RESUMO
BACKGROUND: Preeclampsia (PE) is a hypertensive disorder of pregnancy with various clinical symptoms. However, traditional markers for the disease including high blood pressure and proteinuria are poor indicators of the related adverse outcomes. Here, we performed systematic proteome profiling of plasma samples obtained from pregnant women with PE to identify clinically effective diagnostic biomarkers. METHODS: Proteome profiling was performed using TMT-based liquid chromatography-mass spectrometry (LC-MS/MS) followed by subsequent verification by multiple reaction monitoring (MRM) analysis on normal and PE maternal plasma samples. Functional annotations of differentially expressed proteins (DEPs) in PE were predicted using bioinformatic tools. The diagnostic accuracies of the biomarkers for PE were estimated according to the area under the receiver operating characteristics curve (AUC). RESULTS: A total of 1,307 proteins were identified, and 870 proteins of them were quantified from plasma samples. Significant differences were evident in 138 DEPs, including 71 upregulated DEPs and 67 downregulated DEPs in the PE group, compared with those in the control group. Up-regulated proteins were significantly associated with biological processes including platelet degranulation, proteolysis, lipoprotein metabolism, and cholesterol efflux. Biological processes including blood coagulation and acute-phase response were enriched for down-regulated proteins. Of these, 40 proteins were subsequently validated in an independent cohort of 26 PE patients and 29 healthy controls. APOM, LCN2, and QSOX1 showed high diagnostic accuracies for PE detection (AUC > 0.9 and P < 0.001, for all) as validated by MRM and ELISA. CONCLUSIONS: Our data demonstrate that three plasma biomarkers, identified by systematic proteomic profiling, present a possibility for the assessment of PE, independent of the clinical characteristics of pregnant women.
RESUMO
cAMP response element-binding protein (CREB) regulated transcriptional coactivator 2 (CRTC2) is a critical transcription factor that maintains glucose homeostasis by activating CREB. Energy homeostasis is maintained through multiple pathways; therefore, CRTC2 may interact with other transcription factors, particularly under metabolic stress. CRTC2 liver-specific KO mice were created, and the global proteome, phosphoproteome, and acetylome from liver tissue under high-fat diet conditions were analyzed using liquid chromatography-tandem mass spectrometry and bioinformatics analysis. Differentially regulated proteins (DRPs) were enriched in metabolic pathways, which were subsequently corroborated through animal experiments. The consensus DRPs from these datasets were used as seed proteins to generate a protein-protein interaction network using STRING, and GeneMANIA identified fatty acid synthase as a mutually relevant protein. In an additional local-protein-protein interaction analysis of CRTC2 and fatty acid synthase with DRPs, sterol regulatory element binding transcription factor 1 (SREBF1) was the common mediator. CRTC2-CREB and SREBF1 are transcription factors, and DNA-binding motif analysis showed that multiple CRTC2-CREB-regulated genes possess SREBF1-binding motifs. This indicates the possible induction by the CRTC2-SREBF1 complex, which is validated through luciferase assay. Therefore, the CRTC2-SREBF1 complex potentially modulates the transcription of multiple proteins that fine-tune cellular metabolism under metabolic stress.
RESUMO
Per- and polyfluoroalkyl substances (PFASs) and perfluoroalkyl ether carboxylic acids (PFECAs) are organic chemicals that are widely used in the manufacture of a wide range of human-made products. Many monitoring findings revealed the presence of PFASs and PFECAs in numerous environmental sources, including water, soil, and air, which drew more attention to both chemicals. Because of their unknown toxicity, the discovery of PFASs and PFECAs in a variety of environmental sources was viewed as a cause for concern. In the present study, male mice were given orally one of the typical PFASs, perfluorooctanoic acid (PFOA), and one of the representative PFECAs, hexafluoropropylene oxide-dimer acid (HFPO-DA). The liver index showing hepatomegaly rose significantly after 90 d of exposure to PFOA and HFPO-DA, respectively. While sharing similar suppressor genes, both chemicals demonstrated unique hepatotoxic mechanisms. In different ways, these two substances altered the expression of hepatic stress-sensing genes as well as the regulation of nuclear receptors. Not only are bile acid metabolism-related genes in the liver altered, but cholesterol metabolism-related genes as well. These results indicate that PFOA and HFPO-DA both cause hepatotoxicity and bile acid metabolism impairment with distinct mechanisms.
