Expression of the Gb3/CD77 synthase gene in megakaryoblastic leukemia cells: implication in the sensitivity to verotoxins.

Expression levels of Gb3/CD77 synthase together with Gb3/CD77 antigen were analyzed using human hematopoietic tumor cell lines and normal cells. Among about 40 kinds of cells, Burkitt lymphoma cells showed the highest gene expression concomitant with the expression levels of Gb3/CD77. Unexpectedly, megakaryoblastic leukemia lines also expressed fairly high levels of mRNA of Gb3/CD77 synthase and its product. A megakaryoblastic leukemia line, MEG-01 was sensitive to verotoxins from Escherichia coli O157 and apoptosis was induced via the caspase pathway. We also demonstrated that the cell surface Gb3/CD77 expression was reduced on differentiated MEG-01 although the mRNA level of the alpha1,4Gal-T gene increased. In this case, the localization of Gb3/CD77 was changed from the cell surface to the cytoplasm as stained with a granular pattern, co-localizing with platelet GPIIb-IIIa, indicating that some of them were platelet precursors. Small particles outside of cells also showed similar staining patterns. These results agreed with the previous report that platelets produced in mature megakaryoblasts abundantly contained Gb3/CD77 antigen. Here, we propose the possibility that verotoxins bind immature megakaryoblasts and induce their apoptosis, leading to the arrest of platelet generation in the bone marrow. This may be one of the causes of thrombocytopenia in patients with hemolytic uremic syndrome.     

Two distinct Gb3/CD77 signaling pathways leading to apoptosis are triggered by anti-Gb3/CD77 mAb and verotoxin-1

Globotriasosylceramide (Gb3), a neutral glycosphingolipid, is the B-cell differentiation antigen CD77 and acts as the receptor for most Shiga toxins, including verotoxin-1 (VT-1). We have shown that both anti-Gb3/CD77 mAb and VT-1 induce apoptosis in Burkitt's lymphoma cells. We compared the apoptotic pathways induced by these two molecules by selecting cell lines sensitive to only one of these inducers or to both. In all these cell lines (including the apoptosis-resistant line), VT-1 was transported to the endoplasmic reticulum and inhibited protein synthesis similarly, suggesting that VT-1-induced apoptosis is dissociated from these processes. VT-1 triggered a caspase- and mitochondria-dependent pathway (rapid activation of caspases 8 and 3 associated with a loss of mitochondrial membrane potential (Deltapsim) and the release of cytochrome c from mitochondria). In contrast, the anti-Gb3/CD77 mAb-induced pathway was caspase-independent and only involved partial depolarization of mitochondria. Antioxidant compounds had only marginal effects on VT-1-induced apoptosis but strongly protected cells from anti-Gb3/CD77 mAb-induced apoptosis. VT-1- and anti-Gb3/CD77 mAb-treated cells displayed very different features on electron microscopy. These results clearly indicate that the binding of different ligands to Gb3/CD77 triggers completely different apoptotic pathways.     

Targeted disruption of Gb3/CD77 synthase gene resulted in the complete deletion of globo-series glycosphingolipids and loss of sensitivity to verotoxins

To examine whether globotriaosylceramide (Gb3/CD77) is a receptor for verotoxins (VTs) in vivo, sensitivity of Gb3/CD77 synthase null mutant mice to VT-2 and VT-1 was analyzed. Although wild-type mice died after administration of 0.02 microg of VT-2 or 1.0 microg of VT-1, the mutant mice showed no reaction to doses as much as 100 times that administered to wild types. Expression analysis of Gb3/CD77 in mouse tissues with antibody revealed that low, but definite, levels of Gb3/CD77 were expressed in the microvascular endothelial cells of the brain cortex and pia mater and in renal tubular capillaries. Corresponding to the Gb3/CD77 expression, tissue damage with edema, congestion, and cytopathic changes was observed, indicating that Gb3/CD77 (and its derivatives) exclusively function as a receptor for VTs in vivo. The lethal kinetics were similar regardless of lipopolysaccharide elimination in VT preparation, suggesting that basal Gb3/CD77 levels are sufficient for lethal effects of VTs.     

