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Visceral leishmaniasis (VL) is a clinical form of leishmaniasis with high mortality rates when not treated. Diagnosis suffers from invasive techniques and sub-optimal sensitivities. The current (affordable) treatment with pentavalent antimony as advised by the WHO is possibly harmful to the patient. There is need for an improved diagnosis to prevent possibly unnecessary treatment. N-glycan analysis may aid in diagnosis. We evaluated the N-glycan profiles from active VL, asymptomatic infections (ASYMP) and controls from non-endemic (NC) and endemic (EC) areas. Active VL has a distinct N-glycome profile that associates with disease severity. Our study suggests that the observed glycan signatures could be a valuable additive to diagnosis and assist in identifying possible markers of disease and understanding the pathogenesis of VL. Further studies are warranted to assess a possible future role of blood glycome analysis in active VL diagnosis and should aim at disease specificity.
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While altered protein glycosylation is regarded a trait of oral squamous cell carcinoma (OSCC), the heterogeneous and dynamic glycoproteome of tumor tissues from OSCC patients remain unmapped. To this end, we here employ an integrated multi-omics approach comprising unbiased and quantitative glycomics and glycoproteomics applied to a cohort of resected primary tumor tissues from OSCC patients with (n = 19) and without (n = 12) lymph node metastasis. While all tumor tissues displayed relatively uniform N-glycome profiles suggesting overall stable global N-glycosylation during disease progression, altered expression of six sialylated N-glycans was found to correlate with lymph node metastasis. Notably, glycoproteomics and advanced statistical analyses uncovered altered site-specific N-glycosylation revealing previously unknown associations with several clinicopathological features. Importantly, the glycomics and glycoproteomics data unveiled that comparatively high abundance of two core-fucosylated and sialylated N-glycans (Glycan 40a and Glycan 46a) and one N-glycopeptide from fibronectin were associated with low patient survival, while a relatively low abundance of N-glycopeptides from both afamin and CD59 were also associated with poor survival. This study provides insight into the complex OSCC tissue N-glycoproteome, thereby forming an important resource to further explore the underpinning disease mechanisms and uncover new prognostic glycomarkers for OSCC.
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Carcinoma de Células Escamosas , Neoplasias Bucais , Humanos , Glicosilação , Metástase Linfática , Glicopeptídeos/metabolismo , Proteoma/metabolismo , Polissacarídeos/análiseRESUMO
INTRODUCTION: Aberrant fucosylation is closely related to malignant transformation, cancer detection, and evaluation of treatment efficacy. The fucosylation process requires GDP-L-fucose, fucosyltransferases, and fucosidases. In gastric cancer (GC), fucosylation alterations were associated with tumor formation, metastasis inhibition, and multi-drug resistance. It is not clear whether tissue-specific transplantation antigen P35B (TSTA3) and alpha-L-fucosidase 2 (FUCA2) have any effect on the development of GC. MATERIALS AND METHODS: We used immunohistochemistry to assess the expression of TSTA3 and FUCA2 in 71 gastric adenocarcinoma samples and their relationship with clinicopathological parameters. RESULTS: TSTA3 expression was associated with lower histological grade I and II (P = 0.0120) and intestinal type Lauren classification (P = 0.0120). TSTA3 immunopositivity could predict Lauren's classification. Analysis of mRNA expression in GC validation cohorts corroborates the significant TSTA3 association with histological grade observed in our study. However, no associations were found between TSTA3 staining and overall survival. FUCA2 expression was markedly increased in GC tissues compared with non-tumoral tissues (P < 0.0001) and was associated with surgical staging III and IV (P = 0.0417) and advanced histological grade tumor states (P = 0.0125). CONCLUSIONS: Alterations of FUCA2 and TSAT3 immunoexpression could lay the basis for future studies using cell glycosylation as a biomarker for the planning of therapeutic strategy in primary gastric cancer.
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Adenocarcinoma , Cetona Oxirredutases , Neoplasias Gástricas , Humanos , alfa-L-Fucosidase/metabolismo , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/patologia , Adenocarcinoma/patologia , Biomarcadores , Biomarcadores Tumorais , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Cetona Oxirredutases/genética , Cetona Oxirredutases/metabolismoRESUMO
Diabetes Mellitus (DM) is both, correlated and a known risk factor for colorectal cancer (CRC). Besides favoring the incidence of CRC, DM also accelerates its progression, worsening its prognosis. Previously, hyperglycemia, the DM hallmark, has been shown to lead to aberrant glycosylation of CRC cells, heightening their malignancy both in vivo and in vitro. Here we use mass spectrometry to elucidate the composition and putative structures of N-glycans expressed by MC38 cultured in normoglycemic (LG) and hyperglycemic-like conditions (HG). N-glycans, 67, were identified in MC38 cells cultured in LG and HG. The cells grown in HG showed a greater abundance of N-glycans when compared to LNG cells, without changes in the proportion of sialylated, fucosylated and mannosylated N-glycans. Among the identified N-glycans, 16 were differentially expressed, mostly mannosylated and fucosylated, with a minority of them being sialylated. Metabolomics analysis indicates that the alterations observed in the N-glycosylation may be mostly due to increase of the activated monosaccharides pool, through an increased glucose entrance into the cells. The alterations found here corroborate data from the literature regarding the progression of CRC, advocating for development or repositioning of effective treatments against CRC in diabetic patients.
