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1.
Nat Commun ; 11(1): 6343, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311467

RESUMO

D-mannose is a monosaccharide approximately a hundred times less abundant than glucose in human blood. Previous studies demonstrated that supraphysiological levels of D-mannose inhibit tumour growth and stimulate regulatory T cell differentiation. It is not known whether D-mannose metabolism affects the function of non-proliferative cells, such as inflammatory macrophages. Here, we show that D-mannose suppresses LPS-induced macrophage activation by impairing IL-1ß production. In vivo, mannose administration improves survival in a mouse model of LPS-induced endotoxemia as well as decreases progression in a mouse model of DSS-induced colitis. Phosphomannose isomerase controls response of LPS-activated macrophages to D-mannose, which impairs glucose metabolism by raising intracellular mannose-6-phosphate levels. Such alterations result in the suppression of succinate-mediated HIF-1α activation, imposing a consequent reduction of LPS-induced Il1b expression. Disclosing an unrecognized metabolic hijack of macrophage activation, our study points towards safe D-mannose utilization as an effective intervention against inflammatory conditions.


Assuntos
Interleucina-1beta/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Manose/metabolismo , Manose/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Colite/metabolismo , Colite/patologia , Regulação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Interleucina-1beta/genética , Lipopolissacarídeos/efeitos adversos , Manosefosfatos/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Metabolômica , Monócitos/metabolismo
2.
PLoS One ; 15(11): e0241249, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33170858

RESUMO

OBJECTIVES: Islet transplantation is an emerging treatment option for type 1 diabetes but its application is limited by the shortage of human pancreas donors. Characterization of the N- and O-glycan surface antigens that vary between human and genetically engineered porcine islet donors could shed light on targets of antibody mediated rejection. METHODS: N- and O-glycans were isolated from human and adult porcine islets and analyzed using matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS/MS). RESULTS: A total of 57 porcine and 34 human N-glycans and 21 porcine and 14 human O-glycans were detected from cultured islets. Twenty-eight of which were detected only from porcine islets, which include novel xenoantigens such as high-mannose type N-glycans with core fucosylation and complex-type N-glycans with terminal neuraminic acid residues. Porcine islets have terminal N-glycolylneuraminic acid (NeuGc) residue in bi-antennary N-glycans and sialyl-Tn O-glycans. No galactose-α-1,3-galactose (α-Gal) or Sda epitope were detected on any of the islets. CONCLUSIONS: These results provide important insights into the potential antigenic differences of N- and O-glycan profiles between human and porcine islets. Glycan differences may identify novel gene targets for genetic engineering to generate superior porcine islet donors.


Assuntos
Fucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Manose/metabolismo , Ácidos Neuramínicos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Adulto , Animais , Vias Biossintéticas , Feminino , Glicosilação , Humanos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos
3.
Proc Natl Acad Sci U S A ; 117(43): 26926-26935, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33046650

RESUMO

Influenza virus infections cause a wide variety of outcomes, from mild disease to 3 to 5 million cases of severe illness and ∼290,000 to 645,000 deaths annually worldwide. The molecular mechanisms underlying these disparate outcomes are currently unknown. Glycosylation within the human host plays a critical role in influenza virus biology. However, the impact these modifications have on the severity of influenza disease has not been examined. Herein, we profile the glycomic host responses to influenza virus infection as a function of disease severity using a ferret model and our lectin microarray technology. We identify the glycan epitope high mannose as a marker of influenza virus-induced pathogenesis and severity of disease outcome. Induction of high mannose is dependent upon the unfolded protein response (UPR) pathway, a pathway previously shown to associate with lung damage and severity of influenza virus infection. Also, the mannan-binding lectin (MBL2), an innate immune lectin that negatively impacts influenza outcomes, recognizes influenza virus-infected cells in a high mannose-dependent manner. Together, our data argue that the high mannose motif is an infection-associated molecular pattern on host cells that may guide immune responses leading to the concomitant damage associated with severity.


