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1.
Nat Commun ; 11(1): 3963, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770134

RESUMO

Polysaccharides are the most abundant biomolecules in nature, but are the least understood in terms of their chemical structures and biological functions. Polysaccharides cannot be simply sequenced because they are often highly branched and lack a uniform structure. Furthermore, large polymeric structures cannot be directly analyzed by mass spectrometry techniques, a problem that has been solved for polynucleotides and proteins. While restriction enzymes have advanced genomic analysis, and trypsin has advanced proteomic analysis, there has been no equivalent enzyme for universal polysaccharide digestion. We describe the development and application of a chemical method for producing oligosaccharides from polysaccharides. The released oligosaccharides are characterized by advanced liquid chromatography-mass spectrometry (LC-MS) methods with high sensitivity, accuracy and throughput. The technique is first used to identify polysaccharides by oligosaccharide fingerprinting. Next, the polysaccharide compositions of food and feces are determined, further illustrating the utility of technique in food and clinical studies.


Assuntos
Oligossacarídeos/química , Polissacarídeos/metabolismo , Bactérias/metabolismo , Glucanos/química , Glucanos/metabolismo , Humanos , Lactente , Mananas/química , Mananas/metabolismo , Oxirredução , Polimerização , Fatores de Tempo , Xilanos/química , Xilanos/metabolismo
2.
Nat Commun ; 11(1): 3990, 2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32778659

RESUMO

The molecular mechanisms regulating lymphocyte homing into lymph nodes are only partly understood. Here, we report that B cell-specific deletion of the X-linked gene, Cosmc, and the consequent decrease of protein O-glycosylation, induces developmental blocks of mouse B cells. After transfer into wild-type recipient, Cosmc-null B cells fail to home to lymph nodes as well as non-lymphoid organs. Enzymatic desialylation of wild-type B cells blocks their migration into lymph nodes, indicating a requirement of sialylated O-glycans for proper trafficking. Mechanistically, Cosmc-deficient B cells have normal rolling and firm arrest on high endothelium venules (HEV), thereby attributing their inefficient trafficking to alterations in the subsequent transendothelial migration step. Finally, Cosmc-null B cells have defective chemokine signaling responses. Our results thus demonstrate that Cosmc and its effects on O-glycosylation are important for controlling B cell homing.


Assuntos
Linfócitos B/metabolismo , Linfonodos/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Movimento Celular , Feminino , Glicosilação , Humanos , Imunidade Humoral/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , Polissacarídeos/metabolismo , Transcriptoma , Vênulas
3.
Nat Commun ; 11(1): 4017, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782292

RESUMO

The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states.


Assuntos
Microbioma Gastrointestinal , Hexosaminidases/metabolismo , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Animais , Bactérias/classificação , Bactérias/enzimologia , Bactérias/genética , Bactérias/metabolismo , Cristalografia por Raios X , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hexosaminidases/química , Hexosaminidases/genética , Humanos , Glicoproteínas de Membrana/química , Estrutura Molecular , Mucinas/química , Filogenia , Polissacarídeos/química , Polissacarídeos/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
4.
Nat Commun ; 11(1): 4033, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820167

RESUMO

Peptide hormones and neuropeptides encompass a large class of bioactive peptides that regulate physiological processes like anxiety, blood glucose, appetite, inflammation and blood pressure. Here, we execute a focused discovery strategy to provide an extensive map of O-glycans on peptide hormones. We find that almost one third of the 279 classified peptide hormones carry O-glycans. Many of the identified O-glycosites are conserved and are predicted to serve roles in proprotein processing, receptor interaction, biodistribution and biostability. We demonstrate that O-glycans positioned within the receptor binding motifs of members of the neuropeptide Y and glucagon families modulate receptor activation properties and substantially extend peptide half-lives. Our study highlights the importance of O-glycosylation in the biology of peptide hormones, and our map of O-glycosites in this large class of biomolecules serves as a discovery platform for an important class of molecules with potential opportunities for drug designs.


