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1.
Nat Commun ; 11(1): 5091, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037195

RESUMO

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands. The connections between Siglecs and human disease motivate improved methods to detect Siglec ligands. Here, we describe a new versatile set of Siglec-Fc proteins for glycan ligand detection. Enhanced sensitivity and selectivity are enabled through multimerization and avoiding Fc receptors, respectively. Using these Siglec-Fc proteins, Siglec ligands are systematically profiled on healthy and cancerous cells and tissues, revealing many unique patterns. Additional features enable the production of small, homogenous Siglec fragments and development of a quantitative ligand-binding mass spectrometry assay. Using this assay, the ligand specificities of several Siglecs are clarified. For CD33 (Siglec-3), we demonstrate that it recognizes both α2-3 and α2-6 sialosides in solution and on cells, which has implications for its link to Alzheimer's disease susceptibility. These soluble Siglecs reveal the abundance of their glycan ligands on host cells as self-associated molecular patterns.


Assuntos
Polissacarídeos/análise , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/química , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias da Mama/metabolismo , Células CHO , Cricetulus , Feminino , Células HEK293 , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Células K562 , Espectrometria de Massas , Polissacarídeos/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/isolamento & purificação , Ácidos Siálicos/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismo , Baço/citologia , Baço/metabolismo , Estreptavidina/metabolismo
2.
Org Biomol Chem ; 18(15): 2886-2892, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32236230

RESUMO

Terminal α-2,6-sialylation of N-glycans is a humanized glycosylation that affects the properties and efficacy of therapeutic glycoproteins. Fc di-sialylation (a biantennary N-glycan with two α-2,6-linked sialic acids) of IgG antibodies imparts them with enhanced anti-inflammatory activity and other roles. However, the microheterogeneity of N-glycoforms presents a challenge for therapeutic development. Therefore, controlled sialylation has drawn considerable attention, but direct access to well-defined di-sialylated antibodies remains limited. Herein, a one-pot three-enzyme protocol was developed by engineering a bacterial sialyltransferase to facilitate the modification of therapeutic antibodies with N-acetylneuraminic acid or its derivatives towards optimized glycosylation. To overcome the low proficiency of bacterial sialyltransferase in antibody remodeling, the Photobacterium sp. JT-ISH-224 α-2,6-sialyltransferase (Psp2,6ST) was genetically engineered by terminal truncation and site-directed mutagenesis based on its protein crystal structure. With the optimized reaction conditions and using activity-based screening of various Psp2,6ST variants, a truncated mutant Psp2,6ST (111-511)-His6 A235M/A366G was shown to effectively improve the catalytic efficiency of antibody di-sialylation. Herceptin and the donor substrate promiscuity allow the introduction of bioorthogonal modifications of N-acetylneuraminic acid into antibodies for site-specific conjugation. 2-AB hydrophilic interaction chromatography analysis of the released N-glycans and intact mass characterization confirmed the high di-sialylation of Herceptin via the optimized one-pot three-enzyme reaction. This study established a versatile enzymatic approach for producing highly di-sialylated IgG antibodies. It provides new insights into engineering bacterial sialyltransferase for adaptation to the enzymatic glycoengineering of therapeutic antibodies and the glycosite-specific conjugation of antibodies.


Assuntos
Anticorpos/metabolismo , Photobacterium/enzimologia , Engenharia de Proteínas , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo , Anticorpos/química , Sialiltransferases/genética
3.
Biochemistry ; 59(12): 1242-1251, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32163271

RESUMO

Ganglioside GM3 is a sialylated membrane-based glycosphingolipid that regulates insulin receptor signaling via direct association with the receptor. The level of expression of GM3 synthase (GM3S) and GM3 is increased in tissues of patients with diabetes and murine models of diabetes, and obesity-induced insulin resistance is attenuated in GM3S-deficient mice. Therefore, GM3S has been considered a therapeutic target for type II diabetes; however, no GM3S inhibitors have been reported to date. In this study, we established a high-throughput scintillation proximity assay that can detect GM3S activity to screen GM3S inhibitors from our original chemical library. We also established methods for detecting the activity of GM3S and another sialyltransferase, ST3Gal3, through direct measurement of the enzyme products using an automatic rapid solid-phase extraction system directly coupled to a mass spectrometer. Consequently, we successfully identified two different chemotypes of GM3S-selective inhibitors with a mixed mode of inhibition. We believe that these compounds can be further developed into drugs to treat or prevent diabetes as well as contribute to the development of the ganglioside research field.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Gangliosídeo G(M3)/biossíntese , Ensaios de Triagem em Larga Escala/métodos , Hipoglicemiantes/farmacologia , Sialiltransferases/antagonistas & inibidores , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Diabetes Mellitus Tipo 2/metabolismo , Ensaios Enzimáticos , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Hipoglicemiantes/uso terapêutico , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sialiltransferases/genética , Sialiltransferases/isolamento & purificação , Sialiltransferases/metabolismo , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos
4.
EMBO J ; 39(6): e102214, 2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32030804

