Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry.

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, Pk) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galß1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

CD77 levels over enzyme replacement treatment in Fabry Disease Family (V269M) / Níveis de CD77 ao longo do tratamento de reposição enzimática em família com Doença de Fabry (V269M)

ABSTRACT Introduction: Fabry disease (FD) is a disorder caused by mutations in the gene encoding for lysosomal enzyme α-galactosidase A (α-GAL). Reduced α-GAL activity leads to progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. The recent report of increased expression of CD77 in blood cells of patients with FD indicated that this molecule can be used as a potential marker for monitoring enzyme replacement therapy (ERT). Objective: The purpose of this study was to evaluate the CD77 levels throughout ERT in FD patients (V269M mutation). Methods: We evaluated the fluctuations in PBMC (peripheral blood mononuclear cell) membrane CD77 expression in FD patients undergoing ERT and correlated these levels with those observed in different cell types. Results: A greater CD77 expression was found in phagocytes of patients compared to controls at baseline. Interestingly, the variability in CD77 levels is larger in patients at baseline (340 - 1619 MIF) and after 12 months of ERT (240 - 530 MIF) compared with the control group (131 - 331 MFI). Furthermore, by analyzing the levels of CD77 in phagocytes from patients throughout ERT, we found a constant decrease in CD77 levels. Conclusion: The increased CD77 levels in the phagocytes of Fabry carriers together with the decrease in CD77 levels throughout ERT suggest that measuring CD77 levels in phagocytes is a promising tool for monitoring the response to ERT in FD.

RESUMO Introdução: A doença de Fabry (DF) é um distúrbio causado por mutações no gene que codifica a enzima lisossômica α-galactosidase A (α-GAL). A redução da atividade de α-GAL leva ao acúmulo progressivo de globotriaosilceramida (Gb3), também conhecida como CD77. O recente relato de aumento da expressão de CD77 em células sanguíneas de pacientes com DF indicou que essa molécula pode ser utilizada como um potencial marcador para o monitoramento da terapia de reposição enzimática (TRE). Objetivo: O objetivo deste estudo foi avaliar os níveis de CD77 ao longo da TRE em pacientes com DF (mutação V269M). Métodos: Foram avaliadas as flutuações na expressão de CD77 nas membranas das CMSP (células mononucleares do sangue periférico) em pacientes com DF submetidos à TRE e correlacionados com aqueles observados em diferentes tipos de células. Resultados: Uma maior expressão de CD77 foi encontrada em fagócitos de pacientes em comparação aos controles no início do estudo. Curiosamente, a variabilidade nos níveis de CD77 é maior em pacientes no início do estudo (340 - 1619 MIF) e após 12 meses de TRE (240 - 530 MIF) em comparação com o grupo controle (131 - 331 MFI). Além disso, analisando os níveis de CD77 em fagócitos de pacientes ao longo da TRE, encontramos uma diminuição constante nos níveis de CD77. Conclusão: O aumento nos níveis de CD77 nos fagócitos de portadores de Fabry, juntamente com a diminuição nos níveis de CD77 ao longo da TRE, sugerem que medir os níveis de CD77 nos fagócitos é uma ferramenta promissora para monitorar a resposta à TRE na DF.

Gal epitope expression and immunological properties in iGb3S deficient mice.

The Gal antigen is synthesized by glycoprotein galactosyltransferase alpha 1, 3 (GGTA1) or (and) isoglobotrihexosylceramide 3 synthase (iGb3S). However, whether iGb3S deletion changes Gal epitope expression and immunological properties in animals is still not clear. The objective of this study was to develop iGb3S deficient mice, and characterize their Gal epitope expression and Gal epitope-related immunological properties. iGb3S gene knockout mice were generated on the C57BL/6 background using the bacterial artificial chromosome homology region recombination technique. Gal epitope expression in the iGb3S deficient mice was determined by using a monoclonal anti-Gal antibody. Immunological properties were analyzed by enzyme linked immune sorbent assay. It was found that Gal epitope expression was decreased from 5.19% to 21.74% in the main organs of iGb3S deficient mice, compared with that of C57BL/6 wild type mice, suggesting that the iGb3S gene participated to Gal epitope expression. However, iGb3S deletion alone did not cause significant changes in the immunological properties of iGb3S deficient mice with or without exogenous Gal antigen (Rabbit Red Blood Cell) stimulation. The data from this study suggest that the iGb3S gene likely contributes to Gal epitope expression, but may have a very weak effect on immunological properties of the iGb3S deficient mice.

