RESUMO
INTRODUCTION: N-glycosylation is a ubiquitous and variable posttranslational modification that regulates physiological functions of secretory and membrane-associated proteins and the dysregulation of glycosylation pathways is often associated with cancer growth and metastasis. Prostate-specific membrane antigen (PSMA) is an established biomarker for prostate cancer imaging and therapy. METHODS: Mass spectrometry was used to analyze the distribution of the site-specific glycoforms of PSMA in insect, human embryonic kidney, and prostate cancer cells, and in prostate tissue upon immunoaffinity enrichment. RESULTS: While recombinant PSMA expressed in insect cells was decorated mainly by paucimannose and high mannose glycans, complex, hybrid, and high mannose glycans were detected in samples from human cells and tissue. We noted an interesting spatial distribution of the glycoforms on the PSMA surface-high mannose glycans were the dominant glycoforms at the N459, N476, and N638 sequons facing the plasma membrane, while the N121, N195, and N336 sites, located at the exposed apical PSMA domain, carried primarily complex glycans. The presence of high mannose glycoforms at the former sequons likely results from the limited access of enzymes of the glycosynthetic pathway required for the synthesis of the complex structures. In line with the limited accessibility of membrane-proximal sites, no glycosylation was observed at the N51 site positioned closest to the membrane. CONCLUSIONS: Our study presents initial descriptive analysis of the glycoforms of PSMA observed in cell lines and in prostate tissue. It will hopefully stimulate further research into PSMA glycoforms in the context of tumor staging, noninvasive detection of prostate tumors, and the impact of glycoforms on physicochemical and enzymatic characteristics of PSMA in a tissue-specific manner.
Assuntos
Antígenos de Superfície/metabolismo , Glutamato Carboxipeptidase II/metabolismo , Polissacarídeos , Próstata , Neoplasias da Próstata , Biomarcadores Tumorais/análise , Linhagem Celular , Glicosilação , Humanos , Masculino , Espectrometria de Massas/métodos , Estadiamento de Neoplasias , Polissacarídeos/classificação , Polissacarídeos/metabolismo , Próstata/enzimologia , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-TraducionalRESUMO
In life science fields, database integration is progressing and contributing to collaboration between different research fields, including the glycosciences. The integration of glycan databases has greatly progressed collaboration worldwide with the development of the international glycan structure repository, GlyTouCan. This trend has increased the need for a tool by which researchers in various fields can easily search glycan structures from integrated databases. We have developed a web-based glycan structure search tool, SugarDrawer, which supports the depiction of glycans including ambiguity, such as glycan fragments which contain underdetermined linkages, and a database search for glycans drawn on the canvas. This tool provides an easy editing feature for various glycan structures in just a few steps using template structures and pop-up windows which allow users to select specific information for each structure element. This tool has a unique feature for selecting possible attachment sites, which is defined in the Symbol Nomenclature for Glycans (SNFG). In addition, this tool can input and output glycans in WURCS and GlycoCT formats, which are the most commonly-used text formats for glycan structures.
