Isolation and characterization of the B-cell marker CD20

The integral membrane protein CD20 has been identified as an important therapeutic target in the treatment of non-Hodgkin's lymphoma (NHL). CD20 binding of many antibodies including the therapeutic antibody, rituximab, has been shown to be critically dependent upon the conformation of a loop structure between the third and fourth helical transmembrane regions. In this work, human and murine CD20 proteins expressed in Escherichia coli are shown to be localized with the cell membrane and are purified in nondenaturing detergent solutions. The purified human and murine CD20 proteins have a substantial helical structure as measured by circular dichroism spectroscopy. Only small changes in the secondary structure are observed following the reduction of CD20, with the addition of SDS, or after heating. The rituximab antibody is shown to bind to purified human CD20 with nanomolar affinity. Rituximab binding is abolished by reduction and alkylation of CD20, with data consistent with the proposed antibody epitope being within the disulfide-bonded loop formed between cysteine residues 167 and 183. Disulfide-bond-dependent antibody binding is partially recovered following reoxidation of reduced CD20. Antibody binding is unaffected by mutations of cysteines proposed to be in the intracellular domain of CD20. The affinities of intact rituximab and its Fab fragment to the isolated and purified CD20 are similar to the observed affinity of rituximab Fab for CD20 on the surface of B cells. However, the intact rituximab antibody shows much higher affinity for CD20 on B cells. This suggests that B cells display CD20 in such a way that allows for marked avidity effects to be observed, perhaps through cross-linking of CD20 monomers into lipid rafts, which limits receptor diffusion in the membrane. Such cross-linking may play a role in partitioning CD20 into lipid rafts and in enhancing antibody-dependent B-cell depletion activities of rituximab and other therapeutic anti-CD20 antibodies.     

Dynamic reorganization of chemokine receptors, cholesterol, lipid rafts, and adhesion molecules to sites of CD4 engagement

T cell polarization and redistribution of cellular components are critical to processes such as activation, migration, and potentially HIV infection. Here, we investigate the effects of CD4 engagement on the redistribution and localization of chemokine receptors, CXCR4 and CCR5, adhesion molecules, and lipid raft components including cholesterol, GM1, and glycosyl-phosphatidylinositol (GPI)-anchored proteins. We demonstrate that anti-CD4-coated beads (alpha CD4-B) rapidly induce co-capping of chemokine receptors as well as GPI-anchored proteins and adhesion molecules with membrane cholesterol and lipid rafts on human T cell lines and primary T cells to the area of bead-cell contact. This process was dependent on the presence of cellular cholesterol, cytoskeletal reorganization, and lck signaling. Lck-deficient JCaM 1.6 cells failed to cap CXCR4 or lipid rafts to alpha CD4-B. Biochemical analysis reveals that CXCR4 and LFA-1 are recruited to lipid rafts upon CD4 but not CD45 engagement. Furthermore, we also demonstrate T cell capping of both lipid rafts and chemokine receptors at sites of contact with HIV-infected cells, despite the binding of an HIV inhibitory mAb to CXCR4. We conclude that cell surface rearrangements in response to CD4 engagement may serve as a means to enhance cell-to-cell signaling at the immunological synapse and modulate chemokine responsiveness, as well as facilitate HIV entry and expansion by synaptic transmission.     

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.     

Segregation of CD4 and CXCR4 into Distinct Lipid Microdomains in T Lymphocytes Suggests a Mechanism for Membrane Destabilization by Human Immunodeficiency Virus

