Regulation of the expression of Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids in kidney

Previous studies (Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., and Macher, B. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1369-1373; Galili, U., Shohet, S. B., Korbrin, E., Stults, C. L. M., and Macher, B. A. (1988) J. Biol. Chem. 263, 17755-17762) have established that there is a unique evolutionary distribution of glycoconjugates carrying the Gal alpha 1-3Gal beta 1-4GlcNAc epitope. These glycoconjugates are expressed by cells from New World monkeys and non-primate mammals, but not by cells from humans, Old World monkeys, or apes. The lack of expression of this epitope in the latter species appears to result from the suppression of gene expression for the enzyme UDP-galactose:nLc4Cer alpha 1-3-galactosyltransferase (alpha 1-3GalT) (Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J., and Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297). Although many non-primate species are known to express this carbohydrate epitope, the nature (i.e. glycoprotein or glycosphingolipid) of the glycoconjugate carrying this epitope is only known for a few tissues in a few animal species. Furthermore, it is not known whether all animal species express this epitope in the same tissues. We have investigated these questions by analyzing the glycosphingolipids in kidney from several non-primate animal species. Immunostained thin layer chromatograms of glycosphingolipids from sheep, pig, rabbit, cow, and rat kidney with the Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipid-specific monoclonal antibody, Gal-13, demonstrated that kidney from all of these species except rat contained Gal alpha 1-3Gal beta 1-4GlcNAc neutral glycosphingolipids. A lack of expression of Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids in rat may be due to the lack of expression of the enzyme (alpha 1-3GalT) which catalyzes the formation of the Gal alpha 1-3Gal nonreducing terminal sequence of these compounds or to the lack of expression of glycosyltransferases which are necessary for the synthesis of the neolacto core structure of these compounds. These possibilities were evaluated in two ways. First, the three enzymes (UDP-N-acetylglucosamine:LacCer beta 1-3-N-acetyl-glucosaminyltransferase, UDP-galactose:Lc3Cer beta 1-4-galactosyltransferase, and alpha 1-3GalT) involved in the synthesis of the Gal alpha 1-3Gal beta 1-4GlcNAc glycosphingolipids were assayed using an enzyme-linked immunosorbent assay-based assay system and carbohydrate sequence-specific monoclonal antibodies. Second, TLC immunostaining was done to determine if the glycosphingolipid precursors (i.e. Lc3Cer and nLc4Cer) are expressed in rat kidney. Interestingly, rat kidney had a relatively high level of alpha 1-3GalT activity compared with the other animals tested.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biosynthesis in vitro of a globoside containing a 2-acetamido-2-deoxy-beta-D-galactopyranosyl group (1----3)-linked and Forssman glycolipid by two N-acetylgalactosaminyltransferases from chemically transformed guinea pig cells

Two N-acetylgalactosaminyltransferase activities (GalNAcT-2 and GalNAcT-3) have been characterized in chemically transformed, cultured guinea-pig cell lines (104C1 and 106B). Line 104C1 is a benz[a]pyrene-transformed tumorigenic variant, whereas line 106B is a 7,12-dimethylbenz[a]anthracene-transformed nontumorigenic variant obtained from fetal guinea-pig cells at 43 days of gestation. The GalNAcT-2 (UDP-GalNAc:GbOse3Cer beta-N-acetylgalactosaminyltransferase) isolated from both 104C1 and 106B cells catalyzed the transfer of Gal-NAc from UDP-GalNAc to the 3H-labeled terminal galactose group of Gb3 [( 6-3H]Gal alpha 1----4Gal beta 1----4Glc----Cer). The 3H-labeled globoside was purified and then subjected to exhaustive methylation. After acetolysis, the partially methylated sugars were separated by two-dimensional, thin-layer chromatography. 3H-Label was detected in two major areas, 2,4,6-tri-O-Me-Gal (40%) and 2,3,4,6-tetra-O-Me-Gal (46%). In a separate experiment, 80% of the GalNAc was released when labeled GbOse4Cer [( 3H]GalNAc----Gal alpha 1----4Gal beta 1----4Glc----Cer) was treated with purified clam beta-hexosaminidase. The present results establish the formation of a beta-D-GalpNAc-(1----3) linkage in the terminal region of the biosynthesized globoside. GalNAcT-3 activity (UDP-GalNAc:GbOse4Cer alpha-GalNAc-transferase), which catalyzes the transfer of GalNAc from UDP-[14C]- or -[3H]GalNAc to GbOse4Cer (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc----Cer), was three times higher in 106B cells than in 104C1 cells. The isolated, purified radioactive product formed an immunoprecipitin line against rabbit anti-Forssman antibody.     

