Several reports have indicated that hypoxia, GLI, and connective tissue growth factor (CTGF) contribute to pulmonary fibrosis in idiopathic pulmonary fibrosis. We investigated the participation of mitogen-activated protein kinase kinase (MEK) kinase 1 (MEKK1)/MEK1/ERK1/GLI-1/2 and activator protein-1 (AP-1) signaling in hypoxia-induced CTGF expression in human lung fibroblasts. Hypoxia time-dependently increased CTGF expression, which was attenuated by the small interfering RNA (siRNA) of GLI-1 (GLI-1 siRNA) and GLI-2 (GLI-2 siRNA) in both human lung fibroblast cell line (WI-38) and primary human lung fibroblasts (NHLFs). Moreover, GLI-1 siRNA and GLI-2 siRNA attenuated hypoxia-induced CTGF-luciferase activity, and the treatment of cells with hypoxia induced GLI-1 and GLI-2 translocation. Furthermore, hypoxia-induced CTGF expression was reduced by an MEK inhibitor (PD98059), MEK1 siRNA, ERK inhibitor (U0126), ERK1 siRNA, and MEKK1 siRNA. Both PD98059 and U0126 significantly attenuated hypoxia-induced CTGF-luciferase activity. Hypoxia time-dependently increased MEKK1, ERK, and p38 MAPK phosphorylation. Moreover, SB203580 (a p38 MAPK inhibitor) also apparently inhibited hypoxia-induced CTGF expression. The treatment of cells with hypoxia induced ERK, GLI-1, or GLI-2 complex formation. Hypoxia-induced GLI-1 and GLI-2 translocation into the nucleus was significantly attenuated by U0126. In addition, hypoxia-induced ERK Tyr204 phosphorylation was impeded by MEKK1 siRNA. Moreover, hypoxia-induced CTGF-luciferase activity was attenuated by cells transfected with AP-1 site mutation in a CTGF construct. Exposure to hypoxia caused a time-dependent phosphorylation of c-Jun, but not of c-Fos. Chromatin immunoprecipitation (ChIP) revealed that hypoxia induced the recruitment of c-Jun, GLI-1, and GLI-2 to the AP-1 promoter region of CTGF. Hypoxia-treated cells exhibited an increase in α-smooth muscle actin (α-SMA) and collagen production, which was blocked by GLI-1 siRNA and GLI-2 siRNA. Overall, these data implied that the MEKK1/MEK1/ERK1/GLI-1/GLI-2, and AP-1 pathways mediated hypoxia-induced CTGF expression in human lung fibroblasts. Furthermore, GLI-1 and GLI-2 found to be involved in hypoxia-induced α-SMA and collagen expression. Idiopathic pulmonary fibrosis (IPF) is the most common and lethal form of all interstitial lung diseases (ILDs), with an estimated 5-year survival rate for approximately 20% for affected patients. Patients with IPF have a low quality of life because of dyspnea, chest tightness, and severe dry cough –4. AmongThe hedgehog signaling pathway is highly regulated because it plays a crucial role in embryonic development, tissue patterning, and organogenesis, whereas GLI proteins are the downstream transcriptional factors of this pathway , 10. HedCTGF, a CCN family member, is well known to be a crucial mediator in ILDs, including pulmonary fibrosis . In the Mitogen-activated protein kinase kinase kinase 1 (MEKK1) and ERK regulate chemotaxis, immunocyte recruitment, and inflammatory protein production, in addition to participating in the noncanonical regulation of GLI-1 and GLI-2 proteins . Studies5’-CUA CUG AUA CUC UGG GAU A-3’ (sense1) and 5’-GUC AUA CUC ACG CCU CGA A-3’ (sense2), in addition, GLI-2 siRNA, a mixture containing 2 specific GLI-2 siRNAs, was used with the following sequence: 5’-GAC AUG AGC UCC AUG CUC A-3’ (sense1) and 5’-CGA UUG ACA UGC GAC ACC A-3’ (sense2), as well as MEKK1 siRNA, a mixture containing 2 specific MEKK1 siRNAs, was used with the following sequence: 5’-CAA AGG UGC CAA UUU GCU A -3’ (sense1) and 5’-GGA AUU UCC UGC UGA AUU U -3’ (sense2), which were synthesized by Sigma . Antibodies specific for GLI-2 and α-SMA were obtained from Abcam . An antibody specific for MEK1 was purchased from GeneTex . A Collagen type 1 antibody was purchased from Rockland . All materials for western blotting were acquired from Bio-Rad . The CTGF promoter (-747/+214) luciferase plasmid was gifted by Dr. M.-L. Kuo , pcDNA was gifted by Dr. M.-C. Chen , and pBK-CMV-Lac Z was gifted by Dr. W-W. Lin .A human lung fibroblast cell line (WI-38) was purchased from American Type Culture Collection . The primary normal human lung fibroblasts (NHLFs) were purchased from Lonza . U0126, PD98059, and SB203580 were obtained from Calbiochem-Novabiochem . Lipofectamine 3000 and Lipofectamine Plus reagent, and a minimum essential medium were obtained from Invitrogen Life Technologies . The CTGF ELISA kit was purchased from Assay BioTech . An α-tubulin antibody was acquired from Transduction Laboratories . A GLI-1 antibody was obtained from R&D systems . The siRNAs of MEK1 and ERK1, as well as antibodies specific for the ERK Tyr204 phosphorylation site, ERK; the c-Fos Ser374 phosphorylation site, c-Fos; the c-Jun Ser63 phosphorylation site, c-Jun; the p38 Tyr182 phosphorylation site, p38α; CTGF; and rabbit polyclonal IgG, as well as antirabbit, antimouse, and antigoat IgG-conjugated horseradish peroxidase were acquired from Santa Cruz Biotechnology . GLI-1 siRNA, a mixture containing 2 specific GLI-1 siRNAs, was used with the following sequence: 2 hypoxia, which was achieved using the ProOx model 110 chamber at 37°C, flushed with a gas mixture of 5% CO2/95% N2, purchased from BioSpherix . The oxygen concentration within the chamber was maintained at 1% by using a ProOx 110 oxygen regulator (BioSpherix).A human lung fibroblast (WI-38) cell culture was prepared as described previously . In brie2 hypoxia for 0–48 h. Afterward, the cell starvation medium was collected for further ELISA. Secreted CTGF was detected using the ELISA kit as instructed by the manufacturer.The WI-38 cells were subjected to 1% O5 cells/well) were transfected for 24 h with control siRNA, GLI-1 siRNA, GLI-2 siRNA, MEK1 siRNA, and ERK1 siRNA by using lipofetamine 3000 reagents in accordance with the manufacturer’s instructions. After siRNA transfection, the cells were subjected to hypoxia (1% O2) for the indicated time intervals for western blotting or luciferase activity assays.The WI-38 or NHLF cells (2 × 102) for an additional 24 h, and then the luciferase assay system was used  to assess luciferase activity. The ratio of cells with and without stimulation was referenced to measure the level of luciferase activity induction.The CTGF-luciferase activity assay was previously described . In brieThe WI-38 cells were pretreated with a vehicle (DMSO), PD98059, or U0126 for 20 min, or transfected with GLI-1 siRNA, GLI-2 siRNA, MEK1 siRNA, or ERK1 siRNA for 24 h before they were subjected to hypoxia. Immunoreactivity was detected using western blotting analysis, as previously described .The WI-38 cells were pretreated with a vehicle (DMSO) or U0126 for 20 min in 100-mm dishes before they were stimulated with hypoxia for an additional 2 h. The cell lysates were obtained with a cytoplasmic and nuclear protein extraction kit purchased from TOOLS , which was used in accordance with the manufacturer’s instruction. Immunoreactivity was detected using western blotting analysis.2) for 10 min. The cell lysates were harvested as previously described [The WI-38 cells were incubated in 60-mm dishes before they were subjected to hypoxia (1% Oescribed . Afterwa2) for the indicated time before they were fixed with formaldehyde for an additional 10 min. The c-Jun, GLI-1, and GLI-2 binding to the AP-1 binding site of the CTGF promoter region was detected through a ChIP assay, performed as described previously [5’-GGA TGT ATG TCA GTG GAC AGA-3’, and antisense: 5’-AAG CGC AGT ATT TCC AGC ACC-3’.The WI-38 cells were subjected to hypoxia (1% Oeviously . The pri2) for 2 h. Nuclear GLI-1 and GLI-2 were detected using immunofluorescence staining and confocal microscopy, as described previously [The cells were subjected to hypoxia  or standard deviation (SD) based on at least 3 independent experiments. One-way analysis of variance (ANOVA) was performed followed by the Dunnett test, to analyze the differences between groups. A 2) time-dependently induced CTGF expression, with the peak affecting time at 6 h of hypoxia treatment (2) for 0–48 h, exhibited an increase in hypoxia-induced release of CTGF into the medium in a time dependent manner. After 48 h of stimulation, hypoxia induced an increase in CTGF release from 49.8 ± 3.5 to 108.0 ± 13.7 pg/mL (n = 4)  . Next, w (n = 4) . Moreove (n = 4) . Next, t (n = 4) . In addi (n = 4) . These rAberger et al. 2012) reported that GLI-1 and GLI-2 translocate in the nucleus as the activators or co-activators of genes after a stimulus [ reportedNumerous studies have shown that GLI proteins are MEK/ERK substrates that are mediated by its activity , 23–25. We further evaluate whether hypoxia stimulates ERK activation. The Tyr204 phosphorylation causes an enzymatic stimulation in ERK . Fig 5A ERK is a downstream molecule of MEKK1 that regulates ERK activity . To examYu et al. (2009) found that the CTGF promoter contains several transcriptional factor binding sites  . To deteBolanos et al. (2012) found that GLI-1 and GLI-2 were highly expressed in fibroblasts in pulmonary fibrosis . The oveIncreased CTGF overexpression has been identified in fibrotic tissue and it is crucial in interstitial pulmonary fibrosis. Hypoxia also participates extensively in interstitial pulmonary fibrosis. Severe pulmonary fibrosis leads to reduced ventilation of the lungs and causes hypoxia, resulting in fibroblast proliferation and differentiation , 38–40. The hedgehog pathway and GLI proteins play a major role in cell proliferation and patterning, including fibrosis formation, which can be activated via noncanonical pathways  , 49. StuPasteurella multocida toxin-induced CTGF expression in mouse fibroblasts [2 (hypoxia) induced MEKK1 and ERK phosphorylation. MEKK1 siRNA transfection in cells attenuated hypoxia-induced ERK phosphorylation. Thus, MEKK1 activation was involved in ERK activation and hypoxia-induced CTGF expression in lung fibroblasts. Because ERK is an upstream effector of GLI proteins [Increasingly more data are showing that MEKK1/MEK1/ERK modulates inflammation in airway epithelial cells . MEKK1 iroblasts . The finroblasts . In the proteins , we alsoproteins  and hypoproteins . In the 2) for 0–60 min did not affect c-Fos phosphorylation. These results implied that c-Jun, but not c-Fos, participates in hypoxia-induced CTGF expression. Because the CTGF promoter region does not contain a GLI-1/GLI-2-binding site, we tested whether GLI-1 and GLI-2 cooperated with c-Jun in binding to the AP-1-binding region and promoted CTGF expression. Our ChIP assay results revealed that hypoxia stimulation led to the direct binding of c-Jun, GLI-1, and GLI-2 to the AP-1 promoter region, which cooperated with GLI-1/GLI-2 and mediated hypoxia-induced CTGF expression.Several reports have indicated that AP-1 is crucial for regulating CTGF expression , 55. In The results of our study also implied that GLI-1 and GLI-2 play a crucial role in hypoxia-induced CTGF expression via the MEKK1/MEK1/ERK1 and AP-1 signaling pathways. However, using a fibroblast is not the only approach to simulating the mechanism of IPF. The roles of GLI-1 and GLI-2 in hypoxia-induced CTGF, α-SMA, and collagen expression require further investigation in other cell types . Furthermore, p38 MAPK has been evidenced as a downstream kinase of hypoxia. However, further effort is required to determine the participation of p38 MAPK in hypoxia-induced α-SMA and collagen expression. In our previous study, CTGF was found to mediate CXCL12-induced α-SMA expression . NeverthIn conclusion, we found that hypoxia activated the MEKK1/MEK1/ERK1/GLI-1/GLI-2 signaling pathway, after which it promoted GLI-1, GLI-2, and c-Jun binding to the AP-1 site of the CTGF promoter region to mediate CTGF expression in human lung fibroblasts. The p38 MAPK also participates in hypoxia-induced CTGF expression. Moreover, GLI-1 and GLI-2 mediate hypoxia-induced α-SMA and collagen expression.


Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen–antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis. Neutrophils are innate immune cells that migrate from the circulation to sites of inflammation or infection. Classically, neutrophils are considered the first line of defense since they are the first cells to appear at the affected site, and they display important antimicrobial functions . NeutropHuman neutrophils express constitutively two IgG antibody receptors: FcγRIIa (CD32a) and FcγRIIIb (CD16b) . FcγRIIavia ERK activation. FcγRIIIb was stimulated by specific monoclonal antibodies, and the NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, a MEK-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that, this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.FcγRIIIb cross-linking induced efficient NET formation similarly to Phorbol 12-myristate 13-acetate (PMA) stimulation . This NENeutrophils were isolated from the peripheral blood collected from adult healthy volunteers following a protocol that was approved by the Bioethics Committee at Instituto de Investigaciones Biomédicas – UNAM. All volunteers provided a written informed consent for their blood donation. The procedure for neutrophil isolation was exactly as previously described .PhosSTOP™ phosphatase inhibitor cocktail  were from Roche Diagnostics . PMA and all other chemicals were from Sigma Aldrich . The following antibodies were used: anti-human FcγRI (CD64) mAb 32.2 (ATCC® HB-946™) and anti-human FcγRIIa (CD32a) mAb IV.3  was from Jackson Immuno Research Laboratories Inc. . HRP-conjugated F(ab′)2 goat anti-mouse IgG  and HRP-conjugated F(ab′)2 goat anti-rabbit IgG  were from MP Biomedicals . Rabbit polyclonal anti-phospho-TAK1 (T187)  was from Abcam plc. .Bovine serum albumin (BSA) was from F. Hoffmann-La Roche Ltd. . Piceatannol, a spleen tyrosine kinase (Syk) inhibitor was from Acros Organics . PD98059 and U0126, MEK (ERK kinase) inhibitors were obtained from New England Biolabs  and from Promega , respectively. The antibiotic LL Z1640-2   was from Santa Cruz Biotechnology . GÖ6983, a protein kinase C (PKC) inhibitor, SB 203580, a p38 MAP kinase inhibitor , and 3-(1-methyl-1H-indol-3-yl-methylene)-2-oxo-2,3-dihydro-1H-indole-5-sulfonamide (iSyk), another Syk inhibitor  were from Calbiochem/EMD Millipore . Recombinant Human TGF-β1  was from Peprotech . The cOmplete™ protease inhibitor cocktail  and mAb IV.3 . Rabbit 6 cell/ml in RPMI-1640 medium  containing 500 nM SYTOX® Green , and 100 μl of this cell suspension (1 × 105 PMN) were added to each well of the 96-well plate. The plate was then incubated at 35°C for 20 min in a microplate reader model Synergy HT from BioTek Instruments . Next, 20 μl of 120 nM PMA dissolved in the same RPMI/SYTOX medium were added to each well for a final concentration of 20 nM. After that, the plate was incubated for up to 4 h, reading the fluorescence from the bottom of the plate, using the 485 nm excitation and 528 emission filters, every 5 min. For FcγR stimulation, neutrophils were resuspended at 0.5 × 107 cell/ml in RPMI/SYTOX medium containing 10 μg/ml of the corresponding anti-FcγR antibody and incubated in ice for 20 min. After one wash in PBS, cells were resuspended in the same volume of RPMI/SYTOX medium, and 20 μl of this cell suspension (1 × 105 PMN) were added to each well of the 96-well plate. The plate was then incubated at 35°C for 20 min in a microplate reader. Next, 100 μl of 45 μg/ml goat anti-mouse IgG in RPMI/SYTOX medium were added to each well . Finally, the plate was incubated for up to 4 h, reading the fluorescence every 5 min. For TAK1 inhibition, cells were treated with 10 nM LL Z1640-2 for 30 min before stimulation.Neutrophil extracellular trap formation was quantified by detecting DNA release spectrophotometrically with the DNA-binding dye SYTOX® Green –24. For 7 cells/ml, and 200 μl of the cell suspension were placed in Eppendorf tubes. The corresponding mAb was then added at 10 μg/ml, and the cells were incubated on ice for 30 min. Next, cells were washed twice with 500 μl of PBS. Receptor cross-linking was then induced by resuspending the cells in 100 μl of PBS containing 37 μg/ml of F(ab′)2 goat anti-mouse IgG and incubating them at 37°C for 15 min. For PMN stimulation with PMA or TGF-β, PMN were incubated at 37°C for 15 min with 20 nM PMA or 5 ng/ml TGF-β. In assays where pharmacological inhibitors were used, PMN were pretreated with 10 nM LLZ 640-2 or only with the solvent dimethyl sulfoxide (DMSO) on ice for 30 min before adding the first mAb.PMNs were stimulated by cross-linking Fc receptors with specific mAbs as follows: PMN were resuspended in PBS at 1 × 102 goat anti-rabbit IgG o for 1 h at room temperature. After washing six more times, the membrane was developed with Immobilon Western chemiluminescent HRP substrate  from EMD Millipore  according to the manufacturer’s instructions. Afterward, membranes were stripped with 0.2 M NaOH and reprobed with anti-ERK 1 (1/2000 dilution) or anti-GAPDH (1/1000 dilution) to assess protein loading in PAGE gels.Total protein extracts were obtained by lysing the cells in cold RIPA lysis buffer  supplemented with 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail, which were added just before lysing the cells. Cell lysates were incubated on ice for 20 min, then cleared by centrifugation, and proteins resolved on SDS 10% PAGE. Proteins were then electrotransfered onto polyvinylidine fluoride (PVDF) membranes . Membranes were incubated in blocking buffer   and 0.1% Tween 20 in Tris-buffered saline  overnight at 4°C. Membranes were subsequently probed with the corresponding antibody in blocking buffer for 1 h at room temperature. Anti-phospho-ERK 1 (1/1000 dilution) or anti-phospho TAK1 (1/2000 dilution). Membranes were washed with TBS-Tween six times and incubated with a 1/3000 dilution of HRP-conjugated F(ab′)t-tests using the computer program KaleidaGraph® version 3.6.2 for Mac . Differences were considered statistically different at a value p < 0.05.Quantitative data were expressed as mean ± SEM. Single variable data were compared by paired-sample Student’s Most studies on NETs have used PMA, a potent activator of PKC, to induce the formation of NETs . Direct Others and we have seen that the MEK/ERK signaling pathway is required for both PMA-  and FcγRTransforming growth factor-β-activated kinase 1 was initially identified as a regulator of MAPK in response to TGF-β , thus, wHuman neutrophil expresses constitutively two low-affinity Fcγ receptors, FcγRIIa and FcγRIIIb, and after interferon-γ, they can upregulate FcγRI. Previously, it has been reported that FcγRIIIb is the receptor responsible for NET formation , 23. Thevia Syk.After establishing a role for TAK1 in FcγRIIIb-mediated NET formation, we explored a possible connection from FcγRIIIb to TAK1. Neutrophils were stimulated by FcγRIIIb cross-linking in the presence or absence of two Syk inhibitors. FcγRIIIb-induced TAK1 phosphorylation and also ERK 1 phosphorylation were efficiently blocked by both Syk inhibitors Figure . This reNext, we explored the signaling pathway from TAK1 to ERK. Neutrophils were stimulated by PMA or FcγRIIIb cross-linking in the presence or absence of the TAK1 inhibitor, and ERK 1 activation was detected by Western blotting. First, we confirmed that LL Z1640-2 was inhibiting TAK1 phosphorylation Figure . This ERIn most situations, MEK activation leads to ERK activation, as the former phosphorylates the latter. In order to confirm that this is also the case in the case of FcγRIIIb- or PMA-induced NETosis, neutrophils were treated with the MEK inhibitor PD98059 prior to stimulation. As shown before, cross-linking of FcγRIIIb clearly activated ERK, and this activation was completely blocked by the MEK inhibitor Figure A. SimilaBecause it is well known that TAK1 functions upstream of p38 MAPK pathway rather than ERK , 29 in mvia the MEK/ERK signaling cascade.The MAP3K, TAK1, is activated by different stimuli including cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1, and IL-18, or TLR ligands such as LPS in various cell types , 30–34 aNeutrophil activation is required for the initiation of the several defense mechanisms, including phagocytosis, respiratory burst, release of various microbicidal molecules by degranulation , and theFcγRIIIb is present exclusively on human neutrophils, and it is a GPI-linked receptor, lacking transmembrane and cytoplasmic domains . Despitevia PKC remains unsolved (Figure Although, both stimuli PMA and FcγRIIIb cross-linking initiate signaling that seems to converge at the level of MEK Figure , an impod Figure . Also, td Figure . This idd Figure A. Yet, nIn several cell types, TAK1 functions upstream of p38 MAPK pathway rather than ERK , 29. In In conclusion, to our knowledge, this is the first demonstration that TAK1 can be activated by FcγRIIIb in human neutrophils, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.OA performed most of the experiments for NET formation and analyzed the data. NM prepared the cells and performed Western blots. RC-V generated some of the Western blot data. EU-Q helped with the first draft, performed the statistical analysis, organized the references, and prepared all figures. CR designed the research, mentored all other authors, and wrote the final version of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We have performed western blot and immunohistochemistry to study the expression of the H2S synthesis enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), measurement of enzymatic production of H2S and myographic studies for isometric