Apoptosis of human breast carcinoma cells in the presence of cis-platin and L-/D-PPMP: IV. Modulation of replication complexes and glycolipid: Glycosyltransferases

Apoptosis of human breast carcinoma cells (SKBR-3, MCF-7, and MDA-468) has been observed after treatment of these cells with anti-cancer drug cis-platin and glycosphingolipid biosynthesis inhibitor L- and D-PPMP, respectively. These drugs initiated apoptosis in a dose-dependent manner as measured by phenotypic morphological changes, by binding of a fluorescent phophatidyl serine-specific dye (PSS-380) onto the outer leaflet of the cell membranes, and by activation of caspases, −3, −8, and −9. It was observed that in two hours very little apoptotic process had started but predominant biochemical changes occurred after 6 h. DNA degradation started after 24 hours of drug treatment. However, very little is known about the stability of the ‘`Replication Complexes’’ during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during its replication, repair, and recombination processes. Previously, DNA helicase-III was characterized as a component of the replication complexes isolated from embryonic chicken brains as well as breast and colon carcinoma cells. Helicase activities were measured by a novel method (ROME assay), and DNA polymerase-α activities were determined by regular chain extension of the nicked ACT-DNA, by determining values obtained from +/− aphidicolin-treated incubation mixtures. In all three breast carcinoma cell lines, a common trend was observed: a decrease of activities of DNA polymerase-α and Helicase III. A sharp decrease of activities of the glycolipid sialyltransferases: SAT-2 (CMP-NeuAc; GD3 α2-8 sialyltransferase) and SAT-4 (CMP-NeuAc: GM1a α2-3 sialyltransferase) was observed in the apoptotic carcinoma cells treated with L-PPMP compared with cis-platin.     

Apoptosis of human carcinoma cells in the presence of inhibitors of glycosphingolipid biosynthesis: I. Treatment of Colo-205 and SKBR3 cells with isomers of PDMP and PPMP

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells by anti-cancer drugs and biosynthetic inhibitors of cells surface glycolipids in the human colon carcinoma cells (Colo-205) are of interest in recent years. In our present studies, we have employed different stereoisomers of PPMP and PDMP (inhibit GlcT-glycosyltransferase (GlcT-GLT)) to initiate apoptosis in Colo-205 cells grown in culture in the presence of ³H-TdR and ³H/or ¹⁴C-L-Serine. Our analysis showed that the above reagents (between 1 to 20 μM) initiated apoptosis with induction of Caspase-3 activities and phenotypic morphological changes in a dose-dependent manner. We have observed an increase of radioactive ceramide formation in the presence of a low concentration (1–4 μM) of these reagents in these cell lines. However, high concentrations (4–20 μM) inhibited incorporation of radioactive serine in the higher glycolipids. Colo-205 cells were treated with L-threo-PPMP (0–20 μM) and activities of different GSL: GLTs were estimated in total Golgi-pellets. The cells contained high activity of GalT-4 (UDP-Gal: LcOse3Cer β1-4galactosyltransferase), whereas negligible activity of GalT-3 (UDP-Gal: GM2 β1-3galactosyltransferase) or GM2-synthase activity of the ganglioside pathway was detected. Previously, GLTs involved in the biosynthetic pathway of SA-Le^x formation had been detected in these colon carcinoma (or Colo-205) cells (Basu M et al. Glycobiology 1, 527–35 (1991)). However, during progression of apoptosis in Colo-205 cells with increasing concentrations of L-PPMP, the GalT-4 activity was decreased significantly. These changes in the specific activity of GalT-4 in the total Golgi-membranes could be the resultant of decreased gene expression of the enzyme. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Two glycosphingolipid sialyltransferases are localized in different sub-Golgi compartments in rat liver

A highly purified Golgi preparation from rat liver was fractionated on a sucrose density gradient and the activity of two sialyltransferases, CMP-NeuAc: Gal beta 1----4Glc-Cer (lactosylceramide) alpha-2----3sialyltransferase; Sat-1), and CMP-NeuAc:Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3) Gal beta 1----4Glc-Cer (GM1 ganglioside) alpha 2----3sialyltransferase; SAT-4), involved in the biosynthesis of gangliosides were assayed in the collected fractions. These two activities were recovered in different regions of the gradient; SAT-1 was found in a more dense region than SAT-4. This distribution coincided with that of two N-Asn linked oligosaccharide processing enzymes (UDP-GlcNAc:lysosomal enzyme precursor GlcNAc-1-phosphotransferase and UDP-Gal:ovalbumin galactosyltransferase), assumed as putative markers of cis- and trans-Golgi cisternae, respectively. These findings are consistent with the assembly of ganglioside oligosaccharide chains occurring in different sub-Golgi compartments.     