Recent evidence has suggested that plasma membrane sphingolipids and cholesterol spontaneously coalesce into raft-like microdomains and that specific proteins, including CD4 and some other T-cell signaling molecules, sequester into these rafts. In agreement with these results, we found that CD4 and the associated Lck tyrosine kinase of peripheral blood mononuclear cells and H9 leukemic T cells were selectively and highly enriched in a low-density lipid fraction that was resistant at 0 degrees C to the neutral detergent Triton X-100 but was disrupted by extraction of cholesterol with filipin or methyl-beta-cyclodextrin. In contrast, the CXCR4 chemokine receptor, a coreceptor for X4 strains of human immunodeficiency virus type 1 (HIV-1), was almost completely excluded from the detergent-resistant raft fraction. Accordingly, as determined by immunofluorescence with confocal microscopy, CD4 and CXCR4 did not coaggregate into antibody-induced cell surface patches or into patches of CXCR4 that formed naturally at the ruffled edges of adherent cells. The CXCR4 fluorescent patches were extracted with cold 1% Triton X-100, whereas the CD4 patches were resistant. In stringent support of these data, CD4 colocalized with patches of cholera toxin bound to the raft-associated sphingoglycolipid GM1, whereas CXCR4 did not. Addition of the CXCR4-activating chemokine SDF-1 alpha did not induce CXCR4 movement into rafts. Moreover, binding of purified monomeric gp120 envelope glycoproteins from strains of HIV-1 that use this coreceptor did not stimulate detectable redistributions of CD4 or CXCR4 between their separate membrane domains. However, adsorption of multivalent gp120-containing HIV-1 virion particles appeared to destabilize the local CD4-containing rafts. Indeed, adsorbed HIV-1 virions were detected by immunofluorescence microscopy and were almost all situated in nonraft regions of the cell surface. We conclude that HIV-1 initially binds to CD4 in a raft domain and that its secondary associations with CXCR4 require shifts of proteins and associated lipids away from their preferred lipid microenvironments. Our evidence suggests that these changes in protein-lipid interactions destabilize the plasma membrane microenvironment underlying the virus by at least several kilocalories per mole, and we propose that this makes an important contribution to fusion of the viral and cellular membranes during infection. Thus, binding of HIV-1 may be favored by the presence of CD4 in rafts, but the rafts may then disperse prior to the membrane fusion reaction.     

Statins impair antitumor effects of rituximab by inducing conformational changes of CD20

Background

Rituximab is used in the treatment of CD20⁺ B cell lymphomas and other B cell lymphoproliferative disorders. Its clinical efficacy might be further improved by combinations with other drugs such as statins that inhibit cholesterol synthesis and show promising antilymphoma effects. The objective of this study was to evaluate the influence of statins on rituximab-induced killing of B cell lymphomas.

Methods and Findings

Complement-dependent cytotoxicity (CDC) was assessed by MTT and Alamar blue assays as well as trypan blue staining, and antibody-dependent cellular cytotoxicity (ADCC) was assessed by a ⁵¹Cr release assay. Statins were found to significantly decrease rituximab-mediated CDC and ADCC of B cell lymphoma cells. Incubation of B cell lymphoma cells with statins decreased CD20 immunostaining in flow cytometry studies but did not affect total cellular levels of CD20 as measured with RT-PCR and Western blotting. Similar effects are exerted by other cholesterol-depleting agents (methyl-β-cyclodextrin and berberine), but not filipin III, indicating that the presence of plasma membrane cholesterol and not lipid rafts is required for rituximab-mediated CDC. Immunofluorescence microscopy using double staining with monoclonal antibodies (mAbs) directed against a conformational epitope and a linear cytoplasmic epitope revealed that CD20 is present in the plasma membrane in comparable amounts in control and statin-treated cells. Atomic force microscopy and limited proteolysis indicated that statins, through cholesterol depletion, induce conformational changes in CD20 that result in impaired binding of anti-CD20 mAb. An in vivo reduction of cholesterol induced by short-term treatment of five patients with hypercholesterolemia with atorvastatin resulted in reduced anti-CD20 binding to freshly isolated B cells.

Conclusions

Statins were shown to interfere with both detection of CD20 and antilymphoma activity of rituximab. These studies have significant clinical implications, as impaired binding of mAbs to conformational epitopes of CD20 elicited by statins could delay diagnosis, postpone effective treatment, or impair anti-lymphoma activity of rituximab.     

