RESUMO
Monoclonal antibodies targeting the immune checkpoint PD-1 have provided significant clinical benefit across a number of solid tumors, with differences in efficacy and toxicity profiles possibly related to their intrinsic molecular properties. Here, we report that camrelizumab and cemiplimab engage PD-1 through interactions with its fucosylated glycan. Using a combination of protein and cell glycoengineering, we demonstrate that the two antibodies bind preferentially to PD-1 with core fucose at the asparagine N58 residue. We then provide evidence that the concentration of fucosylated PD-1 in the blood of non-small-cell lung cancer patients varies across different stages of disease. This study illustrates how glycoprofiling of surface receptors and related circulating forms can inform the development of differentiated antibodies that discriminate glycosylation variants and achieve enhanced selectivity, and paves the way toward the implementation of personalized therapeutic approaches.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Inibidores de Checkpoint Imunológico , Receptor de Morte Celular Programada 1 , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Glicosilação , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
Glioblastoma (GB) is the most aggressive primary malignant brain tumor and is associated with short survival. O-GlcNAcylation is an intracellular glycosylation that regulates protein function, enzymatic activity, protein stability, and subcellular localization. Aberrant O-GlcNAcylation is related to the tumorigenesis of different tumors, and mounting evidence supports O-GlcNAc transferase (OGT) as a potential therapeutic target. Here, we used two human GB cell lines alongside primary human astrocytes as a non-tumoral control to investigate the role of O-GlcNAcylation in cell proliferation, cell cycle, autophagy, and cell death. We observed that hyper O-GlcNAcylation promoted increased cellular proliferation, independent of alterations in the cell cycle, through the activation of autophagy. On the other hand, hypo O-GlcNAcylation inhibited autophagy, promoted cell death by apoptosis, and reduced cell proliferation. In addition, the decrease in O-GlcNAcylation sensitized GB cells to the chemotherapeutic temozolomide (TMZ) without affecting human astrocytes. Combined, these results indicated a role for O-GlcNAcylation in governing cell proliferation, autophagy, cell death, and TMZ response, thereby indicating possible therapeutic implications for treating GB. These findings pave the way for further research and the development of novel treatment approaches which may contribute to improved outcomes and increased survival rates for patients facing this challenging disease.
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Cell surface glycosylation has a variety of functions, and its dysregulation in cancer contributes to impaired signaling, metastasis and the evasion of the immune responses. Recently, a number of glycosyltransferases that lead to altered glycosylation have been linked to reduced anti-tumor immune responses: B3GNT3, which is implicated in PD-L1 glycosylation in triple negative breast cancer, FUT8, through fucosylation of B7H3, and B3GNT2, which confers cancer resistance to T cell cytotoxicity. Given the increased appreciation of the relevance of protein glycosylation, there is a critical need for the development of methods that allow for an unbiased interrogation of cell surface glycosylation status. Here we provide an overview of the broad changes in glycosylation at the surface of cancer cell and describe selected examples of receptors with aberrant glycosylation leading to functional changes, with emphasis on immune checkpoint inhibitors, growth-promoting and growth-arresting receptors. Finally, we posit that the field of glycoproteomics has matured to an extent where large-scale profiling of intact glycopeptides from the cell surface is feasible and is poised for discovery of new actionable targets against cancer.
Assuntos
Glicopeptídeos , Neoplasias de Mama Triplo Negativas , Humanos , Glicosilação , Membrana Celular , Proteínas de TransporteRESUMO
Colorectal cancer (CRC) remains a leading cause of cancer-related deaths despite being the most preventable and treatable forms of cancer when caught early through screening. There is an unmet need for novel screening approaches with improved accuracy, less invasiveness, and reduced costs. In recent years, evidence has accumulated around particular biological events that happen during the adenoma-to-carcinoma transition, especially focusing on precancerous immune responses in the colonic crypt. Protein glycosylation plays a central role in driving those responses, and recently, numerous reports have been published on how aberrant protein glycosylation both in colonic tissue and on circulating glycoproteins reflects these precancerous developments. The complex field of glycosylation, which exceeds complexity of proteins by several orders of magnitude, can now be studied primarily because of the availability of new high-throughput technologies such as mass spectrometry and artificial intelligence-powered data processing. This has now opened new avenues for studying novel biomarkers for CRC screening. This review summarizes the early events taking place from the normal colon mucosa toward adenoma and adenocarcinoma formation and associated critical protein glycosylation phenomena, both on the tissue level and in the circulation. These insights will help establish an understanding in the interpretation of novel CRC detection modalities that involve high-throughput glycomics.
