Allele-selective RUNX1 binding regulates P1 blood group status by transcriptional control of A4GALT.

P1 and Pk are glycosphingolipid antigens synthesized by the A4GALT-encoded α1,4-galactosyltransferase, using paragloboside and lactosylceramide as acceptor substrates, respectively. In addition to the compatibility aspects of these histo-blood group molecules, both constitute receptors for multiple microbes and toxins. Presence or absence of P1 antigen on erythrocytes determines the common P1 (P1+Pk+) and P2 (P1-Pk+weak) phenotypes. A4GALT transcript levels are higher in P1 individuals and single-nucleotide polymorphisms (SNPs) in noncoding regions of A4GALT, particularly rs5751348, correlate with P1/P2 status. Despite these recent findings, the molecular mechanism underlying these phenotypes remains elusive. The In(Lu) phenotype is caused by Krüppel-like factor 1 (KLF1) haploinsufficiency and shows decreased P1 levels on erythrocytes. We therefore hypothesized KLF1 regulates A4GALT expression. Intriguingly, P1 -specific sequences including rs5751348 revealed potential binding sites for several hematopoietic transcription factors, including KLF1. However, KLF1 binding did not explain P1 -specific shifts in electrophoretic mobility-shift assays and small interfering RNA silencing of KLF1 did not affect A4GALT transcript levels. Instead, protein pull-down experiments using P1 but not P2 oligonucleotide probes identified runt-related transcription factor 1 (RUNX1) by mass spectrometry. Furthermore, RUNX1 binds P1 alleles selectively, and knockdown of RUNX1 significantly decreased A4GALT transcription. These data indicate that RUNX1 regulates A4GALT and thereby the expression of clinically important glycosphingolipids implicated in blood group incompatibility and host-pathogen interactions.

Clinicopathological significance of ganglioside DSGb5 expression in renal cell carcinoma.

Disialosyl globopentaosylceramide (DSGb5) is a ganglioside originally isolated from tissue extracts of renal cell carcinoma (RCC) with metastasis. Previous in vitro experiments have suggested that DSGb5 promotes metastasis by enhancing the migration of RCC cells and downregulating NK cell cytotoxicity against RCC cells. In this study, we investigated the clinicopathological significance of DSGb5 expression in RCC and outcomes of RCC patients. A total of 156 RCC patients who underwent surgical treatments at our hospital from January 2007 through December 2012 were analyzed in this study. The expression of DSGb5 in RCC specimens was examined by immunohistochemical staining with monoclonal antibody 5F3. The immunostaining intensity of RCC tissues was assessed in comparison with that in benign renal tubules as an internal positive control. The relationship between DSGb5 expression and clinicopathological characteristics was investigated and recurrence free survival following surgery was evaluated. Microvascular invasion was observed in 68% (n = 19/28) and in 45% (n = 58/128) of the DSGb5 high expression group and low expression group, respectively (p = 0.031). Of 156 patients with a median follow up of 51 months, 18 patients (12%) developed metastasis following surgery. Patients in the DSGb5 high expression group showed significantly lower recurrence-free survival as compared with those in the DSGb5 low expression group (log-rank P = 0.047). In the present study, DSGb5 expression was associated with microvascular invasion in RCC tissues, and patients with DSGb5 high expression showed significantly lower recurrence-free survival rates. These findings suggest that DSGb5 expressed in RCC is correlated with metastasis and is a potential predictor for identifying patients who experience metastasis after surgery.

Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi.

Newly synthesized proteins and lipids are transported across the Golgi complex via different mechanisms whose respective roles are not completely clear. We previously identified a non-vesicular intra-Golgi transport pathway for glucosylceramide (GlcCer)--the common precursor of the different series of glycosphingolipids-that is operated by the cytosolic GlcCer-transfer protein FAPP2 (also known as PLEKHA8) (ref. 1). However, the molecular determinants of the FAPP2-mediated transfer of GlcCer from the cis-Golgi to the trans-Golgi network, as well as the physiological relevance of maintaining two parallel transport pathways of GlcCer--vesicular and non-vesicular--through the Golgi, remain poorly defined. Here, using mouse and cell models, we clarify the molecular mechanisms underlying the intra-Golgi vectorial transfer of GlcCer by FAPP2 and show that GlcCer is channelled by vesicular and non-vesicular transport to two topologically distinct glycosylation tracks in the Golgi cisternae and the trans-Golgi network, respectively. Our results indicate that the transport modality across the Golgi complex is a key determinant for the glycosylation pattern of a cargo and establish a new paradigm for the branching of the glycosphingolipid synthetic pathway.

