A missense mutation in connexin26, D66H, causes mutilating keratoderma with sensorineural deafness (Vohwinkel's syndrome) in three unrelated families.

The multiplicity of functions served by intercellular gap junctions is reflected by the variety of phenotypes caused by mutations in the connexins of which they are composed. Mutations in the connexin26 (Cx26) gene ( GJB2 ) at 13q11-q13 are a major cause of autosomal recessive hearing loss (DFNB1), but have also been reported in autosomal dominant deafness (DFNA3). We now report a Cx26 mutation in three families with mutilating keratoderma and deafness [Vohwinkel's syndrome (VS; MIM 124500), as originally described]. VS is characterized by papular and honeycomb keratoderma associated with constrictions of digits leading to autoamputation, distinctive starfish-like acral keratoses and moderate degrees of deafness. In a large British pedigree, we have mapped the defect to the Cx26 locus. All 10 affected members were heterozygous for a non-conservative mutation, D66H, in Cx26. The same mutation was found subsequently in affected individuals from two unrelated Spanish and Italian pedigrees segregating VS, suggesting that D66H in Cx26 is a common mutation in classical VS. This mutation occurs at a highly conserved residue in the first extracellular domain of the Cx26 molecule, and may exert its effects by interfering with assembly into connexons, docking with adjacent cells or gating properties of the gap junction. Our results provide evidence that a specific mutation in Cx26 can impair epidermal differentiation, as well as inner ear function.

Studies on selectin-carbohydrate interactions.

Recruitment of neutrophils to sites of inflammation is now believed to occur through an initial rolling interaction at the luminal surface of activated endothelium and is mediated by a class of mammalian lectins referred to as the selectins. Selectins recognize carbohydrate determinants on co-receptors. It is generally believed that many selectin molecules must bind to many carbohydrate receptor molecules i.e. multivalent binding, to enable sufficient binding strength to elicit the rolling response between the neutrophil and the endothelial cell. One of the approaches to the generation of more potent molecular antagonists of the selectin-mediated cell-cell interaction is to mimic the multivalent interaction in a single compound. Recent experiments utilising conjugated forms of sialyl Lewisx-BSA have explored this feasibility (Welply et al., 1994). In that study, monovalent sLex (sialic acid alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc), the minimum binding determinant for E-selectin, as well as monovalent sialyllactosamine (sialic acid alpha 2-3Gal beta 1-4GlcNAc), a non-binding structure, and the corresponding multivalent BSA-conjugated forms were tested for their ability to inhibit binding of HL-60 cells to immobilised E-selectin. As expected, only sLex and sLex-BSA were found to do so. sLex16-BSA (16 mol tetrasaccharide/mol BSA) showed a dose-dependent inhibition of HL-60 binding with a measured IC50 of 1 microM; demonstrating close to a three-order of magnitude enhancement of inhibitory activity compared to free sLex. This result indicated that multivalent forms of sLex are capable of binding to E-selectin with higher affinity than do monovalent glycans. In another study, fluorescent forms of monovalent sLex were synthesized and used to measure a true thermodynamic dissociation constant for the monovalent sLex:E-selectin interaction of 120 +/- 31 microM (Jacob et.al., 1995).

Binding of sialyl Lewis x to E-selectin as measured by fluorescence polarization.

Fluorescence polarization has been used to directly measure the binding of the tetrasaccharide sialyl Lewisx (sLe(x)[Glc], or NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]Glc) to a soluble form of E-selectin, a member of the class of adhesion molecules that plays an important role in immune-cell response to inflammation. The experiments utilized a fluorescent derivative of sLe(x)[Glc] with fluorescein attached directly to the glucose residue through a beta-glycosidic linkage. The resulting fluorescent sLe(x) was shown to inhibit binding of HL60 cells to immobilized E-selectin and exhibited fluorescence polarization enhancement in the presence of a monovalent form of a recombinant soluble E-selectin-Fc chimera. Thermodynamic dissociation constants of 107 +/- 26 and 120 +/- 31 microM were obtained for the fluorescent sLe(x)[Glc] and the free sLe(x)[Glc] sugars, respectively. These results demonstrate that E-selectin interacts weakly with the minimal carbohydrate recognition determinant sLe(x). Additional binding interactions through the action of the authentic coreceptor or via clustering of the ligand and E-selectin molecules on the respective neutrophil and endothelial cell surfaces may also play a role in the overall cellular binding strength. However, the basic interaction between carbohydrate and protein appears weak, consistent with other carbohydrate-protein interactions studied to date.

