Effects of the glucolipid synthase inhibitor, P4, on functional and phenotypic parameters of murine myeloma cells

This study describes the effects of the glucolipid synthase inhibitor P4, (DL-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol), on various functional and phenotypic parameters of 5T33 murine myeloma cells. Cell recovery was reduced by >85% following incubation of the cells for 3 days in the presence of 4 μM P4 (the IC50 concentration). Both cytostatic and cytotoxic inhibition was observed with tumour cell metabolic activity and clonogenic potential reduced to 42% and 14% of controls, respectively, and viability reduced to 52%. A dose-dependent increase in cells undergoing apoptosis (from 7% to 26%) was also found. P4 induced a decrease in the number of cells expressing H-2 Class I and CD44, and a large increase in cells expressing H-2 Class II and the IgG2b paraprotein. It did not affect surface expression of CD45 or CD54 (ICAM-1). Based on these alterations in tumour cell growth, adhesion molecule expression and potential immunogenicity, it is anticipated that P4 will provide a novel therapeutic approach for the treatment of multiple myeloma. In addition, given that essentially all tumours rely heavily on overexpressed or abnormal glucosphingolipids for growth, development and metastasis, glucolipid synthase inhibitors may prove to be universally effective anti-cancer agents. © 1999 Cancer Research Campaig

Lewis X antigen mediates adhesion of human breast carcinoma cells to activated endothelium. Possible involvement of the endothelial scavenger receptor C-Type lectin

Lewis x (Lex, CD15), also known as SSEA-1 (stage specific embryonic antigen-1), is a trisaccharide with the structure Galβ(1–4)Fucα(1–3)GlcNAc, which is expressed on glycoconjugates in human polymorphonuclear granulocytes and various tumors such as colon and breast carcinoma. We have investigated the role of Lex in the adhesion of MCF-7 human breast cancer cells and PMN to human umbilical endothelial cells (HUVEC) and the effects of two different anti-Lex mAbs (FC-2.15 and MCS-1) on this adhesion. We also analyzed the cytolysis of Lex+-cells induced by anti-Lex mAbs and complement when cells were adhered to the endothelium, and the effect of these antibodies on HUVEC. The results indicate that MCF-7 cells can bind to HUVEC, and that MCS-1 but not FC-2.15 mAb inhibit this interaction. Both mAbs can efficiently lyse MCF-7 cells bound to HUVEC in the presence of complement without damaging endothelial cells. We also found a Lex-dependent PMN interaction with HUVEC. Although both anti-Lex mAbs lysed PMN in suspension and adhered to HUVEC, PMN aggregation was only induced by mAb FC-2.15. Blotting studies revealed that the endothelial scavenger receptor C-type lectin (SRCL), which binds Lex-trisaccharide, interacts with specific glycoproteins of Mr␣∼␣28 kD and 10 kD from MCF-7 cells. The interaction between Lex+-cancer cells and vascular endothelium is a potential target for cancer treatment.Fil: Elola, Maria Teresa. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Capurro, Mariana Isabel. University of Toronto; CanadáFil: Barrio, Maria Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Investigación, Docencia y Prevención del Cáncer; ArgentinaFil: Coombs, Peter J.. Imperial College London; Reino UnidoFil: Taylor, Maureen E.. Imperial College London; Reino UnidoFil: Drickamer, Kurt. Imperial College London; Reino UnidoFil: Mordoh, Jose. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Investigación, Docencia y Prevención del Cáncer; Argentin

Overexpression of α(1,3)-fucosyltransferase VII is sufficient for the acquisition of lung colonization phenotype in human lung adenocarcinoma HAL-24Luc cells

