of the slower enzymatic cleavage of the bond 3Gal-4Glc1 close to the ceramide.
The results from MALDI-TOF MS in the negative ion mode of the sialylated components are presented in Table BII. Examples of possible sialic acid-containing products formed after cleavage with endo-ß-galactosidase are shown in Figure 5 for isomers of (10,8,y,1)Cer and (11,9,y,1)Cer.
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Supporting evidence by EI-MS analysis of degraded permethylated PGCs
To confirm the conclusions from MALDI-TOF MS and FAB-MS concerning terminal sequences of potential interest for bacterial binding two approaches by EI-MS were applied. One was high-temperature GC/MS of reduced and permethylated hydrolysis products (Figure 7), the other was direct inlet EI-MS of two permethylated subfractions after separation of hydrolysis products by ion exchange chromatography (not reproduced). As was possible to interpret from Figure 7, major components from the TIC (total ion chromatogram) provided evidence for linear internal and terminal extensions. Component A, HexN-Hex, (1,1,0)iol corresponding to (1,1,0)i not detected in MALDI-MS, a nonsubstituted trisaccharide, B, (2,1,0)tol, corresponding to (2,1,0)t, and a fucosylated tetrasaccharide, C, (2,1,1)tol, corresponding to (2,1,1)t were found. Furthermore, the C saccharide was evidence for the blood group O determinant, Fuc2Galß4GlcNAc, and no ions were found corresponding to the Lewis x, Galß4(Fuc
3)GlcNAc, or Lewis y, Fuc
2Galß4(Fuc
3)GlcNAc, determinant. Component D, (2,1,1)iol, corresponding to (2,2,1)i may have contained an internal Fuc linked to Gal, but this is inconclusive due to low amounts. No component was found corresponding to a tetrasaccharide with terminal NeuAc, indicating the existence of only nonextended NeuAcGalßGlcNAc, in contrast to extended fucosylated branches (component C, and Table III from FAB-MS). Component E (corresponding to (2,3,0)t) provided evidence for linear extensions on both (1
3)- and (1
6)-linked branches. Component F corresponded to (3,3,0)i from MALDI-MS. No other components from the enzymatic hydrolysis could be chromatographed due to their high molecular weights.
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Composition of the ceramides of the PGCs
Detailed information of the ceramide composition of the PGCs was achieved from the MALDI-MS spectra in the positive ion mode. From the pseudomolecular ions [M+Na]+ of Hex-Cer and Hex-HexN-Hex-Hex-Cer the most abundant ceramides were determined to be (d18:124:0), (d18:124:1), (d18:122:0), (d18:122:1) and smaller amounts of (d18:1-h24:0), (d18:1-h24:1), (d18:126:0), and (d18:126:1). The results are presented in Table IV.
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Discussion |
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The neutral components of the PGCs seemed to have more frequently linear extension of the first N-acetyllactosamine unit since the MALDI spectrum in positive mode was dominated by the ion corresponding to HexN-Hex-Hex-Cer, (2,1,0)Cer. A lower abundance of Hex-Cer supported a relatively slow hydrolysis at lactose. Some evidence existed for a branching at the first Gal.
In negative mode the most abundant ion in the MALDI spectrum could be assigned to the structure (4,3,0,1)Cer with the branching at the second Gal. However, positive ions with an abundance of 24% and 12%, respectively, could be assigned to structures of the neutral glycolipids (4,3,y)Cer, y = 0,1. Here the branching occurred at the second Gal from the ceramide.
For neutral PGCs the first branching point may more frequently be located on the third Gal than on the second Gal from the ceramide. Linear extensions between branches seemed to be frequent in both neutral and sialic acid-containing components. Ions which could be assigned to series where the branching started at the second Gal from the ceramide were observed for both neutral (as series [x + 1,x,y]Cer) and sialic acidcontaining PGCs (as series [x + 1,x,y,1]Cer). Linear extensions took place after the first, second, third, fourth, and fifth branching point, respectively, for the sialic acidcontaining glycolipids belonging to the series (x + 1,x,y,1)Cer. Ions from fully branched structures, unaffected by the enzyme, were not detected in the MALDI spectrum for the neutral components but were observed in the case of the sialic acidcontaining PGCs. These structures followed the series of the form (x + 2,x,y,1)Cer for normal PGCs. One important result in this investigation was the observation of ions which could be assigned to structures with linear extensions at both the (13)- and the (1
6)-linked branches. Further branching may occur along the extended (1
6)-linked branch which may result in a partly dendritic structure of the PGCs.
The full branching of human PGCs was suggested earlier on the basis of analysis by MALDI-TOF MS of intact PGCs, where high degree of fucosylation was shown (Karlsson et al., 1999). Fucose in PGCs of blood group O represents primarily the H-epitope (Fuc
2Galß4GlcNAc) and is located terminally. High-temperature GC/MS documented this and no evidence was obtained for the presence of Lewis determinants.