Enzymatic synthesis of a 6'-sulphated sialyl-Lewisx which is an inhibitor of L-selectin binding to peripheral addressin.

A sulphated form of sialyl-Lewisx, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc6OSO3 beta 1-3Gal, was synthesized enzymatically from a precursor disaccharide, GlcNAc6OSO3 beta 1-3Gal, using sequential steps involving beta 1,4-galactosyltransferase, alpha 2,3-trans-sialidase and recombinant alpha 1,3-fucosyltransferase, respectively. Successful enzymatic fucosylation at the 3 position of the GlcNAc6OSO3 residue demonstrated that fucosyltransferase are capable of generating, in situ, sulphated sialyl Lewisx structures containing sulphate at the 6 position of GlcNAc. The sulphated sialyl-Lewisx pentasaccharide produced by this procedure inhibited binding of a soluble form of L-selectin to 35SO4-labelled peripheral addressin with an IC50 of 0.8 mM, whereas sialyl-Lewisx tetrasaccharide was a weaker inhibitor, displaying an IC50 of 3.2 mM. Hemmerich and Rosen (Biochemistry, 33, 4820-4829, 1994) recently reported the presence of Gal beta 1-4GlcNAcO6SO3 structures on murine peripheral addressin Sgp50, in addition to sialyl Lewisx structures sulphated at the 6-O-galactose position. Based on our data, we suggest that sialyl Lewisx sulphated at the 6-O-GlcNAc position may also exist on receptors and function as a ligand for L-selectin.

Multivalent sialyl-LeX: potent inhibitors of E-selectin-mediated cell adhesion; reagent for staining activated endothelial cells.

Free, monovalent, SLeX (Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)-GlcNAc), SLn (Neu5Ac alpha 2-3Gal beta 1-4GlcNAc) and corresponding BSA-conjugated forms--displaying different ratios of SLeX and SLn to protein--were tested for their ability to inhibit binding of HL-60 cells to immobilized E-selectin. Free SLeX and conjugated SLeX-BSA inhibited cell binding in a dose-dependent manner. SLn and SLn-BSA did not inhibit binding. SLeX16BSA (16 mol tetrasaccharide/mol BSA) and monovalent SLeX inhibited cell binding with measured inhibitory concentrations (IC50S) of 1 microM and 1 mM, respectively, demonstrating a three-order-of-magnitude enhancement of inhibitory activity with the multivalent form of SLeX. A SLex7BSA conjugate was 10-fold less potent than those with 11 or 16 mol SLeX/mol BSA. An assay which measured neutrophil rolling on interleukin (IL)-1 beta-activated human umbilical vein endothelial cells (HUVECs) showed 50% reduction in the number of rolling neutrophils in the presence of 1 microM SLeX16BSA, whereas the level of free, monovalent SLeX oligosaccharide required to produce the same effect was approximately 0.3 mM. SLeX-BSA was found to be an excellent reagent for staining endothelial cells expressing E-selectin. Biotinylated SLeX-BSA in conjunction with Texas red avidin-stained lipopolysaccharide (LPS)-activated HUVECs, and co-incubation of activated cells with anti-E-selectin, specifically blocked staining. The distribution of E-selectin, as determined by binding of SLeX-BSA, was virtually identical with that obtained by binding of anti-E-selectin antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzymatic characterization of beta-D-galactoside alpha 2,3-trans-sialidase from Trypanosoma cruzi.

