Characterization of a human intestinal fucolipid with blood group Lea activity.

A fucolipid that carried human blood group Lea activity was isolated from human small intestine. It contianed fucose, galactose, N-acetyl glucosamine, glucose, and ceramide in a molar ratio of 1:2:1:1:1. After periodate oxidation only 1 molecule of galactose and the N-acetylglucosamine remained. Permethylation of the lipid gave derivatives of a terminal fucose and galactose residue together with 2,4,6-tri-O-methylgalactose and 2,3,6-tri-O-methylglucose. After removal of fucose the lipid could be converted to a ceramide trihexoside with beta-galactosidase, and this, in turn, to ceramide lactoside by the action of beta-N-acetylhexosaminidase. Both enzymes converted the defucosylated derivative to a ceramide monohexoside. The methylated and the methylated and reduced derivatives of the intact lipid gave ions in mass spectrometry for a terminal hexose and deoxyhexose, a terminal trisaccharide of hexose, deoxyhexose and N-acetylhexosamine, and terminal tetra-and pentasaccharides. Ceramide fragments characteristic of hydroxy fatty acids with 16, 22, 23, and 24 carbons were found together with those of phytospingosine as the major long chain base. On the basis of these results and the immunologic activity of the fucolipid, the following structure is proposed: betaGal (1 leads to 3)betaGlcNAc (1 leads to 3)betaGal (1 leads to 4)Glc-ceramide alphaFuc (1 leads to 4).     

Structural studies on glycolipid of shellfish. III. Novel glycolipids from Turbo cornutus

Five kinds of sphingoglycolipids were isolated from Turbo cornutus. Four of them were a series of novel glycolipids consisting only of galactose. The structures of these glycolipids were studied by methylation analysis, periodate oxidation, enzymatic degradation, and proton magnetic resonance spectroscopy. Three glycolipids were characterized as galactosyl(beta 1 leads to 1)ceramide, galactosyl(beta 1 leads to 6)galactosyl(beta 1 leads to 1)ceramide, and galactosyl(beta 1 leads to 6)galactosyl(beta 1 leads to 6)galactosyl(beta 1 leads to 1)ceramide. Data indicating that the 4th glycolipid might be the tetragalactosyl derivative of this series were obtained. The carbohydrate moiety of the 5th glycolipid, in contrast, was composed of fucose, galactose, glucose and N-acetylglycosamine in a molar ratio of 1 : 2 : 1 : 1.     

Enzymic synthesis of a new type of fucose-containing glycolipid with fucosyltransferase of rat ascites hepatoma cell, AH 7974F.

An alpha-fucosyltransferase activity has been demonstrated in rat ascites hepatoma AH 7974F cells catalyzing the transfer of L-fucose to asialo-GM1 prepared from bovine brain GM1 ganglioside to form a fucolipid in the presence of Triton X-100. The radioactive fucolipid was shown to be Fuc-(alpha1 leads to 2)-Gal-(beta1 leads to 3)-GalNAc-(beta1 leads to 4)-Gal-(beta1 leads to 4)-Glc-ceramide from the following results. The radioactive product coincided with authentic blood group H-active fucolipid from AH 7974F cell on thin-layer chromatography. The product formed a precipitation line not only with Ulex europeus lectin but also with eel anti-H serum on agarose gel plates. The terminal 14C-labeled fucose was released by Bacillus fulminans alpha(1 leads to 2)fucosidase as well as Charonia lampas alpha-fucosidase. The optimum pH value for the incorporation of L-fucose into asialo-GM1 was 5.8 in cacodylate/HCl buffer. The fucosyltransferase was highly specific for asialo-GM1.     

Studies on glycosphingolipids of fresh-water bivalves. V. The structure of a novel ceramide octasaccharide containing mannose-6-phosphate found in the bivalve, Corbicula sandai.