force recording. Immunohistochemical assays showed a high CSE expression in the intravesical ureter muscular layer, as well as a strong CSE-immunoreactivity within nerve fibres distributed along smooth muscle bundles. CBS expression, however, was not consistently observed. On ureteral strips precontracted with thromboxane A2 analogue U46619, electrical field stimulation (EFS) and the H2S donor P-(4-methoxyphenyl)-P-4-morpholinylphosphinodithioic acid (GYY4137) evoked frequency- and concentration-dependent relaxations. CSE inhibition with DL-propargylglycine (PPG) reduced EFS-elicited responses and a combined blockade of both CSE and NO synthase (NOS) with, respectively, PPG and NG-nitro-L-arginine (L-NOARG), greatly reduced such relaxations. Endogenous H2S production rate was reduced by PPG, rescued by addition of GYY4137 and was not changed by L-NOARG. EFS and GYY4137 relaxations were also reduced by capsaicin-sensitive primary afferents (CSPA) desensitization with capsaicin and blockade of ATP-dependent K+ (KATP) channels, transient receptor potential A1 (TRPA1), transient receptor potential vanilloid 1 (TRPV1), vasoactive intestinal peptide/pituitary adenylyl cyclase-activating polypeptide (VIP/PACAP) and calcitonin gene-related peptide (CGRP) receptors with glibenclamide, HC030031, AMG9810, PACAP6–38 and CGRP8–37, respectively. These results suggest that H2S, synthesized by CSE, is involved in the inhibitory neurotransmission to the pig intravesical ureter, through an NO-independent pathway, producing smooth muscle relaxation via KATP channel activation. H2S also promotes the release of inhibitory neuropeptides, as PACAP 38 and/or CGRP from CSPA through TRPA1, TRPV1 and related ion channel activation.According to previous observations nitric oxide (NO), as well as an unknown nature mediator are involved in the inhibitory neurotransmission to the intravesical ureter. This study investigates the hydrogen sulfide (H Thus, in rat bladder detrusor, the H2S donor NaHS induces contraction via stimulation of capsaicin-sensitive primary afferents (CSPA), leading to release of tachykinins, such as substance P or neurokinin A 2S produces smooth muscle relaxation. In fact, in the pig bladder neck, H2S, synthesized by CSE, acts as a signaling molecule in the inhibitory neurotransmission, producing smooth muscle relaxation via KATP channel activation and favouring the release of the sensory neuropeptides In the lower urinary tract, HThe density of the autonomic nerve fibers increases progressively from the upper ureter towards the bladder 2S has recently been identified as a powerful inhibitory neurotransmitter in the bladder base 2S role in the distal ureter neurogenic relaxation. Therefore, the current study investigated the involvement of H2S in the inhibitory neurotransmission to the pig intravesical ureter.HAdult pigs of either sex with no lesions in their urinary tract were selected from the Matadero Madrid Norte slaughterhouse. Urinary bladders with attached ureters were removed immediately after the animals were killed, and kept in chilled (4°C) physiological saline solution (PSS). The protocol was carried out in the following 24 h. The adjacent connective and fatty tissues were carefully removed, and longitudinal preparations (4–6 mm long and 2–3 mm wide) of the intravesical ureter were dissected from the bladder Intravesical ureter muscle was homogenized in lysis buffer containing 10 mM Tris-HCl (pH 7.4), 1% SDS, 1 mM sodium vanadate and 0.01% protease inhibitor cocktail . 50 µg protein were separated in a 15% polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). All membranes were blocked by 5% non-fat dry milk for 1 h at room temperature. For immunodetection, membranes were incubated overnight at 4°C with rabbit anti-CSE or anti-CBS  and mouse anti-β-actin  antibodies. Membranes were then washed in 0.05% Tween-20, incubated with HRP-conjugated secondary antibodies  to detect CSE and CBS, for 1h at room temperature, and then washed and visualized by chemiluminescence . Bands for CSE and CBS were normalized to those of β-actin. CSE and CBS expression in urinary bladder neck membranes were included as positive controls.Intravesical ureter segments were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB), for 2 to 4 h at 4°C, and subsequently placed in 30% sucrose in PB for cryoprotection. The tissue was embedded and frozen in OCT compound , and stored at −80°C.Transversal sections 5 µm thick were obtained by means of a cryostat and preincubated in 10% normal goat serum in PB containing 0.3% Triton-X-100, for 2–3 h. Then, sections were incubated with either rabbit anti-CSE or anti-CBS antibodies at 4–8 µg/ml final concentration plus a mouse anti-protein gene product 9.5 , as neuronal marker, diluted 1∶50, during 48 h at 4°C, washed and reacted with the secondary antibodies Alexa Fluor 594 goat-antirabbit (1∶200 dilution) to detect CSE and CBS, and Alexa Fluor 488 goat-antimouse , to detect PGP 9.5, for 2 h at room temperature. The slides were covered with a specific mounting medium with DAPI (Invitrogen), which stains all cell nuclei. Observations were made with a fluorescence microscope (Olympus IX51). No immunoreactivity could be detected in sections incubated in the absence of the primary antiserum 2S endogenous production was measured in intravesical ureter strips following the method previously described in the rat colon 2S donor, respectively, which was connected to a 2 ml second vial containing 0.5 ml of 1% (w/v) zinc acetate. A gas mixture of 95% O2 and 5% CO2 was bubbled from the bottom of the first vial through the incubation solution. The reaction was started by transferring the vials from ice to a water bath at 37°C. The H2S produced in the incubation chamber was then bubbled through the zinc acetate solution and trapped as zinc sulphide. The reaction was stopped at 30 min by injecting 0.5 ml of 50% (w/v) trichloroacetic acid into the incubation solution. Air flow was allowed to continue by an additional 30 min period, to ensure complete trapping of H2S in the zinc acetate solution. The content of the second vial was transferred to test tubes containing 3.5 ml of de-ionized water, 0.4 ml of N, N-dimethyl-p-phenylenediamine sulphate (20 mM) in HCl (7.2 M) and 0.4 ml of FeCl3 (30 mM) in HCl (1.2 M), for performing the methylene blue assay. The absorbance at 670 nm of the resulting solution was measured 20 min later by spectrophotometry . H2S concentration was calculated against a calibration curve of the standard NaHS solutions.H2 in O2, giving a final pH of 7.4. Stretching of 2 g was applied to the preparations and they were allowed to equilibrate for 60 min.The intravesical ureter strips were suspended horizontally with one end connected to an isometric transducer and the other one to a micrometer screw, which regulates the tension applied to the preparations, in a myograph (DMT 820MS) containing PSS gassed with 5% CO2 receptor agonist, EFS was performed by delivering rectangular pulses , at 4 min intervals, from a Cibertec CS20 stimulator . These EFS parameters have previously been used to elicit neurogenic relaxations in the intravesical ureter 2S donor P-(4-methoxyphenyl)-P-4-morpholinylphosphinodithioic acid  addition was obtained. The bath solution was then changed every 15 min for a period of 90 min, the preparations were incubated with the specific treatments for 30 min, and then a second relaxation curve was constructed. The concentration of the agents used was chosen on the basis of previous studies The contractile ability of the strips was determined by exposing them to a 124 mM potassium PSS. In electrical field stimulation (EFS) experiments, noradrenergic neurotransmission and muscarinic receptors were blocked by pre-incubation with guanethidine (10 µM) and atropine (0.1 µM) for 1 h, replacing the solution every 20 min, and these drugs were present throughout the experiment. In strips precontracted with 0.1 µM U46619, a thromboxane ATo desensitize capsaicin-sensitive primary afferents (CSPA), strips were pre-incubated in 10 µM capsaicin for 1 h, replacing the solution every 20 min, and then experiments were conducted in the continuous presence of capsaicin G-nitro-L-arginine (L-NOARG) and O-(carboxymethyl)hydroxylamine (AOAA), NaHS, L-cysteine, pyridoxal 5′-phosphate, zinc acetate trichloroacetic acid, N,N-dimethyl-p-phenylenediamine sulphate, HCl, FeCl3 all from Sigma . Calcitonin gene-related peptide 8-37 (CGRP8-37), capsaicin, glibenclamide, P-(4-methoxyphenyl)-P-4-morpholinylphosphinedithioic acid (GYY4137), 2--N-(4-isopropyl phenyl)acetamide (HC030031), -2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolopyrrolo6-38) and 9,11-dideoxy-9a,11a-methanoepoxy prostaglandin F2α (U46619) from Tocris . AMG9810, AOAA, CGRP8-37, PPG, glibenclamide, GYY4137, HC030031, KT5720, ODQ and PACAP6-38 were dissolved in dimethylsulphoxide. Indomethacin and U46619 were dissolved in ethanol. The other drugs were dissolved in distilled water. The solvents used had no effect on the contractility of the bladder neck preparations.The following drugs were used: (2E)-N--3-[4-phenyl]-2-propenamide (AMG9810), atropine, and DL-propargylglycine (PPG), guanethidine, indomethacin, N2 1.2, NaHCO3 24.9, glucose 11, CaCl2 1.5, KH2PO4 1.2, ethylenediamine tetraacetic acid (EDTA) 0.027. The solution was maintained at 37°C and continuously gassed with 95% O2 and 5% CO2 to maintain pH at 7.4. K+-enriched PSS was PSS in which NaCl was exchanged for KCl on an equimolar basis.The composition of PSS was (mM): NaCl 119, KCl 4.6, MgCl2, where pD2 =  -log EC50 and EC50 is the agonist concentration needed to