Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs - one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I.The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from  N-glycosylation plays a pivotal role in glycoprotein maturation and functioning. Bulky N-glycans located at the surface of secretory and cell membrane glycoproteins molecules are mostly exposed to the extracellular space. These oligosaccharides serve multiple functions acting mainly as stabilizers and protective shields for the glycoprotein outside the cell and as recognition targets in adhesion and immune response modulation. Nevertheless, intracellular membrane glycoproteins facing the lumen of the subcellular organelles may be heavily glycosylated also, suggesting specific functions within the cell. The roles of these oligosaccharides in controlling intracellular processes such as folding, sorting and traffic of proteins have been extensively investigated  and tyrosinase-related protein-2 (TRP-2). These proteins determine melanin synthesis in pigment cells and are important players in mammalian pigmentation A crucial role in the ERQC is played by calnexin (CNX) and calreticulin (CRT). These mannose binding lectins assist tyrosinase during its folding within the ER. We have previously shown that abolishing CNX association by inhibiting the N-glycosylation processing or impairing tyrosinase tethering to the membrane results in a misfolded protein unable to exit the ER Oligosaccharides could be specific recognition elements in the ERAD pathway. An important component of the ERAD pathway is EDEM 1, a mannosidase-like chaperone that regulates the extraction of misfolded polypeptide chains from calnexin cycle and sends them back to the cytosol for proteasomal degradation Streptomyces castaneoglobisporus tyrosinase. We propose an N-glycan distribution covering two opposite sides of the molecule, occurring either in the close proximity or remote of the active site area. While two oligosaccharides located opposite the active site were specific CRT targets, glycosylation mutants analysis provided evidence that a pair of N-glycans covering the active side have been crucial for its ER export towards the secretory pathway. Moreover, while the N-terminus N-glycans are required for the early stages of the folding process, the C-terminus are conditional for completion of its post-translational productive folding.In this paper we investigate the role of conserved individual N-glycans in human tyrosinase maturation and intracellular traffic. To analyze the localization of N-glycans, a 3D model of the central active site unit of human tyrosinase was derived based on the X-ray crystallography data of Human embryonic kidney (HEK 293T) and A375 cells were obtained from the European Collection of Animal Cell Culture . Cells were grown in DMEM, respectively RPMI 160 medium (EuroClone) containing 10% fetal calf serum (Biochrom), 50 units/mL of penicillin and 50 mg/mL of streptomycin (Invitrogen) and maintained at 37°C with 5% CO2. Rabbit anti-calnexin antiserum was a kind gift from Dr. J.J. Bergeron  and the anti-ERGIC 53 antibody was from Dr. Hans-Peter Hauri . The mouse monoclonal anti-hemagglutinin antibody was from Santa Cruz Biotechnology . T311  is a monoclonal antibody IgG2a recognizing human tyrosinase. The cDNA encoding the mouse EDEM fused to an HA-tag in the pCMV-SPORT2 vector was a kind gift from Prof. K. Nagata and Dr. N. Hosokawa . Kifunensine and lactacystin were from Toronto Chemicals, the other reagents were from Sigma.BamHI/XhoI site of pTriEx1.1.1 vector . The sliding glycosylation mutant construct was obtained based on tyrosinase mutant cDNA Δ7 with the following codon changed: Q378N (s7→378). For construction of the triple mutations Δ (N86-N111-N161) and Δ (N290-N337-N371), the mutagenic primers correspond to the middle glycosylation site; for the first two PCR reactions the mutants lacking single glycosylation site were used as template. Unglycosylated mutant Δall was obtained with the mutagenic primers corresponding to the s4-N230 site based on the cDNA sequences of Δ and Δ. Nucleotide sequence of all constructs was confirmed by sequencing.Mutant proteins were obtained based on the human tyrosinase cDNA expression plasmid, pTriEx-WT obtained previously 35S] methionine/cysteine  for 20 minutes and chased for the time specified. Immediately after chase cells were harvested in cold PBS with 20 mM N-ethylmaleimide (NEM). Cells were then lysed with CHAPS lysis buffer  benzene sulfonylfluoride and E-46) for 1 h on ice. In some experiments lactacystin and kifunensine were added during starvation time and maintained during the pulse-chase period.Constructs were transiently transfected in HEK 293 and A375 cells. Semi confluent HEK 293 cells (50–70% confluence) 24 h post-seeding in 6-well dishes were used to transiently express tyrosinase cDNAs (3 µg of DNA/well) using polyethylenimine (PEI) solution  and 90% confluent A375 cells were transfected using Lipofectamine 2000 (Invitrogen) using 7.5 µL of Lipofectamine for 3 µg of DNA. Cells were analyzed 24 h after transfection. For metabolic labeling, transfected cells were starved in the cysteine – methionine free medium for 1 h, pulse labeled with 100–150 µCi of [35S]-labeled cell lysates were centrifuged, and supernatants were incubated with T311 antibodies (1∶50) overnight at 4°C, followed by the addition of 20 µl of protein A-Sepharose and further incubation for 1 h at 4°C. The slurry was washed three times with 0.5% CHAPS in HEPES buffer. Tyrosinase was eluted by boiling the slurry for 5 min in SDS sample buffer with 5% 2-mercaptoethanol or DTT. In some experiments, tyrosinase was eluted from protein with 1% SDS and the protein was resolved by SDS-PAGE under non-reducing conditions, i.e. in the absence of 2-mercaptoethanol. Calnexin co-immunoprecipitations were performed as described previously .Streptomyces castaneoglobisporus (sT)  overlaps with the prokaryal tyrosinase from rus (sT) . This roModel refinement of inserts: aa192–196, aa273–282, aa305–308 and of the N-terminus region within aa142–170 which consisted into bringing locally the structure in compliance with the hT secondary structure prediction, results in an increase of the overall model accuracy from 3.17 Å to 2.48 Å root mean square deviation (rmsd), with the global distance test total score raising from GDT-TS = 54.14 to 62.91. The largest local deviation of the model from an optimal alpha carbon path does not exceed 6 Å rmsd and reach its maximum in the insert aa192–196, a region that does not inflict on the placement of N-glycans, nor onto the location of cysteine residues in the Cys2 domain .All major intrinsically disorder predictors - FoldIndex, disEMBL, IuPred, RONN etc indicate a high propensity for flexibility of the hT sequence in the region aa290–315, while the DLP predictor The model hosts also the last five out of seven glycosylation sites of human tyrosinase, s3:N161, s4:N230, s5:N290, s6:N337 and s7:N371. In sequence these are evenly distributed . HoweverAssessment of the Cys1 sequence properties failed to result into a reliable 3D model. Secondary structure predictors pinpoint along Cys1 four beta strands followed by one helix with 3 or 4 turns toward its C-terminal end. Due to this mainly beta architecture and to the large number of cysteine residues and their pattern, the fold recognition predictors such as Phyre The remarkable conservation of the active site area explains the identity of function between eukarial and prokarial tyrosinases with eukarial enzyme acquiring during evolution specific molecular determinants such as the Cys1 domain, the TM domain and the seven N-linked glycans. To further investigate the role of N-glycans in human tyrosinase function we constructed and characterized tyrosinase glycosylation mutants.all were constructed and analyzed in terms of site occupancy, N-glycan processing and enzymatic activity. The N-glycosylation mutants were expressed in a previously validated mammalian system using kidney embryonic cells Single glycosylation mutants (Δ) at all seven sites, N86, N111, N161, N230, N290, N337 and N371, two triple mutants Δ and Δ and the unglycosylated mutant ΔSix out of seven sites were occupied, as seen from the decrease in mobility caused by the ablation of a consensus site in the single mutants shown in sens) structures of high mannose/hybrid type characteristic to the ER/early Golgi to complex type structures EndoH resistant (res) acquired within the Golgi. Expression of wild type tyrosinase in HEK cells yielded an EndoH sensitive and an EndoH resistant population  derived from While the modeled region did not include the Cys1 domain with the first two N-terminal glycans s1:N86 and s2:N111, it contained the tyrosinase functional unit Cu A-linker-Cu B and the rest of 5 N-glycosylation sites. The model was probed by single and multiple N-glycosylation mutants and the assessment of their role in folding, CNX/CRT interactions, ERAD, intracellular traffic and enzyme activity.We found that only four of the five modeled sites were actually occupied, while Asn at site s5 was not glycosylated. This is consistent with previously reported data The four occupied sites are clustered in two groups, the C-terminal N-sites s6 and s7, located close to the active site, and s3 and s4 on the opposite side of the protein. In sequence these belong to the extended Cu B and extended Cu A, respectively and have in common the fact that they are located on bends which follow or precede regular helices. This is consistent with the very frequent location of N-glycosylation sites at points where any change of the local structure of the polypeptide chain is needed. Such N-sites act as landmarks for ending or starting stretches of regular secondary structure and increase the folding efficiency On the other hand the main structural difference between the two groups is the proximity to the active site and this remarkably correlates with the ability of mutants to recover at 31°C. Both distal N-glycosylation Δ3 and Δ4 mutants are not terminally misfolded as they retain reduced enzymatic activity and the properties of temperature sensitive mutants. In contrast the absence of proximal sites s6 and s7 has far more severe effects on tyrosinase function. Not only that the activity of Δ6 and Δ7 mutants is completely lost, but very low or no recovery is found at 31°C. In addition these C-terminal sites, s6 and s7, are compulsory for tyrosinase ER maturation since in their absence tyrosinase is recycled between the ER and Golgi and eventually retained within the ER and targeted for degradation by EDEM1. The lower stringency for the presence of distal N-glycans at s3 and s4 is consistent with the conservation of these sites during evolution. For example s3 is occasionally missing in tyrosinases or TRP2s of some Salmonidae