RESUMO

Spinal cord microglia contribute to nerve injury-induced neuropathic pain. We have previously demonstrated that toll-like receptor 2 (TLR2) signaling is critical for nerve injury-induced activation of spinal cord microglia, but the responsible endogenous TLR2 agonist has not been identified. Here, we show that nerve injury-induced upregulation of sialyltransferase St3gal2 in sensory neurons leads to an increase in expression of the sialylated glycosphingolipid, GT1b. GT1b ganglioside is axonally transported to the spinal cord dorsal horn and contributes to characteristics of neuropathic pain such as mechanical and thermal hypersensitivity. Spinal cord GT1b functions as an TLR2 agonist and induces proinflammatory microglia activation and central sensitization. Pharmacological inhibition of GT1b synthesis attenuates nerve injury-induced spinal cord microglia activation and pain hypersensitivity. Thus, the St3gal2-GT1b-TLR2 axis may offer a novel therapeutic target for the treatment of neuropathic pain.


Assuntos
Gangliosídeos/metabolismo , Neuralgia/terapia , Traumatismos dos Nervos Periféricos/terapia , Transdução de Sinais , Receptor 2 Toll-Like/agonistas , Animais , Gangliosídeos/antagonistas & inibidores , Regulação da Expressão Gênica , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Neuralgia/etiologia , Traumatismos dos Nervos Periféricos/etiologia , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais , Sialiltransferases/genética , Sialiltransferases/metabolismo , Medula Espinal/metabolismo , Receptor 2 Toll-Like/metabolismo
5.
Mol Med Rep ; 21(3): 1449-1460, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32016470

RESUMO

Fibroblast growth factor receptors (FGFRs) have been implicated in the malignant transformation and chemoresistance of epithelial ovarian cancer; however, the underlying molecular mechanisms are poorly understood. Increased sialyltransferase activity that enhances protein sialylation is an important post­translational process promoting cancer progression and malignancy. In the present study, α2,6­sialyltransferase (ST6Gal­I) overexpression or knockdown cell lines were developed, and FGFR1 was examined to understand the effect of sialylation on migration and drug resistance, and the underlying mechanisms. It was identified that cells with ST6Gal­I overexpression had increased cell viability and migratory ability upon serum deprivation. Moreover, ST6Gal­I overexpression cells had strong resistance to paclitaxel, as demonstrated by low growth inhibition rate and cell apoptosis level. A mechanistic study showed that ST6Gal­I overexpression induced high α2,6­sialylation of FGFR1 and increased the expression of phospho­ERK1/2 and phospho­focal adhesion kinase. Further study demonstrated that the FGFR1 inhibitor PD173047 reduced cell viability and induced apoptosis; however, ST6Gal­I overexpression decreased the anticancer effect of PD173047. In addition, ST6Gal­I overexpression attenuated the effect of Adriamycin on cancer cells. Collectively, these results suggested that FGFR1 sialylation plays an important role in cell migration and drug chemoresistance in ovarian cancer cells.


Assuntos
Antígenos CD/metabolismo , Antineoplásicos/farmacologia , Carcinoma Epitelial do Ovário/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/tratamento farmacológico , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Sialiltransferases/metabolismo , Antígenos CD/genética , Apoptose , Biomarcadores/análise , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Feminino , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Paclitaxel/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Sialiltransferases/genética , Transdução de Sinais
6.
Int J Mol Sci ; 21(3)2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32033474

RESUMO

Gangliosides are constituents of the mammalian cell membranes and participate in the inflammatory response. However, little is known about the presence and enzymatic activity of ganglioside sialyltransferases at the cell surface of macrophages, one of the most important immune cells involved in the innate inflammatory process. In the present study, using biochemical and fluorescent microscopy approaches, we found that endogenous ST8Sia-I is present at the plasma membrane (ecto-ST8Sia-I) of murine macrophage RAW264.7 cells. Moreover, ecto-ST8Sia-I can synthetize GD3 ganglioside at the cell surface in lipopolysaccharide (LPS)-stimulated macrophages even when LPS-stimulated macrophages reduced the total ST8Sia-I expression levels. Besides, cotreatment of LPS with an inhibitor of nitric oxide (NO) synthase recovered the ecto-ST8Sia-I expression, suggesting that NO production is involved in the reduction of ST8Sia-I expression. The diminution of ST8Sia-I expression in LPS-stimulated macrophages correlated with a reduction of GD3 and GM1 gangliosides and with an increment of GD1a. Taken together, the data supports the presence and activity of sialyltransferases at the plasma membrane of RAW264.7 cells. The variations of ecto-ST8Sia-I and ganglioside levels in stimulated macrophages constitutes a promissory pathway to further explore the physiological role of this and others ganglioside metabolism-related enzymes at the cell surface during the immune response.