Assuntos
Fluorocarbonos , Humanos , Camundongos , Masculino , Animais , Fluorocarbonos/toxicidade , Fluorocarbonos/metabolismo , Fígado/metabolismo , Ácidos e Sais BiliaresRESUMO
Fucoidan from brown seaweeds has several biological effects, including preserving intestinal integrity. To investigate the intestinal protective properties of high molecular weight fucoidan (HMWF) from Undaria pinnatifida on intestinal integrity dysfunction caused by methylglyoxal-derived hydroimidazolone-1 (MG-H1), one of the dietary advanced-glycation end products (dAGEs) in the human-colon carcinoma-cell line (Caco-2) cells and ICR mice. According to research, dAGEs may damage the intestinal barrier by increasing gut permeability. The findings of the study showed that HMWF + MG-H1 treatment reduced by 16.8% the amount of reactive oxygen species generated by MG-H1 treatment alone. Furthermore, HMWF + MGH-1 treatment reduced MG-H1-induced monolayer integrity disruption, as measured by alterations in transepithelial electrical resistance (135% vs. 75.5%) and fluorescein isothiocyanate incorporation (1.40 × 10-6 cm/s vs. 3.80 cm/s). HMWF treatment prevented the MG-H1-induced expression of tight junction markers, including zonula occludens-1, occludin, and claudin-1 in Caco-2 cells and mouse colon tissues at the mRNA and protein level. Also, in Caco-2 and MG-H1-treated mice, HMWF plays an important role in preventing receptor for AGEs (RAGE)-mediated intestinal damage. In addition, HMWF inhibited the nuclear factor kappa B activation and its target genes leading to intestinal inflammation. These findings suggest that HMWF with price competitiveness could play an important role in preventing AGEs-induced intestinal barrier dysfunction.
Assuntos
Aldeído Pirúvico , Junções Íntimas , Animais , Células CACO-2 , Claudina-1/genética , Claudina-1/metabolismo , Claudina-1/farmacologia , Fluoresceínas/metabolismo , Fluoresceínas/farmacologia , Humanos , Imidazóis , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Mucosa Intestinal , Isotiocianatos/metabolismo , Isotiocianatos/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Peso Molecular , NF-kappa B/metabolismo , Ocludina/genética , Ocludina/metabolismo , Ocludina/farmacologia , Permeabilidade , Polissacarídeos , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Junções Íntimas/metabolismoRESUMO
Sepsis is an emergent infectious disease and a leading cause of death despite immediate intervention. While Delta neutrophil index (DNI) and myeloperoxidase (MPO) are known as a prodiagnostic marker of sepsis, the preclinical evidence of the best marker of sepsis is unclear. For this, using a well-designed cecal ligation and puncture (CLP)-induced sepsis mouse model, we comparatively measured the level and cost-effectiveness of sepsis biomarkers such as DNI, myeloperoxidase (MPO), procalcitonin (PCT), and tumor necrosis factor-alpha (TNF-α). First, we found that the optimal time point for early detection is at 6 h, 24 h post-CLP. Strikingly, the peak level and fold change of DNI was revealed at 24 h, further showing the best fold change as compared with other biomarker levels. Given the fold change at 6, 24 h, PCT was next to DNI. Third, a cost-effectiveness survey showed that DNI was the best, with PCT next. Further, DNI level was moderate positively associated with PCT (ρ = 0.697, p = 0.012) and TNF-α (ρ = 0.599, p = 0.040). Collectively, these data indicate that DNI in CLP-induced sepsis mice is as effective as the existent inflammatory biomarkers such as MPO, PCT and TNF-α to predict the prognosis of sepsis. This might have clinically important implications that DNI is cost effective, thus quickly and rationally applying to diverse types of imminent sepsis regardless of species. This might be the first report on the validity of DNI in preclinical CLP-induced murine sepsis.
