10 research outputs found
Preparation of the C-ribose-labeled 2′--TOM protected ribonucleoside phosphoramidites –
<p><b>Copyright information:</b></p><p>Taken from "Short, synthetic and selectively C-labeled RNA sequences for the NMR structure determination of protein–RNA complexes"</p><p>Nucleic Acids Research 2006;34(11):e79-e79.</p><p>Published online 28 Jun 2006</p><p>PMCID:PMC1904103.</p><p>© 2006 The Author(s)</p> Abbreviations: Ac = acetyl, Bz = benzoyl, Ibu = isobutyryl, CE = cyanoethyl, DMT = (4,4′-dimethoxy)trityl, TOM = (triisopropylsilyl)oxymethyl. Reagents and conditions: () Adapted from Saito (), detailed procedure in Supplementary Data: 1. FeCl, MgSO, acetone, 20°; 2. pyridinium dichromate, AcO, CHCl, reflux; 3. HIO, THF, 20°; 4. NaBH, THF/EtOH 1:1, 20°; 5. BzCl, pyridine, 20°; 6. AcO, AcOH, HSO, 20°. () 6-Chloro--isobutyrylpurine-2-amine, ,-bis(trimethylsilyl)acetamide (BSA), MeSiOTf, 1,2-dichloroethane, 65°. () 1. Allyl alcohol, DABCO, DBU, 20°; 2. NaOH, THF/MeOH/HO, 0°; 3. DMT-Cl, pyridine, 20°. () Pd(PhP), HNEt, PPh, CHCl, 20°. () Synthesis of : 1. -benzoyladenine, BSA, SnCl, 1,2-dichloroethane, 65°; 2. NaOH, THF/MeOH/HO, 0°; 3. DMT-Cl, pyridine, 20°; synthesis of : 1. uracil, BSA, MeSiOTf, MeCN, 60°; 2. MeNH, EtOH, 20°; 3. DMT-Cl, pyridine, 20°. () BuSnCl, PrNEt, TOM-Cl, 1,2-dichloroethane, 80° according to (). () 1. AcO, DMAP, pyridine 25°; 2. 4-chlorophenyl phosphorodichloridate, 1-1,2,4-triazole, PrNEt, MeCN, 4° → 20°; 3. aqueous NH, dioxane/MeCN, 20°; 4. NaOH, THF/MeOH/HO, 4° 5. AcO, DMF, 20°, according to (). () 2-Cyanoethyldiisopropylphosphoramidochloridite, PrNEt, CHCl, 20°, according to ()
Refining the specificity of the CCL2 lectin.
<p>(A) The chemical and schematic structure of the fucosylated chitobiose (GlcNAcβ1,4[Fucα1,3]GlcNAc-spacer) that was used as ligand for binding studies and structure determination. Indicated is also the B face that is defined as the face on which the carbons are numbered in an anticlockwise order <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706-Taylor1" target="_blank">[69]</a>. (B) Chemical shift deviations upon complex formation at a protein concentration of 0.4 mM at pH 5.7. Overlay of <sup>15</sup>N-HSQC spectra of free CCL2 (blue) and CCL2 bound to one equivalent of fucosylated chitobiose (red). (C) Titration of the amide signal of T111 in CCL2 with fucosylated chitobiose using <sup>15</sup>N-HSQC spectra. The protein∶ligand ratio is displayed on the left. (D) Plot of the chemical shift differences between free and bound CCL2 ( δ = [ δ<sub>HN</sub><sup>2</sup>+(δ<sub>N</sub>/R<sub>scale</sub>)<sup>2</sup> ]<sup>1/2</sup>, R<sub>scale</sub> = 5).</p
Carbohydrate-binding dependent biotoxicity of CCL2.