Assuntos
Adenoma , Neoplasias Colorretais , Lesões Pré-Cancerosas , Humanos , Glicosilação , Inteligência Artificial , Neoplasias Colorretais/patologia , Adenoma/diagnóstico , Adenoma/patologia , Lesões Pré-Cancerosas/patologiaRESUMO
BACKGROUND AND PURPOSE: Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Because pathogen-derived neuraminidase (NEU) stimulates neutrophils, we investigated whether host NEU can be targeted to regulate the neutrophil dysregulation observed in severe infections. EXPERIMENTAL APPROACH: The effects of NEU inhibitors on lipopolysaccharide (LPS)-stimulated neutrophils from healthy donors or COVID-19 patients were determined by evaluating the shedding of surface sialic acids, cell activation, and reactive oxygen species (ROS) production. Re-analysis of single-cell RNA sequencing of respiratory tract samples from COVID-19 patients also was carried out. The effects of oseltamivir on sepsis and betacoronavirus-induced acute lung injury were evaluated in murine models. KEY RESULTS: Oseltamivir and zanamivir constrained host NEU activity, surface sialic acid release, cell activation, and ROS production by LPS-activated human neutrophils. Mechanistically, LPS increased the interaction of NEU1 with matrix metalloproteinase 9 (MMP-9). Inhibition of MMP-9 prevented LPS-induced NEU activity and neutrophil response. In vivo, treatment with oseltamivir fine-tuned neutrophil migration and improved infection control as well as host survival in peritonitis and pneumonia sepsis. NEU1 also is highly expressed in neutrophils from COVID-19 patients, and treatment of whole-blood samples from these patients with either oseltamivir or zanamivir reduced neutrophil overactivation. Oseltamivir treatment of intranasally infected mice with the mouse hepatitis coronavirus 3 (MHV-3) decreased lung neutrophil infiltration, viral load, and tissue damage. CONCLUSION AND IMPLICATIONS: These findings suggest that interplay of NEU1-MMP-9 induces neutrophil overactivation. In vivo, NEU may serve as a host-directed target to dampen neutrophil dysfunction during severe infections.
Assuntos
COVID-19 , Sepse , Humanos , Camundongos , Animais , Oseltamivir/efeitos adversos , Zanamivir/efeitos adversos , Neuraminidase/metabolismo , Neuraminidase/farmacologia , Neutrófilos , Metaloproteinase 9 da Matriz/metabolismo , Espécies Reativas de Oxigênio , Lipopolissacarídeos/farmacologia , Sepse/induzido quimicamenteRESUMO
At cell surface gangliosides might associate with signal transducers proteins, grown factor receptors, integrins, small G-proteins and tetraspanins establishing microdomains, which play important role in cell adhesion, cell activation, motility, and growth. Previously, we reported that GM2 and GM3 form a heterodimer that interacts with the tetraspanin CD82, controlling epithelial cell mobility by inhibiting integrin-hepatocyte growth factor-induced cMet tyrosine kinase signaling. By using molecular dynamics simulations to study the molecular basis of GM2/GM3 interaction we demonstrate, here, that intracellular levels of Ca2+ mediate GM2/GM3 complexation via electrostatic interaction with their carboxyl groups, while hydrogen bonds between the ceramide groups likely aid stabilizing the complex. The presence of GM2/GM3 complex alters localization of CD82 on cell surface and therefore downstream signalization. These data contribute for the knowledge of how glycosylation may control signal transduction and phenotypic changes.
