RESUMO
Protein O-glycosylation is one of the most diverse post-translational modifications. A critical step in the analysis of O-glycomes is the release of glycans from glycoconjugates. Current release methods rely mainly on ß-elimination, which can result in peeling reactions and loss of base-sensitive functionalities leading to misidentification of glycans. To address this challenge, well-defined synthetic glycopeptides were used to establish a robust workflow for the oxidative release of O-glycans suitable for glycomics. Treatment of O-glycopeptides with neutralized hypochlorite resulted in the selective formation of lactic/glycolic acid glycosides, thereby retaining unique information of the parent amino acid (serine/threonine) that is lost by ß-elimination. It locks the glycan in a closed ring configuration, thereby preventing peeling, and furthermore, the carboxylate of the anomeric tag promotes ionization in negative ion mode mass spectrometry, thereby increasing signal intensities. Labile modifications such as sialic acids, sulfates, and acetyl esters are maintained during the release procedure. The promise of the approach was demonstrated by the analysis of O-glycans from bovine submaxillary mucin, which identified mono- and di-O-acetylated sialoglycans as well as previously undetected tri-O-acetylated and sulfated glycans. The use of well-defined glycopeptide standards made it also possible to identify reaction intermediates, which in turn allowed us to postulate a reaction mechanism for oxidative O-glycan release under neutral conditions.
Assuntos
Glicoproteínas , Polissacarídeos , Animais , Bovinos , Glicoproteínas/química , Polissacarídeos/química , Glicosilação , Glicopeptídeos/química , Estresse OxidativoRESUMO
The fucosylation of glycans leads to diverse structures and is associated with many biological and disease processes. The exact determination of fucoside positions by tandem mass spectrometry (MS/MS) is complicated because rearrangements in the gas phase lead to erroneous structural assignments. Here, we demonstrate that the combined use of ion-mobility MS and well-defined synthetic glycan standards can prevent misinterpretation of MS/MS spectra and incorrect structural assignments of fucosylated glycans. We show that fucosyl residues do not migrate to hydroxyl groups but to acetamido moieties of N-acetylneuraminic acid as well as N-acetylglucosamine residues and nucleophilic sites of an anomeric tag, yielding specific isomeric fragment ions. This mechanistic insight enables the characterization of unique IMS arrival-time distributions of the isomers which can be used to accurately determine fucosyl positions in glycans.
Assuntos
Fucose/química , Polissacarídeos/química , Bibliotecas de Moléculas Pequenas/química , Acetilglucosamina/química , Gases/química , Íons/química , Isomerismo , Espectrometria de Massas , Estrutura Molecular , Ácido N-Acetilneuramínico/químicaRESUMO
O-glycosylation is a common post-translational modification that is essential for the defensive properties of mucus barriers. Incomplete and altered O-glycosylation is often linked to severe diseases, such as cancer, cystic fibrosis, and chronic obstructive pulmonary disease. Originating from a nontemplate-driven biosynthesis, mucin-type O-glycan structures are very complex. They are often present as heterogeneous mixtures containing multiple isomers. Therefore, the analysis of complex O-glycan mixtures usually requires hyphenation of orthogonal techniques such as liquid chromatography (LC), ion mobility spectrometry, and mass spectrometry (MS). However, MS-based techniques are mainly qualitative. Moreover, LC separation of O-glycans often lacks reproducibility and requires sophisticated data treatment and analysis. Here we present a mucin-type O-glycomics analysis workflow that utilizes hydrophilic interaction liquid chromatography for separation and fluorescence labeling for detection and quantification. In combination with mass spectrometry, a detailed analysis on the relative abundance of specific mucin-type O-glycan compositions and features, such as fucose, sialic acids, and sulfates, is performed. Furthermore, the average number of monosaccharide units of O-glycans in different samples was determined. To demonstrate universal applicability, the method was tested on mucins from different tissue types and mammals, such as bovine submaxillary mucins, porcine gastric mucins, and human milk mucins. To account for day-to-day retention time shifts in O-glycan separations and increase the comparability between different instruments and laboratories, we included fluorescently labeled dextran ladders in our workflow. In addition, we set up a library of glucose unit values for all identified O-glycans, which can be used to simplify the identification process of glycans in future analyses.