Assuntos
Membrana Celular/metabolismo , Gangliosídeo G(M1)/metabolismo , Gangliosídeos/metabolismo , Macrófagos/metabolismo , Sialiltransferases/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus/metabolismo , Lipogênese/fisiologia , Lipopolissacarídeos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Células RAW 264.7
7.
Int J Mol Sci ; 21(2)2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31947579

RESUMO

We identified and analyzed α2,8-sialyltransferases sequences among 71 ray-finned fish species to provide the first comprehensive view of the Teleost ST8Sia repertoire. This repertoire expanded over the course of Vertebrate evolution and was primarily shaped by the whole genome events R1 and R2, but not by the Teleost-specific R3. We showed that duplicated st8sia genes like st8sia7, st8sia8, and st8sia9 have disappeared from Tetrapods, whereas their orthologues were maintained in Teleosts. Furthermore, several fish species specific genome duplications account for the presence of multiple poly-α2,8-sialyltransferases in the Salmonidae (ST8Sia II-r1 and ST8Sia II-r2) and in Cyprinus carpio (ST8Sia IV-r1 and ST8Sia IV-r2). Paralogy and synteny analyses provided more relevant and solid information that enabled us to reconstruct the evolutionary history of st8sia genes in fish genomes. Our data also indicated that, while the mammalian ST8Sia family is comprised of six subfamilies forming di-, oligo-, or polymers of α2,8-linked sialic acids, the fish ST8Sia family, amounting to a total of 10 genes in fish, appears to be much more diverse and shows a patchy distribution among fish species. A focus on Salmonidae showed that (i) the two copies of st8sia2 genes have overall contrasted tissue-specific expressions, with noticeable changes when compared with human co-orthologue, and that (ii) st8sia4 is weakly expressed. Multiple sequence alignments enabled us to detect changes in the conserved polysialyltransferase domain (PSTD) of the fish sequences that could account for variable enzymatic activities. These data provide the bases for further functional studies using recombinant enzymes.


Assuntos
Sialiltransferases/genética , Vertebrados/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Biologia Computacional/métodos , Evolução Molecular , Peixes/genética , Peixes/metabolismo , Expressão Gênica , Loci Gênicos , Modelos Moleculares , Família Multigênica , Filogenia , Conformação Proteica , Sialiltransferases/química , Sialiltransferases/metabolismo , Relação Estrutura-Atividade , Vertebrados/metabolismo
8.
FASEB J ; 34(1): 881-897, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914669

RESUMO

The α2,3-sialylation of N-glycans is considered important but complicated because the functions of the three ß-galactoside α2,3-sialyltransferases, ST3GAL3, ST3GAL4, and ST3GAL6, could be compensating for one another. To distinguish their specific functions, we established each individual knockout (KO) cell line. Loss of either the ST3GAL3 or ST3GAL6 genes decreased cell proliferation and colony formation, as opposed to the effect in the ST3GAL4 KO cells. The phosphorylation levels of ERK and AKT were significantly suppressed in the ST3GAL6 KO and ST3GAL3 KO cells, respectively. The cell aggregations were clearly observed in the KO cells, particularly the ST3GAL3 KO and ST3GAL6 KO cells, and the expression levels of E-cadherin and claudin-1 were enhanced in both those cell lines, but were suppressed in the ST3GAL4 KO cells. Those alterations were reversed with an overexpression of each corresponding gene in rescued cells. Of particular interest, the α2,3-sialylation levels of ß1 integrin were clearly suppressed in the ST3GAL4 KO cells, but these were increased in the ST3GAL3 KO and ST3GAL6 KO cells, whereas the α2,3-sialylation levels of EGFR were significantly decreased in the ST3GAL6 KO cells. The decrease in α2,3-sialylation increased the α2,6-sialylation on ß1, but not EGFR. Furthermore, a cross-restoration of each of the three genes in ST3GAL6 KO cells showed that overexpression of ST3GAL6 sufficiently rescued the total α2,3-sialylation levels, cell morphology, and α2,3-sialylation of EGFR, whereas the α2,3-sialylation levels of ß1 were greatly enhanced by an overexpression of ST3GAL4. These results clearly demonstrate that the three α2,3-sialyltransferases modify characteristic target proteins and regulate cell biological functions in different ways.