A ceramide octasaccharide containing mannose-6-phosphate was isolated from the fresh-water bivalve Corbicula sandai by solvent fractionation, followed by two types of silicic acid column chromatography, and finally QAE-Sephadex column chromatography. The structural analysis involved the following steps. (a) Gas-liquid chromatography of the component sugars, fatty acids, and long-chain bases. (b) Degradation with HCl and HF to elucidate the sugar sequence. (c) Permethylation analysis coupled with GC-MS to identify the positions of the glycosidic linkages between the sugar units. (d) Chromium trioxide oxidation to determine the anomeric configuration. (e) Smith degradation to determine the site of linkage of the ethanolamine residue. The structure of this novel glycolipid was determined to be: 4-O-MeGalp(bets1 yields 3)-GalNAcp(beta1 yields3)Fucp(alpha1 yields 4)GlcNAcp(beta1 yields 2)Manp(alpha1 yields 3)[Xylp(alpha1 yields 2)][2'-aminoethylphosphoryl(yields 6)]Manp(beta1yields 4)Glcp(beta1 yields 1)ceramide. It is very interesting that fucose was found to be internally linked in this sugar chain. To our knowledge, this is the first example of internal fucose in a glycolipid. The ceramide moiety consisted of normal saturated fatty acids, among which stearic acid was pr     

Chemical structure of monosialoganglioside GM1b biosynthesized in vitro.

The structure of a neuraminidase-labile monosialoganglioside which is formed in vivo from asialoganglioside (galactosyl (beta, 1 in equilibrium 3) N-acetylgalactosaminyl (beta, 1 in equilibrium 4) galactosyl (beta, 1 in equilibrium 4) glucosyl (1 in equilibrium 1) ceramide) and cytidine-5'-monophospho-N-acetylneuraminic acid in the presence of young rat brain sialytransferase has been established. This monosialoganglioside contains a neuraminidase-labile N-acetylneuraminyl group which is linked at position C-3 of the terminal galactosyl unit. This result was obtained by ultramicro scale permethylation of radioactive neuraminidase-labile monosialoganglioside biosynthesized from asialoganglioside labeled with tritium in the terminal galactose.     

Structures and fatty acid compositions of neutral glycosphingolipids of human plasma

Major neutral glycosphingolipids were isolated from human plasma and their structures and fatty acid compositions studied. The four neutral glycosphingolipids of plasma were characterized as Glc beta(1 leads to 1)ceramide, Gal beta(1 leads to 1)- ceramide, Gal beta(1 leads to 4) Glc beta (1 leads to 1)ceramide, Gal alpha(1 leads to 4) Gal beta(1 leads to 4) Glc beta(1 leads to 1)ceramide and GalNAc beta(1 leads to 3) Gal (1 leads to 4) Gal (1 leads to 4) Glc beta(1 leads to 1)-ceramide. The glycosphingolipids contained mostly short chain fatty acids of which most prominent was C16. Erythrocyte glucosylceramide and lactosylceramide exhibited similar fatty acid compositions as their plasma counterparts. Triglycosylceramide and globoside of erythrocytes contained almost exclusively long-chain fatty acids. In lactosylceramide obtained from "p" erythrocytes, an accumulation of long-chain fatty acids was found; this accumulation was not observed, however, in lactosylceramide isolated from "p" plasma. It was concluded that plasma and erythrocyte glycosphingolipids are synthesized at separate sites where short- and long-chain fatty acids, respectively, are available. Plasma and erythrocyte glucosylceramide, and probably a fraction of lactosylceramide, exchange between plasma and erythrocyte pools. The latter conclusion is discussed in the light of the relative roles of carbohydrate and lipid moieties of the glycosphingolipids in maintaining their association with erythrocyte membranes.     

An immunochemical study of the human blood group P1, P, and PK glycosphingolipid antigens.

The erythrocyte PK and P blood group antigens have been identified as ceramide trihexoside (CTH), Gal-(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-Cer, and globoside, GalN-Ac(beta, 1 leads to 3)Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-Cer, respectively, and the following structure has been proposed for the P1 antigen: Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)GlcNAc(beta, 1 leads to 3)Gal(beta, 1 leads to 4)Glc-Cer. Although the P1 and PK determinants have identical terminal disaccharides, CTH did not inhibit anti-P1. The P1 glycolipid and hydatid cyst glycoprotein inhibited the agglutination of P1K erythrocytes by anti-P1 and unabsorbed anti-P1PPK sera, but neither antigen inhibited a specific anti-PK serum. The P1 and PK glycolipids were equally effective in inhibiting the hemagglutinating activity of a lectin with alpha-galactosyl specificity obtained from ova of Salmo trutta. Anti-P sera were inhibited most effectively by human erythrocyte globoside, and to a lesser extent by Forssman glycolipid and rat kidney globoside. In the latter glycolipid the linkage between the internal galactosyl residues is alpha, 1 leads to 3, rather than alpha, 1 leads to 4, as in erythrocyte globoside. No cross-reactions between P and P1 or PK antigens were detected. New hypotheses are offered to explain the genetic regulation and biosynthesis of the P1, P, and PK antigens.     

