alpha1,3 Fucosyltransferase, alpha-L-fucosidase, alpha-D-galactosidase, beta-D-galactosidase, and Le(x) glycoconjugates in developing rat brain.

Fucosyltransferases (FTs) and various glycosidases that are involved in the biosynthesis or degradation of SSEA-1 (Le(x)) antigens and their precursors in the CNS are developmentally regulated. In forebrain and cerebellum with lactosamine (LacNAc) as acceptor the FT activity was maximal at P15-P22, but with the glycolipid substrate paragloboside (nLc4) the maximal activity in cerebellum was obtained at P10-P15. The FT activity, with these substrates, was insensitive to N-ethylmaleimide (NEM) and the glycolipid product had an alpha1,3 linkage (Fuc to GlcNAc) suggesting similarities of the investigated enzyme to the cloned human and rat FT IV. However, the observation of different patterns of FT activity in isoelectrofocused fractions (pH 3.5-10) with different types of acceptors, and the differential expression of Le(x) containing glycolipids and glycoproteins during development strongly suggest the presence of more than one type of FT during development. Data on developmental expression of the hydrolytic enzymes, alpha-L-fucosidase, beta-D-galactosidase and alpha-D-galactosidase, which can potentially hydrolyse SSEA-1 or its precursors, support the notion that SSEA-1 expression is the result of a dynamic balance between the activity of transferases and hydrolases.

Cloning of a rat alpha1,3-fucosyltransferase gene: a member of the fucosyltransferase IV family.

We report the cloning of a rat alpha1,3-fucosyltransferase gene (rFuc-T), isolated from a rat genomic library by a PCR-cross-hybridization based cloning approach using primers derived from the conserved region of human alpha1,3-Fuc-T sequences. Comparison of the rFuc-T predicted amino acid sequence with those of previously cloned human and murine fucosyltransferases showed highest degree of homology to murine Fuc-TIV (87% identity) and human Fuc-TIV (78% identity), with lower homology (41-49% identity) to Fuc-TIII, V, VI, and VII. COS-1 cells transfected with the rFuc-Tgene expressed a fucosyltransferase activity with type 2 (Gal beta1-->4GlcNAc)-containing oligosaccharides and the glycolipid acceptor neolactotetraosylceramide but only low activity with sialylated substrates; the SSEA-1/Le(x) antigen was detected in transfected cells by immunocytochemistry. Based on these results, we surmise that rFuc-T is a member of the fucosyltransferase IV family. Northern blot analysis with a rFuc-T specific probe indicated a major transcript of 4.2 kb most abundantly expressed in rat spleen; minor transcripts of different sizes were detected in several tissues, including rat brain.

Gangliosides in membranes from Torpedo electric organ.

The electric organ membrane has been the subject of many studies, due principally to its rich content of nicotinic acetylcholine receptor (AChR). Knowing its lipid composition is clearly important. Although its major membrane lipids have been characterized, its ganglioside composition has not been as well-described. In this study, gangliosides were characterized in membranes prepared from two species of electric organ, Torpedo californica and T. nobiliana. The ganglioside content of total electric organ membranes and AChR-enriched membranes was similar in both species, accounting for from 0.9 to 1.5% of membrane lipid by weight. However, the AChR-enriched membranes contained significantly less ganglioside relative to AChR than did the total membrane preparations. Five major gangliosides were purified from T. californica and identified as II3NeuNAc-GgOse3 (GM2); II3(NeuNAc)2-GgOse3 (GD2), IV3NeuNAc, II3NeuNAc-GgOse4 (GD1a), IV3NeuNAc, II3(NeuNAc)2-GgOse4 (GT1b), and IV3(NeuNAc)2,II3(NeuNAc)2-GgOse4 (GQ1b). Together these five gangliosides accounted for over 90% of the total ganglioside present in the two membrane preparations from both species. The most abundant ganglioside by far was GM2, which accounted for about one-half of the ganglioside content, followed by GD2. Determination of the N-fatty acid composition was performed on gangliosides purified from T. nobiliana. The lower-order gangliosides, GM2, GD2, and GD1a, contained substantial amounts of very long chain fatty acids (> 20 carbons), including alpha-hydroxynervonic acid (15-21% of total). In contrast, unsubstituted, 14-18 carbon chains accounted for about 90% of the fatty acids on the two higher-order gangliosides, GT1b and GQ1b.

Auditory discrimination reversal learning and assessment of behavioral teratogenesis in rats.