produce half-maximal response. pD2 was estimated by computerized non-linear regression analysis . Results are expressed as a percentage reversal of U46619- or KPSS-induced contraction, and represent the mean ± s.e.m. of n . Differences were analyzed by Student's t-test for paired observations and by analysis of variance and a posteriori Bonferroni method for multiple comparisons. The differences were considered significant with a probability level of P<0.05. P values are shown in the Figure legends.Sensitivity to GYY4137 is expressed in terms of pDBy western blot, a CSE antibody recognized a band of approximately 45 kDa, which corresponded to the expected molecular weight, suggesting CSE protein expression in intravesical ureter smooth muscle Fig. 1A Urothelium-denuded strips of pig intravesical ureter were allowed to equilibrate to a passive tension of 1.5±0.1 g (n = 75 preparations from 47 pigs). U46619 (0.1 µM) induced a sustained contraction above basal tension of 1.7±0.1 g (n = 75).2S donor GYY4137 (0.1 nM–30 µM) induced potent concentration-dependent relaxations , which were not changed as a consequence of urothelium mechanical removal.Under NANC conditions, EFS (0.5–16 Hz) evoked reproducible frequency-dependent relaxations . The H2S plays a role in the inhibitory neurotransmission of the intravesical ureter, ureteral preparations were treated with PPG and AOAA, inhibitors of, respectively, CSE and CBS. PPG (1 mM) reduced EFS-induced relaxations  . The generated H2S level, however, was reduced by CSE blockade with PPG (1 mM)  and restored by addition of the H2S donor, GYY4137 (10 µM)   (CSE protein expression (n = 4 from 4 pigs) Fig. 3D The soluble guanylyl cyclase inhibitor ODQ (5 µM) reduced the EFS relaxations Table 1 + to 80 mM induced a sustained tone of 1.7±0.1 g (n = 6). GYY4137 induced concentration-dependent relaxations on 80 mM K+ PSS-precontracted strips which were lower than those obtained on 0.1 µM U46619-contracted preparations . Glibenclamide (1 µM), a KATP channel inhibitor, reduced both EFS and GYY4137 relaxations (ATP channel involvement in the H2S relaxant responses.Raising extracellular K1) and transient receptor potential vanilloid 1 (TRPV1), respectively, receptors, reduced both EFS and GYY4137 responses. These data indicate that H2S relaxations are partly produced through TRPA1, TRPV1 and/or related ion channel activation-mediated release of inhibitory neuropeptides from CSPA.Capsaicin (10 µM) (6–38 (3 µM) (8–37 (3 µM)  The presence of CSE within nerve fibers widely distributed in the smooth muscle layer of the intravesical ureter. (2) The neurogenic relaxation elicited by EFS was inhibited by PPG. (3) EFS and GYY4137 responses were reduced by blockade of KATP channels, desensitization of CSPA and inhibition of TRPA1 and TRPV1 channels and of PACAP and CGRP receptors.Our results provide morphological and functional evidence that neuronal H2S in the regulation of intravesical ureter blood flow, thus supporting an important role for H2S in vascular tone modulation In the pig intravesical ureter, only CSE expression was consistently observed. Western blot assays showed a band compatible with that expected for CSE in the muscular layer, and immunostaining of ureteral samples revealed a labeling for CSE protein within nerve fibers widely distributed among smooth muscle bundles. The high density and the distribution of CSE immunoreactivity observed in the intravesical ureter agree with that found in pig bladder neck 2S production elicited by PPG and its recovery in response to GYY4137, clearly indicate that neuronally-released endogenous H2S synthesized by CSE is responsible for a considerable part of the NANC inhibitory transmission to the intravesical ureter. The fact that incubation with the NO synthase inhibitor L-NOARG plus PPG abolished the EFS relaxations indicates that, in addition with NO, H2S plays a key role in ureteral inhibitory neurotransmission, and might therefore directly be involved in the regulatory mechanisms of the smooth muscle tone, thus reducing the ureteral resistance during bladder filling. In the intravesical ureter, in addition to the predominant longitudinal smooth muscle fibers, circular and helical fibers have also been described 2S in the distal ureter urodynamic parameters.NO-dependent and independent NANC neurogenic relaxations in the intravesical ureter have previously been reported 2S production for regulating of vascular smooth muscle tension 2S production rate was not modified under conditions of NOS blockade suggests the involvement of a NO-independent pathway in the intravesical ureter endogenous H2S generation. These results agree with those obtained in bladder neck, where both H2S 2S, together with NO, in the intravesical ureter neurogenic relaxation reinforces the role of the autonomic nervous system in the regulation to the distal ureter tension in contrast with the myogenic electrical activity characteristic of the pyeloureteral segments NO has been proposed as an inducer or as a molecular switch for endogenous H2S, both in vivo and in vitro 2 value of 7.7), slow in onset and sustained, which was similar to that previously obtained in bladder neck 2S in the ureteral smooth muscle relaxation. The fact that PPG failed to modify the GYY4137 relaxations may be explained on the basis that PPG is an inhibitor of the endogenous H2S synthesis enzyme CSE, and therefore does not seem probable that it can reduce the responses to the exogenously-added H2S donors. Current results agree with those obtained in bladder neck, where CSE selective blockade did not change the GYY4137 responses 2S produces smooth muscle relaxation via urothelium- or NO-independent mechanisms.In our study, GYY4137, a donor which in the cardiovascular system slowly releases H2+-calmodulin dependent 2S generated from L-cysteine by CSE exerts its biological action by sulfhydrating target proteins, process that may augment guanylyl cyclase activity, thus increasing [cGMP]i and relaxing smooth muscle 2S relaxations. These results agree with those previously described in vascular smooth muscle, where unlike the intracellular signaling responsible for the vasodilator action induced by NO and CO, H2S relaxations were produced in a guanylyl cyclase activation-independent way Like neuronal- and endothelial-NOS, CSE activity is Ca2S has previously been reported to inhibit superoxide anions formation via adenylyl cyclase-PKA pathway in pig pulmonary arterial endothelial cells 2S relaxations.HATP channel activation mediates the H2S-induced relaxation in both vascular and visceral smooth muscle. Thus, KATP channel opening-mediated H2S responses have been described in rat aorta and mesenteric arteries + PSS-precontracted strips. Extracellular [K+] elevation inhibits K+ efflux through membrane K+ channels, and since glibenclamide, a KATP channel inhibitor, reduced the EFS or GYY4137 responses, it seems likely that ionic conductance modifications via KATP channels are involved in H2S relaxations. Interestingly, this signaling pathway is also involved in the neuronal NO-mediated relaxation of the pig intravesical ureter K2S responses.The COX pathway is involved in bladder physiology and pathology, and several studies have demonstrated a role for COX-derived prostanoids in the neural control of bladder smooth muscle tone 2S donors produce contraction of rat detrusor via release of tachykinins such as substance P or neurokinin A from CSPA, by activating non-selective cation channel TRPV1, TRPA1 and/or related ion channels in the sensory nerves 2S relaxant responses are produced, in part, via PACAP 38 and calcitonin gene-related peptide (CGRP) release from CSPA 2S relaxations. The protocol of capsaicin desensitization carried out in our investigation produces an intravesical ureter CSPA functional blockade 1 are recognized as the main target for H2S in sensory neurons 1 selective antagonist, on EFS or GYY4137 responses, suggests the involvement of TRPA1 receptors in H2S relaxations. Moreover, the H2S response reduction produced by blockade of TRPV1 with AMG9810 indicates the mediation of these receptors. The fact that capsaicin inhibition of the EFS or GYY4137 relaxations was higher than that exerted by HC030031 and AMG9810 suggests that in addition to the TRPA1 and TRPV1, the possible role of related ion channels located on sensory neurons. The EFS or GYY4137 response inhibition produced by VIP/PACAP and CGRP receptor blockade, suggests that H2S may promote intravesical ureter smooth muscle relaxation via PACAP 38 and/or CGRP release from CSPA. These results agree with those found in the pig bladder neck, where part of H2S relaxations are indirectly produced via inhibitory neuropeptide release from sensory nerves H2S donors have been proposed as helpful therapeutic tools for unilateral ureteric obstruction-induced renal damage by attenuating fibrosis, oxidative stress and inflammation 2S is responsible for the intravesical ureter NANC inhibitory neurotransmission, thus suggesting that H2S-mediated neurotransmission might be useful as a therapeutic target in the obstructive ureteral pathology and in the vesico-ureteral reflux.H2S, synthesized by CSE, acts as a potent inhibitory neurotransmitter to the pig intravesical ureter through a NO-independent mechanism, producing smooth muscle relaxation via KATP channel activation. H2S also promotes the release of PACAP 38 and CGRP from CSPA through activation of TRPA1, TRPV1 and/or related ion channels in the sensory nerves (2S in the neurogenic relaxation of the intravesical ureter.In conclusion, present results suggest that Hy nerves Fig. 7.