Assuntos
Neutrófilos , Sepse , Animais , Biomarcadores , Modelos Animais de Doenças , Humanos , Camundongos , Punções/efeitos adversos , Estudos Retrospectivos , Sepse/complicações , Sepse/diagnósticoRESUMO
Changes in glycan levels could directly affect the biochemical properties of glycoproteins and thus influence their physiological functions. In order to decode the correlation of glycan prevalence with their physiological contribution, many mass spectrometry (MS) and stable isotope labeling-based methods have been developed for the relative quantification of glycans. In this study, we expand the quantitative glycomic toolbox with the addition of optimized Metabolic Isotope Labeling of Polysaccharides with Isotopic Glucose (MILPIG) approach in baker's yeast (Saccharomyces cerevisiae). We demonstrate that culturing baker's yeast in the presence of carbon-13 labeled glucose (1-13C1) leads to effective incorporation of carbon-13 to both N-linked and O-linked glycans. We established that metabolic incorporation of isotope-labeled glucose at a concentration of 5 mg/mL for three days is required for an accurate quantitative analysis with optimal isotopic cluster distribution of glycans. To validate the robustness of the method, we performed the analysis by 1:1 mixing of normal and isotope-labeled glycans, and obtained excellent linear calibration curves from various analytes. Finally, we quantitated the inhibitory effect of tunicamycin, a N-linked glycosylation inhibitor, to glycan expression profile in yeast.
Assuntos
Glucose/química , Glicômica/métodos , Marcação por Isótopo/métodos , Polissacarídeos/análise , Polissacarídeos/química , Saccharomyces cerevisiae/metabolismo , Calibragem , Isótopos de Carbono/metabolismo , Técnicas de Cultura de Células/métodos , Glicoconjugados/análise , Glicoconjugados/biossíntese , Glicoconjugados/química , Glicosilação , Espectrometria de Massas , Polissacarídeos/biossíntese , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/químicaRESUMO
O-linked ß-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4.
Assuntos
N-Acetilglucosaminiltransferases/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Processamento de Proteína Pós-Traducional , Células-Tronco Embrionárias/metabolismo , Glicosilação , Células HEK293 , Humanos , Ativação TranscricionalRESUMO
Polarized secretion is crucial in many tissues. The conserved protein modification, O-glycosylation, plays a role in regulating secretion. However, the mechanisms by which this occurs are unknown. Here, we demonstrate that an O-glycosyltransferase functions as a novel regulator of secretion and secretory vesicle formation in vivo by glycosylating the essential Golgi/endoplasmic reticulum protein, Tango1 (Transport and Golgi organization 1), and conferring protection from furin-mediated proteolysis. Loss of the O-glycosyltransferase PGANT4 resulted in Tango1 cleavage, loss of secretory granules, and disrupted apical secretion. The secretory defects seen upon loss of pgant4 could be rescued either by overexpression of Tango1 or by knockdown of a specific furin (Dfur2) in vivo. Our studies elucidate a novel regulatory mechanism whereby secretion is influenced by the yin/yang of O-glycosylation and proteolytic cleavage. Moreover, our data have broader implications for the potential treatment of diseases resulting from the loss of O-glycosylation by modulating the activity of specific proteases.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Proteínas de Drosophila/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Subtilisinas/metabolismo , Animais , Calcinose , Catálise , Drosophila melanogaster , Retículo Endoplasmático/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Mucinas/metabolismo , Mutação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Interferência de RNARESUMO
The most abundant N-glycan in plants is the paucimannosidic N-glycan with core ß1,2-xylose and α1,3-fucose residues (Man3XylFuc(GlcNAc)2). Here, we report a mechanism in Arabidopsis thaliana that efficiently produces the largest N-glycan in plants. Genetic and biochemical evidence indicates that the addition of the 6-arm ß1,2-GlcNAc residue by N-acetylglucosaminyltransferase II (GnTII) is less effective than additions of the core ß1,2-xylose and α1,3-fucose residues by XylT, FucTA, and FucTB in Arabidopsis. Furthermore, analysis of gnt2 mutant and 35S:GnTII transgenic plants shows that the addition of the 6-arm non-reducing GlcNAc residue to the common N-glycan acceptor GlcNAcMan3(GlcNAc)2 inhibits additions of the core ß1,2-xylose and α1,3-fucose residues. Our findings indicate that plants limit the rate of the addition of the 6-arm GlcNAc residue to the common N-glycan acceptor as a mechanism to facilitate formation of the prevalent N-glycans with Man3XylFuc(GlcNAc)2 and (GlcNAc)2Man3XylFuc(GlcNAc)2 structures.
