The C-type lectin-like receptors include the Nkrp1 protein family that regulates the activity of natural killer (NK) cells. Rat Nkrp1a was reported to bind monosaccharide moieties in a Ca2+-dependent manner in preference order of GalNac > GlcNAc >> Fuc >> Gal > Man. These findings established for rat Nkrp1a have been extrapolated to all additional Nkrp1 receptors and have been supported by numerous studies over the past two decades. However, since 1996 there has been controversy and another article showed lack of interactions with saccharides in 1999. Nevertheless, several high affinity saccharide ligands were synthesized in order to utilize their potential in antitumor therapy. Subsequently, protein ligands were introduced as specific binders for Nkrp1 proteins and three dimensional models of receptor/protein ligand interaction were derived from crystallographic data. Finally, for at least some members of the NK cell C-type lectin-like proteins, the "sweet story" was impaired by two reports in recent years. It has been shown that the rat Nkrp1a and CD69 do not bind saccharide ligands such as GlcNAc, GalNAc, chitotetraose and saccharide derivatives (GlcNAc-PAMAM) do not directly and specifically influence cytotoxic activity of NK cells as it was previously described.

• MeSH
• buňky NK * chemie   imunologie   metabolismus   MeSH
• CD antigeny * chemie   imunologie   metabolismus   MeSH
• diferenciační antigeny T-lymfocytů * chemie   imunologie   metabolismus   MeSH
• krysa rodu Rattus MeSH
• lektinové receptory NK-buněk - podrodina B * chemie   imunologie   metabolismus   MeSH
• lektiny typu C * chemie   imunologie   metabolismus   MeSH
• lidé MeSH
• oligosacharidy * chemie   imunologie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CD antigeny * MeSH
• CD69 antigen MeSH Prohlížeč
• diferenciační antigeny T-lymfocytů * MeSH
• KLRB1 protein, human MeSH Prohlížeč
• lektinové receptory NK-buněk - podrodina B * MeSH
• lektiny typu C * MeSH
• oligosacharidy * MeSH

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
• Názvy látek
• CD antigeny MeSH
• CD69 antigen MeSH Prohlížeč
• diferenciační antigeny T-lymfocytů MeSH
• Klrb1a protein, rat MeSH Prohlížeč
• lektiny typu C MeSH
• oligopeptidy MeSH
• polysacharidy MeSH
• receptory imunologické MeSH
• rekombinantní proteiny MeSH

The synthetic procedures for a large-scale preparation of o- and p-nitrophenyl 2-acetamido-2-deoxy-α-D-mannopyranoside are described. The synthetic pathway employs the glycosylation of phenol with ManNAc oxazoline, followed by nitration of the aromatic moiety yielding a separable mixture of the o- and p-nitrophenyl derivative in a 2:3 ratio.

• Klíčová slova
• glycosidase, glycosylation, nitrophenyl, oxazoline, α-ManNAc,
• Publikační typ
• časopisecké články MeSH

Fungal β-N-acetylhexosaminidases are enzymes that are used in the chemoenzymatic synthesis of biologically interesting oligosaccharides. The enzyme from Aspergillus oryzae was produced and purified from its natural source and crystallized using the hanging-drop vapour-diffusion method. Diffraction data from two crystal forms (primitive monoclinic and primitive tetragonal) were collected to resolutions of 3.2 and 2.4 Å, respectively. Electrophoretic and quantitative N-terminal protein-sequencing analyses confirmed that the crystals are formed by a complete biologically active enzyme consisting of a glycosylated catalytic unit and a noncovalently attached propeptide.

• MeSH
• Aspergillus oryzae enzymologie   MeSH
• beta-N-acetylhexosaminidasy chemie   metabolismus   MeSH
• glykosylace MeSH
• katalytická doména MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• beta-N-acetylhexosaminidasy MeSH

BACKGROUND: Fungal beta-N-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal beta-N-acetylhexosaminidase. The fungal beta-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined. RESULTS: The complete primary structure of the fungal beta-N-acetylhexosaminidase from Aspergillus oryzae CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the N-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate - chitobiose with a stable value of binding energy during the molecular dynamics simulation. CONCLUSION: Whereas the intracellular bacterial beta-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal beta-N-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and N-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys448 with Cys483 stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.

• MeSH
• Aspergillus oryzae enzymologie   MeSH
• beta-N-acetylhexosaminidasy chemie   izolace a purifikace   metabolismus   MeSH
• dimerizace MeSH
• glykosylace MeSH
• koncentrace vodíkových iontů MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• počítačová simulace * MeSH
• Ramanova spektroskopie metody   MeSH
• spektroskopie infračervená s Fourierovou transformací metody   MeSH
• stabilita enzymů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-N-acetylhexosaminidasy MeSH

NKR-P1 protein is an important activating receptor at the surface of the rat natural killer cells. GlcNAc and chitooligomers were identified as strong activation ligands in vitro and in vivo. Their clustering brings about increase of their affinity to the NKR-P1 by 3-6 orders. Here we describe novel methodology for preparation of neoglycoproteins based on BSA carrying the chitooligomers (n = 2-5). Further on we developed novel methodology of the coupling of glycosylamines via aromatic-SCN activated linker both to protein or synthetic cores. Inhibition studies of chitooligomer glycoconjugates with the NKR-P1 receptor show that our neoglycoproteins are very strong ligands with high binding affinity (-log IC(50) = 13-15). In analogy with our previous observations with GlcNAc clustered on protein or PAMAM backbones the synthetic chitooligomer clusters should provide considerably better ligands in the in vivo antitumor treatment.

• MeSH
• aminy chemie   MeSH
• antigeny povrchové metabolismus   MeSH
• buňky NK metabolismus   MeSH
• chitin chemie   MeSH
• chromatografie metody   MeSH
• glykoproteiny chemická syntéza   metabolismus   MeSH
• imunoblotting MeSH
• inhibiční koncentrace 50 MeSH
• isothiokyanatany chemie   MeSH
• krysa rodu Rattus MeSH
• kyselina N-acetylneuraminová metabolismus   MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C metabolismus   MeSH
• ligandy MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• sérový albumin hovězí chemie   MeSH
• skot MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• thioglykosidy chemická syntéza   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminy MeSH
• antigeny povrchové MeSH
• chitin MeSH
• glykoproteiny MeSH
• isothiokyanatany MeSH
• kyselina N-acetylneuraminová MeSH
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• ligandy MeSH
• sérový albumin hovězí MeSH
• thioglykosidy MeSH