Characterization of solubilized GlcAT-1 (UDP-GlcA: nLcOse4Cer beta 1-3 glucuronyltransferase) activity from embryonic chicken brain and its inhibition by D-erythro-sphingosine

Glycolipid glucuronyltransferase activity (GlcAT-1) has been solubilized and characterized from 19-day-old embryonic chicken brain Golgi-rich membranes. The enzyme catalyzes the biosynthesis in vitro of GlcA beta 1-3nLcOse4Cer glycolipid using neolactetraosylceramide (nLcOse4Cer, Gal beta 1-4GlcNAc beta 1-3Gal beta-1-4Glc-Cer) as the substrate. The membrane-bound enzyme shows optimum activity in the presence of neutral detergents such as Triton CF-54, Triton DF-12, and Nonidet P-40. Approximately 60% of the enzyme activity can be solubilized from the Golgi membrane by Nonidet P-40. The solubilized GlcAT-1 activity is inhibited by different salts such as NaCl, NaBr, NaI, and NaOAc, but not by sodium fluoride (up to 0.4 M concentration). Desialyzed alpha 1 acid glycoprotein (SA alpha 1AGP) can be used as a substrate for glucuronyltransferase. Competition studies between glycolipid (nLcOse4Cer) and glycoprotein SA alpha 1AGP) substrates show a mixed type of inhibition. Phospholipids, in particular phosphatidylglycerol, stimulate solubilized GlcAT-1 activity, while D-erythro-sphingosine, a metabolite of glycosphingolipids, is inhibitory (50% inhibition at 0.8 mM D-erythro-sph). These results demonstrate that both phospholipid as well as sphingosine might be involved in modulating glucuronyltransferase activity.     

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.     

The unusual tetrasaccharide sequence GlcA{beta}-3GalNAc(4-sulfate)({beta}1-4GlcA(2-sulfate){beta}1-3GalNAc(6-sulfate) found in the hexasaccharides prepared by testicular hyaluronidase digestion of shark cartilage chondroitin sulfate D

Eight hexasaccharide fractions were isolated from commercialshark cartilage chondroitin sulfate D by means of gel nitrationchromatography and HPLC on an amine-bound silica column afterexhaustive digestion with sheep testicular hyaluronidase. Capillaryelectrophoresis of the enzymatic digests as well as one- andtwo-dimensional 500 MHz ¹H-NMR spectroscopy demonstrated thatthese hexasaccharides share the common core saccharide structureGlcAß1-3GalNAcß1-4GlcAß1-3GalNAcß1-4GlcAß1-3GalNAcwith three, four, or five sulfate groups in different combinations.Six structures had the same sulfation profiles as those of theunsaturated hexasaccharides isolated from the same source afterdigestion with chondroitinase ABC (Sugahara et al., Eur. J.Biochem., 293, 871–880, 1996) and the other two have notbeen reported so far. In the new components, a D disaccharideunit, GlcA(2-sulfate)ß1-3GalNAc(6-sulfate), characteristicof chondroitin sulfate D was arranged on the reducing side ofan A disaccharide unit, GlcAß1-3GalNAc(4-sulfate),forming an unusual A-D tetrasaccharide sequence, GlcAß1-3GalNAc(4-sulfate)-4GlcA(2-sulfate)ß1-3GaINAc(6-sulfate)which is known to be recognized by the monoclonal antibody MO225.These findings support the notion that the tetrasaccharide sequence,GlcAß1-3GalNAc(4-sulfate)ß1-4GlcAß1-3GalNAc(6-sulfate)is included in the acceptor site of a hitherto unreported 2-O-sulfotransferaseresponsible for its synthesis. The sulfated hexasaccharidesisolated in this study will be useful as authentic oligosaccharideprobes and enzyme substrates in studies of sulfated glycosaminogly-cans. sulfated hexasaccharides chondroitin sulfate D hyaluronidase ¹ H-NMR     

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.     

Biosynthesis in vitro of SA-Lex and SA-diLex by alpha 1-3 fucosyltransferases from colon carcinoma cells and embryonic brain tissues.

