Galectins are proteins of the family of human lectins. By binding terminal galactose units of cell surface glycans, they moderate biological and pathological processes such as cell signaling, cell adhesion, apoptosis, fibrosis, carcinogenesis, and metabolic disorders. The binding of monovalent glycans to galectins is usually relatively weak. Therefore, the presentation of carbohydrate ligands on multivalent scaffolds can efficiently increase and/or discriminate the affinity of the glycoconjugate to different galectins. A library of glycoclusters and glycodendrimers with various structural presentations of the common functionalized N-acetyllactosamine ligand was prepared to evaluate how the mode of presentation affects the affinity and selectivity to the two most abundant galectins, galectin-1 (Gal-1) and galectin-3 (Gal-3). In addition, the effect of a one- to two-unit carbohydrate spacer on the affinity of the glycoconjugates was determined. A new design of the biolayer interferometry (BLI) method with specific AVI-tagged constructs was used to determine the affinity to galectins, and compared with the gold-standard method of isothermal titration calorimetry (ITC). This study reveals new routes to low nanomolar glycoconjugate inhibitors of galectins of interest for biomedical research.
- Klíčová slova
- Biolayer interferometry, Carbohydrate, Click chemistry, Galectin, Glycoconjugate, Multivalency, Transglycosylation,
- MeSH
- galektiny * metabolismus MeSH
- glykokonjugáty * farmakologie chemie MeSH
- lidé MeSH
- ligandy MeSH
- polysacharidy metabolismus MeSH
- sacharidy chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- galektiny * MeSH
- glykokonjugáty * MeSH
- ligandy MeSH
- polysacharidy MeSH
- sacharidy MeSH
Glycan metabolism balance is critical for cell prosperity, and macromolecule glycosylation is essential for cell communication, signaling and survival. Thus, glycotherapy may be a potential cancer treatment. The aim of the present study was to determine whether combined synthetic glycoconjugates (GCs) induce changes in gene expression that alter the survival of colon cancer cells. The current study evaluated the effect of the GCs N‑acetyl‑D‑glucosamine modified polyamidoamine dendrimer and calix[4]arene scaffold on cancer cell proliferation, apoptosis, invasion and sensitivity to immune cell‑mediated killing. Using reverse transcription‑quantitative polymerase chain reaction, the expression of genes involved in the aforementioned processes was measured. It was determined that GCs reduce the expression of the glucosaminyltransferases Mgat3 and Mgat5 responsible for surface glycosylation and employed components of the Wnt signaling pathway Wnt2B and Wnt9B. In addition, the calix[4]arene‑based GC reduced cell colony formation; this was accompanied by the downregulation of the metalloproteinase Mmp3. By contrast, the dendrimer‑based GC affected the expression of the glucose transporter components Sglt1 and Egfr1. Therefore, to the best of our knowledge, the present study is the first to reveal that N‑acetyl‑D‑glucosamine‑dendrimer/calix[4]arene GCs alter mRNA expression in a comprehensive way, resulting in the reduced malignant phenotype of the colon cancer cell line HT‑29.