Assuntos
Glicoproteínas/metabolismo , Interações Hospedeiro-Patógeno , Influenza Humana/metabolismo , Pulmão/metabolismo , Manose/metabolismo , Células A549 , Animais , Metabolismo dos Carboidratos , Feminino , Furões , Glicômica , Glicosilação , Humanos , Vírus da Influenza A Subtipo H1N1 , Lectina de Ligação a Manose/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo
4.
Int J Biol Macromol ; 163: 1649-1658, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32979436

RESUMO

The SARS-CoV-2 spike glycoproteins (SGPs) and human angiotensin converting enzyme 2 (ACE2) are the two key targets for the prevention and treatment of COVID-19. Host cell surface heparan sulfate (HS) is believed to interact with SARS-CoV-2 SGPs to facilitate host cell entry. In the current study, a series of polysaccharides from Saccharina japonica were prepared to investigate the structure-activity relationship on the binding abilities of polysaccharides (oligosaccharides) to pseudotype particles, including SARS-CoV-2 SGPs, and ACE2 using surface plasmon resonance. Sulfated galactofucan (SJ-D-S-H) and glucuronomannan (Gn) displayed strongly inhibited interaction between SARS-CoV-2 SGPs and heparin while showing negligible inhibition of the interaction between SARS-CoV-2 SGPs and ACE2. The IC50 values of SJ-D-S-H and Gn in blocking heparin SGP binding were 27 and 231 nM, respectively. NMR analysis showed that the structure of SJ-D-S-H featured with a backbone of 1, 3-linked α-L-Fucp residues sulfated at C4 and C2/C4 and 1, 3-linked α-L-Fucp residues sulfated at C4 and branched with 1, 6-linked ß-D-galacto-biose; Gn had a backbone of alternating 1, 4-linked ß-D-GlcAp residues and 1, 2-linked α-D-Manp residues. The sulfated galactofucan and glucuronomannan showed strong binding ability to SARS-CoV-2 SGPs, suggesting that these polysaccharides might be good candidates for preventing and/or treating SARS-CoV-2.


Assuntos
Infecções por Coronavirus/virologia , Glucuronatos/metabolismo , Manose/análogos & derivados , Pneumonia Viral/virologia , Polissacarídeos/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Betacoronavirus/química , Betacoronavirus/metabolismo , Sítios de Ligação , Glucuronatos/química , Heparina/química , Heparina/metabolismo , Humanos , Manose/química , Manose/metabolismo , Oligossacarídeos/química , Pandemias , Peptidil Dipeptidase A/metabolismo , Feófitas/química , Polissacarídeos/química , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/química , Relação Estrutura-Atividade
5.
Mol Genet Genomics ; 295(6): 1489-1500, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32948893

RESUMO

Glucose, fructose and mannose are the preferred carbon/energy sources for the yeast Saccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role.


Assuntos
Núcleo Celular/metabolismo , Frutose/metabolismo , Glucose/metabolismo , Hexoquinase/metabolismo , Manose/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Regulação Fúngica da Expressão Gênica , Hexoquinase/genética , Fosforilação , Transporte Proteico , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
6.
Biochim Biophys Acta Gen Subj ; 1864(12): 129709, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32858085

RESUMO

BACKGROUND: In the endoplasmic reticulum (ER), folding of glycoproteins is assisted by a combined action of enzymes and chaperones that leads them to biologically functional structures. In this system, UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) plays an essential role as the "folding sensor" by virtue of its ability to discriminate folding states of client glycoproteins. However, besides its transferase activity, whether UGGT1 possesses any chaperone activity that facilitates protein folding is yet to be addressed. METHODS: We prepared oligomannose-type glycan modified RNase (M9GN2-RNase) by chemoenzymatic means using M9GN-oxazoline and glycan truncated RNase B and analyzed the effect of human UGGT1 (HUGT1) for refolding of the denatured M9GN2-RNase. Refolding was evaluated based on the RNase activity which was measured by the cleavage of the RNA substrate. RESULTS: HUGT1 slightly accelerated the folding of M9GN2-RNase and non-glycosylated RNase A as the same extent. However, HUGT1 remarkably accelerated the folding of M9GN2-RNase in the presence of UDP-Glc. In contrast, neither UDP nor UDP-Gal was effective in enhancing the folding. Additionally, an HUGT1 mutant which lacks the glucosyltransferase activity did not accelerate the protein folding of M9GN2-RNase. CONCLUSIONS: HUGT1has the ability to promote the refolding of denatured protein and the effect would be enhanced when HUGT1 tightly interacts with the client protein via glycan recognition. GENERAL SIGNIFICANCE: Our study provides a possibility that HUGT1 play a role not only in sensing the misfolded glycoprotein but also in promoting folding of glycoproteins in the endoplasmic reticulum glycoprotein quality control.


