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Viviana A. Cavieres and Gonzalo A. Mardones were not included as authors in the original publication [...].
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Glycolipid glycosylation is an intricate process that mainly takes place in the Golgi by the complex interplay between glycosyltransferases. Several features such as the organization, stoichiometry and composition of these complexes may modify their sorting properties, sub-Golgi localization, enzymatic activity and in consequence, the pattern of glycosylation at the plasma membrane. In spite of the advance in our comprehension about physiological and pathological cellular states of glycosylation, the molecular basis underlying the metabolism of glycolipids and the players involved in this process remain not fully understood. In the present work, using biochemical and fluorescence microscopy approaches, we demonstrate the existence of a physical association between two ganglioside glycosyltransferases, namely, ST3Gal-II (GD1a synthase) and ß3GalT-IV (GM1 synthase) with Golgi phosphoprotein 3 (GOLPH3) in mammalian cultured cells. After GOLPH3 knockdown, the localization of both enzymes was not affected, but the fomation of ST3Gal-II/ß3GalT-IV complex was compromised and glycolipid expression pattern changed. Our results suggest a novel control mechanism of glycolipid expression through the regulation of the physical association between glycolipid glycosyltransferases mediated by GOLPH3.
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Glucolípidos , Glicosiltransferasas , Animales , Gangliósido G(M1)/metabolismo , Gangliósidos/metabolismo , Glucolípidos/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Aparato de Golgi/metabolismo , Mamíferos/metabolismo , Fosfoproteínas/metabolismoRESUMEN
Through the presentation of peptide antigens to cytotoxic T lymphocytes, major histocompatibility complex (MHC) class I molecules mediate the adaptive immune response against tumors and viruses. Additional non-immunological functions include the heterotypic association of class I molecules with cell surface receptors, regulating their activities by unknown mechanisms. Also, homotypic associations resulting in class I dimers and oligomers - of unknown function - have been related to pathological outcomes. In this review, we provide an overview of the current knowledge about the occurrence, biochemical nature, and dynamics of homotypic and heterotypic associations of class I molecules at the cell surface with special focus on the molecular species that take part in the complexes and on the evidence that supports novel biological roles for class I molecules. We show that both heterotypic and homotypic class I associations reported in the literature describe not one but several kinds of oligomers with distinctive stoichiometry and biochemical properties.
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The stage-specific embryonic antigen-4 (SSEA-4) is a cell surface glycosphingolipid antigen expressed in early stages of human development. This surface marker is downregulated during the differentiation process but is found re-expressed in several types of tumors, including breast cancer. This feature makes SSEA-4 an attractive target for the development of therapeutic antibodies against tumors. In this work, we first studied the binding and intracellular fate of the monoclonal antibody MC-813-70 directed against SSEA-4. MC-813-70 was found to be rapidly internalized into triple-negative breast cancer cells following binding to its target at the plasma membrane, and to accumulate in acidic organelles, most likely lysosomes. Given the internalization feature of MC-813-70, we next tested whether the antibody was able to selectively deliver the saporin toxin inside SSEA-4-expressing cells. Results show that the immunotoxin complex was properly endocytosed and able to reduce cell viability of breast cancer cells in vitro, either alone or in combination with chemotherapeutic drugs. Our findings indicate that the MC-813-70 antibody has the potential to be developed as an alternative targeted therapeutic agent for cancer cells expressing the SSEA-4 glycolipid.
