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1.
Int J Mol Sci ; 24(10)2023 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-37240090

RESUMO

Changes in protein glycosylation are associated with most biological processes, and the importance of glycomic analysis in the research of disorders is constantly increasing, including in the neurodevelopmental field. We glycoprofiled sera in 10 children with attention-deficit hyperactivity disorder (ADHD) and 10 matching healthy controls for 3 types of samples: whole serum, sera after depletion of abundant proteins (albumin and IgG), and isolated IgG. The analytical methods used were a lectin-based glycoprotein microarray enabling high-throughput glycan analysis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) as a standard method for the identification of glycan structures. For microarray analysis, the samples printed on microarray slides were incubated with biotinylated lectins and detected using the fluorescent conjugate of streptavidin by a microarray scanner. In the ADHD patient samples, we found increased antennary fucosylation, decreased di-/triantennary N-glycans with bisecting N-acetylglucosamine (GlcNAc), and decreased α2-3 sialylation. The results obtained by both independent methods were consistent. The study's sample size and design do not allow far-reaching conclusions to be drawn. In any case, there is a strong demand for a better and more comprehensive diagnosis of ADHD, and the obtained results emphasize that the presented approach brings new horizons to studying functional associations of glycan alterations in ADHD.


Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Criança , Humanos , Glicoproteínas/química , Polissacarídeos/química , Lectinas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Imunoglobulina G/metabolismo
2.
Front Biosci (Landmark Ed) ; 28(2): 39, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36866557

RESUMO

BACKGROUND: Alpha-mannosidosis is a rare lysosomal storage disorder, caused by decreased activity of α-D-mannosidase. This enzyme is involved in the hydrolysis of mannosidic linkages in N-linked oligosaccharides. Due to the mannosidase defect, undigested mannose-rich oligosaccharides (Man2GlcNAc - Man9GlcNAc) accumulating in cells are excreted in large quantities in urine. METHODS: In this work, we determined the levels of urinary mannose-rich oligosaccharides in a patient subjected to novel enzyme replacement therapy. Urinary oligosaccharides were extracted using solid phase extraction (SPE), labeled by fluorescent tag 2-aminobenzamide, and quantified by high-performance liquid chromatography (HPLC) with fluorescence detector (FLD). The identity of peaks was determined by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. In addition, the levels of urinary mannose-rich oligosaccharides were also quantified by 1H nuclear magnetic resonance (NMR) spectroscopy. The data were analyzed using one-tailed paired t-test and Pearson's correlation tests. RESULTS: Compared to levels before the administration of therapy, an approximately two-folds decrease in total mannose-rich oligosaccharides after one month of treatment was observed by NMR and HPLC. After four months, an approximately ten-folds significant decrease in total urinary mannose-rich oligosaccharides was detected, suggesting therapy effectiveness. A significant decrease in the levels of oligosaccharides with 7-9 mannose units was detected by HPLC. CONCLUSIONS: The application of both HPLC-FLD and NMR in quantification of oligosaccharide biomarkers is a suitable approach for monitoring of therapy efficacy in alpha-mannosidosis patients.


Assuntos
alfa-Manosidose , Humanos , Cromatografia Líquida de Alta Pressão , alfa-Manosidose/tratamento farmacológico , Manose , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética
3.
Biomedicines ; 11(2)2023 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-36831116

RESUMO

Congenital disorders of glycosylation (CDG) are a group of rare inherited metabolic disorders caused by a defect in the process of protein glycosylation. In this work, we present a comprehensive glycoprofile analysis of a male patient with a novel missense variant in the SLC35A2 gene, coding a galactose transporter that translocates UDP-galactose from the cytosol to the lumen of the endoplasmic reticulum and Golgi apparatus. Isoelectric focusing of serum transferrin, which resulted in a CDG type II pattern, was followed by structural analysis of transferrin and serum N-glycans, as well as the analysis of apolipoprotein CIII O-glycans by mass spectrometry. An abnormal serum N-glycoprofile with significantly increased levels of agalactosylated (Hex3HexNAc4-5 and Hex3HexNAc5Fuc1) and monogalactosylated (Hex4HexNAc4 ± NeuAc1) N-glycans was observed. Additionally, whole exome sequencing and Sanger sequencing revealed de novo hemizygous c.461T > C (p.Leu154Pro) mutation in the SLC35A2 gene. Based on the combination of biochemical, analytical, and genomic approaches, the set of distinctive N-glycan biomarkers was characterized. Potentially, the set of identified aberrant N-glycans can be specific for other variants causing SLC35A2-CDG and can distinguish this disorder from the other CDGs or other defects in the galactose metabolism.