The monoclonal antibody directed to difucosylated type 2 chain (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc; Y Determinant)

One of the monoclonal (AH-6) antibodies prepared by hybridoma technique against human gastric cancer cell line MKN74 was found to react with a series of glycolipids having the Y determinant (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc). The structure of one such glycolipid isolated from human colonic cancer and from dog intestine was identified as lactodifucohexaosyl-ceramide (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide; IV3,III3Fuc2nLc4Cer). The hapten glycolipid did not react with monoclonal antibodies directed to Lea, Leb, and X-hapten structures, and the AH-6 antibody did not react with the X-hapten ceramide pentasaccharide (Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), H1 glycolipid (Fuc alpha 1 leads to 2Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), nor with glycolipids having the Leb (Fuc alpha 1 leads to 2Gal beta 1 leads to 3[Fuc alpha 1 leads 4]GlcNAc beta 1 leads to R) determinant. The antibody reacted with blood group O erythrocytes, but not with A erythrocytes. Immunostaining of thin layer chromatography with the monoclonal antibody AH-6 indicated that a series of glycolipids with the Y determinant is present in tumors and in O erythrocytes.     

Occurrence of alpha 1-2-fucosylation in membrane glycoproteins of Morris hepatoma 7777 but not in liver. Aberrant type of fucosylation in a malignant tissue.

A comparative study was undertaken to characterize the linkages of L-fucose in N-glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum. After in-vivo radiolabelling of rats with L-[6-3H]fucose, the asparagine-linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride. Two specific alpha-L-fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of L-fucose in the oligosaccharide chains. Fucose alpha 1-2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total L-[3H]fucose in N-glycans), but was not expressed in host liver, kidney cortex and serum. None of the investigated sources contained an appreciable amount of fucose alpha 1-3/4 linked to N-acetyl-D-glucosamine. All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained alpha-fucosyl residues linked at the C6 position of the proximal N-acetyl-D-glucosamine.     

Schistosoma mansoni: inhibition of cercarial "penetration" proteases by components of mammalian blood.

1. Normal human sera and plasma were fractionated in order to identify inhibitors of the "penetration" proteases of Schistosoma mansoni cercariae. 2. The main inhibitor, accounting for 90% of the total activity of serum, appears to be alpha 1-antitrypsin (alpha 1-AT) as identified by separation on DEAE-cellulose and Sephadex, by immunoelectrophoresis and by anticercarial protease activity of purified alpha 1-AT preparations. 3. The inhibition profiles of purified preparations of the 6 known serum antiproteases suggest that the parasite protease is similar to vertebrate chymotrypsin. 4. On a molar basis, the order of inhibitory activity against the cercarial protease is: alpha 1-AT = alpha 2-macroglobulin; C'-1-inactivator; alpha 1-antichymotrypsin. No inhibition was obtained with inter-alpha-inhibitor or antithrombin III.     

Isolation and characterization of the O-glycan chain of the human vitamin-D binding protein

On a highly purified preparation, the structure of the carbohydrate chain of the human vitamin D-binding protein was investigated and two genetic forms of this protein were considered (Gc 2 and Gc 1 proteins). It was found that only the Gc 1 protein (Gc1a isoform) was glycosylated, the glycan moiety representing about 1% of the protein. The structure of this O-glycosidically linked glycan was determined to be: Neu Ac alpha (2 leads to 3) Gal beta (1 leads to 3) GaINAc alpha (1 leads to 0) Ser (or Thr). A tetrasaccharidic O-glycan with two N-acetylneuraminic residues was also characterized. The vitamin D-binding protein is a rare example of a serum protein O-glycosylated only on some genetic forms.     