Assuntos
Gangliosídeo G(M3) , Proteína Kangai-1 , Adesão Celular , Movimento Celular , Proteína Kangai-1/metabolismo , Transdução de SinaisRESUMO
Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase (NEU)-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral NEU inhibitors constrain host NEU activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils. In vivo, treatment with Oseltamivir results in infection control and host survival in peritonitis and pneumonia models of sepsis. Single-cell RNA sequencing re-analysis of publicly data sets of respiratory tract samples from critical COVID-19 patients revealed an overexpression of NEU1 in infiltrated neutrophils. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of cell surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors can serve as host-directed interventions to dampen neutrophil dysfunction in severe infections.
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Dietary habits have been associated with alterations of the human gut resident microorganisms contributing to obesity, diabetes and cancer1. In Western diets, red meat is a frequently eaten food2, but long-term consumption has been associated with increased risk of disease3,4. Red meat is enriched in N-glycolylneuraminic acid (Neu5Gc) that cannot be synthesized by humans5. However, consumption can cause Neu5Gc incorporation into cell surface glycans6, especially in carcinomas4,7. As a consequence, an inflammatory response is triggered when Neu5Gc-containing glycans encounter circulating anti-Neu5Gc antibodies8,9. Although bacteria can use free sialic acids as a nutrient source10-12, it is currently unknown if gut microorganisms contribute to releasing Neu5Gc from food. We found that a Neu5Gc-rich diet induces changes in the gut microbiota, with Bacteroidales and Clostridiales responding the most. Genome assembling of mouse and human shotgun metagenomic sequencing identified bacterial sialidases with previously unobserved substrate preference for Neu5Gc-containing glycans. X-ray crystallography revealed key amino acids potentially contributing to substrate preference. Additionally, we verified that mouse and human sialidases were able to release Neu5Gc from red meat. The release of Neu5Gc from red meat using bacterial sialidases could reduce the risk of inflammatory diseases associated with red meat consumption, including colorectal cancer4 and atherosclerosis13.
Assuntos
Bactérias/enzimologia , Dieta , Microbioma Gastrointestinal , Ácidos Neuramínicos/metabolismo , Neuraminidase/genética , Polissacarídeos/metabolismo , Carne Vermelha/análise , Animais , Bactérias/classificação , Bacteroides/enzimologia , Bacteroides/genética , Clostridiales/enzimologia , Clostridiales/genética , Cristalografia por Raios X , Fezes/química , Fezes/microbiologia , Feminino , Humanos , Masculino , Metagenômica , Camundongos , Camundongos Endogâmicos C57BL , Neuraminidase/metabolismo , Polissacarídeos/químicaRESUMO
CD43 (leukosialin) is a large sialoglycoprotein abundantly expressed on the surface of most cells from the hematopoietic lineage. CD43 is directly involved in the contact between cells participating in a series of events such as signaling, adherence and host parasite interactions. In this study we examined the role of CD43 in the immune response against Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, a potential life-threatening illness endemic in 21 Latin American countries according to the WHO. The acute stage of infection is marked by intense parasitemia and cardiac tissue parasitism, resulting in the recruitment of inflammatory cells and acute damage to the heart tissue. We show here that CD43-/- mice were more resistant to infection due to increased cytotoxicity of antigen specific CD8+ T cells and reduced inflammatory infiltration in the cardiac tissue, both contributing to lower cardiomyocyte damage. In addition, we demonstrate that the induction of acute myocarditis involves the engagement of CD43 cytoplasmic tripeptide sequence KRR to ezrin-radixin-moiesin cytoskeletal proteins. Together, our results show the participation of CD43 in different events involved in the pathogenesis of T. cruzi infection, contributing to a better overall understanding of the mechanisms underlying the pathogenesis of acute chagasic cardiomyopathy.
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Doença de Chagas/metabolismo , Inflamação/patologia , Leucossialina/metabolismo , Miocárdio/patologia , Animais , Antígenos de Protozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Doença de Chagas/imunologia , Doença de Chagas/patologia , Citotoxicidade Imunológica , Suscetibilidade a Doenças , Masculino , Camundongos Endogâmicos C57BL , Mutação/genética , Miocardite/imunologia , Miocardite/parasitologia , Miocardite/patologia , Parasitemia/imunologia , Fagócitos/patologia , Baço/imunologia , Análise de SobrevidaRESUMO
Tumors are formed by several cell types interacting in a complex environment of soluble and matrix molecules. The crosstalk between the cells and extracellular components control tumor fate. Macrophages are highly plastic and diverse immune cells that are known to be key regulators of this complex network, which is mostly because they can adjust their metabolism and reprogram their phenotype and effector function. Here, we review the studies that disclose the central role of metabolism and tumor microenvironment in shaping the phenotype and function of macrophages, highlighting the importance of the hexosamine biosynthetic pathway. We further discuss growing evidence of nutrient-sensitive protein modifications such as O-GlcNAcylation and extracellular glycosylation in the function and polarization of tumor-associated macrophages.
