Gb3 Levels Research Articles

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.

Susceptibility to hemolytic-uremic syndrome relates to erythrocyte glycosphingolipid patterns.

Hemolytic-uremic syndrome (HUS) is usually preceded by enteric infection by Shiga-like toxin-producing Escherichia coli (SLT-EC), but most children with SLT-EC diarrhea do not develop HUS. SLT toxicity depends on entry into the target cell via its host cell glycolipid receptor, globotriaosylceramide (Gb3). The relationship between differential susceptibility to HUS and erythrocyte Gb3 levels, as measured by high-pressure liquid chromatography, was studied. Erythrocytes of children with histories of HUS had lower nonhydroxylated fatty acyl (NFA) Gb3 levels than did erythrocytes of controls (1.6 vs. 2.0 nmol/mL of packed cells); these erythrocytes had lower ratios of NFA-Gb3 to lactosylceramide (0.16) than did erythrocytes of SLT-EC diarrheal patients without subsequent HUS (0.30; P < .003) or of healthy controls (0.28; P < .001). The lower erythrocyte Gb3 levels associated with HUS may reflect a genetic predisposition for differential outcomes of SLT-EC gastroenteritis.

Endothelial heterogeneity in Shiga toxin receptors and responses

This study addresses the basis for regional microvascular susceptibility to bacterial toxins implicated in hemolytic uremic syndrome. The results indicate a relationship between the degree of Shiga toxin sensitivity of human endothelial cells from different sources and the amount of globotriaosylceramide (Gb3) glycosphingolipid receptor for Shiga toxin expressed by these cells. Cell viability and protein synthesis of renal endothelial cells were reduced to 50% by 1 pM Shiga toxin, while umbilical vein cells were not affected by > 1 nM toxin. Similarly, basal levels of Gb3 were approximately 50 times higher in renal endothelial cells than in the umbilical endothelial cells. Pre-exposure of umbilical endothelial cells to tumor necrosis factor-alpha or bacterial lipopolysaccharide increased Gb3 content 4-6-fold coincident with increases in sensitivity to cytotoxic and protein synthesis inhibitory effects of Shiga toxin. Lipopolysaccharide induction of both Gb3 and sensitivity to Shiga toxin cytotoxic action in umbilical endothelial cells was dependent on the structure of lipopolysaccharide. Neither tumor necrosis factor-alpha nor lipopolysaccharide altered the Shiga toxin sensitivity or the Gb3 content of renal endothelial cells. These data indicate that differential endothelial expression of glycolipid receptors for Shiga toxins may be responsible for localized involvement of the kidney in hemolytic uremic syndrome.

Alteration of the glycolipid binding specificity of the pig edema toxin from globotetraosyl to globotriaosyl ceramide alters in vivo tissue targetting and results in a verotoxin 1-like disease in pigs.

All members of the verotoxin (VT) family specifically recognize globo-series glycolipids on the surface of susceptible cells. Those toxins that are associated with human disease, VT1, VT2, and VT2c, bind to globotriaosyl ceramide (Gb3) while VT2e, which is associated with edema disease of swine, binds preferentially to globotetraosyl ceramide (Gb4). We were recently able to identify, using site-directed mutagenesis, amino acids in the binding subunit of these toxins that are important in defining their glycosphingolipid (GSL) binding specificity (Tyrrell, G. J., K. Ramotar, B. Boyd, B. W. Toye, C. A. Lingwood, and J. L. Brunton. 1992. Proc. Natl. Acad. Sci. USA. 89:524). The concomitant mutation of Gln64 and Lys66 in the VT2e binding subunit to the corresponding residues (Glu and Gln, respectively) found in VT2 effectively converted the GSL binding specificity of the mutant toxin from Gb4 to Gb3 in vitro. We now report that the altered carbohydrate recognition of the mutant toxin (termed GT3) has biological significance, resulting in a unique disease after intravascular injection into pigs as compared with classical VT2e-induced edema disease. The tissue localization of radiolabeled GT3 after intravascular injection was elevated in neural tissues compared with VT2e accumulation, while localization of GT3 to the gastrointestinal tract was relatively reduced. Accordingly, the pathological lesions after challenge with GT3 involved gross edema of the cerebrum, cerebellum, and brain stem, while purified VT2e caused hemorrhage and edema of the cerebellum, and submucosa of the stomach and large intestine. In addition, both radiolabeled toxins bound extensively to tissues not directly involved in the pathology of disease. VT2e, unlike GT3 or VT1, bound extensively to red cells, which have high levels of Gb4. The overall tissue distribution of VT2e was thus found to be influenced by regional blood flow to each organ and not solely by the Gb4 levels of these tissues. Conversely, the distribution of GT3 (and VT1), which cleared more rapidly from the circulation, correlated with respective tissue Gb3 levels rather than blood flow. These studies indicate the primary role of carbohydrate binding specificity in determining systemic pathology, suggest that the red cells act as a toxin carrier in edema disease, and indicate that red cell binding does not protect against the pathology of systemic verotoxemia.

Biochemical and clinical analysis of accumulated glycolipids in symptomatic heterozygotes of angiokeratoma corporis diffusum (Fabry's disease) in comparison with hemizygotes.

Angiokeratoma corporis diffusum (Fabry's disease) is an X-linked disorder of glycosphingolipid catabolism. Heterozygous females, although usually asymptomatic, are occasionally as severely afflicted as hemizygous males; recently we identified a heterozygous patient with cardiomyopathy and severe pain in the extremities. In order to elucidate the difference of the clinical features, we analyzed the glycolipid composition of the heart, liver, and kidney obtained from the patient and from a hemizygote. Gas-liquid chromatography revealed that globotriaosylceramide (Gb3) was markedly increased in the heart (32.4 times higher than control) and increased to a lesser extent in the liver and kidney (3.74 and 6.79 times, respectively). The pattern of Gb3 accumulation in the heterozygote, where the highest increases were seen in the heart, was distinct from that in the hemizygote, where elevated levels of Gb3 and Ga2 were found in the kidney. Furthermore, the alpha-galactosidase activity in the heart, liver, and kidney of the heterozygote was 17%, 26%, and 36%, respectively, of normal controls, which correlated well with the accumulation of glycosphingolipid in the heart and with the disease's clinical manifestations. Two other hemizygotic patients, who were identified by low alpha-galactosidase activities, demonstrated the cardiac involvement.