Biosynthesis in vitro of GlcA beta 1-3nLcOse4Cer by a novel glucuronyltransferase (GlcAT-1) from embryonic chicken brain

A novel glucuronyltransferase (GlcAT-1) has been detected in embryonic chicken brains. This enzyme catalyzes the biosynthesis in vitro of glucuronic acid containing glycolipids starting from neolactotetraosylceramide (nLcOse4Cer) and neolactohexaosylceramide (nLcOse6Cer). The activity is present primarily in the Golgi-rich membrane fraction and can be extracted (60%) from the membrane using a neutral detergent, Nonidet P-40, at pH 7.0. The detergent-solubilized GlcAT-1 is stable (70%) at -20 degrees C for at least 4 months. Both membrane-bound GlcAT-1 and solubilized GlcAT-1 show similar pH optima, 6.5-7.0, in HEPES buffer. The Km values were 15 and 200 microM with UDP-[14C] GlcA and nLcOse4Cer, respectively, when the detergent-solubilized supernatant fraction was used as enzyme source. The purified 14C radioactive product that comigrated with chemically characterized GlcA beta 1-3nLcOse4Cer (GlcA-nLc4) also yielded a positive immunostain with monoclonal antibody (human IgM-RI). The anomeric linkage was established as beta-linked GlcA to the terminal galactose of the substrate, as evidenced by 90-99% cleavage of the terminal [14C] GlcA by purified Helix pomatia and limpet glucuronidases. Permethylation studies of the radioactive product obtained from [6-3H]Gal beta 1-4LcOse3Cer and non-radioactive UDP-GlcA showed the presence of 2,4,6-tri-O-methylgalactose in the hydrolyzed enzymatic product. These studies established the structure of the biosynthesized product from nLcOse4Cer as GlcA beta 1-3Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-4Glc-ceramide.     

Alpha 1----3-galactosyltransferase: the use of recombinant enzyme for the synthesis of alpha-galactosylated glycoconjugates

We have reported the isolation and characterization of a bovine cDNA clone containing the complete coding sequence for UDP-Gal:Gal beta 1----4GlcNAc alpha 1----3-galactosyltransferase [Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J. & Shaper, N. L. (1989) J. Biol. Chem. 264, 14290-14297]. Insertion of this cDNA clone into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and subsequent infection of Spodoptera frugiperda (Sf9) insect cells with recombinant virus, resulted in high-level expression of enzymatically active alpha 1----3-galactosyltransferase. The expressed enzyme accounted for about 2% of the cellular protein; the corresponding specific enzyme activity was 1000-fold higher than observed in calf thymus, the tissue with the highest specific enzyme activity reported to date. The recombinant alpha 1----3-galactosyltransferase could be readily detergent-solubilized and subsequently purified by affinity chromatography on UDP-hexanolamine-Sepharose. The recombinant alpha 1----3-galactosyltransferase showed the expected preference for the acceptor substrate N-acetyllactosamine (Gal beta 1----4GlcNAc), and demonstrated enzyme kinetics identical to those previously reported for affinity-purified calf thymus alpha 1----3-galactosyltransferase [Blanken, W. M. & Van den Eijnden, D. H. (1985) J. Biol. Chem. 260, 12927-12934]. In pilot studies, the recombinant enzyme was examined for the ability to synthesize alpha 1----3-galactosylated oligosaccharides, glycolipids and glycoproteins. By a combination of 1H-NMR, methylation analysis, HPLC, and exoglycosidase digestion it was established that, for each of the model compounds, the product of galactose transfer had the anticipated terminal structure, Gal alpha 1----3Gal beta 1----4-R. Our results demonstrate that catalysis by recombinant alpha 1----3-galactosyltransferase can be used to obtain preparative quantities of various alpha 1----3-galactosylated glycoconjugates. Therefore, enzymatic synthesis using the recombinant enzyme is an effective alternative to the chemical synthesis of these biologically relevant compounds.     

