Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Anal Bioanal Chem ; 414(1): 551-559, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34258651

RESUMO

Differential sensing of proteins based on cross-reactive arrays and pattern recognition is a promising technique for the detection and identification of proteins. In this study, a rational biomimetic strategy has been used to prepare sensing materials capable of discriminating structurally similar proteins, such as deletion and point mutants of a cytokine, by mimicking the biological properties of heparan sulfate (HS). Using the self-assembly of two disaccharides, lactose and sulfated lactose at various ratios on the surface of a chip, an array of combinatorial cross-reactive receptors has been prepared. Coupling with surface plasmon resonance imaging (SPRi), the obtained cross-reactive array is very efficient for protein sensing. It is able to detect HS binding proteins (HSbps) such as IFNγ at nanomolar concentrations. Moreover, such a system is capable of discriminating between IFNγ and its mutants with good selectivity.


Assuntos
Citocinas , Heparitina Sulfato , Biomimética , Dissacarídeos , Heparitina Sulfato/química , Ressonância de Plasmônio de Superfície/métodos
2.
Anal Bioanal Chem ; 412(2): 507-519, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31807804

RESUMO

A biosensor device for the detection and characterization of protein-glycosaminoglycan interactions is being actively sought and constitutes the key to identifying specific carbohydrate ligands, an important issue in glycoscience. Mass spectrometry (MS) hyphenated methods are promising approaches for carbohydrate enrichment and subsequent structural characterization. In the study herein, we report the analysis of interactions between the glycosaminoglycans (GAGs) heparin (HP) and heparan sulfate (HS) and various cytokines by coupling surface plasmon resonance imaging (SPRi) for thermodynamic analysis method and MALDI-TOF MS for structural determination. To do so, we developed an SPR biochip in a microarray format and functionalized it with a self-assembled monolayer of short poly(ethylene oxide) chains for grafting the human cytokines stromal cell-derived factor-1 (SDF-1α), monocyte chemotactic protein-1 (MCP-1), and interferon-γ. The thermodynamic parameters of the interactions between these cytokines and unfractionated HP/HS and derived oligosaccharides were successively determined using SPRi monitoring, and the identification of the captured carbohydrates was carried out directly on the biochip surface using MALDI-TOF MS, revealing cytokine preferential affinity for GAGs. The MS identification was enhanced by on-chip digestion of the cytokine-bound GAGs with heparinase, leading to the detection of oligosaccharides likely involved in the binding sequence of GAG ligands. Although several carbohydrate array-based assays have been reported, this study is the first report of the successful analysis of protein-GAG interactions using SPRi-MS coupling.


Assuntos
Glicosaminoglicanos/metabolismo , Dispositivos Lab-On-A-Chip , Proteínas/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Técnicas Biossensoriais , Cinética , Ligantes , Ligação Proteica , Termodinâmica
3.
Glycobiology ; 28(7): 534-541, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29718295

RESUMO

The HS3ST3A1/B1 genes encode two homologous 3-O-sulfotransferases involved in the late modification step during heparan sulfate (HS) biosynthesis. In addition to the single nucleotide polymorphisms (SNPs) rs28470223 (C > T) in the promoter region of both HS3ST3A1 and rs62636623 (Gly/Arg) in the stem region of HS3ST3B1, three missense mutations (rs62056073, rs61729712 and rs9906590) located within the catalytic sulfotransferase domain of 3-OST-B1 are linked and associated to Plasmodium falciparum parasitaemia. To ascertain the functional effects of these SNP associations, we investigated the regulatory effect of rs28470223 and characterized the enzymatic activity of the missense SNP rs61729712 (Ser279Asn) localized at proximity of the substrate binding cleft. The SNP rs28470223 results in decreased promoter activity of HS3ST3A1 in K562 cells, suggesting a reduced in vivo transcription activity of the target gene. A comparative kinetic analysis of wt HS3ST3B1 and the Ser269Asn variant (rs61729712) using a HS-derived oligosaccharide substrate reveals a slightly higher catalytic activity for the SNP variant. These genetic and enzymatic studies suggest that genetic variations in enzymes responsible of HS 3-O-sulfation can modulate their promoter and enzymatic activities and may influence P. falciparum parasitaemia.


Assuntos
Parasitemia/genética , Plasmodium falciparum , Polimorfismo de Nucleotídeo Único , Sulfotransferases/genética , Sítios de Ligação , Linhagem Celular Tumoral , Heparitina Sulfato/metabolismo , Humanos , Mutação de Sentido Incorreto , Ligação Proteica , Sulfotransferases/química , Sulfotransferases/metabolismo
4.
Analyst ; 140(2): 543-50, 2015 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-25408953

RESUMO

The on-line hyphenation of Capillary IsoElectric Focusing (CIEF) with ElectroSpray Ionization Mass Spectrometry (ESI/MS) has been carried out in a non-denaturing detection mode at the CIEF-MS interface. This CIEF-MS coupling methodology relied on the use of 40% glycerol-water medium as anti-convective agent in the CE capillary and the addition of 10 mM ammonium acetate buffer, pH 5, as a volatile aqueous sheath liquid. These CIEF-MS coupling conditions allowed the characterization of the highly basic cytokine human interferon-gamma (IFN-γ) and its detection as a non-covalent homodimer (33,814.3 g mol(-1)) corresponding to the active form of this immune-regulatory protein. An experimental pI value of 9.95 was determined for the human IFN-γ homodimer in these conditions. The CIEF-MS analysis of several variants bearing punctual or deletion mutations within the two D1 and D2 basic clusters at the C-terminal end of IFN-γ revealed the different contribution of these domains to the charge properties of this heparan sulfate-binding protein.


Assuntos
Interferon gama/análise , Focalização Isoelétrica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Resinas Acrílicas/química , Eletroforese Capilar/métodos , Humanos , Interferon gama/genética , Deleção de Sequência/genética
5.
J Am Chem Soc ; 135(25): 9384-90, 2013 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-23734709

RESUMO

The extensive functional repertoire of heparin and heparan sulfate, which relies on their ability to interact with a large number of proteins, has recently emerged. To understand the forces that drive such interactions the binding of heparin to interferon-γ (IFNγ), used as a model system, was investigated. NMR-based titration experiments demonstrated the involvement of two adjacent cationic domains (D1: KTGKRKR and D2: RGRR), both of which are present within the carboxy-terminal sequence of the cytokine. Kinetic analysis showed that these two domains contribute differently to the interaction: D1 is required to form a complex and constitutes the actual binding site, whereas D2, although unable to associate with heparin by itself, increased the association rate of the binding. These data are consistent with the view that D2, through nonspecific electrostatic forces, places the two molecules in favorable orientations for productive binding within the encounter complex. This mechanism was supported by electrostatic potential analysis and thermodynamic investigations. They showed that D1 association to heparin is driven by both favorable enthalpic and entropic contributions, as expected for a binding sequence, but that D2 gives rise to entropic penalty, which opposes binding in a thermodynamic sense. The binding mechanism described herein, by which the D2 domain kinetically drives the interaction, has important functional consequences and gives a structural framework to better understand how specific are the interactions between proteins and heparin.


Assuntos
Aminoácidos/química , Heparitina Sulfato/química , Interferon gama/química , Proteínas/química , Aminoácidos/genética , Sítios de Ligação , Interferon gama/genética , Mutação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...