Biosynthesis of Lewis fucolipid antigens in human colorectal carcinoma cells. Partial characterization of LcOse4Cer and H-1 fucolipid fucosyl transferase acceptor activities

Purified glycolipids were tested for their ability to serve as acceptors of [14C]fucose from GDP-[14C]fucose as catalyzed by cell-free extracts and purified membrane fractions of human colorectal carcinoma cells, SW1116, cultured in serum-free medium. Purified lactotetraosyl ceramide (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or LcOse4Cer) and H-1 glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc-Cer or IV2 Fuc alpha LcOse4Cer) stimulated incorporation of radioactivity into lipid-soluble glycolipid at a rate greater than ten times that of Lea glycolipid [Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4 Fuc alpha LcOse4Cer]. The enzymatic activities in crude and purified membrane fractions were optimized for substrate concentrations (glycolipid and GDP-fucose), detergent requirement (taurocholate), pH, time and protein. The radioactive product of H-1 fucosylation migrated as discrete and distinct bands on high-performance thin-layer chromatograms (HPTLC). Evidence for their identity with Leb fucolipid described previously [Fuc alpha 1----2Gal beta 1----3(Fuc alpha 1----4)GlcNAc beta 1----3Gal beta 1----4Glc-Cer or III4IV2 (Fuc alpha) LcOse4Cer] is presented. The radioactive product of LcOse4Cer fucosylation was mainly Lea fucolipid as determined by co-migration with authentic Lea fucolipid in three HPTLC systems as native and acetylated derivatives. Our results also indicated a low level of H-1 and Leb glycolipid synthesis from LcOse4Cer. On the basis of the optima, linearity for time, and enzyme-limiting conditions, we obtained a 12-19-fold purification of the LcOse4Cer and H-1 fucosyl transferase acceptor activities in three peaks of a sucrose gradient. The peak with the highest specific activity (peak 3) was highest in density and in Na+, K+, ATPase specific activity, although NADH-cytochrome-c reductase and UDP-GalNac transferase were also present in peak 3. The apparent Km values of LcOse4Cer acceptor activity and H-1 acceptor activity in peak 3 were significantly different (p less than 0.01) by statistical tests, 2.4 microM and 0.5 microM, respectively. These apparent Km values were much lower (10(3) X) and the pH optima were lower (4.8-5.3), than the corresponding properties reported for the alpha 1----3/alpha 1----4 fucosyl transferase purified from human milk. Our results suggest a role for the non-glycosidic moieties of the acceptors and/or the tissue-specific or primitive expression of these fucosyl transferase activities.     

Neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii

The neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii were characterized. The most abundant glycolipid was ceramide monosaccharide, followed by ceramide trisaccharide, ceramide tetrasaccharide, and ceramide disaccharide. More complex neutral glycolipids accounted for almost one-third of the total. The total amount of these glycolipids was 0.59 mg/g of dry weight of the ova preparation, a yield which was one-seventh of that of spermatozoa neutral glycolipids. Structural analyses were performed by enzymatic hydrolysis of the glycolipids with exoglycosidases, permethylation experiments, and also immuno-chemical assays. The proposed structures are as follows: ceramide monosaccharides, Gal-Cer and Glc-Cer; ceramide disacharides, Gal(beta 1-4)Gal-Cer, Gal(beta 1-4)Glc-Cer, and Man(beta 1-4)Glc-Cer; ceramide trisaccharide, Man(alpha 1-3)Man(beta 1-4)Glc-Cer; ceramide tetrasaccharides, Man(alpha 1-3)[Xyl(beta 1-2)]Man(beta 1-4)Glc-Cer, GlcNAc(beta 1-2)Man(alpha 1-3)Man(beta 1-4)Glc-Cer, Man(alpha 1-3)[Gal(beta 1-2)]Man(beta 1-4)Glc-Cer, and Man(alpha 1-2?)Man(alpha 1-3)Man(beta 1-4)Glc-Cer. The latter two ceramide tetrasaccharides were new types of glycosphingolipids. The spectrum of ova glycolipids appeared to be more complicated than that of the spermatozoa glycolipids. The ova glycolipids characterized here, with the exception of ceramide tetrasaccharides, contained considerable amounts of 2-hydroxy fatty acids, which were not observed in the spermatozoa glycolipids. The major sphingosine base was C18-sphingenine in all the ova glycolipids as well as in the spermatozoa glycolipids. However, the content of anteiso type of sphingosine base was 2- to 3-fold higher in the ova than in the spermatozoa.     