Topography of glycosyltransferases involved in the initial glycosylations of gangliosides.

We attempted to establish within which organelle UDP-Glc:ceramide beta 1----1'glucosyltransferase (GlcT) is located and moreover to obtain information about its orientation on intracellular membranes as well as that of UDP-Gal:glucosylceramide beta 1----4galactosyltransferase (GalT-2) and CMP-NeuAc:lactosylceramide alpha 2----3sialyltransferase (SAT-1). An extremely purified Golgi apparatus fraction was the only liver fraction where a ceramide-dependent formation of glucosylceramide could be demonstrated. This Golgi fraction, mainly constituted by stacks of intact cisternae which retained the same topographical orientation as in vivo, was then incubated with liposomal dispersions of glycosphingolipid-glycosyltransferase acceptors in reaction mixtures containing all the requirements for enzyme activity but no detergent. Under such conditions, SAT-1 and other late acting glycosyltransferases were over 90% latent, while both GlcT and GalT-2 were just as active as in the detergent-containing assay; they were still inhibited by EDTA. Sepharose-immobilized ceramide and Sepharose-immobilized glucosylceramide were found to be suitable acceptors for GlcT and GalT-2, respectively, still using intact Golgi cisternae as the enzyme source. Moreover, a part of GlcT and GalT-2 activity was released from intact Golgi cisternae upon cathepsin D treatment. These results provide strong evidence that GlcT and GalT-2 face the cytoplasmic side of the Golgi apparatus, whereas SAT-1 and the other late acting enzymes face the luminal side.     

Apoptosis of human carcinoma cells in the presence of inhibitors of glycosphingolipid biosynthesis: I. Treatment of Colo-205 and SKBR3 cells with isomers of PDMP and PPMP

Apoptosis, or programmed cell death, plays an important role in many physiological and diseased conditions. Induction of apoptosis in cancer cells by anti-cancer drugs and biosynthetic inhibitors of cells surface glycolipids in the human colon carcinoma cells (Colo-205) are of interest in recent years. In our present studies, we have employed different stereoisomers of PPMP and PDMP (inhibit GlcT-glycosyltransferase (GlcT-GLT)) to initiate apoptosis in Colo-205 cells grown in culture in the presence of (3)H-TdR and (3)H/or (14)C-L-Serine. Our analysis showed that the above reagents (between 1 to 20 microM) initiated apoptosis with induction of Caspase-3 activities and phenotypic morphological changes in a dose-dependent manner. We have observed an increase of radioactive ceramide formation in the presence of a low concentration (1-4 microM) of these reagents in these cell lines. However, high concentrations (4-20 microM) inhibited incorporation of radioactive serine in the higher glycolipids. Colo-205 cells were treated with L-threo-PPMP (0-20 microM) and activities of different GSL: GLTs were estimated in total Golgi-pellets. The cells contained high activity of GalT-4 (UDP-Gal: LcOse3Cer beta 1-4galactosyltransferase), whereas negligible activity of GalT-3 (UDP-Gal: GM2 beta 1-3galactosyltransferase) or GM2-synthase activity of the ganglioside pathway was detected. Previously, GLTs involved in the biosynthetic pathway of SA-Le(x) formation had been detected in these colon carcinoma (or Colo-205) cells (Basu M et al. Glycobiology 1, 527-35 (1991)). However, during progression of apoptosis in Colo-205 cells with increasing concentrations of L-PPMP, the GalT-4 activity was decreased significantly. These changes in the specific activity of GalT-4 in the total Golgi-membranes could be the resultant of decreased gene expression of the enzyme.     