The substrate specificity, physico-chemical, and kinetic properties of the trans-sialidase from Trypanosoma cruzi have been investigated. The enzyme demonstrates activity towards a wide range of saccharide, glycolipid, and glycoprotein acceptors which terminate with a beta-linked galactose residue, and synthesizes exclusively an alpha 2-3 sialosidic linkage. Oligosaccharides which terminate in Gal beta 1-4(Fuc alpha 1-3)GlcNAc, Gal beta 1-3(Fuc alpha 1-4)GlcNAc, or Gal alpha 1- are not acceptor-substrates. The enzyme utilizes alpha 2,3-linked sialic acid when the donor species is an oligosaccharide and can also transfer, at a low rate, sialic acid from synthetic alpha-sialosides such as p-nitrophenyl-alpha-N-acetylneuraminic acid, but NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)Glc is not a donor-substrate. The trans-sialidase has an apparent pH optimum of 7.9 and a temperature optimum of 13 degrees C. The kinetic properties of the enzyme suggest that the trans-sialylation reaction may occur via a rapid equilibrium random or steady-state ordered mechanism. A method for immobilizing the enzyme is described together with examples of its use for the synthesis of oligosaccharide and glycoprotein precursors of sialyl-Lewis and sialyl-Lewis.

Purification to homogeneity of Charonia lampas alpha-fucosidase by using sequential ligand-affinity chromatography.

An alpha-fucosidase from the liver of the marine gastropod Charonia lampas was purified to homogeneity using a procedure that included cation-exchange and gel-filtration chromatography, chromatofocusing and a final series of affinity-chromatography steps which involved the following gel-immobilized ligands: N-(5-carboxy-1-pentyl)-1,5-dideoxy-1,5-imino-L-fucitol, N-(5-carboxy-1-pentyl)-2-acetamido-1,5-imino-1,2,5-trideoxy-D-glucitol and thio-beta-D-galactoside. The enzyme was found to be a tetrameric glycoprotein with a native Mr of 208,000, and to exist in a number of isoforms displaying pI values in the range 6.0-6.4. Substrate-specificity studies using a number of fucosylated oligosaccharides of the lacto-N and lacto-N-neo series and a synthetic disaccharide confirmed that the enzyme catalyses the hydrolysis of a broad range of fucosidic linkages, and established the following hierarchy of susceptibility: Fuc alpha 2Gal beta 4Glc much much greater than Fuc alpha 6GlcNAc greater than Fuc alpha 2Gal beta 4GlcNAc greater than Gal beta 3(Fuc alpha 4)GlcNAC much much greater than Gal beta 4(Fuc alpha 3)GlcNAc. Similar relative rates of hydrolysis were also demonstrated using biantennary oligosaccharide alditols as substrates which contained fucose linked either alpha 3 or alpha 6 to the N-acetylglucosaminitol residue of the chitobiosyl core.

The use of HPLC-pulsed amperometry for the characterization and assay of glycosidases and glycosyltransferases.

A sensitive and reproducible high performance chromatographic procedure is described for the assay of jack bean beta-galactosidase in which the reaction products are separated on a Dionex AS6 ion exchange column under alkaline conditions and detected by triple-pulsed amperometry. Quantition of the enzyme-released galactose is accomplished by using either fucose or lactose, the substrate, as an internal standard. The validity of the procedure as a general method for the assay and kinetic characterization of exoglycosidases was demonstrated by performing parallel measurements of galactose using an established coupled-enzyme assay, and using these values to calculate Km and Vmax values against lactose. Additional data are presented which establish the applicability of using a similar HPLC approach for the assay of glycosyltransferases.

The isolation by ligand affinity chromatography of a novel form of alpha-L-fucosidase from almond.