and Ictaluridae fishes, and it is always missing from TRP1s while s4 is absent in some Salmonidae fishes or even some Bovidae mammals. By contrast s6 and s7 are strictly conserved in all vertebrate tyrosinases, even though s6 is missing in TRP1 and TRP2 of some lower species. The fundamental role of s7 is also indicated by that it is located on a complex loop connecting the two helices hosting H363, H367, H390 involved copper binding. Furthermore, as seen from Tyrosinase folding starts co-translationally during the polypeptide translocation into the ER lumen. However the completion of a productive folding requires post-translational steps as previously reported The first two N-terminal glycans s1:N81 and s2:N111 that remained structurally unaccounted in our model are located within the Cys1 domain, aa20–140, containing an EGF signature but also a region with high disorder propensity located in-between aa 38–53. They were shown to mediate tyrosinase co-translational folding through their interaction with CNX immediately after the removal of the signal sequence All glycosylation mutants are subjected to degradation through the ERAD pathway with Δ6 and Δ7 sharing some peculiarities. The first step of the intricate timing mechanism for ERAD is the mannosidase I catalyzed cleavage of a mannose residue of proteins retained for longer periods within the ER That N-glycans do not play equivalent roles in protein processing has been shown previously for the cystic fibrosis membrane conductance regulator, a channel protein involved in the pathology of the cystic fibrosis Taken together the herein data indicate that in contrast to the glycosylation of distal N-sites, the presence of N-glycans in precise conserved C-terminal location in close vicinity to the active site is critical for the correct folding of the enzyme. The absence of post-translational modification at these locations alters irreversibly the maturation pathway in a manner recognized by the quality control and degradation ER systems.Figure S1The potential glycosylation site s5 is not occupied in melanoma A 375 cell line. Transfected A375 cells were lysed and postnuclear lysates were subjected to EndoH digestion and blotted with tyrosinase antibody (T311). The Δ5 mutant acquires complex glycans resistant to EndoH and migrates at the same molecular mass as WT tyrosinase.(TIF)Click here for additional data file.Figure S2Association of the Δ1, Δ2, Δ3 and Δ6 mutants with calnexin and calreticulin. Cells were transiently transfected with Δ1, Δ2, Δ3 and Δ6 mutants. 24 h post-transfection cells were pulsed for 20 minutes and chased for 0, 30, 60, 120 min. Cell lysates were sequentially precipitated with anti-calnexin or anti-calreticulin and T311 antibodies . To determine the total amount of tyrosinase an aliquot of the lysate was precipitated with T311 antibodies (Ip TYR). Samples were subjected to 10% SDS PAGE and autoradiographed. One of at least two representative experiments is shown. The ratio CNX (▴) and CRT (•) bound tyrosinase/total tyrosinase over time has been plotted.(TIF)Click here for additional data file.Figure S3Traffic of the single N-glycosylation mutants beyond the ER in the presence of nocodazole. Cells were transiently transfected with Δ1–Δ6 mutants. 5 hours before immunofluorescence cells were incubated or not with nocodazole (NOC), fixed and double-labeled with anti-tyrosinase IgG2a (Tyr) and anti-ERGIC-53 IgG1 (ERGIC-53) monoclonal antibodies. Alexa Fluor 488 anti-monoclonal IgG1 and Alexa Fluor anti-monoclonal IgG2a were used as secondary antibodies. The merged images showing co-localization of tyrosinases with ERGIC-53 are also shown.(TIF)Click here for additional data file.


These activities were inhibited by reducing agents, but no disulphide bonds were detected between RalA and ERp57. Mutation of all four of ERp57’s active site cysteine residues blocked sensitivity to reducing agents, suggesting that redox-dependent conformational changes in ERp57 affect binding to RalA. Mutations in the switch II region of the GTPase domain of RalA specifically reduced or abolished binding to ERp57, but did not block GTP-specific binding to known RalA effectors, the exocyst and RalBP1. Oxidative treatment of A431 cells with H2O2 inhibited cellular RalA activity, and the effect was exacerbated by expression of recombinant ERp57. The oxidative treatment significantly increased the amount of RalA localised to the cytosol. These findings suggest that ERp57 regulates RalA signalling by acting as a redox-sensitive guanine-nucleotide dissociation inhibitor .RalA is a membrane-associated small GTPase that regulates vesicle trafficking. Here we identify a specific interaction between RalA and ERp57, an oxidoreductase and signalling protein. ERp57 bound specifically to the GDP-bound form of RalA, but not the GTP-bound form, and inhibited the dissociation of GDP from RalA  RalA belongs to the Ras family of small GTPases GDIs effectively extract small GTPases from their active location on the surface of membranes by binding to the GTPase domain and by enveloping the C-terminal lipid adduct of the small GTPase, in what is thought to be a two-step process RalA is found on a variety of cellular membranes 2+Studies of the signalling pathways upstream of Ral have almost exclusively concerned its activators. Several GEFs have been described and the most characterised is RalGDS, a Ras effector protein The isolation of organs from animals was done with approval from the Animal Care and Ethics Committee for the Children's Medical Research Institute and The Children's Hospital Westmead , Sydney, Australia.N-acetylcysteine (NAC) and hydrogen peroxide solution were from Sigma Aldrich Pty Ltd.Anti-RalA monoclonal and anti-RalB polyclonal antibodies were obtained from BD Transduction laboratories. Anti-6xHis tag polyclonal antibody was obtained from Santa Cruz Biotechnology, Inc. Anti-ERp57 polyclonal antibody was obtained from StressGen Biotechnologies , and forEscherichia coli and purification using Ni-NTA resin (Qiagen) were carried out according to the manufacturer’s protocol. Large quantities of plasmid DNA for transfection of mammalian cells were prepared using the Wizard Plus Maxipreps DNA purification system (Promega). GST-RalA beads were prepared as previously described 2, 20 mM reduced GSH, followed by dialysis into 20 mM Tris pH 7.4, 150 mM NaCl, 2.5 mM MgCl2.A fragment of human ERp57 cDNA, without the region encoding the N-terminal signal peptide (amino acids 1–23), cloned in a pET9 vector was provided by David Thomas . pGEX-2T-RalA, containing simian RalA cDNA, was provided by Yoshito Kaziro . The RalA and ERp57 coding regions were subcloned into pDONR201 (Invitrogen Corp.) and subjected to site-directed mutagenesis when necessary . Clones were verified by DNA sequencing. The ORFs were further sub-cloned by recombination into pDEST15 , pDEST17  and pcDNA-DEST53  expression vectors (Invitrogen). Expression of 6xHis-ERp57 in v5.2 (Molecular Dynamics).These were performed as described previously Bands of interest were excised from Coomassie Blue-stained polyacrylamide gels. Edman degradation of tryptic peptides was carried out by Newcastle Protein .2) was loaded with 1 µM [3H]-GDP in 10 µl of 20 mM Tris pH 7.4, 50 mM NaCl, 1% Triton X-100, 7.5 mM EDTA, 20 µg/ml leupeptin, 2 mM PMSF, 2x Complete protease inhibitor cocktail (Roche) at 37°C for 20 minutes. 1 M MgCl2 was then added to a final concentration of 10 mM, and the tubes were placed on ice for 5 minutes or until needed. The reaction was started by adding an equal volume of GDI assay buffer  with or without recombinant 6xHis-ERp57.  The reaction was incubated for 10–60 minutes at 37°C. 20 µl aliquots were withdrawn from the bulk reaction at intervals, and then diluted in 500 µl of ice-cold washing buffer  and stored on ice. Samples were filtered through a 0.2 µm nitrocellulose membrane under vacuum in an ice-cold manifold . The membrane was washed three times with 0.5 ml of ice-cold washing buffer under vacuum and then removed and stored in a tray on ice for 3–5 minutes in ice-cold washing buffer. The membrane was blotted with filter paper, air dried, and then cut into individual samples which were incubated with 1 ml of 0.1 M HCl in scintillation vials for 20 minutes at room temperature. Scintillation fluid was added, the vials were shaken well, and then the samples were counted for tritium in a scintillation counter.The method was based on those previously described A431 human epidermal carcinoma cells E. coli as described for GST-RalA 5 cells/dish, and cultured to ∼75–80% confluency overnight. The cells were transfected, and if necessary, serum-starved overnight (as above) and the specified treatments were applied. The cells were washed twice with ice-cold PBS and then lysed on ice by the addition of ice-cold lysis buffer . Cells were scraped and vortexed thoroughly. The lysates were centrifuged at 17,000 g for 10 minutes at 4°C. The supernatant was incubated with 10 µg GST-RalBD protein beads for 30 minutes at 4°C in micro-spin columns, with regular mixing, as previously described 2. Protein was eluted with 30 µl SDS sample buffer, and analysed by SDS-PAGE and Western blotting using the indicated antibodies.The GST-RalBD plasmid construct  was obtained from Larry Feig . GST-RalBD was overproduced in 5 cells per dish, cultured to ∼75–80% confluency, and serum-starved (with or without NAC) overnight. The cells were stimulated with H2O2, and if necessary, washed twice with ice-cold PBS, and lysed on ice in 5 mM Tris pH 7.4 containing 5 mM MgCl2, 20 µg/ml leupeptin and 1 mM PMSF. The cells were scraped, and lysis was assisted by vigorous vortexing and passaging through a 30-gauge needle. The cell lysates were centrifuged at 100,000 rpm for 40 minutes at 4°C in a TLA120 bench-top ultracentrifuge rotor (Beckman). The supernatants (S100 cytosolic fractions) were removed and analysed by SDS-PAGE and Western blotting.A431 cells were seeded into 100 mm tissue culture dishes at ∼2×10≫C. The lysate was then removed by centrifugation using a Micro-spin column. The beads were washed three times with ice-cold lysis buffer (without protease inhibitors), and then twice with ice-cold 20 mM Tris pH 7.4, 2.5 mM MgCl2. 