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Neoplasias do Colo , Hiperglicemia , Glicosilação , Humanos , Monossacarídeos/química , Polissacarídeos/químicaRESUMO
Amphibians secrete a complex array of molecules that shape their interactions with coinhabiting microorganisms and macroscopic predators. Glycans are a rapidly evolving and complex class of biomolecules implicated in intrinsic and extrinsic recognition events. Despite the numerous studies aiming at the biochemical characterization of anuran skin secretions, little is known about protein-linked oligosaccharides, their synthesis pathways, and their homing secreted glycoproteins. In the present report, LC-MS/MS was used to investigate the diversity of N- and O-linked oligosaccharides in the skin secretion of two South American frogs, Pithecopus azureus and Boana raniceps. Additionally, the enzymes responsible for glycan synthesis pathways were evaluated based on their skin tissue transcriptome. Our analyses allowed the annotation of various N- and O-glycan structures commonly found in vertebrate proteins. Paucimannosidic glycans were abundant in the skin secretion of both amphibians; however, hybrid and complex N-glycan structures were detected only in B. raniceps. A good correlation between the structures discovered in glycomic analyses and transcripts encoding enzymes necessary for their synthesis was obtained. Some transcripts such as those of MAN1A2, FUT8, and ST6GALNAC were found solely in B. raniceps. Finally, secreted N- and O- linked glycoproteins were predicted from the transcriptomic data, indicating that proteases and protease inhibitors are putative sources of the glycans described herein. Overall, our results show the presence of oligosaccharides in amphibians skin secretions and suggest that their diversity is species-specific, paving the way for novel perspectives involving amphibian evolution and ecology.
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Glicoproteínas , Espectrometria de Massas em Tandem , Animais , Anuros/metabolismo , Cromatografia Líquida , Glicoproteínas/metabolismo , Glicosilação , Oligossacarídeos/química , Polissacarídeos/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Extracellular vesicles (EVs) are lipid bilayer-enclosed particles involved in intercellular communication, delivery of biomolecules from donor to recipient cells, cellular disposal and homeostasis, potential biomarkers and drug carriers. The content of EVs includes DNA, lipids, metabolites, proteins, and microRNA, which have been studied in various diseases, such as cancer, diabetes, pregnancy, neurodegenerative, and cardiovascular disorders. EVs are enriched in glycoconjugates and exhibit specific glycosignatures. Protein glycosylation is a co- and post-translational modification (PTM) that plays an important role in the expression and function of exosomal proteins. N- and O-linked protein glycosylation has been mapped in exosomal proteins. The purpose of this review is to highlight the importance of glycosylation in EVs proteins. Initially, we describe the main PTMs in EVs with a focus on glycosylation. Then, we explore glycan-binding proteins describing the main findings of studies that investigated the glycosylation of EVs in cancer, pregnancy, infectious diseases, diabetes, mental disorders, and animal fluids. We have highlighted studies that have developed innovative methods for studying the content of EVs. In addition, we present works related to lipid glycosylation. We explored the content of studies deposited in public databases, such as Exocarta and Vesiclepedia. Finally, we discuss analytical methods for structural characterization of glycoconjugates and present an overview of the critical points of the study of glycosylation EVs, as well as perspectives in this field.
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Proteínas de Transporte/metabolismo , Comunicação Celular/fisiologia , Exossomos/metabolismo , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Vesículas Extracelulares/metabolismo , Glicosilação , Humanos , Neoplasias/patologia , Ligação Proteica/fisiologiaRESUMO
BACKGROUND: Type II Congenital Disorders of Glycosylation (CDG-II) are a group of diseases with challenging diagnostics characterized by defects in the processing of glycans in the Golgi apparatus. Mass Spectrometry (MS) has been a valuable tool in the definition of CDG-II subtypes. While some CDG-II subtypes are associated with specific N-glycan structures, others only produce changes in relative levels, reinforcing the demand for quantification methods. METHODS: Plasma samples from control individuals were pooled, derivatized with deuterated iodomethane (I-CD3), and used as internal standards for controls and patients whose glycans were derivatized with iodomethane (I-CH3), followed by MALDI MS, LC-MS and -MS/MS analyses. RESULTS: Total N-glycans from fifteen CDG-II patients were evaluated, and 4 cases with molecular diagnosis were considered in detail: 2ATP6V0A2-CDG siblings, and 2 MAN1B1-CDG patients, one of them carrying a previously undescribed p.Gly536Val mutation. CONCLUSIONS: Our methodology offers a feasible alternative to the current methods for CDG-II diagnosis by MS, which quantify glycan structures as fractions of the total summed signal across a mass spectrum, a strategy that lowers the variability of minor components. Moreover, given its sensitivity for less concentrated yet biologically relevant structures, it might assist the uncovering of novel diagnostic glycans in other CDG-II subtypes.