A single point mutation in the gene encoding Gb3/CD77 synthase causes a rare inherited polyagglutination syndrome.

Rare polyagglutinable NOR erythrocytes contain three unique globoside (Gb4Cer) derivatives, NOR1, NOR(int), and NOR2, in which Gal(α1-4), GalNAc(ß1-3)Gal(α1-4), and Gal(α1-4)GalNAc(ß1-3)Gal(α1-4), respectively, are linked to the terminal GalNAc residue of Gb4Cer. NOR1 and NOR2, which both terminate with a Gal(α1-4)GalNAc- sequence, react with anti-NOR antibodies commonly present in human sera. While searching for an enzyme responsible for the biosynthesis of Gal(α1-4)GalNAc, we identified a mutation in the A4GALT gene encoding Gb3/CD77 synthase (α1,4-galactosyltransferase). Fourteen NOR-positive donors were heterozygous for the C>G mutation at position 631 of the open reading frame of the A4GALT gene, whereas 495 NOR-negative donors were homozygous for C at this position. The enzyme encoded by the mutated gene contains glutamic acid instead of glutamine at position 211 (substitution Q211E). To determine whether this mutation could change the enzyme specificity, we transfected a teratocarcinoma cell line (2102Ep) with vectors encoding the consensus Gb3/CD77 synthase and Gb3/CD77 synthase with Glu at position 211. The cellular glycolipids produced by these cells were analyzed by flow cytometry, high-performance thin-layer chromatography, enzymatic degradation, and MALDI-TOF mass spectrometry. Cells transfected with either vector expressed the P1 blood group antigen, which was absent from untransfected cells. Cells transfected with the vector encoding the Gb3/CD77 synthase with Glu at position 211 expressed both P1 and NOR antigens. Collectively, these results suggest that the C631G mutation alters the acceptor specificity of Gb3/CD77 synthase, rendering it able to catalyze synthesis of the Gal(α1-4)Gal and Gal(α1-4)GalNAc moieties.

Transmembrane BAX inhibitor motif containing (TMBIM) family proteins perturbs a trans-Golgi network enzyme, Gb3 synthase, and reduces Gb3 biosynthesis.

Globotriaosylceramide (Gb3) is a well known receptor for Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae. The expression of Gb3 also affects several diseases, including cancer metastasis and Fabry disease, which prompted us to look for factors involved in its metabolism. In the present study, we isolated two cDNAs that conferred resistance to Stx-induced cell death in HeLa cells by expression cloning: ganglioside GM3 synthase and the COOH terminus region of glutamate receptor, ionotropic, N-methyl-D-asparate-associated protein 1 (GRINA), a member of the transmembrane BAX inhibitor motif containing (TMBIM) family. Overexpression of the truncated form, named GRINA-C, and some members of the full-length TMBIM family, including FAS inhibitory molecule 2 (FAIM2), reduced Gb3, and lactosylceramide was accumulated instead. The change of glycolipid composition was restored by overexpression of Gb3 synthase, suggesting that the synthase is affected by GRINA-C and FAIM2. Interestingly, the mRNA level of Gb3 synthase was unchanged. Rather, localization of the synthase as well as TGN46, a trans-Golgi network marker, was perturbed to form punctate structures, and degradation of the synthase in lysosomes was enhanced. Furthermore, GRINA-C was associated with Gb3 synthase. These observations may demonstrate a new type of posttranscriptional regulation of glycosyltransferases.

Downregulation of alpha-galactosidase A upregulates CD77: functional impact for Fabry nephropathy.