Identification of erythrocyte Gal alpha 1-3Gal glycosphingolipids with a mouse monoclonal antibody, Gal-13

The Gal alpha 1-3Gal structural determinant has been found to have a unique distribution in mammals. Although this determinant is abundantly expressed by erythrocytes and nucleated cells of many mammals, it has not been detected in human cells. However, our previous studies (Galili, U., Rachmilewitz, E. A., Peleg, A., and Flechner, I. (1984) J. Exp. Med. 160, 1519-1531; Galili, U., Clark, M. R., and Shohet, S. B. (1986) J. Clin. Invest. 77, 27-33) have suggested that this epitope is present in small amounts and may be involved in immune-mediated destruction of senescent human erythrocytes. To have a means for exploring this possibility and for studying the species and tissue distribution of this epitope we have raised a monoclonal antibody (Gal-13) which specifically binds to glycoconjugates with a nonreducing terminal Gal alpha 1-3Gal disaccharide. Mice were immunized with rabbit erythrocytes, which express an abundance of glycoconjugates with Gal alpha 1-3Gal epitopes. Clones were screened with a solid-phase binding assay (enzyme-linked immunosorbent assay) for antibodies which bound to ceramide pentahexoside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3-Gal beta Gal beta 1-4Glc1-1Cer) but not to ceramide trihexoside (Gal alpha 1-4Gal beta 1-4Glc1-1Cer). Gal-13 bound to a number of neutral glycosphingolipids from rabbit and bovine erythrocytes. These glycosphingolipids have previously been shown to be a family of linear and branched polylactosamine structures, which have non-reducing terminal Gal alpha 1-3Gal epitopes. The antibody did not bind to the human blood group B glycolipid, Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc1-1Cer, and, therefore, branching at the penultimate galactose blocks Gal-13 binding. However, after removal of the fucose from the B antigen Gal-13 recognized the resulting derivative. Other Gal alpha 1-3Gal glycosphingolipids with an isogloboside or globoside core structure were not recognized by Gal-13 suggesting that the antibody binds to Gal alpha 1-3Gal carried by a lactosamine core structure. Gal-13 has been used to demonstrate that the Gal alpha 1-3Gal ceramide pentahexoside has been evolutionarily conserved in red cells of animals up to the stage of New World monkeys but is not found in Old World monkey red cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lea-active heptaglycosylceramide, a hybrid of type 1 and type 2 chain, and the pattern of glycolipids with Lea, Leb, X (Lex), and Y (Ley) determinants in human blood cell membranes (ghosts). Evidence that type 2 chain can elongate repetitively but type 1 chain cannot

Two major glycolipids reactive with the monoclonal anti-Lea antibody have been isolated from human blood cell membranes. One component was identified as lactofucopentaosyl(II)ceramide and the other as a ceramide heptassaccharide with the structure described below: (formula; see text) The structure includes the Lea determinant (type 1 chain) linked to lactoneotetraosylceramide (type 2 chain); thus, it is regarded to be a hybrid between type 1 and 2 chain. In addition, a minor component having the thin-layer chromatographic mobility of a ceramide nonasaccharide, which was reactive to anti-Lea antibody, was detected. No other component with a thin-layer chromatographic mobility slower than the above components and reactive to the anti-Lea antibody was detected. In contrast, a series of slowly migrating glycolipids having X (Lex) determinant (Gal beta 1----4(Fuc alpha 1----3)GlcNAc) was detected. A similar series of long chain glycolipids having Y (Ley) determinant (Fuc alpha 1----2Gal beta 1----4(Fuc1----3)GlcNAc) was detected in human blood cells; in contrast, only one major Leb glycolipid was found with the mobility of a ceramide hexasaccharide. No glycolipid with a long carbohydrate chain composed exclusively of type 1 chain was detected. Thus, chain elongation may proceed through type 2 chain, but not through type 1 chain. Lea and X (Lex) haptens are distributed equally among blood group A, B, and O red blood cells, whereas the quantity of Leb and Y (Ley) haptens is much lower in A and B blood cells than in O blood cells.     