CRISPR Screen Reveals that EHEC's T3SS and Shiga Toxin Rely on Shared Host Factors for Infection.

Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors-a type III secretion system (T3SS) and Shiga toxins (Stxs)-that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats with Cas9) loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were also targeted. Mutations in these loci not only rescued cells from Stx-mediated cell death, but also prevented cytotoxicity associated with the EHEC T3SS. These mutations interfered with early events associated with T3SS and Stx pathogenicity, markedly reducing entry of T3SS effectors into host cells and binding of Stx. The convergence of Stx and T3SS onto overlapping host targets provides guidance for design of new host-directed therapeutic agents to counter EHEC infection.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors-a type III secretion system (T3SS) and Shiga toxins (Stxs)-that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3) and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogen's key virulence factors.

CD77 levels over enzyme replacement treatment in Fabry Disease Family (V269M).

INTRODUCTION: Fabry disease (FD) is a disorder caused by mutations in the gene encoding for lysosomal enzyme α-galactosidase A (α-GAL). Reduced α-GAL activity leads to progressive accumulation of globotriaosylceramide (Gb3), also known as CD77. The recent report of increased expression of CD77 in blood cells of patients with FD indicated that this molecule can be used as a potential marker for monitoring enzyme replacement therapy (ERT). OBJECTIVE: The purpose of this study was to evaluate the CD77 levels throughout ERT in FD patients (V269M mutation). METHODS: We evaluated the fluctuations in PBMC (peripheral blood mononuclear cell) membrane CD77 expression in FD patients undergoing ERT and correlated these levels with those observed in different cell types. RESULTS: A greater CD77 expression was found in phagocytes of patients compared to controls at baseline. Interestingly, the variability in CD77 levels is larger in patients at baseline (340 - 1619 MIF) and after 12 months of ERT (240 - 530 MIF) compared with the control group (131 - 331 MFI). Furthermore, by analyzing the levels of CD77 in phagocytes from patients throughout ERT, we found a constant decrease in CD77 levels. CONCLUSION: The increased CD77 levels in the phagocytes of Fabry carriers together with the decrease in CD77 levels throughout ERT suggest that measuring CD77 levels in phagocytes is a promising tool for monitoring the response to ERT in FD.

Shiga Toxins Activate the NLRP3 Inflammasome Pathway To Promote Both Production of the Proinflammatory Cytokine Interleukin-1ß and Apoptotic Cell Death.

Shiga toxin (Stx)-mediated immune responses, including the production of the proinflammatory cytokines tumor necrosis-α (TNF-α) and interleukin-1ß (IL-1ß), may exacerbate vascular damage and accelerate lethality. However, the immune signaling pathway activated in response to Stx is not well understood. Here, we demonstrate that enzymatically active Stx, which leads to ribotoxic stress, triggers NLRP3 inflammasome-dependent caspase-1 activation and IL-1ß secretion in differentiated macrophage-like THP-1 (D-THP-1) cells. The treatment of cells with a chemical inhibitor of glycosphingolipid biosynthesis, which suppresses the expression of the Stx receptor globotriaosylceramide and subsequent endocytosis of the toxin, substantially blocked activation of the NLRP3 inflammasome and processing of caspase-1 and IL-1ß. Processing and release of both caspase-1 and IL-1ß were significantly reduced or abolished in Stx-intoxicated D-THP-1 cells in which the expression of NLRP3 or ASC was stably knocked down. Furthermore, Stx mediated the activation of caspases involved in apoptosis in an NLRP3- or ASC-dependent manner. In Stx-intoxicated cells, the NLRP3 inflammasome triggered the activation of caspase-8/3, leading to the initiation of apoptosis, in addition to caspase-1-dependent pyroptotic cell death. Taken together, these results suggest that Stxs trigger the NLRP3 inflammasome pathway to release proinflammatory IL-1ß as well as to promote apoptotic cell death.

Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease.

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed.

A symptomatic Fabry disease mouse model generated by inducing globotriaosylceramide synthesis.