30 µl SDS sample buffer was then added, to elute bound proteins, which were subsequently analysed by SDS-PAGE and Western blotting.For each reaction, a 10 µl bed volume of protein G-agarose beads (Roche) was coupled overnight to 1 µl anti-GFP polyclonal antiserum in 400 µl PBS with rotation at 4°C. A431 cell lysate was prepared as described for the Ral activity assays, and then a 20 µl aliquot was withdrawn for a Western blotting control. The lysates were then incubated with the anti-GFP antibody beads with mixing for 1 hour at 4 m/z  = 1188.59 Da, which yielded a sequence of FV[Met-Ox]QEEFSR. This tryptic peptide corresponds to amino acids 335–343 of ERp57. We also identified the testis-derived p58 that bound to RalA, by both MALDI-TOF MS (data not shown) and Edman sequencing of four tryptic peptides. The peptides sequenced precisely matched the sequence of ERp57 at residues 141–152 (TEDEFKKFISDK), 259–270 (DLLTAYYDVDYE), 306–317 (TFSHELSDFGLE), and 352–362 (FLQEYFDGNLK). Western blotting confirmed that ERp57 bound specifically to RalA-GDP in both synaptosome and testis extracts, while the opposite and expected GTP-dependent binding was observed for the known RalA effector protein RalBP1 in a parallel experiment  agarose in affinity chromatography (“pull-down”) experiments with various Triton X-100 solubilised rat tissue extracts. GST-RalA was loaded with either GTP or GDP immediately prior to use. With this approach we observed a ∼58 kDa protein that bound to GST-RalA in a GDP-dependent manner, which we initially referred to as p58 . The amoperiment .ERp57 is a member of the Protein Disulfide Isomerise (PDI) family, which encompasses a highly versatile group of proteins that have an N-terminal signal sequence and at least one thioredoxin-like domain E. coli, and then used in pull-down experiments with GST-RalA. Like tissue-derived ERp57, purified wild-type 6xHis-ERp57 bound RalA in a GDP-dependent manner  that mediate disulfide exchange in specific substrate proteins, involving the two Cys residues. The b and b' domains lack this motif, but they retain the thioredoxin fold and are involved in substrate binding ERp57 has four thioredoxin-like domains, denoted (from the N- to the C-terminus) ' and a' . The a aS-glutathionylation a' domain ERp57 is a redox-sensitive protein, and cellular oxidative stress elicited with hydrogen peroxide induces oxidation of its active site thiols and a and a' thiol-containing domains of ERp57 in redox-state-dependent binding to RalA, we mutated the pair of active-site Cys residues in either one or both of the a and a' domains to a pair of Ser residues (a or the a' domain (a+a'), abolished the redox-sensitivity of the ERp57-RalA interaction  had no effect . GTP-depNucleotide-dependent conformational changes in RalA are essentially confined to two regions, switch I and switch II, that regulate interactions with RalA effectors Purified GST-RalA and the various mutants were loaded with either GDP or GTP for use in pull-down experiments using rat testis extract. Interestingly, the RalA.D49A mutant efficiently bound ERp57, but the nucleotide dependence of ERp57 binding was completely lost . NucleotThe mutations in RalA’s switch II region  greatly diminished binding to ERp57 . Essentiin vitro assays for GEF, GAP and GDI activities. We did not detect GEF or GAP activity of recombinant ERp57 (data not shown). However, GDI assays, based on monitoring the dissociation of radio-labelled GDP from RalA, revealed a redox-sensitive GDI activity of ERp57. Under control conditions (no ERp57), 21% of the initial RalA-bound 3H-GDP remained after 1 hour .To determine if ERp57 is a regulator of Ral activity, we performed standard r 1 hour . Howeverone hour . We concNext, we examined the concentration dependence of the RalGDI activity of ERp57 in the presence and absence of DTT. The RalGDI activity of 6xHis-ERp57 was concentration dependent , since A431 cells have characterised redox-signalling processes Since oxidation of ERp57’s catalytic Cys residues is associated with maximal RalGDI activity in vitro , and ERp2O2 treatment of serum-starved A431 cells decreased the basal level of RalA activity in a time and concentration dependent manner  2O2-induced RalA inactivation (lanes 3 and 4). None of the treatments altered the total level of cellular RalA  was expressed in A431 cells that were then serum-starved and treated with 1 mM H2O2 for 30 minutes. In cells transfected with the GFP vector (pDEST53) alone, H2O2 decreased RalA activity as we saw previously  binds with high specificity and affinity to RalA through the switch II region of RalA, and this causes a GDI-like effect similar to that observed here Interestingly, the C3 exoenzyme from a and a' domain active-site cysteines for normal binding and activity, implying multiple interaction sites and raising the possibility of a stepwise binding and regulatory activity. Indeed the data in ERp57 shows similar functionality to known GDI proteins. For example, RhoGDI interactions with the small GTPases Cdc42 and Rac1 also map to switch II ERp57 action on RalA shares the general feature of redox-sensitivity with RabGDIs. RabGDI-1 and -2 responded to oxidative treatment of adipocytes by dissociation from membranes, which is a key step in the regulatory activity of GDIs Recently, it was shown that a GDI-like activity of PDEδ is an important contributor to Ras sub-cellular distribution between membranes and cytosol; PDEδ expression increased Ras plasma membrane localisation, activity, and diffusion through the cytosol 2O2) is constrained spatially, rather than influencing the overall redox state of the cell 2O2) showed that supplementary expression of ERp57 significantly exacerbated the inactivation of RalA observed in response to H2O2. Therefore, our data support the notion that a localized oxidative signalling process oxidises ERp57 to result in a strong RalA-ERp57 interaction and GDI activity.Our investigation of endogenous cellular RalA found that it was sensitive to the effects of redox agents, in both its signalling activity and subcellular localisation  and 6. T


To obtain a wide-scale view of the role of ERK in propagating T cell receptor signaling, a quantitative phosphoproteomic analysis of 322 tyrosine phosphorylation sites by mass spectrometry was performed on the human Jurkat T cell line in the presence of U0126, an inhibitor of ERK activation. Relative to controls, U0126-treated cells showed constitutive decreases in phosphorylation through a T cell receptor stimulation time course on tyrosine residues found on upstream signaling proteins , as well as downstream signaling proteins . Additional constitutive decreases in phosphorylation were found on the majority of identified proteins implicated in the regulation of actin cytoskeleton pathway. Although the majority of identified sites on T cell receptor signaling proteins showed decreases in phosphorylation, Tyr598 of ZAP-70 showed elevated phosphorylation in response to U0126 treatment, suggesting differential regulation of this site via ERK feedback. These findings shed new light on ERK’s role in positive feedback in T cell receptor signaling and reveal novel signaling events that are regulated by this kinase, which may fine tune T cell receptor activation.Competing positive and negative signaling feedback pathways play a critical role in tuning the sensitivity of T cell receptor activation by creating an ultrasensitive, bistable switch to selectively enhance responses to foreign ligands while suppressing signals from self peptides. In response to T cell receptor agonist engagement, ERK is activated to positively regulate T cell receptor signaling through phosphorylation of Ser The adaptive immune response relies the T cell receptor (TCR) to discriminate between foreign and self antigen. In canonical T cell activation, signaling events induced by the interaction between a TCR and peptide-major histocompatibility complex (MHC) agonist generates a set of cellular physiological changes that culminate in T cell proliferation, differentiation, and cytokine secretion. Upon activation of the TCR, the Src family protein tyrosine kinases Lck and Fyn phosphorylate the TCR CD3 chain immunoreceptor tyrosine-based activation motifs (ITAMs). Once fully phosphorylated, the ITAMs serve as binding sites for the Syk family protein tyrosine kinase ζ-chain associated protein of 70 kDa (ZAP-70), which is recruited to the TCR. There, ZAP-70 is phosphorylated and activated by the Src kinase Lck. A number of signaling proteins, including the scaffolding proteins linker for activation of T cells (LAT) and SH2 domain-containing leukocyte protein of 76kDa (SLP-76) are subsequently phosphorylated by active ZAP-70. Once phosphorylated, LAT and SLP-76 form a signalosome complex essential for the assembly and activation of downstream signaling proteins. 394, resulting in reduced phosphorylation of the CD3 ζ chains, and attenuation of intracellular signaling by the TCR 59 by ERK Proper T cell discrimination between structurally similar self and foreign antigens is complicated by the continuous signal inputs to the TCR signaling machinery from a plethora of low affinity self antigens. Competing positive and negative feedback pathways constitute one of the central mechanisms utilized to tune the sensitivity of TCR activation to self and foreign ligands 59 Lck will result in SHP-1-mediated dephosphorylation of its activation site, and consequently, global decreases in phosphorylation on the majority of TCR proximal signaling proteins. To elucidate the function of this kinase in regulating the TCR signaling pathway, a wide-scale view of temporal changes in the tyrosine phosphorylation of TCR signaling components is required. Quantitative mass spectrometry-based phosphoproteomics is a powerful means to achieve this goal by enabling the wide-scale identification of sites on proteins phosphorylated in T cells, as well as the quantification of protein phosphorylation dynamics, as demonstrated in various studies The current understanding of ERK’s role in TCR positive feedback signaling has been limited to immunoblot analysis of select TCR proximal proteins. Because of the critical role of Lck in initiation of the TCR signaling cascade, we hypothesize that the loss of ERK-mediated phosphorylation of SerJurkat T cells (E6-1 clone) were obtained from American Tissue Culture Collection . To determine the optimal inhibitor concentration for inhibition of ERK activation, Jurkat T cells were treated with U0126  at various concentrations  at 37°C. Inhibition of MEK1/2 was quantified using Western blots. In phosphoproteomic experiments, prior to stimulation, Jurkat T cells were treated with 20 µM of U0126 for 2.5 hours at 37°C.2 at 37°C. Stable isotope labeling with amino acids in cell culture (SILAC) was performed as described 12C6, 14N4 arginine and 12C6, 14N2 lysine  or 13C6, 15N4 arginine and 13C6, 15N2 lysine  supplemented with 10% heat-inactivated dialyzed FBS (Invitrogen), 2 mM L-glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin in a humidified incubator with 5% CO2 at 37°C for 7 cell doublings. The concentration of lysine and arginine used in SILAC labeling of Jurkat cells in experiments described here was 0.22 mM and 0.38 mM, respectively.Jurkat cells were initially maintained in RPMI 1640 medium  supplemented with 10% heat-inactivated undialyzed FBS , 2 mM L-glutamine, 100 U/ml penicillin G, and 100 µg/ml streptomycin (HyClone) in