Sub-Golgi distribution in rat liver of CMP-NeuAc GM3- and CMP-NeuAc:GT1b alpha 2----8sialyltransferases and comparison with the distribution of the other glycosyltransferase activities involved in ganglioside biosynthesis

Using a sucrose density gradient fractionation of a highly purified Golgi apparatus from rat liver, we determined the sub-Golgi distribution of CMP-NeuAc:GM3 ganglioside alpha 2----8sialyltransferase (GM3-SAT) and CMP-NeuAc:GT1b ganglioside alpha 2----8sialyltransferase (GT1b-SAT), in comparison with that of the other glycosyltransferase activities involved in ganglioside biosynthesis. While GM3-SAT was recovered in several density fractions, GT1b-SAT was mainly found on less dense sub-Golgi membranes; this indicates that these two activities are physically separate. Moreover, with regard to the monosialo pathway, CMP-NeuAc:lactosylceramide alpha 2----3sialyltransferase, UDP-GalNAc:GM3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GM2 ganglioside beta 1----3galactosyltransferase, and CMP-NeuAc:GM1 ganglioside alpha 2----3sialyltransferase were resolved from more dense to less dense fractions, respectively. In the disialo pathway, UDP-GalNAc:GD3 ganglioside beta 1----4N-acetylgalactosaminyltransferase, UDP-Gal:GD2 ganglioside beta 1----3galactosyltransferase and CMP-NeuAc:GD1b ganglioside alpha 2----3sialyltransferase co-distributed with the corresponding activities of the monosialo pathway. These last results indicate that many Golgi glycosyltransferases involved in ganglioside biosynthesis are localized in the order in which they act.     

Solubilized glycosyltransferases and biosynthesis in vitro of glycolipids

The assembly of most of the ceramide-linked glycolipids (GSLs) in eukaryotic cells occurs in Golgi bodies. At least 18 different glycolipid:glycosyltransferases (GSL:GLTs) have been characterized, 10 of which have been solubilized. These GLTs can be classified into 2 distinct groups: 1) GLTs dedicated to either Dol-P-P-sugar(s) or ceramide-linked sugar(s); and 2) GLTs with dual loyalties (i.e., they compete with glycolipid- and glycoprotein-bound oligosaccharides). Studies with solubilized and purified GalNAcT-1 and GalNAcT-2 from embryonic chicken brains prove that GalNAcT-1 (UDP-GalNAc:GM3 beta 1-4GalNAcT) is specific for GSL, whereas GalNAcT-2 (UDP-GalNAc:Gb3 beta 1-3GalNAcT) can transfer to an oligosaccharide containing the alpha-linked terminal galactose. Similarly, GalT-3 (UDP-Gal:GM2 beta 1-3GalT) is more specific for ganglio-oligosaccharide and GalT-4 (UDP-Gal:Lc3 beta 1-4GalT) can transfer galactose to N-acetylglucosamine linked to p-nitrophenol, glycolipid or glycoprotein. Both GalT-3 and GalT-4 have been separated and purified from embryonic chicken brains. Studies with solubilized SAT-4 and SAT-3, from bovine spleen and embryonic chicken brains, respectively, suggest the existence of 2 different gene-expressed alpha 2-3SATs. The newly discovered FucT-3 (GDP-Fuc:NeuGc-iLc6-alpha 1-3FucT) from human colon carcinoma (Colo-205) has also been solubilized and separated from other GSL:GLTs. Using a new activity gel-Western blot combined technique, the molecular mass of this FucT-3 was determined to be 105 kDa.     

Purification, properties, and immunological characterization of GalT-3 (UDP-galactose: GM2 ganglioside, β1-3 galactosyltransferase) from embryonic chicken brain