Assuntos
Macrófagos/imunologia , Macrófagos/metabolismo , Neoplasias/imunologia , Neoplasias/metabolismo , Animais , Glicosilação , Humanos , FenótipoRESUMO
While infectious agents have typical host preferences, the noninvasive enteric bacterium Vibrio cholerae is remarkable for its ability to survive in many environments, yet cause diarrheal disease (cholera) only in humans. One key V. cholerae virulence factor is its neuraminidase (VcN), which releases host intestinal epithelial sialic acids as a nutrition source and simultaneously remodels intestinal polysialylated gangliosides into monosialoganglioside GM1. GM1 is the optimal binding target for the B subunit of a second virulence factor, the AB5 cholera toxin (Ctx). This coordinated process delivers the CtxA subunit into host epithelia, triggering fluid loss via cAMP-mediated activation of anion secretion and inhibition of electroneutral NaCl absorption. We hypothesized that human-specific and human-universal evolutionary loss of the sialic acid N-glycolylneuraminic acid (Neu5Gc) and the consequent excess of N-acetylneuraminic acid (Neu5Ac) contributes to specificity at one or more steps in pathogenesis. Indeed, VcN was less efficient in releasing Neu5Gc than Neu5Ac. We show enhanced binding of Ctx to sections of small intestine and isolated polysialogangliosides from human-like Neu5Gc-deficient Cmah-/- mice compared to wild-type, suggesting that Neu5Gc impeded generation of the GM1 target. Human epithelial cells artificially expressing Neu5Gc were also less susceptible to Ctx binding and CtxA intoxication following VcN treatment. Finally, we found increased fluid secretion into loops of Cmah-/- mouse small intestine injected with Ctx, indicating an additional direct effect on ion transport. Thus, V. cholerae evolved into a human-specific pathogen partly by adapting to the human evolutionary loss of Neu5Gc, optimizing multiple steps in cholera pathogenesis.
Assuntos
Evolução Biológica , Cólera/microbiologia , Suscetibilidade a Doenças , Células Epiteliais/metabolismo , Oxigenases de Função Mista/fisiologia , Ácidos Neuramínicos/metabolismo , Vibrio cholerae/classificação , Animais , Cólera/metabolismo , Cólera/patologia , Células Epiteliais/patologia , Feminino , Humanos , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Intestino Delgado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade da Espécie , Vibrio cholerae/patogenicidadeRESUMO
Epithelial to mesenchymal transition (EMT) is a developmental program reactivated by tumor cells that leads to the switch from epithelial to mesenchymal phenotype. During EMT, cells are transcriptionally regulated to decrease E-cadherin expression while expressing mesenchymal markers such as vimentin, fibronectin, and N-cadherin. Growing body of evidences suggest that cells engaged in EMT undergo a metabolic reprograming process, redirecting glucose flux toward hexosamine biosynthesis pathway (HBP), which fuels aberrant glycosylation patterns that are extensively observed in cancer cells. HBP depends on nutrient availability to produce its end product UDP-GlcNAc, and for this reason is considered a metabolic sensor pathway. UDP-GlcNAc is the substrate used for the synthesis of major types of glycosylation, including O-GlcNAc and cell surface glycans. In general, the rate limiting enzyme of HBP, GFAT, is overexpressed in many cancer types that present EMT features as well as aberrant glycosylation. Moreover, altered levels of O-GlcNAcylation can modulate cell morphology and favor EMT. In this review, we summarize some of the current knowledge that correlates glucose metabolism, aberrant glycosylation and hyper O-GlcNAcylation supported by HBP that leads to EMT activation. Developmental Dynamics 247:481-491, 2018. © 2017 Wiley Periodicals, Inc.