4.
Anal Chim Acta ; 1234: 340512, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36328727

RESUMO

In this study, we applied MXene as column cartridge for N-glycan enrichment from human samples with a focus on the analysis of sialic acid linkages using a derivatisation protocol followed by glycan analysis via Matrix Assisted Laser Desorption Ionisation-Time Of Flight Mass Spectrometry (MALDI-TOF-MS). The MXene-based cartridge enriches a higher number of glycans (i.e., sialylated and bisecting N-glycans) when compared to the commercial HILIC columns. We demonstrate the strong potential of MXene as a stationary phase in MS glycomic analysis.


Assuntos
Ácido N-Acetilneuramínico , Polissacarídeos , Humanos , Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
5.
Am J Med Genet A ; 185(11): 3494-3501, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34467644

RESUMO

Congenital disorder of glycosylation type Ig (ALG12-CDG) is a rare inherited metabolic disease caused by a defect in alpha-mannosyltransferase 8, encoded by the ALG12 gene (22q13.33). To date, only 15 patients have been diagnosed with ALG12-CDG globally. Due to a newborn Slovak patient's clinical and biochemical abnormalities, the isoelectric focusing of transferrin was performed with observed significant hypoglycosylation typical of CDG I. Furthermore, analysis of neutral serum N-glycans by mass spectrometry revealed the accumulation of GlcNAc2Man5-7 and decreased levels of GlcNAc2Man8-9, which indicated impaired ALG12 enzymatic activity. Genetic analysis of the coding regions of the ALG12 gene of the patient revealed a novel homozygous substitution mutation c.1439T>C p.(Leu480Pro) within Exon 10. Furthermore, both of the patient's parents and his twin sister were asymptomatic heterozygous carriers of the variant. This comprehensive genomic and glycomic approach led to the confirmation of the ALG12 pathogenic variant responsible for the clinical manifestation of the disorder in the patient described.


Assuntos
Defeitos Congênitos da Glicosilação/genética , Predisposição Genética para Doença , Manosiltransferases/genética , Polissacarídeos/genética , Defeitos Congênitos da Glicosilação/epidemiologia , Defeitos Congênitos da Glicosilação/patologia , Feminino , Testes Genéticos , Glicosilação , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Mutação de Sentido Incorreto/genética , Fenótipo , Polissacarídeos/metabolismo , Eslováquia/epidemiologia , Transferrina/genética
6.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360993

RESUMO

The ferroxidase ceruloplasmin (CP) plays a crucial role in iron homeostasis in vertebrates together with the iron exporter ferroportin. Mutations in the CP gene give rise to aceruloplasminemia, a rare neurodegenerative disease for which no cure is available. Many aspects of the (patho)physiology of CP are still unclear and would benefit from the availability of recombinant protein for structural and functional studies. Furthermore, recombinant CP could be evaluated for enzyme replacement therapy for the treatment of aceruloplasminemia. We report the production and preliminary characterization of high-quality recombinant human CP in glycoengineered Pichia pastoris SuperMan5. A modified yeast strain lacking the endogenous ferroxidase has been generated and employed as host for heterologous expression of the secreted isoform of human CP. Highly pure biologically active protein has been obtained by an improved two-step purification procedure. Glycan analysis indicates that predominant glycoforms HexNAc2Hex8 and HexNAc2Hex11 are found at Asn119, Asn378, and Asn743, three of the canonical four N-glycosylation sites of human CP. The availability of high-quality recombinant human CP represents a significant advancement in the field of CP biology. However, productivity needs to be increased and further careful glycoengineering of the SM5 strain is mandatory in order to evaluate the possible therapeutic use of the recombinant protein for enzyme replacement therapy of aceruloplasminemia patients.