Assuntos
Acetilglucosamina/metabolismo , Arabidopsis/metabolismo , Polissacarídeos/biossíntese , Arabidopsis/química , Arabidopsis/genética , Sequência de Carboidratos , Dados de Sequência Molecular , Polissacarídeos/químicaRESUMO
Archaea, one of three major evolutionary lineages of life, encode proteasomes highly related to those of eukaryotes. In contrast, archaeal ubiquitin-like proteins are less conserved and not known to function in protein conjugation. This has complicated our understanding of the origins of ubiquitination and its connection to proteasomes. Here we report two small archaeal modifier proteins, SAMP1 and SAMP2, with a beta-grasp fold and carboxy-terminal diglycine motif similar to ubiquitin, that form protein conjugates in the archaeon Haloferax volcanii. The levels of SAMP-conjugates were altered by nitrogen-limitation and proteasomal gene knockout and spanned various functions including components of the Urm1 pathway. LC-MS/MS-based collision-induced dissociation demonstrated isopeptide bonds between the C-terminal glycine of SAMP2 and the epsilon-amino group of lysines from a number of protein targets and Lys 58 of SAMP2 itself, revealing poly-SAMP chains. The widespread distribution and diversity of pathways modified by SAMPylation suggest that this type of protein conjugation is central to the archaeal lineage.
Assuntos
Proteínas Arqueais/metabolismo , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Deleção de Genes , Glicilglicina/metabolismo , Haloferax volcanii/genética , Haloferax volcanii/metabolismo , Imunoprecipitação , Espectrometria de Massas , Dados de Sequência Molecular , Nitrogênio/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Alinhamento de Sequência , Enxofre/metabolismo , Ubiquitinação , Ubiquitinas/químicaRESUMO
N-glycosylation is a major modification of glycoproteins in eukaryotic cells. In Arabidopsis, great progress has been made in functional analysis of N-glycan production, however there are few studies in monocotyledons. Here, we characterized a rice (Oryza sativa L.) osmogs mutant with shortened roots and isolated a gene that coded a putative mannosyl-oligosaccharide glucosidase (OsMOGS), an ortholog of α-glucosidase I in Arabidopsis, which trims the terminal glucosyl residue of the oligosaccharide chain of nascent peptides in the endoplasmic reticulum (ER). OsMOGS is strongly expressed in rapidly cell-dividing tissues and OsMOGS protein is localized in the ER. Mutation of OsMOGS entirely blocked N-glycan maturation and inhibited high-mannose N-glycan formation. The osmogs mutant exhibited severe defects in root cell division and elongation, resulting in a short-root phenotype. In addition, osmogs plants had impaired root hair formation and elongation, and reduced root epidemic cell wall thickness due to decreased cellulose synthesis. Further analysis showed that auxin content and polar transport in osmogs roots were reduced due to incomplete N-glycosylation of the B subfamily of ATP-binding cassette transporter proteins (ABCBs). Our results demonstrate that involvement of OsMOGS in N-glycan formation is required for auxin-mediated root development in rice.
