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1.
West Afr J Med ; 37(3): 231-236, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32476116

RESUMO

BACKGROUND: Hypertensive disorders of pregnancy associated with potentially fatal outcomes are common obstetrics occurrences. Early diagnosis, management and prediction of outcomes are challenges to be surmounted especially in developing countries. Biomarkers are emerging as useful tools for diagnosis and prognostication in varying health conditions. Elevated levels of serum copeptin and Brain Natriuretic Peptide (BNP) are associated with adverse perinatal outcomes and may serve as potential biomarkers utilized during routine antenatal care. OBJECTIVE: To determine the level and clinical value of copeptin and BNP as biomarkers of hypertensive disorders of pregnancy among Nigerian pregnant women. METHODS: This case-control study comprised 156 consenting pregnant women equally grouped into those with chronic hypertension (CH), gestational hypertension (GH), and preeclampsia (PE) as cases and normotensives as controls. Pregnant women were recruited from the antenatal clinic, University College Hospital, Nigeria. Blood pressures were measured and blood (10ml) was drawn from patients, serum and plasma obtained accordingly while other data were collected using interviewer administered questionnaire and medical records. Serum copeptin and plasma BNP levels were measured using enzyme-linked immunosorbent assay. Data was analysed with SPSS version 20.0 and statistical significance was set at p < 0.05. RESULTS: The mean levels of SBP and DBP were significantly higher in CH (155.41±2.14; 102.36±2.0 mmHg), GH (150.49±0.82; 98.67±0.56 mmHg), and PE (153.92±1.47; 98.92±0.61 mmHg), compared to controls (101.85±1.9; 66.77±1.24 mmHg). Mean serum copeptin and plasma BNP were significantly higher in women with GH (21.25±1.31pmol/L; 223.05±14.95pg/mL) and PE (22.47±1.01pmol/L; 253.99±17.69pg/mL) compared with controls (9.05±1.01pmol/L; 48.63±2.50pg/mL) (p<0.05). There was no significant difference in the mean levels of copeptin and BNP in CH compared with controls (p>0.05). The ROC curve for copeptin gave an AUC of 0.829 (p= 0.000) with a cut off value of 10.15pmol/ L while the AUC for BNP was 0.902 (p= 0.000) with a cut off value of 50.81pg/mL. CONCLUSION: Serum copeptin and plasma BNP levels were significantly higher in GH and PE and may be used as markers of hypertensive disorders of pregnancy among Nigerian pregnant women.


Assuntos
Glicopeptídeos/sangue , Hipertensão Induzida pela Gravidez/diagnóstico , Peptídeo Natriurético Encefálico/sangue , Pré-Eclâmpsia/diagnóstico , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Humanos , Hipertensão Induzida pela Gravidez/sangue , Hipertensão Induzida pela Gravidez/epidemiologia , Nigéria/epidemiologia , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/epidemiologia , Valor Preditivo dos Testes , Gravidez
2.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 32(5): 590-594, 2020 May.
Artigo em Chinês | MEDLINE | ID: mdl-32576353