Qualitative auditory discrimination procedures were used to evaluate discrimination acquisition and reversal learning in rats. Twelve adult rats prenatally exposed to ethanol (ETOH) and 12 unexposed isocaloric controls (CON) were given training with a positively reinforced successive discrimination procedure. Most ETOH subjects were impaired relative to CON subjects on accuracy during early training sessions and the number of sessions required to meet an 80% accuracy criterion. Some ETOH subjects were also impaired on the rate of learning over a series of repeated discrimination reversals. Individual differences in reversal learning rates varied more widely with ETOH subjects than with CON subjects. Our results indicate that the auditory discrimination procedures may find application in assessments of behavioral teratogenesis.

Dexamethasone modulates rat renal brush border membrane phosphate transporter mRNA and protein abundance and glycosphingolipid composition.

Glucocorticoids are important regulators of renal phosphate transport. This study investigates the role of alterations in renal brush border membrane (BBM) sodium gradient-dependent phosphate transport (Na-Pi cotransporter) mRNA and protein abundance in the dexamethasone induced inhibition of Na-Pi cotransport in the rat. Dexamethasone administration for 4 d caused a 1.5-fold increase in the Vmax of Na-Pi cotransport (1785 +/- 119 vs. 2759 +/- 375 pmol/5 s per mg BBM protein in control, P < 0.01), which was paralleled by a 2.5-fold decrease in the abundance of Na-Pi mRNA and Na-Pi protein. There was also a 1.7-fold increase in BBM glucosylceramide content (528 +/- 63 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02). To determine whether the alteration in glucosylceramide content per se played a functional role in the decrease in Na-Pi cotransport, control rats were treated with the glucosylceramide synthase inhibitor, D-threo-1-phenyl-2-decanoyl-amino-3-morpholino-1-propanol (PDMP). The resultant 1.5-fold decrease in BBM glucosylceramide content (199 +/- 19 vs. 312 +/- 41 ng/mg BBM protein in control, P < 0.02) was associated with a 1.4-fold increase in Na-Pi cotransport activity (1422 +/- 73 vs. 1048 +/- 85 pmol/5 s per mg BBM protein in control, P < 0.01), and a 1.5-fold increase in BBM Na-Pi protein abundance. Thus, dexamethasone-induced inhibition of Na-Pi cotransport is associated with a decrease in BBM Na-Pi cotransporter abundance, and an increase in glucosylceramide. Since primary alteration in BBM glucosylceramide content per se directly and selectively modulates BBM Na-Pi cotransport activity and Na-Pi protein abundance, we propose that the increase in BBM glucosylceramide content plays an important role in mediating the inhibitory effect of dexamethasone on Na-Pi cotransport activity.

Globotriaosylceramide, Gb3, is an alternative functional receptor for Shiga-like toxin 2e.

We reexamined the binding specificity of the Shiga-like toxin variant associated with porcine edema disease, SLT2e, which is reported to be more cytotoxic for Vero cells than for HeLa cells, by using receptor-deficient cells and a liposomal insertion system for purified glycolipids. We found that SLT2e preferentially uses globotetraosylceramide as a receptor but can also cause cytotoxicity by using globotriaosylceramide, the SLT2 receptor. We conclude that the differential cytotoxicity of SLT2e on HeLa and Vero cells is a function of both the receptor preference of the toxin and the specific glycolipid content of the target cells being used.

Developmentally regulated Gb3 galactosyltransferase and alpha-galactosidase determine Shiga toxin receptors in intestine.

The receptor for Shiga toxin on rabbit intestinal microvillus membranes (MVMs) has been identified as a developmentally regulated glycolipid, globotriaosylceramide [galactose alpha 1-4 galactose beta 1-4 glucose beta 1-1 ceramide (Gb3)]. MVM Gb3 levels increase markedly in the third week of life, concomitant with fluid secretory responses to the toxin. To study mechanisms controlling developmental regulation of MVM Gb3, we measured the specific synthetic Gb3 galactosyltransferase and degradative alpha-galactosidase activities and subcellular distribution of Gb3 at various ages. Quantitative high-performance liquid chromatography demonstrated a similar developmental pattern in both microsomal and MVM Gb3, indicating that late expression of MVM Gb3 is not due to delayed migration of Gb3 from the microsomal to the MVM. The specific Gb3 galactosyltransferase activity increased with age, with a sharp increase seen at 18 days of age, whereas alpha-galactosidase activity followed an inverse pattern. Thus, regulation of both synthetic and degradative pathways for Gb3 appears to explain the observed changes in Gb3 levels with age. The nature of the signals for developmental regulation of Gb3 levels is unknown, as are the physiological consequences of altered MVM glycolipid composition other than mediating response to Shiga toxin.

Expression of glycosphingolipids in serum-free primary cultures of mouse kidney cells: male-female differences and androgen sensitivity.