Evaluation of an amino acid residue critical for the specificity and activity of human Gb3/CD77 synthase

Human Gb3/CD77 synthase (α1,4-galactosyltransferase) is the only known glycosyltransferase that changes acceptor specificity because of a point mutation. The enzyme, encoded by A4GALT locus, is responsible for biosynthesis of Gal(α1–4)Gal moiety in Gb3 (CD77, P^k antigen) and P1 glycosphingolipids. We showed before that a single nucleotide substitution c.631C > G in the open reading frame of A4GALT, resulting in replacement of glutamine with glutamic acid at position 211 (substitution p. Q211E), broadens the enzyme acceptor specificity, so it can not only attach galactose to another galactose but also to N-acetylgalactosamine. The latter reaction leads to synthesis of NOR antigens, which are glycosphingolipids with terminal Gal(α1–4)GalNAc sequence, never before described in mammals. Because of the apparent importance of position 211 for enzyme activity, we stably transfected the 2102Ep cells with vectors encoding Gb3/CD77 synthase with glutamine substituted by aspartic acid or asparagine, and evaluated the cells by quantitative flow cytometry, high-performance thin-layer chromatography and real-time PCR. We found that cells transfected with vectors encoding Gb3/CD77 synthase with substitutions p. Q211D or p. Q211N did not express P^k, P1 and NOR antigens, suggesting complete loss of enzymatic activity. Thus, amino acid residue at position 211 of Gb3/CD77 synthase is critical for specificity and activity of the enzyme involved in formation of P^k, P1 and NOR antigens. Altogether, this approach affords a new insight into the mechanism of action of the human Gb3/CD77 synthase.     

Efficient generation of useful monoclonal antibodies reactive with globotriaosylceramide using knockout mice lacking Gb3/CD77 synthase

Efficient generation of useful monoclonal antibodies (mAbs) with high performance in cancer therapeutics has been expected. Generation of mAbs reactive with globotriaosylceramide (Gb3/CD77) was compared between A/J mice and Gb3/CD77 synthase-deficient (A4GalT-knockout) mice by immunizing Gb3-liposome. Specificity and functions of established antibodies were examined by ELISA, TLC- immunostaining, cytotoxicity of cancer cells and immunoblotting. Compared with results with conventional mice, better generation of mAbs with higher functions has been achieved with A4GalT-knockout mice, i.e. acquisition of IgG class antibodies, activities in antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and aggregation activity toward a Burkitt’s lymphoma line Ramos. Binding of mAb k52 induced tyrosine phosphorylation of several proteins in Ramos cells. One of the strongest phosphorylation bands turned out to be c-Cbl. Pretreatment of B cell lines with mAbs resulted in the attenuation of BCR-mimicking signaling. All these results suggested that A4GalT-knockout mice are very useful to generate mAbs against globo-series glycolipids, and that suppressive signaling pathway driven by endogenous Gb3-ligand molecules might be present in B cells.     

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(β1–3)Gal(α1–4), and Gal(α1–4)GalNAc(β1–3)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.     

The gene coding for the human T-lymphocyte CD2 antigen is located on chromosome 1p

Summary A cDNA clone encoding the human T lymphocyte sheep erythrocyte receptor [the CD2 (T11) antigen] was used as a probe to define the chromosomal location of the gene. The signal, revealed by hybridisation to Southern blots of genomic DNA from somatic cell hybrids, showed a high degree of concordance for human chromosome 1. In particular, the hybrid F4Sc13C19 which contained the short arm only of human chromosome 1 was positive. The location of the CD2 gene to 1p13 was confirmed by in situ hybridisation.     