The binding specificity of normal and variant rat Kupffer cell (lectin) receptors expressed in COS cells.

A receptor uniquely found on the surface of rat Kupffer cells was shown previously to bind oligosaccharides terminating in galactose, N-acetylgalactosamine, and fucose. To analyze further the binding specificity of the receptor, receptor-mediated adhesion of transfected COS cells to immobilized glycolipids of known structure was measured. The glycolipid Gb4Cer (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1Cer) was the best ligand. Gb5Cer (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1Cer) and LacCer (Gal beta 1-4Glc beta 1Cer) bound more weakly (five times less than Gb4Cer) and Gb3Cer (Gal alpha 1-4Gal beta 1-4Glc beta 1Cer), and g3Cer(GalNAc beta 1-4Gal beta 1-4Glc beta 1Cer) bound even more weakly (60 times less than Gb4Cer). Gangliosides did not support adhesion of transfected cells. The adhesion of COS cells transfected with plasmids encoding variants of the receptor was also examined. In each variant, either tryptophan 498 or 523, which are conserved in most C-type lectins, was replaced by one of several amino acids. Variants that retained binding activity had the same specificity as the normal receptor. Differences between variants were noted, however, in maximal levels of adhesion and these differences correlated with altered expression of the receptor variants in COS cells.     

Diplococcal beta-galactosidase with a specificity reacting to beta 1-4 linkage but not to beta 1-3 linkage as a useful exoglycosidase for the structural elucidation of glycolipids

Diplococcal beta-galactosidase, which is known to be useful for the structural studies of glycoprotein-linked oligosaccharides, was found to show the same substrate specificity in cleaving Gal beta 1-4 linkages of glycolipids as that of the oligosaccharides. The optimum conditions of beta-galactosidase in the 80% ammonium sulfate precipitates of the culture medium of Streptococcus (Diplococcus) pneumoniae were determined with nLcOse4Cer radiolabeled by the galactose oxidase-NaB3H4 procedure. Detergent was required for the highest activity, and different combinations of several buffers and detergents showed different properties in stimulating beta-galactosidase, and in enhancing or suppressing N-acetyl-beta-hexosaminidase which was contaminated in the enzyme preparation. The optimum pH was found to be at 6.5, and specific activity and Km were 8.1 nmol/mg protein/h and 1 nmol, respectively. While more than 70% of beta-galactose was liberated from LacCer and nLcOse4Cer within 1 h under the optimum conditions to form GlcCer and nLcOse3Cer, respectively, none was liberated from LcOse4Cer, GalCer, GgOse4Cer, GbOse3Cer, IV3 alpha GalnLcOse4Cer, and Il3NeuAcGgOse4Cer, showing the substrate specificity solely to Gal beta 1-4 linkage.     

Biosynthesis in vitro of Ii core glycosphingolipids from neolactotetraosylceramide by beta 1-3- and beta 1-6-N-acetylglucosaminyltransferases from mouse T-lymphoma

The N-acetylglucosaminyltransferases probably involved in the biosynthesis in vitro of Ii core glycosphingolipids have been solubilized from a membrane preparation of mouse lymphoma P-1798 and partially characterized. The detergent-extracted membrane supernatant contains both beta 1-3- and beta 1-6-N-acetylglucosaminyltransferase activities that transfer [3H]GlcNAc from UDP-[3H]GlcNAc to the terminal galactose of neolactotetraosylceramide (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; nLcOse4ceramide), to form the Ii core structures. The linkage of [3H]N-acetylglucosamine incorporated into the terminal galactose of nLcOse4Cer was determined from identification of 2,4,6-tri-O-methyl[3H]galactose and 2,3,4-tri-O-methyl[3H]galactose after hydrolysis of the permethylated enzymatic products, GlcNAc beta-[3H]Gal-GlcNAc-Gal-Glc-ceramide. In addition to the presence of beta-N-acetylglucosaminyltransferases, we have detected a galactosyltransferase activity in this soluble supernatant fraction that catalyzes the transfer of [14C]galactose from UDP-[14C]galactose to lactotriaosylceramide (GlcNAc beta 1-3Gal beta 1-4Glc-ceramide; LcOse3ceramide) to form nLcOse4ceramide, the acceptor in the N-acetylglucosaminyltransferase-catalyzed reaction.     