Anderson-Fabry disease, an inherited deficiency in the lysosomal enzyme alpha-galactosidase A, is characterized by the progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. We sought to clarify the pathogenesis of Fabry disease by establishing a cell model of this disorder. The expression of alpha-galactosidase A was transiently silenced by RNA interference in HK2 and primary human renal epithelial cells and stably silenced in HK2 cells by retroviral transfection with small hairpin RNA. All of the silenced cells had histological similarities to cells of patients with Fabry disease. The cells had reduced viability, significant accumulation of intracellular Gb3, and a modest but significant increase in membranous Gb3 expression compared to nonsilenced cells. When silenced HK2 cells were reconstituted with agalsidase-alpha, a protein used for enzyme replacement therapy, they decreased their membranous CD77 expression to levels indistinguishable from those of nonsilenced cells. Because plasma and urinary Gb3 levels are not reliable biomarkers for Fabry disease, our study suggests that membranous CD77 levels mirror Gb3 tissue load and that CD77 expression levels may be used to monitor the efficacy of enzyme replacement therapy.

Escherichia coli shiga-like toxins induce apoptosis and cleavage of poly(ADP-ribose) polymerase via in vitro activation of caspases.

Shiga-like toxin-producing Escherichia coli causes hemorrhagic colitis and hemolytic-uremic syndrome in association with the production of Shiga-like toxins, which induce cell death via either necrosis or apoptosis. However, the abilities of different Shiga-like toxins to trigger apoptosis and the sequence of intracellular signaling events mediating the death of epithelial cells have not been completely defined. Fluorescent dye staining with acridine orange and ethidium bromide showed that Shiga-like toxin 1 (Stx1) induced apoptosis of HEp-2 cells in a dose- and time-dependent manner. Stx2 also induced apoptosis in a dose-dependent manner. Apoptosis induced by Stx1 (200 ng/ml) and apoptosis induced by Stx2 (200 ng/ml) were maximal following incubation with cells for 24 h (94.3% +/- 1.8% and 81.7% +/- 5.2% of the cells, respectively). Toxin-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, cell shrinkage, and the formation of apoptotic bodies, as assessed by transmission electron microscopy. Stx2c induced apoptosis weakly even at a high dose (1,000 ng/ml for 24 h; 26.7% +/- 1.3% of the cells), whereas Stx2e did not induce apoptosis of HEp-2 cells. Thin-layer chromatography confirmed that HEp-2 cells express the Stx1-Stx2-Stx2c receptor, globotriaosylceramide (Gb3), but not the Stx2e receptor, globotetraosylceramide (Gb4). Western blot analysis of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme, demonstrated that incubation with Stx1 and Stx2 induced cleavage, whereas incubation with Stx2e did not result in cleavage of PARP. A pan-caspase inhibitor (Z-VAD-FMK) and a caspase-8-specific inhibitor (Z-IETD-FMK) eliminated, in a dose-dependent fashion, the cleavage of PARP induced by Shiga-like toxins. Caspase-8 activation was confirmed by detection of cleavage of this enzyme by immunoblotting. Cleavage of caspase-9 and the proapoptotic member of the Bcl-2 family BID was also induced by Stx1, as determined by immunoblot analyses. We conclude that different Shiga-like toxins induce different degrees of apoptosis that correlates with toxin binding to the glycolipid receptor Gb3 and that caspases play an integral role in the signal transduction cascade leading to toxin-mediated programmed cell death.

Isogloboside biosynthesis in metastatic R3230AC cells results from a decreased GM3 synthase activity.

We have determined that the production of a metastasis-associated neutral glycosphingolipid, isogloboside (iGb(4)Cer, GalNAcbeta1-3Galalpha1-3Galbeta1-4Glcbeta1-O-ceramide) is associated with the loss of G(M3) synthase activity. Assays for neutral glycosphingolipid-forming glycosyltransferases in cells producing various levels of iGb(4)Cer revealed no consistent differences that could account for the difference in iGb(4)Cer biosynthesis. However, comparison of the activity of G(M3) synthase in homogenates of these two cell types revealed that cells that did not synthesize iGb(4)Cer had activity significantly greater than that of cells possessing this antigen. Furthermore, somatic cell hybrids generated using clones of the iGb(4)Cer -producing and nonproducing cell lines lacked iGb(4)Cer while possessing high levels of G(M3) synthase activity. When iGb(4)Cer-producing cells were transfected with a G(M3) synthase expression vector, all of the resultant clones were negative for iGb(4)Cer production. The results of these studies clearly show that the presence of G(M3) synthase prevents the formation of iGb(4)Cer in these cells.

Expression of globo-series gangliosides in human renal cell carcinoma.

