Chemical and immunological identification of glycolipid-based blood group ABH and Lewis antigens in human kidney

A polar non-acid glycolipid fraction has been isolated from human kidney. It was shown by thin-layer chromatography to be a mixture of glycolipids having more than four carbohydrate residues. Immunological testing revealed strong blood group Lea and A activity together with weak Leb, P1 and B activity. Mass spectrometry of the permethylated and permethylated-reduced (LiAlH4) glycolipid fraction showed that the two major components were a five sugar fucolipid (isomer of Lea) and a glycolipid having four hexoses and one N-acetylhexosamine. In addition, blood group Leb, B and A type hexaglycosylceramides were present. Evidence for small amounts of more complex glycolipids was also found. Acid degradation and gas chromatography of the native fraction revealed fucose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. This is the first chemical isolation and characterization of complex blood group active glycolipids in human kidney. The existence of these molecules is discussed in view of their possible role as transplantation antigens.     

Modes of shedding of glycosphingolipids from mouse lymphoma cells

To characterize the process by which glycolipids are shed from cell membranes, the cellular and supernatant glycolipids were compared from a variant of the mouse lymphoma L5178Y which had been selected for strong expression of the neutral glycolipid gangliotriaosylceramide (GgOse3Cer). This glycolipid was present in three forms which differed in their fatty acid composition. Whereas the major cell-associated form of GgOse3Cer contained C24 fatty acids, the predominant form shed into the culture supernatant contained C16 fatty acids. Ultracentrifugation of the culture medium yielded a pellet with a GgOse3Cer profile similar to that of the cells and a supernatant enriched in the C16 fatty acid form. Gel filtration of the culture medium revealed two GgOse3Cer-containing pools. The first was excluded from Sepharose CL-2B and had a GgOse3Cer profile similar to that of the cells, while the second migrated with proteins in the range of 25,000-500,000 daltons and was enriched in the C16 fatty acid form. These results suggest two forms in which glycolipids are released from cell membranes. The first is in a large complex, possibly a membrane vesicle, which retains the glycolipid profile of the membrane of intact cells while the second form appears to result from the preferential release of particular glycolipid components.     

Polarity of the Forssman glycolipid in MDCK epithelial cells

To determine whether epithelial plasma membrane glycolipids are polarized in a manner analogous to membrane proteins, MDCK cells grown on permeable filters were analyzed for the expression of Forssman ceramide pentasaccharide, the major neutral glycolipid in these cells. In contrast to a recent report which described exclusive apical localization of the Forssman glycolipid (Hansson, G.C., Simons, K. and Van Meer, G. (1986) EMBO J. 5, 483-489), immunofluorescence and immunoelectron microscopic staining revealed the Forssman glycolipid on both the apical and basolateral surfaces of polarized cells. Immunoblots indicated that the Forssman antigen was detectable only on glycolipids and not on proteins. Analysis of metabolically labeled glycolipids released into the apical and basal culture medium, either as shed membrane vesicles or in budding viruses, also demonstrated the presence of the Forssman glycolipid on both apical and basolateral membranes of polarized cells. Quantitation of the released glycolipid indicated that the Forssman glycolipid was concentrated in the apical membrane. These results are consistent with previous reports which described quantitative enrichment of glycolipids in the apical domain of several epithelia.     

Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. I. Can structure of the phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids

A number of eukaryotic surface glycoproteins, including the variant surface glycoproteins of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosyl-phosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. The rapidity of this carboxyl-terminal modification suggests the existence of a prefabricated precursor glycolipid that can be transferred en bloc to the polypeptide. We have reported the purification and partial characterization of a candidate precursor glycolipid (P2) and of a compositionally similar glycolipid (P3) from T. brucei (Menon, A. K., Mayor, S., Ferguson, M. A. J., Duszenko, M., and Cross, G. A. M. (1988) J. Biol. Chem. 263, 1970-1977). The primary structure of the glycan portions of P2 and P3 have now been analyzed by a combination of selective chemical fragmentation and enzymatic glycan sequencing at the subnanomolar level. The glycans were generated by deamination, NaB3H4 reduction, and dephosphorylation of glycolipids purified from different trypanosome variants. Glycan fragments derived from biosynthetically labeled glycolipids were also analyzed. The cumulative data strongly suggest that P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The structural similarities suggest that GPI membrane anchors are derived from common precursor glycolipids that become variably modified during or after addition to newly synthesized proteins.     