An alpha-fucosidase has been extracted from almond meal and purified 163,000-fold to apparent homogeneity using a novel affinity ligand, N-(5-carboxy-1-pentyl)-1,5-dideoxy-1,5-imino-L-fucitol, coupled to Affi-Gel 102. Substrate specificity studies demonstrate that the enzyme hydrolyzes the alpha-fucosidic linkages in Gal(beta 1----3)(Fuc(alpha 1----4]GlcNAc(beta 1----3)Gal(beta 1----4)Glc and Gal(beta 1----4)(Fuc(alpha 1----3]GlcNAc(beta 1----3)Gal(beta 1----4)Glc at similar rates but is unable to hydrolyze Fuc(alpha 1----2)Gal, Fuc(alpha 1----6)GlcNAc, or the synthetic substrate, p-nitrophenyl alpha-L-fucopyranoside. Hence, the enzyme closely resembles an alpha-fucosidase I isolated previously from a commercial preparation of partially purified almond beta-glucosidase (Ogata-Arakawa, M., Muramatsu, T., and Kobata, A. (1977) Arch. Biochem. Biophys. 181, 353-358). However, native and subunit relative molecular masses of 106,000 and 54,000 respectively, different charge and hydrophobicity properties, and the absence of stimulation by NaCl clearly distinguish this enzyme, designated alpha-fucosidase III, from other almond alpha-fucosidases reported previously.

A novel carbohydrate, differentiation antigen on fucogangliosides of human myeloid cells recognized by monoclonal antibody VIM-2.

A mouse monoclonal antibody, VIM-2, specific for human blood cells of myelomonocytic lineage, was found to bind to a series of minor gangliosides isolated from the cells of patients with chronic myelogenous leukemia (Uemura, K., Macher, B.A., DeGregorio, M., Scudder, P., Buehler, J., Knapp, W., and Feizi, T. (1985) Biochim. Biophys. Acta 846, 26-36). TLC immunostaining studies with the VIM-2 antibody of gangliosides from normal human neutrophils, acute myeloid leukemia, and chronic myelogenous leukemia cells showed that the total amount and the ratio of the VIM-2 gangliosides varies among these different myeloid cells and appears to be related to the level of cellular differentiation. Purification of these gangliosides from chronic myelogenous leukemia cells was aided by a sensitive enzyme-linked immunosorbent assay procedure used in conjunction with high performance liquid chromatography. Structures for two of the immunoreactive gangliosides (a ceramide decasaccharide, VIII3NeuAcV3-Fuc-nLc8Cer and a ceramide dodecasaccharide X3-NeuAcVII3Fuc-nLc10Cer) are proposed from negative ion fast atom bombardment mass spectrometry of the native gangliosides, methylation analysis, and the combined use of glycosidase treatment and TLC immunostaining with carbohydrate sequence specific antibodies. The VIM-2 antigen was thus characterized as involving the sialofucooligosaccharide sequence.

500 MHz 1H n.m.r. of oligosaccharides of N-acetyl-lactosamine-type released from human erythrocyte glycopeptides by endo-beta-galactosidase.

500 MHz 1H n.m.r. spectroscopy has been used in structural studies of three linear and five branched oligosaccharides of N-acetyl-lactosamine-type that were released from desialylated blood group O erythrocyte glycopeptides by treatment with the endo-beta-galactosidase of Bacteroides fragilis followed by reduction. The following oligosaccharide alditols were characterized: (formula; see book)

Characterisation of oligosaccharides released from human-blood-group O erythrocyte glycopeptides by the endo-beta-galactosidase of Bacteroides fragilis. A study of the enzyme susceptibility of branched poly(N-acetyllactosamine) structures.