Human immunodeficiency virus type 1 uses lipid raft-colocalized CD4 and chemokine receptors for productive entry into CD4(+) T cells

In this report, we describe a crucial role of lipid raft-colocalized receptors in the entry of human immunodeficiency virus type 1 (HIV-1) into CD4(+) T cells. We show that biochemically isolated detergent-resistant fractions have characteristics of lipid rafts. Lipid raft integrity was required for productive HIV-1 entry as determined by (i) semiquantitative PCR analysis and (ii) single-cycle infectivity assay using HIV-1 expressing the luciferase reporter gene and pseudotyped with HIV-1 HXB2 envelope or vesicular stomatitis virus envelope glycoprotein (VSV-G). Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) relocalized raft-resident markers to a nonraft environment but did not significantly change the surface expression of HIV-1 receptors. MbetaCD treatment inhibited productive infection of HIV-1 by 95% as determined by luciferase activity in cells infected with HXB2 envelope-pseudotyped virus. In contrast, infection with VSV-G-pseudotyped virus, which enters the cells through an endocytic pathway, was not suppressed. Biochemical fractionation and confocal imaging of HIV-1 receptor distribution in live cells demonstrated that CD4, CCR5, and CXCR4 colocalized with raft-resident markers, ganglioside GM1, and glycosylphosphatidylinositol-anchored CD48. While confocal microscopy analysis revealed that HIV-1 receptors localized most likely to the same lipid microdomains, sucrose gradient analysis of the receptor localization showed that, in contrast to CD4 and CCR5, CXCR4 was associated preferentially with the nonraft membrane fraction. The binding of HIV-1 envelope gp120 to lipid rafts in the presence, but not in the absence, of cholesterol strongly supports our hypothesis that raft-colocalized receptors are directly involved in virus entry. Dramatic changes in lipid raft and HIV-1 receptor redistribution were observed upon binding of HIV-1 NL4-3 to PM1 T cells. Colocalization of CCR5 with GM1 and gp120 upon engagement of CD4 and CXCR4 by HIV-1 further supports our observation that HIV-1 receptors localize to the same lipid rafts in PM1 T cells.     

Lipid cosorting mediated by shiga toxin induced tubulation

To maintain cell membrane homeostasis, lipids must be dynamically redistributed during the formation of transport intermediates, but the mechanisms driving lipid sorting are not yet fully understood. Lowering sphingolipid concentration can reduce the bending energy of a membrane, and this effect could account for sphingolipid depletion along the retrograde pathway. However, sphingolipids and cholesterol are enriched along the anterograde pathway, implying that other lipid sorting mechanisms, such as protein-mediated sorting, can dominate. To characterize the influence of protein binding on the lipid composition of highly curved membranes, we studied the interactions of the B-subunit of Shiga toxin (STxB) with giant unilamellar vesicles containing its glycosphingolipid receptor [globotriaosylceramide (Gb3)]. STxB binding induced the formation of tubular membrane invaginations, and fluorescence microscopy images of these highly curved membranes were consistent with co-enrichment of Gb3 and sphingolipids. In agreement with theory, sorting was stronger for membrane compositions close to demixing. These results strongly support the hypothesis that proteins can indirectly mediate the sorting of lipids into highly curved transport intermediates via interactions between lipids and the membrane receptor of the protein.     

Fatty acid-dependent globotriaosyl ceramide receptor function in detergent resistant model membranes

Glycosphingolipid (GSL) fatty acid strictly regulates verotoxin 1 (VT1) and the HIV adhesin, gp120 binding to globotriaosyl ceramide within Gb(3)/cholesterol detergent resistant membrane (DRM) vesicle constructs and in Gb(3) water-air interface monolayers in a similar manner. VT2 bound Gb(3)/cholesterol vesicles irrespective of fatty acid composition, but VT1 bound neither C18 nor C20Gb(3)vesicles. C18/C20Gb(3) were dominant negative in mixed Gb(3) fatty acid isoform vesicles, but including C24:1Gb(3) gave maximal binding. VT1 bound C18Gb(3) vesicles after cholesterol removal, but C20Gb(3)vesicles required sphingomyelin in addition for binding. HIV-1gp120 also bound C16, C22, and C24, but neither C18 nor C20Gb(3) vesicles. C18 and C20Gb(3) were, in mixtures without C24:1Gb(3), dominant negative for gp120 vesicle binding. Gp120/VT1bound C18 and C24:1Gb(3) mixtures, although neither isoform bound alone. Monolayer surface pressure measurement showed VT1, but not VT2, bound Gb(3) at cellular DRM surface pressures, and confirmed loss of VT1 and gp120 (but not VT2) specific C18Gb(3) binding. We conclude fatty-acid mediated fluidity within simple model GSL/cholesterol DRM can selectively regulate GSL carbohydrate-ligand binding.     