Assuntos
Glucosiltransferases/metabolismo , Polissacarídeos/metabolismo , Redobramento de Proteína , Ribonucleases/metabolismo , Glicosilação , Humanos , Manose/metabolismo , Desnaturação Proteica , Dobramento de Proteína , Especificidade por Substrato
7.
Biochim Biophys Acta Biomembr ; 1862(11): 183412, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32710850

RESUMO

Mannose transporters constitute a superfamily (Man-PTS) of the Phosphoenolpyruvate Carbohydrate Phosphotransferase System (PTS). The membrane complexes are homotrimers of protomers consisting of two subunits, IIC and IID. The two subunits without recognizable sequence similarity assume the same fold, and in the protomer are structurally related by a two fold pseudosymmetry axis parallel to membrane-plane (Liu et al. (2019) Cell Research 29 680). Two reentrant loops and two transmembrane helices of each subunit together form the N-terminal transport domain. Two three-helix bundles, one of each subunit, form the scaffold domain. The protomer is stabilized by a helix swap between these bundles. The two C-terminal helices of IIC mediate the interprotomer contacts. PTS occur in bacteria and archaea but not in eukaryotes. Man-PTS are abundant in Gram-positive bacteria living on carbohydrate rich mucosal surfaces. A subgroup of IICIID complexes serve as receptors for class IIa bacteriocins and as channel for the penetration of bacteriophage lambda DNA across the inner membrane. Some Man-PTS are associated with host-pathogen and -symbiont processes.


Assuntos
Proteínas de Bactérias , Bacteriocinas , Bacteriófagos , Bactérias Gram-Positivas , Manose , Fosfotransferases , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacteriocinas/química , Bacteriocinas/metabolismo , Bacteriófagos/química , Bacteriófagos/metabolismo , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/metabolismo , Bactérias Gram-Positivas/virologia , Manose/química , Manose/metabolismo , Fosfotransferases/química , Fosfotransferases/metabolismo , Conformação Proteica em alfa-Hélice , Domínios Proteicos
8.
Biochim Biophys Acta Gen Subj ; 1864(9): 129637, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32442478

RESUMO

BACKGROUND: C-mannosylation is the one of glycosylations. Microfibril-associated glycoprotein 4 (MFAP4), an important protein for tissue homeostasis and cell adhesion, contains a consensus sequence of C-mannosylation in its fibrinogen C-terminal domain. In this study, we sought to demonstrate that fibrinogen C-terminal domain is a new substrate domain for C-mannosylation. METHODS: We established an MFAP4-overexpresssing HT1080 cell line and purified recombinant MFAP4 protein from the conditioned medium for LC-MS/MS analysis. Subcellular localization of MFAP4 was observed under confocal fluorescence microscope. RESULTS: We found that MFAP4 is C-mannosylated at Trp235 in the fibrinogen C-terminal domain by LC-MS/MS. To determine the functions of the C-mannosylation of MFAP4, we established a C-mannosylation-defective mutant MFAP4-overexpresssing HT1080 cell line and measured its secretion of MFAP4. The secretion of MFAP4 decreased significantly in the C-mannosylation-defective mutant MFAP4-overexpresssing cell line versus wild-type cells. Moreover, co-transfection experiments indicated that C-mannosylated MFAP4 accelerated its secretion. CONCLUSIONS: Our results demonstrate that the fibrinogen C-terminal domain is a novel C-mannosylation domain and that the C-mannosylation of MFAP4 is important for its secretion. GENERAL SIGNIFICANCE: These results suggest that C-mannosylation has a role for dominant effect for MFAP4 secretion.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibrinogênio/química , Fibrinogênio/metabolismo , Glicoproteínas/metabolismo , Manose/metabolismo , Linhagem Celular Tumoral , Humanos , Domínios Proteicos , Transporte Proteico
9.
Biochim Biophys Acta Gen Subj ; 1864(8): 129632, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32416197