Assuntos
Bases de Dados Factuais , Internet , Polissacarídeos/genética , Software , Disciplinas das Ciências Biológicas , Humanos , Polissacarídeos/química , Polissacarídeos/classificação , Polissacarídeos/ultraestruturaRESUMO
Breast cancer is the most prevalent cancer in women. Early detection of this disease improves survival and therefore population screenings, based on mammography, are performed. However, the sensitivity of this screening modality is not optimal and new screening methods, such as blood tests, are being explored. Most of the analyses that aim for early detection focus on proteins in the bloodstream. In this study, the biomarker potential of total serum N-glycosylation analysis was explored with regard to detection of breast cancer. In an age-matched case-control setup serum protein N-glycan profiles from 145 breast cancer patients were compared to those from 171 healthy individuals. N-glycans were enzymatically released, chemically derivatized to preserve linkage-specificity of sialic acids and characterized by high resolution mass spectrometry. Logistic regression analysis was used to evaluate associations of specific N-glycan structures as well as N-glycosylation traits with breast cancer. In a case-control comparison three associations were found, namely a lower level of a two triantennary glycans and a higher level of one tetraantennary glycan in cancer patients. Of note, various other N-glycomic signatures that had previously been reported were not replicated in the current cohort. It was further evaluated whether the lack of replication of breast cancer N-glycomic signatures could be partly explained by the heterogenous character of the disease since the studies performed so far were based on cohorts that included diverging subtypes in different numbers. It was found that serum N-glycan profiles differed for the various cancer subtypes that were analyzed in this study.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Polissacarídeos/sangue , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , Polissacarídeos/classificação , Polissacarídeos/metabolismoRESUMO
Broad-spectrum antivirals are more needed than ever to provide treatment options for novel emerging viruses and for viruses that lack therapeutic options or have developed resistance. A large number of viruses rely on charge-dependent non-specific interactions with heparan sulfate (HS), a highly sulfated glycosaminoglycan (GAG), for attachment to cell surfaces to initiate cell entry. As such, inhibitors targeting virion-HS interactions have potential to have broad-spectrum antiviral activity. Previous research has explored organic and inorganic small molecules, peptides, and GAG mimetics to disrupt virion-HS interactions. Here we report antiviral activities against both enveloped (the herpesvirus human cytomegalovirus) and non-enveloped (adenovirus) DNA viruses for four defined marine sulfated glycans: a sulfated galactan from the red alga Botryocladia occidentalis; a sulfated fucan from the sea urchin Lytechinus variegatus, and a sulfated fucan and a fucosylated chondroitin sulfate from the sea cucumber Isostichopus badionotus. As evidenced by gene expression, time of addition, and treatment/removal assays, all four novel glycans inhibited viral attachment and entry, most likely through interactions with virions. The sulfated fucans, which both lack anticoagulant activity, had similar antiviral profiles, suggesting that their activities are not only due to sulfation content or negative charge density but also due to other physicochemical factors such as the potential conformational shapes of these carbohydrates in solution and upon interaction with virion proteins. The structural and chemical properties of these marine sulfated glycans provide unique opportunities to explore relationships between glycan structure and their antiviral activities.
Assuntos
Adenoviridae/efeitos dos fármacos , Antivirais/farmacologia , Citomegalovirus/efeitos dos fármacos , Polissacarídeos/farmacologia , Sulfatos/química , Organismos Aquáticos/química , Linhagem Celular , Heparitina Sulfato/farmacologia , Humanos , Polissacarídeos/química , Polissacarídeos/classificação , Rodófitas/química , Vírion/efeitos dos fármacos , Ligação Viral/efeitos dos fármacosRESUMO
Chronic lesions in the limbs of farm animals cause lameness due to chronic infection and inflammation. Exploratory treatments for chronic wounds in humans may be suitable for adaptation into the field of animal care. Specifically, antimicrobial linear polysaccharides like oxidized regenerated cellulose (ORC) and chitin/chitosan are biodegradable hemostats that are being explored to promote healing of chronic wounds but have not been directly compared using the same biological specimen. Despite their current use in humans, linear polysaccharides possess features that may preclude their use as biodegradable bandages. For example, ORC promotes inflammation when it remains in vivo and chitin/chitosan stimulate size-dependent proinflammatory responses. In order to assess the use of these materials to treat chronic wounds we have compared their effects on cellular toxicity and in stimulating the production of proinflammatory cytokines by bovine epidermal fibroblasts. While neither polysaccharide increased cell mortality, on average, they caused minor alterations in expression of proinflammatory cytokines from cells isolated from different animals. Both polysaccharides reduced expression of proinflammatory cytokines stimulated by microbial lipopolysaccharide. We conclude that the polysaccharides used in this study are relatively inert and may improve healing of chronic epidermal wounds in farm animals.