Assuntos
Sialiltransferases/metabolismo , Caderinas/metabolismo , Linhagem Celular , Proliferação de Células/fisiologia , Glicosilação , Humanos , Fosforilação/fisiologia , Transporte Proteico , Sialiltransferases/genética
9.
Int J Mol Sci ; 21(3)2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31979110

RESUMO

In the scenario of personalized medicine, targeted therapies are currently the focus of cancer drug development. These drugs can block the growth and spread of tumor cells by interfering with key molecules involved in malignancy, such as receptor tyrosine kinases (RTKs). MET and Recepteur d'Origine Nantais (RON), which are RTKs frequently overactivated in gastric cancer, are glycoprotein receptors whose activation have been shown to be modulated by the cellular glycosylation. In this work, we address the role of sialylation in gastric cancer therapy using an innovative 3D high-throughput cell culture methodology that mimics better the in vivo tumor features. We evaluate the response to targeted treatment of glycoengineered gastric cancer cell models overexpressing the sialyltransferases ST3GAL4 or ST3GAL6 by subjecting 3D spheroids to the tyrosine kinase inhibitor crizotinib. We show here that 3D spheroids of ST3GAL4 or ST3GAL6 overexpressing MKN45 gastric cancer cells are less affected by the inhibitor. In addition, we disclose a potential compensatory pathway via activation of the Insulin Receptor upon crizotinib treatment. Our results suggest that cell sialylation, in addition of being involved in tumor progression, could play a critical role in the response to tyrosine kinase inhibitors in gastric cancer.


Assuntos
Crizotinibe/farmacologia , Receptores Proteína Tirosina Quinases/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/metabolismo , Sialiltransferases/metabolismo
10.
Transplantation ; 104(4): 675-681, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31634326

RESUMO

Although xenografts are one of the most attractive strategies for overcoming the shortage of organ donors, cellular rejection by macrophages is a substantial impediment to this procedure. It is well known that macrophages mediate robust immune responses in xenografts. Macrophages also express various inhibitory receptors that regulate their immunological function. Recent studies have shown that the overexpression of inhibitory ligands on porcine target cells results in the phosphorylation of tyrosine residues on intracellular immunoreceptor tyrosine-based inhibitory motifs on macrophages, leading to the suppression of xenogenic rejection by macrophages. It has also been reported that myeloid-derived suppressor cells, a heterogeneous population of immature myeloid cells, suppress not only NK and cytotoxic T lymphocyte cytotoxicity but also macrophage-mediated cytotoxicity. This review is focused on the recent findings regarding strategies for inhibiting xenogenic rejection by macrophages.


Assuntos
Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Imunidade Celular , Macrófagos/imunologia , Transplante Heterólogo/efeitos adversos , Animais , Antígeno CD47/genética , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Xenoenxertos/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/transplante , Fagocitose , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/imunologia , Proteína D Associada a Surfactante Pulmonar/metabolismo , Sialiltransferases/genética , Sialiltransferases/imunologia , Sialiltransferases/metabolismo , Transdução de Sinais , Resultado do Tratamento
11.
J Neurochem ; 152(3): 333-349, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31608978

RESUMO

In humans, variations in the polysialic acid-producing enzyme ST8SIA2 and disturbances in the cortical inhibitory system are associated with neurodevelopmental psychiatric disorders such as schizophrenia and autism. In mice, the ST8SIA2-dependent formation of polysialic acid during embryonic development is crucial for the establishment of interneuron populations of the medial prefrontal cortex. However, the spatial pattern and the neurodevelopmental mechanisms of interneuron changes caused by loss of ST8SIA2 function have not been fully characterized. Here, we use immunohistochemical analysis to demonstrate that densities of parvalbumin-positive interneurons are not only reduced in the medial prefrontal cortex, but also in the adjacent motor and somatosensory cortices of St8sia2-deficient male mice. These reductions, however, were confined to the rostral parts of the analyzed region. Mice with conditional knockout of St8sia2 under the interneuron-specific Lhx6 promoter, but not mice with a deletion under the Emx1 promoter that targets cortical excitatory neurons and glia, largely recapitulated the area-specific changes of parvalbumin-positive interneurons in the anterior cortex of St8sia2-/- mice. Live imaging of interneuron migration in slice cultures of the developing cortex revealed a comparable reduction of directional persistence accompanied by increased branching of leading processes in slice cultures obtained from St8sia2-/- embryos or from embryos with interneuron-specific ablation of St8sia2. Together, the data demonstrate a cell-autonomous impact of ST8SIA2 on cortical interneuron migration and the distribution of parvalbumin-positive interneurons in the anterior cortex. This provides a neurodevelopmental mechanism for how dysregulation of ST8SIA2 may lead to disturbed inhibitory balance as observed in schizophrenia and autism.