RESUMO

BACKGROUND: Mindin (spondin2), a secretory protein related to neural development and immunity, is a member of thrombospondin type I repeat (TSR) superfamily proteins, and has a unique glycosylation of C-mannosylation in its structure. However, it remains unclear whether C-mannosylation plays a functional role in the biosynthesis of mindin in cells. METHODS: Protein C-mannosylation was analyzed by mass spectrometry. Mindin expression was examined by immunoblot and immunofluorescence analyses in COS-7 cells transfected with the expression vectors for wild type (mindin-WT) or C-mannosylation-defective mutant of mindin (mindin-mutF). The redox status was examined in mindin by using 4-acetoamide-4'-maleimidylstilbene-2,2'-disulfonate. RESULTS: When mindin cDNA was expressed in COS-7 cells, C-mannosylation of mindin was confirmed at Trp257 by mass spectrometry. In cells expressing a mindin-mutF, secretion of the mutant was significantly inhibited compared with mindin-WT. In immunofluorescence analysis, mindin-mutF was accumulated in the endoplasmic reticulum (ER), whereas mindin-WT was detected in the Golgi. In addition, mindin-mutF showed an enhanced interaction with calreticulin, an ER-resident chaperone, in cells. In cells, reduced forms were increased in mindin-mutF, compared with a mostly oxidized form of mindin-WT. In the presence of chemical chaperones such as dimethylsulfoxide or 4-phenylbutyrate, inhibited secretion of mindin-mutF was ameliorated in cells, although redox-dependent folding was not affected. CONCLUSIONS: C-Mannosylation of mindin facilitates its secretion especially through modulating disulfide bond formation in mindin in cells. GENERAL SIGNIFICANCE: These results suggest that C-mannosylation plays a functional role in the redox-dependent folding and transport of TSR superfamily proteins in cells.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Manose/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Glicosilação , Camundongos , Chaperonas Moleculares/metabolismo , Células NIH 3T3 , Coelhos
10.
Proc Natl Acad Sci U S A ; 117(22): 11931-11939, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32424105

RESUMO

Cell surfaces are often decorated with glycoconjugates that contain linear and more complex symmetrically and asymmetrically branched carbohydrates essential for cellular recognition and communication processes. Mannose is one of the fundamental building blocks of glycans in many biological membranes. Moreover, oligomannoses are commonly found on the surface of pathogens such as bacteria and viruses as both glycolipids and glycoproteins. However, their mechanism of action is not well understood, even though this is of great potential interest for translational medicine. Sequence-defined amphiphilic Janus glycodendrimers containing simple mono- and disaccharides that mimic glycolipids are known to self-assemble into glycodendrimersomes, which in turn resemble the surface of a cell by encoding carbohydrate activity via supramolecular multivalency. The synthetic challenge of preparing Janus glycodendrimers containing more complex linear and branched glycans has so far prevented access to more realistic cell mimics. However, the present work reports the use of an isothiocyanate-amine "click"-like reaction between isothiocyanate-containing sequence-defined amphiphilic Janus dendrimers and either linear or branched oligosaccharides containing up to six monosaccharide units attached to a hydrophobic amino-pentyl linker, a construct not expected to assemble into glycodendrimersomes. Unexpectedly, these oligoMan-containing dendrimers, which have their hydrophobic linker connected via a thiourea group to the amphiphilic part of Janus glycodendrimers, self-organize into nanoscale glycodendrimersomes. Specifically, the mannose-binding lectins that best agglutinate glycodendrimersomes are those displaying hexamannose. Lamellar "raft-like" nanomorphologies on the surface of glycodendrimersomes, self-organized from these sequence-defined glycans, endow these membrane mimics with high biological activity.


Assuntos
Biomimética/métodos , Dendrímeros/síntese química , Glicoconjugados/síntese química , Nanopartículas/química , Membrana Celular/química , Glicolipídeos/química , Interações Hidrofóbicas e Hidrofílicas , Isotiocianatos/metabolismo , Lectinas/metabolismo , Manose/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Pesquisa Médica Translacional/métodos
11.
Anal Chim Acta ; 1109: 69-77, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32252907

RESUMO

Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme. We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied. Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished. We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batches.