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Inmunotoxinas/farmacología , Saporinas/farmacología , Antígenos Embrionarios Específico de Estadio/inmunología , Neoplasias de la Mama Triple Negativas/inmunología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/inmunología , Adenocarcinoma/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Femenino , Humanos , Inmunotoxinas/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Saporinas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Ganglioside glycosyltransferases (GGTs) are type II membrane proteins bearing a short N-terminal cytoplasmic tail, a transmembrane domain (TMD), and a lumenal catalytic domain. The expression and activity of these enzymes largely determine the quality of the glycolipids that decorate mammalian cell membranes. Many glycosyltransferases (GTs) are themselves glycosylated, and this is important for their proper localisation, but few if any other post-translational modifications of these proteins have been reported. Here, we show that the GGTs, ST3Gal-V, ST8Sia-I, and ß4GalNAcT-I are S-acylated at conserved cysteine residues located close to the cytoplasmic border of their TMDs. ST3Gal-II, a GT that sialylates glycolipids and glycoproteins, is also S-acylated at a conserved cysteine located in the N-terminal cytoplasmic tail. Many other GTs also possess cysteine residues in their cytoplasmic regions, suggesting that this modification occurs also on these GTs. S-acylation, commonly known as palmitoylation, is catalysed by a family of palmitoyltransferases (PATs) that are mostly localised at the Golgi complex but also at the endoplasmic reticulum (ER) and the plasma membrane. Using GT ER retention mutants, we found that S-acylation of ß4GalNAcT-I and ST3Gal-II takes place at different compartments, suggesting that these enzymes are not substrates of the same PAT. Finally, we found that cysteines that are the target of S-acylation on ß4GalNAcT-I and ST3Gal-II are involved in the formation of homodimers through disulphide bonds. We observed an increase in ST3Gal-II dimers in the presence of the PAT inhibitor 2-bromopalmitate, suggesting that GT homodimerisation may be regulating S-acylation.
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N-Acetilgalactosaminiltransferasas/metabolismo , Procesamiento Proteico-Postraduccional , Sialiltransferasas/metabolismo , Acilación , Secuencia de Aminoácidos , Animales , Células CHO , Línea Celular , Secuencia Conservada , Cricetulus , Cisteína/metabolismo , Dimerización , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Mutación , N-Acetilgalactosaminiltransferasas/química , N-Acetilgalactosaminiltransferasas/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Filogenia , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Sialiltransferasas/química , Sialiltransferasas/genética , beta-Galactosida alfa-2,3-SialiltransferasaAsunto(s)
Anticuerpos Monoclonales/metabolismo , Membrana Celular/metabolismo , Endocitosis/inmunología , Gangliósido G(M1)/metabolismo , Gangliósidos/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/inmunología , Animales , Afinidad de Anticuerpos , Autoantígenos/inmunología , Autoantígenos/metabolismo , Transporte Biológico , Células CHO , Línea Celular Tumoral , Membrana Celular/inmunología , Cricetulus , Endosomas/inmunología , Endosomas/metabolismo , Gangliósido G(M1)/inmunología , Gangliósidos/inmunología , Expresión Génica , Humanos , Cinética , Ratones , Ratones Noqueados , Imagen Molecular , Neuronas/citología , Neuronas/inmunología , Neuronas/metabolismo , Unión Proteica , Imagen de Lapso de TiempoRESUMEN
ST3Gal-II, a type II transmembrane protein, is the main mammalian sialyltransferase responsible for GD1a and GT1b ganglioside biosynthesis in brain. It contains two putative N-glycosylation sites (Asn(92) and Asn(211)). Whereas Asn(92) is only conserved in mammalian species, Asn(211) is highly conserved in mammals, birds and fish. The present study explores the occupancy and relevance for intracellular trafficking and enzyme activity of these potential N-glycosylations in human ST3Gal-II. We found that ST3Gal-II distributes along the Golgi complex, mainly in proximal compartments. By pharmacological, biochemical and site-directed mutagenesis, we observed that ST3Gal-II is mostly N-glycosylated at Asn(211) and that this co-translational modification is critical for its exit from the endoplasmic reticulum and proper Golgi localization. The individual N-glycosylation sites had different effects on ST3Gal-II enzymatic activity. Whereas the N-glycan at position Asn(211) seems to negatively influence the activity of the enzyme using both glycolipid and glycoprotein as acceptor substrates, the single N-glycan mutant at Asn(92) had only a moderate effect. Lastly, we demonstrated that the N-terminal ST3Gal-II domain containing the cytosolic, transmembrane and stem region (amino acids 1-51) is able to drive a protein reporter out of the endoplasmic reticulum and to retain it in the Golgi complex. This suggests that the C-terminal domain of ST3Gal-II depends on N-glycosylation to attain an optimum conformation for proper exit from the endoplasmic reticulum, but it does not represent an absolute requirement for Golgi complex retention of the enzyme.