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.     

Molecular cloning of globotriaosylceramide/CD77 synthase, a glycosyltransferase that initiates the synthesis of globo series glycosphingolipids

The expression cloning of a cDNA for globotriaosylceramide (Gb3)/CD77 synthase (alpha1,4-galactosyltransferase) was achieved using an anti-Gb3 antibody and mouse L cells as a recipient cell line for the transfection. The isolated cDNA clone designated pVTR1 predicted a type II membrane protein with 19 amino acids of cytoplasmic domain, 26 amino acids of transmembrane region, and a catalytic domain with 308 amino acids. Introduction of the cDNA clone into L cells resulted in the neosynthesis of Gb3/CD77, and the extracts of the transfectant cells showed alpha1, 4-galactosyltransferase activity only on lactosylceramide and galactosylceramide. In Northern blotting, a 2.3-kilobase mRNA was strongly expressed in heart, kidney, spleen, and placenta and weakly in colon, small intestine, and brain. Transfection of the cDNA into L cells resulted in the constitution of sensitivity to the apoptosis with Shiga-like toxins (verotoxins). Since Gb3/CD77 synthase initiates the synthesis of globo series glycolipids, the isolation of this cDNA will make possible further investigations into the function of its important series of glycolipids.     

Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains

Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.     

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.     

Apoptosis of bystander T cells induced by human immunodeficiency virus type 1 with increased envelope/receptor affinity and coreceptor binding site exposure

Apoptosis of uninfected bystander CD4(+) T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) pathogenesis. The viral and host mechanisms that lead to bystander apoptosis are not well understood. To investigate properties of the viral envelope glycoproteins (Env proteins) that influence the ability of HIV-1 to induce bystander apoptosis, we used molecularly cloned viruses that differ only in specific amino acids in Env. The ability of these strains to induce bystander apoptosis was tested in herpesvirus saimiri-immortalized primary CD4(+) T cells (CD4/HVS), which resemble activated primary T cells. Changes in Env that increase affinity for CD4 or CCR5 or increase coreceptor binding site exposure enhanced the capacity of HIV-1 to induce bystander apoptosis following viral infection or exposure to nonreplicating virions. Apoptosis induced by HIV-1 virions was inhibited by CD4, CXCR4, and CCR5 antibodies or by the CXCR4 inhibitor AMD3100, but not the fusion inhibitor T20. HIV-1 virions with mutant Envs that bind CXCR4 but are defective for CD4 binding or membrane fusion induced apoptosis, whereas CXCR4 binding-defective mutants did not. These results demonstrate that HIV-1 virions induce apoptosis through a CXCR4- or CCR5-dependent pathway that does not require Env/CD4 signaling or membrane fusion and suggest that HIV-1 variants with increased envelope/receptor affinity or coreceptor binding site exposure may promote T-cell depletion in vivo by accelerating bystander cell death.     

Globotriaosyl ceramide modulates interferon-α-induced growth inhibition and CD19 expression in Burkitt's lymphoma cells

Previous studies have indicated that globotriaosyl ceramide (Gb3 or CD77) plays a role in -interferon signal transduction and CD19-mediated homotypic adhesion in B cell lines derived from Burkitt's lymphoma. These roles for Gb3 may involve the proteins IFNAR-1 (subunit 1 of the interferon- receptor) and CD19, respectively, both of which have potential Gb3-binding sites in their extracellular domains which resemble those of the verotoxin (Shiga toxin and Shiga-like toxin) B subunit. The majority of this work was performed using wild-type Daudi cells and a single, Gb3-deficient mutant cell line, VT500. In the present investigations, these and additional Daudi-derived cells with varying degrees of sensitivity to interferon- were examined for Gb3 expression, interferon-induced growth inhibition and CD19 expression. The degree of interferon-induced growth inhibition and CD19 expression correlated with Gb3 expression in the various cell lines tested. In addition, reconstitution of the VT500 cell line with Gb3 but not other glycolipids partially restored the sensitivity of cells to IFN-induced growth inhibition. The degree to which reconstitution restored sensitivity to growth inhibition was similar to the results of previous studies in which Gb3 reconstitution restored sensitivity to verotoxin-induced cytotoxicity. These results demonstrate that Gb3 is specifically required for IFN-induced growth inhibition in Daudi cells and provide further evidence of a role for Gb3 in CD19 expression and function in these cells.     