Specificity of lung surfactant protein SP-A for both the carbohydrate and the lipid moieties of certain neutral glycolipids.

Binding specificity of the major surfactant protein SP-A from human and dog lung has been investigated. Radiobinding experiments have shown that both proteins bind in a Ca(2+)-dependent manner to galactose, mannose, fucose, and glucose linked to bovine serum albumin. These results are in accord with a previous study in which monosaccharides were linked to agarose (Haagsman, H. P., Hawgood, S., Sargeant, T., Buckley, D., White, R. T., Drickamer, K., and Benson, B. J. (1987) J. Biol. Chem. 262, 13877-13880). Chromatogram overlays in conjunction with in situ liquid secondary ion mass spectrometry (TLC-LSIMS) of several purified glycosphingolipids and neoglycolipids as well as binding assays with glycolipids immobilized on plastic wells, demonstrate recognition of galactose (human and dog SP-A), glucose, and lactose (human SP-A) in association with specific lipids. In addition, the occurrence of several neutral and acidic glycosphingolipids in human and rat extracellular surfactants and rat alveolar type II cells is described. Selected components among the neutral glycolipids are bound by radiolabeled human SP-A; these are identified by TLC-LSIMS as predominantly ceramide mono- and disaccharides (human surfactant) and ceramide tri- and tetrasaccharides (rat surfactant and type II cells). A recombinant carbohydrate recognition domain (CRD) of human SP-A inhibits the binding of human SP-A to galactosyl ceramide and to galactose- and mannose-bovine serum albumin, indicating that the CRD is directly involved in the binding of SP-A to these ligands. These results provide evidence for a novel type of binding specificity for proteins that have Ca(2+)-dependent CRDs and raise the possibility that glycosphingolipids are endogenous ligands for SP-A.     

Binding specificity of monoclonal antibodies to ganglioside, Fuc-GM1

The binding specificity of thirteen mouse monoclonal antibodies reacting with Fuc-GM1, Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)-Gal beta 1-4Glc beta 1-1Cer, a ganglioside found to be associated with small cell lung carcinoma (O. Nilsson et al. (1984) Glycoconjugate J. 1, 43-49) was studied. The results are based upon radioimmunodetection of their binding to structurally related glycolipids adsorbed to microtiter plates or chromatographed on thin-layer plates. Four of thirteen antibodies reacted only with Fuc-GM1 and both the fucose and the sialic residues were necessary for binding. Optimal binding was obtained when the sialic acid was N-acetylneuraminic acid. When this sialic acid residue was substituted with N-glycoloylneuraminic acid the binding activity was reduced and up to 10-times more Fuc-GM1 was needed for detection. The ceramide composition did not influence the binding. The other nine monoclonal antibodies cross-reacted with glycolipids containing structures closely related to Fuc-GM1 and differed from the specific ones by recognizing a smaller portion of the carbohydrate moiety in Fuc-GM1. These results indicate that anticarbohydrate monoclonal antibodies, recognizing structures involving a large proportion of the sugar in the glycolipid, possess a high specificity and might be useful for detection of tumor-associated ganglioside antigen.     

Identification of a defence mechanism in vivo against the leakage of enterokinase into the blood.

1. The serum proteinase inhibitors alpha 1-antitrypsin, alpha 2-macroglobulin, inter-alpha-trypsin inhibitor and C1-esterase inhibitor were found not to affect the catalytic activity of human enterokinase, whereas bovine trypsin activity was modified essentially as expected. Enterokinase was also not inhibited by Trasylol (trypsin inhibitor from bovine lung) or bovine pancreatic trypsin inhibitor. No other component in human or mouse serum complexing with enterokinase was identified. 2. Human enterokinase administered intravenously into mice was rapidly cleared from the circulation with a half-life of 2.5 min. This removal was not the result of the difference in species, since partially purified mouse enterokinase was cleared at the same rate as the human enzyme. Clearance was mediated by recognition of the carbohydrate portion of enterokinase and not through specific recognition of its catalytic site. Immunofluorescent staining showed that the enzyme accumulated in the liver. Attempts to block the clearance by the simultaneous infusion of competing glycoproteins suggested that enterokinase was taken up by hepatocytes. Of the glycoproteins tested only two, human lactoferrin (terminal fucosyl alpha 1 leads to 3 N-acetylglucosamine) and bovine asialo-fetuin (terminal galactosyl beta 1 leads to 4 N-acetylglucosamine) were weakly competitive. Two inhibitors of endocytosis, Intralipid and Triton WR1339, failed to delay the removal of enterokinase. It is proposed that enterokinase is cleared from the circulation by an as yet uncharacterized hepatocyte receptor.     