RESUMO

OBJECTIVE: To explore the value of serum copeptin, S100B protein and uric acid (UA) levels in the prognosis of children with traumatic brain injury (TBI). METHODS: Eighty-six children with TBI admitted to Danzhou People's Hospital from January 1st, 2017 to December 31st, 2019 were selected. The gender, age, body mass index (BMI), cause of TBI, and baseline data of body temperature, heart rate (HR) and respiratory rate (RR) were collected, and the serum copeptin, S100B protein and UA levels were recorded on the 1st, 3rd and 5th day after admission. According to 28-day prognosis, the children were divided into survival group and death group. According to the Glasgow coma score (GCS) at admission, the children were divided into moderate group (GCS score 9-12) and severe group (GCS score 3-8). The changes of serum copeptin, S100B protein and UA level of each group were observed on the 1st, 3rd and 5th day after admission. Receiver operator characteristic (ROC) curve was drawn to analyze the value of serum copeptin, S100B protein and UA in predicting the death of TBI children in 28 days. Pearson correlation method was used to analyze the correlation between GCS score and serum copeptin, S100B protein and UA in dead children during 28 days. RESULTS: Eighty-six children were enrolled in the final analysis, 35 died and 51 survived in 28 days, with 28 in moderate coma and 58 in severe coma. There was no significant difference in the baseline data between the different prognosis groups. The serum copeptin, S100B protein and UA levels of the children in the death group on the 1st day after admission were significantly higher than those in the survival group, and showed an increasing trend with the extension of hospitalization. The differences between the death group and the survival group on the 5th day were still statistically significant [copeptin (µg/L): 19.37±6.50 vs. 8.06±2.14, S100B protein (µg/L): 9.52±2.86 vs. 3.75±0.97, UA (µmol/L): 527.40±273.84 vs. 255.38±143.75, all P < 0.01]. The levels of serum copeptin, S100B protein and UA in the severe group also increased over time, and were significantly higher than those in the moderate group [5-day copeptin (µg/L): 17.84±4.73 vs. 9.15±2.42, 5-day S100B protein (µg/L): 8.80±2.40 vs. 4.17±1.16, 5-day UA (µmol/L): 494.72±262.53 vs. 276.20±150.37, all P < 0.01]. ROC curve analysis showed that serum copeptin, S100B protein and UA at each time point after admission had predictive value for 28-day death of TBI children, and the most predictive value was on the 3rd day, and the area under ROC curve (AUC) predicted by the combination of three parameters were significantly higher than that predicted by a single one (AUC: 0.940 vs. 0.852, 0.837, 0.793, Z values were 5.392, 5.704 and 6.612, respectively, all P < 0.05), with the sensitivity and specificity of 96.3% and 88.5%, respectively. Pearson correlation analysis showed that serum copeptin, S100B protein and UA were significantly negatively correlated with GCS score on the 3rd day after admission in 28-day dead TBI children (r values were -0.862, -0.827, -0.758, respectively, all P < 0.01). CONCLUSIONS: The increase of serum copeptin, S100B protein and UA levels is related to the severity and prognosis of TBI children. The combination of serum copeptin, S100B protein and UA on the 3rd day after admission has a better value in predicting the death of TBI children.


Assuntos
Lesões Encefálicas Traumáticas , Subunidade beta da Proteína Ligante de Cálcio S100/sangue , Criança , Glicopeptídeos , Humanos , Prognóstico , Curva ROC , Ácido Úrico
3.
FASEB J ; 34(6): 7253-7264, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32367579

RESUMO

Drug repurposing is potentially the fastest available option in the race to identify safe and efficacious drugs that can be used to prevent and/or treat COVID-19. By describing the life cycle of the newly emergent coronavirus, SARS-CoV-2, in light of emerging data on the therapeutic efficacy of various repurposed antimicrobials undergoing testing against the virus, we highlight in this review a possible mechanistic convergence between some of these tested compounds. Specifically, we propose that the lysosomotropic effects of hydroxychloroquine and several other drugs undergoing testing may be responsible for their demonstrated in vitro antiviral activities against COVID-19. Moreover, we propose that Niemann-Pick disease type C (NPC), a lysosomal storage disorder, may provide new insights into potential future therapeutic targets for SARS-CoV-2, by highlighting key established features of the disorder that together result in an "unfavorable" host cellular environment that may interfere with viral propagation. Our reasoning evolves from previous biochemical and cell biology findings related to NPC, coupled with the rapidly evolving data on COVID-19. Our overall aim is to suggest that pharmacological interventions targeting lysosomal function in general, and those particularly capable of reversibly inducing transient NPC-like cellular and biochemical phenotypes, constitute plausible mechanisms that could be used to therapeutically target COVID-19.