The expression of neutral glycosphingolipids was examined in primary kidney cell cultures derived from adult male and female beige mutant mice (C57BL/6J;bgj/bgj) with enrichment for proximal tubule cells during preparation of explants and using defined serum-free medium for the culture conditions. Cells proliferated for 7 days in vitro to provide confluent or nearly confluent monolayers of epithelial-type growth indicative of proximal tubule cells. The male vs female differences in neutral glycosphingolipids seen in the kidney in vivo were retained in these 7 day cultures. Cultures derived from males contained galacto- and digalactosylceramides whereas those from females did not express these types of glycolipids. Also, male cells had higher ratios of sphingosine: phytosphingosine containing species in Nfa (non-hydroxy fatty acid) globotriaosylceramide and in glucosylceramide than females. The shift in sphingosine: phytosphingosine to male ratios in Nfa globotriaosylceramide and in glucosylceramide could be stimulated in female kidney cells by treatment with 10(-5) M testosterone or 5 alpha-dihydrotestosterone. The male-specific expression of neutral glycosphingolipids, then, appears to be stable character of male-type differentiation in mouse kidney that is passed on during proliferation in culture. Female kidney cells retain an ability to respond to androgens with specific changes in neutral glycosphingolipid expression during 7 days of growth in vitro in serum-free conditions, but do not respond with the induction of the male-specific glycolipids galacto- and digalactosylceramides as seen in vivo.

Pathogenesis of Shigella diarrhea: XVII. A mammalian cell membrane glycolipid, Gb3, is required but not sufficient to confer sensitivity to Shiga toxin.

Shiga toxin recognizes a galactose-alpha 1-->4-galactose terminal glycolipid, globotriaosylceramide (Gb3), in sensitive mammalian cells and is translocated by endocytosis to the cytoplasm, where it blocks protein synthesis. To determine if Gb3 is both required and sufficient for toxicity, Gb3 content in cells was altered by blocking key biosynthetic or degradative path enzymes with specific inhibitors. The resulting decrease or increase in cellular Gb3 was associated with a decrease or increase in binding of and response to Shiga toxin. Toxin-resistant Gb3-deficient variants of sensitive cells fused with liposomes containing Gb3 but not globotetraosylceramide (Gb4) became susceptible, whereas fusion of Gb3 liposomes to naturally resistant Gb3-deficient CHO cells increased toxin binding but not cytotoxicity. These data demonstrate that Gb3 is required, but not sufficient, for the action of Shiga toxin and suggest the existence of a toxin translocation mechanism linked to surface glycolipids that is not expressed in CHO cells.

The expression of H-like blood group glycolipids in small cell carcinoma of the lung.

Monoclonal antibody SM1 has been shown to be preferentially reactive with small cell carcinoma of the lung (SCCL) cell lines by fluorescent and radioimmunoassay membrane staining (1). Using solid phase indirect radioimmunoassay, the antigen is not detected in non-SCCL lung carcinomas histologically classified as squamous carcinoma, adenocarcinoma or large cell carcinoma, and other tumors, viz; pheochromocytoma, a mesoderm derived lymphoblastic leukemia cell line or in normal human brain, heart, liver, colon, endothelial tissues of the aorta and blood vessels, skin, omentum, muscle, lung parenchyma and is weakly reactive with bronchial mucosa, pancreas, and kidney. The membrane antigens detected by SM1 were isolated from small cell carcinoma of the lung (SCCL) cell line, SW2, using anion exchange chromatography and thin layer chromatography, and were further analysed by exoglycosidase and endoglycosidase treatments followed by chemical staining and immunostaining with SM1 and other antibodies. We show here that SM1 antibody reacts with a group of fucose-containing neutral glycolipids and gangliosides many of which are cross-reactive with antibodies to H antigens.

The accumulation and metabolism of glycosphingolipids in primary kidney cell cultures from beige mice.