The sialyl-fucosyl-lactosamine-epitope present in sialyl (SA)-Lex (NeuAc alpha 2-3Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta 1-3Gal beta 1-4Glc-Cer), a carcinoembryonic antigen, has been recognized recently as a ligand for the binding of leukocyte-endothelial cell adhesion molecule 1 (LECAM-1) to myeloid and tumour cell surfaces. We have recently detected the presence of an alpha 1-3 fucosyltransferase (FucT-3) activity in both embryonic chicken brain (ECB) and human colon carcinoma cells (Colo-205) which catalyses the biosynthesis in vitro of SA-Lex and SA-diLex. Fucosyltransferase activities from both sources are stimulated in the presence of divalent cations (Mn2+, Mg2+, Ca2+, Co2+ and Fe2+), although absolute metal requirement is not observed. Substrate specificity studies with this partially purified (ECB, 3000-fold; Colo-205, 100-fold) novel FucT-3 indicate the preference for terminally sialyl-substituted glycolipid acceptors, as observed by the lower Km values when sialyl-neolactotetraosyl ceramide, LM1, (Neu-Gc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4 Glc-Cer; Km = 0.048 mM) and sialyl-norhexaosylceramide, NeuGc-nLc6, (Neu-Gc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer; Km = 0.032 mM) were used as substrates. Fucosyltransferase from Colo-205 requires the presence of the acyl group of the ceramide moiety and an acetyl group on glucosamine in the acceptor glycolipid since lyso-LM1 was found to be completely inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymatic in vitro synthesis of radiolabeled pentasaccharides GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc/Glc and the isomeric Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc/Glc.

Four radiolabeled pentasaccharides, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4Glc, and Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc, were prepared in virtually pure form. They were obtained by partial enzymic beta 1,4-galactosylations of the appropriate tetrasaccharide acceptors or by partial enzymic degalactosylations of the appropriate hexasaccharides, followed by paper chromatographic separations. All four pentasaccharides contain two nonidentical distal branches, making them valuable primers for enzymatic in vitro synthesis of larger oligo(N-acetyllactosaminoglycans).     

Effect of a fatty acid moiety of phospholipid and ceramide on purified GalT-3 (UDP-Gal:GM2 beta 1-3 galactosyltransferase) activity from embryonic chicken brain

Galactosyltransferase, GalT-3 (UDP-Gal:GM2 beta 1-3 galactosyltransferase) has been characterized and solubilized from 19-day-old embryonic chicken brain, and purified to over 2000-fold using mixed-modal chromatography on a omega-aminohexyl Sepharose column and affinity chromatography on a UDP-hexanolamine Sepharose column. The activity of purified GalT-3 was modulated by phospholipids in vitro with stimulation observed specifically with dipalmitoyl phosphatidylethanolamine (PE). All natural phospholipids tested (PE, PC and PI) inhibited GalT-3 activity. Enzyme activity was affected by the structure of the phospholipid vesicle. It was stabilized by the hexagonal (dipalmitoyl PE) structure and inhibited by the bilayer (dielaidoyl PE) structure. The long-chain fatty acid moiety of the glycosphingolipid substrate, GM2, was found to be necessary for optimum enzyme activity. In the absence of fatty acid, the modified substrates, lyso-GM2 and acetyl-GM2, had a 10-fold increased Km and a 4-8 fold decreased Vmax compared to the normal substrate. We postulate that GalT-3 belongs to a group of glycosyltransferases having recognition for both the carbohydrate as well as the hydrophobic domains (HY-CARS) of their substrates and that the fatty acid moiety of either the substrate (GM2) or a heterotropic effector (phospholipid) plays an important role in regulating the activity of this enzyme.     

Three-dimensional structure of a glycosphingolipid having a novel carbohydrate linkage, Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta, determined by theoretical calculations

The novel glycosphingolipid, SEGLx (Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta Cer), which was identified by us (Kawakami Y, et al. (1993) J Biochem 114: 677-83), shows a characteristic spectrum on 1H-NMR analysis, in which the anomeric proton resonances of a reducing end galactose and a glucose are split. To elucidate the structural characteristics of SEGLx, we determined its three-dimensional (3D) structure by means of computer simulation, involving such techniques as molecular mechanics (MM2), the semiempirical molecular orbital method (AM1), molecular dynamics (Amber), and computer 3D modelling. With the hypothesis that all OH group(s) of a ceramide participate in intramolecular hydrogen bonds, two kinds of stable conformers, horizontal and right-angled ones, were formed, depending on the ceramide species. The present findings suggest that the chemical species of both the long chain base and fatty acid moieties, mainly the occurrence of OH group(s), affect the chemical shifts of the anomeric proton resonances not only of the reducing terminal galactose but also the penultimate glucose through the formation of intramolecular hydrogen bonds. Computer simulation through theoretical calculation and 3D modelling was shown to be the best means of confirming the results obtained by experimental analysis.     