a humidified incubator with 5% CO8 cells/ml in PBS. For each stimulation time point, 1×108 cells were treated with OKT3 and OKT4 antibodies  at a concentration of 2.5 µg/ml of each antibody for 30 seconds at 37°C. Cells were then cross-linked with 22 µg/ml of goat anti-mouse IgG  and incubated at 37°C for 0, 2.5, 5, or 10 minutes. In total, 4 biological replicates of each time course stimulation were prepared for each condition.Prior to TCR stimulation, Jurkat cells cultured in heavy SILAC medium were treated with 20 µM U0126 for 2.5 hours, and cells cultured in light SILAC medium were treated with a vehicle control (0.1% DMSO). To stimulate the TCR, cells were reconstituted at a concentration of 1×10To halt the stimulation, cells were placed in lysis buffer  and incubated for 20 minutes at 4°C. Lysates were then sonicated at a 30 watt output with 2 bursts of 30 seconds each and cleared at 20,000×g for 15 minutes at 4°C. Protein concentrations were measured by the DC Protein Assay . Once protein concentrations were determined, cell lysates from inhibitor-treated and DMSO-treated cells were combined in a 1∶1 protein concentration ratio, reduced with 45 mM DTT for 20 minutes at 60°C, and alkylated with 100 mM iodoacetamide for 15 minutes at room temperature (RT) in the dark. Cell lysates were then diluted 4-fold with 20 mM HEPES buffer, pH 8.0 and digested with TPCK-treated trypsin  in a 1∶1 (w/w) trypsin:protein ratio overnight at RT. Tryptic peptides were acidified to pH 2.0 with 20% trifluoroacetic acid (TFA), cleared at 1,800×g for 5 minutes at RT, and desalted using C18 Sep-Pak plus cartridges  as described Peptide immunoprecipitation was performed using p-Tyr-100 phosphotyrosine antibody beads . Lyophilized peptides from each time point were reconstituted in ice-cold immunoaffinity purification (IAP) buffer  and dissolved through gentle shaking for 30 minutes at RT and brief sonication in a sonicator water bath. Prior to peptide immunoprecipitation, a 5 pmol fraction of synthetic phosphopeptide LIEDAEpYTAK was added to each time point sample as an exogenous quantitation standard. Peptide solutions were then cleared at 1,800×g for 5 minutes at RT, combined with p-Tyr-100 phosphotyrosine antibody beads, and incubated for 2 hours at 4°C. Beads were washed 3 times with IAP buffer and twice with cold Milli-Q water, and eluted with 0.15% TFA. Samples were then desalted using Zip-Tip C18 columns .59)  and anti-phosphotyrosine antibody, clone 4G10 (EMD Millipore), the following primary antibodies were from Cell Signaling Technologies: anti-phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204), anti-p44/42 MAPK (ERK1/2), anti-phospho-ZAP-70 (Tyr493), anti-ZAP-70, anti-GAPDH, and anti-Lck. Bound antibodies were detected with anti-rabbit IgG and anti-mouse IgG directly conjugated to an IRDye (Li-Cor). Bands were visualized and quantified using the Odyssey CLx Imaging System (Li-Cor).Total cellular protein from 9 M urea cell lysates was diluted in sample loading buffer  for each proteomic sample. Equal amounts of protein, as measured by the DC Protein Assay, were separated by 4–20% gradient SDS-PAGE on a Precise Tris-HEPES gel , and electroblotted onto an Immobilon membrane (EMD Millipore). The membrane was blocked with Odyssey blocking buffer  and then incubated with primary antibody in blocking buffer. With the exception of anti-Lck  After the database search, resulting peptide assignments were filtered down to 1% false discovery rate (FDR) by a logistic spectral score Relative quantitation of phosphopeptide abundance was performed via calculation of select ion chromatogram (SIC) peak areas for heavy and light SILAC-labeled phosphopeptides identified from 4 biological replicate experiments. Peak areas were calculated by inspection of SICs using software programmed in Microsoft Visual Basic 6.0 based on Xcalibur Development kit 2.0 SR2 (Thermo Fisher Scientific). Quantitative data was calculated automatically for every assigned phosphopeptide using the ICIS algorithm available in the Xcalibur XDK.For label-free comparison of phosphopeptide abundance in DMSO-treated control cells across the time course of TCR stimulation, individual time point SICs were normalized to an exogenously spiked standard phosphopeptide LIEDAEpYTAK peak area in the same time point. The LIEDAEpYTAK phosphopeptide was added at the same amount in each time point and replicate sample and accompanied cellular phosphopeptides through peptide immunoprecipitation and reversed-phase elution into the mass spectrometer. A label-free data heatmap was generated for comparison of phosphopeptides in DMSO-treated control cells through the time course of receptor stimulation, representing the average of 4 biological replicate experiments. The magnitude of change of the heatmap color was calculated from the natural log of the ratio of the fold change of each individual phosphopeptide peak area compared with the geometric mean for that phosphopeptide across all time points. In the heatmap representation, the geometric mean of a given phosphopeptide across all time points was set to the color black. A blue color represented below average abundance, while yellow represented above average abundance for each unique phosphopeptide. Blanks in the heatmap indicated that a clearly defined SIC peak was not observed for that phosphopeptide in any of the replicate analyses for that time point. The heatmap colors were generated from the average of the LIEDAEpYTAK standard phosphopeptide normalized SICs in the four replicate experiments. The coefficient of variation (CV) of the 4 biological replicates was calculated for each heatmap square. P values were calculated for each time point compared to the time point with the minimal average peak area for that phosphopeptide. A q value is defined as the measure of the minimum FDR at which a test can be called significant In a second type of heatmap, SILAC ratios from the average of 4 biological replicate experiments corresponding to phosphopeptide abundance differences between inhibitor-treated and DMSO-treated control cells across the time course of receptor stimulation were represented. For the SILAC heatmaps, a black color represented a ratio of 1 between the two cell treatments for a given phosphopeptide at that time point. A red color represented less abundance, and green represented higher abundance of the given phosphopeptide in the inhibitor-treated cells compared with the DMSO-treated control cells. The magnitude of change of the heatmap color was calculated as described 59 Lck were quantified by Western blot. Phosphorylation of Ser59 Lck increased in response to TCR stimulation in 0.1% DMSO treated cells. As expected, U0126-treated cells showed no increase in the phosphorylation of this site in response to TCR stimulation  in each time point. SILAC ratios were also calculated by comparison of the SIC peak areas of phosphopeptides from inhibitor-treated cells to their DMSO-treated counterparts. For each sequenced phosphopeptide, two different visual representations of quantitative data in the form of heatmaps were generated to reflect either the label-free or SILAC ratio data.To ascertain biologically relevant conclusions, stringent statistical tests were performed on the SIC peak areas of confidently assigned phosphopeptides that assessed the significance of quantification of phosphopeptide abundance across replicate observations. Q values assessed the significance of phosphopeptide abundance changes across the 4 time points of TCR stimulation in label-free heatmaps or between U0126-treated and DMSO-treated measurements for each time point in SILAC heatmaps. Statistical significance was only attributed to quantified phosphopeptides with q values less than 0.02. The reproducibility of SILAC analysis among replicate experiments was confirmed by evaluating the correlation of log2 transformed SILAC ratios between each biological replicate in scatter plots . A complThe resulting phosphoproteomic data presents a wide-scale view of the temporal changes of tyrosine phosphorylation events following TCR stimulation and inhibition of ERK activation. In all, from the SILAC experiments, 322 non-redundant tyrosine phosphopeptides on 243 proteins were identified at 1% FDR, of which 243 showed statistically significant U0126-responsive changes . Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed on identified sites  [35], . PhA wide-scale quantitative phosphoproteomic analysis of U0126-treated Jurkat T cells has been performed with the objective of elucidating the phosphorylation events that characterize ERK feedback in TCR signaling. Using a combination of label-free and SILAC quantification techniques, subtle fluctuations of cellular tyrosine signaling networks in response to ERK perturbation were captured and quantified over time. From this analysis, new insights into the critical role of ERK feedback in the regulation of the dynamic tyrosine phosphoproteome after TCR engagement in Jurkat T cells have been provided. Future studies will apply the unbiased and wide-scale approach demonstrated here to primary T cells with the hopes of better understanding the role of ERK positive feedback in TCR signaling.Figure S1Titration to determine optimal conditions for U0126 inhibition of MEK1/2. Jurkat T cells were treated with various concentrations  of the MEK1/2 inhibitor for multiple incubation periods . 0 µM samples were treated with 0.1% DMSO, as this was the background control. Inhibition was determined using immunoblots. After U0126 treatment and TCR stimulation, cell lysates were separated by SDS-PAGE and immunoblotted with a phospho-ERK1/2 specific antibody. Densitometric analysis was performed on relative levels of phospho-ERK1/2. Shown is the mean ± S.D. from 3 biological replicate experiments.(TIF)Click here for additional data file.Figure S2Quantification of ERK levels after inhibitor treatment. Jurkat T cells incubated with either 0.1% DMSO or 20 µM U0126 for 2.5 hours were separated by SDS-PAGE and immunobloted with antibodies against ERK1/2 and GAPDH. Densitometric analysis was performed on relative levels of ERK1/2. Shown is the mean ± S.D. from 4 biological replicate experiments.(TIF)Click here for additional data file.Figure S3Assessment of the reproducibility of SILAC ratios amongst the four biological replicate experiments. Scatter plots of SILAC ratios (log2 transformed) from four replicate experiments demonstrated good correlation and thus reproducibility.(TIF)Click here for additional data file.Dataset S1Quantitative and statistic analysis of all identified phosphopeptides. Sequence and phosphorylation site assignment of all identified phosphopeptides with their corresponding SIC peak areas and statistics  from both U0126-treated and DMSO-treated control Jurkat T cells.(XLS)Click here for additional data file.