Assuntos
Antivirais/farmacocinética , Betacoronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos , Endossomos/virologia , Hidroxicloroquina/farmacologia , Lisossomos/virologia , Doença de Niemann-Pick Tipo C/patologia , Pneumonia Viral/tratamento farmacológico , Proteína ADAM17/fisiologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/farmacologia , Alanina/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Benzilisoquinolinas/farmacologia , Benzilisoquinolinas/uso terapêutico , Transporte Biológico , Catepsina L/fisiologia , Endocitose , Endossomos/efeitos dos fármacos , Endossomos/fisiologia , Glicopeptídeos/farmacologia , Glicopeptídeos/uso terapêutico , Humanos , Hidroxicloroquina/farmacocinética , Hidroxicloroquina/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lipídeos de Membrana/metabolismo , Microdomínios da Membrana/fisiologia , Doença de Niemann-Pick Tipo C/metabolismo , Oxisteróis/metabolismo , Pandemias , Peptidil Dipeptidase A/metabolismo , Receptores Virais/metabolismo , Serina Endopeptidases/fisiologia , Triazóis/farmacologia , Triazóis/uso terapêutico , Internalização do Vírus/efeitos dos fármacos
4.
Science ; 369(6501): 330-333, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32366695

RESUMO

The emergence of the betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), represents a considerable threat to global human health. Vaccine development is focused on the principal target of the humoral immune response, the spike (S) glycoprotein, which mediates cell entry and membrane fusion. The SARS-CoV-2 S gene encodes 22 N-linked glycan sequons per protomer, which likely play a role in protein folding and immune evasion. Here, using a site-specific mass spectrometric approach, we reveal the glycan structures on a recombinant SARS-CoV-2 S immunogen. This analysis enables mapping of the glycan-processing states across the trimeric viral spike. We show how SARS-CoV-2 S glycans differ from typical host glycan processing, which may have implications in viral pathobiology and vaccine design.


Assuntos
Betacoronavirus/química , Polissacarídeos/química , Glicoproteína da Espícula de Coronavírus/química , Sítios de Ligação , Infecções por Coronavirus , Glicopeptídeos/química , Glicopeptídeos/imunologia , Glicosilação , Humanos , Espectrometria de Massas , Modelos Moleculares , Oligossacarídeos/química , Pandemias , Pneumonia Viral , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
5.
J Chromatogr A ; 1621: 461039, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32295703

RESUMO

Type 1 autoimmune pancreatitis (AIP) is a kind of IgG4-related disease in which higher IgG4 and total IgG levels have been found in patient serum. Due to the similar imaging features and laboratory parameters between AIP and pancreatic ductal adenocarcinoma (PDAC), a differential diagnosis is still challenging. Since IgG profiles can be potential bio-signatures for disease, we developed and validated a method which coupled on-bead enzymatic protein elution process to an efficient UHPLC-MS/MS method to determine IgG subclass and glycosylation. A stable-isotope labeled IgG was incorporated as internal standard to achieve accurate quantification. For calibration curves, the correlation coefficients for total IgG and the four IgG subclasses were higher than 0.995. Intraday (n = 5) and interday (n = 3) precisions of the peak area ratios of LLOQ, low, medium, and high QC samples were all less than 6.6% relative standard deviation (% RSD), and the accuracies were between 93.5 and 114.9%. Calibration curves, precision, and accuracy were also evaluated for 26 IgG glycopeptides. The method was applied to samples from healthy controls and patients with AIP and PDAC. Distinct IgG patterns were discovered among the groups, and 7 glycopeptides showed high potential in differentiating AIP and PDAC. The results demonstrated that the developed method is suitable for multi-feature analysis of human IgG, and the discovered IgG profiles can be used as bio-signatures for AIP and PDAC.


Assuntos
Pancreatite Autoimune/imunologia , Carcinoma Ductal Pancreático/imunologia , Cromatografia Líquida de Alta Pressão/métodos , Glicopeptídeos/análise , Imunoglobulina G/sangue , Neoplasias Pancreáticas/imunologia , Espectrometria de Massas em Tandem/métodos , Pancreatite Autoimune/diagnóstico , Carcinoma Ductal Pancreático/diagnóstico , Diagnóstico Diferencial , Glicosilação , Humanos , Imunoglobulina G/classificação , Imunoglobulina G/metabolismo , Neoplasias Pancreáticas/diagnóstico
6.
Chem Commun (Camb) ; 56(18): 2767-2770, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32022009

RESUMO

We synthesized "glyco-arylopeptides", whose folding structure significantly changes depending on the kind of saccharide in their side chain. The saccharide moiety interacts with the main chain via hydrogen bonding, and the non-natural polypeptides form two well-defined architectures-(P)-31- and (M)-41-helices-depending on the length of the saccharide chains and even the configuration of a single stereo-genic center in the epimers.