Wheat germ agglutinin chromatography of GlcNac beta 1-3(GlcNAc beta 1-6)Gal and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, obtained by in vitro synthesis and by partial cleavage of teratocarcinoma poly-N-acetyllactosaminoglycans

GlcNAc beta 1-3(GlcNAc beta 1-6) [14C(U)]Gal and GlcNAc beta 1-3(GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc were prepared by in vitro synthesis. They were characterized by enzymatic sequencing, by partial acid hydrolysis, and by periodate oxidation experiments. The two saccharides were isolated also from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine embryonal carcinoma cells (line PC 13). The tetrasaccharide was retarded in a column of agarose-linked wheat germ agglutinin; the trisaccharide was strongly bound. Chromatography in this column separated the trisaccharide into two distinct peaks, which represented interconvertible molecules. Together with our previous data on linear teratocarcinoma saccharides, these findings show that affinity chromatography with immobilized wheat germ agglutinin can be advantageously used in fractionating radiolabeled oligo-N-acetyllactosaminoglycans and saccharides related to them.     

A novel pentaglycosylceramide in ostrich liver, IV4-beta-Gal-nLc4Cer, with terminal Gal(beta1-4)Gal, a xenoepitope recognized by human natural antibodies

Thin layer chromatograms of ostrich liver neutral glycosphingolipids were immunostained with human sera. In addition to the expected staining of the Forssman pentaglycosylceramide by some sera, more polar and less abundant unknown glycolipids could be stained. Among them, the shortest carbohydrate chain glycolipid was purified and structurally characterized by mass spectrometry, proton NMR and methylation analysis. It was a novel pentaglycosylceramide of the neolactoseries terminated with the Gal(beta1-4)Gal determinant which is not expressed in mammalian species. Human antibodies affinity-purified on a synthetic Gal(beta1-4)Gal(beta1-4)Glc-Sepharose column recognized the newly characterized Gal(beta1-4)Gal-terminated pentaglycosylceramide, and, in addition, longer chain glycolipids. Occurrence of antibodies directed at the Gal(beta1-4)Gal epitope was studied by ELISA on 108 human sera. Anti-Gal(beta1-4)Gal antibodies were predominantly IgM, and their distribution was similar to that of anti-Gal(alpha1-3)Gal and anti-Forssman IgMs. It was concluded that anti-Gal(beta1-4)Gal are natural antibodies, not previously identified in man. They can be considered as xenoantibodies directed at species which express Gal(beta1-4)Gal-terminated carbohydrate chains.     

Biosynthesis of a novel recombinant peptide derived from hPTH(1-34)

A recombinant human parathyroid hormone fragment, Pro-Pro-[Arg(11)]hPTH(1-34)-Pro-Pro (MW=4550 Da), was developed by substituting Arg for Leu at position 11 and adding Pro-Pro at the carboxyl terminus. Following a single injection (0.5-13.5 μg/bird) of the rhPTH fragment, the serum calcium level in chickens increased 12-42% (P<0.05) after 1h as determined by the Parson's Chicken Assay. The functional activity of Pro-Pro-[Arg(11)]hPTH(1-34)-Pro-Pro may be due to removal of the N-terminus Pro-Pro- by X-prolyl dipeptidyl peptidase IV (DPPIV) in vivo, increasing its activity compared to Pro-Pro-hPTH(1-34). This artificial rhPTH fragment could be used to increase calcium mobilization and potentially improve bone health.     

Specific inhibition of binding of antistasin and [A103,106,108] antistasin 93-119 to sulfatide (Gal(3-SO4)beta 1-1Cer) by glycosaminoglycans.