Assuntos
Ácidos Indolacéticos/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Polissacarídeos/metabolismo , alfa-Glucosidases/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Bases , Transporte Biológico , Divisão Celular , Tamanho Celular , Parede Celular/genética , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Glicosilação , Microscopia Confocal , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Oryza/genética , Oryza/crescimento & desenvolvimento , Filogenia , Epiderme Vegetal/citologia , Epiderme Vegetal/genética , Epiderme Vegetal/metabolismo , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Reação em Cadeia da Polimerase Via Transcriptase Reversa , alfa-Glucosidases/classificação , alfa-Glucosidases/genéticaRESUMO
We report here an unliganded receptor structure in the common gamma-chain (γ(c)) family of receptors and cytokines. The crystal structure of the unliganded form of the interleukin-7 alpha receptor (IL-7Rα) extracellular domain (ECD) at 2.15 Å resolution reveals a homodimer forming an "X" geometry looking down onto the cell surface with the C termini of the two chains separated by 110 Å and the dimer interface comprising residues critical for IL-7 binding. Further biophysical studies indicate a weak association of the IL-7Rα ECDs but a stronger association between the γ(c)/IL-7Rα ECDs, similar to previous studies of the full-length receptors on CD4(+) T cells. Based on these and previous results, we propose a molecular mechanism detailing the progression from the inactive IL-7Rα homodimer and IL-7Rα-γ(c) heterodimer to the active IL-7-IL-7Rα-γ(c) ternary complex whereby the two receptors undergo at least a 90° rotation away from the cell surface, moving the C termini of IL-7Rα and γ(c) from a distance of 110 Å to less than 30 Å at the cell surface. This molecular mechanism can be used to explain recently discovered IL-7- and γ(c)-independent gain-of-function mutations in IL-7Rα from B- and T-cell acute lymphoblastic leukemia patients. The mechanism may also be applicable to other γ(c) receptors that form inactive homodimers and heterodimers independent of their cytokines.
Assuntos
Interleucina-7/metabolismo , Transdução de Sinais , Dimerização , Interleucina-7/química , Ligantes , Ligação Proteica , Conformação Proteica , Difração de Raios XRESUMO
Adipose tissue is both an energy storage depot and an endocrine organ. The impaired regulation of the secreted proteins of adipose tissue, known as adipocytokines, observed during obesity contributes to the onset of whole-body insulin resistance and the pathobiology of type 2 diabetes mellitus (T2DM). In addition, the global elevation of the intracellular glycosylation of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc) via either genetic or pharmacological methods is sufficient to induce insulin resistance in both cultured cells and animal models. The elevation of global O-GlcNAc levels is associated with the altered expression of many adipocytokines. We have previously characterized the rodent adipocyte secretome during insulin sensitive and insulin resistant conditions. Here, we characterize and quantify the secretome and glycome of primary human adipocytes during insulin responsive and insulin resistant conditions generated by the classical method of hyperglycemia and hyperinsulinemia or by the pharmacological manipulation of O-GlcNAc levels. Using a proteomic approach, we identify 190 secreted proteins and report a total of 20 up-regulated and 6 down-regulated proteins that are detected in both insulin resistant conditions. Moreover, we apply glycomic techniques to examine (1) the sites of N-glycosylation on secreted proteins, (2) the structures of complex N- and O-glycans, and (3) the relative abundance of complex N- and O-glycans structures in insulin responsive and insulin resistant conditions. We identify 91 N-glycosylation sites derived from 51 secreted proteins, as well as 155 and 29 released N- and O-glycans respectively. We go on to quantify many of the N- and O-glycan structures between insulin responsive and insulin resistance conditions demonstrating no significant changes in complex glycosylation in the time frame for the induction of insulin resistance. Thus, our data support that the O-GlcNAc modification is involved in the regulation of adipocytokine secretion upon the induction of insulin resistance in human adipocytes.
RESUMO
The Oct1 transcription factor is a potent regulator of stress responses, metabolism, and tumorigenicity. Although Oct1 is regulated by phosphorylation and ubiquitination, the presence and importance of other modifications is unknown. Here we show that Oct1 is modified by O-linked ß-N-acetylglucosamine (O-GlcNAc) moieties. We map two sites of O-GlcNAcylation at positions T255 and S728 within human Oct1. Under anchorage-independent overgrowth conditions, Oct1 associates 3-fold more strongly with the Gadd45a promoter and mediates transcriptional repression. Increased binding correlates with quantitative reductions in Oct1 nuclear periphery-associated puncta, and a reduced association with lamin B1. The O-GlcNAc modification sites are important for both Gadd45a repression and anchorage-independent survival. In contrast to chronic overgrowth conditions, following acute nutrient starvation Oct1 mediates Gadd45a activation. The O-GlcNAc sites are also important for Gadd45a activation under these conditions. We also, for the first time, identify specific Oct1 ubiquitination sites. The findings suggest that Oct1 integrates metabolic and stress signals via O-GlcNAc modification to regulate target gene activity.