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Retículo Endoplásmico/enzimología , Aparato de Golgi/enzimología , Sialiltransferasas/metabolismo , Animales , Asparagina/genética , Asparagina/metabolismo , Células CHO , Cricetinae , Cricetulus , Retículo Endoplásmico/genética , Evolución Molecular , Glicosilación , Aparato de Golgi/genética , Humanos , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Sialiltransferasas/genética , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
Gangliosides are sialic acid-containing glycolipids expressed on plasma membranes from nearly all vertebrate cells. The expression of ganglioside GD3, which plays essential roles in normal brain development, decreases in adults but is up regulated in neuroectodermal and epithelial derived cancers. R24 antibody, directed against ganglioside GD3, is a validated tumor target which is specifically endocytosed and accumulated in endosomes. Here, we exploit the internalization feature of the R24 antibody for the selective delivery of saporin, a ribosome-inactivating protein, to GD3-expressing cells [human (SK-Mel-28) and mouse (B16) melanoma cells and Chinese hamster ovary (CHO)-K1 cells]. This immunotoxin showed a specific cytotoxicity on tumor cells grew on 2D monolayers, which was further evident by the lack of any effect on GD3-negative cells. To estimate the potential antitumor activity of R24-saporin complex, we also evaluated the effect of the immunotoxin on the clonogenic growth of SK-Mel-28 and CHO-K1(GD3+) cells cultured in attachment-free conditions. A drastic growth inhibition (>80-90%) of the cell colonies was reached after 3 days of immunotoxin treatment. By the contrary, colonies continue to growth at the same concentration of the immuntoxin, but in the absence of R24 antibody, or in the absence of both immunotoxin and R24, undoubtedly indicating the specificity of the effect observed. Thus, the ganglioside GD3 emerge as a novel and attractive class of cell surface molecule for targeted delivery of cytotoxic agents and, therefore, provides a rationale for future therapeutic intervention in cancer.
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Anticuerpos/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Gangliósidos/metabolismo , Inmunotoxinas/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/metabolismo , Animales , Células CHO , Proliferación Celular , Cricetinae , Cricetulus , Endosomas/metabolismo , Gangliósidos/inmunología , Humanos , Inmunotoxinas/farmacocinética , Ratones , Microscopía Confocal , Microscopía Fluorescente , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacocinética , Saporinas , Sales de Tetrazolio , TiazolesRESUMEN
Altered networks of gene regulation underlie many pathologies, including cancer. There are several proteins in cancer cells that are turned either on or off, which dramatically alters the metabolism and the overall activity of the cell, with the complex machinery of enzymes involved in the metabolism of glycolipids not being an exception. The aberrant glycosylation of glycolipids on the surface of the majority of cancer cells, associated with increasing evidence about the functional role of these molecules in a number of cellular physiological pathways, has received considerable attention as a convenient immunotherapeutic target for cancer treatment. This has resulted in the development of a substantial number of passive and active immunotherapies, which have shown promising results in clinical trials. More recently, antibodies to glycolipids have also emerged as an attractive tool for the targeted delivery of cytotoxic agents, thereby providing a rationale for future therapeutic interventions in cancer. This review first summarizes the cellular and molecular bases involved in the metabolic pathway and expression of glycolipids, both in normal and tumor cells, paying particular attention to sialosylated glycolipids (gangliosides). The current strategies in the battle against cancer in which glycolipids are key players are then described.