Verotoxin 1 from Escherichia coli affects Gb3/CD77+ bovine lymphocytes independent of interleukin-2, tumor necrosis factor-alpha,and interferon-alpha

Verotoxin (VT)-induced immunomodulation has been implicated in the ability of VT-producing Escherichia coli (VTEC) to cause persistent infections in cattle. VT1, also referred to as Shiga toxin 1, is a potent cytotoxin that modulates cytokine secretions and functions. This prompted the current investigation to examine whether the inhibiting effect of VT1 on bovine lymphocytes correlates with the expression of the cellular VT1 receptor Gb3/CD77 or is mediated instead via perturbation of cytokine secretion. Using blood mononuclear cells stimulated by mitogens as a model, VT1 significantly blocked lymphoblast transformation and proliferation in the BoCD8+ T cell and BoCD21+ B cell population. In contrast, VT1 dramatically reduced the number of viable Gb3/CD77+ blast cells within all subpopulations identified (BoCD2+, BoCD4+, BoCD8+, WC1+ [i.e., gammadelta T cells] BoCD21+, and BoCD25+). Similar effects of VT1 were observed when the culture medium was supplemented with selected cytokines: tumor necrosis factor-alpha-sensitizing endothelial cells against VT1, interferon-alpha (IFN-alpha) as bovine IFN-alpha receptors are partially homologous to the B-subunit of VT1, and interleukin-2 that is critical for lymphocyte proliferation in vitro. The addition of these cytokines was neither able to mimic nor to overcome the effects of VT1. Therefore, it is concluded that VT1 directly acts on bovine lymphocytes rather than inducing a cytokine-mediated effect. VT1 considerably affects all main bovine lymphocyte subpopulations, implicating that the immune system is a predominant target for VT1 in cattle.     

The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer.

SMT3 is an essential Saccharomyces cerevisiae gene encoding a 11.5 kDa protein similar to the mammalian ubiquitin-like protein SUMO-1. We have found that Smt3p, like SUMO-1 and ubiquitin, can be attached to other proteins post-translationally and have characterized the processes leading to the activation of the Smt3p C-terminus for conjugation. First, the SMT3 translation product is cleaved endoproteolytically to expose Gly98, the mature C-terminus. The presence of Gly98 is critical for Smt3p's abilities to be conjugated to protein substrates and to complement the lethality of a smt3Delta strain. Smt3p undergoes ATP-dependent activation by a novel heterodimeric enzyme consisting of Uba2p, a previously identified 71 kDa protein similar to the C-terminus of ubiquitin-activating enzymes (E1s), and Aos1p (activation of Smt3p), a 40 kDa protein similar to the N-terminus of E1s. Experiments with conditional uba2 mutants showed that Uba2p is required for Smt3p conjugation in vivo. Furthermore, UBA2 and AOS1 are both essential genes, providing additional evidence that they act in a distinct pathway whose role in cell viability is to conjugate Smt3p to other proteins.     

The yeast gene COQ5 is differentially regulated by Mig1p,Rtg3p and Hap2p

In vivo electroporation (EP) is gaining momentum for drug and gene delivery. In particular, DNA transfer by EP to muscle tissue can lead to highly efficient long-term gene expression. We characterized a vascular effect of in vivo EP and its consequences for drug and gene delivery. Pulses of 10-20,000 micros and 0.1-1.6 kV/cm were applied over hind- and forelimb of mice and perfusion was examined by dye injection. The role of a sympathetically mediated vasoconstrictory reflex was investigated by pretreatment with reserpine. Expression of a transferred gene (luciferase), permeabilization (determined using (51)Cr-EDTA), membrane resealing and effects on perfusion were compared to assess the significance of the vascular effects. Above the permeabilization threshold, a sympathetically mediated Raynaud-like phenomenon with perfusion delays of 1-2 min was observed. Resolution of this phase followed kinetics of membrane resealing. Above a second threshold, irreversible permeabilization led to long perfusion delays. These vascular reactions (1) affect kinetics of drug delivery, (2) predict efficient DNA transfer, which is optimal during short perfusion delays, and (3) might explain electrocardiographic ST segment depressions after defibrillation as being caused by vascular effects of EP of cardiac muscle.     