Effects of O-linked glycosylation on the cell surface expression and stability of decay-accelerating factor, a glycophospholipid-anchored membrane protein

Decay accelerating factor (DAF) is a glycophospholipid-anchored membrane glycoprotein that protects mammalian host cells from inadvertant complement lysis. The effects of inhibiting mucin-type O-glycosylation on the cell surface expression of DAF were studied by introducing an expression vector for human DAF into wild-type Chinese hamster ovary and ldlD cells. The ldlD cells express reversible defects in the addition of galactose and N-acetylgalactosamine (GalNAc) to oligosaccharide chains on glycoproteins and glycolipids. Mucin-type O-glycosylation of proteins is inhibited in ldlD cells and can be selectively corrected by the addition of GalNAc to the culture medium. The attachment of a phosphatidylinositol phospholipase C-sensitive glycolipid anchor to DAF and its efficient sorting to the cell surface in ldlD cells were independent of galactose and GalNAc additions to glycolipids and proteins. Attachment of galactose and GalNAc to DAF's glycolipid anchor were apparently not required for its normal function. However, in the absence of O-glycosylation DAF was proteolytically cleaved soon after reaching the cell surface, and a large fragment of DAF was released into the culture medium. This rapid proteolysis/release resulted in the expression of very low steady state levels of O-glycosylation-deficient DAF as measured by immunoblotting. These results, in conjunction with those obtained from studies of three other membrane glycoproteins expressed in ldlD cells, suggest that O-linked sugars on membrane glycoproteins may frequently play a role in determining the level of cell surface expression of these proteins.     

Glycosphingolipids of human large intestine: detailed structural characterization with special reference to blood group compounds and bacterial receptor structures

Non-acid glycosphingolipid expression was studied in the large intestines from four individuals with the A1Le(a-b+), BLe(a-b+), and OLe(a-b+) blood group phenotypes. In the A1Le(a-b+) case, specimens were taken from the ascending and sigmoid parts of the large intestine in order to compare the expression of glycolipids in the proximal and distal regions of the intestine. In one blood group OLe(a-b+) individual, epithelial cells were isolated from the residual stroma to compare the glycolipid compositions in these two tissue compartments. GlcCer, GalCer, LacCer, Gb3Cer, and Gb4Cer were the major compounds in all three individuals, as shown by mass spectrometry, proton NMR spectroscopy, and degradation studies. The Lea-5 glycolipid was the major complex blood group glycolipid in all individuals, except in the proximal ascending part of the large intestine of the A1Le(a-b+) case, in which the Leb-6 glycolipid was predominant. There were trace amounts of blood group ABH glycolipids, in agreement with the ABO blood group phenotypes of the donors, Lewis antigens with more than six sugar residues in the carbohydrate chain, and blood group X and Y glycolipid antigens. The epithelial cells were dominated by monoglycosylceramides and the Lea-5 glycolipid, while only trace amounts of di-, tri-, and tetraglycosylceramide structures were present. No reactivity was seen in the epithelial cell fraction with Gal alpha 1-4Gal specific Escherichia coli, anti-Pk, or anti-P antibodies, indicating the absence of the glycolipid-borne Gal alpha 1-4Gal sequence in human large intestinal epithelial cells.     

Novel fucolipids accumulating in human adenocarcinoma. III. A hybridoma antibody (FH6) defining a human cancer-associated difucoganglioside (VI3NeuAcV3III3Fuc2nLc6)

A new fucoganglioside, 6B, which accumulates in human colonic adenocarcinoma but is absent in normal colonic mucosa, was isolated from a monosialoganglioside fraction of colonic adenocarcinomas. The structure of this ganglioside was identified as shown below by methylation analysis, direct probe mass spectrometry, and enzymatic degradation followed by examination of the degradation products with specific monoclonal antibodies. (formula; see text) The hybridoma (FH6) secreting a monoclonal IgM antibody directed to this glycolipid was selected by reactivity of the antibody with this ganglioside and lack of reactivity with other glycolipids having a closely related structure, such as sialosyllactoneotetraosylceramide (IV3NeuAcnLc4), sialosyllactofucopentaosy(III)ceramide (IV3NeuAcIII3FucnLc4), sialosyllactofucopentaosy(II)ceramide (sialosyl-Lea glycolipid; IV3NeuAcIII4FucLc4), and 6C fucoganglioside (sialosyl 2 leads to 6 fucoganglioside; VI6NeuAcIII3FucnLc6). The antibody was highly reactive with a large variety of human cancer cells, but was less reactive or did not react with a variety of normal cells.     