- Klíčová slova
- N-acetyl-glucosaminyl-transferase, WNT, colon cancer cell, glycosylation, polyamidoamine dendrimer, calix[4]arene,
- MeSH
- apoptóza genetika MeSH
- buňky HT-29 MeSH
- glukosa metabolismus MeSH
- glykokonjugáty farmakologie MeSH
- lidé MeSH
- molekuly buněčné adheze genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorové kmenové buňky účinky léků metabolismus MeSH
- nádory tračníku genetika metabolismus MeSH
- proliferace buněk MeSH
- proteiny usnadňující transport glukosy genetika MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- stanovení celkové genové exprese MeSH
- testy nádorových kmenových buněk MeSH
- transkriptom MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glukosa MeSH
- glykokonjugáty MeSH
- molekuly buněčné adheze MeSH
- proteiny usnadňující transport glukosy MeSH
Recognition of glycosylation patterns is one of the basic features of innate immunity. Ability of C-type lectin-like receptors such as NKR-P1 to bind saccharide moieties has become recently a controversial issue. In the present study, binding assay with soluble fluorescently labeled recombinant rat NKR-P1A and mouse NKR-P1C proteins revealed apparently no affinity to the various neoglycoproteins. Lack of functional linkage between NKR-P1 and previously described saccharide binder was supported by the fact, that synthetic N-acetyl-D-glucosamine octabranched dendrimer on polyamidoamine scaffold (GN8P) did not change gene expression of NKR-P1 isoforms in C57BL/6 and BALB/c mice divergent in the NK gene complex (both in vitro and in vivo). Surprisingly, N-acetyl-D-glucosamine-coated tetrabranched polyamido-amine dendrimer specifically binds to NKT cells and macrophages but not to NK cells (consistently with changes in cytokine patterns). Despite the fact that GN8P has been tested as an immunomodulator in anti-cancer treatment animal models for many years, surprisingly no changes in cytokine profiles in serum relevant to anti-cancer responses using B16F10 and CT26 harboring mouse strains C57BL/6 and BALB/c are observed. Our results indicate possible indirect involvement of NK cells in GN8P mediated immune responses.
- Klíčová slova
- Anti-tumor immunity, C-type lectin related protein, Carbohydrate dendrimer, Clr, GN4P-A: GlcNAc4-PAMAM-ATTO 565, GN4P-NH(2)-GlcNAc(4)-PAMAM, GN4P: GlcNAc4-PAMAM, GN8P: GlcNAc8-PAMAM, GlcNAc, Gzmb, Macrophages, N-acetyl-d-glucosamine, N-acetyl-d-glucosamine-coated octabranched polyamidoamine dendrimer, N-acetyl-d-glucosamine-coated tetrabranched polyamidoamine dendrimer, N-acetyl-d-glucosamine-coated tetrabranched polyamidoamine dendrimer fluorescently labeled with ATTO 565, N-acetyl-d-glucosamine-coated tetrabranched polyamidoamine dendrimer with free NH(2) group, NK cells, NKG2D, NKR-P1, NKR-P1 receptors, NKT cells, PAMAM dendrimer, PMA, Prf, SBA, SMC, granzyme B, natural killer group 2, member D, natural killer receptor protein 1, perforin, phorbol 12-myristate 13-acetate, polyamidoamine dendrimer, soybean agglutinin, spleen mononuclear cell,
- MeSH
- acetylglukosamin imunologie metabolismus MeSH
- buňky NK imunologie metabolismus MeSH
- dendrimery metabolismus MeSH
- experimentální nádory farmakoterapie genetika imunologie MeSH
- exprese genu účinky léků imunologie MeSH
- glykokonjugáty imunologie metabolismus farmakologie MeSH
- interferon gama krev genetika imunologie MeSH
- krysa rodu Rattus MeSH
- kultivované buňky MeSH
- lektinové receptory NK-buněk - podrodina B genetika imunologie metabolismus MeSH
- lektiny typu C genetika imunologie metabolismus MeSH
- makrofágy imunologie metabolismus MeSH
- myši inbrední BALB C MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- NKT buňky imunologie metabolismus MeSH
- oligosacharidy imunologie metabolismus MeSH
- polyaminy imunologie metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protein - isoformy genetika imunologie metabolismus MeSH
- průtoková cytometrie MeSH
- slezina cytologie imunologie metabolismus MeSH
- TNF-alfa krev genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- acetylglukosamin MeSH
- dendrimery MeSH
- glykokonjugáty MeSH
- interferon gama MeSH
- lektinové receptory NK-buněk - podrodina B MeSH
- lektiny typu C MeSH
- oligosacharidy MeSH
- Poly(amidoamine) MeSH Prohlížeč
- polyaminy MeSH
- protein - isoformy MeSH
- TNF-alfa MeSH
We investigated the role of natural killer (NK) cells and CD161, their primary C-type-lectin-like receptor in rheumatoid arthritis (RA). Samples were compared with healthy donors (HD), dermatomyositic (DM), polymyositic (PM), and osteoarthritic (OA) patients. RA, PM, and DM NK cell cytotoxicities significantly decreased relative to the HD and OA NK cells (p<0.0001). These results correlated with an increased expression of NK cell inhibitory receptor CD161, in active disease RA patients. We demonstrated that NK cells are able to respond to mutated citrullinated vimentin (MCV), an RA-specific autoantigen, leading to increases in both PAD4 enzyme and CD161 mRNA expression. MGAT5 glycosidase involvement was detected in GlcNAc metabolism within the synoviocytes of RA patients. Our findings reveal a functional relationship between CD161 expression and NK cell cytotoxicity as well as reactivity to glycans and MCV, thus providing new insight into the pathogenesis of RA and confirming the involvement of surface glycosylation.