Preparative in vitro generation of lacto-series type 1 chain glycolipids catalyzed by beta 1----3-galactosyltransferase from human colonic adenocarcinoma Colo 205 cells

Lacto-series glycolipids, comprising two isomeric types distinguished as type 1 or 2 based upon the linkage of the terminal galactose of the chains, form the basis for a diversity of cell surface antigens expressed on cells. Experimentally, type 2 chain precursors are generally more abundant in tissues for extractive purposes to yield rather large quantities of material compared to the type 1 chain structures. Conditions have been defined for in vitro conversion of terminal Gal beta 1----4GlcNAc linkages of type 2 chain precursors to yield type 1 lacto-series chain based terminal Gal beta 1----3GlcNAc structures in 5- to 10-mg amounts or higher. The terminal galactose of underivatized type 2 chain structures is removed by hydrolysis with jack bean beta-galactosidase followed by transfer of galactose in beta 1----3 linkage catalyzed by a beta 1----3-galactosyltransferase from human colonic adenocarcinoma Colo 205 cells which was first depleted of beta 1----4-galactosyltransferase by chromatography on alpha-lactalbumin-Sepharose. Scaled-up reaction mixtures provided a final yield of product after isolation of about 90% from the immediate Lc3Cer precursor in the 5-mg product range. The biosynthetic product was subjected to extensive chemical analysis by 1H NMR and mass spectrometric methods. These results indicated the presence of a high purity terminal Gal beta 1----3-linked product. The amount of material was sufficient for nondestructive characterization by 2-D NMR, with subsequent confirmation of structure by +FAB-MS and methylation analysis by GC-MS. The results indicate an effective means to rapidly generate lacto-series type 1 precursors in vitro as a superior alternative to direct tissue extractive procedures.     

Identification of UDP-galactose: lactose (lactosylceramide) alpha-4 and beta-3 galactosyltransferases in human kidney

Two galactosyltransferases were identified in human kidney microsomes which both transfer galactose from UDP Gal to lactose as well as to lactosylceramide. Using a solubilized and a partially purified enzyme preparation sufficient product could be obtained for detailed structural analysis. The trisaccharide products were isolated by gel permeation chromatography and separated by preparative high performance thin layer chromatography. The anomeric configuration of the transferred galactose was determined by specific glycosidase digestion and the linkage was identified by methylation and gas-liquid-chromatography. The glycolipid products were not separated but analyzed directly, before and after alpha or beta galactosidase digestion, by methylation, hydrolysis and thin layer chromatography. Into both acceptor substrates galactose was incorporated in alpha 1-4 (30%) and beta 1-3 (70%) linkages. The alpha 1-4 galactosyltransferase is responsible for the synthesis of the Pk antigen Gal alpha 1-4 Gal beta 1-4 Glc-ceramide in human kidney. The beta 1-3 galactosyltransferase has not previously been identified.     

Acidic glycosphingolipids of human amnion

Six major acidic glycosphingolipids were isolated from human amnion using DEAE Sephadex A-25 and silica beads column chromatography. The structures of these glycosphingolipids were determined by methylation analysis, TLC immunostaining and/or negative ion FAB-MS, and were concluded to be II3 alpha NeuAcLacCer(GM3), IV3 alpha NeuAcnLc4-Cer (sialyl[alpha 2-3]paragloboside), IV6 alpha NeuAcnLc4Cer (sialyl[alpha 2-6]paragloboside), IV3 alpha NeuAcIII4 alpha FucLc4Cer (sialyl Lea), VI3 alpha NeuAcnLc6Cer (i-ganglioside) and II3 alpha (NeuAc alpha 2----8NeuAc)LacCer (GD3). In addition, several minor glycosphingolipids were detected with specific monoclonal antibodies, including glycolipids with NeuAc alpha 2-3Gal beta 1-4GlcNAc-beta 1- or NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1- determinant. Our results show that the glycosphingolipids of human amnion are characterized by having mainly type II chain analogues and onco-fetal antigens.     