Gangliosides have been shown to be involved in development, differentiation, oncogenesis, and cancer progression. We investigated immunohistochemical expression of globo-series gangliosides in human renal cell carcinoma (RCC) and whether their expression is related to the clinical course. The expression of globo-series gangliosides was evaluated in fresh-frozen sections of 55 primary renal tumors and 8 metastatic deposits using monoclonal antibodies RM1 and RM2, which define monosialosyl and disialosyl galactosylgloboside, respectively. The immunoreactivity of primary tumors to RM1 and/or RM2 was correlated with the clinicopathological data. Cumulative incidence of metastasis detected at initial diagnosis and during the follow-up period was significantly higher in the cases whose primary tumors were RM1/RM2-positive (RM1 and/or RM2-positive) than in the RM1/ RM2-negative (neither RM1 nor RM2-positive) cases (P < 0.05). During the follow-up period, metastasis developed in none of the RM1/RM2-negative cases which had not shown metastasis at initial diagnosis. High nuclear grade was observed only in the RM1/RM2-positive cases. The RM1/RM2-positive rate of the metastatic deposits was higher than that of the primary tumors. Furthermore, a metastatic deposit obtained from one of the cases whose primary tumors were equivocal for RM1/ RM2 was extensively stained by RM1 and RM2. These results indicate that globo-series gangliosides may be one of the biochemical indicators related to the metastatic potential of human RCC.

In vitro biosynthesis of GbOse4Cer (globoside) and GM2 ganglioside by the (1-->3) and (1-->4)-N-acetyl beta-D-galactosaminyltransferases from embryonic chicken brain. Solubilization, purification, and characterization of the transferases.

(1-->4)-N-Acetyl-beta-D-galactosaminyltransferase (GalNAcT-1) and (1-->3)-N-acetyl-beta-D-galactosaminyltransferase (GalNAcT-2), which are involved in the in vitro biosynthesis of GM2 and GbOse4Cer glycosphingolipids, respectively, have been solubilized and separated by differential detergent extraction from a membrane preparation of 19-day-old embryonic chicken brain. The separated GalNAcT-1 activity had a pH optima of 7.8-8.0, and the separated GalNAcT-2 activity a single pH optimum of 7.2. Furthermore, the partially purified GalNAcT-2 preparation catalyzed the transfer of N-acetylgalactosamine from UDP-D-[3H]GalNAc to only GbOse3Cer and nLcOse5Cer. Both GalNAcT-1 and GalNAcT-2 activities were purified to approximately 316- and 428-fold, respectively, by use of UDP-hexanolamine-Sepharose 4B affinity-column chromatography. However, the partially purified GalNAcT-1 preparation appeared to be active only with GM3, lactosylceramide, and lactotriaosylceramide. The proposed linkage of the N-acetylgalactosamine unit incorporated into GM3 is beta-D-GalpNAc-(1-->4)-GM3 from the isolation of [3H]threitol after hydrolysis of the desialylated, lead tetraacetate-treated, enzymic product, beta-D-GalpNAc-(1-->4)-beta-D-[6-3H]Galp-(1-->4)-beta-D-Glcp-(1-->1)-Cer . In addition, beta-D-GalpNAc-(1-->3)-GbOse3Cer was produced, as shown by the identification of 2,4,6-tri-O-methyl-galactose after permethylation and hydrolysis of the GalNAcT-2 enzymic product, GalpNAc-[6-3H]Galp--->Gal-->Glc-->Cer.

Brefeldin A induced inhibition of de novo globo- and neolacto-series glycolipid core chain biosynthesis in human cells. Evidence for an effect on beta 1-->4galactosyltransferase activity.

De novo synthesis of neolacto-series glycolipids has been studied in human cell lines via metabolic labeling of ceramide with [3H]serine. Intense labeling of ceramide mono- and dihexoside glycolipids occurred with labeling of progressively longer chain derivatives with increasing time. Most of the label was recovered in neutral glycolipids with about 5% of the total labeling in the ganglioside fraction. Experiments done using cell treatment with 2.5 micrograms/ml brefeldin A resulted in a stimulation in the total amount of labeling, accumulation of a neutral glycolipid identified as Lc3 due to inhibited transfer of the neolacto-series core chain terminal beta-Gal residue, and a corresponding inhibition of labeling of longer chain neutral glycolipids in all cell lines. Brefeldin A also blocked synthesis of the globo-series precursor, Gb3, longer chain sialylated structures such as IV3NeuAcnLc4, but not de novo GM3 synthesis. Brefeldin A treatment had no effect on cellular beta 1-->3N-acetylglucosaminyl-, beta 1-->4galactosyl-, or alpha 1-->3fucosyltransferase specific activities, nor was it inhibitory in beta 1-->4galactosyltransferase assays in vitro. The results describe brefeldin A-induced blocks in globo- and neolacto-series glycolipid biosynthesis, consistent with differential localization of enzymes in intracellular membranes. In particular, the results suggest that the beta 1-->4galactosyltransferase in these cells is either not redistributed by brefeldin A or is otherwise rendered nonfunctional.