Assuntos
Citocinas/genética , Citocinas/imunologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Inflamação/imunologia , Lipopolissacarídeos/farmacologia , Polissacarídeos/farmacologia , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Celulose Oxidada/farmacologia , Quitina/farmacologia , Polissacarídeos/classificação , Pele/citologia , CicatrizaçãoRESUMO
Seaweed oligosaccharides possess great bioactivities. However, different microbial strains are required to degrade multiple polysaccharides due to their limited biodegradability, thereby increasing the cost and complexity of production. Shewanella sp. WPAGA9 was isolated from deep-sea sediments in this study. According to the genomic and biochemical analyses, the extracellular fermentation broth of WPAGA9 had versatile degradation abilities for three typical seaweed polysaccharides including agar, carrageenan, and alginate. The maximum enzyme activities of the extracellular fermentation broth of WPAGA9 were 71.63, 76.4, and 735.13 U/ml for the degradation of agar, alginate, and carrageenan, respectively. Moreover, multiple seaweed oligosaccharides can be produced by the extracellular fermentation broth of WPAGA9 under similar optimum conditions. Therefore, WPAGA9 can simultaneously degrade three types of seaweed polysaccharides under similar conditions, thereby greatly reducing the production cost of seaweed oligosaccharides. This finding indicates that Shewanella sp. WPAGA9 is an ideal biochemical tool for producing multiple active seaweed oligosaccharides at low costs and is also an important participant in the carbon cycle process of the deep-sea environment.
Assuntos
Fermentação , Sedimentos Geológicos/microbiologia , Polissacarídeos/metabolismo , Alga Marinha/metabolismo , Shewanella/química , Shewanella/metabolismo , Ágar/metabolismo , Alginatos/metabolismo , Carragenina/metabolismo , Oceanos e Mares , Oligossacarídeos/metabolismo , Polissacarídeos/classificação , Shewanella/enzimologia , Shewanella/isolamento & purificaçãoRESUMO
Glycans serve important roles in signaling events and cell-cell communication, and they are recognized by lectins, viruses and bacteria, playing a variety of roles in many biological processes. However, there was no system to organize the plethora of glycan-related data in the literature. Thus GlyTouCan (https://glytoucan.org) was developed as the international glycan repository, allowing researchers to assign accession numbers to glycans. This also aided in the integration of glycan data across various databases. GlyTouCan assigns accession numbers to glycans which are defined as sets of monosaccharides, which may or may not be characterized with linkage information. GlyTouCan was developed to be able to recognize any level of ambiguity in glycans and uniquely assign accession numbers to each of them, regardless of the input text format. In this manuscript, we describe the latest update to GlyTouCan in version 3.0, its usage, and plans for future development.
Assuntos
Biologia Computacional/métodos , Bases de Dados Factuais , Polissacarídeos/classificação , Software , Humanos , Cooperação Internacional , Internet , Polissacarídeos/análise , Polissacarídeos/química , Terminologia como AssuntoRESUMO
Phaeocystis globosa has become one of the primary causes of harmful algal bloom in coastal areas of southern China in recent years, and it poses a serious threat to the marine environment and other activities depending upon on it (e.g., aquaculture, cooling system of power plants), especially in the Beibu Gulf. We found colonies of P. globosa collected form Guangxi (China) were much larger than those obtained from Shantou cultured in lab. To better understand the causes of giant colonies formation, colonial cells collected from P. globosa GX strain (GX-C) and ST strain (ST-C) were separated by filtration. Morphological observations, phylogenetic analyses, rapid light-response curves, fatty acid profiling and transcriptome analyses of two type cells were performed in the laboratory. Although no differences in morphology and 18S rRNA sequences of these cells were observed, the colonies of GX strain (4.7 mm) are 30 times larger than those produced by the ST strain (300 µm). The rapid light-response curve of GX-C was greater than that of ST-C, consistent with the upregulated photosynthetic system, while the fatty acid content of GX-C was lower than that of ST-C, also consistent with the downregulated synthesis of fatty acids and the upregulated degradation of fatty acids. In summary, the increased energy generated by GX-C is allocated to promote the secretion of extracellular polysaccharides for colony formation. We performed a physiological and molecular assessment of the differences between the GX-C and ST-C strains, providing insights into the mechanisms of giant colonies formation in P. globosa.