RESUMO
The dense O-glycosylation of mucins plays an important role in the defensive properties of the mucus hydrogel. Aberrant glycosylation is often correlated with inflammation and pathology such as COPD, cancer, and Crohn's disease. The inherent complexity of glycans and the diversity in the O-core structure constitute fundamental challenges for the analysis of mucin-type O-glycans. Due to coexistence of multiple isomers, multidimensional workflows such as LC-MS are required. To separate the highly polar carbohydrates, porous graphitized carbon is often used as a stationary phase. However, LC-MS workflows are time-consuming and lack reproducibility. Here we present a rapid alternative for separating and identifying O-glycans released from mucins based on trapped ion mobility mass spectrometry. Compared to established LC-MS, the acquisition time is reduced from an hour to two minutes. To test the validity, the developed workflow was applied to sputum samples from cystic fibrosis patients to map O-glycosylation features associated with disease.
Assuntos
Mucinas , Espectrometria de Massas em Tandem , Humanos , Mucinas/metabolismo , Espectrometria de Massas em Tandem/métodos , Reprodutibilidade dos Testes , Polissacarídeos/química , GlicosilaçãoRESUMO
Keratan sulfate (KS) is a glycosaminoglycan that is widely expressed in the extracellular matrix of various tissue types, where it is involved in many biological processes. Herein, we describe a chemo-enzymatic approach to preparing well-defined KS oligosaccharides by exploiting the known and newly discovered substrate specificities of relevant sulfotransferases. The premise of the approach is that recombinant GlcNAc-6-O-sulfotransferases (CHST2) only sulfate terminal GlcNAc moieties to give GlcNAc6S that can be galactosylated by B4GalT4. Furthermore, CHST1 can modify the internal galactosides of a poly-LacNAc chain; however, it was found that a GlcNAc6S residue greatly increases the reactivity of CHST1 of a neighboring and internal galactoside. The presence of a 2,3-linked sialoside further modulates the site of modification by CHST1, and a galactoside flanked by 2,3-Neu5Ac and GlcNAc6S is preferentially sulfated over the other Gal residues. The substrate specificities of CHST1 and 2 were exploited to prepare a panel of KS oligosaccharides, including selectively sulfated N-glycans. The compounds and several other reference derivatives were used to construct a microarray that was probed for binding by several plant lectins, Siglec proteins, and hemagglutinins of influenza viruses. It was found that not only the sulfation pattern but also the presentation of epitopes as part of an O- or N-glycan determines binding properties.
RESUMO
Influenza viruses can move across the surface of host cells while interacting with their glycocalyx. This motility may assist in finding or forming locations for cell entry and thereby promote cellular uptake. Because the binding to and cleavage of cell surface receptors forms the driving force for the process, the surface-bound motility of influenza is expected to be dependent on the receptor density. Surface gradients with gradually varying receptor densities are thus a valuable tool to study binding and motility processes of influenza and can function as a mimic for local receptor density variations at the glycocalyx that may steer the directionality of a virus particle in finding the proper site of uptake. We have tracked individual influenza virus particles moving over surfaces with receptor density gradients. We analyzed the extracted virus tracks first at a general level to verify neuraminidase activity and subsequently with increasing detail to quantify the receptor density-dependent behavior on the level of individual virus particles. While a directional bias was not observed, most likely due to limitations of the steepness of the surface gradient, the surface mobility and the probability of sticking were found to be significantly dependent on receptor density. A combination of high surface mobility and high dissociation probability of influenza was observed at low receptor densities, while the opposite occurred at higher receptor densities. These properties result in an effective mechanism for finding high-receptor density patches, which are believed to be a key feature of potential locations for cell entry.
Assuntos
Influenza Humana , Orthomyxoviridae , Humanos , Receptores Virais/química , Receptores Virais/metabolismo , Receptores de Superfície Celular , Vírion/metabolismoRESUMO
Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor associated with the pathogenesis of autoimmune diseases. Allosteric inhibition of RORγt is conceptually new, unique for this specific nuclear receptor, and offers advantages over traditional orthosteric inhibition. Here, we report a highly efficient in silico-guided approach that led to the discovery of novel allosteric RORγt inverse agonists with a distinct isoxazole chemotype. The the most potent compound, 25 (FM26), displayed submicromolar inhibition in a coactivator recruitment assay and effectively reduced IL-17a mRNA production in EL4 cells, a marker of RORγt activity. The projected allosteric mode of action of 25 was confirmed by biochemical experiments and cocrystallization with the RORγt ligand binding domain. The isoxazole compounds have promising pharmacokinetic properties comparable to other allosteric ligands but with a more diverse chemotype. The efficient ligand-based design approach adopted demonstrates its versatility in generating chemical diversity for allosteric targeting of RORγt.