Assuntos
Movimento Celular/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Interneurônios/metabolismo , Sialiltransferases/metabolismo , Animais , Interneurônios/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
12.
Cardiovasc Res ; 116(1): 114-126, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30854566

RESUMO

AIMS: Sialylation is up-regulated during the development of cardiac hypertrophy. Sialyltransferase7A (Siat7A) mRNA is consistently over-expressed in the hypertrophic left ventricle of hypertensive rats independently of genetic background. The aims of this study were: (i) to detect the Siat7A protein levels and its roles in the pathological cardiomyocyte hypertrophy; (ii) to elucidate the effect of sialylation mediated by Siat7A on the transforming-growth-factor-ß-activated kinase (TAK1) expression and activity in cardiomyocyte hypertrophy; and (iii) to clarify hypoxia-inducible factor 1 (HIF-1) expression was regulated by Siat7A and transactivated TAK1 expression in cardiomyocyte hypertrophy. METHODS AND RESULTS: Siat7A protein level was increased in hypertrophic cardiomyocytes of human and rats subjected to chronic infusion of angiotensin II (ANG II). Delivery of adeno-associated viral (AAV9) bearing shRNA against rat Siat7A into the left ventricular wall inhibited ventricular hypertrophy. Cardiac-specific Siat7A overexpression via intravenous injection of an AAV9 vector encoding Siat7A under the cardiac troponin T (cTNT) promoter aggravated cardiac hypertrophy in ANG II-treated rats. In vitro, Siat7A knockdown inhibited the induction of Sialyl-Tn (sTn) antigen and cardiomyocyte hypertrophy stimulated by ANG II. Mechanistically, ANG II induced the activation of TAK1-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling in parallel to up-regulation of Siat7A in hypertrophic cardiomyocytes. Siat7A knockdown inhibited activation of TAK1-NF-κB pathway. Interestingly, HIF-1α expression was increased in cardiomyocytes stimulated by ANG II but decreased after Siat7A knockdown. HIF-1α knockdown efficiently decreased TAK1 expression. ChIP and luciferase assays showed that HIF-1α transactivated the TAK1 promoter region (nt -1285 to -1274 bp) in the cardiomyocytes following ANG II stimulus. CONCLUSION: Siat7A was up-regulated in hypertrophic myocardium and promoted cardiomyocyte hypertrophy via activation of the HIF-1α-TAK1-NF-κB pathway.


Assuntos
Angiotensina II , Hipertrofia Ventricular Esquerda/enzimologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Miócitos Cardíacos/enzimologia , Sialiltransferases/metabolismo , Função Ventricular Esquerda , Remodelação Ventricular , Animais , Linhagem Celular , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Humanos , Hipertrofia Ventricular Esquerda/induzido quimicamente , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Esquerda/prevenção & controle , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , MAP Quinase Quinase Quinases/genética , Masculino , Miócitos Cardíacos/patologia , Interferência de RNA , Ratos Wistar , Sialiltransferases/genética , Transdução de Sinais
13.
J Biotechnol ; 307: 87-97, 2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31697975

RESUMO

Alpha-1-antitrypsin (A1AT) is an abundant serum inhibitor of serine proteases. A1AT deficiency is a common genetic disorder which is currently treated with augmentation therapies. These treatments involve weekly injections of patients with purified plasma-derived A1AT. Such therapies can be extremely expensive and rely on plasma donors. Hence, large-scale production of recombinant A1AT (rA1AT) could greatly benefit these patients, as it could decrease the cost of treatments, reduce biosafety concerns and ensure quantitative and qualitative controls of the protein. In this report, we sought to produce α2,6-sialylated rA1AT with our cumate-inducible stable CHO pool expression system. Our different CHO pools could reach volumetric productivities of 1,2 g/L. The human α2,6-sialyltransferase was stably expressed in these cells in order to mimic elevated α2,6-sialylation levels of native A1AT protein. Sialylation of the recombinant protein was stable over the duration of the fed-batch production phase and was higher in a pool where cells were sorted and enriched by FACS based on cell-surface α2,6-sialylation. Addition of ManNAc to the cell culture media during production enhanced both α2,3 and α2,6 A1AT sialylation levels whereas addition of 2F-peracetylfucose potently inhibited fucosylation of the protein. Finally, we demonstrated that rA1AT proteins exhibited human neutrophil elastase inhibitory activities similar to the commercial human plasma-derived A1AT.