- MeSH
- autoantigeny farmakologie MeSH
- autoimunitní nemoci imunologie MeSH
- autoprotilátky krev imunologie MeSH
- buňky NK cytologie účinky léků imunologie metabolismus MeSH
- cytotoxicita imunologická účinky léků imunologie MeSH
- dermatomyozitida imunologie MeSH
- dospělí MeSH
- exprese genu účinky léků genetika MeSH
- glukokortikoidy terapeutické užití MeSH
- glykokonjugáty farmakologie MeSH
- hydrolasy genetika MeSH
- imunosupresiva terapeutické užití MeSH
- lektinové receptory NK-buněk - podrodina B agonisté genetika metabolismus MeSH
- leukocyty mononukleární cytologie účinky léků imunologie metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- N-acetylglukosaminyltransferasy genetika MeSH
- osteoartróza imunologie MeSH
- peptidylarginindeiminasa typu 4 MeSH
- peptidylarginindeiminasy MeSH
- počet buněk MeSH
- polymyozitida imunologie MeSH
- polysacharidy farmakologie MeSH
- revmatoidní artritida diagnóza farmakoterapie imunologie MeSH
- senioři MeSH
- synoviální tekutina cytologie metabolismus MeSH
- vimentin farmakologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase MeSH Prohlížeč
- autoantigeny MeSH
- autoprotilátky MeSH
- glukokortikoidy MeSH
- glykokonjugáty MeSH
- hydrolasy MeSH
- imunosupresiva MeSH
- lektinové receptory NK-buněk - podrodina B MeSH
- N-acetylglukosaminyltransferasy MeSH
- PADI4 protein, human MeSH Prohlížeč
- peptidylarginindeiminasa typu 4 MeSH
- peptidylarginindeiminasy MeSH
- polysacharidy MeSH
- vimentin MeSH
1. Haemagglutinating activity (HA) was found and characterized in lysate of the spirochete Borrelia recurrentis. 2. The highest HA was observed using native rabbit red blood cells (RBC), especially oxidized rabbit RBC. 3. In a haemagglutination inhibition test the HA showed an affinity with monosaccharides D-glucosamine, D-galactosamine and N-acetyl-D-mannosamine and several glycoproteins and polysaccharides. 4. An inhibitory effect was also achieved by mouse monoclonal antibody H 9724, polyclonal antibodies from B. recurrentis infected mice and with rabbit polyclonal antibodies directed against B. recurrentis HA. 5. B. recurrentis proteins components of 29, 33, 41 and 55 kDa were identified by immunoblotting as structural subunits of complex binding activity of a new agglutinin.
- MeSH
- Borrelia imunologie MeSH
- galaktosamin farmakologie MeSH
- glukosamin farmakologie MeSH
- glykokonjugáty farmakologie MeSH
- hemaglutinační testy MeSH
- hemaglutininy chemie izolace a purifikace metabolismus MeSH
- hexosaminy farmakologie MeSH
- imunoblotting MeSH
- králíci MeSH
- molekulová hmotnost MeSH
- monoklonální protilátky MeSH
- testy inhibice hemaglutinace MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- galaktosamin MeSH
- glukosamin MeSH
- glykokonjugáty MeSH
- hemaglutininy MeSH
- hexosaminy MeSH
- monoklonální protilátky MeSH
- N-acetylmannosamine MeSH Prohlížeč