Assuntos
Lipase/metabolismo , Manose/metabolismo , Aspergillus niger/enzimologia , Cromatografia Líquida , Glicosilação , Interações Hidrofóbicas e Hidrofílicas , Lipase/química , Manose/química , Espectrometria de Massas
12.
Comp Biochem Physiol B Biochem Mol Biol ; 243-244: 110437, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32247057

RESUMO

Salinity is an important abiotic factor for aquatic organisms. In fish, changes in salinity affect physiological responses and alter the immune system. Takifugu rubripes is an important economic marine fish, and mechanisms of T. rubripes adaptation to salinity changes need to be further documented. In this study, a transcriptome sequencing technique was used to analyse genes that were differentially expressed in the T. rubripes gill after low-salinity stress for 30 d, and differential gene expression was further validated by quantitative real-time PCR (qPCR). After assembly, 385 differentially expressed genes (DEGs) were identified, including 182 upregulated genes and 203 downregulated genes. The DEGs were assigned to Gene Ontology (GO) classes with a total of 1647 functional terms. Most DEGs were assigned to biological process (984; 59.8%) followed by molecular function (445; 27.0%) and cellular component (218; 13.2%). Further KEGG analysis allocated 385 DEGs to 95 KEGG pathways. After q-value correction, 7 pathways (Glycolysis/Gluconeogenesis; Biosynthesis of amino acids; Carbon metabolism; Fructose and mannose metabolism; Pentose phosphate pathway; Metabolism of xenobiotics by cytochrome P450; and Glycine, serine and threonine metabolism) remained significant. qPCR results indicated that the transcripts of six selected genes sharply increased after 30 d of low-salinity stress. Low-salinity stress obviously increased SLC39A6, SLC5A9, NKAα1, CYP1A1, CYP1B1, and GSTA expression. In contrast, the genes encoding Aldoaa, GPI, FBP2 and GAPDH exhibited downregulation. In addition, three solute carrier (SLC) genes selected from the DEGs were further studied for differential expression patterns after low-salinity exposure, and the results showed that the SLCs were upregulated in T. rubripes after 72 h of low-salinity exposure. This investigation provides data for understanding the molecular mechanisms of fish responses to low-salinity stress and provides a reference for rationally setting salinity levels in aquaculture.


Assuntos
Estresse Salino/genética , Transdução de Sinais/genética , Takifugu/metabolismo , Transcriptoma/genética , Aclimatação/genética , Aminoácidos/metabolismo , Animais , Frutose/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Ontologia Genética , Gluconeogênese/genética , Glicólise/genética , Manose/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Takifugu/genética
13.
Mol Immunol ; 121: 144-158, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32222585

RESUMO

Most of therapeutic monoclonal antibodies belong to the immunoglobulin G1 (IgG1) family; they interact with the Fcγ receptors (FcγRs) at the surface of immune cells to trigger effector functions. The IgG1-Fc N-glycans impact the interaction with FcγRs and are considered a critical quality attribute. Pioneer studies on FcγR N-glycans have unveiled an additional complexity in that the N-glycan linked on the Asn-162 of FcγRIIIa was shown to be directly involved in the strong affinity for afucosylated IgG1. The last few years have thus seen the emergence of many studies investigating the complex influence of FcγRIIIa N-glycans on the interaction with IgG1 through their glycosylation sites or their glycoprofiles. In this context, we performed site-directed mutagenesis along with glycoengineering on FcγRs (FcγRI, FcγRIIaH131/b and FcγRIIIaV158/F158) in an effort to elucidate the impact of FcγRs N-glycans on the interaction with IgG1. Furthermore, we assessed their binding to various trastuzumab glycoforms with an enhanced surface plasmon resonance assay. The FcγRIIIa N-glycans had the highest impact on the interaction with IgG1. More specifically, the N162 glycan positively influenced the affinity (15-fold) for afucosylated IgG1 while the N45 glycan presented a negative impact (2-fold) regardless of the IgG1 glycoforms. Interestingly, only the FcγRIIIa glycoprofile had an impact on the interaction with IgG1 with a 1.5-fold affinity increase when FcγRIIIa displays high-mannose glycans. These results provide invaluable insights into the complex and strong influence of N-glycosylation upon FcγRs/IgG1 binding and are instrumental to further understand the impact of FcγRs N-glycosylation in their natural forms.