<p>(A) Schematic representation of N-glycan structures in plants, insects and nematodes. Upper panel, left: Typical paucimannosidic plant N-glycan, highly abundant in HRP. Upper panel right: Fucosylated paucimannosidic N-glycan present in <i>D. melanogaster</i>. Lower panel: Fucose biosynthesis and N-glycan structure in <i>C. elegans</i>. Genes coding for enzymes involved in the fucose biosynthesis (lower panel, left) and fucose transfer to the core of N-glycans in <i>C. elegans</i> (lower panel, right) are indicated in dashed boxes. (B) Toxicity of recombinant <i>E. coli</i> expressing CCL2 (black bars) towards <i>C. elegans</i> wildtype (N2) and various fucosylation mutants. Error bars indicate standard errors of the mean. Asterisks (*) show cases where all data were 0. Significant differences were observed between the vector control and CCL2 for N2 (n = 10, p = 0.013), <i>fut-1(ok892)</i> (n = 10, p = 0.013) and <i>fut-6(ok475)</i> (n = 10, p = 0.013) worms, but not for <i>bre-1(ye4)</i> (n = 10, p = 0.329) or <i>fut-6(ok475)fut-1(ok892)</i> (n = 10, p = 0.329). (C) Fluorescence microscopy of <i>C. elegans</i> feeding on <i>E. coli</i> expressing a dTomato-CCL2 fusion protein, showing the grinder and anterior part of the intestine. (D) Toxicity of purified CCL2 towards <i>D. melanogaster</i> quantified as number of developed pupae (gray bars) or flies (black bars). BSA was included as control. Error bars indicate standard errors of the mean. Development of pupae and flies treated with CCL2 were significantly different from the control (pupae: n = 10, p = 0.013; flies: n = 10, p = 0.013). (E) Toxicity of <i>E. coli</i> expressing different CCL2 variants with mutations in residues involved in carbohydrate binding towards <i>C. elegans</i> wildtype (N2). Vector control and CCL2 wildtype (WT) were included as controls. Asterisks (*) show cases where all data were 0. Error bars indicate standard error of the mean. W78A, Y92A and W94A were significantly different from WT control (n = 10, p = 0.013), whereas L87A, N91A, V93A were not (n = 10, p = 1.0).</p
Potential intermolecular protein–carbohydrate hydrogen bonds based on the orientations and positions of the carbohydrate in the complex structure.
<p>Note that no intermolecular hydrogen bond constraints were used during the calculations.</p
Sequence conservation among CCL2-like proteins and comparison to two typical representatives of fungi and plants.
<p>Sequence alignment of several fungal and plant R-type lectins. CCL2_A: CCL2 of <i>C. cinerea</i> strain AmutBmut; CCL2_O: CCL2 of <i>C. cinerea</i> strain Okayama7; CCL1_A: CCL1 of <i>C. cinerea</i> strain AmutBmut; CCL1_O: CCL1 of <i>C. cinerea</i> strain Okayama7; PP_L1: <i>Postia placenta</i> lectin 1 (Pospl1_130016); PP_L2: <i>Postia placenta</i> lectin 2 (Pospl1_121916); SL_L1: <i>Serpula lacrymans</i> lectin 1 (SerlaS7_144703); CP_L1: <i>Coniophora puteana</i> lectin 1 (Conpu1_119225); PO_L1: <i>Pleurotus ostreatus</i> lectin 1 (PleosPC9_89828); PO_L2: <i>Pleurotus ostreatus</i> lectin 2 (PleosPC15_1043947); PO_L3: <i>Pleurotus ostreatus</i> lectin 3 (PleosPC9_64199); PO_L4: <i>Pleurotus ostreatus</i> lectin 4 (PleosPC15_1065820); DS_L1: <i>Dicomitus squalis</i> lectin 1 (Dicsq1); AO_L1: <i>Arthrobotrys oligospora</i> lectin 1 (s00075g2); LB_L1: <i>Laccaria bicolor</i> lectin 1 (Lbic_330799); LB_L2: <i>Laccaria bicolor</i> lectin 2 (Lbic_327918); MOA: <i>Marasmius oreades</i> agglutinin; SNA-II: <i>Sambucus nigra</i> agglutinin/ribosome inactivating protein type II. The distantly related canonical R-type lectins MOA (fungal, 14% sequence identity) and SNA-II (plant, 13% sequence identity) were included in the alignment based on comparison of their 3D structures <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706-Grahn2" target="_blank">[70]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706-Maveyraud1" target="_blank">[71]</a>. The Clustal X color scheme was used. Residues involved in the carbohydrate recognition are indicated at the bottom for CCL2, MOA and SNA-II. The secondary structure of CCL2 and the conservation is indicated as well. The alignment was generated with Jalview <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706-Waterhouse1" target="_blank">[72]</a>.</p
Solution structure of the CCL2 lectin in the absence of a ligand determined by NMR spectroscopy.