A 1-3 galactosyltransferase (GalT-3; UDP-Gal; GM2 1-3galactosyltransferase) was purified over 5100-fold from 19-day-old embryonic chicken brain homogenate employing detergent solubilization, -lactalbumin Sepharose, Q-Sepharose, UDP-hexanolamine Sepharose, and GalNAc1-4Gal-Synsorb column chromatography. The purified enzyme was resolved into two bands on reducing gels with apparent molecular weights of 62 kDa and 65 kDa, respectively. GalT-3 activity was also localized in the same regions by activity gel analysis and sucrose-density gradient centrifugation of a detergent-solubilized extract of 19-day-old embryonic chicken brain. Purified GalT-3 exhibited apparentK _mS of 33 µm, 22 µm and 14.4mM with respect to the substrates GM2, UDP-galactose, and MnCl₂, respectively. Substrate specificity studies with the purified enzyme and a variety of glycosphingolipids, glycoproteins, and synthetic substrates revealed that the enzyme was highly specific only for the glycosphingolipid acceptors, GM2 and GgOse3Cer (asialo-GM2). Ovine-asialo-agalacto submaxillary mucin inhibited the transfer of galactose to GM2 but did not act as an acceptor in the range of concentrations tested. Polyclonal antibodies raised against purified GalT-3 inhibited GalT-3 activityin vitro and Western-immunoblot analysis of purified GalT-3 showed immunopositive bands at 62 and 65 kDa.Abbreviations CNS central nervous system - GM1 monosialotetraosylganglioside, Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer - GM2 monosialotriaosylganglioside, GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer - DSS detergent solubilized supernatant - ECB embryonic chicken brain - TBS Tris-buffered saline     

Biosynthesisin vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core

The galactosyltransferase, GalT-4, which catalyses the biosynthesisin vitro of neolactotetraosylceramide, nLcOse4Cer (Gal1-4GleNAc1-3Gal1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcNAc1-3Gal1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn²⁺). TheK _m values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 µm, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-1-acid glycoprotein orN-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc1-1-3Gal1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for anN-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH₂1-3Gal1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while theK _m andV _max of the reacetylated substrate (GlcNac1-3Gal1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.Abbreviations EDTA ethylenediamine tetraacetate - ME -mercaptoethanol - PEG polyethylene glycol - PBS phosphate buffered saline - Suc sucrose - Mn²⁺ manganese - Gal galactose - GlcNAc N-acetylglucosamine - UDP-Gal Uridine diphosphate galactose - Ab antibody - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ECB embryonic chicken brain - Cer ceramide - nLc4 or NlcOse4Cer Gal1-4GleNac1-3Gal1-4Glc-Cer, neoLactotetraosylceramide - Lc3 or LcOse3Cer GlcNac1-3Gal1-4Glc-Cer, lactotriaosylceramide - iLc5 iLcOse5Cer, GlcNAc1-3nLcOse4Cer - nLc6 nLcOse6Cer, Gal1-4iLcOse5Cer - SA^–Gal^–₁AGP asialo-agalacto1-acid glycoprotein - TLC thin layer chromatography     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

Effects of lectin activation on sialyltransferase activities in human lymphocytes

The effects of phytohemagglutinin (PHA) stimulation on the activities of sialyltransferase 1 (SAT-1), and sialyltransferase 3 (SAT-3), in human lymphocytes were investigated in vitro. For SAT-1 and SAT-3, respectively, the apparent Km values with variable CMP-NeuAc concentrations were 0.19 and 0.015 mM and with variable LacCer were 0.075 and 0.17 mM. Progressive increases in the activities of SAT-1 and SAT-3 were detected in lymphocytes stimulated with PHA, whereas no increase was observed in control lymphocytes incubated in culture medium alone. These increased activities occurred within 18-36 h of incubation and preceded optimum lymphocyte proliferation. Intact lymphocytes were needed for the lectin-stimulated increase of sialyltransferase activities because neither concanavalin A nor phytohemagglutinin added to the broken cell preparation modulated SAT-1 activity. The glycolipid products formed as a result of these enzymatic reactions in the presence of endogenous and exogenous acceptors were tentatively identified by thin-layer chromatography and autofluorography. The addition of exogenous LacCer to the SAT-1 assay resulted in the radiolabeling of a small amount of ganglioside GM1b (3.4%), but GM3 was the major labeled product (96%). When GgOse4Cer was added to the SAT-3 assay, 32% GM3 and 24.6% GM1b were detected while 44% consisted of glycolipids not labeled in assays performed without exogenous acceptors. Of the radioactivity transferred to endogenous acceptors, 81.3% was in GM3 and 14.6% in GM1b. These results demonstrate that the modulation of sialyltransferase activity occurs earlier than cellular activation.     