The proto-oncogene, c-Abl encodes a ubiquitously expressed tyrosine kinase that critically governs the cell death response induced by genotoxic agents such as ionizing radiation and cisplatin. The catalytic function of Abl, which is essential for executing DNA damage response (DDR), is normally tightly regulated but upregulated several folds upon IR exposure due to ATM-mediated phosphorylation on S465. However, the mechanism/s leading to activation of Abl's apoptotic activity is currently unknown.KXXK--X3-5--SGS) is acetylated following DNA damage. We further observed that the S465 phosphorylated Abl is acetyl modified during DNA damage. Signifying the modification, cells expressing the non acetylatable K921R mutant displayed attenuated apoptosis compared to wild-type in response to IR or bleomycin treatment. WT-Abl induced apoptosis irrespective of new protein synthesis. Furthermore, upon γ-irradiation K921R-Abl displayed reduced chromatin binding compared to wild type. Finally, loss of Abl K921 acetylation in Tip60-knocked down cells and co-precipitation of Abl with Tip60 in DNA damaged cells identified Tip60 as an Abl acetylase.We investigated the role of acetyl modification in regulating apoptotic activity of Abl and the results showed that DNA strand break-inducing agents, ionizing radiation and bleomycin induced Abl acetylation. Using mass spectrophotometry and site-specific acetyl antibody, we identified Abl K921, located in the DNA binding domain, and conforming to one of the lysine residue in the consensus acetylation motif (Collective data showed that DNA damage-induced K921 Abl acetylation, mediated by Tip60, stimulates transcriptional-independent apoptotic activity and chromatin-associative property thereby defining a new regulatory mechanism governing Abl's DDR function. Cells were lysed in 1× lysis buffer (described above) and incubated with the anti-Flag monoclonal antibody-conjugated M2 agarose beads (Sigma). The bound material was resolved on a 5-15% SDS-PAGE and the Abl band corresponding to ~130 kDa was excised, incubated with trypsin, and the products were run through a Thermo Finnigan LCQ DECA XP Plus ion trap mass spectrometer interfaced with a Shimadzu Binary HPLC to carry out liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.Anti-Tip60 immune-complexes were obtained by incubating lysates formed from mock and irradiated HeLa cells with 5 ul of the antibody as described above. The immune-complex was washed twice in HAT assay buffer  and incubated with GST-Abl fusion proteins (0.5 μg) in the presence of 0.5 μCi [acetyl-1-14C] (370 kBq) and cold Acetyl CoA (10 μM). The reaction was carried out in 60 μl of HAT buffer for 30 min at 30°C and terminated using 3× SDS sample buffer. Reaction mixture was resolved on a 5-15% SDS-PAGE, transferred onto Immobilon-P and exposed to autoradiography after flurography.g for 5 minutes. Nuclei were washed with Buffer A and lysed in 200 μL of Buffer B containing 3 μΜ EDTA, 0.2 μΜ EGTA, 1 μΜ DTT, and protease inhibitors followed by incubation on ice for 10 minutes. The lysate was centrifuged at 1,700 × g for 4 min. to separate soluble nuclear proteins from chromatin. The isolated chromatin was washed with Buffer B and pelleted by centrifugation at 10,000 ×g for 1 minute. Chromatin was resuspended in SDS-sample buffer and sheared by sonication and resolved on 5-15%SDS-PAGE. The proteins were transferred onto Immobilon-P and western blotted against the indicated antibody.Chromatin fractionation was performed as described by Mendez and Stillman  with minFor quantitation of apoptosis, cells were stained with Annexin V-FITC2 (Phamingen) and analyzed by FACS Calibur flow cytometer. Approximately, 10,000 total events were collected and then subsequently analyzed for the percentage of Annexin v-FITC (FLH1-H)-positive, propidium iodide (FL2-H)-negative cells.t test . Differences with a P value of <0.05 were considered statistically significant.Data are expressed as means ± SD and obtained by combining data from separate experiments. Differences between control and treated groups were analyzed using ATM: Ataxia Telangiectasia Mutated; IR: ionizing radiation; UV: ultraviolet radiation; shRNA: short hairpin RNA; HLB: HMG-1 like box.The authors declare that they have no competing interests.ZJ and RK performed most of the experiments and helped in the preparation of the manuscript. SJ helped in site-directed mutagenesis and flow cytometry. TKP helped through reagents, discussions and editing of the manuscript. RB conceived the study, participated in its design and coordination, prepared the manuscript and supervised the project. All authors read and approved the final manuscript.MS analysis of trypsin digested products of Abl. A. Flag-Abl immunopurified from lysates of IR (5Gy) exposed cells were subjected to in gel digestion following resolution by SDS-PAGE. The trypsin digested products were subjected to MS analysis on an LCQ Deca XP Plus.Click here for fileMS/MS of the peptide ion at m/z 477.8, corresponding to the (M+3H)3+ species is shown.Click here for filep300 acetyl-transferase is dispensable for IR-induced K921 Abl acetylation. Mock-treated and IR (5 Gy) exposed HCT116 and HCT116/p300-/-cells were harvested 4 h later. Lysates were adjusted for equal protein content and probed with K921 site specific anti-acetyl-Abl (top panel), anti-Abl (middle panel) or anti-p300 antibody (bottom panel).Click here for fileα-amaninitin on IR-induced Abl acetylationEffect of CHX and . + Abl3T3 cells were exposed to IR (4.3Gy) in the presence or absence of CHX or α-amanitin and harvested at the indicated time points. Lysates were probed with K921 site specific acetyl-Abl (top panel) or Abl (Flag) antibody (bottom panel).Click here for file


The majority of immediate type adverse reactions to local anaesthetics seem to be non-IgE-mediated. We report a case of a 31-year-old woman, who developed conjunctivitis and conjunctival erythema immediately after intrauterine application of a local anaesthetic. Skin prick testing and intradermal testing were done with lidocaine, mepivacaine, and procaine. Intradermal testing showed positive reactions to mepivacaine (1 : 10), undiluted lidocaine, and procaine (1 : 10 and undiluted). Specific IgE could be detected against mepivacaine, but not against latex. Serum tryptase was in the normal range. In order to rule out the exceptional case of a true IgE-mediated reaction, allergy testing with local anaesthetics is still required in the workup of patients. Many immediate-type adverse reactions to local anaesthetics (LAs) are described worldwide although the vast majority seems to be IgE independent . The patWe report the case of a 31-year-old woman, who had developed conjunctivitis and conjunctival erythema immediately after the intrauterine application of an unknown LAs in the process of an abortion. In Austria, only three LA are available without the addition of epinephrine . Hence, we tested with the following marketed LA: lidocaine , mepivacaine , and procaine  at increasing concentrations of 1 : 100, 1 : 10, and undiluted. These were followed by intradermal tests (IDTs) of 0.03 mL at 1 : 10 concentration and undiluted LA. Specific IgE against lidocaine, mepivacaine, tetracaine, and articaine was measured using a classical RAST assay . Specific IgE against latex and chlorhexidine was assessed by ImmunoCAP . In addition, serum tryptase was determined with ImmunoCAP to rule out an underlying mast cell disease. Astonishingly, the patient developed wheals to IDTs of the diluted amide-type LA mepivacaine (at a 1 : 10 concentration) and the undiluted lidocaine , Table 1A possible IgE-mediated mechanism was further supported by an elevated signal in the nonstandardized RAST to mepivacaine . The summary of the results is reported in Total serum IgE was normal, and the latex ImmunoCAP remained negative. Serum tryptase was within the normal range excluding mast cell activation syndrome and mastocytosis.Herein, we report the rare case of a possible true IgE-mediated type 1 reaction to LAs. The patient had positive IDTs to two different LAs of the amide type and one of an ester type, a reaction that was reproducible at another control visit. In the case of lidocaine, we could only detect a positive reaction with the undiluted solution which, however, can reportedly induce false positive reactions [There were some recent publications about LA hypersensitivity. Bhole et al. pointed out the importance of other allergic elicitors such as chlorhexidine and latex . In a NoTaking together, we describe the rare case of a possible IgE-mediated reaction to an amide-type LA with cross-reactivity to an ester-type LA. Despite the dominance of non-IgE-mediated mechanisms and the less frequent non-LA type 1 allergens eliciting hypersensitivity reactions to LA, the existence of true IgE-mediated reactions cannot be completely ruled out at first hand. Hence, we think that allergy testing with LA is still required in the work-up of these patients.