Assuntos
Hormônios Peptídicos/química , Hormônios Peptídicos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Idoso , Animais , Linhagem Celular , Desenho de Fármacos , Feminino , Glicosilação , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Neuropeptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Estabilidade Proteica , Ratos , Suínos
5.
Proc Natl Acad Sci U S A ; 117(33): 20223-20234, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759215

RESUMO

Nano-sized archaeota, with their small genomes and limited metabolic capabilities, are known to associate with other microbes, thereby compensating for their own auxotrophies. These diminutive and yet ubiquitous organisms thrive in hypersaline habitats that they share with haloarchaea. Here, we reveal the genetic and physiological nature of a nanohaloarchaeon-haloarchaeon association, with both microbes obtained from a solar saltern and reproducibly cultivated together in vitro. The nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh is an aerotolerant, sugar-fermenting anaerobe, lacking key anabolic machinery and respiratory complexes. The nanohaloarchaeon cells are found physically connected to the chitinolytic haloarchaeon Halomicrobium sp. LC1Hm. Our experiments revealed that this haloarchaeon can hydrolyze chitin outside the cell (to produce the monosaccharide N-acetylglucosamine), using this beta-glucan to obtain carbon and energy for growth. However, LC1Hm could not metabolize either glycogen or starch (both alpha-glucans) or other polysaccharides tested. Remarkably, the nanohaloarchaeon's ability to hydrolyze glycogen and starch to glucose enabled growth of Halomicrobium sp. LC1Hm in the absence of a chitin. These findings indicated that the nanohaloarchaeon-haloarchaeon association is both mutualistic and symbiotic; in this case, each microbe relies on its partner's ability to degrade different polysaccharides. This suggests, in turn, that other nano-sized archaeota may also be beneficial for their hosts. Given that availability of carbon substrates can vary both spatially and temporarily, the susceptibility of Halomicrobium to colonization by Ca Nanohalobium can be interpreted as a strategy to maximize the long-term fitness of the host.


Assuntos
Halobacteriaceae/fisiologia , Nanoarchaeota/fisiologia , Polissacarídeos/metabolismo , Simbiose/fisiologia , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Técnicas de Cocultura , Regulação da Expressão Gênica em Archaea , Genoma Arqueal , Genômica , Filogenia
6.
Proc Natl Acad Sci U S A ; 117(34): 20597-20606, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788370

RESUMO

The major histocompatibility complex class-I (MHC-I) peptide-loading complex (PLC) is a cornerstone of the human adaptive immune system, being responsible for processing antigens that allow killer T cells to distinguish between healthy and compromised cells. Based on a recent low-resolution cryo-electron microscopy (cryo-EM) structure of this large membrane-bound protein complex, we report an atomistic model of the PLC and study its conformational dynamics on the multimicrosecond time scale using all-atom molecular dynamics (MD) simulations in an explicit lipid bilayer and water environment (1.6 million atoms in total). The PLC has a layered structure, with two editing modules forming a flexible protein belt surrounding a stable, catalytically active core. Tapasin plays a central role in the PLC, stabilizing the MHC-I binding groove in a conformation reminiscent of antigen-loaded MHC-I. The MHC-I-linked glycan steers a tapasin loop involved in peptide editing toward the binding groove. Tapasin conformational dynamics are also affected by calreticulin through a conformational selection mechanism that facilitates MHC-I recruitment into the complex.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Calreticulina/metabolismo , Microscopia Crioeletrônica , Antígenos de Histocompatibilidade Classe I/ultraestrutura , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/ultraestrutura , Simulação de Dinâmica Molecular , Polissacarídeos/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo
7.
PLoS One ; 15(8): e0237666, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822385

RESUMO

Before fertilization, sperm bind to epithelial cells of the oviduct isthmus to form a reservoir that regulates sperm viability and capacitation. The sperm reservoir maintains optimum fertility in species, like swine, in which semen deposition and ovulation may not be well synchronized. We demonstrated previously that porcine sperm bind to two oviductal glycan motifs, a biantennary 6-sialylated N-acetyllactosamine (bi-SiaLN) oligosaccharide and 3-O-sulfated Lewis X trisaccharide (suLeX). Here, we assessed the ability of these glycans to regulate sperm Ca2+ influx, capacitation and affect sperm lifespan. After 24 h, the viability of sperm bound to immobilized bi-SiaLN and suLeX was higher (46% and 41% respectively) compared to viability of free-swimming sperm (10-12%). Ca2+ is a central regulator of sperm function so we assessed whether oviduct glycans could affect the Ca2+ influx that occurs during capacitation. Using a fluorescent intracellular Ca2+ probe, we observed that both oviduct glycans suppressed the Ca2+ increase that occurs during capacitation. Thus, specific oviduct glycans can regulate intracellular Ca2+. Because the increase in intracellular Ca2+ was suppressed by oviduct glycans, we examined whether glycans affected capacitation, as determined by protein tyrosine phosphorylation and the ability to undergo a Ca2+ ionophore-induced acrosome reaction. We found no discernable suppression of capacitation in sperm bound to oviduct glycans. We also detected no effect of oviduct glycans on sperm motility during capacitation. In summary, LeX and bi-SiaLN glycan motifs found on oviduct oligosaccharides suppress the Ca2+ influx that occurs during capacitation and extend sperm lifespan but do not affect sperm capacitation or motility.