Background: The Streptococcus pneumoniae sialidase NanC produces a nonspecific inhibitor of hydrolytic sialidases.Results: The NanC crystal structure is presented in complex with mechanistically relevant ligands.Conclusion: A constricted and hydrophobic active site produces 2-deoxy-2,3-didehydro-N-acetylneuraminic acid  via a covalent intermediate and direct proton abstraction by a catalytic aspartic acid.Significance: Insights into an unusual reaction mechanism will aid the design of sialidase inhibitors. Streptococcus pneumoniae is an important human pathogen that causes a range of disease states. Sialidases are important bacterial virulence factors. There are three pneumococcal sialidases: NanA, NanB, and NanC. NanC is an unusual sialidase in that its primary reaction product is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid , a nonspecific hydrolytic sialidase inhibitor. The production of Neu5Ac2en from α2–3-linked sialosides by the catalytic domain is confirmed within a crystal structure. A covalent complex with 3-fluoro-β-N-acetylneuraminic acid is also presented, suggesting a common mechanism with other sialidases up to the final step of product formation. A conformation change in an active site hydrophobic loop on ligand binding constricts the entrance to the active site. In addition, the distance between the catalytic acid/base (Asp-315) and the ligand anomeric carbon is unusually short. These features facilitate a novel sialidase reaction in which the final step of product formation is direct abstraction of the C3 proton by the active site aspartic acid, forming Neu5Ac2en. NanC also possesses a carbohydrate-binding module, which is shown to bind α2–3- and α2–6-linked sialosides, as well as N-acetylneuraminic acid, which is captured in the crystal structure following hydration of Neu5Ac2en by NanC. Overall, the pneumococcal sialidases show remarkable mechanistic diversity while maintaining a common structural scaffold. Streptococcus pneumoniae is a common resident of the human nasopharynx. Although carriage is normally asymptomatic, the bacterium can cause severe illness, particularly in the very young, the elderly, and the immunocompromised, and can be triggered following infection with the influenza virus  domain of unknown function that protrudes from the catalytic domain –26. NanC–N-acetylneuraminic acid (Neu5Ac). NanB is an intramolecular trans-sialidase generating 2,7-anhydro-Neu5Ac as a reaction product (iK of 2 μm (Vibrio cholerae (iK of 2–3 mm (N-acetylneuraminic acid (3F-β-Neu5Ac), and the viral neuraminidase inhibitor oseltamivir carboxylate (OC). In addition, complexes with the substrate α2–3-sialyllactose (3′SL), α2–6-sialyllactose (6′SL), and Neu5Ac in the CBM binding site of the catalytic domain are described. Together, these data provide a structural basis for the unusual production of Neu5Ac2en by this enzyme.The pneumococcal sialidases vary in substrate specificities, mechanism, and kinetic parameters. Although NanA can cleave a range of sialic acid substrates including those with α2–3, α2–6, and α2–8 glycosidic linkages , 27, Nan product , 25. Des of 2 μm , 28. In  of 2 μm , the hum of 2 μm , 31, andcholerae . Neu5Ac24YW0, 4YW1, 4YW2, 4YW3, and 4YW5 were obtained from scheme A, and PDB codes 4YZ1, 4YZ2, 4YZ4, and 4YZ5 were obtained from scheme B as described below. Differences between the two schemes will be highlighted and referred to as A or B throughout the methods description.Coordinates for all crystal structures have been deposited in the Protein Data Bank. Two separate cloning, expression, and purification schemes were used. PDB codes S. pneumoniae TIGR4 nanC gene was expressed in the pet21b vector  was used as a template for PCR amplification of the NanC protein-coding region. The In-Fusion® cloning method was used to insert the PCR product into a pOPINF plasmid vector  expression strain (Novagen) in autoinduction media . The cultures were initially grown at 37 °C, followed by prolonged growth at lowered temperatures . The cells were harvested by centrifugation, resuspended in lysis buffer  supplemented with DNase I (10–20 μg/ml) and Complete protease inhibitor mixture tablets (Roche), and lysed using a constant flow cell disrupter. Insoluble components were removed by centrifugation. Soluble lysate was loaded onto nickel-Sepharose columns  equilibrated in  lysis buffer. Bound protein was washed extensively using lysis buffer  and eluted using an imidazole step gradient .Recombinant plasmids (A and B) were expressed in m Tris-HCl, pH 7.5, and loaded onto a 5-ml SP-FF cation-exchange column . The bound protein was washed using 20 mm Tris-HCl, pH 7.5, 100 mm NaCl and eluted using 20 mm Tris-HCl, 300 mm NaCl. The major peak from  was concentrated and subjected to size exclusion chromatography using a 120-ml Sephacryl S-200 column  equilibrated in 20 mm Tris, pH 7.5 (A) or 20 mm Tris, pH 7.5, 200 mm NaCl, 5% glycerol (B). In scheme B, post size exclusion, the protein was subjected to His6 tag cleavage by incubation with 0.1% (w/w) human rhinovirus B 3C protease , followed by reverse His tag purification.In scheme A, the eluted protein fraction was diluted 1:2 with 20 mm KH2PO4 at 20 mg/ml (A) and 20% PEG3350, 0.25 m ammonium sulfate, 0.1 m Hepes, pH 8.0, at 10 mg/ml (B). These initial crystals were optimized by varying crystallization droplet size and protein to precipitant ratio , incorporation of additives ; B, 7.5% ethanol or isopropanol), and microseeding (A)  ethylene glycol. Additional cryoprotectant was not necessary for scheme A crystals.Promising crystals were obtained from 16% PEG8000, 20% glycerol, 40 mding (A) . AdditivS. pneumoniae NanB structure . Data collected in-house were reduced using the HKL2000 package . Synchroode 2VW2 ) as the ode 2VW2  and improde 2VW2  and refiode 2VW2  (B). Theode 2VW2  and MolPode 2VW2  servers 6 tag cleavage using TEV protease and reverse His tag purification . Heat of dilution experiments were performed, and the resulting isotherm was subtracted from the experimental conditions.The NanC CBM was subcloned into the pEHISTEV vector using primers GGCGCCATGGCTCAGGAGACTGAAACTTCTG and TGGTGCTCGAGTTTACATCTTTTTAACAGTTTCTTC and purified according to scheme A with the addition of Hisfication . ITC expa). The CBM presents a CBM family 40 (CBM40) β-sandwich fold  shared by viral, bacterial, and mammalian sialidases. Asp boxes, motifs typical of bacterial sialidases , followed by a catalytic domain  Neu5Ac complexes were achieved by soaking NanC crystals with Neu5Ac and also by extended soaks (>20 min) with Neu5Ac2en, thus confirming that NanC can hydrate Neu5Ac2en to Neu5Ac. Following short soaks (2 min) with 3′SL and soaks with 6′SL, electron density was observed for the sialyl and galactosyl rings; the glucosyl rings were not visible in electron density. Key interacting residues of the CBM binding site are Arg-237 and Arg-161, which provide electrostatic interactions with the Neu5Ac carboxylate group; Glu-159, which hydrogen bonds to the O4 hydroxyl group; and Arg-151, which hydrogen bonds to the carbonyl of the N-acetyl group . In addition, Phe-149, Leu-128, and Leu-170 form a hydrophobic pocket that accommodates the terminal methyl of the N-acetyl group. Observed protein to ligand interactions are limited to the terminal Neu5Ac residue. Thus the affinities of 3′SL and 6′SL for the NanC CBM were very similar, with dK values of 1.48 mm (b) and 1.60 mm (c), respectively, as measured by isothermal titration calorimetry (ITC). The binding site of the CBM is on the same side as the active site of the catalytic domain and the protein face on which they reside displays an overall positive charge (b and c). These two factors combined could have a role in orienting the active site in relation to the negatively charged host-bound substrate.Structures of the CBM have been obtained in complex with Neu5Ac, 3′SL, and 6′SL Figs. 2a and a–i 1.48 mm b and 1.6 1.60 mm c, respece charge , b and cc–f, h, and i). The NanC active site is a narrow constricted cleft, similar to NanB (a). These residues partially block the active site entrance providing specificity for α2–3-linked substrates as opposed to α2–6.Structures of NanC have been determined with Neu5Ac2en, 3F-β-Neu5Ac, and OC bound at the catalytic active site . The negatively charged active site shares key features of structurally characterized bacterial, mammalian and parasitic sialidases in general and the pneumococcal sialidases in particular (2352–b and c). These include an arginine triad, comprised of Arg-290, Arg-600, and Arg-662, which orients the ligand in the active site via electrostatic interactions with the Neu5Ac2en carboxylate (a and b). The hydrophobic pocket  accommodates the N-acetyl group mirroring the CBM. The ligand sits on top of the catalytic pair of Glu-584 and Tyr-695 and beneath the general acid base Asp-315. The latter residue is substantially closer to the C2 of Neu5Ac2en than in NanA or NanB Neu5Ac2en complexes: 3.2 Å in comparison with 4.2 and 4.5 Å, respectively  presented continuous electron density from the ligand C2 position to Tyr-695 (a and b). 3F-β-Neu5Ac was covalently bound to Tyr-695 in an unstrained 2C5 chair conformation. Equivalent complexes have also been observed in the C. perfringens sialidase NanI, the Trypanosoma rangeli sialidase TrSA, and the Trypanosoma cruzi trans-sialidase TcTS (a). 2,3F-Neu5Ac was also found bound in the CBM binding site (h).NanC crystals soaked with 2,3-difluoro- Tyr-695 , a and base TcTS , 60, 61.ase TcTS a. 2,3F-Ning site h.a). The Cα of Phe-396 advances 2.1 Å into the active site, and the side chain changes conformation, moving out of the active site to accommodate the ligand. The net result of the movement of the 396 loop is that it partially constricts the active site (b and c). The movement appears to be stabilized by hydrophobic interactions between Phe-396 and the C9 carbon of Neu5Ac2en because no hydrogen bonds or other stabilizing interactions are formed between the residues of the 396 loop and Neu5Ac2en (a).The most significant structural change observed upon binding and/or formation of Neu5Ac2en is the substantial shift in Phe-316 and Phe-396 of the hydrophobic pocket and the loop upon which the latter resides  a. The Cαive site , b and ceu5Ac2en a.i and a and b). Compared with Neu5Ac2en, OC undergoes a translation of ∼0.8 Å, along a plane approximately defined by the ligand ring, out of the active site. This is likely to be caused by the electrostatic repulsion between the 4-amino group of OC and Arg-309. In comparison with the Neu5Ac2en complex, arginine triad interactions, and hydrogen bonds from Asp-372 to both the 4-amino group and the N-acetyl amine group are maintained. The 4-amino group induces a pivot in Asp-315 introducing a new hydrogen-bonding partner. The Neu5Ac2en interactions with Ser-582 and Tyr-553 via the glycerol group are lost. Furthermore, the Neu5Ac2en glycerol group makes favorable hydrophobic interactions with Phe-396 (a), whereas the OC pentyl ether group is unable to reproduce this interaction, and the 396 loop does not move into the active site (a). This is predicted to be due to steric clashing between Phe-396 and the pentyl group.Although a poor inhibitor , OC bindFigs. 3 Phe-396 a, whereaive site a. This id), and a covalent intermediate using a fluorinated substrate analogue (h) and the influenza viral neuraminidase inhibitor OC (i). In addition, we have captured complexes of the CBM with Neu5Ac, 3′SL, and 6′SL .NanC is an unusual sialidase in that its primary reaction product is Neu5Ac2en, a nonspecific sialidase inhibitor. We have captured crystal structures of the catalytic site of NanC in complex with its first product, Neu5Ac2en d, and a analogue h and thebitor OC i. In adda).The presented crystal structures provide insight into the NanC reaction specificity and mechanism. Following substrate selection, the substrate is orientated through electrostatic interactions between the Neu5Ac carboxylic acid and the arginine triad. This brings the C2 position into proximity with Tyr-695, which can perform a nucleophilic attack. This step appears to be common to all three pneumococcal sialidases . Tyr-695Fig. 8c). When longer soaks were performed, Neu5Ac2en is still observed in the active site, and cleaved Neu5Ac was observed in the CBM binding site (d). When short Neu5Ac2en soaks were performed, the CBM binding site was empty, whereas bound Neu5Ac became apparent when the soaking time was increased. This is consistent with 1H NMR experiments where Neu5Ac2en is formed after 0.5 h and is hydrated to Neu5Ac over a further period of 6 h .When the pool of substrate is exhausted and Neu5Ac2en is in excess, NanC can hydrate Neu5Ac2en to Neu5Ac. When short 3′SL soaks are performed, electron density is observed for uncleaved 3′SL in the CBM binding site with Neu5Ac2en in the active site c. When ling site d. When sd of 6 h . During d et al. , 52. In d et al. b.d). This advanced position would allow the C3 proton to be abstracted directly. In NanA and NanB, there is space for attack at the C2 position by an activated water molecule forming Neu5Ac, and the C6 glycerol moiety forming 2,7-anhydro-Neu5Ac, respectively, which is not the case in NanC. Mutation of Asp-315 to Ala abolishes catalytic activity . This conformational change is a key feature because the movement constricts the opening to the active site in a manner that would make it almost watertight (c). Mutating NanC Phe-396 to the hydrophilic Asn, as is found in NanB, leads to the production of a mixture of both Neu5Ac2en and Neu5Ac . The V. cholerae sialidase also has a sialic acid binding CBM; however, the affinities for 3′SL and 6′SL are much tighter, at 18 and 19 μm, respectively . This has also been seen in the intramolecular trans-sialidases NanB . In comparison with NanA and NanC, the equivalent position in NanB or Neu2 is hydrophilic (iK greater than 6 mm). This is in stark contrast to the low micromolar inhibition observed for Neu5Ac2en and zanamivir, which can contribute numerous hydrogen bonding interactions to the pocket via the C6 glycerol groups (2F0Z). These variations in protein-ligand interactions highlight the difficulties in translating structure based drug developments between sialidases, because although the active sites are often similar, their ability to bind individual inhibitors differs substantially.OC is an antiviral agent designed to target the influenza virus neuraminidase. It has also demonstrated efficacy against ry tract , 73. Nan or NanC . The man or NanC . In part or NanC . Convers or NanC a. In comrophilic , 25, 56.l groups , 56, 74 The results described here present a structurally rationalized and novel mechanism for the unusual production of Neu5Ac2en by the NanC sialidase and completes the structural description of pneumococcal sialidase catalysis. The mechanistic diversity among the three sialidases, NanA, NanB, and NanC, is achieved through subtle amino acid substitutions, positional shifts of conserved residues, and active site plasticity; the active site core is otherwise preserved. These changes hint at the evolutionary pressure to adapt to environmental niches, which vary in substrate type and quantity and underline the importance of the sialidases for bacterial nutrition and successful host cell invasion. In summary, these works highlight the importance of thorough structural characterization of sialidases; although they may appear, superficially, very similar, they show significant mechanistic diversity.G. L. T. and M. A. W. conceived and supervised the work. C. D. O. and P. L. separately expressed, crystallized, and solved the crystal structure of NanC according to schemes A and B, respectively. C. D. O. performed the ITC analysis. O. S. and M. A. W. performed initial crystallographic analysis. J. A. P., G. L. T., and M. A. W. contributed to data analysis. C. D. O. and P. L. led writing of the paper with contributions from J. A. P., G. L. T., and M. A. W. All authors analyzed the results and approved the final version of the manuscript.