Elevation of 4 beta-galactosyltransferase activity for paragloboside synthesis in sera of patients with cancer.

Galactosyltransferase activities in sera of cancer patients were determined by assaying the formation of paragloboside from UDP-galactose and lactotriaosylceramide immobilized on microtiter plates by means of the enzyme-linked immunosorbent assay using a monoclonal antibody, H-11, directed to paragloboside. Enzyme properties were as follows. Optimum pH was 6.8 in cacodylate buffer, and Km values were 2 microM for lactotriaosylceramide and 29 microM for UDP-galactose. The enzyme activity was inhibited by the addition of alpha-lactalbumin. Glucose (20 mM) inhibited the enzyme activity in the presence of alpha-lactalbumin (0.1 mg/ml) but not in its absence. These enzyme properties are similar to those of bovine milk galactosyltransferase, indicating that the enzyme in the sera might be lactose synthetase. The enzyme activities in sera from patients with cancer, patients with benign disease, or a reference sample group were assayed. The activity was below the limit of detection (5.5 pmol/25 microliters serum/2 h) in the reference sample group. Remarkable elevations of the enzyme activity were observed with high incidence in patients with cancer, especially those with blood cancer (100%). A high incidence was observed in the progressive stage, and the enzyme activity was detected at stage 1 in lung, esophagus, stomach, colorectal, and testis cancer. The enzyme activity in sera from patients with benign disease was elevated in 22% of the patients. After effective therapies, the enzyme activity decreased to below the limit of detection. Release of the galactosyltransferase into culture medium of cancer cells could be demonstrated. These observations suggest that the galactosyltransferase is released from cancer tissue into the circulation. The present method for the assay of galactosyltransferase may be useful for the detection of patients with cancer and for monitoring neoplastic recurrence after therapy.

Decreased biosynthesis of Forssman glycolipid after retinoic acid-induced differentiation of mouse F9 teratocarcinoma cells. Lectin-affinity chromatography of the glycolipid-derived oligosaccharide.

Glycolipids synthesized by the mouse teratocarcinoma F9 cells and F9 cells (RA/F9 cells) induced to differentiate by a 3-day treatment with 0.1 microM all-trans-retinoic acid were analyzed. Both F9 cells and RA/F9 cells were incubated in media containing either D-[6-3H]galactose or D-[6-3H]glucosamine; the metabolically-radiolabeled glycolipids were isolated and the oligosaccharides were released from the glycolipids by ozonolysis and alkali fragmentation. From both cells, a single major pentasaccharide was isolated from the mixture of neutral [3H]oligosaccharides by affinity chromatography on a column of immobilized Helix pomatia agglutinin. The structure of this oligosaccharide was analyzed by methylation analysis and specific exoglycosidase treatments and identified as the Forssman pentasaccharide alpha-D-GalpNAc-(1----3)-beta-D-GalpNAc-(1----4)-alpha-D-Galp-(1----4)-b eta-D- Galp-(1----4)-D-Glc. There was a 3-4-fold decreased amount of the Forssman pentasaccharide from RA/F9 cells relative to F9 cells. In contrast, there were no major differences between these cells in the levels of globoside, the precursor to Forssman glycolipid. To investigate the basis for the decline in Forssman glycolipid synthesis upon differentiation, the activity of UDP-D-Gal-NAc:GbOse4Cer alpha-(1----3)-N-acetyl-D-galactosaminyltransferase (Forssman synthase) was determined in extracts of both the F9 and RA/F9 cells. The specific activity of Forssman synthase was approximately 70% lower in differentiated relative to the nondifferentiated cells. These data demonstrated that F9 cells synthesize authentic Forssman glycolipid, and that its expression and the activity of Forssman synthase were decreased following induced cellular differentiation.