Desialylated human blood group O erythrocyte glycopeptides were digested with the endo-beta-galactosidase of Bacteroides fragilis and the enzyme-released products reduced with NaBH4 and purified by Bio-Gel P-4 chromatography. Three linear and six branched oligosaccharides of poly(N-acetylllactosamine) type, which together accounted for 90% of the oligosaccharide alditols, were characterised by fast-atom-bombardment mass spectrometry and gas-liquid chromatography/mass spectrometry. Linkage and composition data were obtained for the remaining material. The salient findings were (a) the branched oligosaccharide alditols each contained the sequence: (Formula: see text) and (b) there was no evidence for the terminal branch-point sequence: (Formula: see text). Together these observations indicate that, as with erythrocyte glycolipids described previously [Scudder, P., Hanfland, P., Uemura, K. & Feizi, T. (1984) J. Biol. Chem. 259, 6586-6592], the endo-beta-galactosidase of Bacteroides fragilis cannot hydrolyse branch-point beta-galactosidic linkages on erythrocyte membrane glycopeptides.

Erythrocyte nucleoside and sugar transport. Endo-beta-galactosidase and endoglycosidase-F digestion of partially purified human and pig transporter proteins.

Nucleoside- and glucose-transport proteins isolated from human erythrocyte membranes were photoaffinity-labelled with [3H]nitrobenzylthioinosine and [3H]cytochalasin B, respectively, and subjected to endo-beta-galactosidase or endoglycosidase-F digestion. Without enzyme treatment the two radiolabelled transporters migrated on SDS/polyacrylamide gels with the same apparent Mr (average) of 55,000. Apparent Mr (average) values after endo-beta-galactosidase digestion were 47,000 and 48,000 for the nucleoside and glucose transporters respectively, and 44,000 and 45,000 respectively after endoglycosidase-F digestion. In contrast, endo-beta-galactosidase had no effect on the electrophoretic mobility of the nucleoside transporter isolated from pig erythrocytes. This transport system exhibited a higher Mr than the human protein, endoglycosidase-F treatment decreasing its apparent Mr (average) from 64,000 to 57,000. It is concluded that the human and pig erythrocyte nucleoside transporters are glycoproteins containing N-linked oligosaccharide. The data provide evidence of substantial carbohydrate and polypeptide differences between the human and pig erythrocyte nucleoside transporters, but evidence of molecular similarities between the human erythrocyte nucleoside and glucose transporters.

Sulphate groups are involved in the antigenicity of keratan sulphate and mask i antigen expression on their poly-N-acetyllactosamine backbones. An immunochemical and chromatographic study of keratan sulphate oligosaccharides after desulphation or nitrosation.

Conditions were established for desulphation of hexa-, octa-, deca- and larger oligosaccharides derived from corneal keratan sulphate after treatment with endo-beta-galactosidase. The antigenicities of the desulphated oligosaccharides were compared with those of the native oligosaccharides in chromatogram binding, plastic-plate binding or inhibition of binding assays using a novel microimmunochemical approach with oligosaccharide-lipid conjugates (neoglycolipids). The results clearly show that sulphate residues are essential components of the antigenic determinant(s) recognised by three monoclonal antibodies to keratan sulphate, 5-D-4, 1-B-4 and MZ15, but they mask the i antigen activity of the linear poly-(N-acetyllactosamine) backbones of this glycosaminoglycan. Immunochemical assays, before and after beta-N-acetylglucosaminidase treatment of desulphated linear hexa-, octa- and decasaccharides derived from keratan sulphate, indicate that for reaction with one anti-i antibody, Den, there is an absolute requirement for the non-reducing beta-galactosyl residue of the i antigen structure to be in the terminal position, but with a second anti-i antibody, Tho, there is in addition some reactivity with the i antigen structure having an N-acetylglucosamine residue at the non-reducing end. The chromatographic properties after desulphation or nitrosation of a minor keratan sulphate oligosaccharide (a dodecasaccharide), which reacts especially well with antibody 5-D-4, have provided the first evidence for the presence of glucosamine residues that may be N-sulphated in corneal keratan sulphate.

Isolation and characterization of sulphated oligosaccharides released from bovine corneal keratan sulphate by the action of endo-beta-galactosidase.