Assuntos
Ceruloplasmina/genética , Microbiologia Industrial/métodos , Engenharia de Proteínas/métodos , Saccharomycetales/genética , Ceruloplasmina/metabolismo , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales/metabolismo
7.
J Biotechnol ; 314-315: 34-40, 2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32298669

RESUMO

Glycosylation of therapeutic glycoproteins significantly affects their physico-chemical properties, bioactivity and immunogenicity. The determination of glycan composition is highly important regarding their development and production. Therefore, there is a demand for analytical techniques enabling rapid and reliable glycoprofiling of therapeutic proteins. For the investigation of changes in glycan structures, we have employed two platforms: lectin-based protein microarray, and MALDI-MS. In lectin-based microarray analysis, the samples of IgA were printed on the microarray slide, incubated with the set of lectins with various specificity and evaluation of changes in glycosylation was based on differences in reactivity of samples with lectins. MALDI-MS was used for N-glycan analysis of IgA1 samples. IgAs are effective as therapeutic agents in defense against viruses that use sialic acid as a receptor. Dimeric IgA1 antibodies were produced by stable cell line IgA1/2G9 on the basal medium at different conditions (different supplementation and feeding) and we also evaluated the effect of different conditions on lactate production, which correlates with IgA productivity. Decrease of lactate levels was observed during supplementation with succinic acid, asparagine, or with mannose feeding. We found by lectin-based microarray analysis that the metabolic shift from glutamine to asparagine or feeding with glucose caused increase of high mannose type glycans what was confirmed also by MALDI-MS. Among other changes in IgA glycosylation determined by lectin-based protein microarray were, for example, reduced galactosylation after supplementation with succinic acid and increase of both sialylation and galactosylation after supplementation with glutamine and feeding with mannose. The elucidation of mechanism of determined changes requires further investigation, but the described analytical approach represent effective platform for determination, screening and evaluation of glycosylation of therapeutic proteins.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina A/química , Lectinas/química , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/biossíntese , Células CHO , Cricetulus , Meios de Cultura/química , Meios de Cultura/metabolismo , Glicosilação , Imunoglobulina A/análise , Imunoglobulina A/biossíntese , Polissacarídeos/análise , Polissacarídeos/química , Análise Serial de Proteínas/métodos , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
8.
Mech Ageing Dev ; 183: 111151, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589880

RESUMO

Structural changes of glycans are observed in different (patho)physiological conditions. Human placental membrane (glyco)proteins were isolated from the first and third trimester placentas of mothers at different ages. By using lectin microarray, we demonstrated that the placental membrane N-glycome contains several N-glycan groups: high mannose, asialylated and sialylated biantennary moieties, bisected, core fucosylated, fucosylated at other positions (bearing terminal and/or antennary Fuc), α2-6 and α2-3 sialylated structures. Employing MALDI-TOF MS enabled identification of over sixty different N-glycan structures in all samples, with 17 moieties exceeding the relative abundance of 2%. The major MS peaks originated from: 1) biantennary complex type N-glycan with a bisecting GlcNAc residue and 2) a core Fuc paucimannosidic and high mannose type structures M3-M9. Age of mothers and the stage of placental development affected N-glycome. The work presented in this article is the first comprehensive mass spectrometric study of the N-glycome of human placental membrane proteins. Our results may be seen as the baseline which can serve for future MALDI MS profiling of the placental membrane N-glycome in different pathophysiological conditions.


Assuntos
Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Placenta/metabolismo , Proteínas da Gravidez/metabolismo , Adulto , Feminino , Humanos , Gravidez
9.
Proteomics ; 16(24): 3085-3095, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-26920336

RESUMO

The construction of a sensitive electrochemical lectin-based immunosensor for detection of a prostate specific antigen (PSA) is shown here. Three lectins with different carbohydrate specificities were used in this study to glycoprofile PSA, which is the most common biomarker for prostate cancer (PCa) diagnosis. The biosensor showed presence of α-L-fucose and α-(2,6)-linked terminal sialic acid within PSA´s glycan with high abundance, while only traces of α-(2,3)-linked terminal sialic acid were found. MALDI TOF/TOF mass spectrometry was applied to validate results obtained by the biosensor with a focus on determination of a type of sialic acid linkage by two methods. The first direct comparison of electrochemical immunosensor assay employing lectins for PSA glycoprofiling with mass spectrometric techniques is provided here and both methods show significant agreement. Thus, electrochemical lectin-based immunosensor has potential to be applied for prostate cancer diagnosis.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , Ácido N-Acetilneuramínico/análise , Antígeno Prostático Específico/análise , Anticorpos Imobilizados/química , Impedância Elétrica , Humanos , Lectinas/química , Limite de Detecção , Masculino , Neoplasias da Próstata/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
10.
J Biol Chem ; 290(28): 17206-17, 2015 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-26018083