Assuntos
Cálcio/metabolismo , Oviductos/metabolismo , Polissacarídeos/metabolismo , Espermatozoides/metabolismo , Suínos/fisiologia , Animais , Sobrevivência Celular , Feminino , Masculino , Análise do Sêmen , Capacitação Espermática , Motilidade Espermática
8.
PLoS Biol ; 18(8): e3000788, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841232

RESUMO

Women with bacterial vaginosis (BV), an imbalance of the vaginal microbiome, are more likely to be colonized by potential pathogens such as Fusobacterium nucleatum, a bacterium linked with intrauterine infection and preterm birth. However, the conditions and mechanisms supporting pathogen colonization during vaginal dysbiosis remain obscure. We demonstrate that sialidase activity, a diagnostic feature of BV, promoted F. nucleatum foraging and growth on mammalian sialoglycans, a nutrient resource that was otherwise inaccessible because of the lack of endogenous F. nucleatum sialidase. In mice with sialidase-producing vaginal microbiotas, mutant F. nucleatum unable to consume sialic acids was impaired in vaginal colonization. These experiments in mice also led to the discovery that F. nucleatum may also "give back" to the community by reinforcing sialidase activity, a biochemical feature of human dysbiosis. Using human vaginal bacterial communities, we show that F. nucleatum supported robust outgrowth of Gardnerella vaginalis, a major sialidase producer and one of the most abundant organisms in BV. These results illustrate that mutually beneficial relationships between vaginal bacteria support pathogen colonization and may help maintain features of dysbiosis. These findings challenge the simplistic dogma that the mere absence of "healthy" lactobacilli is the sole mechanism that creates a permissive environment for pathogens during vaginal dysbiosis. Given the ubiquity of F. nucleatum in the human mouth, these studies also suggest a possible mechanism underlying links between vaginal dysbiosis and oral sex.


Assuntos
Proteínas de Bactérias/genética , Disbiose/microbiologia , Fusobacterium/metabolismo , Gardnerella vaginalis/metabolismo , Neuraminidase/genética , Polissacarídeos/metabolismo , Vaginose Bacteriana/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Técnicas de Tipagem Bacteriana , Disbiose/patologia , Feminino , Fusobacterium/genética , Fusobacterium/isolamento & purificação , Fusobacterium/patogenicidade , Gardnerella vaginalis/genética , Gardnerella vaginalis/isolamento & purificação , Gardnerella vaginalis/patogenicidade , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/genética , Neuraminidase/metabolismo , RNA Ribossômico 16S/genética , Ácidos Siálicos/metabolismo , Simbiose/genética , Vagina/microbiologia , Vaginose Bacteriana/patologia
9.
Nat Protoc ; 15(8): 2668-2704, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32681150

RESUMO

The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocalyx is thus essential to understanding cell physiology and elucidating its role in promoting health and disease. This protocol describes how to comprehensively characterize the glycocalyx N-glycans and O-glycans of glycoproteins, as well as intact glycolipids in parallel, using the same enriched membrane fraction. Profiling of the glycans and the glycolipids is performed using nanoflow liquid chromatography-mass spectrometry (nanoLC-MS). Sample preparation, quantitative LC-tandem MS (LC-MS/MS) analysis, and data processing methods are provided. In addition, we discuss glycoproteomic analysis that yields the site-specific glycosylation of membrane proteins. To reduce the amount of sample needed, N-glycan, O-glycan, and glycolipid analyses are performed on the same enriched fraction, whereas glycoproteomic analysis is performed on a separate enriched fraction. The sample preparation process takes 2-3 d, whereas the time spent on instrumental and data analyses could vary from 1 to 5 d for different sample sizes. This workflow is applicable to both cell and tissue samples. Systematic changes in the glycocalyx associated with specific glycoforms and glycoconjugates can be monitored with quantitation using this protocol. The ability to quantitate individual glycoforms and glycoconjugates will find utility in a broad range of fundamental and applied clinical studies, including glycan-based biomarker discovery and therapeutics.