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Análise Química do Sangue/métodos , Defeitos Congênitos da Glicosilação/sangue , Polissacarídeos/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adolescente , Criança , Pré-Escolar , Defeitos Congênitos da Glicosilação/genética , Feminino , Genótipo , Humanos , Lactente , Masculino , MutaçãoRESUMO
The complexity of snake venoms has long been investigated to explore a myriad of biologically active proteins and peptides that are used for immobilizing or killing prey, and are responsible for the pathological effects observed on envenomation. Glycosylation is the main post-translational modification (PTM) of viperid venoms but currently there is little understanding of how protein glycosylation impacts the variation of venom proteomes. We have previously reported that Bothrops venom glycoproteomes contain a core of components that markedly define their composition and parallel their phylogenetic classification. Here we extend those observations to eight Bothrops species evaluating the N-glycomes by LC-MS as assigned cartoon structures and detailing those structures separately as methylated analogs using ion-trap mass spectrometry (MSn). Following ion disassembly through multiple steps provided sequence and linkage isomeric details that characterized 52 unique compositions in Bothrops venoms. These occurred as 60 structures, of which 26 were identified in the venoms of the Jararaca Complex (B. alcatraz, B. insularis, and B. jararaca), 20 in B. erythromelas, B. jararacussu, B. moojeni and B. neuwiedi venoms, and 22 in B. cotiara venom. Further, quantitative analysis of these N-glycans showed variable relative abundances in the venoms. For the first time a comprehensive set of N-glycan structures present in snake venoms are defined. Despite the fact that glycosylation is not template-defined, the N-glycomes of these venoms mirror the phylogeny cladograms of South American bothropoid snakes reported in studies on morphological, molecular data and feeding habits, exhibiting distinct molecular signatures for each venom. Considering the complexity of N-glycan moieties generally found in glycoproteins, characterized by different degrees of branching, isomer structures, and variable abundances, our findings point to these factors as another level of complexity in Bothrops venoms, features that could dramatically contribute to their distinct biological activities.
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Association between altered glycosylation patterns and poor prognosis in cancer points glycans as potential specific tumor markers. Most proteins are glycosylated and functionally arranged on cell surface and extracellular matrix, mediating interactions and cellular signaling. Thereby, aberrant glycans may be considered a pathological phenotype at least as important as changes in protein expression for cancer and other complex diseases. As most serum glycoproteins have hepatic origin, liver disease phenotypes, such as hepatocellular carcinoma (HCC), may present altered glycan profile and display important modifications. One of the prominent obstacles in HCC is the diagnostic in advanced stages when patients have several liver dysfunctions, limiting treatment options and life expectancy. The characterization of glycomic profiles in pathological conditions by means of mass spectrometry (MS) may lead to the discovery of early diagnostic markers using non-invasive approaches. MS is a powerful analytical technique capable of elucidating many glycobiological issues and overcome limitations of the serological markers currently applied in clinical practice. Therefore, MS-based glycomics of tumor biomarkers is a promising tool to increase early detection and monitoring of disease.
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Glycomics turned out to be a very extensive project where its subdivision is consequently emerging. This is seen by the growing number of terminologies used to define subprojects concerning particular classes of bioactive carbohydrates. Sulfated fucans (SFs) and sulfated galactans (SGs) are relatively new classes of sulfated polysaccharides (SPs) that occur mostly in marine organisms, and exhibit a broad range of medicinal effects. Their structures are taxonomically dependent, and their therapeutic actions include benefits in inflammation, coagulation, thrombosis, angiogenesis, cancer, oxidation, and infections. Some red algae, marine angiosperm and invertebrates express SPs of unique structures composed of regular repeating oligomeric units of well-defined sulfation patterns. This fine pattern of structural regularity is quite rare among any naturally occurring long SPs, and enables accurate structure-biofunction correlations. Seeing that, fucanomics and galactanomics may comprise distinguished glycomics subprojects. We hereby discuss the relevance that justifies the international recognition of these subprojects in the current glycomics age associated with the beneficial outcomes that these glycans may offer in drug development.