Neutral glycosphingolipids and gangliosides from spleen T lymphoblasts of genetically different inbred mouse strains

The gangliosides GM1b, GalNAc-GM1b and GD1α are typical compounds of concanavalin A stimulated splenic T lymphoblasts of CBA/J inbred mice. Their structural characterization has been described in previous studies. The intention of this work was the comparative TLC immunostaining analysis of the glycosphingolipid composition of lectin stimulated splenic T lymphoblasts obtained from six genetically different inbred mouse strains. The strains examined were AKR, BALB/c, C57BL/6, CBA/J, DBA/2 and WHT/Ht, which are commonly used for biochemical and immunological studies. The neutral glycosphingolipid GgOse4Cer, the precursor for GM1b-type gangliosides, was expressed by all six strains investigated. AKR, C57BL/6 and DBA/2 showed high and BALB/c, CBA/J and WHT/Ht diminished expression in T lymphoblasts, based on single cell calculation. The gangliosides GM1b and GalNAc-GM1b, elongation products of GgOse4Cer, displayed strain-specific differences in their intensities, which were found to correlate with the intensities of GgOse4Cer expression of the same strains. Concerning sialic acid substitution of gangliosides, GM1b and GalNAc-GM1b predominantly carry N-acetylneuraminic acid, whereas choleragenoid receptors GM1a and Gal-GalNAc-GM1b, which are also expressed by all six strains, are characterized by dominance of N-glycolylneuraminic acid. Two highly polar gangliosides, designated with X and Y, which have not been previously recognized in murine lymphoid tissue, were detected by positive anti-GalNAc-GM1b antibody and choleragenoid binding, respectively. Both gangliosides were restricted to AKR, DBA/2 and C57BL/6 mice. The other three strains BALB/c, CBA/J and WHT/Ht are lacking these structures. In summary, the GM1b-type pathway is quite active in all six strains analysed in this study. Strain-specific genetic variations in T lymphoblast gangliosides were observed with the occurrence of gangliosides X and Y. This study and data from other groups strongly indicate for GM1b-type gangliosides a functional association with T cell activation and leukocyte mediated reactions. Abbreviations: ConA, concanavalin A; GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) [48] and the ganglioside nomenclature system of Svennerholm [49] for GM1a-type gangliosides. Glucosylceramide or GlcCer, Glcβ1-1Cer; lactosylceramide or LacCer, Galβ1-4Glcβ1-1Cer; gangliotriaosylceramide or GgOse3Cer or Gg3, GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliotetraosylceramide or GgOse4Cer or Gg4, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliopentaosylceramide or GgOse5Cer, GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliohexaosylceramide or GgOse6Cer, Galβ1-3GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer. GM3, II3NeuAc-LacCer; GM1 or GM1a, II3NeuAc-GgOse4Cer; GM1b, IV3NeuAc-GgOse4Cer; GalNAc-GM1b, IV3NeuAc-GgOse5Cer; GD1a, IV3NeuAc, II3NeuAc-GgOse4Cer; GD1b, II3(NeuAc)2-GgOse4Cer; GD1c, IV3(NeuAc)2-GgOse4Cer; GD1α, IV3NeuAc, III6NeuAc-GgOse4Cer. Only NeuAc-substituted gangliosides are presented in this list of abbreviations This revised version was published online in November 2006 with corrections to the Cover Date.     

Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's Iymphoma and germinal centre B cells

A new monoclonal antibody (TU-1) directed against the Galα1-4Galβ1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer (Galα1-4Galβ1-4Glcβ1-1′Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Galβ1-4Glcβ1-1′Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1′Cer), galactosylparagloboside (Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer), sulfatide (HSO3-3Galβ1-1′Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes. Abbreviations: ATL, adult T-cell leukaemia; BSA, bovine serum albumin; Cer, ceramide; DPPC, L-α-dipalmitoylphosphatidylcholine; EBV, Epstein-Barr virus; FCS, fetal calf serum; GalCer, Galβ1-1′Cer; GlcCer, Glcβ1-1′Cer; LacCer, Galβ1-4Glcβ1-1′Cer; Gb3Cer, Galα1-4Galβ1-4Glcβ1-1′Cer; Iyso-Gb3Cer, Galα1-4Galβ1-4Glc1-1′Sph; Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glc1-1′Cer; galactosylparagloboside, Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; Gg3Cer, GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; Gg4Cer, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; GM3, Neu5Acα2-3Galβ1-4Glcβ1-1′Cer; GM2, GalNAcβ1-4(Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; GM1a, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1a, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1b, Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GT1b, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MAb, monoclonal antibody; MPL, monophosphoryl lipid A; P1 antigen, Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; PVP, polyvinylpyrolidone; Sph, sphingosine; sulfatide, HSO3-Galβ1-1′Cer; TDM, trehalose dimycolate; TLC, thin-layer chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

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.     