Assuntos
Haptófitas/classificação , Proliferação Nociva de Algas/fisiologia , Redes e Vias Metabólicas/genética , Fotossíntese/genética , Transcriptoma , China , Ácidos Graxos/biossíntese , Ácidos Graxos/classificação , Haptófitas/genética , Haptófitas/metabolismo , Haptófitas/efeitos da radiação , Proliferação Nociva de Algas/efeitos da radiação , Luz , Transdução de Sinal Luminoso , Redes e Vias Metabólicas/efeitos da radiação , Oceano Pacífico , Fotossíntese/efeitos da radiação , Filogenia , Polissacarídeos/biossíntese , Polissacarídeos/classificação , RNA Ribossômico 18S/genéticaRESUMO
Characterizing polysaccharides with large molecular weights and isomeric heterogeneity with mass spectrometry (MS) is generally difficult. In this work, we demonstrate how coupling size exclusion chromatography (SEC) and high-resolution MS with source-induced dissociation (SID) can be used for the separation and direct structural evaluation of intact polysaccharides. The analytical method was successfully developed using dextran standards up to 3755 kDa. This method was used to separate naturally occurring plant polysaccharides based on size, after which numerous polysaccharide fragments were identified from the resulting MS spectra. The results provided strong evidence for structural diversity, complexity, and heterogeneity among polysaccharides. MS showed superior sensitivity and reliability for the polysaccharides in eluted fractions when compared to a refractive index detector. Putative compositions for the fragments were proposed based on exact mass values. The work demonstrated that SEC-SID-MS is a feasible alternative for obtaining valuable structural information from the analysis of intact polysaccharides.
Assuntos
Astragalus propinquus/química , Codonopsis/química , Dendrobium/química , Extratos Vegetais/química , Polissacarídeos/isolamento & purificação , Wolfiporia/química , Cromatografia em Gel/instrumentação , Cromatografia em Gel/métodos , Humanos , Peso Molecular , Polissacarídeos/química , Polissacarídeos/classificação , Pós , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
The effect of partial acid hydrolysis on the physical and chemical properties of galactomannan, arabinoxylan, and xyloglucan was investigated. Polysaccharides were treated at 50 °C with hydrochloric acid for 3-48 h. Portions of isopropanol (i-PrOH) were added sequentially to the hydrolyzates, resulting in fractions that were collected by centrifugation. As expected, a significant reduction of weight-average molecular weight (Mw) was observed with increasing hydrolysis time. Fractional precipitation was successfully applied to collect at least one polymer fraction with dispersity (D) close to one for each polysaccharide. The monosaccharide composition analysis showed that the partial hydrolysis usually lowered the relative amount of side chains, with the exception of galactomannan, where the composition remained largely unaffected. Estimation of the polymer conformation in solution, through evaluation of the Mark-Houwink parameter coefficient (α), confirmed that acid hydrolysis influenced the polysaccharides' conformation. It was demonstrated that acid treatment in dilute solution followed by fractional isopropanol precipitation is a method, extendible to a variety of polysaccharides, to obtain materials of decreased molecular weight and low dispersity with slightly altered overall composition and conformation.
Assuntos
Carboidratos/química , Mananas/química , Monossacarídeos/química , Polissacarídeos/química , Carboidratos/classificação , Carboidratos da Dieta/análise , Galactose/análogos & derivados , Ácido Clorídrico , Hidrólise , Peso Molecular , Polissacarídeos/classificação , ÁguaRESUMO
Virus infection is an intricate process that requires the concerted action of both viral and host cell components. Entry of viruses into cells is initiated by interactions between viral proteins and cell-surface receptors. Various cell-surface glycans function as initial, usually low-affinity attachment factors, providing a first anchor of the virus to the cell surface, and further facilitate high-affinity binding to virus-specific cell-surface receptors, while other glycans function as specific entry receptors themselves. It is now possible to rapidly identify specific glycan receptors using different techniques, define atomic-level structures of virus-glycan complexes, and study these interactions at the single-virion level. This review provides a detailed overview of the role of glycans in viral infection and highlights experimental approaches to study virus-glycan binding along with specific examples. In particular, we highlight the development of the atomic force microscope to investigate interactions with glycans at the single-virion level directly on living mammalian cells, which offers new perspectives to better understand virus-glycan interactions in physiologically relevant conditions.