Assuntos
Glicopeptídeos/química , Oligossacarídeos/química , Peptídeos/síntese química , Teoria da Densidade Funcional , Estrutura Molecular , Peptídeos/química , Dobramento de Proteína
7.
Cancer Immunol Immunother ; 69(5): 703-716, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32034426

RESUMO

Cancer is one of the main causes of mortality worldwide and a major public health concern. Among various strategies, therapeutic vaccines have been developed to stimulate anti-tumoral immune responses. However, in spite of extensive studies, this approach suffers from a lack of efficacy. Recently, we designed the MAG-Tn3 vaccine, aiming to induce antibody responses against Tn, a tumor-associated carbohydrate antigen. The Tn antigen is of interest because it is expressed by several adenocarcinomas, but not normal cells. The fully synthetic glycopeptide vaccine MAG-Tn3 is composed of four arms built on three adjacent Tn moieties associated with the tetanus toxin-derived peptide TT830-844 CD4+ T-cell epitope. This promiscuous CD4+ T-cell epitope can bind to a wide range of HLA-DRB molecules and is thus expected to activate CD4+ T-cell responses in a large part of the human population. The MAG-Tn3 vaccine was formulated with the GSK-proprietary immunostimulant AS15, composed of CpG7909, MPL, and QS21, which has been shown to stimulate both innate and humoral responses, in addition to being well tolerated. Here, seven patients with localized breast cancer with a high-risk of relapse were immunized with the MAG-Tn3 vaccine formulated with AS15. The first results of phase I clinical trial demonstrated that all vaccinated patients developed high levels of Tn-specific antibodies. Moreover, these antibodies specifically recognized Tn-expressing human tumor cells and killed them through a complement-dependent cytotoxicity mechanism. Overall, this study establishes, for the first time, the capacity of a fully synthetic glycopeptide cancer vaccine to induce specific immune responses in humans.


Assuntos
Anticorpos Antineoplásicos/sangue , Antígenos Glicosídicos Associados a Tumores/imunologia , Neoplasias da Mama/terapia , Vacinas Anticâncer/imunologia , Recidiva Local de Neoplasia/prevenção & controle , Adjuvantes Imunológicos/administração & dosagem , Adulto , Idoso , Animais , Anticorpos Antineoplásicos/imunologia , Antígenos Glicosídicos Associados a Tumores/administração & dosagem , Antígenos Glicosídicos Associados a Tumores/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Feminino , Glicopeptídeos/administração & dosagem , Glicopeptídeos/genética , Glicopeptídeos/imunologia , Humanos , Imunogenicidade da Vacina , Injeções Intramusculares , Células Jurkat , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/imunologia , Resultado do Tratamento , Vacinação/métodos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
8.
Anal Bioanal Chem ; 412(7): 1509-1520, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32002580

RESUMO

Highly specific enrichment of N-linked glycopeptides from complex biological samples is crucial prior to mass spectrometric analysis. In this work, a hydrophilic metal-organic framework composite is prepared by the growth of UiO-66-NH2 on graphene sheets, followed by its post-synthetic modification to attach boronic acid to form GO@UiO-66-PBA. The fabrication of graphene oxide-MOF composite results in enhanced surface area with improved thermal and chemical stability. The synthesized MOF nanocomposite is characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and BET. A crystalline structure with high porosity offering large surface area and good hydrophilicity of the nanocomposite assists as an enrichment tool in glycoproteomics. The GO@UiO-66-PBA nanocomposite selectively enriches N-linked glycopeptides from tryptic digests of horseradish peroxidase (HRP) and immunoglobulin (IgG). GO@UiO-66-PBA nanoparticles show a low detection limit (1 fmol) and good specificity (1:200), reusability and reproducibility for N-linked glycopeptide enrichment from IgG digest. The binding capacity of GO@UiO-66-PBA is 84 mg/g for protein concentration, with a good recovery of 86.5%. A total of 372 N-linked glycopeptides corresponding to different glycoproteins are identified from only 1 µL of human serum digest. Thus, the presented research work can be an efficient separation platform for N-linked glycopeptide enrichment from complex samples, which can be extended to cost-effective routine analysis. Graphical abstract.