Assuntos
Plasticidade Celular , Transição Epitelial-Mesenquimal , Redes e Vias Metabólicas , Animais , Glicosilação , Hexosaminas/biossíntese , HumanosRESUMO
The immunomodulatory receptor Siglec-3/CD33 influences risk for late-onset Alzheimer's disease (LOAD), an apparently human-specific post-reproductive disease. CD33 generates two splice variants: a full-length CD33M transcript produced primarily by the "LOAD-risk" allele and a shorter CD33m isoform lacking the sialic acid-binding domain produced primarily from the "LOAD-protective" allele. An SNP that modulates CD33 splicing to favor CD33m is associated with enhanced microglial activity. Individuals expressing more protective isoform accumulate less brain ß-amyloid and have a lower LOAD risk. How the CD33m isoform increases ß-amyloid clearance remains unknown. We report that the protection by the CD33m isoform may not be conferred by what it does but, rather, from what it cannot do. Analysis of blood neutrophils and monocytes and a microglial cell line revealed that unlike CD33M, the CD33m isoform does not localize to cell surfaces; instead, it accumulates in peroxisomes. Cell stimulation and activation did not mobilize CD33m to the surface. Thus, the CD33m isoform may neither interact directly with amyloid plaques nor engage in cell-surface signaling. Rather, production and localization of CD33m in peroxisomes is a way of diminishing the amount of CD33M and enhancing ß-amyloid clearance. We confirmed intracellular localization by generating a CD33m-specific monoclonal antibody. Of note, CD33 is the only Siglec with a peroxisome-targeting sequence, and this motif emerged by convergent evolution in toothed whales, the only other mammals with a prolonged post-reproductive lifespan. The CD33 allele that protects post-reproductive individuals from LOAD may have evolved by adaptive loss-of-function, an example of the less-is-more hypothesis.
Assuntos
Doença de Alzheimer/genética , Predisposição Genética para Doença , Macrófagos/metabolismo , Microglia/metabolismo , Neutrófilos/metabolismo , Polimorfismo de Nucleotídeo Único , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Alelos , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Motivos de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/patologia , Humanos , Lipopolissacarídeos/toxicidade , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Microglia/citologia , Microglia/imunologia , Microglia/patologia , N-Formilmetionina Leucil-Fenilalanina/toxicidade , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuraminidase/metabolismo , Neuraminidase/toxicidade , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/patologia , Peroxissomos/efeitos dos fármacos , Peroxissomos/metabolismo , Peroxissomos/patologia , Filogenia , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico/efeitos dos fármacos , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/química , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genéticaRESUMO
One of the most consistent epidemiological associations between diet and human disease risk is the impact of red meat consumption (beef, pork, and lamb, particularly in processed forms). While risk estimates vary, associations are reported with all-cause mortality, colorectal and other carcinomas, atherosclerotic cardiovascular disease, type II diabetes, and possibly other inflammatory processes. There are many proposed explanations for these associations, some long discussed in the literature. Attempts to explain the effects of red meat consumption have invoked various red meat-associated agents, including saturated fat, high salt intake, Trimethylamine-N-oxide (TMAO) generation by microbiota, and environmental pollutants contaminating red meat, none of which are specific for red meat. Even the frequently mentioned polycyclic aromatic carcinogens arising from high temperature cooking methods are not red meat specific, as these are also generated by grilling poultry or fish, as well as by other forms of cooking. The traditional explanations that appear to be more red meat specific invoke the impact of N-nitroso compounds, heme iron, and the potential of heme to catalyze endogenous nitrosation. However, heme can be denatured by cooking, high levels of plasma hemopexin will block its tissue delivery, and much higher amounts of heme likely originate from red blood cell breakdown in vivo. Therefore, red meat-derived heme could only contribute to colorectal carcinoma risk, via direct local effects. Also, none of these mechanisms explain the apparent human propensity i.e., other carnivores have not been reported at high risk for all these diseases. A more recently proposed hypothesis involves infectious agents in beef from specific dairy cattle as agents of colorectal cancer. We have also described another mechanistic explanation for the human propensity for risk of red-meat associated diseases that is consistent with most observations: metabolic incorporation of a non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) into the tissues of red meat consumers and the subsequent interaction with inflammation-provoking antibodies against this "xenoautoantigen". Overall, we conclude that while multiple mechanisms are likely operative, many proposed theories to date are not specific for red meat, and that the viral and xenoautoantigen theories deserve further consideration. Importantly, there are potential non-toxic dietary antidotes, if the xenoautoantigen theory is indeed correct.