A series of oligosaccharides has been isolated from the keratan sulphate peptidoglycan (3 M NaCl fraction) of bovine cornea after digestion with the endo-beta-galactosidase of Bacteroides fragilis. Structural information on the major oligosaccharides was obtained from (a) their susceptibilities to endo-beta-galactosidase before and after desulphation, (b) their elution positions on a column of Bio-Gel P-4 and retention times on a high-performance anion-exchange column and (c) negative-ion fast-atom-bombardment mass spectrometry. More than 75% of the oligosaccharides were sulphated unbranched poly(N-acetyllactosamine) sequences, (-3/4GlcNAc beta 1-3Gal beta 1-)n, and approximately 3% was the neutral disaccharide, GlcNAc beta 1-3Gal. The sulphated disaccharide, GlcNAc-SO-3 beta 1-3Gal, accounted for almost 35% of the oligosaccharide material while 40% consisted of four oligosaccharides, unbranched tetra-, hexa-, octa- and decasaccharides of poly(N-acetyllactosamine) type, having 3, 5, 7 and 9 sulphate residues respectively. Proton nuclear magnetic resonance studies at 500 MHz (Hounsell, E. F., et al. following paper in this journal) have shown that a sulphate residue is attached to the C-6 position of each N-acetylglucosamine and each internal galactose residue of these four oligosaccharides which express to varying degrees the antigenic determinants recognised by three monoclonal antibodies to keratan sulphate (Mehmet, H. et al., paper which follows the next paper in this journal).

The antigenic determinants recognized by three monoclonal antibodies to keratan sulphate involve sulphated hepta- or larger oligosaccharides of the poly(N-acetyllactosamine) series.

The carbohydrate determinants of keratan sulphate recognized by three monoclonal antibodies (5-D-4, 1-B-4 and MZ15) have been investigated by solid-phase radioimmunoassay using bovine corneal keratan sulphate as the immobilized reference antigen. The antibodies appeared highly specific for sulphated poly(N-acetyllactosamine) sequences, for their binding was strongly inhibited by preparations of keratan sulphate, but not by glycoproteins with non-sulphated poly(N-acetyllactosamine) sequences of I and i antigen types, a desulphated keratan sulphate hexasaccharide, an array of neutral and sulphated mono- and disaccharides and other glycosaminoglycans. Inhibition of binding assays using a series of structurally characterized sulphated di, tetra-, hexa-, octa- and decasaccharides, and partially characterized larger oligosaccharides, isolated from bovine corneal keratan sulphate after digestion with endo-beta-galactosidase (see preceding two papers in this journal) showed that the smallest oligosaccharide reactive with all three antibodies was the linear pentasulphated hexasaccharide, E-II although antibody 1-B-4 reacted with a tetrasulphated analogue. The heptasulphated octasaccharide, G-III, was more active; among the structurally characterized keratan sulphate oligosaccharides the nonasulphated decasaccharide, I-IV, was the most active. Thus, the hepta- and octasaccharide sequences, indicated by brackets below are proposed as candidate antigenic structures recognized by the three monoclonal antibodies. (Formula: see text). Antibody 5-D-4 differs from the other two antibodies in reacting relatively strongly with a minor oligosaccharide which chromatographs as a hexasulphated octasaccharide, G-I, and most strongly with a minor sulphated, linear dodecasaccharide, J-II, which has been partially characterized [Tang, P.W., Scudder, P., Mehmet, H., Hounsell, E. F. & Feizi, T., unpublished results] and may contain N-sulphated glucosamine residues.

1H-NMR studies at 500 MHz of a neutral disaccharide and sulphated di-, tetra-, hexa- and larger oligosaccharides obtained by endo-beta-galactosidase treatment of keratan sulphate.