Assuntos
Polissacarídeos/metabolismo , Vírion/fisiologia , Ligação Viral , Internalização do Vírus , Humanos , Microscopia de Força Atômica/métodos , Polissacarídeos/classificação , Polissacarídeos/genética , Ligação Proteica , Receptores Virais/metabolismo , Proteínas Virais/metabolismoRESUMO
Biological functions of N-glycans are frequently related to their unique branching patterns. Multistage mass spectrometry (MSn) has become the primary method for glycan structural analysis. However, selection of the best fragment as the precursor for the next round of product-ion scanning is important but difficult. We have previously proposed the concept and designed the approach of glycan intelligent precursor selection (GIPS) to guide MSn experiments, but its use in N-glycans is not straightforward as some N-glycans are of high similarity in branching patterns. In the present work we introduced new elements to GIPS to improve its performance in N-glycan branching pattern analysis. These include a hypothesis and significance test, based on Bayes factor, and DPbiased as a new precursor selection strategy. The improved GIPS was successfully applied to identification of individual N-glycans, and incorporated into MALDI-MS N-glycan profiling for assignment of N-glycans obtained from glycoproteins and complex human serum.
Assuntos
Glicoproteínas/química , Polissacarídeos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Glicoproteínas/sangue , Humanos , Estrutura Molecular , Polissacarídeos/química , Polissacarídeos/classificaçãoRESUMO
Echinoderms are among the most primitive deuterostomes and have been used as model organisms to understand chordate biology because of their close evolutionary relationship to this phylogenetic group. However, there are almost no data available regarding the N-glycomic capacity of echinoderms, which are otherwise known to produce a diverse set of species-specific glycoconjugates, including ones heavily modified by fucose, sulfate, and sialic acid residues. To increase the knowledge of diversity of carbohydrate structures within this phylum, here we conducted an in-depth analysis of N-glycans from a brittle star (Ophiactis savignyi) as an example member of the class Ophiuroidea. To this end, we performed a multi-step N-glycan analysis by HPLC and various exoglyosidase and chemical treatments in combination with MALDI-TOF MS and MS/MS. Using this approach, we found a wealth of hybrid and complex oligosaccharide structures reminiscent of those in higher vertebrates as well as some classical invertebrate glycan structures. 70% of these N-glycans were anionic, carrying either sialic acid, sulfate, or phosphate residues. In terms of glycophylogeny, our data position the brittle star between invertebrates and vertebrates and confirm the high diversity of N-glycosylation in lower organisms.
Assuntos
Glicômica/métodos , Polissacarídeos/química , Estrelas-do-Mar/metabolismo , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Glicosídeo Hidrolases/metabolismo , Glicosilação , Oligossacarídeos/química , Filogenia , Polissacarídeos/classificação , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estrelas-do-Mar/classificaçãoRESUMO
The current hydrocolloid industry requires new techniques for biomass characterization, which can quickly and ecologically characterize contained sugars. This work proposes the use of Fourier Transform Infrared microspectroscopy in combination with multivariate methods, to localize and identify the main carbohydrates and other components present in fresh brown seaweeds, avoiding time-consuming samples pre-treatments. Infrared images of Macrocystis pyrifera samples were analyzed by Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) and Principal Component Analysis (PCA) as chemometrics techniques to identify the compounds. MCR-ALS was the best strategy, delivering pure spectra of chemical compound that PCA did not. The carbohydrates identified by this method were 1-3-ß-glucans divided into endofibers and laminarin; two types of fucoidans (rich in fucose or mannuronic acid), alginate and mannitol, besides other compounds such as proteins. This technique represents an opportunity for the hydrocolloid industry for a modern, rapid and environmentally-friendly characterization of macroalgal biomass to enhance its use.
Assuntos
Carboidratos/isolamento & purificação , Polissacarídeos/isolamento & purificação , Alga Marinha/química , Espectroscopia de Infravermelho com Transformada de Fourier , Alginatos/química , Carboidratos/química , Análise dos Mínimos Quadrados , Análise Multivariada , Polissacarídeos/química , Polissacarídeos/classificação , Análise de Componente Principal , Açúcares/química , Açúcares/isolamento & purificaçãoRESUMO
Among the earliest deuterostomes, the echinoderms are an evolutionary important group of ancient marine animals. Within this phylum, the holothuroids (sea cucumbers) are known to produce a wide range of glycoconjugate biopolymers with apparent benefits to health; therefore, they are of economic and culinary interest throughout the world. Other than their highly modified glycosaminoglycans (e.g. fucosylated chondroitin sulfate and fucoidan), nothing is known about their protein-linked glycosylation. Here we used multistep N-glycan fractionation to efficiently separate anionic and neutral N-glycans before analyzing the N-glycans of the black sea cucumber (Holothuria atra) by MS in combination with enzymatic and chemical treatments. These analyses showed the presence of various fucosylated, phosphorylated, sialylated, and multiply sulfated moieties as modifications of oligomannosidic, hybrid, and complex-type N-glycans. The high degree of sulfation and fucosylation parallels the modifications observed previously on holothuroid glycosaminoglycans. Compatible with its phylogenetic position, H. atra not only expresses vertebrate motifs such as sulfo- and sialyl-Lewis A epitopes but displays a high degree of anionic substitution of its glycans, as observed in other marine invertebrates. Thus, as for other echinoderms, the phylum- and order-specific aspects of this species' N-glycosylation reveal both invertebrate- and vertebrate-like features.