Assuntos
Elastase de Leucócito/antagonistas & inibidores , Sialiltransferases/metabolismo , alfa 1-Antitripsina/metabolismo , Animais , Medicamentos Biossimilares/metabolismo , Células CHO , Cricetulus , Humanos , Proteínas Recombinantes , Sialiltransferases/genética , alfa 1-Antitripsina/genética
14.
Cancer Immunol Res ; 8(2): 167-178, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31831633

RESUMO

Patients with ulcerative colitis have an increased risk of developing colitis-associated colon cancer (CACC). Changes in glycosylation of the oncoprotein MUC1 commonly occur in chronic inflammation, including ulcerative colitis, and this abnormally glycosylated MUC1 promotes cancer development and progression. It is not known what causes changes in glycosylation of MUC1. Gene expression profiling of myeloid cells in inflamed and malignant colon tissues showed increased expression levels of inflammatory macrophage-associated cytokines compared with normal tissues. We analyzed the involvement of macrophage-associated cytokines in the induction of aberrant MUC1 glycoforms. A coculture system was used to examine the effects of M1 and M2 macrophages on glycosylation-related enzymes in colon cancer cells. M2-like macrophages induced the expression of the glycosyltransferase ST6GALNAC1, an enzyme that adds sialic acid to O-linked GalNAc residues, promoting the formation of tumor-associated sialyl-Tn (sTn) O-glycans. Immunostaining of ulcerative colitis and CACC tissue samples confirmed the elevated number of M2-like macrophages as well as high expression of ST6GALNAC1 and the altered MUC1-sTn glycoform on colon cells. Cytokine arrays and blocking antibody experiments indicated that the macrophage-dependent ST6GALNAC1 activation was mediated by IL13 and CCL17. We demonstrated that IL13 promoted phosphorylation of STAT6 to activate transcription of ST6GALNAC1. A computational model of signaling pathways was assembled and used to test IL13 inhibition as a possible therapy. Our findings revealed a novel cellular cross-talk between colon cells and macrophages within the inflamed and malignant colon that contributes to the pathogenesis of ulcerative colitis and CACC.See related Spotlight on p. 160.


Assuntos
Colite Ulcerativa/imunologia , Colite/complicações , Colo/imunologia , Neoplasias do Colo/imunologia , Glicopeptídeos/metabolismo , Células Mieloides/imunologia , Sialiltransferases/genética , Linhagem Celular Tumoral , Colite/imunologia , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Colo/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Biologia Computacional , Citocinas/genética , Citocinas/metabolismo , Glicosilação , Humanos , Inflamação/metabolismo , Interleucina-13/metabolismo , Ativação de Macrófagos/imunologia , Fator de Transcrição STAT6/metabolismo , Sialiltransferases/metabolismo , Transdução de Sinais
15.
Glycobiology ; 30(2): 95-104, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31584066

RESUMO

Three missense variants of ST3GAL3 are known to be responsible for a congenital disorder of glycosylation determining a neurodevelopmental disorder (intellectual disability/epileptic encephalopathy). Here we report a novel nonsense variant, p.Y220*, in two dichorionic infant twins presenting a picture of epileptic encephalopathy with impaired neuromotor development. Upon expression in HEK-293T cells, the variant appears totally devoid of enzymatic activity in vitro, apparently accumulated with respect to the wild-type or the missense variants, as detected by western blot, and in large part properly localized in the Golgi apparatus, as assessed by confocal microscopy. Both patients were found to efficiently express the CA19.9 antigen in the serum despite the total loss of ST3GAL3 activity, which thus appears replaceable from other ST3GALs in the synthesis of the sialyl-Lewis a epitope. Kinetic studies of ST3GAL3 revealed a strong preference for lactotetraosylceramide as acceptor and gangliotetraosylceramide was also efficiently utilized in vitro. Moreover, the p.A13D missense variant, the one maintaining residual sialyltransferase activity, was found to have much lower affinity for all suitable substrates than the wild-type enzyme with an overall catalytic efficiency almost negligible. Altogether the present data suggest that the apparent redundancy of ST3GALs deduced from knock-out mouse models only partially exists in humans. In fact, our patients lacking ST3GAL3 activity synthesize the CA19.9 epitope sialyl-Lewis a, but not all glycans necessary for fine brain functions, where the role of minor gangliosides deserves further attention.