Another important observation was, that fucose in human PGCs may be located on di- or monolactosamine terminal extensions while sialic acid is present only on monolactosamine branches. Worth noticing is that both Fuc and NeuAc may be present in the same molecules. The present work was based on the assumption that endo-ß-galactosidase is cleaving linear but not branched poly-N-acetyllactosamine structures (Figure 1). According to Scudder et al. (1983, 1984) branching Gal in the neolacto chains remain resistant to hydrolysis even at very high enzyme concentrations (up to 2.5 U/ml). Our own results confirm that the endo-ß-galactosidase specificity is very restricted.
In summary, human erythrocyte PGCs are microheterogenous within terminal and internal parts of the poly-N-acetyllactosamine chain. The first branch may be located on the first, second or third Gal and linear extensions may occur between branches. The linear extensions may occur along both the (13)- and the (1
6)-linked branch probably followed by further branching resulting in a partly dendritic structure. There may be fully branched regions within the poly-N-acetyllactosamine chain and there are examples of structures which are fully branched. A summarizing formula is shown in Figure 8.
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may hypothetically provide the binding epitope for H.pylori, including part of NeuAc and branching characteristics.
This structure is probably not common to all sialic acid-containing PGCs in the mixture since digestion with endo-ß-galactosidase revealed components belonging to the series (x + 1,x,y,1)Cer observed in the MALDI mass spectrum (see Figure 3). These components have consecutive branching with the second branch at the third Gal and not at the fourth Gal as the structure above. However, as the component (4,3,0,1)Cer was dominating in the MALDI mass spectrum in negative mode and the internal oligosaccharide (3,3,0,1)i was present, some of the human PGC components in the series (x + 2,x,y,1)Cer may have the structure above as an internal part of the molecules (see for example the isomers of [10,8,y,1]Cer and [11,9,y,1]Cer in Figure 5).
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Materials and methods |
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MALDI-TOF MS
MALDI mass spectra were acquired on a TofSpec-E time-of-flight mass spectrometer (Micromass, Manchester, England) equipped with delayed extraction and a nitrogen laser (337 nm, 4 ns pulse, LSI, Boston, MA) operated in the reflectron mode at ±20 kV acceleration voltage. Matrix and calibrations used as described previously (Karlsson et al., 1999).
FAB-MS
FAB-MS was performed on a JEOL SX-102A spectrometer (JEOL, Tokyo, Japan) in positive ion mode and using 6 keV Xenon atoms. 1 µl of the sample dissolved in chloroform/methanol (1:1) was added to the matrix that consisted of a mixture of glycerol/3-nitrobenzyl alcohol (1:1). Mass spectrometric conditions: a) acceleration voltage +10 kV; mass range scanned, m/z 1002400; linear magnet scan with scan time, 25 s; resolution, 1200 (m/m, 10% valley definition); b) acceleration voltage +8 kV; mass range scanned, m/z 20003200; linear magnet scan with scan time, 10.5 s; resolution, 1200 (m/
m, 10% valley definition).
EI-MS
High-temperature GC/MS was performed on a Hewlett-Packard 5890-II gas chromatograph coupled to a JEOL SX-102A spectrometer (JEOL, Tokyo, Japan). Other conditions were as described previously (Karlsson et al., 1994). Direct inlet EI-MS was performed on a JEOL SX-102A mass spectrometer (JEOL, Tokyo, Japan) using the in-beam technique (Breimer et al., 1980
). The electron energy was 70 eV, the trap current 300 µA and the acceleration voltage +10 kV. The temperature in the ion source was programmed from 150°C to 410°C at a rate of 15°C/min.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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Ciucanu,I. and Kerek,F. (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res., 131, 209217.[ISI]
Dabrowski,J., Dabrowski,U., Bermel,W., Kordowicz,M. and Hanfland,P. (1988) Structure elucidation of the blood group B like and blood group I active octaantenary ceramide tetracontasaccharide from rabbit erythrocyte membranes by two-dimensional 1H NMR spectroscopy at 600 MHz. Biochemistry, 27, 51495155.[Medline]
Dejter-Juszynski,M., Harpaz,N., Flowers,H.M. and Sharon,N. (1978) Blood group ABH-specific macroglycolipids of human erythrocytes: isolation in high yields from a crude membrane glycoprotein fraction. Eur. J. Biochem., 83, 363373.[Abstract]
Domon,B. and Costello,C.E. (1988) A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconjugate J., 5, 397409.[ISI]
Dunn,B.E., Cohen,H. and Blaser,M. (1997) Helicobacter pylori. Clin. Microbiol. Rev., 10, 720741.[ISI]
Egge,H., Kordowicz,M., Peter-Katalinic J. and Hanfland,P. (1985) Immunochemistry of II-active oligo- and polyglycosylceramides from rabbit erythrocyte membranes. J. Biol. Chem., 260, 49274935.[Abstract]
Johansson,L., Johansson,P. and Miller-Podraza,H. (1999) Neu5Ac3Gal is part of the Helicobacter pylori binding epitope in polyglycosylceramides of human erythrocytes. Eur. J. Biochem., 266, 559565.