Purification of uridine diphosphate-galactose:glucosyl ceramide, beta 1-4 galactosyltransferase from human kidney.

A galactosyltransferase that transfers galactose from UDP-galactose to glucosylceramide was purified 440-fold to apparent homogeneity from normal human kidney "buffy coat" preparation employing detergent extraction, ultrafiltration, and Sepharose Q column chromatography. On reducing and nonreducing gels, the enzyme resolved into two bands with apparent molecular weights on the order of 60,000 and 58,000, respectively. The activity of the enzyme was also associated with these two bands following separation on polyacrylamide gels. Analytical isoelectric focusing revealed that the pI of this enzyme is approximately 4.55. Product characterization and substrate specificity studies employing chromatography, enzymatic digestion with various glycosidases, and use of a variety of glycosphingolipid substrates revealed that the major product synthesized by this enzyme was Cer1-1 beta Glc4-1Gal, and Cer1-1 beta Glc was the preferred substrate. Digestion of the 60- and 58-kDa proteins with Staphylococcus aureus (V-8) protease revealed at least six peptides having identical electrophoretic migration. This finding suggests that the two proteins may be related to each other. Western immunoblot assays revealed that the antibody against UDP-galactose:GlcCer, beta 1-4 galactosyltransferase (GalT-2) but not galactosyltransferase UDP-Gal:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyltransferase (EC 2.4.1.38) (B-GT) immunoprecipitated (recognized) the kidney GalT-2. In contrast, antibody against B-GT did not immunoprecipitate GalT-2. Thus our data indicate that GalT-2 and B-GT are two distinct enzymes. The availability of the enzyme GalT-2 and corresponding antibody will allow functional studies in the near future.     

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.     

Three-dimensional structure of the oligosaccharide terminus of globotriaosylceramide and isoglobotriaosylceramide in solution. A rotating-frame NOE study using hydroxyl groups as long-range sensors in conformational analysis by 1H-NMR spectroscopy

Spatial structures of the oligosaccharide parts of globotriaosylceramide, Gal(alpha 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer (Cer = ceramide) and isoglobotriaosylceramide, Gal(alpha 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer were investigated in (C2H3)2SO solution by means of laboratory and rotating frame NOE, hydroxyl protons being used as long-range sensors defining the distance constraints. Both oligosaccharides were found to exist in more than one conformation interconverting rapidly on the NMR time scale. The conformation of the Gal(alpha 1-4)Gal(beta 1-4)Glc beta trisaccharide dissolved in 2H2O appeared to be the same as that of the corresponding part of the glycosphingolipid in (C2H3)2SO solution.     

Biosynthetic pathways for the Leb and Y glycolipids in the gastric carcinoma cell line KATO III as analyzed by a novel assay

The biosynthetic pathways for the difucosylated type 1 and 2 glycolipids, Leb and Y, respectively, were investigated in the gastric carcinoma cell line KATO III, using a novel chromatogram binding assay. The type of fucosylation obtained was deduced from the binding pattern of monoclonal antibodies specific for the biosynthesized glycolipid products using microsomal fractions as the source of enzyme, pure glycolipids and non-radioactive GDP-fucose as acceptor and donor substrates, respectively. The Leb glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer) was synthesized mainly via the blood group H, type 1, precursor (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3LacCer). However, the Lea glycolipid (Gal beta 1----3GlcNAc(4----1 alpha Fuc)beta 1----3LacCer) also served as a precursor for the alpha 1----2 fucosyltransferase, thus allowing conversion of Lea to Leb. This biosynthetic route represents either an "aberrant" specificity of the Fuc alpha 1----2 transferase associated with these gastric carcinoma cells and/or a new member of the alpha 1----2 fucosyltransferase family. The Y glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) was synthesized exclusively via the classical pathway using the blood group H type 2 glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc beta 1----3LacCer) as precursor. The X glycolipid (Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) did not serve as an acceptor substrate for the alpha 1----2 fucosyltransferase(s) present. The use of non-radioactive sugar-nucleotides as donor substrate, defined glycolipid precursors as acceptor substrates and of specific monoclonal anti-glycolipid antibodies for detection provides a rapid and highly specific assay for analyzing biosynthetic pathways of glycosyltransferases.     