Assuntos
Glicocálix/metabolismo , Glicômica/métodos , Linhagem Celular , Humanos , Polissacarídeos/metabolismo
10.
PLoS One ; 15(7): e0236518, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702033

RESUMO

Thermophilic microorganisms and their enzymes have been utilized in various industrial applications. In this work, we isolated and characterized thermophilic anaerobic bacteria with the cellulose and hemicellulose degrading activities from a tropical dry deciduous forest in northern Thailand. Out of 502 isolated thermophilic anaerobic soil bacteria, 6 isolates, identified as Thermoanaerobacterium sp., displayed an ability to utilize a wide range of oligosaccharides and lignocellulosic substrates. The isolates exhibited significant cellulase and xylanase activities at high temperature (65°C). Among all isolates, Thermoanaerobacterium sp. strain R63 exhibited remarkable hydrolytic properties with the highest cellulase and xylanase activities at 1.15 U/mg and 6.17 U/mg, respectively. Extracellular extract of Thermoanaerobacterium sp. strain R63 was thermostable with an optimal temperature at 65°C and could exhibit enzymatic activities on pH range 5.0-9.0. Our findings suggest promising applications of these thermoanaerobic bacteria and their potent enzymes for industrial purposes.


Assuntos
Celulose/metabolismo , Polissacarídeos/metabolismo , Microbiologia do Solo , Thermoanaerobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Biomassa , Celulase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Filogenia , Especificidade por Substrato , Thermoanaerobacterium/classificação , Thermoanaerobacterium/enzimologia , Thermoanaerobacterium/isolamento & purificação
11.
Blood Adv ; 4(13): 2967-2978, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32609845

RESUMO

Thrombocytopenia is a common complication of influenza virus infection, and its severity predicts the clinical outcome of critically ill patients. The underlying cause(s) remain incompletely understood. In this study, in patients with an influenza A/H1N1 virus infection, viral load and platelet count correlated inversely during the acute infection phase. We confirmed this finding in a ferret model of influenza virus infection. In these animals, platelet count decreased with the degree of virus pathogenicity varying from 0% in animals infected with the influenza A/H3N2 virus, to 22% in those with the pandemic influenza A/H1N1 virus, up to 62% in animals with a highly pathogenic A/H5N1 virus infection. This thrombocytopenia is associated with virus-containing platelets that circulate in the blood. Uptake of influenza virus particles by platelets requires binding to sialoglycans and results in the removal of sialic acids by the virus neuraminidase, a trigger for hepatic clearance of platelets. We propose the clearance of influenza virus by platelets as a paradigm. These insights clarify the pathophysiology of influenza virus infection and show how severe respiratory infections, including COVID-19, may propagate thrombocytopenia and/or thromboembolic complications.


Assuntos
Plaquetas/virologia , Vírus da Influenza A/patogenicidade , Influenza Humana/complicações , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Trombocitopenia/etiologia , Animais , Plaquetas/metabolismo , Plaquetas/patologia , Modelos Animais de Doenças , Furões , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/patogenicidade , Vírus da Influenza A Subtipo H3N2/fisiologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Virus da Influenza A Subtipo H5N1/fisiologia , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Influenza Humana/patologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/complicações , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Trombocitopenia/metabolismo , Trombocitopenia/patologia , Trombocitopenia/virologia , Internalização do Vírus
12.
Nat Commun ; 11(1): 3285, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620774

RESUMO

The early life human gut microbiota exerts life-long health effects on the host, but the mechanisms underpinning its assembly remain elusive. Particularly, the early colonization of Clostridiales from the Roseburia-Eubacterium group, associated with protection from colorectal cancer, immune- and metabolic disorders is enigmatic. Here, we describe catabolic pathways that support the growth of Roseburia and Eubacterium members on distinct human milk oligosaccharides (HMOs). The HMO pathways, which include enzymes with a previously unknown structural fold and specificity, were upregulated together with additional glycan-utilization loci during growth on selected HMOs and in co-cultures with Akkermansia muciniphila on mucin, suggesting an additional role in enabling cross-feeding and access to mucin O-glycans. Analyses of 4599 Roseburia genomes underscored the preponderance and diversity of the HMO utilization loci within the genus. The catabolism of HMOs by butyrate-producing Clostridiales may contribute to the competitiveness of this group during the weaning-triggered maturation of the microbiota.