At a clinically relevant concentration of 1 μM, fluoxetine significantly increased astroglial pHi from 7.05 to 7.34 after 3 weeks and from 7.18 to 7.58 after 4 weeks of drug treatment. Stimulation of NHE1 is a result of transporter phosphorylation mediated by several intracellular signaling cascades that include MAPK/ERK1/2, PI3K/AKT and ribosomal S6 kinase (RSK). Fluoxetine stimulated phosphorylation of ERK1/2, AKT and RSK in a concentration dependent manner. Positive crosstalk exists between two signal pathways, MAPK/ERK1/2 and PI3K/AKT activated by fluoxetine since ERK1/2 phosphrylation could be abolished by inhibitors of PI3K, LY294002 and AKT, triciribine, and AKT phosphorylation by inhibitor of MAPK, U0126. As a result, RSK phosphorylation was not only inhibited by U0126 but also by inhibitor of LY294002. The NHE1 phoshorylation resulted in stimulation of NHE1 activity as revealed by the NH4Cl-prepulse technique; the increase of NHE1 activity was dependent on fluoxetine concentration, and could be inhibited by both U0126 and LY294002. Our findings suggest that regulation of astrocytic pHi and brain pH may be one of the mechanisms underlying fluoxetine action.Clinical evidence suggest astrocytic abnormality in major depression (MD) while treatment with anti-psychotic drugs affects astroglial functions. Astroglial cells are involved in pH homeostasis of the brain by transporting protons  and bicarbonate (through the sodium-bicarbonate co-transporter NBC or the chloride-bicarbonate exchanger AE). Here we show that chronic treatment with fluoxetine increases astroglial pH Numerous molecular cascades expressed  in astrocytes control interstitial concentration of principal ions, regulate movement and metabolism of major neurotransmitters, supply neurones with energy substrates and contribute to tissue defence through, for example, secreting scavengers of reactive oxygen species  in neural cells is decreased in the euthymic state as measured by 31P-MRS . The medium was exchanged with fresh medium of similar composition on day 3, and subsequently, every 3–4 days. From day 3, the serum concentration was reduced to 10%, and after 2 weeks, 0.25 mM dibutyryl cyclic AMP (dBcAMP) was included in the medium, which leads to a morphological and functional differentiation of astrocytes  by vortexing and filtering through nylon meshes with pore sizes of 80 and subsequently 10 μm as previously described , cells were loaded with 5 μM BCECF-AM for 30 min at 37°C. After washing twice with HEPES buffer, fluorescence was excited at 440 and 490 nm and emission monitored at 530 nm. The fluorescence ratio (490/440 nm) was determined and calibrated to yield pHi values by interpolation between the measured fluorescence ratios in a similar culture after 20 min of exposure to one of 3 calibration buffers . The calibration buffer also contained 56 μM nigericin, an H+/K+ exchanger, in order to equilibrate pHi with extracellular pH. Steady-state pHi was determined as the average of five individual readings, each from six 20 s intervals  were used to detect pHd Acosta  and Mannd Acosta . After w4Cl-prepulse technique  during the last twenty-seven 25 s intervals was calculated by subtracting the pHi after about 2 min (five cycles) exposure to NH4Cl from the pHi measured every minute during the recovery for total of 10 min, and NHE activity was determined as ΔpHi/Δt  and horse serum from Invitrogen . Fluoxetine, SB204741 (N-(1-methyl-5-indolyl)-N′-(3-methyl-5-isothiazolyl) urea), triciribine hydrate and first antibodies, raised against β-actin were also purchased from Sigma . The U0126  and LY294002 (2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) were obtained from Calbiochem . Santa Cruz Biotechnology  supplied first antibodies, raised against ERK (K-23), sc-94, against phosphorylated ERK (E-4), sc-7383, against phosphorylated AKT1/2/3 (Ser 473), sc-33437 and against phosphorylated RSK1/2 (Thr 359/Ser 363), sc-12898-R. Protein A/G PLUS-agarose, sc-2003 were also purchased from Santa Cruz Biotechnology . The rabbit polyclonal antibody against AKT and phosphor-(Ser) 14-3-3 binding motif antibody were purchased from Cell Signaling Technology . Chemicon  supplied the first antibody, raised against NHE1. The second antibody goat anti-mouse IgG HRP conjugate (W4021) was from Promega  and goat anti-rabbit IgG HRP conjugate (sc-2004) from Santa Cruz Biotechnology . ECL detection reagents were from Amersham Biosciences, Buckinghamshire, UK. Invitrogen Corp.  provided BCECF-AM, an acetoxymethyl (AM) ester of 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF).P < 0.05.Differences between multiple groups were evaluated by one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD) multiple comparison test for unequal replications. The level of significance was set at i inreased from 7.05 ± 0.13 (n = 32) to 7.48 ± 0.06 (n = 32) after 3 days of treatment  to 7.34 ± 0.02 (n = 165), and for 4 weeks from 7.18 ± 0.02 (n = 114) to 7.58 ± 0.01 (n = 153). Alkalinization of astroglial cytoplasm indicates, most likely, an increase in H+ extrusion which, arguably, may result from an increase in activity of NHE1 that represents the main pathway for proton efflux.Chronic treatment with fluoxetine caused a concentration- and time-dependent intracellular alkalinization Figure . In astrt Figure . When cet Figure , with a The activity of NHE1 is known to be regulated by the transporter phosphorylation  treatment of astroglial cells with fluoxetine indeed triggered ERK6 Figure . Similarr Figure  indicatir Figure  and by 1r Figure .Fluoxetine also potentiated the phosphorylation of RSK in cultured astrocytes. Treatment of cells with 1 and 10 μM fluoxetine for 20 min induced a significant increase of RSK phosphorylation Figure . The levi changes in individual cells following acidification induced by 20 mM NH4Cl  the NHE1 phosphorylation; (iii) the interaction of MAPK/ERK1/2 and PI3K/AKT signal pathways; (iv) the RSK phosphorylation; and (v) the NHE1 activation following acidic stress.Proton homeostasis and maintenance of intra- and extracellular pH in the brain is critically important for normal activity of neural cells and therefore for brain functions. Decrease in brain pH is associated with variety of pathological conditions, such as ischemia and epilepsy , thus generating diacyglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3). The fluoxetine-induced and 5-HT2B receptor-mediated ERK1/2 phosphorylation as well as the effect of fluoxetine on epidermal growth factor receptor (EGFR) phosphorylation could be abolished by (i) AG1478, an inhibitor of the EGFR tyrosine kinase and by (ii) GM6001, a potent and broad-based inhibitor of Zn2+-activated metalloproteinases (Li et al., 1/2. The RSK belongs to highly conserved Ser/Thr kinases that are involved in many cellular processes (Anjum and Blenis, As expected, both NHE1 and RSK were phosphorylated by acute treatment with fluoxetine, suggesting RSK was involved in NHE1 phosphorylation. This was suppressed by inhibition of either MAPK/ERK2 Figure . The NHE1/2 and AKT in astrocytes. Conceptually, the PI3K/AKT and Raf/MAPK/ERK1/2 represent two parallel signal pathways, although, the interactions between PI3K/AKT and Raf/MAPK/ERK1/2 cascades may occur at several different stages and could be either positive or negative. In the present work we found that PI3K/AKT and MAPK/ERK1/2 signaling potentiated each other (Figure 1/2 in ammonium-induced ERK1/2 phosphorylation in astrocytes, since ERK1/2 phosphorylation was inhibited by both the MAK inhibitor U0126 and PI3K/AKT inhibitor, LY294002 (Dai et al., 1/2 pathway at/or upstream of RAS (Hawes et al., 1/2 potentiates the PI3K/AKT signaling pathway.We found that fluoxetine at clinically relevant concentrations induced phosphorylation of both ERKr Figure . Previoui increases in astrocytes chronically treated with fluoxetine in a time- and concentration-dependent manner. This reflects changes in pH homeostasis that may influence brain pH and astrocytic functions and may contribute to the drug action. Changes in pHi significantly affects astroglial physiology. Addition of lactate acid or a decrease of pHi inhibits communications through gap junction and facilitates internalization of Cx43 in astrocytes (Anders, i (Pappas et al., i, for instance, inhibited conversion of inactive phosphorylase b to active phosphorylase a, thus decreasing glycogenolysis (Danforth, 2+]i rises (Ozawa, + concentration (Hof et al., +, glutamate and adenosine is also dependent on glycogenolysis (Xu et al., The pH Anders, . Astrocys Ozawa,  by varioLP conceived and designed experiments; JR, DS, QB collected and analyzed the data; LP and AV interpreted the data and wrote the paper. All authors commented on the manuscript and have approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


An unusual cause of an allergic reaction to food is the passive transfer of specific IgE through blood product transfusions. We report two separate allergic reactions due to passive transfer of food specific IgE from a pooled platelet transfusion.A non-atopic 8 year old boy received multiple blood product transfusions as part of his treatment for meduloblastoma. He subsequently experienced anaphylaxis to salmon. Within minutes of eating salmon, he developed angioedema of the lip, facial erythema, throat discomfort and low blood pressure. Before this episode, he regularly ate fish with no reaction. The passive transfer of food specific IgE was suspected and he was advised to carry an epinephrine auto-injector and avoid all vertebrate fish. Specific IgE to salmon by ImmunoCAP was positive. Follow-up was arranged to follow his specific IgE to salmon with the expectation that his allergy would resolve. One week after his anaphylactic episode to fish, he developed an allergic reaction to peanuts. He ate a chocolate peanut butter cup and within 10 minutes he vomited, developed angioedema of the lip and experienced lethargy. Previously, he routinely ate peanuts without any symptoms. Skin prick testing showed positive results to peanut, salmon, mixed fish, and tree nut mix. He had a positive ImmunoCAP to peanut. Approximately 6 months later, he had undetectable ImmunoCAP results to both salmon and peanut. He resumed consumption of salmon and peanuts with no reaction. As part of the adverse event investigation by Canadian Blood Services all donors associated with the reaction were contacted and one donor stated that they have a severe allergy to peanuts, tree nuts, shellfish, and all fish including salmon. This information implicated one specific pooled platelet transfusion in which the platelets were suspended in the plasma of the atopic donor. The donor has been excluded from future donations.To our knowledge, this is the first reported case of two allergic reactions to food documented to be caused by passive transfer of food-specific IgE from pooled platelets. This case shows that if a passive allergy from a transfusion occurs, consideration should be given to look for additional passively transferred specific IgE. Knowledge of the allergies in the blood donors can assist in the investigations and avoidance instructions.


