In the shadow of SARS-CoV-2, influenza seems to be an innocent virus, although new zoonotic influenza viruses evolved by mutations may lead to severe pandemics. According to WHO, there is an urgent need for better antiviral drugs. Blocking viral hemagglutinin with multivalent N-acetylneuraminic acid derivatives is a promising approach to prevent influenza infection. Moreover, dual inhibition of both hemagglutinin and neuraminidase may result in a more powerful effect. Since both viral glycoproteins can bind to neuraminic acid, we have prepared three series of amphiphilic self-assembling 2-thio-neuraminic acid derivatives constituting aggregates in aqueous medium to take advantage of their multivalent effect. One of the series was prepared by the azide-alkyne click reaction, and the other two by the thio-click reaction to yield neuraminic acid derivatives containing lipophilic tails of different sizes and an enzymatically stable thioglycosidic bond. Two of the three bis-octyl derivatives produced proved to be active against influenza viruses, while all three octyl derivatives bound to hemagglutinin and neuraminidase from H1N1 and H3N2 influenza types.
- MeSH
- chřipka lidská * farmakoterapie MeSH
- hemaglutininové glykoproteiny viru chřipky metabolismus MeSH
- hemaglutininy farmakologie MeSH
- kyselina N-acetylneuraminová farmakologie metabolismus MeSH
- kyseliny neuraminové MeSH
- lidé MeSH
- neuraminidasa metabolismus MeSH
- virus chřipky A, podtyp H1N1 * MeSH
- virus chřipky A, podtyp H3N2 MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In recent decades, it has become clear that most of human proteins are glycosylated and that protein glycosylation plays an important role in health and diseases. At present, simple, fast and inexpensive methods are sought for clinical applications and particularly for improved diagnostics of various diseases, including cancer. We propose a label- and reagent-free electrochemical method based on chronopotentiometric stripping (CPS) analysis and a hanging mercury drop electrode for the detection of interaction of sialylated protein biomarker a prostate specific antigen (PSA) with two important lectins: Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA). Incubation of PSA-modified electrode with specific SNA lectin resulted in an increase of CPS peak H of the complex as compared to this peak of individual PSA. By adjusting polarization current and temperature, PSA-MAA interaction can be either eliminated or distinguished from the more abundant PSA-SNA complex. CPS data were in a good agreement with the data obtained by complementary methods, namely surface plasmon resonance and fluorescent lectin microarray. It can be anticipated that CPS will find application in glycomics and proteomics.
Bile acids have been implicated in cholestatic liver damage, primarily due to their detergent effect on membranes and induction of oxidative stress. Gangliosides can counteract these harmful effects by increasing the rigidity of the cytoplasmic membrane. Induction of haem oxygenase (HMOX) has been shown to protect the liver from increased oxidative stress. The aim of this study was to determine the changes in the synthesis and distribution of liver gangliosides following bile duct ligation (BDL), and to assess the effects of HMOX both on cholestatic liver injury and ganglioside metabolism. Compared to controls, BDL resulted in a significant increase in total as well as complex gangliosides and mRNA expression of corresponding glycosyltransferases ST3GalV, ST8SiaI and B3GalTIV. A marked shift of GM1 ganglioside from the intracellular compartment to the cytoplasmic membrane was observed following BDL. Induction of oxidative stress by HMOX inhibition resulted in a further increase of these changes, while HMOX induction prevented this effect. Compared to BDL alone, HMOX inhibition in combination with BDL significantly increased the amount of bile infarcts, while HMOX activation decreased ductular proliferation. We have demonstrated that cholestasis is accompanied by significant changes in the distribution and synthesis of liver gangliosides. HMOX induction results in attenuation of the cholestatic pattern of liver gangliosides, while HMOX inhibition leads to the opposite effect.