Leech-derived antistasin is a potent anticoagulant and antimetastatic protein that binds sulfatide (Gal(3-SO4)beta 1-1Cer) and sulfated polysaccharides. In this study, the synthetic fragment [A103,106,108] antistasin 93-119, which corresponds to the carboxyl terminus, showed specific and saturable binding to sulfatide. Binding was competitively blocked by glycosaminoglycans (GAGs) in the order: dextran sulfate 5000 congruent to dextran sulfate 500,000 greater than heparin greater than dermatan sulfate much greater than chondroitin sulfates A and C. This rank order of inhibitory potency was identical to that observed with whole antistasin. We suggest that residues 93-119 of antistasin represent a critical domain for binding GAGs and sulfated glycolipids.     

Escherichia coli beta-galactosidase unexpectedly cleaves the hexasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc without branch specificity

The branch specificity of Escherichia coli beta-galactosidase (EC 3.2.1.23) was studied by analyzing the cleavage of the branched hexasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc (1). This hexasaccharide was cleaved to pentasaccharides Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6) [14C(U)]Gal beta 1-4GlcNAc (3) and GlcNAc beta 1-3(Gal-beta 1-4GlcNAc beta 1-6) [14C(U)]Gal beta 1-4GlcNAc (4) without any appreciable branch specificity. Even the further conversions of the pentasaccharides 3 and 4 into the tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc seemed to proceed at similar rates, without any appreciable branch specificity. In marked contrast to the hexasaccharide 1, the pentasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)[14C(U)]Gal (2), missing the reducing end GlcNAc, is known to be cleaved selectively at the 6-branch; this finding was confirmed in the present study. The different behaviour of hexasaccharide 1 and pentasaccharide 2 reflects differences in the reactivity of their 6-branches; the preferred conformations of these closely related molecules may be quite different.     

Human urokinase contains GalNAc beta (1-4)[Fuc alpha (1-3)]GlcNAc beta (1-2) as a novel terminal element in N-linked carbohydrate chains.

Structural analysis of enzymically released N-linked carbohydrate chains of human urokinase (urinary-type plasminogen activator) by 1H NMR spectroscopy and FAB-MS demonstrated that the N-linked oligosaccharides on the only N-glycosylation site contain diantennary structures with the novel GalNAc beta (1-4) [Fuc alpha (1-3)]GlcNAc beta (1-2) element in the upper or the lower branch.     

Human Lewis {alpha}1->3/4fucosyltransferase: specificity of fucose transfer to GlcNAc{beta}1->3Gal{beta}1->4Glc{beta}1->1Cer (LcOse3Cer)

Biosynthesis of the Le^x series of carbohydrate antigens proceedsby fucose transfer in 13-linkage to the penultimate GlcNAc residueof a neolacto-series oligosaccharide acceptor, a reaction catalysedby multiple enzymes expressed in human tissues. Particularlybroad acceptor specificity, including the ability to catalysefucose transfer to both lacto- and neolacto-series acceptorsas well as the precursor Lc₃ structure (where Lc₃ lactotriaosylceramide,is GlcNAcß13Galß14Glcß1Cer), existsfor one human fucosyltransferase form, the Lewis 13/4fucosyltransferase(FucT-III). To determine if fucose transfer to Lc₃may representan alternate early step in Le^xor Le^a antigen biosynthesis withthis enzyme, the chemical structure of the fucosylated Lc₃ reactionproduct formed by the Lewis 13/4fucosyltransferase from Colo205 cells has been defined. Transfer of [¹⁴C]fucose to Lc₃ yieldeda labelled product migrating as a tetrasaccharide on thin layerchromatography plates. This product remained an acceptor forboth ß13- and ß14-galactosyl transfer onthe terminal GlcNAc residue. The product was degraded to a fucosylatedtrisaccharide derivative by bovine kidney ß-N-acetylglucosaminidase.Fast atom bombardment mass spectrometry and methylation analysisconfirmed that the product was composed exclusively of the followingstructure containing a fucose linked to the 3-position of theinternal Glc residue: GlcNAcß13Galß14Glcß11Cer Such a structure does not represent an intermdiate in Le^xorLe^a antigen biosynthesis. Thus, the evidence suggests that Le^xor Le^a antigen synthesis results exclusively from fucosylationof complete core chains. fucosyltransferase lacto-series LcOse₃Cer Lewis antigen transfer specificity