Pagophilus groenlandicus) have evolved life history strategies to exploit seasonal sea ice as a breeding platform. As such, individuals are prepared to deal with fluctuations in the quantity and quality of ice in their breeding areas. It remains unclear, however, how shifts in climate may affect seal populations. The present study assesses the effects of climate change on harp seals through three linked analyses. First, we tested the effects of short-term climate variability on young-of-the year harp seal mortality using a linear regression of sea ice cover in the Gulf of St. Lawrence against stranding rates of dead harp seals in the region during 1992 to 2010. A similar regression of stranding rates and North Atlantic Oscillation (NAO) index values was also conducted. These analyses revealed negative correlations between both ice cover and NAO conditions and seal mortality, indicating that lighter ice cover and lower NAO values result in higher mortality. A retrospective cross-correlation analysis of NAO conditions and sea ice cover from 1978 to 2011 revealed that NAO-related changes in sea ice may have contributed to the depletion of seals on the east coast of Canada during 1950 to 1972, and to their recovery during 1973 to 2000. This historical retrospective also reveals opposite links between neonatal mortality in harp seals in the Northeast Atlantic and NAO phase. Finally, an assessment of the long-term trends in sea ice cover in the breeding regions of harp seals across the entire North Atlantic during 1979 through 2011 using multiple linear regression models and mixed effects linear regression models revealed that sea ice cover in all harp seal breeding regions has been declining by as much as 6 percent per decade over the time series of available satellite data.Harp seals ( We are currently witnessing significant changes in high latitude ecosystems, manifested most noticeably by rapid declines in the extent of summer ice, significant reductions in perennial ice cover, and declines in sea ice thickness in Arctic and Antarctic regions Rapid changes in temperature and ice conditions in Arctic ecosystems pose significant challenges for marine mammals that use sea ice as a platform for breeding and social activity  rely on seasonal sea ice as a substrate for pupping and nursing their young Harp seals  is the dominant pattern in climate variability across the North Atlantic, shaping environmental conditions from Canada to Russia The purpose of the present study was threefold. First, we tested the hypothesis that light ice years in the Northwest Atlantic result in increased neonatal mortality of seals by regressing the number of recorded strandings of dead harp seal pups in the Northeastern United States This series of analyses allows us to 1) establish the links between ice cover, NAO conditions and patterns in first year seal mortality over time, 2) provide a novel perspective on previously observed changes in harp seal populations in relation to climate variability and 3) characterize the longer-term warming signal present in sea ice variability in the breeding regions of harp seals across the North Atlantic.To illustrate the recent links between sea ice cover and seal mortality, we used stranding data for dead harp seal pups extracted from the US Northeastern Region stranding dataset covering marine mammal strandings in the Northeastern US from 1993 to 2010 For the retrospective analysis of NAO conditions and sea ice, we considered patterns in sea ice cover in two breeding regions of harp seals – the Gulf of St. Lawrence in eastern Canada, and the White Sea region between Norway and Russia – in relation to historical variability in the NAO . These tWe then conducted a wider examination of sea ice cover across all four breeding locations of harp seals  in Boulder, Colorado, for 1979 to 2011 during February and March, the breeding season of harp seals. These values were derived from NASA Nimbus-7 Scanning Multi-channel Microwave Radiometer (SSMR) data (1979–1987), Defense Meteorological Satellite Program (DMSP) Special Sensor Microwave/Imager (SSM/I) data  NAO index values . These data are based on the difference of normalized sea level pressure between Lisbon, Portugal and Stykkisholmur/Reykjavik, Iceland since 1864.The effects of changing ice conditions on seal neonatal mortality rates have not been investigated consistently over time, although previous studies indicate that when ice conditions are heavy mortality is generally low (e.g. 1.1–1.4%) Satellite-derived values of sea ice concentration were employed to further assess the relationship between winter NAO values and sea ice concentrations identified previously in To investigate the interaction of the NAO with long term trends in ice concentration we initially constructed 3-dimensional wire plots of multiple linear regressions of sea ice concentration against NAO index value, and year for all four breeding regions using the lattice package in R 2 = 0.37), where lighter ice conditions correlate with increased numbers of stranded dead seals. The regression of NAO index values and seal mortality (2 = 0.16). See A total of 365 dead harp seals specifically identified and reported as YOY animals stranded in the Northeastern US during 1993 to 2010. The distribution of standard lengths was normal  and the mean length of YOY animals was 105 cm with a SD of 9.5 cm. Using the upper range of lengths (115 cm) and avoiding animals listed specifically as sub-adults regardless of standard length, we then identified a total of 693 YOY seals in the stranding database and extracted these records for linear regression modeling. The linear regression of seal strandings against ice cover in the Gulf of St. Lawrence (the most proximate breeding location) is presented in ortality  revealedThe relationships between sea ice conditions and NAO index in the White Sea and Gulf of St. Lawrence breeding regions during March 1979 to 2011 are presented in The general trajectory of the winter NAO index from 1865 to 2011 is illustrated in A series of three-dimensional wire plots that display the linear relationships between sea ice cover in harp seal breeding regions during the month of February as a function of NAO index over our entire time series (1979 to 2011) are presented in The results of our mixed effects models reveal a statistically significant annual decline of sea ice cover in all four breeding regions during the study period, regardless of variation in NAO conditions. Both monthly model coefficients were significant at the 95% percent confidence interval. February ice concentration declined at a rate of 0.63±0.12% per year from 1979 to 2011 . The modEubaleana glacialis) in the same region The linear regressions of ice cover in the Gulf of St. Lawrence and NAO index values against stranding rates of YOY seals in the Northeastern US provide a useful proxy for changes in seal mortality associated with reductions in ice cover driven by NAO variability in the Northwestern Atlantic. Strandings data from the same region have been used previously to assess spatial patterns in stranding rates of ice seals North Atlantic Oscillation conditions greatly affect sea ice dynamics in harp seal breeding regions. When the winter NAO is negative, sea ice cover tends to be below average in the breeding locations of harp seals on the east coast of Canada and this has contributed to significant mortality of neonatal harp seals through a combination of interrupted nursing, starvation, cold stress and crushing by shifting floes when prematurely forced into the water by the rapid melting and break-up of ice While natural mortality rates for this population  have not been produced for the range of ice/NAO conditions captured by our times series, our linear regression of ice cover and stranded dead harp seals in the Northeastern US provides further evidence that young harp seals fare poorly in light ice years.This is not a recent phenomenon. For example, harp seals were forced to prematurely vacate whelping patches in the Gulf of St. Lawrence during the winter of 1966 Harp seals numbers now appear to be plateauing in the Northwest Atlantic off eastern Canada There have been dramatic changes in the trajectory of harp seal populations across the North Atlantic over time, and these fluctuations have never been fully explained. Several hypotheses have been offered to explain fluctuations in harp seal abundance across the Atlantic during our study period, including overexploitation, by-catches and changes in prey availability A large reduction in harp seal abundance in the Northwest Atlantic occurred during the 1950s through the early 1970s, with estimates ranging between 50 and 66% declines in seal numbers Contrary to the above relationship in the Northwest Atlantic, sea ice conditions in the Northeast Atlantic (White Sea) are out of phase with the NAO ; during Hunting mortality may also affect the population dynamics of harp seals across the Atlantic, especially when viewed as a cumulative stressor on reproductive output of a population. Harp seals are hunted commercially and for subsistence purposes across their range. In the Northwest Atlantic, total landed catches of harp seals during the extended negative NAO regime (1950 to 1972) were relatively high – ranging up to 389,410 seals annually. This hunt focused on newborn whitecoat pups The results of our retrospective analysis reveal that harp seal populations across the Atlantic appear to have fluctuated in synchrony with NAO trends and associated ice conditions. As such, we hypothesize that changing ice conditions may have contributed to the population dynamics of harp seals across the North Atlantic, most likely through periods of sustained reproductive failure or success, coupled with hunting and other environmental factors. Further research is required to assess how combinations of removals by sealers and recent (1996 to 2011) ice-induced increases in neonatal mortality will affect current and future harp seal populations.Our mixed-effects model effectively extracts the long-term warming signal from our time series of sea ice cover values that is dominated by NAO variability. Our model indicates that sea ice in all harp seal breeding regions is in decline regardless of the influence of the NAO, at a rate of approximately 6% per decade in February, and 4% per decade in March (this lower rate of decline likely stems from the fact that the majority of sea ice loss happens in February). This overall decline is less dramatic than recently