Fabry disease is a lysosomal storage disorder in which neutral glycosphingolipids, predominantly Gb3 (globotriaosylceramide), accumulate due to deficient α-Gal A (α-galactosidase A) activity. The GLAko (α-Gal A-knockout) mouse has been used as a model for Fabry disease, but it does not have any symptomatic abnormalities. In the present study, we generated a symptomatic mouse model (G3Stg/GLAko) by cross-breeding GLAko mice with transgenic mice expressing human Gb3 synthase. G3Stg/GLAko mice had high Gb3 levels in major organs, and their serum Gb3 level at 5-25 weeks of age was 6-10-fold higher than that in GLAko mice of the same age. G3Stg/GLAko mice showed progressive renal impairment, with albuminuria at 3 weeks of age, decreased urine osmolality at 5 weeks, polyuria at 10 weeks and increased blood urea nitrogen at 15 weeks. The urine volume and urinary albumin concentration were significantly reduced in the G3Stg/GLAko mice when human recombinant α-Gal A was administered intravenously. These data suggest that Gb3 accumulation is a primary pathogenic factor in the symptomatic phenotype of G3Stg/GLAko mice, and that this mouse line is suitable for studying the pathogenesis of Fabry disease and for preclinical studies of candidate therapies.

Large scale enzymatic synthesis of oligosaccharides and a novel purification process.

Herein we report the practical chemo enzymatic synthesis of trisaccharide and derivatives of iGb3 and Gb3, and a novel purification process using immobilized yeast to remove the monosaccharide from the reaction mixture. High purity oligosaccharide compounds were achieved in large scale. This study represents a facile enzymatic synthesis of and novel purification process of oligosaccharide.

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.

A novel, promoter-based, target-specific assay identifies 2-deoxy-D-glucose as an inhibitor of globotriaosylceramide biosynthesis.

Abnormal biosynthesis of globotriaosylceramide (Gb3) is known to be associated with Gb3-related diseases, such as Fabry disease. The Gb3 synthase gene (Gb3S) codes for alpha1,4-galactosyltransferase, which is a key enzyme involved in Gb3 biosynthesis in vivo. Transcriptional repression of Gb3S is a way to control Gb3 biosynthesis and may be a suitable target for the treatment of Gb3-related diseases. To find a transcriptional inhibitor for Gb3S, we developed a convenient cell-based chemical screening assay system by constructing a fusion gene construct of the human Gb3S promoter and a secreted luciferase as reporter. Using this assay, we identified 2-deoxy-D-glucose as a potent inhibitor for the Gb3S promoter. In cultured cells, 2-deoxy-D-glucose markedly reduced endogenous Gb3S mRNA levels, resulting in a reduction in cellular Gb3 content and a corresponding accumulation of the precursor lactosylceramide. Moreover, cytokine-induced expression of Gb3 on the cell surface of endothelial cells, which is closely related to the onset of hemolytic uremic syndrome in O157-infected patients, was also suppressed by 2-deoxy-D-glucose treatment. These results indicate that 2-deoxy-D-glucose can control Gb3 biosynthesis through the inhibition of Gb3S transcription. Furthermore, we demonstrated the general utility of our novel screening assay for the identification of new inhibitors of glycosphingolipid biosynthesis.

Globotriaosylceramide-expressing Burkitt's lymphoma cells are committed to early apoptotic status by rhamnose-binding lectin from catfish eggs.

Silurus asotus (catfish) egg lectin (SAL) has a strong affinity to Gal alpha-linked carbohydrate chains of not only glycoproteins but also glycosphingolipids such as globotriaosylceramide (Gb3). SAL uniformly bound to surfaces of Gb3-expressing (Gb3+) Burkitt's lymphoma cells, while Gb3 molecules were interspersed on the surfaces of Gb3+ cells. After a short period of treating Raji and Daudi cells with SAL, each cell size was 10 and 25% smaller than that of untreated cells, respectively. Treatment of Gb3+ cells with SAL caused an increase in binding of annexin V, however, neither caspase activation nor DNA fragmentation was observed after treatment with SAL for 22 h. Since SAL did not induce cell death in Gb3+ cells, SAL may function as an inducer of early apoptotic signal. We have revealed that SAL did not bind to D-threo-1-phenyl-2-decanoylamino-3-morphorino-1-propanol (D-PDMP)-treated Raji cells, and no cell shrinkage was observed in Gb3-deficient Raji cells treated with SAL, indicating that Gb3 localized in the glycosphingolipid-enriched microdomain (GEM) was involved in SAL-induced cell shrinkage through activation of voltage-gated potassium channel Kv1.3, and that the glycoprotein ligands on Gb3-deficient Raji cells treated with SAL were not included in this phenomenon. These results suggest that SAL leads the cells to early apoptotic status via binding to Gb3 existing in GEM, and that this binding is a prerequisite condition to induce early stage of apoptosis.