UDP-galactose:lactosylceramide α-galactosyltransferase activity in human placenta

The activity of UDP-Gal: LacCer galactosyltransferase in human placenta was studied by using crude homogenate and Triton CF-54 extract as the source of enzyme. Transfer of galactose to lactosylceramide was optimal in the presence of 0.1% Triton CF-54 and Mn²⁺ at pH 6.3, and the reaction product was susceptible to -galactosidase.Abbreviations LacCer lactosylceramide (Gal1-4Glc1-1Cer) - Gb₃ globotriaosylceramide (Gal1-4Gal1-4Glc1-1Cer) - Gb₄ globoside (GalNAc1-3Gal1-4Gal1-4Glc1-1Cer) - TLC thin-layer chromatography - GC/MS gas chromatography/mass spectrometry - NMR nuclear magnetic resonance - EDTA ethylenediamine tetraacetic acid - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate     

Susceptibility of infant mice to F5 (K99)E. coli infection: Differences in glycosyltransferase activities in intestinal mucosa of inbred CBA and DBA/2 strains

EnterotoxigenicEscherichia coli (ETEC) strains expressing F5 (K99) fimbriae cause diarrhoea in the young animal through adhesion to specific sialoglycolipids of the small intestine surface. We studied here an infant mouse diarrhoea model, as CBA infant mice are susceptible to F5-positive ETEC infection, whereas DBA/2 ones are resistant. In an attempt to determine an enzymatic basis for susceptibility and resistance, we investigated the intestine ganglioside pattern in relation to the activity of glycosyltransferases responsible for the globo- and ganglio-series. We observed that the intestine of susceptible CBA infant mice displayed a characteristic sialoglycolipid pattern containing mainly the F5 receptors. The two murine strains differed in the relative activities of galactosyltransferases (GbOse₃Cer and GM1 synthases),N-acetylgalactosylaminyltransferases (GA2 and GM2 synthases) and sialyltransferases (GM3 and GD3 synthases). An elevated GM3-synthase activity was observed in the intestine of susceptible CBA infant mice, at the age of high susceptibility. Hence, we conclude that the marked specificity of mouse type correlated with susceptibility and resistance to F5-positive ETEC infection which could be controlled through the regulation of glycosyltransferase activities.Abbreviations NeuAc N-acetylneuraminic acid - NeuGc N-glycolylneuraminic acid - Glc glucose - GalNAc N-acetylgalactosamine - Gal galactose - Car ceramide - LacCer lactosylceramide (Galß-4Glcß1-1Cer) - GA2 asialo-GM2 (GgOse₃Cer) - GA1 asialo-GM1 (GgOse₄Cer) - NeuAc/NeuGc-GMla II³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/NeuGc-GM1a IV³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/NeuGc-GM2 II³ NeuAc/neuGc-GgOse₃Cer - NeuAc/NeuGc-GM3, II³ NeuAc/NeuGc-LacCer; NeuAc/NeuGc-GD1a, IV³ NeuAc/NeuGc, II³ NeuAc/NeuGc-GgOse₄Cer; NeuAc/NeuGc-GD1b II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GD1c IV³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GD2, II³ (NeuAc/NeuGc)₂-GgOse₃Cer; NeuAc/NeuGc-GD3, II³ (NeuAc/NeuGc)₂-Lac Cer; NeuAc/NeuGcGT1a IV³ (NeuAc/NeuGc)₂, II³ NeuAc/NeuGc-GgOse₄Cer - NeuAc/neuGc-GT1b IV³ NeuAc/NeuGc, II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GT1c II³ (NeuAc/NeuGc)₃-GgOse₄Cer; NeuAc/NeuGc-GT2, II³ (NeuAc/NeuGc)₃-GgOse₃Cer - NeuAc/NeuGc-GT3 II³ (NeuAc/NeuGc)₃-Lac Cer - NeuAc/NeuGc-GQ1b IV³ (NeuAc/NeuGc)₂, II³ (NeuAc/NeuGc)₂-GgOse₄Cer - NeuAc/NeuGc-GQ1c IV³ NeuAc/NeuGc, II³ (NeuAc/NeuGc)₃-GgOse₄Cer - NeuAc/NeuGc-GP1c IV³ (NeuAc/NeuGc)₂, II³ (NeuAc/NeuGc)₃-GgOse₄Cer - GD, GT and GQ di-, tri- and tetra-sialoglangliosides. NeuGc-SPG, IV³ NeuGc-nLcOse₄Cer. Glycosyltransferases assayed in this work areN-acetylgalactosaminyltransferases - UDP-GalNAc lactosylceramide 1-4N-acetylgalactosaminyltransferase or GA2 synthase (EC 2.4.1-) and UDP-GalNAc:(N-acetylneuraminyl)-lactosylceramide 1-4N-acetylgalactosaminyltransferase or GM2 synthase (EC 2.4.1.92) - sialyltransferases CMP-N-acetylneuraminate: lactosylceramide 2–3 sialyltransferase (sialyltransferases I and IV) or GM3 synthase (EC 2.4.99.-) and CMP-N-acetylneuraminate:(N-acetylneuraminyl) lactosylceramide 2-8 sialyltransferase (sialyltransferase II) or GD3 synthase (EC 24.99.8) - galactosyltransferases UDP-galactose:N-acetylgalactosaminyl-(N-acetylneuraminyl) lactosylceramide 1-3 galactosyltransferase (galactosyltransferase II) or GM1a synthase (EC 2.4.1.62) and UDP-galactose:lactosylceramide 1-4 galactosyltransferase or GbOse₃Cer synthase (EC 2.4.1-)     