Metastatic human lung adenocarcinoma HAL-8Luc cells display an enhanced expression of alpha(1,3)-fucosyltransferases (alpha(1,3)-Fuc-Ts) compared with their non-metastatic counterpart HAL-24Luc cells. This correlates with an increased surface expression of Lewis(x) (Le(x))- and Lewis(a) (Le(a))-related molecules and an in vitro enhanced adhesive capacity to E-selectin-expressing endothelial cells (Martin-Satué et al (1998). Cancer Res 58: 1544-1550). In the present work we have stably transfected HAL-24Luc cells with the cDNAs for the alpha(1,3)-Fuc-TIV and VII enzymes and analysed by flow cytometry the expression of Le(x), sialyl-Le(x), sialyl-Le(x) dimeric, Le(a) and sialyl-Le(a). Fuc-TVII transfectants exclusively overexpress sialyl-Le(x) while Fuc-TIV-transfected cells only overexpress the Le(x) oligosaccharide. We show that solely Fuc-TVII transfectants are able to adhere to interleukin-1beta-stimulated HUVEC monolayers. We also demonstrate that Fuc-TVII overexpression in HAL-24Luc cells is sufficient for the acquisition of the lung colonization phenotype. This is the first report directly showing the contribution of an alpha(1,3)-Fuc-T to the metastatic behaviour of human lung adenocarcinoma cells

Centralized Modularity of N-Linked Glycosylation Pathways in Mammalian Cells

Glycosylation is a highly complex process to produce a diverse repertoire of cellular glycans that are attached to proteins and lipids. Glycans are involved in fundamental biological processes, including protein folding and clearance, cell proliferation and apoptosis, development, immune responses, and pathogenesis. One of the major types of glycans, N-linked glycans, is formed by sequential attachments of monosaccharides to proteins by a limited number of enzymes. Many of these enzymes can accept multiple N-linked glycans as substrates, thereby generating a large number of glycan intermediates and their intermingled pathways. Motivated by the quantitative methods developed in complex network research, we investigated the large-scale organization of such N-linked glycosylation pathways in mammalian cells. The N-linked glycosylation pathways are extremely modular, and are composed of cohesive topological modules that directly branch from a common upstream pathway of glycan synthesis. This unique structural property allows the glycan production between modules to be controlled by the upstream region. Although the enzymes act on multiple glycan substrates, indicating cross-talk between modules, the impact of the cross-talk on the module-specific enhancement of glycan synthesis may be confined within a moderate range by transcription-level control. The findings of the present study provide experimentally-testable predictions for glycosylation processes, and may be applicable to therapeutic glycoprotein engineering

Structure Determination of Oligosaccharides Isolated from A + , H + and A − H − Hog-Submaxillary-Gland Mucin Glyoproteins, by 360-MHz 1 H-NMR Spectroscopy, Permethylation Analysis and Mass Spectrometry

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65915/1/j.1432-1033.1981.tb05545.x.pd

Structural analysis of three novel trisaccharides isolated from the fermented beverage of plant extracts

<p>Abstract</p> <p>Background</p> <p>A fermented beverage of plant extracts was prepared from about fifty kinds of vegetables and fruits. Natural fermentation was carried out mainly by lactic acid bacteria (<it>Leuconostoc </it>spp.) and yeast (<it>Zygosaccharomyces </it>spp. and <it>Pichia </it>spp.). We have previously examined the preparation of novel four trisaccharides from the beverage: <it>O</it>-β-D-fructopyranosyl-(2->6)-<it>O</it>-β-D-glucopyranosyl-(1->3)-D-glucopyranose, <it>O</it>-β-D-fructopyranosyl-(2->6)-<it>O</it>-[β-D-glucopyranosyl-(1->3)]-D-glucopyranose, <it>O</it>-β-D-glucopyranosyl-(1->1)-<it>O</it>-β-D-fructofuranosyl-(2<->1)-α-D-glucopyranoside and <it>O</it>-β-D-galactopyranosyl-(1->1)-<it>O</it>-β-D-fructofuranosyl-(2<->1)- α-D-glucopyranoside.</p> <p>Results</p> <p>Three further novel oligosaccharides have been found from this beverage and isolated from the beverage using carbon-Celite column chromatography and preparative high performance liquid chromatography. Structural confirmation of the saccharides was provided by methylation analysis, MALDI-TOF-MS and NMR measurements.</p> <p>Conclusion</p> <p>The following novel trisaccharides were identified: <it>O</it>-β-D-fructofuranosyl-(2->1)-<it>O</it>-[β-D-glucopyranosyl-(1->3)]-β-D-glucopyranoside (named "3^G-β-D-glucopyranosyl β, β-isosucrose"), <it>O</it>-β-D-glucopyranosyl-(1->2)-<it>O</it>-[β-D-glucopyranosyl-(1->4)]-D-glucopyranose (4¹-β-D-glucopyranosyl sophorose) and <it>O</it>-β-D-fructofuranosyl-(2->6)-<it>O</it>-β-D-glucopyranosyl-(1->3)-D-glucopyranose (6²-β-D-fructofuranosyl laminaribiose).</p