- MeSH
- biologické markery metabolismus MeSH
- cholestáza enzymologie genetika metabolismus patologie MeSH
- cytoplazma metabolismus MeSH
- gangliosidy metabolismus MeSH
- hemová oxygenasa (decyklizující) metabolismus MeSH
- intracelulární membrány metabolismus MeSH
- játra enzymologie metabolismus patologie MeSH
- kyselina N-acetylneuraminová metabolismus MeSH
- ligace MeSH
- messenger RNA genetika metabolismus MeSH
- oxidační stres * MeSH
- potkani Wistar MeSH
- proliferace buněk MeSH
- tělesná hmotnost MeSH
- velikost orgánu MeSH
- žlučové cesty patologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Galactose-deficient O-glycans in the hinge region (HR) of immunoglobulin A1 (IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). O-Glycans of circulatory IgA1 consist of N-acetylgalactosamine (GalNAc) with a β1,3-linked galactose; both sugars may be sialylated. In patients with IgAN, α2,6-sialylated GalNAc is a frequent form of the galactose-deficient O-glycans. Prior analyses of IgA1-producing cells had indicated that α2,6-sialyltransferase II (ST6GalNAc-II) is likely responsible for sialylation of GalNAc of galactose-deficient IgA1, but direct evidence is missing. METHODS: We produced a secreted variant of recombinant human ST6GalNAc-II and an IgA1 fragment comprised of Cα1-HR-Cα2. This IgA1 fragment and a synthetic HR peptide with enzymatically attached GalNAc residues served as acceptors. ST6GalNAc-II activity was assessed in vitro and the attachment of sialic acid to these acceptors was detected by lectin blot and mass spectrometry. RESULTS: ST6GalNAc-II was active with both acceptors. High-resolution mass spectrometry analysis revealed that up to three sialic acid residues were added to the GalNAc residues of the HR glycopeptide. CONCLUSIONS: Our data provide direct evidence that ST6GalNAc-II can sialylate GalNAc of galactose-deficient IgA1. As serum levels of galactose-deficient IgA1 with sialylated glycoforms are increased in IgAN patients, our data explain the corresponding part of the biosynthetic pathway.
- MeSH
- autoantigeny imunologie MeSH
- galaktosa nedostatek MeSH
- glykosylace MeSH
- hmotnostní spektrometrie MeSH
- IgA nefropatie enzymologie imunologie patologie MeSH
- imunoglobulin A metabolismus MeSH
- kultivované buňky MeSH
- kyselina N-acetylneuraminová metabolismus MeSH
- lidé MeSH
- rekombinantní proteiny imunologie metabolismus MeSH
- sialyltransferasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
The Rho GTPase Rac1 is a multifunctional protein working through different effector pathways. The emerging physiological significance of glycanlectin recognition gives reason to testing the possibility for an influence of modulation of Rac1 expression on these molecular aspects. Using human colon adenocarcinoma (SW620) cells genetically engineered for its up- and down-regulation (Rac1+ and Rac1- cells) along with wild-type and mock-transfected control cells, the questions are addressed whether the presence of adhesion/growth-regulatory galectins and distinct aspects of cell surface glycosylation are affected. Proceeding from RT-PCR data to Western blotting after two-dimensional gel electrophoresis and flow cytofluorimetry with non-crossreactive antibodies against six members of this lectin family (i.e. galectins-1, -3, -4, -7, -8 and -9), a reduced extent of the presence of galectins-1, -7 and -9 was revealed in the case of Rac1 cells. Application of these six galectins as probes to determination of cell reactivity for human lectins yielded relative increases in surface labelling of Rac1- cells with galectins-1, -3 and -7. Examining distinct aspects of cell surface glycosylation with a panel of 14 plant/fungal lectins disclosed a decrease in α2,6-sialylation of N-glycans and an increase in PNA-reactive sites (i.e. non-sialylated core 1 O-glycans), two alterations known to favour reactivity for galectins-1 and -3. Thus, manipulation of Rac1 expression selectively affects the expression pattern within the galectin network at the level of proteins and distinct aspects of cell surface glycosylation.