Complexing of glycolipids and their transfer between membranes by the activator protein for degradation of lysosomal ganglioside GM2

The lysosomal degradation of ganglioside GM2 by hexosaminidase A depends on the presence of the specific activator protein which mediates the interaction between micellar or membrane-bound ganglioside and water-soluble hydrolase. The mechanism and the glycolipid specificity of this activator were studied in more detail. 1. It could be shown with three different techniques (isoelectric focusing, centrifugation and electrophoresis) that the activator protein extracts glycolipid monomers from micelles or liposomes to give water-soluble complexes with a stoichiometry of 1 mol of glycolipid/mol of activator protein. Liposome-bound ganglioside GM2 is considerably more stable against extraction and degradation than micellar ganglioside. 2. In the absence of enzyme the activator acts in vitro as glycolipid transfer protein, transporting glycolipids from donor to acceptor membranes. 3. The activator protein is rather specific for ganglioside GM2. Other glycolipids (GM3 GM1, GD1a and GA2) form less stable complexes with the activator and are transferred at a slower rate (except for ganglioside GM1) than ganglioside GM2.     

Forssman glycolipid variants of dog gastric mucosa. Structure of a branched ceramide octasaccharide.

A fucose-containing ceramide octasaccharide exhibiting Forssman antigenic activity, and reacting in human H anti-H and anti-A systems, was isolated from water-soluble glycolipids of dog gastric mucosa. Defucosylation of the glycolipid resulted in the loss of H-activity, but had no effect on its Forssman nor blood-group A antigenic activity. The branched structure of glycolipid was identified by partial acid hydrolysis, sequential degradation with specific glycosidases and comparison of the permethylation products of the native and enzyme-degraded compound. The structure of this glycolipid is proposed to be: formula.     

Point mutational analysis of the liganding site in human glycolipid transfer protein. Functionality of the complex

Mammalian glycolipid transfer proteins (GLTPs) facilitate the selective transfer of glycolipids between lipid vesicles in vitro. Recent structural determinations of the apo- and glycolipid-liganded forms of human GLTP have provided the first insights into the molecular architecture of the protein and its glycolipid binding site (Malinina, L., Malakhova, M. L., Brown, R. E., and Patel, D. J. (2004) Nature 430, 1048-1053). In the present study, we have evaluated the functional consequences of point mutation of the glycolipid liganding site of human GLTP within the context of a carrier-based mechanism of glycolipid intermembrane transfer. Different approaches were developed to rapidly and efficiently assess the uptake and release of glycolipid by GLTP. They included the use of glass-immobilized, glycolipid films to load GLTP with glycolipid and separation of GLTP/glycolipid complexes from vesicles containing glycolipid (galactosylceramide or lactosylceramide) or from monosialoganglioside dispersions by employing nickel-nitrilotriacetic acid-based affinity or gel filtration strategies. Point mutants of the sugar headgroup recognition center (Trp-96, Asp-48, Asn-52) and of the ceramide-accommodating hydrophobic tunnel (Phe-148, Phe-183, Leu-136) were analyzed for their ability to acquire and release glycolipid ligand. Two manifestations of point mutation within the liganding site were apparent: (i) impaired formation of the GLTP/glycolipid complex; (ii) impaired acquisition and release of bound glycolipid by GLTP. The results are consistent with a carrier-based mode of GLTP action to accomplish the intermembrane transfer of glycolipid. Also noteworthy was the inefficient release of glycolipid by wtGLTP into phosphatidylcholine acceptor vesicles, raising the possibility of a function other than intermembrane glycolipid transfer in vivo.     