Assuntos
Holothuria/metabolismo , Polissacarídeos/química , Sulfatos/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Ecossistema , Glicosídeo Hidrolases/metabolismo , Glicosilação , Holothuria/classificação , Filogenia , Polissacarídeos/classificação , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The polysaccharide was first successfully isolated from the leaf of Dendrobium officinale by hot water extraction and alcohol precipitation and further purified using DEAE-52 and Sephadex G-100 chromatography. The structure of LDOP-1 was characterized by HPLC, GPC, and FT-IR and NMR spectroscopy, and its protective effect on LPS-induced GES-1 cell injury was analyzed. Results showed that LDOP was a homogeneous polysaccharide with average molecular weight of 91.8 kDa and consisted of Man, Gla, Glc, Glc acid, and Ara at a molar ratio of 2.0:1.3:1.6:1.7:0.7. LDOP had two types of residues, including 1,6-linked α-d-Glup and 1,4-linked α-d-Manp. Activity studies indicated that LDOP-1 can significantly suppress the release of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 from LPS-induced GES-1 cell injury, decreased the protein expressions of TLR4, phospho-NF-κB, ASC, NLRP3, cleaved-IL-1ß, IL-6, and Bax, increased the protein expression of Bcl-2, and downregulated the ratios of cleaved caspase-1 to pro-caspase-1, phospho-IκBα to IκBα, and phospho-NF-кB to NF-κB. These findings strongly suggested that LDOP can prevent LPS-induced GES-1 cell injury by inhibiting the release of inflammatory cytokines regulated via the TLR4/NF-κB signal pathways.
Assuntos
Dendrobium/química , Inflamação/tratamento farmacológico , Lipopolissacarídeos/química , Polissacarídeos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Interleucina-6/genética , Lipopolissacarídeos/farmacologia , Espectroscopia de Ressonância Magnética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Polissacarídeos/classificação , Transdução de Sinais/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de Fourier , Receptor 4 Toll-Like/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Muco-obstructive lung diseases (MOLDs), like cystic fibrosis and chronic obstructive pulmonary disease, affect a spectrum of subjects globally. In MOLDs, the airway mucus becomes hyperconcentrated, increasing osmotic and viscoelastic moduli and impairing mucus clearance. MOLD research requires relevant sources of healthy airway mucus for experimental manipulation and analysis. Mucus collected from endotracheal tubes (ETT) may represent such a source with benefits, e.g., in vivo production, over canonical sample types such as sputum or human bronchial epithelial (HBE) mucus. Ionic and biochemical compositions of ETT mucus from healthy human subjects were characterized and a stock of pooled ETT samples generated. Pooled ETT mucus exhibited concentration-dependent rheologic properties that agreed across spatial scales with reported individual ETT samples and HBE mucus. We suggest that the practical benefits compared with other sample types make ETT mucus potentially useful for MOLD research.