- MeSH
- 2D gelová elektroforéza MeSH
- buněčná membrána metabolismus MeSH
- fenotyp MeSH
- galektiny genetika metabolismus MeSH
- glykosylace MeSH
- kyselina N-acetylneuraminová metabolismus MeSH
- lektiny metabolismus MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- nádory tračníku genetika metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- polysacharidy metabolismus MeSH
- průtoková cytometrie MeSH
- rac1 protein vázající GTP metabolismus MeSH
- regulace genové exprese u nádorů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
IgA nephropathy (IgAN), the most common primary glomerulonephritis, is characterized by renal immunodeposits containing IgA1 with galactose-deficient O-glycans (Gd-IgA1). These immunodeposits originate from circulating immune complexes consisting of anti-glycan antibodies bound to Gd-IgA1. As clinical disease onset and activity of IgAN often coincide with mucosal infections and dysregulation of cytokines, we hypothesized that cytokines may affect IgA1 O-glycosylation. We used IgA1-secreting cells derived from the circulation of IgAN patients and healthy controls and assessed whether IgA1 O-glycosylation is altered by cytokines. Of the eight cytokines tested, only IL-6 and, to a lesser degree, IL-4 significantly increased galactose deficiency of IgA1; changes in IgA1 O-glycosylation were robust for the cells from IgAN patients. These cytokines reduced galactosylation of the O-glycan substrate directly via decreased expression of the galactosyltransferase C1GalT1 and, indirectly, via increased expression of the sialyltransferase ST6GalNAc-II, which prevents galactosylation by C1GalT1. These findings were confirmed by siRNA knockdown of the corresponding genes and by in vitro enzyme reactions. In summary, IL-6 and IL-4 accentuated galactose deficiency of IgA1 via coordinated modulation of key glycosyltransferases. These data provide a mechanism explaining increased immune-complex formation and disease exacerbation during mucosal infections in IgAN patients.
- MeSH
- buněčné linie MeSH
- cytokiny * farmakologie MeSH
- dospělí MeSH
- galaktosa metabolismus nedostatek MeSH
- galaktosyltransferasy * metabolismus MeSH
- genový knockdown MeSH
- glykosylace účinky léků MeSH
- IgA nefropatie enzymologie patologie MeSH
- imunoglobulin A * metabolismus MeSH
- interleukin-4 farmakologie MeSH
- interleukin-6 farmakologie MeSH
- kyselina N-acetylneuraminová metabolismus MeSH
- lidé MeSH
- malá interferující RNA metabolismus MeSH
- polysacharidy metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
The presence of sialylated structures in tick organs was observed previously using lectin staining. Recently, we demonstrated the presence of sialylated N-glycans using mass spectrometry in tick salivary glands and the gut. However, we proposed a host (blood) origin for these glycans and mapped the transport of sialylated molecules from the gut to the salivary glands. In this report, we performed quantitation of whole sialic acid and of metabolically incorporated sialic acid (N-azido neuraminic acid) in Ixodes ricinus tick samples. We show that the majority of sialylated molecules in the adult tick originate in the host (blood) and are not synthesized by the tick. Similar results were observed for tick cell cultures. The almost complete absence of tick sialylated molecules and the specific transport and localization of host structures into the tick salivary glands and the saliva raises many questions on the role of these molecules in the physiology and, specifically, the blood-feeding of ticks.
- MeSH
- buněčné linie MeSH
- glykoproteiny krev metabolismus MeSH
- interakce hostitele a parazita * MeSH
- klíště metabolismus fyziologie MeSH
- kyselina N-acetylneuraminová metabolismus MeSH
- sliny metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We describe the detection of sialylated N-linked glycans in partially fed Ixodes ricinus tick females using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry. Sialylated glycans were detected in salivary glands as well as in tick guts and we propose the host origin of these structures. In addition, we mapped the transport of sialylated structures from the blood meal through the gut to the salivary glands using electron microscopy. Specific localization of sialylated glycans to basement membranes of salivary glands was observed. Finally, the influence of the sample preparation methods for electron microscopy on ultrastructure and immunogold labeling was evaluated.
- MeSH
- epitopy MeSH
- gastrointestinální trakt metabolismus MeSH
- imunohistochemie MeSH
- klíště metabolismus MeSH
- kyselina N-acetylneuraminová metabolismus MeSH
- malpighické trubice metabolismus MeSH
- polysacharidy metabolismus MeSH
- slinné žlázy metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- transmisní elektronová mikroskopie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- MeSH
- bazocelulární karcinom metabolismus patologie MeSH
- buněčná diferenciace MeSH
- financování organizované MeSH
- galektiny metabolismus MeSH
- kyselina N-acetylneuraminová metabolismus MeSH
- Langerhansovy buňky metabolismus patologie MeSH
- lektiny metabolismus MeSH
- lidé MeSH
- mezibuněčná komunikace MeSH
- molekuly buněčné adheze nervové MeSH
- nádory hlavy a krku metabolismus patologie MeSH
- rostlinné lektiny metabolismus MeSH
- spinocelulární karcinom metabolismus patologie MeSH
- vrozené poruchy metabolismu sacharidů MeSH
- Check Tag
- lidé MeSH