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.

Enzyme replacement therapy stabilizes obstructive pulmonary Fabry disease associated with respiratory globotriaosylceramide storage.

Fabry disease is an X-linked glycosphingolipidosis caused by a deficiency of α-galactosidase A, a lysosomal enzyme. Symptoms in hemizygous males and heterozygous females are due to lysosomal storage of globotriaosylceramide in the central and peripheral nervous system, vascular endothelium, cardiac valves and myocytes, gastrointestinal tract, and renal epithelium. Pulmonary involvement is also a recognized manifestation of Fabry disease, but histopathological evidence of pulmonary lysosomal storage is scant. We report a 51-year-old woman with a G43R α-galactosidase A mutation and normal spirometry testing 2.5 years prior to presentation, who experienced a dry, nonproductive cough that persisted despite treatment with antibiotics and bronchodilators. Spirometry demonstrated a mixed restrictive/obstructive pattern as well as impaired gas exchange. Patchy ground-glass pulmonary interstitial infiltrates were found on plain radiography and computerized tomography. She underwent an open lung biopsy that demonstrated peribronchiolar fibrosis and smooth-muscle hyperplasia. Prominent inclusion bodies of the bronchiolar/arteriolar smooth muscle and endothelium were present. Electron microscopy indicated the inclusion bodies were lamellated zebra bodies consistent with globotriaosylceramide storage. Enzyme replacement therapy (ERT) with agalsidase-beta was instituted. Since initiation of therapy, she occasionally has a dry cough but markers of obstructive lung disease have remained stable in the past 4 years. This report demonstrates that pulmonary involvement in Fabry disease is due to lysosomal storage, and suggests that ERT is capable of stabilizing pulmonary Fabry disease. However, progressive worsening of her total lung capacity indicates that ERT cannot reverse the ongoing process of fibrosis also seen in Fabry disease.

Epithelial and mesenchymal cells in the bovine colonic mucosa differ in their responsiveness to Escherichia coli Shiga toxin 1.

Bovine colonic crypt cells express CD77 molecules that potentially act as receptors for Shiga toxins (Stx). The implication of this finding for the intestinal colonization of cattle by human pathogenic Stx-producing Escherichia coli (STEC) remains undefined. We used flow cytometric and real-time PCR analyses of primary cultures of colonic crypt cells to evaluate cell viability, CD77 expression, and gene transcription in the presence and absence of purified Stx1. A subset of cultured epithelial cells had Stx receptors which were located mainly intracellularly, with a perinuclear distribution, and were resistant to Stx1-induced apoptosis and Stx1 effects on chemokine expression patterns. In contrast, a population of vimentin-positive cells, i.e., mesenchymal/nonepithelial cells that had high numbers of Stx receptors on their surface, was depleted from the cultures by Stx1. In situ, CD77(+) cells were located in the lamina propria of the bovine colon by using immunofluorescence staining. A newly established vimentin-positive crypt cell line with high CD77 expression resisted the cytolethal effect of Stx1 but responded to Stx1 with a significant increase in interleukin-8 (IL-8), GRO-alpha, MCP-1, and RANTES mRNA. Combined stimulation with lipopolysaccharide and Stx1 increased IL-10 mRNA. Our results show that bovine colonic crypt cells of epithelial origin are resistant to both the cytotoxic and modulatory effects of Stx1. In contrast, some mucosal mesenchymal cells, preliminarily characterized as mucosal macrophages, are Stx1-responsive cells that may participate in the interaction of STEC with the bovine intestinal mucosa.

Human colorectal tumors and metastases express Gb3 and can be targeted by an intestinal pathogen-based delivery tool.