In the preceding paper in this journal, the major oligosaccharides obtained by endo-beta-galactosidase digestion of bovine corneal keratan sulphate were identified as a neutral disaccharide, GlcNAc beta 1-3Gal, and sulphated di-, tetra-, hexa-, octa- and decasaccharides based on the sequence (-3/4GlcNAc beta 1-3Gal beta 1-)n having 1, 3, 5, 7 and 9 sulphate groups, respectively. In the present study, these oligosaccharides have been analysed by 500-MHz 1H-NMR spectroscopy using spin-decoupling and two-dimensional correlated spectroscopy experiments. The NMR data confirm the beta-configuration of all the interglycosidic linkages and are consistent with an alternating sequence of----4GlcNAc and----3Gal, a non-reducing-end N-acetylglucosamine residue and a reducing-end galactose residue. The NMR data have also established that a sulphate group is linked to the C6 position of all sugar residues except the reducing-end galactose as follows: (Formula: see text). The signals of the protons attached to the sulphated carbon atoms show marked downfield shifts (approximately 0.4 ppm from equivalent protons of non-sulphated carbon atoms), while the protons at C5 vicinal to sulphated atoms show a change of 0.1-0.2 ppm and other protons of the sulphated monosaccharides show smaller changes in chemical shift (0.01-0.1 ppm). The proton at C4 of the non-sulphated reducing-end galactose linked at C3 also shows a significant change in chemical shift (0.03 ppm).

Novel antigenic specificity involving the blood group antigen, Lea, in combination with onco-developmental antigen, SSEA-1, recognized by two monoclonal antibodies to human milk-fat globule membranes.

Two monoclonal antibodies to human milk-fat globule membranes, which recognize an epithelial antigen designated MAM-3c, were found to bind strongly to epithelial glycoproteins derived from non-secretors. Further investigations, using purified glycoproteins and structurally defined oligosaccharides, established that the optimal antigenic structure for both antibodies involves the Type 1 based blood group antigen, Lea, in combination with the Type 2 based onco-developmental antigen, SSEA-1, (Formula: see text) as in lacto-N-difucohexaose II. The antibodies may also react with the corresponding monofucosyl structures lacking the 3- or 4- linked fucose residues and to a lesser extent with the afucosyl tetrasaccharide sequence as in lacto-N-tetraose. The Lea and SSEA-1 antigens are known to occur on human epithelial glycoproteins. However, this is the first report of an antigenic specificity involving a combination of the Type 1 and Type 2 based fuco-oligosaccharides and occurring on epithelial glycoproteins.

Glycosphingolipid carriers of carbohydrate antigens of human myeloid cells recognized by monoclonal antibodies.

Six monoclonal antibodies with known specificities for the carbohydrate antigens i, X or Y, and seven anti-myeloid antibodies (determinants unknown) selected for their differing reaction patterns with human leucocytes were tested in chromatogram binding assays for reactions with myeloid cell glycolipids derived from normal human granulocytes and chronic myelogenous leukemia cells. Antigenicities were found exclusively on minor glycolipids which were barely or not at all detectable with orcinol-sulphuric acid stain. Among these, a neutral glycosphingolipid bound the anti-i antibody Den and chromatographed as the ceramide octasaccharide, Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer. Several species of neutral glycosphingolipids with six to more than ten monosaccharides were detected which carry the X antigen and others the Y antigen: Gal beta 1----4(Fuc alpha 1----3)GlcNAc and Fuc alpha 1----2Gal beta 1----4(Fuc alpha 1----3)GlcNAc, respectively. In addition, three new types of carbohydrate specificities were detected among the myeloid cell glycolipids. Two were associated with neutral glycolipids: the first, recognised by anti-myeloid antibodies VIM-1 and VIM-10, was expressed on a distinct set of glycolipids with six or more monosaccharides, and the second, recognized by VIM-8, was expressed on glycolipids with more than ten monosaccharides. The third specificity, recognised by the anti-myeloid antibody VIM-2, was expressed on slow migrating sialoglycolipids with backbone structures of the poly-N-acetyllactosamine type that are susceptible to degradation with endo-beta-galactosidase. Thus, we conclude that the i and Y antigens occur among the glycolipids of normal myeloid and chronic myelogenous leukemia cells and that a high proportion of hybridoma antibodies raised against differentiation antigens of myeloid cells are directed at carbohydrate structures.