The intracellular voyage of cholera toxin: going retro

Cholera toxin (CT) and related AB₅-subunit toxins move from the plasma membrane through the trans-Golgi and endoplasmic reticulum (ER) to the cytosol of host cells. The toxins exploit a specific glycolipid pathway rather than a protein pathway. They bind glycolipids that associate with lipid rafts at the cell surface, which carry the toxins retrograde to the Golgi and ER. In the ER, the A1-chain of the CT unfolds and enters the cytosol by hijacking the cellular machinery that enables misfolded proteins to cross the membrane for degradation by the proteasome, a process termed retro-translocation. Upon entering the cytosol, the A1-chain rapidly refolds, avoids the proteasome and induces toxicity.     

Glycolipid depletion using a ceramide analogue (PDMP) alters growth, adhesion, and membrane lipid organization in human A431 cells.

Glycolipids were depleted from the membranes of human A431 cells using 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glucosylceramide synthetase. After 6 days of culture in the presence of 5 microM D-threo-PDMP, glycolipid content was reduced to approximately 5% of control levels. By contrast, synthesis per cell of phosphatidylcholine, sphingomyelin, triglycerides, and glycoprotein was relatively unchanged in PDMP-treated cells. In parallel with glycolipid depletion, PDMP-treated cells exhibited a rapid loss of epithelial cell morphology, a reduced rate of cell growth, and inhibition of cell-substrate adhesion. The effects of D-threo-PDMP on cell morphology and substrate adhesion were blocked by exogenous GM3 addition and were not observed with L-threo-PDMP (a relatively inactive enantiomer). Fluorescence photobleaching and recovery (FPR) was used to investigate the hypothesis that glycolipids influence cell behavior, in part, by changing the diffusion characteristics of membrane proteins and lipids. Diffusion coefficients and mobile fractions of two integral membrane proteins, the EGF receptor and a class I MHC antigen, did not differ significantly between control and PDMP-treated cells. Diffusion coefficients of lipid probes, NBD-PC and fluorescent GM1 ganglioside, were similarly unaffected by glycolipid depletion. However, lipid probes did show a significant increase in mobile fraction (the fraction of lipids that are free to diffuse) in PDMP-treated cells. This increase was blocked by culturing cells in the presence of exogenous GM3 ganglioside. The results suggest that glycolipids play a role in the formation of lipid domains in A431 cell membranes. Glycolipid-mediated changes in membrane lipid organization may influence receptor activation and transmembrane signaling, leading to changes in cell growth, morphology, and adhesion.     

A comparison of membrane components of normal and transformed BALB/c cells.

Membrane glycolipids, glycoproteins, and surface proteins of normal and transformed BALB/c cell lines have been compared. Several virally and spontaneously transformed cell lines showed differences in membrane components compared to normal A31 cells. These differences consisted of increased amounts of simpler gangliosides, absence of the large external transformation sensitive (LETS) protein, and the appearance of a major new glycoprotein band of about 105 000 molecular weight. In contrast, the spontaneously transformed cell line that caused the fastest growing tumors in vivo and the most rapid animal death (3T12T) did not have these changes. A31 and 3T12T glycolipid profiles appear similar as did glycoproteins and cell surface proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When Pronase-generated glycopeptides were analyzed by Sephadex G-50 chromatography, and enrichment in faster-eluting species was seen in two killing tumor lines (c5T and 3T12T) compared to A31. Regressing tumor lines (MSC, c5) did not show this change. Isolated membrane glycoproteins yield glycopeptides of different sized after Pronase digestion. In addition, several 3T12T glycoproteins yield glycopeptides that are larger than those from the corresponding glycoproteins of A31 cells. It appears that glycopeptide alterations associated with transformation occur in several membrane glycoproteins.     

Monoclonal antibodies recognizing epitopes carried on both glycolipids and glycoproteins of the human milk fat globule membrane.