Biosynthesis of terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R oligosaccharide sequences on glycoconjugates. Purification and acceptor specificity of a UDP-Gal:N-acetyllactosaminide alpha 1----3-galactosyltransferase from calf thymus

A UDP-Gal:Gal beta 1----4GlcNAc-R alpha 1----3- and a UDP-Gal:GlcNAc-R beta 1----4-galactosyltransferase have been purified 44,000- and 101,000-fold, respectively, from a Triton X-100 extract of calf thymus by affinity chromatography on UDP-hexanolamine-Sepharose and alpha-lactalbumin-Sepharose in a yield of 25-40%. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a major polypeptide species with a molecular weight of 40,000 and a minor form at Mr 42,000 for the alpha 1----3-galactosyltransferase and a major polypeptide with Mr 51,000 for the beta 1----4-galactosyltransferase. Analytical gel filtration on Sephadex G-100 yielded a monomeric form for each of the galactosyltransferases with Mr 43,000 and 59,000 respectively, in addition to peaks of activity at higher molecular weights. Isoelectric focussing of the alpha 1----3-galactosyltransferase revealed a significant charge heterogeneity with forms varying in pI values between 5.0 and 6.5. Acceptor specificity studies indicated that the purified alpha 1----3-galactosyltransferase was free from contaminating galactosyltransferase activities such as those involved in the synthesis of Gal beta 1----4GlcNAc-R and Gal beta 1----3GalNAc-R sequences, the blood group B determinant, the Pk antigen, trihexosylceramide, and ganglioside GM1. The alpha 1----3-galactosyltransferase appeared to be highly active with glycoproteins, oligosaccharides, and glycolipids having a terminal Gal beta 1----4GlcNAc beta 1----unit such as asialo-alpha 1-acid glycoprotein (Km = 1.25 mM), Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3Man beta 1----4GlcNAc (Km = 0.57 mM), and paragloboside. The action of the alpha 1----3-galactosyltransferase was found to be mutually exclusive with that of the NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase from bovine colostrum. In addition alpha 1----3-fucosylation of the N-acetylglucosamine residue in the preferred disaccharide acceptor structure completely blocked galactosylation of the alpha 1----3-galactosyltransferase.     

Biosynthesis of marsupial milk oligosaccharides: characterization and developmental changes of two galactosyltransferases in lactating mammary glands of the tammar wallaby, Macropus eugenii

Tammar wallaby (Macropus eugenii) mammary glands contain two galactosyltransferases of which the first, 4 beta GalT, is a UDP-galactose:N-acetylglucosaminyl beta 1----4-galactosyltransferase equivalent to the A protein of the lactose synthase of eutherian mammals. The second enzyme, 3 beta GalT, is a UDP-galactose:lactose beta 1----3-galactosyltransferase, not previously identified in mammary glands of any species, which catalyses the formation of Gal beta 1----3 Gal beta 1----4 Glc from lactose. The two enzyme activities, as well as the lactose synthase activity, have been characterised with respect to the effects of pH, apparent Km values, effects of bovine and tammar alpha-lactalbumins, heat sensitivity and identity of products. Studies on the substrate specificity and heat sensitivity of the 3 beta GalT activity suggest that this enzyme may catalyse the beta-galactosylation of Gal beta 1----3Gal beta 1----4Glc as well as of lactose. The activity of the 3 beta GalT, unlike that of the 4 beta GalT, changes dramatically during the course of lactation in parallel with similar changes in the carbohydrate content of tammar milk.     

Complete enzymic synthesis of the mucin-type sialyl Lewis x epitope, involved in the interaction between PSGL-1 and P-selectin

Sialyl Lewis x (sLe(x)) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-alpha-D-galactosaminide (GalNAc(alpha1-pNp as core substrate, the sLe(x)-oligosaccharide Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)[Gal(bet a1-3)]GalNAc(alpha1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(beta1-3)GalNAc(alpha1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of beta3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 beta6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc(alpha1-pNp in a yield of >85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk beta4-galactosyltransferase 1 (EC 2.4.1.38) (yield of >85%), then sialylated using CMP-Neu5Ac and purified recombinant alpha3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human alpha3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 micromol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure.     