Complex -Glycans Influence the Spatial Arrangement of Voltage Gated Potassium Channels in Membranes of Neuronal-Derived Cells

The intrinsic electrical properties of a neuron depend on expression of voltage gated potassium (Kv) channel isoforms, as well as their distribution and density in the plasma membrane. Recently, we showed that N-glycosylation site occupancy of Kv3.1b modulated its placement in the cell body and neurites of a neuronal-derived cell line, B35 neuroblastoma cells. To extrapolate this mechanism to other N-glycosylated Kv channels, we evaluated the impact of N-glycosylation occupancy of Kv3.1a and Kv1.1 channels. Western blots revealed that wild type Kv3.1a and Kv1.1 α-subunits had complex and oligomannose N-glycans, respectively, and that abolishment of the N-glycosylation site(s) generated Kv proteins without N-glycans. Total internal reflection fluorescence microscopy images revealed that N-glycans of Kv3.1a contributed to its placement in the cell membrane while N-glycans had no effect on the distribution of Kv1.1. Based on particle analysis of EGFP-Kv proteins in the adhered membrane, glycosylated forms of Kv3.1a, Kv1.1, and Kv3.1b had differences in the number, size or density of Kv protein clusters in the cell membrane of neurites and cell body of B35 cells. Differences were also observed between the unglycosylated forms of the Kv proteins. Cell dissociation assays revealed that cell-cell adhesion was increased by the presence of complex N-glycans of Kv3.1a, like Kv3.1b, whereas cell adhesion was similar in the oligomannose and unglycosylated Kv1.1 subunit containing B35 cells. Our findings provide direct evidence that N-glycans of Kv3.1 splice variants contribute to the placement of these glycoproteins in the plasma membrane of neuronal-derived cells while those of Kv1.1 were absent. Further when the cell membrane distribution of the Kv channel was modified by N-glycans then the cell-cell adhesion properties were altered. Our study demonstrates that N-glycosylation of Kv3.1a, like Kv3.1b, provides a mechanism for the distribution of these proteins to the cell body and outgrowths and thereby can generate different voltage-dependent conductances in these membranes

Oligosaccharide Binding Proteins from Bifidobacterium longum subsp. infantis Reveal a Preference for Host Glycans

Bifidobacterium longum subsp. infantis (B. infantis) is a common member of the infant intestinal microbiota, and it has been characterized by its foraging capacity for human milk oligosaccharides (HMO). Its genome sequence revealed an overabundance of the Family 1 of solute binding proteins (F1SBPs), part of ABC transporters and associated with the import of oligosaccharides. In this study we have used the Mammalian Glycan Array to determine the specific affinities of these proteins. This was correlated with binding protein expression induced by different prebiotics including HMO. Half of the F1SBPs in B. infantis were determined to bind mammalian oligosaccharides. Their affinities included different blood group structures and mucin oligosaccharides. Related to HMO, other proteins were specific for oligomers of lacto-N-biose (LNB) and polylactosamines with different degrees of fucosylation. Growth on HMO induced the expression of specific binding proteins that import HMO isomers, but also bind blood group and mucin oligosaccharides, suggesting coregulated transport mechanisms. The prebiotic inulin induced other family 1 binding proteins with affinity for intestinal glycans. Most of the host glycan F1SBPs in B. infantis do not have homologs in other bifidobacteria. Finally, some of these proteins were found to be adherent to intestinal epithelial cells in vitro. In conclusion, this study represents further evidence for the particular adaptations of B. infantis to the infant gut environment, and helps to understand the molecular mechanisms involved in this process