The targeting of solid tumors requires delivery tools that resist intracellular and extracellular inactivation, and that are taken up specifically by tumor cells. We have shown previously that the recombinant nontoxic B-subunit of Shiga toxin (STxB) can serve as a delivery tool to target digestive tumors in animal models. The aim of this study was to expand these experiments to human colorectal cancer. Tissue samples of normal colon, benign adenomas, colorectal carcinomas, and liver metastases from 111 patients were obtained for the quantification of the expression of the cellular STxB receptor, the glycosphingolipid globotriaosyl ceramide (Gb(3) or CD77). We found that compared with normal tissue, the expression of Gb(3) was strongly increased in colorectal adenocarcinomas and their metastases, but not in benign adenomas. Short-term primary cultures were prepared from samples of 43 patients, and STxB uptake was studied by immunofluorescence microscopy. Of a given tumor sample, on average, 80% of the cells could visibly bind STxB, and upon incubation at 37 degrees C, STxB was transported to the Golgi apparatus, following the retrograde route. This STxB-specific intracellular targeting allows the molecule to avoid recycling and degradation, and STxB could consequently be detected on tumor cells even 5 days after initial uptake. In conclusion, the targeting properties of STxB could be diverted for the delivery of contrast agents to human colorectal tumors and their metastases, whose early detection and specific targeting remains one of the principal challenges in oncology.

Alteration of gene expression induced by Silurus asotus lectin in Burkitt's lymphoma cells.

Silurus asotus lectin (SAL) is a member of the rhamnose-binding lectin (RBL) family, and recognizes globotriaosylceramide (Gb3) on the cell surface of Burkitt's lymphoma cell lines, such as Raji and Daudi cells. The variation of gene expression in the treatment of both cells with SAL was analyzed using the differential display (DD) method with combination of 16 kinds of arbitary primers and 3 kinds of anchor primers. Treatment of Raji cells with SAL down-regulated mitochondria-associated granulocyte-macropharge colony-stimulating factor (GM-CSF) signaling molecule (Magmas) gene, and up-regulated N-myc downstream regulated gene (NDRG) 3. On the other hand, treatment of Daudi cells with SAL down-regulated Rad50 gene. Since Magmas gene expression was repressed in SAL-treated Raji cells, but did not change in SAL-treated D-threo-1-phenyl-2-decanoylamino-3-morphorino-1-propanol (PDMP)-pretreated Raji cells, it was clear that the expression of Magmas, NDRG3 or Rad50 was regulated by SAL binding to Gb3.

Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog.

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.

Characteristic expression of Lewis-antigenic glycolipids in human ovarian carcinoma-derived cells with anticancer drug-resistance.

By comparing ovarian carcinoma-derived KF28 cells with the corresponding anticancer drug-resistant cells, the taxol- and cisplatin-resistant properties were found to be closely related with MDR1 and BSEP, and MRP2 transporters, respectively. In addition to the transporters expression, the amounts of glycolipids, particularly their longer carbohydrate structures, in the resistant cells increased to 3-4-fold of those in the sensitive cells due to enhanced transcription of the respective glycosyltransferases. The major glycolipids in the sensitive and resistant cells were GlcCer and Gb(3)Cer, respectively, and extension of the carbohydrate structure into Lewis antigen characteristically occurred in the resistant cells. Le(b), which was not detected in the cisplatin-resistant cells, was present in the taxol-resistant cells, while Le(x) was present in the cisplatin-resistant cells at a higher concentration than in the taxol-resistant cells. 2-Hydroxy fatty acids were significantly abundant in glycolipids of the resistant cells, but they were not detected in free ceramides or sphingomyelin, indicating that the enhanced synthesis of glycolipids in the resistant cells was not linked with the removal pathway for virulent ceramides derived from sphingomyelin. The resistant cells with abundant glycolipids exhibited lower membrane fluidity than the KF28 cells, and this property might be involved in the anticancer drug-resistance.

Chewing the fat on natural killer T cell development.

Natural killer T cells (NKT cells) are selected in the thymus by self-glycolipid antigens presented by CD1d molecules. It is currently thought that one specific component of the lysosomal processing pathway, which leads to the production of isoglobotrihexosylceramide (iGb3), is essential for normal NKT cell development. New evidence now shows that NKT cell development can be disrupted by a diverse range of mutations that interfere with different elements of the lysosomal processing and degradation of glycolipids. This suggests that lysosomal storage diseases (LSDs) in general, rather than one specific defect, can disrupt CD1d antigen presentation, leading to impaired development of NKT cells.