Assuntos
Ácidos Borônicos/química , Glicopeptídeos/química , Estruturas Metalorgânicas/química , Grafite/química , Peroxidase do Rábano Silvestre/química , Humanos , Microscopia Eletrônica de Varredura , Reprodutibilidade dos Testes
9.
Anal Bioanal Chem ; 412(7): 1497-1508, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32025769

RESUMO

Analysis of glycoprotein sialylation is challenging due to the relatively low abundance of sialylated glycopeptides (SGPs) in complex biosamples and low signals of SGPs in mass spectrometry. In this study, a mesoporous poly-melamine-formaldehyde (mPMF) polymer was prepared and utilized as the high-efficiency sorbent for SGPs. The mPMF polymer featured high surface area (755.4 m2 g-1) and high density of amine and triazine functional groups. This polymer demonstrated high enrichment selectivity (resistant to 100 molar fold interference of BSA) and superior adsorption capacity (560 mg g-1) for SGPs. The high performance of mPMF toward SGPs ascribes to the unique physicochemical properties of mPMF and high density of accessible binding sites for glycopeptides. Further application of mPMF to HeLa S3 cell lysate resulted in 576 characterized glycopeptides with 218 unique glycosylation sites. This finding provides a new choice of promising extraction approach for characterization of protein glycosylation. Graphical abstract A mesoporous poly-melamine-formaldehyde (mPMF) polymer was prepared and utilized as the high-efficiency enrichment sorbent for sialylated glycopeptides (SGPs).


Assuntos
Glicopeptídeos/química , Ácido N-Acetilneuramínico/química , Polímeros/química , Triazinas/química , Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas/métodos , Porosidade
11.
Talanta ; 209: 120563, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892091

RESUMO

In this study is described an on-line titanium dioxide solid-phase extraction capillary electrophoresis-mass spectrometry (TiO2-SPE-CE-MS) method for the analysis of the glycopeptide glycoforms obtained from the tryptic digests of recombinant human erythropoietin (rhEPO). The O126-glycopeptide of rhEPO was used to optimize the methodology given its importance in quality control of biopharmaceuticals and doping analysis. Several aspects that affect the selective retention and elution, peak efficiency and electrophoretic separation of the O126 glycoforms were investigated to maximize detection sensitivity while minimizing non-specific retention of peptides. Under the optimized conditions, the microcartridge lifetime was around 10 analyses and repeatability was acceptable (%RSD values of 9-11% and 6-11% for migration times and peak areas, respectively). The method was linear between 0.5 and 50 mg L-1 and 10-50 mg L-1 for O126 glycoforms containing NeuAc and NeuGc, respectively, and limits of detection (LODs) were up to 100 times lower than by CE-MS. Although optimized for O-glycopeptides, the method proved also successful for preconcentration of N83-glycopeptides, without compromising the separation between glycopeptide glycoforms with different number of sialic acids. Tryptic digests of other glycoproteins (i.e. human apolipoprotein CIII (APO-C3) and bovine alpha-1-acid glycoprotein (bAGP)) were also analyzed, demonstrating the applicability to glycopeptides with different glycan composition and nature.