Biosynthesis of eukaryotic cell surface glycosphingolipids using solubilized glycosyltransferases

Two fucsyltransferases (FucT-2 and FucT-3) have been solubilized from Golgi-rich membrane fraction of bovine spleen, using a cationic detergent. FucT-3 was distinguished from FucT-2 by comparing their kinetic parameters and heat stability. FucT-2 and FucT-3 lost activity (85 %) and (5 %), respectively, when heated at 55°C for 10 sec. Two galactosyltransferases (GalT-3 and GalT-4) and two sialyltransferases (SAT-2 and SAT-3) have also been solubilized from embryonic chicken brain membranes using nonionic detergents. Affinity chromatography and microisoelectric focusing were used to separate these enzymes into functionally pure fractions. Anomeric and positional linkages in some of the products (LM1 and LD1c) have also been established. The terminal NeuAc(α2-8) linkage in GD3 and LD1c was established by identification of the partially methylated penultimate [Ac-¹⁴C]sialic acid.     

Structure and Mechanism of Vacuolar Na<Superscript>+</Superscript>-Translocating ATPase From <Emphasis Type="Italic">Enterococcus hirae</Emphasis>

V-type Na⁺-ATPase from Entercoccus hirae consists of nine kinds of subunits (NtpA₃, B₃, C₁, D₁, E₁₋₃, F₁₋₃, G₁, I₁, and K₁₀) which are encoded by the ntp operon. The amino acid sequences of the major subunits, A, B, and K (proteolipid), were highly similar to those of A, B, and c subunits of eukaryotic V-ATPases, and those of β, α, and c subunits of F-ATPases. We modeled the A and B subunits by homology modeling using the structure of β and α subunits of F-ATPase, and obtained an atomic structure of NtpK ring by X-ray crystallography. Here we briefly summarize our current models of the whole structure and mechanism of the E. hirae V-ATPase.     

Glycoconjugate histochemistry of the digestive tract of <Emphasis Type="Italic">Triturus carnifex</Emphasis> (Amphibia,Caudata)

Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4) _n oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal, and (GlcNAc β1,4) _n oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3 GalNAc, (GlcNAc β1,4) _n oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4) _n oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H⁺K⁺-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc β1,4) _n oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.