Assuntos
Imunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Animais , Células CHO , Cricetulus , Glicosilação , Células HEK293 , Humanos , Imunoglobulina G/imunologia , Manose/metabolismo , Mutagênese Sítio-Dirigida , Polissacarídeos/metabolismo , Engenharia de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Receptores de IgG/genética , Receptores de IgG/imunologia
14.
PLoS One ; 15(3): e0230358, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32208424

RESUMO

Endothelial dysfunction is a critical event in vascular inflammation characterized, in part, by elevated surface expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is heavily N-glycosylated, and like other surface proteins, it is largely presumed that fully processed, complex N-glycoforms are dominant. However, our recent studies suggest that hypoglycosylated or high mannose (HM)-ICAM-1 N-glycoforms are also expressed on the cell surface during endothelial dysfunction, and have higher affinity for monocyte adhesion and regulate outside-in endothelial signaling by different mechanisms. Whether different ICAM-1 N-glycoforms are expressed in vivo during disease is unknown. In this study, using the proximity ligation assay, we assessed the relative formation of high mannose, hybrid and complex α-2,6-sialyated N-glycoforms of ICAM-1 in human and mouse models of atherosclerosis, as well as in arteriovenous fistulas (AVF) of patients on hemodialysis. Our data demonstrates that ICAM-1 harboring HM or hybrid epitopes as well as ICAM-1 bearing α-2,6-sialylated epitopes are present in human and mouse atherosclerotic lesions. Further, HM-ICAM-1 positively associated with increased macrophage burden in lesions as assessed by CD68 staining, whereas α-2,6-sialylated ICAM-1 did not. Finally, both HM and α-2,6-sialylated ICAM-1 N-glycoforms were present in hemodialysis patients who had AVF maturation failure compared to successful AVF maturation. Collectively, these data provide evidence that HM- ICAM-1 N-glycoforms are present in vivo, and at levels similar to complex α-2,6-sialylated ICAM-1 underscoring the need to better understand their roles in modulating vascular inflammation.


Assuntos
Aterosclerose/patologia , Endotélio Vascular/patologia , Inflamação/patologia , Molécula 1 de Adesão Intercelular/imunologia , Isoformas de Proteínas/análise , Adulto , Idoso , Animais , Artérias/citologia , Artérias/patologia , Derivação Arteriovenosa Cirúrgica/efeitos adversos , Aterosclerose/imunologia , Modelos Animais de Doenças , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Epitopos/análise , Epitopos/imunologia , Epitopos/metabolismo , Feminino , Glicosilação , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/imunologia , Molécula 1 de Adesão Intercelular/análise , Molécula 1 de Adesão Intercelular/metabolismo , Macrófagos/imunologia , Masculino , Manose/metabolismo , Camundongos , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Ácido N-Acetilneuramínico/metabolismo , Isoformas de Proteínas/metabolismo , Adulto Jovem
15.
Proc Natl Acad Sci U S A ; 117(14): 7633-7644, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32213588

RESUMO

Membrane-bound oligosaccharides form the interfacial boundary between the cell and its environment, mediating processes such as adhesion and signaling. These structures can undergo dynamic changes in composition and expression based on cell type, external stimuli, and genetic factors. Glycosylation, therefore, is a promising target of therapeutic interventions for presently incurable forms of advanced cancer. Here, we show that cholangiocarcinoma metastasis is characterized by down-regulation of the Golgi α-mannosidase I coding gene MAN1A1, leading to elevation of extended high-mannose glycans with terminating α-1,2-mannose residues. Subsequent reshaping of the glycome by inhibiting α-mannosidase I resulted in significantly higher migratory and invasive capabilities while masking cell surface mannosylation suppressed metastasis-related phenotypes. Exclusive elucidation of differentially expressed membrane glycoproteins and molecular modeling suggested that extended high-mannose glycosylation at the helical domain of transferrin receptor protein 1 promotes conformational changes that improve noncovalent interaction energies and lead to enhancement of cell migration in metastatic cholangiocarcinoma. The results provide support that α-1,2-mannosylated N-glycans present on cancer cell membrane proteins may serve as therapeutic targets for preventing metastasis.