Assuntos
Aterosclerose , Neoplasias Colorretais , Dieta/efeitos adversos , Ácido N-Acetilneuramínico , Carne Vermelha/efeitos adversos , Animais , Aterosclerose/epidemiologia , Bovinos , Humanos , Modelos Biológicos , Ácido N-Acetilneuramínico/efeitos adversos , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Fatores de RiscoRESUMO
Lectin galactoside-binding soluble 3 binding protein (LGALS3BP, also called Mac-2 binding protein) is a heavily glycosylated secreted molecule that has been shown previously to be up-regulated in many cancers and has been implicated in tumor metastatic processes, as well as in other cell adhesion and immune functions. The CD33-related subset of sialic acid-binding immunoglobulin-like lectins (Siglecs) consists of immunomodulatory molecules that have recently been associated with the modulation of immune responses to cancer. Because up-regulation of Siglec ligands in cancer tissue has been observed, the characterization of these cancer-associated ligands that bind to inhibitory CD33-related Siglecs could provide novel targets for cancer immunomodulatory therapy. Here we used affinity chromatography of tumor cell extracts to identify LGALS3BP as a novel sialic acid-dependent ligand for human Siglec-9 and for other immunomodulatory Siglecs, such as Siglec-5 and Siglec-10. In contrast, the mouse homolog Siglec-E binds to murine LGALS3BP with lower affinity. LGALS3BP has been observed to be up-regulated in human colorectal and prostate cancer specimens, particularly in the extracellular matrix. Finally, LGALS3BP was able to inhibit neutrophil activation in a sialic acid- and Siglec-dependent manner. These findings suggest a novel immunoinhibitory function for LGALS3BP that might be important for immune evasion of tumor cells during cancer progression.
Assuntos
Antígenos de Neoplasias/biossíntese , Biomarcadores Tumorais/biossíntese , Proteínas de Transporte/biossíntese , Neoplasias Colorretais/metabolismo , Glicoproteínas/biossíntese , Fatores Imunológicos/biossíntese , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Células HEK293 , Humanos , Fatores Imunológicos/genética , Fatores Imunológicos/imunologia , Masculino , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Evasão Tumoral , Regulação para Cima/imunologiaRESUMO
Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development, several issues regarding their pluripotency, differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study, we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that, upon spontaneous differentiation, iPS-MBMC present high amounts of terminal ß-galactopyranoside residues, pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation, prompting an ectoderm commitment. Exposed ß-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface, which could modulate intercellular or intracellular interactions. Together, our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and, therefore, the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Glicoproteínas de Membrana/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Ácido N-Acetilneuramínico/metabolismoRESUMO
Growing evidences indicate that aberrant glycosylation can modulate tumor cell invasion and metastasis. The process termed "epithelial-mesenchymal transition" (EMT) provides a basic experimental model to shed light on this complex process. The EMT involves a striking decline in epithelial markers, accompanied by enhanced expression of mesenchymal markers, culminating in cell morphology change and increased cell motility. Few recent studies have established the participation glycosylation during EMT. Studies now come into knowledge brought to light the involvement of a site-specific O-glycosylation in the IIICS domain of human oncofetal fibronectin (onfFN) during the EMT process. Herein we show that high glucose induces EMT in A549 cells as demonstrated by TGF-ß secretion, cell morphology changes, increased cellular motility and the emergence of mesenchymal markers. The hyperglycemic conditions increased onfFN protein levels, promoted an up regulation of mRNA levels for ppGalNAc-T6 and FN IIICS domain, which contain the hexapeptide (VTHPGY) required for onfFN biosynthesis. Glucose effect involves hexosamine (HBP) biosynthetic pathway as overexpression of glutamine: fructose-6-phosphate amidotransferase increases mesenchymal markers, onfFN levels and mRNA levels for FN IIICS domain. In summary, our results demonstrate, for the first time that the metabolism of glucose through HBP promotes O-glycosylation of the oncofetal form of FN during EMT modulating tumorogenesis.