Assuntos
Acetilglucosamina/metabolismo , Fator de Transcrição Brn-3A/metabolismo , Ativação Transcricional , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Proteínas de Ciclo Celular/genética , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Immunoblotting , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Brn-3A/genéticaRESUMO
Factors influencing the sour taste of coffee and the properties of chlorogenic acid are not yet fully understood. This study aimed to evaluate the impact of roasting degree on pH-associated changes in coffee bean extract and the thermal stability of chlorogenic acid. Coffee bean extract pH decreased up to a chromaticity value of 75 but increased with higher chromaticity values. Ultraviolet-visible spectrophotometry and structural analysis attributed this effect to chlorogenic and caffeic acids. Moreover, liquid chromatography-mass spectrometry analysis identified four chlorogenic acid types in green coffee bean extract. Chlorogenic acid isomers were eluted broadly on HPLC, and a chlorogenic acid fraction graph with two peaks, fractions 5 and 9, was obtained. Among the various fractions, the isomer in fraction 5 had significantly lower thermal stability, indicating that thermal stability differs between chlorogenic acid isomers.
RESUMO
Mass spectrometry-based approaches encompass a powerful collection of tools for the analysis biological molecules, including glycans and glycoconjugates. Unlike most traditional bioanalytical methods focusing on these molecules, mass spectrometry is especially suited for multiplexing, by utilizing stable-isotope labeling. Indeed, stable isotope-based multiplexing can be regarded as the gold-standard approach in reducing noise and uncertainty in quantitative mass spectrometry and quantitative analyses generally. The increasing sophistication and depth of biological questions being asked continue to challenge the practitioners of mass spectrometry method development. To understand the biological relevance of glycans, many stable isotope labeling-based mass spectrometry methods have been developed. Based on the duplex MILPIG (metabolic isotope labeling of polysaccharides with isotopic glucose), we establish here a novel triplex isotope labeling method using baker's yeast as the model system. Two differentially isotope-labeled glucoses (medium: 1-13C1 and heavy: 1,2-13C2), in addition to natural abundance glucose (light), were successfully used to label each monosaccharide ring in N-linked glycans in three different cell culture conditions, that, after sample mixing, resulted in a predictable triplet spectrum amenable for relative quantitation. We demonstrate excellent accuracy and precision of relative quantitation for a 1:1:1 mixture of glycans labeled in such a fashion. In addition, we applied triplex MILPIG to interrogate differential N-glycan profiles in tunicamycin-treated and control yeast cells and show that different N-glycans respond differently to tunicamycin.
Assuntos
Glucose , Saccharomyces cerevisiae , Tunicamicina/farmacologia , Polissacarídeos/análise , Marcação por Isótopo/métodos , IsótoposRESUMO
The severe phenotypic effects of altered glycosylation in the congenital muscular dystrophies, including Walker-Warburg syndrome, muscle-eye-brain disease, Fukuyama congenital muscular dystrophy, and congenital muscular dystrophy 1D, are caused by mutations resulting in altered glycans linked to proteins through O-linked mannose. A glycosyltransferase that branches O-Man, N-acetylglucosaminyltransferase Vb (GnT-Vb), is highly expressed in neural tissues. To understand the expression and function of GnT-Vb, we studied its expression during neuromorphogenesis and generated GnT-Vb null mice. A paralog of GnT-Vb, N-acetylglucosaminyltransferase (GnT-V), is expressed in many tissues and brain, synthesizing N-linked, ß1,6-branched glycans, but its ability to synthesize O-mannosyl-branched glycans is unknown; conversely, although GnT-Vb can synthesize N-linked glycans in vitro, its contribution to their synthesis in vivo is unknown. Our results showed that deleting both GnT-V and GnT-Vb results in the total loss of both N-linked and O-Man-linked ß1,6-branched glycans. GnT-V null brains lacked N-linked, ß1,6-glycans but had normal levels of O-Man ß1,6-branched structures, showing that GnT-Vb could not compensate for the loss of GnT-V. By contrast, GnT-Vb null brains contained normal levels of N-linked ß1,6-glycans but low levels of some O-Man ß1,6-branched glycans. Therefore, GnT-V could partially compensate for GnT-Vb activity in vivo. We found no apparent change in α-dystroglycan binding of glycan-specific antibody IIH6C4 or binding to laminin in GnT-Vb null mice. These results demonstrate that GnT-V is involved in synthesizing branched O-mannosyl glycans in brain, but the function of these branched O-mannosyl structures is unresolved using mice that lack these glycosyltransferases.