Karlsson,H., Karlsson,N. and Hansson,G.C. (1994) High-temperature gas chromatography and gas chromatography-mass spectrometry of glycoprotein and glycosphingolipid oligosaccharides. Mol. Biotech., 1, 165180.[Medline]
Karlsson,H., Johansson,L., Miller-Podraza,H. and Karlsson, K-A. (1999) Fingerprinting of large oligosaccharides linked to ceramide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: highly heterogeneous polyglycosylceramides of human erythrocytes with receptor activity for Helicbacter pylori. Glycobiology, 9, 765778.
Karlsson,K.-A. (1998) Meaning and therapeutic potential of microbial recognition of host glycoconjugates. Mol. Microbiol., 29, 111.[ISI][Medline]
Karlsson,K.-A. (2000) The human gastric colonizer Helicobacter pylori: a challenge for hostparasite glycobiology. Glycobiology, accepted.
Koscielak,J., Miller-Podraza,H., Krauze,R. and Piasek,A. (1976) Isolation and characterization of poly (glycosyl)ceramides (megaloglycolipids) with A, H and I blood-group activitoes. Eur. J. Biochem., 71, 918.[Abstract]
Koscielak,J., Zdebska,E., Wilczynska,Z., Miller-Podraza,H. and Dzierzkowa-Borodej,W. (1979) Immunochemistry of Ii-active glycosphingolipids of erythrocytes. Eur. J. Biochem., 96, 331337.[Abstract]
Levery,S.B., Nudelman,E.D., Salyan,M.E.K. and Hakomori,S.-i. (1989) Novel tri- and tetrasialosylpoly-N-acetyllactosaminyl gangliosides of human placenta: structure determination of pentadeca- and eicosaglycosylceramides by methylation analysis, fast atom bombardment mass spectrometry and 1H NMR spectroscopy. Biochemistry, 28, 77727781.[ISI][Medline]
Liukkonen,J., Haataja,S., Tikkanen,K., Kelm,S. and Finne,J. (1992) Identification of N-acetylneuraminyl2,3poly-N-acetyllactosamine glycans as the receptors of sialic acid-binding Streptococcus suis strains. J. Biol. Chem., 267, 2110521111.
Loomes,L.M., Uemura,K.-i., Childs,R.A., Paulson,J.C., Rogers,G.N., Scudder,P.R., Michalski,J.-C., Hounsell,E.F., Taylor-Robinson,D. and Feizi,T. (1984) Erythrocyte receptors for Mycoplasma pneumoniae are sialylated oligosaccharides of Ii antigen type. Nature, 307, 560563.[ISI][Medline]
Miller-Podraza,H., Andersson,C. and Karlsson,K.-A. (1993) New method for the isolation of of polyglycosylceramides from human eruthrocyte membranes. Biochim. Biophys. Acta, 1168, 330339.[ISI][Medline]
Miller-Podraza,H., Milh Abul,M., Bergström,J. and Karlsson,K.-A. (1996) Recognition of glycoconjugates by Helicobacter pylori: an apparently high-affinity binding of human polyglycosylceramides, a second sialic acid-based specificity. Glycoconjugate J., 13, 453460.[ISI][Medline]
Miller-Podraza,H., Stenhagen,G., Larsson,T., Andersson,C. and Karlsson,K.-A. (1997) Screening for the presence of polyglycosylceramides in various tissues:partial characterization of blood group-active complex glycosphingolipids of rabbit and dog small intestines. Glycoconjugate J., 14, 231239.[ISI][Medline]
Scudder,P., Uemura,K.-i., Dolby,J., Fukuda,M.N. and Feizi,T. (1983) Isolation and characterization of an endo-ß-galactosidase from Bacteroides fragilis. Biochem J., 213, 485494.[ISI][Medline]
Scudder,P., Hanfland,P., Uemura,K.-i. and Feizi,T. (1984) Endo-ß-galactosidase of Bacteroides fragilis and Escherichia freundii hydrolyze linear but not branched oligosaccharide domains of glycolipids of the neolacto series. J. Biol. Chem., 259, 65866592.
Slomiany,B.L., Slomiany,A., Galicki,N.I. and Kojima,K. (1980) Complex fucolipids of hog gastric mucosa. Structure of the eikosahexoside. Eur. J. Biochem., 113, 2732.[Abstract]
Zdebska,E.,Krauze,R. and Koscielak,J. (1983) Structure and blood-group I activity of poly (glycosyl)ceramides. Carbohydr. Res., 120, 113130.[ISI][Medline]