observed conditions in eastern Canada (2000 to 2010), and it appears that the large scale effects of recent warming at high latitudes may have been obscured in the Northwest Atlantic in part by the effects of a consistently positive winter NAO regime on seasonal sea ice development and persistence during 1980 to 2000.Our results are the first to illustrate that ice cover in the breeding habitats for harp seals across the North Atlantic has been in decline since the beginning of the satellite record of ice conditions (1979). Along with observed increases in YOY mortality in the Northwest Atlantic harp seal herds indicated above, the consistent decline in seasonal sea ice cover across the North Atlantic has also resulted in a recent sharp reduction in pup production in the White Sea harp seal population Recent records of harp seals whelping off East Greenland (the West Ice) indicate that some response of the animals to phenological shifts in breeding habitat due to warming may be occurring Cystophora cristata) may be especially at risk. The Northwest Atlantic stock appears to be stable at present, but the Northeast Atlantic stock, which breeds on the West Ice, off the east coast of Greenland, has declined by 85–90% over the last 40–60 years, prompting a listing of this species as Vulnerable on the IUCN Red List of Threatened Species Harp seals have evolved to use seasonal sea ice as a breeding substrate and have adopted specific life history traits to succeed in this environment. Considering this, they are well suited to deal with natural shifts in climate, including the effects of the NAO on sea ice conditions. However, these animals may not be well adapted to absorb the cumulative effects of human influences (primarily hunting and to a lesser extent by-catch – which can be high in some years


Background. The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT) has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol) allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Methods. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtained. Recognition of linear IgG-epitopes of Bet v 1 and Bet v 2 and the capacity of these IgG-antibodies to block binding of human-IgE was determined. Results. Serum from rabbits immunized with native extracts recognised 11 linear epitopes from Bet v 1, while that from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from native immunized animals, and 9 from Dpg-Pol immunized one. Dpg-Pol and native immunized serum did not always recognise the same epitopes, but specific-IgG from both could block human-IgE binding sites for native extract. Conclusions. Depigmented-polymerized birch extract stimulates the synthesis of specific IgG-antibodies which recognize common but also novel epitopes compared with native extracts. IgG-antibodies induced by Dpg-Pol effectively inhibit human-IgE binding to allergens which may be part of the mechanism of action of SIT. Specific allergen immunotherapy (SIT) to the offending allergens confers clinical benefits in patients suffering from allergic diseases and is the only effective treatment with capacity to modify the evolution of the disease. Different markers have been suggested over the years to measure the immunological effect of the vaccines and to predict clinical benefits. Whilst no marker is yet sufficiently validated for routine clinical monitoring of SIT, induction of allergen-specific IgG antibodies (mainly IgG4 subclass) with the capacity to block allergen-IgE interaction has long been suggested as a potential mechanism for SIT  and moreMapping sequential and conformational IgG epitopes is becoming a potent tool for the exploration of the immunological response to allergens. However, little is known about the importance of such epitopes and their effect from an immunological point of view . In the in vivo against individual allergens [Depigmented allergoids are allergenic extracts subjected to a mild acid treatment following by a polymerization with glutaraldehyde in order to reduce the allergenicity while preserving their immunogenicity. The resulting product is a molecule containing individual allergens in chains which have a high molecular weight. The clinical efficacy of depigmented allergoids has been clearly demonstrated in double blind placebo controlled clinical trials by reduction in symptoms and medication scores –10. Morellergens  and theillergens . Few datllergens . The objectives of this study were to investigate the epitope specificity of the antibody response induced in rabbits after immunization with depigmented-polymerized (Dpg-Pol) allergenic extracts of birch, using native extracts as control, and to determine the capacity of the generated IgG antibodies to block the allergen epitopes recognized by human IgE antibodies.Betula alba  pollen  was extracted in phosphate-buffered saline (PBS), 0.01 M pH 7.4. After centrifugation, the supernatant was collected, sterile filtered, dialyzed against highly purified water, filtered, frozen, and lyophilised. Lyophilised native extract was reconstituted in highly purified water and the pH reduced using a mild acid treatment in order to separate the low molecular weight substances. The extract was dialyzed in membranes with a cut-off of 3.5 kDa  and all this material removed. The pH was again adjusted to physiological conditions, and the extract was sterile filtered, frozen, and lyophilised. Depigmented extract was reconstituted in PBS 0.01 M pH 7.4 and polymerised with glutaraldehyde. The resulting material was then dialyzed in 100 kDa dialysis membranes , filtered, and lyophilised. Finally native and Dpg-Pol extracts were individually dissolved (260 μg of protein/mL) and adsorbed to aluminum hydroxide .A native extract from birch pollen  and its corresponding Dpg-Pol extract were manufactured following previously described methods. Briefly, 100 g of defatted B. alba adsorbed onto aluminum hydroxide (3%). Rabbits were immunized with 3 subcutaneous injections with 130 μg of protein per dose, containing 15 μg and 23 μg of Bet v 1 for native and Dpg-Pol extract, respectively, following a previously described methodology [Two New Zealand white rabbits were immunized with native and two with Dpg-Pol allergen extracts of hodology . Preimmune serum was immediately collected before the first immunization. Twenty-one days after the first immunization booster 1 was administered. Booster 2 was administered 51 days after the first immunization. Rabbits were bled after 71 days and the serum samples collected. Individual and pooled sera from both rabbits immunized with each extract were kept at −20°C. The rabbit immunization was conducted at the Vivotecnia Research facilities . All the procedures were approved by the Institutional Review Board of Vivotecnia Research and followed the local ethical rules for animal experimentation.Specific pool of sera was prepared after mixing the same quantity of 15 individual serum samples from allergic patients, clinically diagnosed as allergic to birch and with positive specific IgE to birch (78.9 kUA/l), rBet v 1 (95.6 kUA/l), and rBet v 2 (2.37 kUA/l). Serum samples were acquired from Plasmalab International  which operates in full compliance of Food and Drug Administration regulations. All serum samples were previously tested for carbohydrates recognition by CAP , using bromelin (nAna c 2) in solid phase. In all cases this assay was negative.B. alba extract (20 μg/mL), rBet v 1, and rBet v 2 (1 μg/mL) . Serum samples were diluted from 1 : 5,000 to 1 : 320,000 in serial dilutions and incubated for 2 hours. After washing, the secondary antibody consisting of Goat anti-Rabbit-IgG-Peroxidase   was added. Finally microplates were washed, the reaction developed, and microplates read at 450 nm. Two independent experiments were performed, and measures were done in duplicate.Specific IgG to birch, Bet v 1, and Bet v 2 were measured in serum samples from rabbits immunized with native and Dpg-Pol extracts by direct ELISA. Briefly, microplates  were coated with native http://www.uniprot.org/). Linear synthetic peptides covalently bound to a cellulose membrane by the C-terminus (SPOTs) were commercially obtained from Sigma Genosys . A total of 25 peptides containing 12 aminoacids and 1 containing 10 aminoacids were prepared with Bet v 1, and 24 peptides containing 12 aminoacids and 1 containing 13 aminoacids were prepared with Bet v 2. All of them were synthetized overlapping by 6 aminoacids. Synthetic peptides are shown in Sequences of Bet v 1 (P15494) and Bet v 2 (P25816) were obtained from the UniProtKB/Swiss-Prot data bank  for 10 minutes. Afterwards they were blocked with blocking buffer  for 2 hours. After washing, pool of sera obtained from native or Dpg-Pol immunized rabbits was individually incubated  overnight. After the incubation with the secondary antibody, Goat anti-Rabbit-IgG-PO (Nordic Immunology), membranes were finally developed with RapidStep ECL Reagent  and peptides identified by chemoluminescence with ChemiDoc XRS .μg/mL) and incubated at room temperature overnight. Sera from native and Dpg-Pol immunized rabbits were incubated with serial dilutions of native and Dpg-Pol extracts. After 2 hours, inhibited samples were incubated with coated microplates for 2 hours. After washing Goat anti-Rabbit-IgG-PO  (Nordic Immunology) was added and finally developed and read at 450 nm.Microplates were coated with native and Dpg-Pol extracts (20 μg was loaded in each lane. Membranes were blocked for 2 hours with PBS 0.05% tween-1% skimmed milk. Serum samples from immunized rabbits (2 with native and 2 with Dpg-Pol extracts) were independently incubated at a dilution 1/400 for 2 hours with native birch extract (650 μg). Afterwards inhibited serum samples were incubated with membranes overnight. After washing, membranes were incubated with anti-Rabbit-IgG-PO  (Nordic Immunology) and finally developed. Native serum samples without inhibition and those inhibited with bovine serum albumin (BSA) were used as controls.Native extract was electrophoretically (SDS-PAGE) separated and electrotransferred onto a P-Immobilon membrane . A total of 65 The ability of native and Dpg-Pol extracts to induce IgG antibodies with capacity for blocking IgE epitopes of birch extract and rBet v 1 was investigated by ELISA inhibition experiments.B. alba extract (20 μg/mL) and rBet v 1 (1 μg/mL) (Indoor Biotechnologies), blocked with 1% BSA-PBS-T, and incubated with native and Dpg-Pol rabbit sera and their preimmune sera. After washing, plates were incubated with the human pool of sera (1/10 dilution). Bound IgE was detected with anti-human-IgE-PO . Optical densities were measured at 450 nm.Briefly, microplates (Nunc Maxisorp) were coated, respectively, with native i/ODP) × 100. ODi and ODP correspond to the optical densities after preincubation with the rabbit's immune sera and the corresponding preimmune sera, respectively [Percentage of inhibition was calculated as follows: percentage of IgE binding = 100 − (ODectively .Both groups of rabbits immunized with native (rabbit 978 and 979) and Dpg-Pol (rabbit 976 and 977) extracts showed high titres of specific IgG antibodies against native extract and Bet v 1 and Bet v 2 purified allergens . SpecifiSerum samples obtained from rabbits immunized with native and Dpg-Pol extracts recognised different IgG linear epitopes .With respect to Bet v 1, serum samples from rabbits immunized with native extracts recognised 11 epitopes while serum samples from Dpg-Pol immunized animals recognised 8 epitopes. In case of Bet v 2, 8 epitopes were recognized from animals immunized with native extracts and 9 epitopes from Dpg-Pol immunized animals. Summarizing, Dpg-Pol immunized serum samples did not always recognize the same epitopes as those recognized by native immunized serum samples but recognized other epitopes of the native allergens as shown in Membrane was incubated with the pool of preimmune sera, and no peptide was recognized (data not shown).