The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A) was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-D-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.
- MeSH
- CD antigeny metabolismus MeSH
- diferenciační antigeny T-lymfocytů metabolismus MeSH
- krysa rodu rattus MeSH
- lektiny typu C metabolismus MeSH
- lidé MeSH
- oligopeptidy chemická syntéza farmakologie MeSH
- polysacharidy chemická syntéza farmakologie MeSH
- receptory imunologické metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This paper describes an efficient oxime ligation strategy to prepare multivalent conjugates wherein peptides alone or in combination with carbohydrate or oxime groups were coupled to a cyclopeptide scaffold. To demonstrate the versatility of this approach, two classes of conjugates have been prepared. In one class, we attached two or four peptide sequences to the cyclopeptide core together with free oxime groups, while the second class contains an additional substitution with four or two monosaccharides. The well-defined structure of these conjugates was confirmed by high-resolution mass spectrometry.
This work reveals new structural relationships in the complex process of the interaction between activation receptors of natural killer cells (rat NKR-P1, human CD69) and novel bivalent carbohydrate glycomimetics. The length, glycosylation pattern and linker structure of receptor ligands were examined with respect to their ability to precipitate the receptor protein from solution, which simulates the in vivo process of receptor aggregation during NK cell activation. It was found that di-LacdiNAc triazole compounds show optimal performance, reaching up to 100% precipitation of the present protein receptors, and achieving high immunostimulatory activities without any tendency to trigger activation-induced apoptosis. In the synthesis of the compounds tested, two enzymatic approaches were applied. Whereas a β-N-acetylhexosaminidase could only glycosylate one of the two acceptor sites available with yields below 10%, the Y284L mutant of human placental β1,4-galactosyltransferase-1 worked as a perfect synthetic tool, accomplishing even quantitative glycosylation at both acceptor sites and with absolute regioselectivity for the C-4 position. This work insinuates new directions for further ligand structure optimisation and demonstrates the strong synthetic potential of the mutant human placental β1,4-galactosyltransferase-1 in the synthesis of multivalent glycomimetics and glycomaterials.
- MeSH
- aktivace lymfocytů účinky léků imunologie MeSH
- beta-N-acetylhexosaminidasy metabolismus MeSH
- biomimetika metody MeSH
- buňky NK chemie účinky léků imunologie metabolismus MeSH
- CD antigeny imunologie metabolismus MeSH
- diferenciační antigeny T-lymfocytů imunologie metabolismus MeSH
- galaktosyltransferasy genetika metabolismus MeSH
- imunoprecipitace MeSH
- krysa rodu rattus MeSH
- lektiny typu C agonisté imunologie metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- molekulární mimikry MeSH
- mutace MeSH
- placenta enzymologie MeSH
- polysacharidy chemická syntéza farmakologie MeSH
- receptory buněk NK agonisté imunologie metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- těhotenství MeSH
- vazba proteinů účinky léků imunologie MeSH
- vazebná místa účinky léků imunologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We have recently identified a new class of high affinity ligands for CD69 leukocyte membrane receptor, carboxylated calixarenes. Of the three compounds investigated here, thiacalix[4]arene had the highest affinity for CD69 in direct binding assays, and proved to be the most specific inhibitor of CD69 identified so far in receptor precipitation and cellular activation experiments. Carboxylated calixarenes also proved effective at protection of CD69(high) lymphocytes from apoptosis triggered by a multivalent ligand or antibody. Thus, carboxylated calixarenes set a new paradigm for noncarbohydrate ligands for CD69 making them attractive for protection of killer cells in combined animal tumor therapies.
- MeSH
- apoptóza MeSH
- CD antigeny metabolismus MeSH
- diferenciační antigeny T-lymfocytů metabolismus MeSH
- kalixareny chemie metabolismus MeSH
- krysa rodu rattus MeSH
- kyseliny karboxylové chemie MeSH
- lektiny typu C metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- publikace stažené z tisku MeSH
Human placental beta1,4-galactosyltransferase-I (EC 2.4.1.38) transfers the galactosyl moiety from UDP-Gal to various GlcNAc or Glc acceptors in vivo. Here, we describe the construction of its Y284L mutant as a His(6)propeptide-catbeta4GalT1 construct, in which the Gal-transferase activity was totally abolished in favor of its GalNAc-transferase activity. We used this mutant in the synthesis of three mono- and bivalent LacdiNAc glycomimetics with good yields. These compounds proved to be powerful ligands of two activation receptors of natural killer cells, NKR-P1 and CD69. A synthetic bivalent tethered di-LacdiNAc is the best currently known precipitation agent for both of these receptors and has promising potential for the development of immunoactive glycodrugs.
- MeSH
- bakteriální proteiny metabolismus MeSH
- Campylobacter jejuni enzymologie MeSH
- CD antigeny metabolismus MeSH
- diferenciační antigeny T-lymfocytů metabolismus MeSH
- epimerázy sacharidů metabolismus MeSH
- galaktosyltransferasy genetika metabolismus MeSH
- glykokonjugáty biosyntéza chemická syntéza metabolismus MeSH
- laktosa analogy a deriváty biosyntéza chemická syntéza metabolismus MeSH
- lektinové receptory NK-buněk - podrodina B metabolismus MeSH
- lektiny typu C MeSH
- lidé MeSH
- mutace MeSH
- placenta enzymologie MeSH
- substrátová specifita MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- publikace stažené z tisku MeSH
A series of calixarenes substituted with 2-acetamido-2-deoxy-beta-D-glucopyranose linked by a thiourea spacer was prepared and tested for binding activity to heterogeneously expressed activation receptors of the rat natural killer cells NKR-P1, and the receptor CD69 (human NK cells, macrophages). In the case of NKR-P1, the binding affinity of beta-D-GlcNAc-substituted calixarenes carrying two or four sugar units was in a good agreement with the inhibitory potencies of the linear chitooligomers (chitobiose to chitotetraose) reported previously. The influence of GlcNAc substitution of the calixarene skeleton on binding affinity for CD69 receptor was more profound and the 5,11,17,23-tetrakis[N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-thioureido]-25,26,27,28-tetrapropoxycalix[4]arene (cone) (1) proved to be the best CD69 ligand identified to date. Lower GlcNAc substitution led to dramatic decrease of the binding activity (by about 1.5 order of magnitude per one GlcNAc unit). The immunostimulating activity results with the newly synthesized GlcNAc tetramers on calixarene scaffolds exhibited stimulation of natural cytotoxicity of human PBMC in concentrations 10(-4) and 10(-8)M. These calix-sugar compounds were superior to the previously tested PAMAM-GlcNAc(8)5.
- MeSH
- acetylglukosamin analogy a deriváty farmakologie MeSH
- aktivace lymfocytů účinky léků MeSH
- antitumorózní látky farmakologie chemická syntéza MeSH
- buňky NK imunologie účinky léků MeSH
- financování organizované MeSH
- glykokonjugáty chemická syntéza terapeutické užití MeSH
- kalixareny chemie MeSH
- kinetika MeSH
- leukocyty mononukleární účinky léků MeSH
- lidé MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- nádory farmakoterapie imunologie MeSH
- T-lymfocyty imunologie MeSH
- Check Tag
- lidé MeSH