Intracellular targeting of the endoplasmic reticulum/nuclear envelope by retrograde transport may determine cell hypersensitivity to verotoxin via globotriaosyl ceramide fatty acid isoform traffic

The pentameric B subunit of verotoxin (VT) mediates the attachment to cell surface globotriaosyl ceramide (Gb3) to facilitate receptor-mediated endocytosis of the toxin. In highly toxin-sensitive tumor cells, the holotoxin and VT1 B subunit is targeted intracellularly to elements of the endoplasmic reticulum (ER)/nuclear membrane. In less sensitive cells, the toxin is targeted to components of the Golgi apparatus. We have studied two cell systems: the induced VT hypersensitivity of human astrocytoma cell lines cultured in the presence of sodium butyrate (compared to sodium propionate and capronate) and the increased VT sensitivity of multiple drug-resistant mutants as compared to parental human ovarian carcinoma cells. In both cases, a difference in the intracellular retrograde transport of the receptor-bound internalized toxin to the ER/nuclear envelope, as opposed to the Golgi, correlated with a >1,000-fold increase in cell sensitivity to VT. This change in intracellular routing may be due to sorting of Gb3 fatty acid isoforms, since nuclear targeting was found in turn to correlate with the preferential synthesis of Gb3 containing shorter chain (primarily C16) fatty acid species. We propose that the isoform-dependent traffic of Gb3 from the cell surface to the ER/nuclear membrane provides a new signal transduction pathway for Gb3 binding proteins.     

Shiga Toxin Receptor Gb3Cer/CD77: Tumor-Association and Promising Therapeutic Target in Pancreas and Colon Cancer

Background

Despite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas.

Methodology/Principal Findings

Tissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors.

Conclusions/Significance

Enhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.     

Evaluation of prototype transmembrane 4 superfamily protein complexes and their relation to lipid rafts

Recent literature suggests that tetraspanin proteins (transmembrane 4 superfamily; TM4SF proteins) may associate with each other and with many other transmembrane proteins to form large complexes that sometimes may be found in lipid rafts. Here we show that prototype complexes of CD9 or CD81 (TM4SF proteins) with alpha(3)beta(1) (an integrin) and complexes of CD63 (a TM4SF protein) with phosphatidylinositol 4-kinase (PtdIns 4-K) may indeed localize within lipid raft-like microdomains, as seen by three different criteria. First, these complexes localize to low density light membrane fractions in sucrose gradients. Second, CD9 and alpha(3) integrin colocalized with ganglioside GM1 as seen by double staining of fixed cells. Third, CD9-alpha3beta1 and CD81-alpha3beta1 complexes were shifted to a higher density upon cholesterol depletion from intact cells or cell lysate. However, CD9-alpha3beta1, CD81-alpha3beta1, and CD63-PtdIns 4-K complex formation itself was not dependent on localization into raftlike lipid microdomains. These complexes did not require cholesterol for stabilization, were maintained within well solubilized dense fractions from sucrose gradients, were stable at 37 degrees C, and were small enough to be included within CL6B gel filtration columns. In summary, prototype TM4SF protein complexes (CD9-alpha3beta1, CD81-alpha3beta1, and CD63-PtdIns 4-K) can be solubilized as discrete units, independent of lipid microdomains, although they do associate with microdomains resembling lipid rafts.     