In the normal C57BL/6J male mouse a specific subset of the kidney glycosphingolipids which is associated with multilamellar bodies of lysosomal origin and represents about 10% of the total kidney glycolipids, is excreted into the urine each day. This excretion is blocked and glycosphingolipids accumulate in the kidney of bgJ/bgJ mutants of this strain. To examine this process in vitro, glycosphingolipid metabolism and excretion were studied in beige mouse kidney cell cultures. Primary kidney cell cultures from male C57BL/6J control and bgJ/bgJ beige mutants were grown in D-valine medium and glycosphingolipids labeled with [3H]palmitate. As we have shown previously, the giant lysosomes of altered morphology were maintained in cultures of the beige kidney cells. Beige-J and control cells synthesized the same types of glycosphingolipids, but the mutant cells had quantitatively higher levels of these compounds than control cells, as determined by high performance liquid chromatography. Beige-J cells incorporated more [3H]palmitate into glycosphingholipids than control cells on a cpm/mg protein basis and the specific activity (cpm/pmole glycosphingolipid) was lower in beige cells. Medium from beige-J cells accumulated more glycosphingolipids than that from control cells in a 24 h period. The glycosphingolipids released into the medium as determined by HPLC were primarily non-lysosomal types and both control and mutant cells retained the glycosphingolipids associated with lysosomal multilamellar bodies excreted in vivo. The elevated levels of lysosomal glycosphingolipids and the dysmorphic lysosomes in primary cultures of beige cells, then, are not caused by a mutant block in secretion of lysosomes.

Detailed histopathological examination contributes to the assessment of Escherichia coli urovirulence.

To determine the usefulness of detailed histopathological evaluation in the assessment of urovirulence of different Escherichia coli strains in a mouse model of ascending, unobstructed urinary tract infection, and to evaluate the relationship between susceptibility to urinary tract infection and renal levels of P fimbrial receptor glycolipids in different mouse strains, female Swiss Webster and Balb/c mice were inoculated transurethrally with one of four different well-characterized wild type E. coli strains or with an E. coli K-12 laboratory strain, and renal glycolipid levels were determined for both mouse strains. In Swiss Webster mice, each of the wild type E. coli strains was more virulent than the laboratory strain by both microbiological and histopathological criteria. Despite the common origin and identical virulence factor profiles of the three urosepsis isolates studied, one was significantly less urovirulent than the other two. Paradoxically, this strain stimulated a greater degree of leukocytosis than did the more urovirulent strains. Balb/c mice were significantly more susceptible to infection with this strain than were Swiss Webster mice, a difference possibly contributed to by the significantly higher renal levels of P fimbrial receptor glycolipids in Balb/c mice. Detailed histopathological analysis revealed significant differences in virulence between bacterial strains, and in susceptibility to infection between different mouse strains, that were inapparent from culture results alone.

Rapid isolation of monosialogangliosides from bovine brain gangliosides by selective-overload chromatography.

A procedure for rapid isolation of monosialogangliosides from purified bovine brain gangliosides has been developed. It utilizes the selective difference in association between monosialogangliosides and polysialogangliosides for the ion-exchange resin Q-Sepharose. When the ion-exchange column is overloaded with a bovine brain ganglioside mixture in the proper ganglioside to column bed-volume ratio, the polysialogangliosides are selectively retained by the column while the monosialogangliosides emerge with the void volume without the use of salt for elution. With the critical ganglioside to bed-volume ratio (1 g:8.32 ml), and an appropriate column bed-height to column radius ratio of 6.9, monosialogangliosides are reproducibly obtained in high purity with greater than 90% yield. The method has been used at both the analytical and preparative scale. We call this separation technique selective-overload chromatography.

Lipid composition of lysosomal multilamellar bodies of male mouse urine.

Previous studies from our laboratory have shown that male C57BL/6J mice excrete into the urine multilamellar lysosomal bodies that contain specific neutral glycosphingolipids. These mice excrete approximately 20-30% of their kidney glycolipids each day. The significance and function of this secretion of multilamellar lysosomal organelles is unknown. To characterize these excreted bodies further, we report here their neutral lipid and phospholipid composition. The bodies were collected by differential centrifugation, extracted with chloroform-methanol, and lipids were fractionated into neutral lipids, glycolipids, and phospholipids. The neutral lipids consisted primarily of cholesterol, dolichol, and ubiquinone. The phospholipid fraction consisted primarily of a single molecular species of phosphatidylcholine. This lipid which comprises more than 90% of the total phospholipids was found to contain 16:0 ether and C22:6 n-3 fatty acid as determined by gas-liquid chromatography-mass spectrometry. The glycosphingolipids as reported previously consisted primarily of galabiosylceramides and globotriaosylceramides. This membrane lipid composition is different from any previously reported cellular organelle.

UDP-galactose:globoside galactosyltransferase in murine kidney.

