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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.
Gene ; 761: 145049, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32791092

RESUMO

Breast cancer (BRCA) is a highly heterogeneous disease due to the complicated microenvironment in the tumor, making the treatment benefits varied. Therefore, this study aims to identify a gene signature in the tumor microenvironment (TME) associated with the prognosis of BRCA patients. We downloaded the immune, stromal, and proliferation (ISP)-associated genes from the literature on BRCA. mRNA expression and clinical information obtained from The Cancer Genome Atlas (TCGA) were performed to identify the initial biomarker. Furthermore, we validated the robustness of the gene signature in the independent validation data set GSE20685. A four-gene signature in TME, including CD74, MMP9, RPA3, and SHCBP1, was constructed to predict the overall survival of BRCA. The survival time of the high-risk group was significantly worse than that of the low-risk group. Univariate and multivariate Cox regression analysis showed that our four-gene ISP signature was an independent prognostic factor in TCGA and GSE20685 data sets. The AUC suggested that our four-gene ISP signature was comparable to TNM classification at predicting the overall survival of BRCA patients. Interestingly, BRCA patients with high-risk scores were more likely to be associated with stromal and proliferation of cancer. In contrast, those with high-risk scores were more likely to be associated with tumor immunity-related pathway. We found an innovative biomarker in TME to predict the prognosis of BRCA. This signal might reflect the imbalance of TME and provide potential biomarkers for the individualized and precise treatment of BRCA.


Assuntos
Neoplasias da Mama/genética , Microambiente Tumoral/genética , Adulto , Antígenos CD/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/metabolismo , Proteínas de Ligação a DNA/genética , Bases de Dados Genéticas , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Haplótipos/genética , Humanos , Metaloproteinase 9 da Matriz/genética , Pessoa de Meia-Idade , Prognóstico , RNA Longo não Codificante/genética , Fatores de Risco , Proteínas Adaptadoras da Sinalização Shc/genética , Sialiltransferases/genética
4.
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
5.
Biochem J ; 477(6): 1179-1201, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32141499

RESUMO

Fucosyltransferase 8 (FUT8) and ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) are glycosyltransferases that catalyze α1,6-fucosylation and α2,6-sialylation, respectively, in the mammalian N-glycosylation pathway. They are aberrantly expressed in various human diseases. FUT8 is non-glycosylated but is responsible for the fucosylation of ST6GAL1. However, the mechanism for the interaction between these two enzymes is unknown. In this study, we show that serum levels of α2,6-sialylated N-glycans are increased in Fut8-/- mice, whereas the mRNA and protein levels of ST6GAL1 are unchanged in mouse live tissues. The level of α2,6-sialylation on IgG was also enhanced in Fut8-/- mice along with ST6GAL1 catalytic activity increase in both serum and liver. Moreover, it was observed that ST6GAL1 prefers non-fucosylated substrates. Interestingly, increased core fucosylation accompanied by a reduction in α2,6-sialylation, was detected in rheumatoid arthritis patient serum. These findings provide new insight into the interactions between FUT8 and ST6GAL1.


Assuntos
Antígenos CD/genética , Fucosiltransferases/deficiência , Fucosiltransferases/genética , Sialiltransferases/deficiência , Sialiltransferases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Feminino , Fucose/genética , Fucose/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Humanos , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade
6.
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
7.
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
8.
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
9.
PLoS One ; 15(2): e0229269, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084196

RESUMO

Psychiatric disorders including depression and anxiety comprise a broad range of conditions with different symptoms. We have developed a mouse model of depression/anxiety in mice deficient in the St3gal4 gene. In this study, we performed a comparative analysis of urinary volatile organic compounds (VOCs) in St3gal4-deficient (St3gal4-KO) and wild-type mice using gas chromatography-mass spectrometry, and we screened 18 putative VOCs. Principal component analysis (PCA) based on these VOCs identified a major group of 11 VOCs, from which two groups were clarified by hierarchical clustering analysis. One group including six VOCs (pentanoic acid, 4-methyl-, ethyl ester; 3-heptanone, 6-methyl; benzaldehyde; 5,9-undecadien-2-ol, 6,10-dimethyl; and unknown compounds RI1291 and RI1237) was correlated with the startle response (r = 0.620), which is related to an unconscious defensive response. The other group including two VOCs (beta-farnesene and alpha-farnesene) comprised pheromones which increased in KO mice. Next, male mice underwent a social behavior test with female mice in the estrus stage, showing reduced access of KO male mice to female mice. Comparative analysis of urinary VOCs before and after encounters revealed that the six VOCs were not changed by these encounters. However, in WT mice, the two farnesenes increased after the encounters, reaching the level observed in KO mice, which was not altered following the encounter. Taken together, these results indicated that St3gal4 was involved in modulating urinary VOCs. Moreover, VOC clusters discovered by comparison of St3gal4-KO mice with WT mice were correlated with differential emotional behaviors.