Assuntos
Eletroforese Capilar/métodos , Eritropoetina/análise , Glicopeptídeos/análise , Extração em Fase Sólida/métodos , Titânio/química , Biomarcadores/análise , Eletroforese Capilar/instrumentação , Desenho de Equipamento , Eritropoetina/isolamento & purificação , Glicopeptídeos/isolamento & purificação , Humanos , Espectrometria de Massas/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/isolamento & purificação , Extração em Fase Sólida/instrumentação
12.
Nat Chem Biol ; 16(3): 351-360, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31932717

RESUMO

Polypeptide GalNAc-transferase T3 (GalNAc-T3) regulates fibroblast growth factor 23 (FGF23) by O-glycosylating Thr178 in a furin proprotein processing motif RHT178R↓S. FGF23 regulates phosphate homeostasis and deficiency in GALNT3 or FGF23 results in hyperphosphatemia and familial tumoral calcinosis. We explored the molecular mechanism for GalNAc-T3 glycosylation of FGF23 using engineered cell models and biophysical studies including kinetics, molecular dynamics and X-ray crystallography of GalNAc-T3 complexed to glycopeptide substrates. GalNAc-T3 uses a lectin domain mediated mechanism to glycosylate Thr178 requiring previous glycosylation at Thr171. Notably, Thr178 is a poor substrate site with limiting glycosylation due to substrate clashes leading to destabilization of the catalytic domain flexible loop. We suggest GalNAc-T3 specificity for FGF23 and its ability to control circulating levels of intact FGF23 is achieved by FGF23 being a poor substrate. GalNAc-T3's structure further reveals the molecular bases for reported disease-causing mutations. Our findings provide an insight into how GalNAc-T isoenzymes achieve isoenzyme-specific nonredundant functions.


Assuntos
Fatores de Crescimento de Fibroblastos/química , N-Acetilgalactosaminiltransferases/metabolismo , Animais , Células CHO , Cricetulus , Fatores de Crescimento de Fibroblastos/metabolismo , Glicopeptídeos/química , Glicosilação , Humanos , Isoenzimas/metabolismo , Lectinas/metabolismo , N-Acetilgalactosaminiltransferases/fisiologia , Treonina/metabolismo
13.
Nat Commun ; 11(1): 303, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949166

RESUMO

α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.


Assuntos
Distrofias Musculares/metabolismo , Açúcares de Nucleosídeo Difosfato/química , Açúcares de Nucleosídeo Difosfato/metabolismo , Pentosiltransferases/química , Pentosiltransferases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Glicopeptídeos , Células HEK293 , Humanos , Modelos Moleculares , Distrofias Musculares/genética , Pentosiltransferases/genética , Fosfatos/metabolismo , Polissacarídeos/metabolismo , Conformação Proteica , Domínios Proteicos , Ribitol/metabolismo
16.
Anal Chim Acta ; 1098: 181-189, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31948582

RESUMO

Protein N-glycosylation plays crucial roles in many biological processes and has close association with the occurrence and development of various cancers. Therefore, it is necessary to analyze the abnormal changes of N-glycopeptides in complex biological samples for biomarker discovery. However, due to their low abundance and poor ionization, N-glycopeptides identification in complex samples by mass spectrometry (MS) is still a challenging task. In this work, a novel magnetic hydrophilic material was prepared by serial functionalization of ultra-thin two-dimensional molybdenum disulfide with Fe3O4 nanoparticles, gold nanowire and glutathione (MoS2-Fe3O4-Au/NWs-GSH) for efficient N-glycopeptides enrichment. The advantage of using the new nanocomposite is threefold. First, the introduction of magnetic Fe3O4 nanoparticles efficiently simplifies the enrichment process. Second, the gold nanowire modification enlarges the surface area of the nanocomposites to facilitate interaction with N-glycopeptides. Third, the employment of highly hydrophilic glutathione leads to specific HILIC-based retention of N-glycopeptides. Low femtomolar detection sensitivity and 1:1000 enrichment selectivity can be achieved using MoS2-Fe3O4-Au/NWs-GSH enrichment and bio-mass spectrometry analysis. Successful applications in human urine exosome and serum proteins were demonstrated by the enrichment and identification of 1250 and 489 N-glycopeptides, respectively. This remarkable data set of N-glycoproteome indicates the application potential of the novel nanocomposites for N-glycopeptides enrichment in complex biological samples and for related glycoproteome studies.