RESUMO

The ability of different glycosphingolipids (GSLs) to activate type I natural killer T cells (NKT cells) has been known for 2 decades. The possible therapeutic use of these GSLs has been studied in many ways; however, studies are needed in which the efficacy of promising GSLs is compared under identical conditions. Here, we compare five unique GSLs structurally derived from α-galactosylceramide. We employed biophysical and biological assays, as well as x-ray crystallography to study the impact of the chemical modifications of the antigen on type I NKT cell activation. Although all glycolipids are bound by the T cell receptor of type I NKT cells in real time binding assays with high affinity, only a few activate type I NKT cells in in vivo or in vitro experiments. The differences in biological responses are likely a result of different pharmacokinetic properties of each lipid, which carry modifications at different parts of the molecule. Our results indicate a need to perform a variety of assays to ascertain the therapeutic potential of type I NKT cell GSL activators.


Assuntos
Galactosilceramidas/química , Galactosilceramidas/imunologia , Células T Matadoras Naturais/imunologia , Animais , Apresentação de Antígeno , Antígenos CD1d/química , Antígenos CD1d/metabolismo , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Galactosilceramidas/metabolismo , Glicoesfingolipídeos/química , Glicoesfingolipídeos/imunologia , Glicoesfingolipídeos/metabolismo , Humanos , Cinética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Células T Matadoras Naturais/classificação , Receptores de Antígenos de Linfócitos T alfa-beta/química , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Ressonância de Plasmônio de Superfície
11.
J Immunol ; 191(6): 2916-25, 2013 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-23960235

RESUMO

NKT cells, a unique type of regulatory T cells, respond to structurally diverse glycolipids presented by CD1d. Although it was previously thought that recognition of glycolipids such as α-galactosylceramide (α-GalCer) by the NKT cell TCR (NKTCR) obeys a key-lock principle, it is now clear this interaction is much more flexible. In this article, we report the structure-function analysis of a series of novel 6''-OH analogs of α-GalCer with more potent antitumor characteristics. Surprisingly, one of the novel carbamate analogs, α-GalCer-6''-(pyridin-4-yl)carbamate, formed novel interactions with the NKTCR. This interaction was associated with an extremely high level of Th1 polarization and superior antitumor responses. These data highlight the in vivo relevance of adding aromatic moieties to the 6''-OH position of the sugar and additionally show that judiciously chosen linkers are a promising strategy to generate strong Th1-polarizing glycolipids through increased binding either to CD1d or to NKTCR.


Assuntos
Galactosilceramidas/imunologia , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Neoplasias Experimentais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Glicolipídeos/química , Glicolipídeos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície
12.
FEMS Microbiol Lett ; 284(2): 231-6, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18510561

RESUMO

Light and starvation are two principal environmental stimuli inducing conidiation in the soil micromycete Trichoderma spp. We observed that volatiles produced by conidiating colonies of Trichoderma spp. elicited conidiation in colonies that had not been induced previously by exposure to light. The inducing effect of volatiles was both intra- and interspecific. Chemical profiles of the volatile organic compounds (VOCs) produced by the nonconidiated colonies grown in the dark and by the conidiating colonies were compared using solid-phase microextraction of headspace samples followed by tandem GC-MS. The conidiation was accompanied by increased production of eight-carbon compounds 1-octen-3-ol and its analogs 3-octanol and 3-octanone. When vapors of these compounds were applied individually to dark-grown colonies, they elicited their conidiation already at submicromolar concentrations. It is concluded that the eight-carbon VOCs act as signaling molecules regulating development and mediating intercolony communication in Trichoderma.


Assuntos
Cetonas/metabolismo , Octanóis/metabolismo , Esporos Fúngicos/fisiologia , Trichoderma/metabolismo , Trichoderma/fisiologia , Cromatografia Gasosa , Escuridão , Volatilização
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