Modulation of Forssman glycosphingolipid expression by murine macrophages: coinduction with class II MHC antigen by the lymphokines IL4 and IL6.

In contrast to murine spleen M phi, resident peritoneal M phi from health mice express very little Forssman glycolipid antigen (Fo). The following experiments suggest that Fo expression by peritoneal M phi may be associated with inflammation. Balb/c and CBA/J mice were given inflammatory stimuli by i.p. injection of live BCG, thioglycollate (TG), Corynebacterium parvum (CP), proteose peptone (PP), or LPS. Control animals received pyrogen-free saline. Expression of Fo and Ia antigen by peritoneal M phi was determined by immunofluorescence after 4 d. Application of TG or CP led to an up to 30-fold increase in Fo+, Ia+ double positive M phi over that in control animals. LPS caused mainly an increase in the percentage of double-positive M phi, whereas no effects were seen in BCG or PP treated animals. To clarify the possible involvement of cytokines in this process and to identify these, the effects of LPS and various cytokines on in vitro induction of Fo and Ia expression were studied in further experiments. LPS, IL6, and IL4 caused induction of up to 15% Fo+ and Ia+ M phi after a 4 d culture period. M phi colony stimulating factor (M-CSF) from lung-conditioned medium was also moderately active. IL1, TNF, and IL2 had no influence, whereas IFN-gamma only induced Ia. For a successful in vitro induction of Fo and Ia, a prior priming of the mice with PP appeared mandatory. This suggests that only M phi of a certain developmental stage can acquire Fo under the influence of the appropriate cytokines. The data may provide the first evidence for cytokine-mediated modulation of a glycolipid antigen of known chemical structure.

Biosynthesis of sialosyllactotetraosylceramide in human colorectal carcinoma cells.

A monosialoganglioside, IV3-NeuNAcLcOse4Cer, has recently been detected in colorectal carcinoma cells, small cell lung carcinoma cells, embryonal carcinoma cells and in human brain extracts. We report here the presence of a CMP-sialic: LcOse4Cer sialyl transferase activity in subcellular membrane fractions of the human colorectal carcinoma. SW1116, which recognizes the non-reducing terminal galactosyl moiety of lactotetraosylceramide. A convenient method for structural analysis of picomolar quantities of the radioactive enzymatic product(s) using bacterial endoglycoceramidase, sialidase and a viral sialidase is presented.

Lipid composition of PC12 pheochromocytoma cells: characterization of globoside as a major neutral glycolipid.

We have studied the lipid composition of PC12 pheochromocytoma cells cultured in the presence and absence of nerve growth factor (NGF). Neutral and acidic lipid fractions were isolated by column chromatography on DEAE-Sephadex and analyzed by high-performance thin-layer chromatography (HPTLC). The total lipid concentration was approximately 220 micrograms/mg of protein, and the concentration of neutral glycolipids was 1.6-1.8 microgram/mg of protein for both NGF-treated and untreated cells. The neutral glycolipid fraction contained a major component, which accounted for approximately 80% of the total and which was characterized as globoside on the basis of HPTLC mobility, carbohydrate analysis, fast atom bombardment mass spectrometry, and mild acid hydrolysis. The major fatty acids of globoside were C16:0 (10%), C18:0 (16%), C22:0 (23%), C24:1 (17%), and C24:0 (24%). C18 sphingenine accounted for almost all of the long-chain bases. The other neutral glycolipids were tentatively identified as glucosylceramide (15%), lactosylceramide (4%), and globotriosylceramide (4.5%). The concentration of ganglioside sialic acid was approximately 0.34 and 0.18 microgram/mg of protein for cells grown in the presence and absence of NGF, respectively. Although there was an increase in ganglioside concentration in NGF-treated cells, NGF did not produce any differential effects on the relative proportions of the individual gangliosides. Several of the gangliosides appear to contain fucose, and one of these was tentatively identified as fucosyl-GM1. Brain-type gangliosides of the ganglio series were also detected by an HPTLC-immunostaining method. However, the fatty acid and long chain base compositions of PC12 cell gangliosides (and their TLC mobility) differ from those of brain gangliosides.(ABSTRACT TRUNCATED AT 250 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.