Characterization of the binding of propionibacterium granulosum to glycosphingolipids adsorbed on surfaces. An apparent recognition of lactose which is dependent on the ceramide structure

The binding properties of a strain of Propionibacterium granulosum derived from human skin was investigated with reference to glycosphingolipids separated on thin layer chromatograms or coated in microtiter wells using externally (125I) and metabolically [( 35S]methionine) labeled bacteria. Binding was found to lactosylceramide (LacCer; Gal beta 1-4Glc beta 1-Cer) but not to glycolipids lacking the lactose sequence (i.e. Glc beta 1-Cer, Gal beta 1-Cer or Gal alpha 1-4Gal beta 1-Cer). In microtiter wells, binding occurred at 50 ng and became half-maximal at the theoretical value for a monomolecular layer of LacCer (i.e. 100-200 ng/well). The bacteria also bound to glycolipids with various substitutions (e.g. GalNAc beta 1-4, Gal beta 1-3GalNAc beta 1-4, Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4, Gal alpha 1-3, GlcNAc beta 1-3, Gal beta 1-3GlcNAc beta 1-3, Gal beta 1-4GlcNAc beta 1-3, and Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3) at the Gal of LacCer, although only those species with GalNAc beta 1-4 or Gal beta 1-3GalNAc beta 1-4 were as active as LacCer itself. Glycolipids with other additions (e.g. Gal alpha 1-4 and NeuAc alpha 2-3) were negative. For unsubstituted LacCer, the binding required either a trihydroxy base or 2-hydroxy fatty acid, specifying the epithelial type of ceramide; LacCer composed of a dihydroxy base and nonhydroxy fatty acid was negative. This is interpreted as indicating that the proper presentation of the binding epitope depends on the ceramide structure. The relevance of this to biological membranes has not yet been established. Neither free lactose (up to 20 mg/ml) nor lactose-bovine serum albumin (5 mg/ml) prevented the binding of bacteria to LacCer, two facts that support the solid-phase binding data demonstrating a low affinity binding and the crucial importance of a particular lactose epitope.     

Biosynthesis in vitro of disialosylneolactotetraosylceramide by a solubilized sialyltransferase from embryonic chicken brain

A sialyltransferase involved in the biosynthesis in vitro of LD1c (NeuAc alpha 2-8NeuGc alpha 2-3Gal beta 1-4Glc-NAc beta 1-3Gal beta 1-4Glc-Cer) has been characterized from 9 to 11-day-old embryonic chicken brains. The CMP-[14C]NeuAc:LM1(alpha 2-8)sialyltransferase (SAT-2) sedimented (75%) at the junction of 0.75 and 1.2 M on a discontinuous sucrose density gradient when still membrane bound. In addition to the biosynthesis of LD1c, the detergent-solubilized (0.4% Nonidet P-40) preparation also catalyzes the transfer of sialic acid to O-8 of sialic acid in GM3 to form GD3 (NeuAc alpha 2-8NeuAc alpha 2 - 3Gal beta 1 - 4Glc - Cer). Substrate inhibition studies indicated that these two reactions are probably catalyzed by the same enzyme, SAT-2. The kinetic parameters of SAT-2 activity were determined. The Km values were 70 and 63 microM with CMP-[14C]NeuAc and LM1, respectively, when the detergent-solubilized supernatant fraction was used as enzyme source. The (alpha 2-8)-linkage between the terminal and penultimate sialic acids was determined using nonradioactive CMP-NeuAc and [Ac-14C]LM1 as substrates (Higashi, H., and Basu, S. (1982) Anal. Biochem. 120, 159-164) for the enzyme, followed by identification of the permethylated [14C]sialic acid of the product by radioautography. At 0.5 mM N-ethylmaleimide, the SAT-2 activity was inhibited 50% whereas SAT-1 and SAT-3 activities (Basu, M., Basu, S., Stoffyn, A., and Stoffyn, P. (1982) J. Biol. Chem. 257, 12765-12769) remained uninhibited.