There are increased levels of stage-specific embryonic antigens-3 and -1 (SSEA-3 and SSEA-1) globo-series glycolipids in male versus female DBA/2 and C57BL/6 kidneys, respectively. To determine what enzymatic steps may be responsible for these differences, the activity and properties of UDP-galactose:globoside galactosyltransferase were studied in male and female mouse kidney microsomes. This enzyme participates in the biosynthesis of galactosylgloboside, SSEA-3 glycolipid; the reaction product was identified by high performance thin-layer chromatography (HPTLC) immunostaining. In C57BL/6 mice, the specific activity of the enzyme, in the presence of CHAPS, was 2-fold greater in the male than that in the female. Optimum pH for the enzyme from both sexes was about 5.6, and Mn2+ was essential for maximal activity. Fifty percent of the male and female enzyme activity was lost after preincubating the microsomes for 1 min at 55 degrees C; thereafter, the enzyme from female microsomes had a slower rate of denaturation. The Km for globoside in presence of sodium cholate for both male and female was 0.035 mM, but it was approximately 2-fold greater for the female in presence of CHAPS. The enzyme in male and female microsomes was differentially activated by CHAPS and cholate. The results suggest the presence of an enzyme modulator in these membranes. In DBA/2 mice, the enzyme activity was about 2-fold greater in males than that in the female. The specific activity of the enzyme in the two strains was of a similar magnitude.(ABSTRACT TRUNCATED AT 250 WORDS)

Fucosylated oligosaccharides of human milk protect suckling mice from heat-stabile enterotoxin of Escherichia coli.

Human milk protects suckling mice from the diarrheagenic effects of heat-stabile enterotoxin of Escherichia coli (ST). To identify the human milk fraction responsible for this protection, pooled skimmed, deproteinated milk was passed through charcoal, whereupon lactose was separated from the oligosaccharides. The oligosaccharides contained ST-protective activity; the lactose did not. The neutral, but not the acidic, fraction exhibited protective activity against ST (22% vs. 57% mortality, respectively; P less than .001). The fucosylated, but not the nonfucosylated, subfractions of the neutral fraction contained the factor protective against ST (35% vs. 50% mortality, respectively; P less than .05). An oligosaccharide isolation scheme based on different principles produced confirmatory results. The commercially available neutral fucosylated oligosaccharides of human milk did not significantly protect the mice from the effects of ST. Thus, the protective factor against ST seems to be a minor neutral fucosyloligosaccharide of human milk.

Characterization of porcine omental lipids.

Porcine omental lipid extracts were fractionated and the major lipid components characterized. Approximately 97% of the chloroform/methanol extract consisted of triglycerides containing primarily 16:0, 18:0, 18:1, and 18:2 fatty acids. Small quantities of free fatty acids, cholesterol, di- and monoglycerides were also detected. The phospholipid fraction, obtained by solvent partition and Unisil column chromatography and characterized by high performance liquid chromatography (HPLC) and HPLC-mass spectrometry, was found to consist primarily of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. The neutral glycolipids, isolated by solvent partition and Unisil column chromatography and identified by HPTLC and HPLC, were found to consist primarily of di-, tri- and tetraosylceramides. The complex glycolipid fraction, obtained from Folch upper phase solvent partition and characterized by HPTLC and immunoblotting, was found to consist primarily of ganglio-, globo-, and neolacto- neutral glycolipids and ganglio-, globo-, neolacto- and fucosylated gangliosides.

Quantitation of the rabbit intestinal glycolipid receptor for Shiga toxin. Further evidence for the developmental regulation of globotriaosylceramide in microvillus membranes.

Shiga toxin, produced by Shigella dysenteriae 1, causes enterotoxic, cytotoxic, and neurotoxic effects, which may be mediated by a glycolipid receptor, globotriaosylceramide, Gb3. To study the relationship of this receptor and toxin effects, globotriaosylceramide was quantitated and further characterized in rabbit small intestinal microvillus membranes at various ages. Glycolipids were extracted from rabbit microvillus membranes, purified on Unisil columns, and quantitated by high-performance liquid chromatography. The major glycolipid peaks were hydroxylated fatty acid-containing glucosylceramide, lactosylceramide, and globotriaosylceramide. There was a marked increase of globotriaosylceramide levels with age, ranging from 0.02 to 16.2 pmol/micrograms microvillus membrane protein in neonates and adults, respectively. The globotriaosylceramide peak was susceptible to alpha-galactosidase treatment, which produced an elevation in the lactosylceramide peak, but markedly reduced globotriaosylceramide content in 34-day-old rabbits. Binding of iodinated Shiga toxin to globotriaosylceramide was documented on high-performance thin-layer chromatography plates by autoradiography. The glycolipid receptor for Shiga toxin in rabbit microvillus membranes is thus a hydroxylated fatty acid-containing globotriaosylceramide. This moiety is virtually absent in neonates and gradually increases with age. Quantitative differences in globotriaosylceramide may be the underlying basis for the age-specific differences in functional responsiveness of rabbit intestinal tissue to Shiga toxin.

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis.

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le(x)(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.