The molecules of the human milk fat globule membrane (MFGM) which bind four murine monoclonal antibodies (LICR LON M3, M8, M18 and M24) raised against the human MFGM have been identified. By using 'Western' blotting [Burnette (1981) Anal. Biochem. 112, 195-203] it was shown that each antibody reacted with a different set of proteins. M3 and M24 were similar in their pattern of reaction with the membrane proteins, but were quite distinct from M8 and M18, which also differed from each other. Glycopeptides prepared from the MFGM by exhaustive Pronase digestion were able to inhibit partially the binding of M3 and M24, and prevent totally the binding of M8 and M18, to the MFGM in an enzyme-linked immunoabsorbent assay. Oligosaccharides obtained by the deproteination of human milk also completely inhibited the binding of M3, M18 and M24 to the MFGM. However, the binding of M8 was not inhibited by these saccharides, and therefore M8 may not be recognizing a simple carbohydrate determinant. By using an enzyme-linked assay, M8 and M18 were shown not to bind to MFGM glycolipid, whereas M3 and M24 did, and this was confirmed by overlaying thin layer chromatograms of MFGM lipids with these antibodies. Both M3 and M24 showed a similar complex pattern of reaction, binding to more than one glycolipid moiety. By these means all four antibodies have been shown to react with antigens which involve carbohydrate side chains carried on different proteins, and two were also shown to react with such determinants on glycolipids.     

Specificity of binding of a strain of uropathogenic Escherichia coli to Gal alpha 1----4Gal-containing glycosphingolipids

A strain of Escherichia coli originally isolated from urine of a patient with acute pyelonephritis was studied in detail for binding to glycosphingolipids. Bacteria labeled metabolically with [14C]glucose were layered over a glycolipid chromatogram and bound bacteria were detected by autoradiography. The detection was down to a few ng of glycolipid (pmol level) under these assay conditions. At a test level of 500 ng all glycolipids (more than a dozen molecular species analyzed) with Gal alpha 1----4Gal as an internal or terminal part bound the bacteria strongly while glycolipids known to lack this sequence were negative. Conformational analysis using hard sphere calculations including the exo-anomeric effect showed a bend in the saccharide chain at this disaccharide with a largely hydrophobic surface of the convex side, probably being part of the binding epitope. Mixtures of glycolipids isolated from a human ureter scraping and from urinary sediments bound bacteria in the 2- to 7-sugar interval. Thus, this infectious strain of E. coli recognizes glycolipids being present in epithelial cells lining the urinary tract.     

Effects of bile salts on glucosylceramide containing membranes

The glycolipid transfer protein (GLTP) is capable of transporting glycolipids from a donor membrane, through the aqueous environment, to an acceptor membrane. The GLTP mediated glycolipid transfer from sphingomyelin membranes is very slow. In contrast, the transfer is fast from membranes composed of phosphatidylcholine. The lateral glycolipid membrane organization is known to be driven by their tendency to mix non-randomly with different membrane lipids. Consequently, the properties of the membrane lipids surrounding the glycolipids play an important role in the ability of GLTP to bind and transfer its substrates. Since GLTP transfer of glycolipids is almost nonexistent from sphingomyelin membranes, we have used this exceptionality to investigate if membrane intercalators can alter the membrane packing and induce glycolipid transfer. We found that the bile salts cholate, deoxycholate, taurocholate and taurodeoxycholate, cause glucosylceramide to become transferrable by GLTP. Other compounds, such as single chain lipids, ceramide and nonionic surfactants, that have membrane-perturbing effects, did not affect the transfer capability of GLTP. We speculate that the strong hydrogen bonding network formed in the interfacial region of glycosphingolipid-sphingomyelin membranes is disrupted by the membrane partition of the bile salts causing the glycosphingolipid to become transferrable.     

MmpS4 promotes glycopeptidolipids biosynthesis and export in Mycobacterium smegmatis

The MmpS family (mycobacterial membrane protein small) includes over 100 small membrane proteins specific to the genus Mycobacterium that have not yet been studied experimentally. The genes encoding MmpS proteins are often associated with mmpL genes, which are homologous to the RND (resistance nodulation cell division) genes of Gram-negative bacteria that encode proteins functioning as multidrug efflux system. We showed by molecular genetics and biochemical analysis that MmpS4 in Mycobacterium smegmatis is required for the production and export of large amounts of cell surface glycolipids, but is dispensable for biosynthesis per se. A new specific and sensitive method utilizing single-chain antibodies against the surface-exposed glycolipids was developed to confirm that MmpS4 was dispensable for transport to the surface. Orthologous complementation demonstrated that the MmpS4 proteins are exchangeable, thus not specific to a defined lipid species. MmpS4 function requires the formation of a protein complex at the pole of the bacillus, which requires the extracytosolic C-terminal domain of MmpS4. We suggest that MmpS proteins facilitate lipid biosynthesis by acting as a scaffold for coupled biosynthesis and transport machinery.     