Multigram-scale synthesis of a sulfation-site programmed dodecasaccharide is described. CXCL8- and CXCL12-mediated in vitro and in vivo biology is shown to be regulated by a single sulfation site change. in vitro and in vivo biology mediated by the two important chemokines, CXCL12 (SDF1α) or CXCL8 (IL-8).The multigram-scale synthesis of a sulfation-site programmed heparin-like dodecasaccharide is described. Evaluation alongside dodecasaccharides lacking this single glucosamine O6-sulfation, or having per-O6-sulfation, shows that site-specific modification of the terminal glucosamine dramatically interconverts regulation of  N-substituted glucosamine (GlcN) and a hexuronic acid, which is either glucuronic acid (GlcA) or the more conformationally-flexible iduronic acid (IdoA). Chains of approximately 50 and up to ∼200 monosaccharides are covalently linked to core proteins to form HS proteoglycans (HSPGs) that are embedded in the cell membrane or extracellular matrix, where they mediate interactions key to coordination of signaling, proliferation and stem-cell differentiation.1 HS binds over a hundred proteins2 and plays ubiquitous regulatory roles in angiogenesis, inflammation and stem-cell differentiation and thus HS-related oligosaccharides offer numerous potential applications as therapeutics.3 Following the discovery of the specific interaction between heparin and anti-thrombin III approximately thirty years ago and the major medical importance of heparin, there has been a continued search for H/HS-like structures with defined biological structural specificity for other targets. The success of Arixtra (Fondparinux) as a synthetic anti-coagulant LMWH heparin (5-mer) has illustrated the clinical potential for pure short synthetic heparin-related fragments.4 Much important biological regulation is effected by longer sequences and the great heterogeneity of natural extracts makes synthetic routes essential, however, particularly progression to in vivo biology of any homogenous longer heparin-like oligosaccharides has been precluded by lack of scalable syntheses. We recently exploited large-scale iduronate synthesis6 to generate gram-scale quantities of structurally-defined per-6-OH heparin-like dodecasaccharide 1.7 This iterative synthetic strategy was also applied to synthesis of the new homogeneously per-6-O-sulfated heparin-like dodecamer 3.8 We report here application to synthesis of the first example of a site-specifically mono-6-O-sulfated heparin-like dodecasaccharide, 2. Provision of 2 on approximately 2 gram scale firmly establishes this approach as a practicable general route to various long heparin-like oligosaccharides on scales exceeding other GAG 12-mer syntheses by two orders of magnitude and underpins the practicability of delivering long synthetic GAGs for in vivo biology.The glycosylaminoglycan (GAG) heparan sulfate (HS) is a linear oligosaccharide polymer of alternating 11 Identifying new, discrete synthetic HS structures with defined biological effects is central to understanding GAG microstructure-specificity and the potential for discovery of new biomedical H/HS mimetics.12Click here for additional data file.HS is organized into domains of high and low sulfation, with high sulfation domains consisting predominantly of GlcN- and O6, and IdoA O2 sulfation. These higher sulfation domains are principal effector parts of HS, binding ligand cytokines leading to receptor activation.14 HS-binding chemokines CXCL8 (IL-8), CCL1915 and CXCL1218 (SDF-1α) are involved in angiogenesis, metastasis and inflammation and are thus high value biomedical targets. Specific effector HS ligands for these chemokines remain undefined, and identification of specific structures which differentially modulate biological activity of these chemokines would provide new insight into GAG-chemokine chemical biology and potential for new drug developments.GAGs are critically involved in chemokine biology, regulating oligomerization and phenotypic effects.O-sulfation is important for differential effects of H/HS oligosaccharides,19 and as a regulator of the biological activity of several HS-dependent cytokines, such as CXCL820 and CXCL1221 as well as FGF222 and VEGF.23 Prior work has indicated that homogenous sulfation levels and locations can affect the binding preferences of other GAGs to target proteins.25 Access to site-specific variations of longer HS sequences is essential to investigate whether site-specific 6-O-sulfation variations can determine biological effect in longer native-like ligands.Considerable evidence indicates that the overall level of glucosamine-6-1–3), and most particularly our capacity to deliver much larger scale syntheses than previously available, provides a tool-kit to interrogate the effects of both level and site-specificity of defined sulfation in vitro and in vivo. We report here that the presence or absence of a single site-specific sulfate interconverts CXCL8 or CXCL12 inhibitory effects between 1 and 2, both in vitro and in vivo. These dramatic differential effects are not evident for the per-6-O-sulfated dodecasaccharide analogue 3, therefore indicating here that charge density is not a critical mediator of cytokine-mediated effects, as previously suggested.22The syntheses of a series of three sulfation-programmed oligosaccharides , 1–3, an2 with an additional O6-sulfate at one specific terminus was achieved by capitalizing on the robust scalability demonstrated for prior synthesis of homogenous dodecasaccharides.7 Thus, 10-mer acceptor 4 was converted into the protected 12-mer 6 by coupling with O6-modified disaccharide donor 5,8 which programmes for subsequent GlcN 6-O-sulfation at the non-reducing terminal unit only  and subsequent debenzylation/azide reduction using Pd(OH)2/C generated ∼2 g of the site-specifically GlcN-6-O-sulfated penultimate dodecasaccharide 8. Final N-sulfation afforded the novel target dodecasaccharide 2 on ∼2 g scale, demonstrating the generic scalability of this iterative synthetic approach.8Generation of novel HS 12-mer nit only , by tyinThe homogeneity of all materials was unambiguously confirmed by LCMS, MS and 800 MHz HSQC NMR analyses. Most particularly, ESI mass spectral data were fully consistent with a mono-O6-sulfated species, whilst the HSQC data provided confirmation of single site of sulfation significantly differentiated from the main-chain C6 methylene units .O-sulfated dodecasaccharide 2 alongside fully 6-O-desulfated 12-mer, 17 and the fully 6-O-sulfated 12-mer 3,8 provided a toolkit to evaluate the biological effects of modifying a single sulfation site at the non-reducing terminal glucosamine in long synthetic heparin-like species, as well as control evaluation of full O6-sulfation both in a otherwise identical synthetic 12-mer (3) and a digest HS 12-mer. It must be emphasized that whilst all synthetic sequences shared the same terminal 4-O-sulfate, digest HS – the regular standard in biological assays – also lacks a 4-OH as digests contain a terminal unsaturation, and thus there is no rationale to consider the inclusion or absence of a 4-OH relevant, as this is invariably also a synthetic construct from other synthetic routes. Further, it is noted that the only variable between the synthetic species were 6-O-sulfation changes and any hypothetical relative effects from a 4-O-sulfate should therefore be discounted.‡Migration and pERK signaling assays compared synthetic 4-OS-terminated per-6-OS sequence 3 with commercial digest-derived dp12 ESI Fig. S25 This is further supported by our migration and pERK signaling assays comparing synthetic 4-OS-terminated per-6-OS sequence 3 with commercial digest-derived dp12  or a single, non-reducing end GlcN-O6 sulfate (2). The fully 6-O-sulfated dodecasaccharide 3 did not show this inhibition switching, and its behaviour was very similar to that of de-polymerised digest dp12 derived from commercial heparin  was effected with site-specifically sulfated 2 while the most effective inhibitor of CXCL8 was 1, reducing STAT3 phosphorylation >80% at 1 and 10 μg ml–1  were observed in vivo. CXCL8 or CXCL12 and oligosaccharide-impregnated sponges were implanted subcutaneously in mice to determine the effects of the oligosaccharides on chemokine-induced biology . These findings indicate that other significant site-specific sulfation-determined effects await discovery and biomedical exploitation.In summary, we demonstrate that an efficient iterative chemical strategy now extends to multigram scale synthesis of a site-specifically sulfated dodecasaccharide establishing this as a general gram-plus scale access to differentiated, structure-programmable synthetic dodecasaccharides. This has thereby also enabled the first Cancer Research UK (C2075/A9106) and the UK Medical Research Council (G0601746 and G902173) are thanked for project grant funding (GCJ and JMG). The EPSRC National Mass Spectrometry Service, Swansea are thanked for mass spectroscopic analyses, and Dr Matthew Cliff (MIB NMR Facility) and Rehana Sung (LCMS).