Assuntos
Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibronectinas/metabolismo , Glucose/farmacologia , Sequência de Aminoácidos , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Hexosaminas/biossíntese , Humanos , Hiperglicemia/patologia , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Dados de Sequência Molecular , Transferases de Grupos Nitrogenados/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Commonly found at the outermost ends of complex carbohydrates in extracellular medium or on outer cell membranes, sialic acids play important roles in a myriad of biological processes. Mammals synthesize sialic acid through a complex pathway, but Trypanosoma cruzi, the agent of Chagas' disease, evolved to obtain sialic acid from its host through a trans-sialidase (TcTS) reaction. Studies of the parasite cell surface architecture and biochemistry indicate that a unique system comprising sialoglycoproteins and sialyl-binding proteins assists the parasite in several functions including parasite survival, infectivity, and host-cell recognition. Additionally, TcTS activity is capable of extensively remodeling host cell glycomolecules, playing a role as virulence factor. This review presents the state of the art of parasite sialobiology, highlighting how the interplay between host and parasite sialic acid helps the pathogen to evade host defense mechanisms and ensure lifetime host parasitism.
RESUMO
Upon activation, cytotoxic CD8(+) T lymphocytes are desialylated exposing beta-galactose residues in a physiological change that enhances their effector activity and that can be monitored on the basis of increased binding of the lectin peanut agglutinin. Herein, we investigated the impact of sialylation mediated by trans-sialidase, a specific and unique Trypanosoma transglycosylase for sialic acid, on CD8(+) T cell response of mice infected with T. cruzi. Our data demonstrate that T. cruzi uses its trans-sialidase enzyme to resialylate the CD8(+) T cell surface, thereby dampening antigen-specific CD8(+) T cell response that might favor its own persistence in the mammalian host. Binding of the monoclonal antibody S7, which recognizes sialic acid-containing epitopes on the 115-kDa isoform of CD43, was augmented on CD8(+) T cells from ST3Gal-I-deficient infected mice, indicating that CD43 is one sialic acid acceptor for trans-sialidase activity on the CD8(+) T cell surface. The cytotoxic activity of antigen-experienced CD8(+) T cells against the immunodominant trans-sialidase synthetic peptide IYNVGQVSI was decreased following active trans-sialidase-mediated resialylation in vitro and in vivo. Inhibition of the parasite's native trans-sialidase activity during infection strongly decreased CD8(+) T cell sialylation, reverting it to the glycosylation status expected in the absence of parasite manipulation increasing mouse survival. Taken together, these results demonstrate, for the first time, that T. cruzi subverts sialylation to attenuate CD8(+) T cell interactions with peptide-major histocompatibility complex class I complexes. CD8(+) T cell resialylation may represent a sophisticated strategy to ensure lifetime host parasitism.
Assuntos
Antígenos de Protozoários/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Peptídeos/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença de Chagas/enzimologia , Doença de Chagas/genética , Doença de Chagas/imunologia , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Glicosilação , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Leucossialina/genética , Leucossialina/imunologia , Leucossialina/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ácido N-Acetilneuramínico/genética , Ácido N-Acetilneuramínico/imunologia , Neuraminidase/imunologia , Peptídeos/genética , Peptídeos/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Sialiltransferases/genética , Sialiltransferases/imunologia , Sialiltransferases/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
We herein describe the synthesis and characterization of nine new 1,3,4-thiadiazolium-2-phenylamine chlorides derived from natural piperine. We evaluate their toxic effects against the different evolutive forms of Trypanosoma cruzi, and the host cell (murine macrophages). The results obtained show that mesoionic hydrochloride MI possesses the best activity profile. Compound MI may be a prototype for use in the development of a new chemotherapeutic agent with high efficiency, which may be employed in the treatment of Chagas' disease.