A processive lipid glycosyltransferase in the small human pathogen Mycoplasma pneumoniae: involvement in host immune response

The human pathogen Mycoplasma pneumoniae has a very small genome but with many yet not identified gene functions, e.g. for membrane lipid biosynthesis. Extensive radioactive labelling in vivo and enzyme assays in vitro revealed a substantial capacity for membrane glycolipid biosynthesis, yielding three glycolipids, five phosphoglycolipids, in addition to six phospholipids. Most glycolipids were synthesized in a cell protein/lipid-detergent extract in vitro; galactose was incorporated into all species, whereas glucose only into a few. One (MPN483) of the three predicted glycosyltransferases (GTs; all essential) was both processive and promiscuous, synthesizing most of the identified glycolipids. These enzymes are of a GT-A fold, similar to an established structure, and belong to CAZy GT-family 2. The cloned MPN483 could use both diacylglycerol (DAG) and human ceramide acceptor substrates, and in particular UDP-galactose but also UDP-glucose as donors, making mono-, di- and trihexose variants. MPN483 output and processitivity was strongly influenced by the local lipid environment of anionic lipids. The structure of a major beta1,6GlcbetaGalDAG species was determined by NMR spectroscopy. This, as well as other purified M. pneumoniae glycolipid species, is important antigens in early infections, as revealed from ELISA screens with patient IgM sera, highlighting new aspects of glycolipid function.     

Glycolipid-lectin interactions: reactivity of lectins from Helix pomatia, Wisteria floribunda, and Dolichos biflorus with glycolipids containing N-acetylgalactosamine

The autoradiographic detection of 125I-labeled lectins binding to glycolipids on thin-layer chromatograms can be used to rapidly analyze total glycolipid extracts of cells or tissues for specific oligosaccharide structures. The Helix pomatia lectin which binds with high affinity to terminal alpha-linked GalNAc residues did not bind to globoside (terminal beta 1-3GalNAc) but did bind the ganglioside GM2 and its asialo derivative which have terminal beta 1-4GalNAc residues. The lectin from Dolichos biflorus bound specifically to the Forssman glycolipid with relatively low affinity. The lectin from Wisteria floribunda was bound to Forssman glycolipid, globoside, and the asialo derivative of the ganglioside GM2. The interactions of these lectins with the glycolipid-derived, 3H-labeled oligosaccharides was also analyzed by affinity chromatography. The results indicated that the reactivity of multivalent carbohydrate-binding proteins with polyvalent surfaces of glycolipids is strong enough to permit detection of low-affinity interactions that may not be observed in binding assays that are based on carbohydrate-protein interactions in solution. The autoradiographic analysis of 125I-Helix pomatia lectin binding to thin-layer chromatograms of total lipid extracts from human erythrocyte membranes detected the quantitative differences in the A-active glycolipids from type A1 and A2 cells.     

Biosynthesis of Lewis antigenic glycolipid by cell-free extracts of human intestinal tumor cells cultured in serum-free medium

Extracts of the human intestinal tumor cell line SW1116 were able to stimulate the incorporation of (14C) fucose from GDP-(14C) fucose into organically soluble glycolipid. The reaction required a purified glycolipid preparation from human meconium as lipid acceptor. The active glycolipid co-migrated with standard globoside on high performance thin-layer chromatography (HPTLC) and had molecular species (M + H) under fast-atom bombardment mass spectrometry of 1199, 1245 and 1269. Globoside itself was inactive and asialo GM1b had low activity. The radioactive products co-purified with Lewis a and Lewis b and co-migrated principally (60-90%) with Lewis b monoclonal antibody binding cellular glycolipids on HPTLC. Analysis of fucosidase digests suggested the presence of two different fucosyl-hexose linkages one of which was susceptible to cleavage. We conclude that the data are consistent with fucosylation of lactotetraosyl ceramide to Lewis a and Lewis b antigenic glycolipids.