Biosynthesis of the sialyl-Lex determinant carried by type 2 chain glycosphingolipids (IV3NeuAcIII3FucnLc4, VI3NeuAcV3FucnLc6, and VI3NeuAcIII3V3Fuc2nLc6) in human lung carcinoma PC9 cells

The pathway for synthesis of three glycosphingolipids bearing a common sialyl-Lex determinant (NeuAc alpha 2----3Gal beta 1----4[Fuc alpha 1----3]GlcNac beta 1----R) from their type 2 lactoseries precursors has been studied using the 0.2% Triton X-100-soluble fraction from human lung carcinoma PC9 cells. Two enzymes were found to be required for their synthesis: (i) an alpha 1----3 fucosyltransferase, the properties of which have been characterized as being similar to the enzyme from human small cell lung carcinoma NCI-H69 cells (Holmes, E. H., Ostrander, G. K., and Hakomori, S. (1985) J. Biol. Chem. 260, 7619-7627); and (ii) an alpha 2----3 sialyltransferase that was efficiently solubilized by 0.2% Triton X-100 and required divalent metal ions and 0.3% Triton CF-54 for optimal activity at pH 5.9 in cacodylate buffer. Biosynthesis of the sialyl-Lex determinant was shown to proceed via sialylation of nLc6 and nLc4, followed by alpha 1----3 fucosylation at the penultimate GlcNAc residues, based on the following: (i) transfer of NeuAc by PC9 cell sialyltransferase was found only when the nonfucosylated acceptors nLc4 and nLc6 were added, and none of the glycolipids with Lex structure (III3FucnLc4; V3FucnLc6; III3V3Fuc2nLc6) were sialylated; and (ii) the PC9 cell fucosyltransferase was active with both neutral and ganglioside neolacto (type 2 chain) acceptors. Transfer of fucose to VI3NeuAcnLc6 yielded mono- and difucosyl derivatives, whereas only a monofucosyl derivative was obtained when VI6NeuAcnLc6 was the acceptor. This is most probably due to different conformations at the terminus of the two acceptor gangliosides. The fucosyltransferase was incapable of transferring fucose to sialyl 2----3 lactotetraosylceramide (IV3NeuAcLc4).     

Incorporation of the hexose analogue 2-deoxy-D-galactose into membrane glycoproteins in HepG2 cells.

The incorporation of 2-deoxy-D-galactose into the oligosaccharide moieties of glycoproteins and the consequences of 2-deoxy-D-galactose treatment on the fucosylation of glycoproteins were investigated in the human hepatoma cell line HepG2. Using different methods, it was shown that treatment of HepG2 cells with 2-deoxy-D-galactose leads to an incorporation of 2-deoxy-D-galactose and a decrease of L-fucose incorporation into the oligosaccharides of glycoproteins. The extent of labeling by L-[3H]fucose was determined by removing L-[3H]fucose from labeled cells with the aid of a purified alpha 1,2-fucosidase from Aspergillus niger. Using this method, it was shown that 2-deoxy-D-galactose markedly inhibits alpha 1,2-fucosylation. Measurement of the amount of 2-deoxy-D-galactose incorporated, however, showed that replacement of D-galactose by 2-deoxy-D-galactose does not entirely account for the decrease in alpha 1,2-fucosylation. In addition, a hitherto unreported compensatory increase of alpha 1,3/alpha 1,4-fucosylation was found to occur when alpha-1,2-fucosylation was inhibited by treatment with 2-deoxy-D-galactose.