Assuntos
Proteínas Sanguíneas/urina , Dissulfetos/química , Exossomos/química , Glutationa/química , Glicopeptídeos/química , Nanopartículas de Magnetita/química , Molibdênio/química , Cromatografia Líquida de Alta Pressão , Humanos , Interações Hidrofóbicas e Hidrofílicas
17.
Am J Physiol Renal Physiol ; 318(3): F702-F709, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31961713

RESUMO

Alcohol consumption influences sodium-water homeostasis. However, the effect of alcohol on vasopressin levels is controversial. The aim of the present study was to evaluate physiological changes of alcohol consumption on the stable vasopressin surrogate marker copeptin. In addition, we aimed at investigating the effect of additional sodium and/or water consumption on plasma sodium, osmolality, and copeptin levels. Ten healthy men underwent four interventions in random order: 1) beer consumption only, 2) beer consumption with additional water, 3) beer consumption with additional stock, or 4) water consumption only. Fluid consumption was equal between interventions and calculated to reach a blood alcohol concentration of 0.8‰ in the beer interventions. Blood and urinary samples were taken at six time points over the observation period of 720 min. The primary end point was the mean difference in copeptin levels 90 min after the start of fluid consumption, which showed no in-between group differences (P = 0.4). However, a higher total urinary volume excretion in all alcohol compared with water interventions was observed (P = 0.01). Furthermore, plasma copeptin, sodium, and urinary osmolality levels increased significantly at the end of the observation period in all alcohol compared with water-only interventions (P = 0.02). In conclusion, initial copeptin suppression does not differ between alcohol or water interventions but seems to be prolonged in the alcohol interventions. This leads to increased volume loss followed by a counterregulation with increased copeptin levels and water retention after 720 min in alcohol compared with interventions. Additional sodium and/or water consumption with alcohol did not change the observed alcohol-induced effects.


Assuntos
Cerveja , Glicopeptídeos/metabolismo , Sódio/sangue , Equilíbrio Hidroeletrolítico , Água , Adulto , Consumo de Bebidas Alcoólicas , Ingestão de Líquidos , Glicopeptídeos/genética , Homeostase/efeitos dos fármacos , Humanos , Masculino , Adulto Jovem
18.
Biomarkers ; 25(2): 137-143, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31902247

RESUMO

Background: Takotsubo cardiomyopathy (TTC) is a syndrome of acute non-coronary heart failure with similar symptoms and electrocardiograms to acute anterior ST-elevation myocardial infarction (STEMI). Little is known about the pathophysiology of TTC. We assessed admission plasma concentrations of biomarkers reflecting neuroendocrine response (copeptin, mid-regional-pro-adrenomedullin, pro-atrial-natriuretic-peptide, soluble thrombomodulin (sTM), syndecan-1) and inflammation (suppression-of-tumorigenicity 2 (ST2), high-sensitive C-reactive-protein) in TTC patients and compared to patients with acute anterior STEMI.Materials and methods: Twenty TTC patients were matched with 40 STEMI patients by age, gender and left ventricular ejection fraction. Blood was sampled upon hospital admission immediately before acute coronary angiography.Results: The groups had similar comorbidities. TTC patients had higher plasma concentrations of sTM: 7.94 (5.89;9.61) vs. 6.42 (5.50;7.82)ng/ml, p = 0.04 and ST2 (53 (32;157) vs. 45 (31;55)ng/ml, p = 0.008) and higher heart rate: 101 ([Formula: see text]33) vs. 76([Formula: see text]14)bpm, p = 0.0001, but lower concentrations of copeptin (10.4 (7.6;39) vs. 92.3 (13;197)pmol/l, p < 0.05) and troponin T (348 (98;759) vs. 1190 (261;4105)ng/l, p = 0.04).Conclusion: TTC patients had higher plasma concentrations of sTM and ST2, higher heart rate and lower copeptin and troponin T concentrations compared to acute anterior STEMI patients. This study contributes to the hypothesis that TTC patients have endothelial cell damage and are hemodynamically more stable than patients with acute anterior STEMI on admission.