Assuntos
Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Manose/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/patologia , Feminino , Glicosilação , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Metástase Neoplásica , Fenótipo , Multimerização Proteica
16.
Food Chem ; 315: 126264, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32006867

RESUMO

A previous study revealed that Saccharomyces cerevisiae mcd4Δ, a cell wall mutant with a defect in the synthesis of the glycosylphosphatidylinositol anchor, has a strong macrophage activation ability. In this study, remarkable emulsion formation after cell suspensions of mcd4Δ and anp1Δ (which exhibit an extreme reduction of mannan) were mixed with oil was found. Moreover, the relationship between cell wall mutation and emulsion formation was investigated, suggesting that och1Δ with a defect in the formation of N-linked glycans also had a strong emulsification ability and that high molecular weight materials released from the cells were involved in emulsion formation. Furthermore, two strains (asc1Δ and scp160Δ) with a strong emulsification ability without a large decrease in mannan content were also found from the wide screening of strains that exhibit an emulsifying activity using more than 5000 gene-deficient strains. These results provide valuable information for the development of a yeast-derived emulsifier.


Assuntos
Membrana Celular/química , Parede Celular/química , Emulsificantes/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Parede Celular/genética , Emulsões/química , Glucose/metabolismo , Ativação de Macrófagos , Mananas/metabolismo , Manose/metabolismo , Camundongos , Mutação , Polissacarídeos/química , Células RAW 264.7 , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Nat Commun ; 11(1): 899, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060313

RESUMO

The human gut microbiota plays a central role not only in regulating the metabolism of nutrients but also promoting immune homeostasis, immune responses and protection against pathogen colonization. The genome of the Gram-negative symbiont Bacteroides thetaiotaomicron, a dominant member of the human intestinal microbiota, encodes polysaccharide utilization loci PULs, the apparatus required to orchestrate the degradation of a specific glycan. EndoBT-3987 is a key endo-ß-N-acetylglucosaminidase (ENGase) that initiates the degradation/processing of mammalian high-mannose-type (HM-type) N-glycans in the intestine. Here, we provide structural snapshots of EndoBT-3987, including the unliganded form, the EndoBT-3987-Man9GlcNAc2Asn substrate complex, and two EndoBT-3987-Man9GlcNAc and EndoBT-3987-Man5GlcNAc product complexes. In combination with alanine scanning mutagenesis and activity measurements we unveil the molecular mechanism of HM-type recognition and specificity for EndoBT-3987 and an important group of the GH18 ENGases, including EndoH, an enzyme extensively used in biotechnology, and for which the mechanism of substrate recognition was largely unknown.


Assuntos
Bacteroides thetaiotaomicron/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides thetaiotaomicron/química , Bacteroides thetaiotaomicron/enzimologia , Bacteroides thetaiotaomicron/genética , Microbioma Gastrointestinal , Regulação Bacteriana da Expressão Gênica , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Humanos , Manose/química , Manose/metabolismo , Especificidade por Substrato
18.
Molecules ; 25(4)2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-32059500

RESUMO

The expression of different glycans at the cell surface dictates cell interactions with their environment and other cells, being crucial for the cell fate. The development of the central nervous system is associated with tremendous changes in the cell glycome that is tightly regulated. Herein, we have employed biorthogonal Cu-free click chemistry to image temporal distribution of different glycans in live mouse hippocampal neurons during their maturation in vitro. We show development-dependent glycan patterns with increased fucose and decreased mannose expression at the end of the maturation process. We also demonstrate that this approach is biocompatible and does not affect glycan transport although it relies on an administration of modified glycans. The applicability of this strategy to tissue sections unlocks new opportunities to study the glycan dynamics under more complex physiological conditions.