Structure-dependent pseudoreceptor intracellular traffic of adamantyl globotriaosyl ceramide mimics

The verotoxin (VT) (Shiga toxin) receptor globotriaosyl ceramide (Gb(3)), mediates VT1/VT2 retrograde transport to the endoplasmic reticulum (ER) for cytosolic A subunit access to inhibit protein synthesis. Adamantyl Gb(3) is an amphipathic competitive inhibitor of VT1/VT2 Gb(3) binding. However, Gb(3)-negative VT-resistant CHO/Jurkat cells incorporate adaGb(3) to become VT1/VT2-sensitive. CarboxyadaGb(3), urea-adaGb(3), and hydroxyethyl adaGb(3), preferentially bound by VT2, also mediate VT1/VT2 cytotoxicity. VT1/VT2 internalize to early endosomes but not to Golgi/ER. AdabisGb(3) (two deacyl Gb(3)s linked to adamantane) protects against VT1/VT2 more effectively than adaGb(3) without incorporating into Gb(3)-negative cells. AdaGb(3) (but not hydroxyethyl adaGb(3)) incorporation into Gb(3)-positive Vero cells rendered punctate cell surface VT1/VT2 binding uniform and subverted subsequent Gb(3)-dependent retrograde transport to Golgi/ER to render cytotoxicity (reduced for VT1 but not VT2) brefeldin A-resistant. VT2-induced vacuolation was maintained in adaGb(3)-treated Vero cells, but vacuolar membrane VT2 was lost. AdaGb(3) destabilized membrane cholesterol and reduced Gb(3) cholesterol stabilization in phospholipid liposomes. Cholera toxin GM1-mediated Golgi/ER targeting was unaffected by adaGb(3). We demonstrate the novel, lipid-dependent, pseudoreceptor function of Gb(3) mimics and their structure-dependent modulation of endogenous intracellular Gb(3) vesicular traffic.     

Low cholesterol triggers membrane microdomain-dependent CD44 shedding and suppresses tumor cell migration

CD44 is a cell surface adhesion molecule for hyaluronan and is implicated in tumor invasion and metastasis. Proteolytic cleavage of CD44 plays a critical role in the migration of tumor cells and is regulated by factors present in the tumor microenvironment, such as hyaluronan oligosaccharides and epidermal growth factor. However, molecular mechanisms underlying the proteolytic cleavage on membranes remain poorly understood. In this study, we demonstrated that cholesterol depletion with methyl-β-cyclodextrin, which disintegrates membrane lipid rafts, enhances CD44 shedding mediated by a disintegrin and metalloproteinase 10 (ADAM10) and that cholesterol depletion disorders CD44 localization to the lipid raft. We also evaluated the effect of long term cholesterol reduction using a statin agent and demonstrated that statin enhances CD44 shedding and suppresses tumor cell migration on a hyaluronan-coated substrate. Our results indicate that membrane lipid organization regulates CD44 shedding and propose a possible molecular mechanism by which cholesterol reduction might be effective for preventing and treating the progression of malignant tumors.     

Distinct mechanisms of CD4+ and CD8+ T-cell activation and bystander apoptosis induced by human immunodeficiency virus type 1 virions

Apoptosis of uninfected bystander T cells contributes to T-cell depletion during human immunodeficiency virus type 1 (HIV-1) infection. HIV-1 envelope/receptor interactions and immune activation have been implicated as contributors to bystander apoptosis. To better understand the relationship between T-cell activation and bystander apoptosis during HIV-1 pathogenesis, we investigated the effects of the highly cytopathic CXCR4-tropic HIV-1 variant ELI6 on primary CD4(+) and CD8(+) T cells. Infection of primary T-cell cultures with ELI6 induced CD4(+) T-cell depletion by direct cell lysis and bystander apoptosis. Exposure of primary CD4(+) and CD8(+) T cells to nonreplicating ELI6 virions induced bystander apoptosis through a Fas-independent mechanism. Bystander apoptosis of CD4(+) T cells required direct contact with virions and Env/CXCR4 binding. In contrast, the apoptosis of CD8(+) T cells was triggered by a soluble factor(s) secreted by CD4(+) T cells. HIV-1 virions activated CD4(+) and CD8(+) T cells to express CD25 and HLA-DR and preferentially induced apoptosis in CD25(+)HLA-DR(+) T cells in a CXCR4-dependent manner. Maximal levels of binding, activation, and apoptosis were induced by virions that incorporated MHC class II and B7-2 into the viral membrane. These results suggest that nonreplicating HIV-1 virions contribute to chronic immune activation and T-cell depletion during HIV-1 pathogenesis by activating CD4(+) and CD8(+) T cells, which then proceed to die via apoptosis. This mechanism may represent a viral immune evasion strategy to increase viral replication by activating target cells while killing immune effector cells that are not productively infected.