Assuntos
Butiratos/metabolismo , Clostridiales/metabolismo , Leite Humano/metabolismo , Mucinas/metabolismo , Oligossacarídeos/metabolismo , Bifidobacterium/metabolismo , Clostridiales/genética , Colo/microbiologia , Eubacterium/metabolismo , Microbioma Gastrointestinal/fisiologia , Humanos , Lactente , Recém-Nascido , Metabolismo/fisiologia , Leite Humano/química , Polissacarídeos/metabolismo , Verrucomicrobia/metabolismo , Desmame
13.
PLoS One ; 15(6): e0234989, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32598367

RESUMO

Alterations in glycosylation are seen in many types of cancer, including colorectal cancer (CRC). Glycans, the sugar moieties of glycoconjugates, are involved in many important functions relevant to cancer and can be of value as biomarkers. In this study, we have used mass spectrometry to analyze the N-glycan profiles of 35 CRC tissue samples and 10 healthy tissue samples from non-CRC patients who underwent operations for other reasons. The tumor samples were divided into groups depending on tumor location (right or left colon) and stage (II or III), while the healthy samples were divided into right or left colon. The levels of neutral and acidic N-glycan compositions and glycan classes were analyzed in a total of ten different groups. Surprisingly, there were no significant differences in glycan levels when all right- and left-sided CRC samples were compared, and few differences (such as in the abundance of the neutral N-glycan H3N5) were seen when the samples were divided according to both location and stage. Multiple significant differences were found in the levels of glycans and glycan classes when stage II and III samples were compared, and these glycans could be of value as candidates for new markers of cancer progression. In order to validate our findings, we analyzed healthy tissue samples from the right and left colon and found no significant differences in the levels of any of the glycans analyzed, confirming that our findings when comparing CRC samples from the right and left colon are not due to normal variations in the levels of glycans between the healthy right and left colon. Additionally, the levels of the acidic glycans H4N3F1P1, H5N4F1P1, and S1H5N4F1 were found to change in a cancer-specific but colon location-nonspecific manner, indicating that CRC affects glycan levels in similar ways regardless of tumor location.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Colorretais/metabolismo , Glicômica , Polissacarídeos/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Colo/patologia , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Glicosilação , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto Jovem
14.
PLoS One ; 15(6): e0234145, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497126

RESUMO

Oxidative stress and inflammation determine retinal ganglion cell degeneration, leading to retinal impairment and vision loss. Müller glial cells regulate retinal repair under injury, through gliosis. Meanwhile, reactive gliosis can turn in pathological effects, contributing to neurodegeneration. In the present study, we tested whether Cord Blood Serum (CBS), rich of growth factors, might improve the viability of Müller cells under in vitro damage. BDNF, NGF, TGF-α, GDNF and EGF levels were measured in CBS samples by Human Magnetic Luminex Assay. CBS effects were evaluated on rat (rMC-1) and human (MIO-M1) Müller cells, under H2O2 and IL-1ß damage. Cells grown with FBS or CBS both at 5% were exposed to stress and analyzed in terms of cell viability, GFAP, IL-6 and TNF-α expression. CBS was also administrated after treatment with K252a, inhibitor of the neurotrophin receptor Trk. Cell viability of rMC-1 and MIO-M1 resulted significantly improved when pretreated with CBS and exposed to H2O2 and IL-1ß, in comparison to the standard culture with FBS. Accordingly, the gliosis marker GFAP resulted down-regulated following CBS priming. In parallel, we observed a lower expression of the inflammatory mediators in rMC-1 (TNF-α) and MIO-M1 (IL-6, TNF- α), especially in presence of inflammatory damage. Trk inhibition through K252a administration impaired the effects of CBS under stress conditions on MIO-M1 and rMC-1 viability, not significantly different from FBS condition. CBS is enriched with neurotrophins and its administration to rMC-1 and MIO-M1 attenuates the cytotoxic effects of H2O2 and IL-1ß. Moreover, the decrease of the main markers of gliosis and inflammation suggests a promising use of CBS for neuroprotection aims. This study is a preliminary basis that prompts future investigations to deeply explore and confirm the CBS potential.