<p>The side (A) and top (B) view of the most representative structure out of 20 structures is shown. The three pseudo symmetric sections of the β-trefoil fold corresponding to residues S9–N60, S61–S100 and G101–V142 are colored green, yellow and orange, respectively. Characteristic regions are labeled according to Renko et al. for better orientation <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706-Renko1" target="_blank">[26]</a>. (C) Chemical shift deviations mapped on the structure of CCL2 in the same orientation as in A. Chemical shifts of residues in red experience a combined NH chemical shift deviation >0.4 ppm, for residues in pink >0.15 ppm. (D) Secondary structure and subdomain borders displayed on the protein sequence. The same color code as in A and B is used. Bold residues are forming the hydrophobic core of the protein.</p
Binding of CCL2 wild-type to different carbohydrates and CCL2 variants to GlcNAcβ1,4[Fucα1,3]GlcNAcβ1-sp (sp: spacer O-[CH<sub>2</sub>]<sub>5</sub>COOH) measured with isothermal titration calorimetry and NMR spectroscopy at 299K.
a<p>nd: not determined.</p>b<p>The increased affinity of N91A might be an artifact caused by interaction of the artificial carbohydrate spacer O-(CH<sub>2</sub>)<sub>5</sub>-COOH with residue 91. Whereas the spacer might sterically clash with N91, Ala in this position could form favorable van-der-Waals interactions. In the case of natural N-glycans, where the reducing GlcNAc is linked to Asn of a glycoprotein projecting away from CCL2 (upper right corner of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat-1002706-g005" target="_blank">Figure 5D</a>), Asn is likely to be favored at this position of CCL2 due to the potential formation of H-bonds.</p
NMR structure determination statistics of CCL2 in the free form and in complex with the fucosylated chitobiose (GlcNAcβ1,4[Fucα1,3]GlcNAc-spacer, the spacer [CH<sub>2</sub>]<sub>5</sub>COOH was truncated in the structure calculations to a methyl group.).
*<p>Phi values for prolines were omitted.</p>**<p>Pairwise r.m.s. deviation was calculated among 20 refined structures.</p
Thermodynamic binding parameters.
<p>(A) ITC experiment of wild type CCL2 binding to GlcNAcβ1,4[Fucα1,3]GlcNAc-spacer. The raw calorimetric output is shown on top, the fitted binding isotherm at the bottom. The protein concentration in the cell was 70 µM, carbohydrate concentration in the syringe was 2.4 mM. (B) Thermodynamic binding parameters of CCL2 (in red) in comparison to other lectins with a focus on high affinity binding. Anti Le<sup>X</sup> Fab: Fab fragment of the monoclonal antibody 291-2G3-4; ConA: concavalin A from jack bean seeds (<i>Canavalia ensiformis</i>); CTB: cholera-toxin B subdomain; GS4: <i>Griffonia simplicifolia</i> lectin 4; MOA: <i>Marasmius oreades</i> agglutinin; RSL: <i>Ralstonia solanacearum</i> fucose-binding lectin; TeNT: tetanus neurotoxin; WBA II: winged bean (<i>Psophocarpus tetragonolobus</i>) acidic agglutinin. For simplicity, lectins that use Ca<sup>2+</sup> for carbohydrate recognition are not displayed. Details for each correlation are found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002706#ppat.1002706.s019" target="_blank">Table S6</a>. Data points in blue are discussed in the text.</p
Isolation and differential expression of <i>C. cinerea</i> CCL1 and CCL2.
<p>(A) Specific binding of CCL2 to horseradish peroxidase (HRP). Coomassie-stained SDS-PAGE showing Input, Flow through and Bound (Beads) fractions of a soluble protein extract from <i>C. cinerea</i> fruiting bodies upon affinity-chromatography using immobilized HRP. The Bound fraction was released by boiling the HRP-sepharose beads in Lämmli sample buffer. The loaded protein amount of the Bound fraction (Beads) corresponds to two equivalents of Input and Flow through fractions. Sizes of the marker proteins are indicated. (B) Immunoblot comparing expression levels of CCL2 between vegetative mycelium and fruiting bodies of <i>C. cinerea</i>. Equal amounts of total protein were loaded in each lane. A polyclonal antiserum raised in rabbits against purified CCL2 was used for detection. (C) Comparisons of relative expression ratio (or fold up-regulation) of the genes encoding CCL1 and CCL2 by qRT-PCR in fruiting bodies relative to vegetative mycelium. Error bars represent standard deviation of the mean.</p