Assuntos
Química Click , Hipocampo/crescimento & desenvolvimento , Neurônios/metabolismo , Polissacarídeos/metabolismo , Animais , Diferenciação Celular/genética , Fucose/metabolismo , Regulação da Expressão Gênica/genética , Glicosilação , Hipocampo/metabolismo , Manose/metabolismo , Camundongos , Polissacarídeos/genética , Espectrometria de Massas em Tandem
19.
J Nanobiotechnology ; 18(1): 24, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32000788

RESUMO

BACKGROUND: Targeted delivery of virus-associated antigens to professional antigen-presenting cells (APCs) is considered as an efficient strategy to enhance the pyrophytic effect of vaccines against rhabdovirus disease. MATERIALS AND METHODS: In this study, we constructed a targeted carbon nanotubes-based vaccine deliver system (SWCNTs-MG) which can recognize the signature receptor (mannose) of APCs. An environmentally and economically important disease called spring viremia of carp (SVC) was studied as a model to evaluate the feasibility of single-walled carbon nanotubes (SWCNTs) conjugated with mannosylated antigen for rhabdovirus prevention. RESULTS: Results showed that SWCNTs-MG could cross into fish body and present to internal immune-related tissues through gill, muscle and intestine within 6 h immersed vaccination. With further modification of mannose moiety, the obtained nanovaccine showed enhanced uptake by carp macrophages and immune-related tissues, which would then trigger strong immune responses against spring viremia of carp virus (SVCV) infection. Moreover, the survival rate of fish vaccinated with SWCNTs-MG (30 mg/L) was 63.5% after SVCV infection, whereas it was 0% for the control group. CONCLUSION: This study not only provide a theoretical basis and research template for the application of targeted nanovaccine system in aquatic animals, but also play an important role in supporting development of healthy aquaculture and ensuring the safety of aquatic products and ecology.


Assuntos
Células Apresentadoras de Antígenos/metabolismo , Portadores de Fármacos/química , Manose/metabolismo , Nanotubos de Carbono/química , Infecções por Rhabdoviridae/prevenção & controle , Vacinas de DNA/metabolismo , Animais , Carpas , Permeabilidade da Membrana Celular , Liberação Controlada de Fármacos , Doenças dos Peixes/epidemiologia , Doenças dos Peixes/virologia , Humanos , Imunização , Macrófagos/metabolismo , Terapia de Alvo Molecular , Rhabdoviridae/efeitos dos fármacos , Infecções por Rhabdoviridae/virologia , Distribuição Tecidual , Vacinação
20.
J Virol ; 94(9)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32102878

RESUMO

Semen is the primary transmission vehicle for various pathogenic viruses. Initial steps of transmission, including cell attachment and entry, likely occur in the presence of semen. However, the unstable nature of human seminal plasma and its toxic effects on cells in culture limit the ability to study in vitro virus infection and inhibition in this medium. We found that whole semen significantly reduces the potency of antibodies and microbicides that target glycans on the envelope glycoproteins (Envs) of HIV-1. The extraordinarily high concentration of the monosaccharide fructose in semen contributes significantly to the effect by competitively inhibiting the binding of ligands to α1,2-linked mannose residues on Env. Infection and inhibition in whole human seminal plasma are accurately mimicked by a stable synthetic simulant of seminal fluid that we formulated. Our findings indicate that, in addition to the protein content of biological secretions, their small-solute composition impacts the potency of antiviral microbicides and mucosal antibodies.IMPORTANCE Biological secretions allow viruses to spread between individuals. Each type of secretion has a unique composition of proteins, salts, and sugars, which can affect the infectivity potential of the virus and inhibition of this process. Here, we describe HIV-1 infection and inhibition in whole human seminal plasma and a synthetic simulant that we formulated. We discovered that the sugar fructose in semen decreases the activity of a broad and potent class of antiviral agents that target mannose sugars on the envelope protein of HIV-1. This effect of semen fructose likely reduces the efficacy of such inhibitors to prevent the sexual transmission of HIV-1. Our findings suggest that the preclinical evaluation of microbicides and vaccine-elicited antibodies will be improved by their in vitro assessment in synthetic formulations that simulate the effects of semen on HIV-1 infection and inhibition.


Assuntos
Frutose/metabolismo , Frutose/farmacologia , Sêmen/metabolismo , Adulto , Anti-Infecciosos/farmacologia , Antivirais/antagonistas & inibidores , Antivirais/farmacologia , Linhagem Celular Tumoral , Produtos do Gene env/metabolismo , Genes env/genética , Células HEK293 , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Masculino , Manose/metabolismo , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Sêmen/virologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
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