Assuntos
Células Ependimogliais/citologia , Células Ependimogliais/efeitos dos fármacos , Sangue Fetal/metabolismo , Soro/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Ependimogliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/genética , Humanos , Estresse Oxidativo/efeitos dos fármacos , Polissacarídeos/metabolismo , Ratos , Fator de Necrose Tumoral alfa/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(25): 14209-14219, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513731

RESUMO

The physical dimensions of proteins and glycans on cell surfaces can critically affect cell function, for example, by preventing close contact between cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call cell surface optical profilometry (CSOP), to quantify the height of key multidomain proteins on a model cell, as well as to capture average protein and glycan heights on native cell membranes. We show that average height of a protein is significantly smaller than its contour length, due to thermally driven bending and rotation on the membrane, and that height strongly depends on local surface and solution conditions. We find that average height increases with cell surface molecular crowding but decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope, based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native cell surface using antibodies and lectins. This versatile method for profiling cell surfaces has the potential to advance understanding of the molecular landscape of cells and the role of the molecular landscape in cell function.


Assuntos
Membrana Celular/química , Proteínas de Membrana/química , Polissacarídeos/química , Anticorpos , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Epitopos , Imunofluorescência , Células HEK293 , Humanos , Lectinas , Bicamadas Lipídicas , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Polissacarídeos/metabolismo , Domínios Proteicos
16.
Ecotoxicol Environ Saf ; 202: 110901, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32593805

RESUMO

This study aimed to investigate the role of Platycodon grandiflorus polysaccharide (PGPS) in chromium (VI)-induced autophagy in a chicken embryo fibroblast cell lines (DF-1 cells). DF-1 cells were exposed to Cr (VI), PGPSt, and Cr (VI) + PGPSt, and their effects on cell viability, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and autophagy-related proteins were examined. The results showed that the cell viability was reduced after Cr (VI) treatment, and 3-MA, CsA or PGPSt suppressed this decrease. Cr (VI) treatment increased the ROS levels and decreased the MMP, thereby enhancing the expression of mitochondrial autophagy marker proteins (PINK1, Parkin, and LC3-II), inhibiting mitophagy autophagy protein TOMM20 expression, and promoting the degradation of autophagy-related marker p62. These changes led to exceeding mitochondrial autophagy and cell trauma and could be mitigated by PGPSt. Overall, our research showed that Cr (VI) can induce exceeding mitochondrial autophagy in DF-1 cells, whereas PGPSt can improve Cr (VI)-induced mitochondrial autophagy by inhibiting ROS and restoring MMP.


Assuntos
Cromo/toxicidade , Platycodon/fisiologia , Polissacarídeos/metabolismo , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromo/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitofagia , Extratos Vegetais , Platycodon/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases
17.
Nat Commun ; 11(1): 2694, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483155

RESUMO

Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface.


Assuntos
Toxinas Bacterianas/toxicidade , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Polissacarídeos/metabolismo , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacocinética , Sítios de Ligação , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HEK293 , Heparina/química , Heparina/metabolismo , Humanos , Insetos/microbiologia , Antígenos CD15/química , Antígenos CD15/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Morganella morganii/patogenicidade , Photorhabdus/patogenicidade , Polissacarídeos/química , Xenorhabdus/patogenicidade
18.
Plant Mol Biol ; 103(4-5): 581-596, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32409993

RESUMO

KEY MESSAGE: N-glycans play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects. Asparagine-linked (Asn/N-) glycosylation is one of the most prevalent and complex protein modifications and the associated N-glycans play crucial roles on protein folding and secretion. The studies have shown that many glycoproteins hold multiple N-glycans, yet little is known about the redundancy of N-glycans on a protein. In this study, we used BRI1 to decipher the roles of N-glycans on protein secretion and function. We found that all 14 potential N-glycosylation sites on BRI1 were occupied with oligosaccharides. The elimination of single N-glycan had no obvious effect on BRI1 secretion or function except N154-glycan, which resulted in the retention of BRI1 in the endoplasmic reticulum (ER), similar to the loss of multiple highly conserved N-glycans. To misfolded bri1, the absence of N-glycans next to local structural defects enhanced the ER retention and the artificial addition of N-glycan could help the misfolded bri1-GFPs exiting from the ER, indicating that the N-glycans might serve as steric hindrance to protect the structure defects from ER recognition. We also found that the retention of misfolded bri1-9 by lectins and chaperones in the ER relied on the presence of multiple N-glycans distal to the local defects. Our findings revealed that the N-glycans might play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Polissacarídeos/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/fisiologia , Alcaloides/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Glicoproteínas/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosilação , Modelos Moleculares , Oligossacarídeos/metabolismo , Plantas Geneticamente Modificadas , Conformação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Plântula , Sementes/citologia , Sementes/metabolismo , Transdução de Sinais/genética
19.
Am J Physiol Lung Cell Mol Physiol ; 319(1): L148-L158, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32432921