Assuntos
Antígenos Glicosídicos Associados a Tumores , Erros Inatos do Metabolismo dos Carboidratos , Epilepsia , Regulação da Expressão Gênica , Mutação de Sentido Incorreto , Sialiltransferases , Gêmeos Dizigóticos , Antígenos Glicosídicos Associados a Tumores/biossíntese , Antígenos Glicosídicos Associados a Tumores/genética , Erros Inatos do Metabolismo dos Carboidratos/genética , Erros Inatos do Metabolismo dos Carboidratos/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Feminino , Humanos , Lactente , Masculino , Sialiltransferases/genética , Sialiltransferases/metabolismo
16.
Medicina (Kaunas) ; 55(12)2019 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-31795149

RESUMO

BACKGROUND AND OBJECTIVES: Sialylation plays important roles in tumor progression. Our present study aimed to demonstrate the alteration of sialylation and its role in cholangiocarcinoma (CCA). MATERIALS AND METHODS: The α2,3- and α2,6-sialylation in CCA tissue was analyzed by lectin-histochemistry using Maackia amurensis lectin-II (MAL-II) and Sambucus nigra agglutinin (SNA). CCA cell lines were treated with the pan-sialylation inhibitor 3Fax-peracetyl-Neu5Ac (3F-Sia) followed by proliferation and chemosensitivity assays. RESULTS: MAL-II binding α2,3-Sialylated Glycan (MAL-SG) and SNA binding α2,6-Sialylated Glycan (SNA-SG) were both elevated in CCA compared with hyperplastic/dysplastic (HP/DP) and normal bile ducts (NBD). The positive staining for MAL-SG or SNA-SG were found in 82% (61/74) of the CCA cases. Higher expression of MAL-SG in CCA was associated with shorter survival of the patients. The median survival of patients with high and low MAL-SG were 167 and 308 days, respectively, with overall survival of 233 days, suggesting the involvement of MAL-SG in CCA progression. MAL-SG expression of CCA cell lines was markedly decreased after treatment with 3F-Sia for 48 to 72 h. While proliferation of CCA cells were not affected by 3F-Sia treatment, their susceptibility to 5-fluorouracil (5-FU) was significantly enhanced. These results suggest that sialylation is involved in the development of 5-FU resistance and the sialylation inhibitor 3F-Sia can be used as a chemosensitizer for CCA. CONCLUSIONS: Sialylation is critically involved in the development of chemoresistance of CCA, and sialylation inhibitors may be used as a chemosensitizer in CCA treatment.


Assuntos
Antimetabólitos Antineoplásicos/farmacocinética , Neoplasias dos Ductos Biliares/mortalidade , Colangiocarcinoma/mortalidade , Fluoruracila/farmacocinética , Polissacarídeos/metabolismo , Sialoglicoproteínas/metabolismo , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/metabolismo , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Humanos , Maackia , Lectinas de Plantas , Sialiltransferases/metabolismo
17.
Sci Rep ; 9(1): 17993, 2019 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-31784620

RESUMO

Overexpression of hST3Gal1 leads to hypersialylation of cell-surface glycoconjugates, a cancer-associated condition that promotes cell growth, migration and invasion. Upregulation of this enzyme in ovarian cancer is linked to cancer progression and metastasis, contributing also to chemotherapy resistance. Strategies for preventing metastasis include the inhibition of hST3Gal1, which demands structure-based studies on its strict regioselectivity and substrate/donor preference. Herein we describe the contribution of various residues constituting donor CMP-Neu5Ac and acceptor Galß1-3GalNAc-R binding sites to catalysis. Removal of hydrogen bonds and/or stacking interactions among substrates and residues Y191, Y230, N147, S148 and N170 affected the enzyme's activity to a different extent, revealing the fine control needed for an optimal catalytic performance. To gain further understanding of the correlation among structure, activity and stability, the in vitro role of hST3Gal1 disulphide bonds was analysed. As expected, disruption of the Glycosyltransferase family 29 (GT29) invariant bond C142-C281, as well as the ST3Gal1 subfamily conserved disulphide C61-C139 inactivates the enzyme. While disulphide C59-C64 is not essential for function, its absence reduces the activity (kcat) for donor and acceptor substrates to about 67 and 72%, respectively, and diminishes the enzyme's melting temperature (Tm) by 7 °C.


Assuntos
Dissulfetos/química , Sialiltransferases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Sequência Conservada , Humanos , Ligação de Hidrogênio , Estrutura Molecular , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Sialiltransferases/química , Sialiltransferases/isolamento & purificação , Relação Estrutura-Atividade , Especificidade por Substrato
18.
Mol Med Rep ; 20(5): 4499-4506, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31702036

RESUMO

The aberrant expression of sialyltransferase has a role in cell differentiation, neoplastic transformation and the progression of various types of cancer. Our previous studies have shown that high expression of ß­galactoside­α2,3­sialyltransferase III (ST3Gal3) in the metastatic ovarian cancer cell line HO8910PM attenuated cisplatin­induced apoptosis. The present study demonstrated that paclitaxel­induced chemoresistance in ovarian cancer cells upregulated the expression of ST3Gal3 and reduced the activity of caspase­8/3. The results of the present study revealed that the endogenous levels of ST3Gal3 mRNA and protein were significantly higher in HO8910PM cells compared with SKOV3 cells. A higher expression of ST3Gal3 was correlated with an increased resistance to paclitaxel, while the downregulation of ST3Gal3 resulted in paclitaxel­induced apoptosis. Paclitaxel upregulated ST3Gal3 expression at the mRNA and protein levels in HO8910PM cells, but not in SKOV3 cells. Silencing of ST3Gal3 by small interfering RNA reversed these effects and increased the protein levels of caspase­8/3, which may contribute to paclitaxel­induced apoptosis. The results of the present study suggested that ST3Gal3 was a target for paclitaxel­-related resistance during ovarian cancer chemotherapy.


Assuntos
Caspase 3/metabolismo , Caspase 8/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas , Paclitaxel/farmacologia , Sialiltransferases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia
19.
Theranostics ; 9(24): 7431-7446, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31695778

RESUMO

Malignant transformation of gastric cells is accompanied by the deregulated expression of glycosyltransferases leading to the biosynthesis of tumor-associated glycans such as the sialyl-Lewis X antigen (SLex). SLex presence on cell surface glycoconjugates increases the invasive capacity of gastric cancer cells and is associated with tumor metastasis. ST3Gal IV enzyme is involved in the synthesis of SLex antigen and overexpressed in gastric carcinomas. Herein, we identified the glycoproteins carrying SLex in gastric cancer cells overexpressing ST3Gal IV enzyme and evaluated their biomarker potential for gastric carcinoma. Methods: SLex modified glycoproteins were identified applying western blot and mass spectrometry. Immunoprecipitation, proximity ligation assay (PLA), E-selectin binding assay and CRISPR/cas9 knockout experiments were performed to characterize the presence of SLex on the identified glycoprotein. Protein N-glycans of the SLex protein carrier were in deep analyzed by porous-graphitized-carbon liquid-chromatography and tandem mass spectrometry glycomics. In silico expression analysis of α2-3 sialyltransferase ST3Gal IV and SLex protein carrier was performed and the conjoint expression of the SLex modified glycoproteins evaluated by immunohistochemistry and PLA in a series of gastric carcinomas. Results: Carcinoembryonic antigen (CEA; CEACAM5) was identified and validated by different methodologies as a major carrier of SLex. N-glycomics of CEA revealed that complex N-glycans are capped with α2-3 linked sialic acid (Neu5Acα2-3Galß1-4GlcNAc). Data set analysis of ST3Gal IV and CEA showed that ST3Gal IV expression was associated with patient´s poor survival, whereas CEA did not show any prognostic value. The co-expression of both CEA and SLeX was observed in 86,3% of gastric carcinoma cases and 74,5% of the total cases displayed the conjoint CEA+SLex in situ PLA expression. This expression was associated with clinicopathological features of the tumors, including infiltrative pattern of tumor growth, presence of venous invasion and patient's poor survival. CEA immunoprecipitation from gastric carcinoma tissues also confirmed the presence of SLex. Conclusion: CEA is the major glycoprotein carrying SLex in gastric carcinoma and the conjoint detection of CEA-SLex is associated with aggressive tumor features highlighting its PLA detection as a biomarker of gastric cancer patient prognosis for theranostic applications.


Assuntos
Biomarcadores Tumorais/metabolismo , Antígeno Carcinoembrionário/metabolismo , Antígeno Sialil Lewis X/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Glicômica , Glicoproteínas/metabolismo , Humanos , Invasividade Neoplásica , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/farmacologia , Prognóstico , Sialiltransferases/metabolismo , Análise de Sobrevida
20.
Nat Commun ; 10(1): 5404, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776339

RESUMO

Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N-glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli. We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications.


Assuntos
Sistema Livre de Células/metabolismo , Engenharia de Proteínas/métodos , Proteínas/metabolismo , Antígenos CD/metabolismo , Escherichia coli/genética , Glicoproteínas/biossíntese , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Humanos , Redes e Vias Metabólicas , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Proteínas/genética , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo
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