Ribonuclease H1 (RNase H) enzymes are well-conserved endonucleases that are present in all domains of life and are particularly important in the life cycle of retroviruses as domains within reverse transcriptase. Despite extensive study, especially of the E. coli homolog, the interaction of the highly negatively charged active site with catalytically required magnesium ions remains poorly understood. In this work, we describe molecular dynamics simulations of the E. coli homolog in complex with magnesium ions, as well as simulations of other homologs in their apo states. Collectively, these results suggest that the active site is highly rigid in the apo state of all homologs studied and is conformationally preorganized to favor the binding of a magnesium ion. Notably, representatives of bacterial, eukaryotic, and retroviral RNases H all exhibit similar active-site rigidity, suggesting that this dynamic feature is only subtly modulated by amino acid sequence and is primarily imposed by the distinctive RNase H protein fold. This active-site sequence motif and requirement for Mg2+ is widely shared with other nucleases, suggesting a common catalytic mechanism1.Ribonuclease H1 (RNase H) proteins are well-conserved endonucleases that are found in all domains of life and cleave the RNA strand of an RNA-DNA duplex substrate. The RNase H active site canonically consists of a highly conserved DED(D) motif , three tEscherichia coli (ecRNH), in which this active-site motif is represented as D10, E48, D70, and D1342 , and one associated with D10, E48, and D70 (denoted the B site)7 7 ; the B sve site .2+ with ecRNH, monitored independently by1H and25Mg2+ NMR, suggests that only a single ion binds to the protein in the absence of substrate5. The identified binding site has relatively weak affinity;dK has been reported in the micromolar5 to low millimolar range12. The second site may be occupied only upon binding of substrate8, possibly due to the presence of high local concentration in the ion cloud of the highly negatively charged nucleic acid molecule. Conformational changes in the active site upon binding the first ion have been suggested as well, with the second site proposed as being responsible for the attenuation of activity at high ion concentrations13. Collectively, these results have been used to propose both a one-metal14 and a two-metal16 catalytic mechanism. Computational work using the quantum mechanics/molecular mechanics (QM/MM) method applied to theBacillus halodurans19 andHomo sapiens20 complexes generally supports the two-metal mechanism.Experimental evidence from nuclear magnetic resonance (NMR) studies locates the area surrounding the active site as the region most susceptible to perturbation upon interaction with ions . Titrati21. For this reason, inhibitors of retroviral RNase H domains, particularly that of HIV, have been widely reported22, although none to date have reached clinical use. Most such inhibitors interact with the active site in the metal-bound state22 and therefore must be selective for retroviral RNase H domains to find clinical utility. The HIV RNase H domain (hivRNH) has been reported to bind two metal ions even in the absence of substrate23, although the reason for this difference in behavior between hivRNH and ecRNH is not clear. TheHomo sapiens RNase H domain (hsRNH) has not been structurally characterized in the absence of substrate and its binding behavior is less well understood. However, it has higher sequence identity and is more structurally similar to ecRNH than hivRNH.RNase H domains are widely distributed in cellular organisms, but also occur as a component of the reverse transcriptase protein found in retroviruses, in which they are required for viral proliferationapo state for the binding of a single Mg2+ ion24. However, experimental constraints prevent the detailed observation of the protein’s dynamic behavior in the presence of a bound ion at ps-ns timescale. The present work aims to more fully understand the dynamics of ecRNH in the Mg2+-bound state through molecular dynamics simulations of ecRNH in the presence of single Mg2+ ions in various positions in the active site as suggested by crystallographic studies. In addition, the dynamic behavior of the active site in theapo state is compared with homologs from other organisms.Combined nuclear magnetic resonance (NMR) and molecular dynamics (MD) studies of the behavior of the ecRNH active site residues suggest that the residues of the ecRNH active site are preorganized in the3) or by prediction using the H++26 pKa predictor. Unless otherwise specified, all simulations were performed at a pH of 5.5 to recapitulate the conditions used in prior NMR experiments on ecRNH28. Crystallographic water molecules were removed from all structures prior to solvation using Schrodinger’s Maestro tool, version 8.5 or 9.1, as distributed in the Desmond software package. Simulations were performed as described29 using Desmond academic release 3 or source release 2.4.2.130. Proteins were described with the Amber99SB force field31, solvated with TIP3P water32 in a cubic box with a 10Å buffer region from solute to box boundary, and neutralized with Cl− ions. Bonds to hydrogen atoms were constrained using the M-SHAKE algorithm33. Simulations containing Mg2+ ions used the Aqvist parameter set34. Electrostatics were calculated with the PME method using a 9Å cutoff. All simulations used a 2.5fs inner timestep on a 1-1-3 RESPA cycle and were performed in the NVT ensemble using a Nosé-Hoover thermostat after equilibration to constant box volume for 5ns in the NPT ensemble. All simulations described in this work were run for 100ns unless otherwise noted. These simulation conditions applied to theapo state of RNase H homologs have previously been shown to reproduce NMR data well35.For each initial protein structure, protonation states for titratable residues were assigned either by experimental measurement  structures used for initiating trajectories are listed, along with their resolutions and any system-specific preparation steps, in2+-bound state (PDB ID 1RDD)6. However, the position of the ion identified in this structure is not stable in simulation and exits the binding site immediately upon initiation of the trajectory. The ion transiently interacts with the protein at a variety of sites on the protein surface over the course of the 89ns trajectory but never returns to its original position in the active site  exhibits disorder in both the active-site and handle loops7, the ion positions were instead modeled into the apo ecRNH structure (PDB ID 2RN2) by superposition. For the model of the B-site Mg2+ ion, the rotamer of E48 was also corrected to match the orientation observed in the 1G15 structure. For comparison to an alternative homolog, the Mg2+ ion in the B site was also modeled into the RNase H structure from the thermophilic bacteriumThermus thermophilus , whose structure was also solved in the absence of divalent ions38.Additional simulations were carried out under the same conditions for single Mg2+ ions were found to be stably associated with the ecRNH active site in both simulations, despite the fact that the ions were modeled into a structure that did not originally contain them  from E48 (the unique participant in the B site)24, comparisons of the two trajectories provide an additional opportunity to distinguish between these two sites.Of the four conserved catalytic residues, D134 is known to be somewhat dispensable; catalytic activity is retained, though reduced, by substitutions with N or H, which also increase thermostabilityAlthough single metal ions in both sites were stably bound to the protein, the RMSD over the course of each 100ns trajectory was larger for the ion in the A site (1.2Å) compared to the B site (0.6Å), which in turn is similar to the RMSD of a 30ns control simulation of ttRNH with an ion modeled into the B site (0.6Å). Additionally, a small amount of motion in the direction of the B site was observed for the ion in the A site; the initial and final positions differ by 1.7Å . , between the XMRV ∆C mutant compared to the HIV homolog , or between any of the retroviral domains compared to ecRNH . This re2+ ions. We observe that the well-studied RNase H homolog fromE. coli contains a conformationally preorganized active site that is highly rigid on the ps-ns timescale in the presence of a single Mg2+ ion, which is likely located at the B site crystallographically identified by examining the Mn2+ complex. Additionally, we examined theapo state dynamics of the active site—previously validated by comparison to NMR data in the case of theE. coli homolog24—and found that similar patterns of active-site rigidity are present in all homologs examined, including representatives of bacterial, eukaryotic, and retroviral RNases H. This result suggests that active site dynamics are only subtly modulated by amino acid sequence and are primarily imposed by the characteristic protein fold. Although it has long been recognized that RNases H share similar topologies and active-site conformations with other endonucleases46, the present work extends this observation from static crystal structures to dynamics on the ps-ns timescale.In this work we aimed to use molecular dynamics simulations to understand the dynamic behavior of the RNase H family in complex with catalytically required MgHomo sapiens RNase H homolog and theThermus thermophilus argonaute protein (a distant RNase H homolog with similar active-site architecture) in complex with two Mg2+ ions and substrate analogs also observe high active-site rigidity47; this is consistent with the present data and implies that active-site preorganization is a general property of this larger family of nucleases. Although selective inhibitors of the HIV RNase H domain have been developed based on the hypothesis that the metal ion dependence of the HIV domain’s catalytic mechanism differs from that of the human homolog15, it is likely that the physical origin of this selectivity is not dependent on the active-site conformations sampled on the ps-ns timescale.Simulations of thedoi:10.5281/zenodo.843148ZENODO: Molecular dynamics derived side chain order parameters for Asp, Glu, Asn, and Gln residues in ribonucleases H, and molecular dynamics trajectories for E. coli ribonuclease H. et al., JACS 2014 -ref 24) it would be an excellent topic paper in the area of protein structure and function for an undergraduate or graduate seminar course. This is an exciting paper which beautifully demonstrates the value of MD simulations coupled with experimental data for understanding structure-dynamic-function mechanisms. The research appears to be carefully executed, with proper controls, and the paper is well written. Together with related papers , it would be helpful to document the active-site sequence variation across these homologs to support this conclusion.all simulations were performed at a pH of 5.5 to recapitulate the conditions used in prior NMR experiments on ecRNH”. What is the pH optimum of enzyme function?  Is there any information about “physiological pH”. Based on the pKa’s of active site ionizable groups, how might the pH selected for this study impact the conclusions.The authors state “The work is suitable for publication inI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. A new Figure 8 has been added to the paper, showing a structure-based sequence alignment for all of the bacterial , eukaryotic (hsRNH), and retroviral  RNase H structures used to initiate simulations as part of this work.The pH optimum of the RNase H reaction was previously mentioned only in the Introduction (refs 3 and 4). We have added a comment in the Methods section on the motivation for the choice of pH 5.5 in the NMR experiments and the impact of this on the simulations. In brief, the active site residues are expected to be unprotonated in the presence of Mg2+ at both the experimental pH of 5.5 and the reaction optima of 7.5-9.5.Thank you for your thoughtful comments on our paper. In response to your suggestions, we have published a version 2 of the paper which addresses both of your points, specifically: The authors set out to use molecular dynamics simulations to the binding of a required magnesium ion on Ribonuclease H1, utilizing a number of distinct structures including those crystallized in different conditions or forms (Apo or different types of Holo forms) and orthologs. The main result found by the authors is that the binding site is very rigid and pre-organized in the presence of a single magnesium ion. The authors show that this observation holds for all orthologs studied. Based on this, the authors suggest that 'the active site dynamics are only subtly modulated by amino acid sequence and are primarily imposed by the characteristic protein fold'. It is indeed interesting that most RNases not only conserve the structure of the binding site but also dynamic properties in the ns-ps range. However, I feel that showing a multiple sequence alignment of the RNase H family highlighting the members used in this study as well as the positions that constitute the substrate and metal binding sites would considerably help understand that indeed this pre-organization comes about despite divergent binding-site sequences. Without seeing such an alignment is hard to accept that the rigidity of the binding-site is not affected by changes in binding-site sequence. If the binding-site sequences turn out to be highly conserved upon analysis of the multiple sequence alignment, it may be necessary to perform mutations in silico in the binding-site to support the conclusion above.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you for your thoughtful comments on our paper. In response to your suggestion, we have published a version 2 of the paper containing a new Figure 8, which shows a structure-based multiple sequence alignment for all RNases H examined (see reviewer response above). The positions of the active-site residues and the substrate-binding regions have been highlighted on the MSA to facilitate comparison of the sequences within these regions. The point you raise can be subtle - since some sequence similarity is required for maintenance of the overall fold, rather than for specific substrate interactions - so we have edited the text of the Conclusions section for clarity and changed the final sentence of the Abstract to read "...may primarily be imposed...". We have also updated the Conclusions section with a new reference to a recent paper on the evolution of the RNase H superfamily (ref 50).