Assuntos
Biomarcadores/sangue , Infarto do Miocárdio com Supradesnível do Segmento ST/diagnóstico , Cardiomiopatia de Takotsubo/diagnóstico , Idoso , Diagnóstico Diferencial , Células Endoteliais/patologia , Feminino , Glicopeptídeos/sangue , Frequência Cardíaca , Hemodinâmica , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/sangue , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio com Supradesnível do Segmento ST/sangue , Cardiomiopatia de Takotsubo/sangue , Trombomodulina/sangue , Troponina T/sangue
19.
J Chromatogr A ; 1610: 460545, 2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31551124

RESUMO

The facile enrichment of glycopeptides or glycoproteins poses great challenges for glycoproteomic research. In this study, a novel hydrophilic material, named zwitterionic hydrophilic L-cysteine derivatized straticulate-C3N4 composites (LCAC), were synthesized and evaluated for the enrichment of N-glycopeptides. LCAC exhibited good biocompatibility, excellent hydrophilicity and selectivity, by virtue of the large surface of C3N4 and the zwitterionic property offered by cysteine. LCAC demonstrated excellent performance for N-glycopeptide enrichment with the sensitivity of 0.033 fmol/µL, selectivity of 1:100, and high recovery rate (∼85%). The performance of LCAC was demonstrated by the identification of 35 N-glycopeptides from IgG, as well as capturing 1809 human urine N-glycopeptides corresponding to 876 N-glycoproteins. Comparing the LCAC with our developed phenylboronic acid functionalized material showed a certain complementary due to the different binding mechanism. The simple production and enhanced hydrophilic properties make the material a promising choice for glycoproteomics researches.


Assuntos
Cisteína/química , Glicopeptídeos/isolamento & purificação , Glicoproteínas/isolamento & purificação , Nitrilos/química , Cromatografia de Afinidade , Glicopeptídeos/urina , Glicoproteínas/urina , Humanos , Interações Hidrofóbicas e Hidrofílicas , Sensibilidade e Especificidade
20.
J Chromatogr A ; 1610: 460546, 2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31570191

RESUMO

Selective enrichment of glycopeptides from complex sample with hydrophilic interaction liquid chromatography (HILIC) method, followed by cleavage of N-glycans by PNGase F to expose an easily detectable mark on the former glycosylation sites is used extensively as a sample preparation for comprehensive glycoproteome analysis. However, the coenrichment of hydrophilic nonglycosylated peptides and the released N-glycans seriously affect the identification of deglycopeptides with nano-LC-MS/MS. Here, we developed a new method for highly efficient and specific enrichment of human plasma N-glycopeptides using HILIC-PNGaseF-HILIC workflow (HPH). The first HILIC enriches the N-glycopeptides from the complex peptide mixtures. After the enriched N-glycopeptides are deglycosylated with PNGase F, the second HILIC captures the coenrichment of hydrophilic nonglycosylated peptides and the N-glycans, and then further enriches the deglycosylated peptides. The glycopeptide enrichment efficiency can be notably improved by employing HPH, evaluated by the highly recovery (more than 93.6%) and specific capturing glycopeptides from tryptic digest of IgG and BSA up to the molar ratios of 1:200. Meanwhile, we found that the alkylated proteins with IAA can affect the enrichment efficiency for N-glycopeptides with HILIC method. Moreover, after optimism the protein digestion, this novel HPH strategy allowed for the identified 722 N-glycopeptides within 202 unique glycoproteins from 1 µL human plasma digest using PNGase F in H216O. Meanwhile, this new HPH strategy identified an average 501 N-glycopeptides within averagely 134 unique glycoproteins from 1 µL human plasma digest using PNGase F in H218O. The enhanced glycopeptide detection was promoted by a substantial depletion of nonglycosylated peptides in the second HILIC. It was found that 52.2% more N-glycosylation peptides were identified by the HPH strategy compared with the using one HILIC enrichment alone.


Assuntos
Cromatografia Líquida/métodos , Glicopeptídeos/sangue , Glicoproteínas/sangue , Proteoma/análise , Glicopeptídeos/isolamento & purificação , Glicoproteínas/isolamento & purificação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteoma/isolamento & purificação , Proteômica
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