Dynamic interplay between the neutral glycosphingolipid CD77/Gb3 and the therapeutic antibody target CD20 within the lipid bilayer of model B lymphoma cells

The centroblast-specific differentiation marker CD77 (Gb(3)), is the receptor for Shiga-like toxin (SLT). The dynamic relationship between Gb(3)/CD77 and key B-cell membrane proteins was studied in Burkitt's lymphoma cells with a focus on CD20. Engagement of Gb(3)/CD77 with SLT-B reduced the amount of CD20 and CXCR4 available, but levels of BCR, MHC Class II, CD21, CD27 and CD54 remained unchanged. Cholesterol depletion promoted a decrease in the number of sites accessed by CD20, CXCR4 and Gb(3)/CD77 antibodies. Constitutive localisation of Gb(3)/CD77 to lipid rafts was unperturbed by either SLT-B binding or cholesterol depletion, whereas the opposite was true for CD20. The effects were specific to SLT-B, highlighted by the inability of cholera toxin B-subunit to alter CD20 availability. Thus, the binding of Gb(3)/CD77 by its cognate ligand transmits information within the lipid bilayer of model lymphoma cells to impact the behaviour of selective proteins, most notably CD20, via a mechanism influenced by the level of cholesterol within the membrane.     

Structure of the human erythrocyte blood group P1 glycosphingolipid.

A glycosphingolipid with blood group P1 activity was extracted from an acetone powder of human erythrocyte stroma with chloroform-methanol. It was purified by chromatography on columns of silicic acid and by preparative thin-layer chromatography of the fully acetylated and deacetylated glycolipid. The purified glycolipid contained galactose, N-acetylglucosamine, and glucose in a molar ratio of 3:1:1. Treatment of the P1 glycolipid with fig alpha-galactosidase released a single galactosyl residue and destroyed the blood group activity, and the alpha-galactosidase product had the same chromatographic mobility as paragloboside. Substitution sites on the neutral sugars of the P1 glycolipid and the alpha-galactosidase product were established by identification of methylated alditol acetates, and substitution on N-acetylglucosamine was determined by identification of methyl glycoside derivatives. The terminal nonreducing disaccharide of the P1 glycolipid is Gal(alpha, 1 leads to 4)Gal. N-Acetylglucosamine was identified as the next sugar in sequence by mass spectrometric analysis of the permethylated P1 glycolipid. On the assumption that the glucose residue is linked to ceramide, we propose the following structure for the P1 glycolipid: Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-NAc(beta, 1 leads to 4)Glc-Cer.     

The p88 molecular chaperone is identical to the endoplasmic reticulum membrane protein, calnexin.

We previously described a novel molecular chaperone (designated p88) that participates in the assembly of murine class I histocompatibility molecules (Degen, E., and Williams, D. B. (1991) J. Cell Biol. 112, 1099-1115). Our findings suggest that p88 may either promote proper assembly of class I molecules or retain them, probably within the endoplasmic reticulum (ER), until assembly of the ternary complex of heavy chain, beta 2-microglobulin, and peptide ligand is complete. In this report, we compare p88 to calnexin, a calcium-binding 90-kDa phosphoprotein of the ER membrane (Wada, I., Rindress, D., Cameron, P. H., Ou, W.-J., Doherty, J.-J., II, Louvard, D., Bell, A.W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610). We show that p88 and calnexin share antigenic epitopes defined by a polyclonal anti-calnexin antiserum. Furthermore, both proteins were immunoprecipitated in association with an intracellularly retained variant of the class I H-2Kb molecule. Since p88 and calnexin were also indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, were resistant to digestion with endoglycosidase H, and exhibited virtually identical patterns of peptide fragments following digestion with either V8 protease or trypsin, we conclude that p88 and calnexin represent the same protein. The identification of the p88 chaperone as a phosphorylated, calcium-binding protein of the ER membrane suggests possible means whereby its interaction with class I molecules may be regulated.