RESUMO

Surfactant protein D (SP-D) is a C-type lectin that participates in the innate immune defense of lungs. It binds pathogens through its carbohydrate recognition domain in a calcium-dependent manner. Human surfactant protein D (hSP-D) has been routinely obtained from bronchoalveolar lavage of patients suffering from pulmonary alveolar proteinosis (PAP) and from amniotic fluid (AF). As a consequence of the disease, hSP-D obtained from PAP is found in higher amounts and is mainly composed of higher order oligomeric forms. However, PAP-hSP-D has never been directly compared with nonpathological human protein in terms of structure and biological activity. Moreover, the quantitative distribution of the different hSP-D oligomeric forms in human protein obtained from a natural source has never been evaluated. In this work, we have determined the quantitative distribution of AF-hSP-D oligomers, characterized the sugars attached through the N-glycosylation site of the protein, and compared the activity of hSP-D from AF and PAP with respect to their ability to bind and agglutinate bacteria. We have found that fuzzy balls (40%) are the most abundant oligomeric form in AF-hSP-D, very closely followed by dodecamers (33%), with both together constituting 73% of the protein mass. The glycan attached to the N-glycosylation site was found to be composed of fucose, galactose, sialic acid, and N-acetylglucosamine. Finally, in the functional assays performed, hSP-D obtained from PAP showed higher potency, probably as a consequence of its higher proportion of large oligomers compared with hSP-D from AF.


Assuntos
Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/metabolismo , Líquido Amniótico/metabolismo , Asparagina/metabolismo , Ligação Competitiva , Cromatografia de Afinidade , Feminino , Glicosilação , Humanos , Polissacarídeos/metabolismo , Gravidez , Ligação Proteica , Multimerização Proteica , Proteinose Alveolar Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/isolamento & purificação , Relação Estrutura-Atividade
20.
World J Microbiol Biotechnol ; 36(5): 73, 2020 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-32385754

RESUMO

Liometopum apiculatum is a species of ants widely distributed in arid and semi-arid ecosystems where there is a relative food shortage compared with tropical ecosystems. L. apiculatum has established an ecological balance involving symbiotic interactions, which have allowed them to survive through mechanisms that are still unknown. Therefore, the aim of this study was to explore the metabolic potential of isolated bacteria from L. apiculatum using enzymatic activity assay and substrate assimilation. Results revealed a complex bacteria consortium belonging to Proteobacteria, Firmicutes, and Actinobacteria phylum. Most of the isolated bacteria showed activities associated with biopolymers degradation, from them Exiguobacterium and B. simplex showed the highest amylolytic activity (27 U/mg protein), while A. johnsonii and B. pumulis showed the highest cellulolytic and xylanolytic activities (1 and 2.9 U/mg protein, respectively). By other hand, some microorganisms such as S. ficaria, E. asburiae, P. agglomerans, A. johnsonii, S. rubidaea, S. marcescens, S. warneri, and M. hydrocarbonoxydans were able to grow up to 1000 mg/L of phthalates esters. These results not only revealed the important contribution of the symbionts in L apiculatum ants feeding habits, but also have shown a promising source of enzymes with potential biotechnological applications such as lignocellulosic biomass hydrolysis and bioremediation processes.


Assuntos
Formigas/microbiologia , Bactérias/isolamento & purificação , Bactérias/metabolismo , Biodegradação Ambiental , Microbiota/fisiologia , Animais , Bactérias/classificação , Bactérias/enzimologia , Biomassa , Celulose/metabolismo , Hábitos , Hidrólise , Larva/microbiologia , Lignina/metabolismo , Polissacarídeos/metabolismo , Simbiose , Xilanos/metabolismo
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