Assuntos
Muco/química , Potássio/análise , Reologia/métodos , Sódio/análise , Traqueia/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Cátions Monovalentes , Feminino , Voluntários Saudáveis , Humanos , Intubação Intratraqueal , Masculino , Pessoa de Meia-Idade , Polissacarídeos/classificação , Polissacarídeos/isolamento & purificação , Potássio/metabolismo , Proteínas/classificação , Proteínas/isolamento & purificação , Sódio/metabolismo , Escarro/química , Traqueia/fisiologiaRESUMO
"The totality is not, as it were, a mere heap, but the whole is something besides the parts."-Aristotle. We built a classifier that uses the totality of the glycomic profile, not restricted to a few glycoforms, to differentiate samples from two different sources. This approach, which relies on using thousands of features, is a radical departure from current strategies, where most of the glycomic profile is ignored in favor of selecting a few features, or even a single feature, meant to capture the differences in sample types. The classifier can be used to differentiate the source of the material; applicable sources may be different species of animals, different protein production methods, or, most importantly, different biological states (disease vs healthy). The classifier can be used on glycomic data in any form, including derivatized monosaccharides, intact glycans, or glycopeptides. It takes advantage of the fact that changing the source material can cause a change in the glycomic profile in many subtle ways: some glycoforms can be upregulated, some downregulated, some may appear unchanged, yet their proportion-with respect to other forms present-can be altered to a detectable degree. By classifying samples using the entirety of their glycan abundances, along with the glycans' relative proportions to each other, the "Aristotle Classifier" is more effective at capturing the underlying trends than standard classification procedures used in glycomics, including PCA (principal components analysis). It also outperforms workflows where a single, representative glycomic-based biomarker is used to classify samples. We describe the Aristotle Classifier and provide several examples of its utility for biomarker studies and other classification problems using glycomic data from several sources.
Assuntos
Glicômica/métodos , Glicopeptídeos/classificação , Glicoproteínas/classificação , Cirrose Hepática/diagnóstico , Monossacarídeos/classificação , Polissacarídeos/classificação , Biomarcadores/análise , Glicopeptídeos/isolamento & purificação , Glicopeptídeos/metabolismo , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Glicosilação , Humanos , Cirrose Hepática/metabolismo , Monossacarídeos/isolamento & purificação , Monossacarídeos/metabolismo , Polissacarídeos/isolamento & purificação , Polissacarídeos/metabolismo , Análise de Componente Principal , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Terminologia como AssuntoRESUMO
Changes of α-2,3-/α-2,6-linked sialic acids (SAs) in sialylglycans have been found to be closely related with some diseases. However, accurate quantification of sialylglycans at the isomeric level remains challenging due to their instability, structural complexity, and low mass spectrometry (MS) detection sensitivity. Herein, we propose an analytical strategy named "glycoqueuing", which allows sequential chromatographic elution and high-sensitivity MS quantification of various sialylglycan isomers based on isotopic labeling followed by analysis via online reversed-phase high performance liquid chromatography coupling with MS (RP-HPLC-MS). The new method was validated by detailed structural identification and quantification of fetal bovine serum (FBS) N-linked sialylglycan isomers, during which many branching isomers were successfully differentiated, and 28 sialylglycan compositions with Neu5Gc residues were analyzed. The method was successfully applied to isomer-specific, quantitative comparison of sialylated N-glycans between bovine and rabbit immunoglobulin G (IgG) and the search for serum sialylated N-glycan biomarker candidates of hepatocellular carcinoma, during which a 55% increase of α-2,6-sialylated fucosylated N-glycans was revealed, demonstrating the great applicability and potential clinical usage of the method.
Assuntos
Carcinoma Hepatocelular/sangue , Imunoglobulina G/sangue , Neoplasias Hepáticas/sangue , Polissacarídeos/química , Ácidos Siálicos/química , Animais , Sequência de Carboidratos , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/patologia , Bovinos , Cromatografia de Fase Reversa , Fucose/química , Glicosilação , Humanos , Imunoglobulina G/química , Imunoglobulina G/classificação , Isomerismo , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/patologia , Polissacarídeos/classificação , Coelhos , Ácidos Siálicos/classificação , Coloração e Rotulagem/métodos , Espectrometria de Massas em TandemRESUMO
The structural diversity of glycans on cells-the glycome-is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic, and structural basis for glycan binding in a natural context. The cell-based glycan array is self-renewable and reports glycosyltransferase genes required (or blocking) for interactions through logical sequential biosynthetic steps, which is predictive of structural glycan features involved and provides instructions for synthesis, recombinant production, and genetic dissection strategies. Broad utility of the cell-based glycan array is demonstrated, and we uncover higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins.