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1.
Adv Exp Med Biol ; 1325: 3-24, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495528

RESUMO

N-glycosylation is a highly conserved glycan modification, and more than 7000 proteins are N-glycosylated in humans. N-glycosylation has many biological functions such as protein folding, trafficking, and signal transduction. Thus, glycan modification to proteins is profoundly involved in numerous physiological and pathological processes. The N-glycan precursor is biosynthesized in the endoplasmic reticulum (ER) from dolichol phosphate by sequential enzymatic reactions to generate the dolichol-linked oligosaccharide composed of 14 sugar residues, Glc3Man9GlcNAc2. The oligosaccharide is then en bloc transferred to the consensus sequence N-X-S/T (X represents any amino acid except proline) of nascent proteins. Subsequently, the N-glycosylated nascent proteins enter the folding step, in which N-glycans contribute largely to attaining the correct protein fold by recruiting the lectin-like chaperones, calnexin, and calreticulin. Despite the N-glycan-dependent folding process, some glycoproteins do not fold correctly, and these misfolded glycoproteins are destined to degradation by proteasomes in the cytosol. Properly folded proteins are transported to the Golgi, and N-glycans undergo maturation by the sequential reactions of glycosidases and glycosyltransferases, generating complex-type N-glycans. N-Acetylglucosaminyltransferases (GnT-III, GnT-IV, and GnT-V) produce branched N-glycan structures, affording a higher complexity to N-glycans. In this chapter, we provide an overview of the biosynthetic pathway of N-glycans in the ER and Golgi.


Assuntos
Glicoproteínas , Dobramento de Proteína , Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Lectinas , Polissacarídeos/metabolismo
2.
Science ; 373(6556): 774-779, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385392

RESUMO

The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.


Assuntos
Lycopersicon esculentum/parasitologia , Oxigenases de Função Mista/metabolismo , Pectinas/metabolismo , Phytophthora infestans/enzimologia , Doenças das Plantas/parasitologia , Solanum tuberosum/parasitologia , Cobre , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Modelos Moleculares , Oxirredução , Phytophthora infestans/genética , Phytophthora infestans/patogenicidade , Folhas de Planta/parasitologia , Polissacarídeos/metabolismo , Conformação Proteica , Domínios Proteicos , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
4.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361718

RESUMO

Several classes of polysaccharides have been described to have hypocholesterolemic potential, namely cholesterol bioaccessibility and bioavailability. This review will highlight the main mechanisms by which polysaccharides are known to affect cholesterol homeostasis at the intestine, namely the effect (i) of polysaccharide viscosity and its influence on cholesterol bioaccessibility; (ii) on bile salt sequestration and its dependence on the structural diversity of polysaccharides; (iii) of bio-transformations of polysaccharides and bile salts by the gut microbiota. Different quantitative structure-hypocholesterolemic activity relationships have been explored depending on the mechanism involved, and these were based on polysaccharide physicochemical properties, such as sugar composition and ramification degree, linkage type, size/molecular weight, and charge. The information gathered will support the rationalization of polysaccharides' effect on cholesterol homeostasis and highlight predictive rules towards the development of customized hypocholesterolemic functional food.


Assuntos
Anticolesterolemiantes/química , Ácidos e Sais Biliares/química , Colesterol/química , Alimento Funcional/análise , Mucosa Intestinal/metabolismo , Polissacarídeos/química , Anticolesterolemiantes/metabolismo , Anticolesterolemiantes/uso terapêutico , Ácidos e Sais Biliares/metabolismo , Disponibilidade Biológica , Biotransformação , Colesterol/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Estrutura Molecular , Peso Molecular , Polissacarídeos/metabolismo , Polissacarídeos/uso terapêutico , Eletricidade Estática
5.
Nat Commun ; 12(1): 4975, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404791

RESUMO

Plant cell wall hydrolysates contain not only sugars but also substantial amounts of acetate, a fermentation inhibitor that hinders bioconversion of lignocellulose. Despite the toxic and non-consumable nature of acetate during glucose metabolism, we demonstrate that acetate can be rapidly co-consumed with xylose by engineered Saccharomyces cerevisiae. The co-consumption leads to a metabolic re-configuration that boosts the synthesis of acetyl-CoA derived bioproducts, including triacetic acid lactone (TAL) and vitamin A, in engineered strains. Notably, by co-feeding xylose and acetate, an enginered strain produces 23.91 g/L TAL with a productivity of 0.29 g/L/h in bioreactor fermentation. This strain also completely converts a hemicellulose hydrolysate of switchgrass into 3.55 g/L TAL. These findings establish a versatile strategy that not only transforms an inhibitor into a valuable substrate but also expands the capacity of acetyl-CoA supply in S. cerevisiae for efficient bioconversion of cellulosic biomass.


Assuntos
Parede Celular/metabolismo , Engenharia Metabólica , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Biomassa , Reatores Biológicos , Fermentação , Lignina , Pironas/metabolismo , Saccharomyces cerevisiae/genética , Vitamina A/metabolismo , Xilose/metabolismo
6.
J Gen Virol ; 102(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34424155

RESUMO

Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein's surface. Here we used IBV propagated in embryonated hens' eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.


Assuntos
Coronavirus/fisiologia , Polissacarídeos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Alcaloides/química , Alcaloides/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Cromatografia Líquida , Biologia Computacional/métodos , Coronavirus/efeitos dos fármacos , Infecções por Coronavirus/veterinária , Regulação Viral da Expressão Gênica , Glicosilação/efeitos dos fármacos , Vírus da Bronquite Infecciosa/fisiologia , Modelos Moleculares , Conformação Molecular , Peso Molecular , Testes de Neutralização , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/química , Doenças das Aves Domésticas/virologia , Transporte Proteico , Espectrometria de Massas por Ionização por Electrospray , Glicoproteína da Espícula de Coronavírus/genética , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
7.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360627

RESUMO

Fucosylation is involved in a wide range of biological processes from cellular adhesion to immune regulation. Although the upregulation of fucosylated glycans was reported in diseased corneas, its implication in ocular surface disorders remains largely unknown. In this study, we analyzed the expression of a fucosylated glycan on the ocular surface in two mouse models of dry eye disease (DED), the NOD.B10.H2b mouse model and the environmental desiccating stress model. We furthermore investigated the effects of aberrant fucosylation inhibition on the ocular surface and DED. Results demonstrated that the level of type 2 H antigen, an α(1,2)-fucosylated glycan, was highly increased in the cornea and conjunctiva both in NOD.B10.H2b mice and in BALB/c mice subjected to desiccating stress. Inhibition of α(1,2)-fucosylation by 2-deoxy-D-galactose (2-D-gal) reduced corneal epithelial defects and increased tear production in both DED models. Moreover, 2-D-gal treatment suppressed the levels of inflammatory cytokines in the ocular surface and the percentages of IFN-γ+CD4+ cells in draining lymph nodes, whereas it did not affect the number of conjunctival goblet cells, the MUC5AC level or the meibomian gland area. Together, the findings indicate that aberrant fucosylation underlies the pathogenesis of DED and may be a novel target for DED therapy.


Assuntos
Túnica Conjuntiva/metabolismo , Córnea/metabolismo , Síndromes do Olho Seco/etiologia , Galactose/análogos & derivados , Antígenos H-2/metabolismo , Animais , Túnica Conjuntiva/efeitos dos fármacos , Córnea/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/metabolismo , Fucose/metabolismo , Galactose/farmacologia , Galactose/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Polissacarídeos/metabolismo
8.
Molecules ; 26(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34443447

RESUMO

Okara is a soybean transformation agri-food by-product, the massive production of which currently poses severe disposal issues. However, its composition is rich in seed storage proteins, which, once extracted, can represent an interesting source of bioactive peptides. Antimicrobial and antifungal proteins and peptides have been described in plant seeds; thus, okara is a valuable source of compounds, exploitable for integrated pest management. The aim of this work is to describe a rapid and economic procedure to isolate proteins from okara, and to produce an enzymatic proteolyzed product, active against fungal plant pathogens. The procedure allowed the isolation and recovery of about 30% of okara total proteins. Several proteolytic enzymes were screened to identify the proper procedure to produce antifungal compounds. Antifungal activity of the protein digested for 24 h with pancreatin against Fusarium and R. solani mycelial growth and Pseudomonas spp was assessed. A dose-response inhibitory activity was established against fungi belonging to the Fusarium genus. The exploitation of okara to produce antifungal bioactive peptides has the potential to turn this by-product into a paradigmatic example of circular economy, since a field-derived food waste is transformed into a source of valuable compounds to be used in field crops protection.


Assuntos
Antifúngicos/farmacologia , Enzimas/metabolismo , Proteínas de Plantas/metabolismo , Plantas/microbiologia , Polissacarídeos/metabolismo , Liofilização , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Peso Molecular , Proteólise/efeitos dos fármacos , Alimentos de Soja , Espectrofotometria Ultravioleta , Tripsina/metabolismo , Inibidores da Tripsina/farmacologia
9.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445134

RESUMO

Coxsackievirus A24 variant (CVA24v) is the primary causative agent of the highly contagious eye infection designated acute hemorrhagic conjunctivitis (AHC). It is solely responsible for two pandemics and several recurring outbreaks of the disease over the last decades, thus affecting millions of individuals throughout the world. To date, no antiviral agents or vaccines are available for combating this disease, and treatment is mainly supportive. CVA24v utilizes Neu5Ac-containing glycans as attachment receptors facilitating entry into host cells. We have previously reported that pentavalent Neu5Ac conjugates based on a glucose-scaffold inhibit CVA24v infection of human corneal epithelial cells. In this study, we report on the design and synthesis of scaffold-replaced pentavalent Neu5Ac conjugates and their effect on CVA24v cell transduction and the use of cryogenic electron microscopy (cryo-EM) to study the binding of these multivalent conjugates to CVA24v. The results presented here provide insights into the development of Neu5Ac-based inhibitors of CVA24v and, most significantly, the first application of cryo-EM to study the binding of a multivalent ligand to a lectin.


Assuntos
Antivirais/farmacologia , Infecções por Coxsackievirus/dietoterapia , Enterovirus Humano C/efeitos dos fármacos , Ácido N-Acetilneuramínico/farmacologia , Conjuntivite Hemorrágica Aguda/tratamento farmacológico , Conjuntivite Hemorrágica Aguda/metabolismo , Conjuntivite Hemorrágica Aguda/virologia , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/virologia , Glucose/metabolismo , Humanos , Lectinas/metabolismo , Ligantes , Polissacarídeos/metabolismo , Receptores Virais/metabolismo
10.
Molecules ; 26(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34443386

RESUMO

Lectins facilitate cell-cell contact and are critical in many cellular processes. Studying lectins may help us understand the mechanisms underlying tissue regeneration. We investigated the localization of an R-type lectin in a marine annelid (Perinereis sp.) with remarkable tissue regeneration abilities. Perinereis nuntia lectin (PnL), a galactose-binding lectin with repeating Gln-X-Trp motifs, is derived from the ricin B-chain. An antiserum was raised against PnL to specifically detect a 32-kDa lectin in the crude extracts from homogenized lugworms. The antiserum detected PnL in the epidermis, setae, oblique muscle, acicula, nerve cord, and nephridium of the annelid. Some of these tissues and organs also produced Galactose (Gal) or N-acetylgalactosamine (GalNAc), which was detected by fluorescent-labeled plant lectin. These results indicated that the PnL was produced in the tissues originating from the endoderm, mesoderm, and ectoderm. Besides, the localizing pattern of PnL partially merged with the binding pattern of a fluorescent-labeled mushroom lectin that binds to Gal and GalNAc. It suggested that PnL co-localized with galactose-containing glycans in Annelid tissue; this might be the reason PnL needed to be extracted with haptenic sugar, such as d-galactose, in the buffer. Furthermore, we found that a fluorescein isothiocyanate-labeled Gal/GalNAc-binding mushroom lectin binding pattern in the annelid tissue overlapped with the localizing pattern of PnL. These findings suggest that lectin functions by interacting with Gal-containing glycoconjugates in the tissues.


Assuntos
Anelídeos/metabolismo , Organismos Aquáticos/metabolismo , Lectinas/metabolismo , Animais , Misturas Complexas , Ligantes , Polissacarídeos/metabolismo
11.
Postepy Biochem ; 67(2): 104-116, 2021 06 30.
Artigo em Polonês | MEDLINE | ID: mdl-34378887

RESUMO

Changes in glycosylation pattern of cell surface, body fluids and extracellular matrix glycoconjugates is a characteristic feature of tumor cell malignancy. These changes are the result of mutations of tumor-associated genes as well as epigenetic changes in the tumor environment, including nutrient influx, hypoxia, cytokine expression and stimulation of chronic inflammation. The unique set of cell surface glycoantigens on neoplastic cells is recognized by endogenous lectins located in the extracellular matrix, vascular endothelium, on leukocytes or platelets, and has an impact on disrupting basic cellular processes, such as intercellular recognition, cell-cell adhesion or cell-ECM interaction. These changes have a critical impact on the migration, invasive and metastatic potential of neoplastic cells and modulate the immune response. This unique pattern of sugar antigens on the cancer cells can be a vaulable marker to identify them, determine the stage of the disease as well as be a target of anti-cancer therapy.


Assuntos
Neoplasias , Adesão Celular , Matriz Extracelular/metabolismo , Glicosilação , Humanos , Neoplasias/metabolismo , Polissacarídeos/metabolismo
12.
Theranostics ; 11(16): 7970-7983, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34335974

RESUMO

The novel ß-coronavirus, SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has infected more than 177 million people and resulted in 3.84 million death worldwide. Recent epidemiological studies suggested that some environmental factors, such as air pollution, might be the important contributors to the mortality of COVID-19. However, how environmental exposure enhances the severity of COVID-19 remains to be fully understood. In the present report, we provided evidence showing that mdig, a previously reported environmentally-induced oncogene that antagonizes repressive trimethylation of histone proteins, is an important regulator for SARS-CoV-2 receptors neuropilin-1 (NRP1) and NRP2, cathepsins, glycan metabolism and inflammation, key determinants for viral infection and cytokine storm of the patients. Depletion of mdig in bronchial epithelial cells by CRISPR-Cas-9 gene editing resulted in a decreased expression of NRP1, NRP2, cathepsins, and genes involved in protein glycosylation and inflammation, largely due to a substantial enrichment of lysine 9 and/or lysine 27 trimethylation of histone H3 (H3K9me3/H3K27me3) on these genes as determined by ChIP-seq. Meanwhile, we also validated that environmental factor arsenic is able to induce mdig, NRP1 and NRP2, and genetic disruption of mdig lowered expression of NRP1 and NRP2. Furthermore, mdig may coordinate with the Neanderthal variants linked to an elevated mortality of COVID-19. These data, thus, suggest that mdig is a key mediator for the severity of COVID-19 in response to environmental exposure and targeting mdig may be the one of the effective strategies in ameliorating the symptom and reducing the mortality of COVID-19.


Assuntos
COVID-19/metabolismo , COVID-19/virologia , Dioxigenases/metabolismo , Histona Desmetilases/metabolismo , Neuropilina-1/metabolismo , Proteínas Nucleares/metabolismo , Polissacarídeos/metabolismo , SARS-CoV-2/metabolismo , Células Epiteliais Alveolares/metabolismo , Animais , COVID-19/epidemiologia , Catepsinas/metabolismo , Linhagem Celular , Células Cultivadas , Dioxigenases/biossíntese , Dioxigenases/genética , Exposição Ambiental , Histona Desmetilases/biossíntese , Histona Desmetilases/genética , Histonas/metabolismo , Humanos , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Pandemias , Ratos , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo
13.
Nat Commun ; 12(1): 4070, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34210959

RESUMO

Mucins are a large family of heavily O-glycosylated proteins that cover all mucosal surfaces and constitute the major macromolecules in most body fluids. Mucins are primarily defined by their variable tandem repeat (TR) domains that are densely decorated with different O-glycan structures in distinct patterns, and these arguably convey much of the informational content of mucins. Here, we develop a cell-based platform for the display and production of human TR O-glycodomains (~200 amino acids) with tunable structures and patterns of O-glycans using membrane-bound and secreted reporters expressed in glycoengineered HEK293 cells. Availability of defined mucin TR O-glycodomains advances experimental studies into the versatile role of mucins at the interface with pathogenic microorganisms and the microbiome, and sparks new strategies for molecular dissection of specific roles of adhesins, glycoside hydrolases, glycopeptidases, viruses and other interactions with mucin TRs as highlighted by examples.


Assuntos
Mucinas/metabolismo , Membrana Mucosa/metabolismo , Polissacarídeos/genética , Polissacarídeos/metabolismo , Engenharia Genética , Glicosilação , Células HEK293 , Humanos , Microbiota , Mucina-1/genética , Mucina-1/metabolismo
14.
Biomolecules ; 11(6)2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207636

RESUMO

Proteomics-large-scale studies of proteins-has over the last decade gained an enormous interest for studies aimed at revealing proteins and pathways involved in disease. To fully understand biological and pathological processes it is crucial to also include post-translational modifications in the "omics". To this end, glycomics (identification and quantification of glycans enzymatically or chemically released from proteins) and glycoproteomics (identification and quantification of peptides/proteins with the glycans still attached) is gaining interest. The study of protein glycosylation requires a workflow that involves an array of sample preparation and analysis steps that needs to be carefully considered. Herein, we briefly touch upon important steps such as sample preparation and preconcentration, glycan release, glycan derivatization and quantification and advances in mass spectrometry that today are the work-horse for glycomics and glycoproteomics studies. Several proteins related to Alzheimer disease pathogenesis have altered protein glycosylation, and recent glycomics studies have shown differences in cerebrospinal fluid as well as in brain tissue in Alzheimer disease as compared to controls. In this review, we discuss these techniques and how they have been used to shed light on Alzheimer disease and to find glycan biomarkers in cerebrospinal fluid.


Assuntos
Doença de Alzheimer/metabolismo , Glicoproteínas/análise , Polissacarídeos/análise , Doença de Alzheimer/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Biomarcadores/metabolismo , Líquido Cefalorraquidiano/química , Líquido Cefalorraquidiano/metabolismo , Cromatografia Líquida/métodos , Glicômica/métodos , Glicoproteínas/líquido cefalorraquidiano , Glicoproteínas/metabolismo , Glicosilação , Humanos , Polissacarídeos/líquido cefalorraquidiano , Polissacarídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
15.
Methods Mol Biol ; 2350: 313-329, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331294

RESUMO

We describe a multiplexed imaging mass spectrometry approach especially suitable for fibrosis research. Fibrosis is a process characterized by excessive extracellular matrix (ECM) secretion. Buildup of ECM impairs tissue and organ function to promote further progression of disease. It is an ongoing analytical challenge to access ECM for diagnosis and therapeutic treatment of fibrosis. Recently, we reported the use of the enzyme collagenase type III to target the ECM proteome in thin histological tissue sections of fibrotic diseases including hepatocellular carcinoma, breast cancer, prostate cancer, lung cancer and aortic valve stenosis. Detection of collagenase type III peptides by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows localization of ECM peptide sequences to specific regions of fibrosis. We have further identified that the ECM proteome accessed by collagenase type III has on average 3.7 sites per protein that may be differentially N-glycosylated. N-glycosylation is a major posttranslational modification of the ECM proteome, influencing protein folding, secretion, and organization. Understanding both N-glycosylation signaling and regulation of ECM expression significantly informs on ECM signaling in fibrosis.


Assuntos
Biomarcadores , Matriz Extracelular/metabolismo , Histocitoquímica/métodos , Espectrometria de Massas/métodos , Polissacarídeos/metabolismo , Fibrose/metabolismo , Fibrose/patologia , Glicosilação , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Pesquisa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fluxo de Trabalho
16.
Virology ; 562: 142-148, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34325286

RESUMO

SARS-CoV, MERS-CoV, and potentially SARS-CoV-2 emerged as novel human coronaviruses following cross-species transmission from animal hosts. Although the receptor binding characteristics of human coronaviruses are well documented, the role of carbohydrate binding in addition to recognition of proteinaceous receptors has not been fully explored. Using natural glycan microarray technology, we identified N-glycans in the human lung that are recognized by various human and animal coronaviruses. All viruses tested, including SARS-CoV-2, bound strongly to a range of phosphorylated, high mannose N-glycans and to a very specific set of sialylated structures. Examination of two linked strains, human CoV OC43 and bovine CoV Mebus, reveals shared binding to the sialic acid form Neu5Gc (not found in humans), supporting the evidence for cross-species transmission of the bovine strain. Our findings, revealing robust recognition of lung glycans, suggest that these receptors could play a role in the initial stages of coronavirus attachment and entry.


Assuntos
COVID-19/virologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Polissacarídeos/metabolismo , SARS-CoV-2/metabolismo , Animais , Bovinos , Humanos , Pulmão/metabolismo , Manose/química , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Ácido N-Acetilneuramínico/química , Fosforilação , Análise Serial de Proteínas , Ligação Proteica , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/fisiologia
17.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298931

RESUMO

Rhus potaninii Maxim is an economically and medicinally important tree species in China. It produces galls (induced by aphids) with a high abundance of tannins. Here, we discuss the histology, cellular structures and their distribution, and the macromolecular components of secretive glandular trichomes on the leaves of R. potaninii. A variation in the density of glandular trichomes and tomenta was found between the adaxial and abaxial sides of a leaf in different regions and stages of the leaf. The glandular trichomes on R. potaninii trees comprise a stalk with no cellular structure and a head with 8-15 cells. Based on staining, we found that the secretion of glandular trichomes has many polysaccharides, phenolic compounds, and acidic lipids but very few neutral lipids. The dense glandular trichomes provide mechanical protection for young tissues; additionally, their secretion protects the young tissues from pathogens by a special chemical component. According to transcriptome analysis, we found enhanced biosynthetic and metabolism pathways of glycan, lipids, toxic amino acids, and phenylpropanoids. This shows a similar tendency to the staining. The numbers of differentially expressed genes were large or small; the averaged range of upregulated genes was greater than that of the downregulated genes in most subpathways. Some selectively expressed genes were found in glandular trichomes, responsible for the chitinase activity and pathogenesis-related proteins, which all have antibacterial activity and serve for plant defense. To our knowledge, this is the first study showing the components of the secretion from glandular trichomes on the leaf surface of R. potaninii.


Assuntos
Regulação da Expressão Gênica de Plantas/genética , Expressão Gênica/genética , Folhas de Planta/genética , Rhus/genética , Transcriptoma/genética , Tricomas/genética , Regulação para Baixo/genética , Perfilação da Expressão Gênica/métodos , Lipídeos/genética , Fenol/metabolismo , Polissacarídeos/metabolismo , Tricomas/metabolismo , Regulação para Cima/genética
18.
Molecules ; 26(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200965

RESUMO

Glycosylation is the most prevalent and varied form of post-translational protein modifications. Protein glycosylation regulates multiple cellular functions, including protein folding, cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. In particular, membrane proteins are frequently highly glycosylated, which is both linked to physiological processes and of high relevance in various disease mechanisms. The cellular glycome is increasingly considered to be a therapeutic target. Here we describe a new strategy to compare membrane glycoproteomes, thereby identifying proteins with altered glycan structures and the respective glycosites. The workflow started with an optimized procedure for the digestion of membrane proteins followed by the lectin-based isolation of glycopeptides. Since alterations in the glycan part of a glycopeptide cause mass alterations, analytical size exclusion chromatography was applied to detect these mass shifts. N-glycosidase treatment combined with nanoUPLC-coupled mass spectrometry identified the altered glycoproteins and respective glycosites. The methodology was established using the colon cancer cell line CX1, which was treated with 2-deoxy-glucose-a modulator of N-glycosylation. The described methodology is not restricted to cell culture, as it can also be adapted to tissue samples or body fluids. Altogether, it is a useful module in various experimental settings that target glycan functions.


Assuntos
Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Glucose/metabolismo , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Polissacarídeos/metabolismo , Proteômica/métodos
19.
Int J Mol Sci ; 22(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206740

RESUMO

N-Glycosylations are an important post-translational modification of proteins that can significantly impact cell function. Terminal sialic acid in hybrid or complex N-glycans has been shown to be relevant in various types of cancer, but its role in non-malignant cells remains poorly understood. We have previously shown that the motility of human bone marrow derived mesenchymal stromal cells (MSCs) can be modified by altering N-glycoforms. The goal of this study was to determine the role of sialylated N-glycans in MSCs. Here, we show that IFN-gamma or exposure to culture media low in fetal bovine serum (FBS) increases sialylated N-glycans, while PDGF-BB reduces them. These stimuli alter mRNA levels of sialyltransferases such as ST3Gal1, ST6Gal1, or ST3Gal4, suggesting that sialylation of N-glycans is regulated by transcriptional control of sialyltransferases. We next show that 2,4,7,8,9-pentaacetyl-3Fax-Neu5Ac-CO2Me (3F-Neu5Ac) effectively inhibits sialylations in MSCs. Supplementation with 3F-Neu5Ac increases adhesion and migration of MSCs, as assessed by both videomicroscopy and wound/scratch assays. Interestingly, pre-treatment with 3F-Neu5Ac also increases the survival of MSCs in an in vitro ischemia model. We also show that pre-treatment or continuous treatment with 3F-Neu5Ac inhibits both osteogenic and adipogenic differentiation of MSCs. Finally, secretion of key trophic factors by MSCs is variably affected upon exposure to 3F-Neu5Ac. Altogether, our experiments suggest that sialylation of N-glycans is tightly regulated in response to environmental cues and that glycoengineering MSCs to reduce sialylated N-glycans could be beneficial to increase both cell migration and survival, which may positively impact the therapeutic potential of the cells.


Assuntos
Movimento Celular , Células-Tronco Mesenquimais/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polissacarídeos/metabolismo , Sialiltransferases/metabolismo , Adipócitos/metabolismo , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Interferon gama/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Osteoblastos/citologia , Sialiltransferases/antagonistas & inibidores
20.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206350

RESUMO

The monolayer character of two-dimensional materials predestines them for application as active layers of sensors. However, their inherent high sensitivity is always accompanied by a low selectivity. Chemical functionalization of two-dimensional materials has emerged as a promising way to overcome the selectivity issues. Here, we demonstrate efficient graphene functionalization with carbohydrate ligands-chitooligomers, which bind proteins of the lectin family with high selectivity. Successful grafting of a chitooligomer library was thoroughly characterized, and glycan binding to wheat germ agglutinin was studied by a series of methods. The results demonstrate that the protein quaternary structure remains intact after binding to the functionalized graphene, and that the lectin can be liberated from the surface by the addition of a binding competitor. The chemoenzymatic assay with a horseradish peroxidase conjugate also confirmed the intact catalytic properties of the enzyme. The present approach thus paves the way towards graphene-based sensors for carbohydrate-lectin binding.


Assuntos
Grafite/química , Lectinas/metabolismo , Polissacarídeos/química , Peroxidase do Rábano Silvestre , Lectinas/análise , Polissacarídeos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína
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