Assuntos
Ansiedade/urina , Depressão/urina , Metabolômica , Compostos Orgânicos Voláteis/urina , Animais , Ansiedade/metabolismo , Depressão/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Camundongos , Sialiltransferases/deficiência , Sialiltransferases/genética , Compostos Orgânicos Voláteis/metabolismo
10.
Am J Psychiatry ; 177(6): 526-536, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32046534

RESUMO

OBJECTIVE: Attention deficit hyperactivity disorder (ADHD) is a common, highly heritable neuropsychiatric disorder. ADHD often co-occurs with intellectual disability, and shared overlapping genetics have been suggested. The aim of this study was to identify novel ADHD genes by investigating whether genes carrying rare mutations linked to intellectual disability contribute to ADHD risk through common genetic variants. Validation and characterization of candidates were performed using Drosophila melanogaster. METHODS: Common genetic variants in a diagnostic gene panel of 396 autosomal intellectual disability genes were tested for association with ADHD risk through gene set and gene-wide analyses, using ADHD meta-analytic data from the Psychiatric Genomics Consortium for discovery (N=19,210) and ADHD data from the Lundbeck Foundation Initiative for Integrative Psychiatric Research for replication (N=37,076). The significant genes were functionally validated and characterized in Drosophila by assessing locomotor activity and sleep upon knockdown of those genes in brain circuits. RESULTS: The intellectual disability gene set was significantly associated with ADHD risk in the discovery and replication data sets. The three genes most consistently associated were MEF2C, ST3GAL3, and TRAPPC9. Performing functional characterization of the two evolutionarily conserved genes in Drosophila melanogaster, the authors found that their knockdown in dopaminergic (dMEF2) and circadian neurons (dTRAPPC9) resulted in increased locomotor activity and reduced sleep, concordant with the human phenotype. CONCLUSIONS: This study reveals that a large set of intellectual disability-related genes contribute to ADHD risk through effects of common alleles. Utilizing this continuity, the authors identified TRAPPC9, MEF2C, and ST3GAL3 as novel ADHD candidate genes. Characterization in Drosophila suggests that TRAPPC9 and MEF2C contribute to ADHD-related behavior through distinct neural substrates.


Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Locomoção/genética , Fatores de Regulação Miogênica/genética , Sialiltransferases/genética , Adulto , Idoso , Animais , Ritmo Circadiano , Neurônios Dopaminérgicos/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Humanos , Deficiência Intelectual/genética , Fatores de Transcrição MEF2/genética , Masculino , Pessoa de Meia-Idade , Neurônios/metabolismo , Sono/genética
11.
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
12.
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
13.
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
14.
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
15.
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
16.
J Oral Pathol Med ; 49(3): 253-259, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31747460

RESUMO

OBJECTIVE: Aberrant glycosylation, mainly sialylation and fucosylation, is recently considered as a major hallmark of cancer. Aberrant sialylation has long been associated with various neoplastic diseases. However, role of aberrant sialylation in oral cancer is still in its infancy. The present study aimed to examine mRNA expressions of α-2, 3, α-2, 6 sialyltransferase (ST) families and sialidase in 160 human oral cancer tissues. METHODS: mRNA expression of ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST3GAL6, ST6GAL1, and neuraminidase 3 (NEU3) was analyzed by RT-qPCR in 80 paired malignant and adjacent normal tissues from oral cancer patients. RESULTS: The results indicated significant (P ≤ .05) down-regulation of various STs (ST3GAL1, ST3GAL2, ST3GAL3, ST3GAL4, ST3GAL6, and ST6GAL1) and sialidases (NEU3) in malignant tissues as compared to adjacent normal tissues. Higher mRNA levels of ST3GAL2 and ST3GAL3 were significantly associated with advanced stage of the disease, lymph node involvement, and perineural invasion, which denote their role in progression and metastasis of oral cancer. Present study also revealed altered sialylation patterns according to anatomical site of the disease and tobacco habit. CONCLUSION: The study demonstrated significant role of elevated mRNA levels of ST3GAL2 and ST3GAL3 in disease progression and metastasis of oral carcinoma.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Sialiltransferases/genética , Adulto , Idoso , Carcinoma de Células Escamosas/enzimologia , Progressão da Doença , Feminino , Glicosilação , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/enzimologia , Neuraminidase/genética
17.
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
18.
J Natl Cancer Inst ; 112(4): 356-368, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31286138

RESUMO

BACKGROUND: Tumor growth can be addicted to vital oncogenes, but whether long noncoding RNAs (lncRNAs) are essential to cancer survival is largely uncharacterized. METHODS: We retrieved Gene Expression Omnibus datasets to identify lncRNA overexpression in 257 cancers vs 196 normal tissues and analyzed the association of ST8SIA6-AS1 (termed Aurora A/Polo-like-kinase 1 [PLK1]-associated lncRNA, APAL) with the clinical outcomes of multiple types of cancer from public RNA sequencing and microarray datasets as well as from in-house cancer cohorts. Loss- and gain-of-function experiments were performed to explore the role of APAL in cancers in vitro and in vivo. RNA pulldown and RNA immunoprecipitation were used to investigate APAL-interacting proteins. All statistical tests were two-sided. RESULTS: APAL is overexpressed in multiple human cancers associated with poor clinical outcome of patients. APAL knockdown causes mitotic catastrophe and massive apoptosis in human breast, lung, and pancreatic cancer cells. Overexpressing APAL accelerates cancer cell cycle progression, promotes proliferation, and inhibits chemotherapy-induced apoptosis. Mechanism studies show that APAL links up PLK1 and Aurora A to enhance Aurora A-mediated PLK1 phosphorylation. Notably, targeting APAL inhibits the growth of breast and lung cancer xenografts in vivo (MCF-7 xenografts: mean tumor weight, control = 0.18 g [SD = 0.03] vs APAL locked nucleic acids = 0.07 g [SD = 0.02], P < .001, n = 8 mice per group; A549 xenografts: mean tumor weight control = 0.36 g [SD = 0.10] vs APAL locked nucleic acids = 0.10 g [SD = 0.04], P < .001, n = 9 mice per group) and the survival of patient-derived breast cancer organoids in three-dimensional cultures. CONCLUSIONS: Our data highlight the essential role of lncRNA in cancer cell survival and the potential of APAL as an attractive therapeutic target for a broad-spectrum of cancers.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Longo não Codificante/metabolismo , Sialiltransferases/genética , Células A549 , Animais , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/genética , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Mitose/fisiologia , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Antissenso/genética , RNA Longo não Codificante/genética
19.
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
20.
Elife ; 82019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31872800

RESUMO

Lymph nodes (LNs) are a common site of metastasis in solid cancers, and cutaneous melanomas show inherent properties of LN colonization. However, interactions between LN stroma and pioneer metastatic cells during metastatic colonization remain largely uncharacterized. Here we studied mice implanted with GFP-expressing melanoma cells to decipher early LN colonization events. We show that Siglec1-expressing subcapsular sinus (SCS) macrophages provide anchorage to pioneer metastatic cells. We performed in vitro co-culture to demonstrate that interactions between hypersialylated cancer cells and Siglec1 drive the proliferation of cancer cells. When comparing the transcriptome profile of Siglec1-interacting cancer cells against non-Siglec1-interacting cancer cells, we detected enrichment in positive regulators of cell cycle progression. Further, knockout of St3gal3 sialyltransferase compromised the metastatic efficiency of tumor cells by reducing α-2,3-linked sialylation. Thus, the interaction between Siglec1-expressing SCS macrophages and pioneer metastatic cells drives cell cycle progression and enables efficient metastatic colonization.


Assuntos
Metástase Linfática/genética , Melanoma Experimental/genética , Melanoma/genética , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Neoplasias Cutâneas/genética , Animais , Carcinogênese/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática/patologia , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Melanoma/patologia , Melanoma Experimental/patologia , Camundongos , Sialiltransferases/genética , Neoplasias Cutâneas/patologia
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