Assuntos
Encéfalo/enzimologia , Regulação Enzimológica da Expressão Gênica , N-Acetilglucosaminiltransferases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Polissacarídeos/metabolismo , Animais , Glicosilação , Humanos , Camundongos , Camundongos Knockout , Distrofias Musculares/enzimologia , Distrofias Musculares/genética , N-Acetilglucosaminiltransferases/genética , Proteínas do Tecido Nervoso/genética , Polissacarídeos/genéticaRESUMO
O-Linked glycosylation is a functionally and structurally diverse type of protein modification present in many tissues and across many species. α-Dystroglycan (α-DG), a protein linked to the extracellular matrix, whose glycosylation status is associated with human muscular dystrophies, displays two predominant types of O-glycosylation, O-linked mannose (O-Man) and O-linked N-acetylgalactosamine (O-GalNAc), in its highly conserved mucin-like domain. The O-Man is installed by an enzyme complex present in the endoplasmic reticulum. O-GalNAc modifications are initiated subsequently in the Golgi apparatus by the UDP-GalNAc polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T) enzymes. How the presence and position of O-Man influences the action of the ppGalNAc-Ts on α-DG and the distribution of the two forms of glycosylation in this domain is not known. Here, we investigated the interplay between O-Man and the addition of O-GalNAc by examining the activity of the ppGalNAc-Ts on peptides and O-Man-containing glycopeptides mimicking those found in native α-DG. These synthetic glycopeptides emulate intermediate structures, not otherwise readily available from natural sources. Through enzymatic and mass spectrometric methods, we demonstrate that the presence and specific location of O-Man can impact either the regional exclusion or the site of O-GalNAc addition on α-DG, elucidating the factors contributing to the glycosylation patterns observed in vivo. These results provide evidence that one form of glycosylation can influence another form of glycosylation in α-DG and suggest that in the absence of proper O-mannosylation, as is associated with certain forms of muscular dystrophy, aberrant O-GalNAc modifications may occur and could play a role in disease presentation.
Assuntos
Acetilgalactosamina/metabolismo , Distroglicanas/metabolismo , Manose/metabolismo , Complexos Multienzimáticos/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismo , Acetilgalactosamina/genética , Animais , Linhagem Celular , Distroglicanas/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Glicosilação , Humanos , Manose/genética , Camundongos , Complexos Multienzimáticos/genética , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , N-Acetilgalactosaminiltransferases/genética , Uridina Difosfato N-Acetilgalactosamina/genéticaRESUMO
The abundance and structural diversity of glycans on glycoproteins and glycolipids are highly regulated and play important roles during vertebrate development. Because of the challenges associated with studying glycan regulation in vertebrate embryos, we have chosen to study mouse embryonic stem (ES) cells as they differentiate into embryoid bodies (EBs) or into extraembryonic endodermal (ExE) cells as a model for cellular differentiation. We profiled N- and O-glycan structures isolated from these cell populations and examined transcripts encoding the corresponding enzymatic machinery for glycan biosynthesis in an effort to probe the mechanisms that drive the regulation of glycan diversity. During differentiation from mouse ES cells to either EBs or ExE cells, general trends were detected. The predominance of high mannose N-glycans in ES cells shifted to an equal abundance of complex and high mannose structures, increased sialylation, and increased α-Gal termination in the differentiated cell populations. Whereas core 1 O-glycan structures predominated in all three cell populations, increased sialylation and increased core diversity characterized the O-glycans of both differentiated cell types. Increased polysialylation was also found in both differentiated cell types. Differences between the two differentiated cell types included greater sialylation of N-glycans in EBs, whereas α-Gal-capped structures were more prevalent in ExE cells. Changes in glycan structures generally, but not uniformly, correlated with alterations in transcript abundance for the corresponding biosynthetic enzymes, suggesting that transcriptional regulation contributes significantly to the regulation of glycan expression. Knowledge of glycan structural diversity and transcript regulation should provide greater understanding of the roles of protein glycosylation in vertebrate development.