μg of protein was obtained, with a sigmoidal curve between 0% and 100% of inhibition. However, after incubation of native extract with Dpg-Pol immunized serum it was not possible to calculate the 50% inhibition, because the inhibition curve did not reach total inhibition (Inhibition experiments using serum from native and Dpg-Pol immunized rabbits with native and polymerized extracts showed differences in the IgG response to the two extracts. When native extract was incubated with native immunized serum samples and inhibited itself, a 50% inhibition point of 7.96 hibition . Similarhibition .When native extract was used in solid phase, serum samples from native and Dpg-Pol immunized animals were capable of inhibiting practically the 100% of the extract (When native extract and purified Bet v 1 were inhibited with serum samples from native and Dpg-Pol immunized animals and afterwards incubated with the pool of human sera, the results demonstrated that human IgE binding epitopes of the native extract and those of rBet v 1 were blocked by rabbit IgG antibodies induced by both native and Dpg-Pol extract. A curve of inhibition between 0% and 100% was obtained. The percentage of IgE inhibition was calculated according to the formula previously described. For a rabbit sera dilution of 1 : 2 serum samples with native induced antibodies inhibited IgE binding to the whole extract by 88.8% while serum samples with Dpg-Pol induced antibodies inhibited by 94.5%. IgG induced by native and Dpg-Pol extracts inhibited IgE binding to Bet v 1 by 94.3 and 96.4%, respectively .The clinical efficacy of allergen immunotherapy has been related to induction of IgG antibodies that block IgE-allergen interaction . The abiin vivo, broadly similar to that for native allergen extract. However, the polymerization with glutaraldehyde may also modify the tertiary and quaternary structure of the molecules as we also observed recognition of specific IgG antibodies to new regions or epitopes in the individual allergens. Besides, the polymerization reaction modifies conformational epitopes that represent the majority of allergen IgE epitopes [in vivo and in vitro [In general terms it is accepted that exogenous antigens are captured by antigen presenting cells, processed in small peptides, combined with MHC class II molecules, and finally presented to different cells . Howeverepitopes , and as in vitro , 21 compAccording to these concepts of creation of new structures with new IgG epitopes after polymerization, ELISA inhibition experiments have always shown different curves when native and Dpg-Pol extracts are compared. In our study, comparing the sigmoidal curves obtained using serum from native or Dpg-Pol immunized animals, we observed a different inhibition pattern capacity when they were used with native or Dpg-Pol extracts in solid phase or as inhibitors. That means that the induced antibodies are recognising different structures in the molecules, although in both cases the inhibition capacity was comparable and correlates perfectly when each serum sample is inhibited with its corresponding inducing extract. But, though the formation of new epitopes with capacity to stimulate new specific antibodies is important for the improvement of the immunological effect from an allergic point of view, the real benefit of these new antibodies is their capacity to block IgE epitopes, where IgE is binding to allergens inducing the allergenic response. The capacity of IgG induced by both Dpg-Pol and native birch pollen extract to block human IgE-allergen interaction was demonstrated in our study by combining human and rabbit samples following the method previously described by Ball et al. . IgE binIn this study we used experimental animals because they have not previously been exposed to birch pollen. It would be of interest to study epitope specificity of IgG4 induced in patients given immunotherapy with Dpg-Pol extracts, but this would be more complex because of varied immune response and the presence of IgG antibodies before treatment. We have previously demonstrated increases in allergen-specific IgG4 in patients treated with Dpg-Pol extracts , and we In summary, mapping peptides can be a potent tool for the identification of specific regions related to the immunological response of allergenic vaccines. According to our results, depigmented-polymerized allergenic molecules acquire a different structure, from an immunological point of view, stimulating the synthesis of most of the IgG induced by native extracts and also specific antibodies, which are not induced by native extracts, with capacity to recognized new linear sequences in Bet v 1 and Bet v 2. This immunological response produces blocking antibodies which not only have capacity to inhibit IgG antibodies produced after the immunization with native extracts, but also are able to block specific regions of IgE binding sites in allergens. These results explain and are in agreement with the presence of blocking antibodies demonstrated in clinical trials.


Pleurotus cystidiosus.Angiotensin I-converting enzyme (ACE) inhibitors have been reported to reduce mortality in patients with hypertension. Compared to chemosynthetic drugs, ACE inhibitors derived from natural sources such as food proteins are believed to be safer for consumption and to have fewer adverse effects. Some edible mushrooms have been reported to significantly reduce blood pressure after oral administration. In addition, mushrooms are known to be rich in protein content. This makes them a potential source of ACE inhibitory peptides. Hence, the objective of the current study was to isolate and characterise ACE inhibitory peptides from an edible mushroom, P. cystidiosus based on the bioassay guided purification steps, i.e. ammonium sulphate precipitation, reverse phase high performance liquid chromatography and size exclusion chromatography. Active fraction was then analysed by LC-MS/MS and potential ACE inhibitory peptides identified were chemically synthesized. Effect of in vitro gastrointestinal digestions on the ACE inhibitory activity of the peptides and their inhibition patterns were evaluated.ACE inhibitory proteins were isolated from P. cystidiosus with molecular masses of 679.53 and 546.36 Da, respectively. Both peptides exhibited potentially high ACE inhibitory activity with IC50 values of 62.8 and 277.5 μM, respectively. SEC chromatograms and BIOPEP analysis of these peptides revealed that the peptide sequence of the hexapeptide, AHEPVK, was stable throughout gastrointestinal digestion. The pentapeptide, GPSMR, was hydrolysed after digestion and it was predicted to release a dipeptide ACE inhibitor, GP, from its precursor. The Lineweaver-Burk plot of AHEPVK showed that this potent and stable ACE inhibitor has a competitive inhibitory effect against ACE.Two potential ACE inhibitory peptides, AHEPVK and GPSMR were identified from P. cystidiosus could be potential ACE inhibitors. Although these peptides had lower ACE inhibitory activity compared to commercial antihypertensive drugs, they are derived from mushroom which could be easily obtained and should have no side effects. Further in vivo studies can be carried out to reveal the clear mechanism of ACE inhibition by these peptides.The present study indicated that the peptides from  These din vivo,8.Pleurotus cornucopiae, Lyophyllum decastes, P. nebrodensis, Grifola frondosa, P. sajor-caju and Lentinula edodes[G. frondosa, P. cornucopiae, Pholiota adiposa and Tricholoma giganteum[P. cystidiosus has exhibited the most potent ACE inhibitory activity. Proteomic analysis of P. cystidiosus has shown that it contains potential ACE inhibitory peptides[P. cystidiosus.Mushrooms have received increasing attention in recent years because of their health-stimulating properties and medicinal effects. Some edible mushrooms have been reported to significantly reduce blood pressure after oral administration. Examples are la edodes-16. The la edodes. Thus, tgiganteum-21. Amonpeptides. TherefoP. cystidiosus were obtained from Gano Farm Sdn. Bhd. and authenticated by morphology and molecular methods by experts in the Mushroom Research Centre, University of Malaya, Malaysia. Herbarium voucher specimen (KLU-M 1234) was deposited in the Kuala Lumpur Herbarium, University of Malaya. Culture for this species was deposited at Mushroom Research Centre culture collection, University of Malaya and was assigned a culture code (KUM 61204). All solvents and chemicals used in this study were of analytical and HPLC grade. Acetonitrile and trifluoroacetic acid (TFA) were obtained from Merck . ACE from rabbit lung, hippuryl-L-histidyl-L-leucine (HHL) and gastrointestinal proteases  were purchased from Sigma-Aldrich .Sporocarps (or fruiting bodies) of P. cystidiosus was done based on a previous study[P. cystidiosus were cleaned, sliced and blended with distilled water at a ratio of 1:2 (w/v). The mixture was filtered and centrifuged to remove unwanted debris. Proteins were precipitated out from the water extract using ammonium sulphate at 10-100% salt saturation. Precipitated proteins showing the highest ACE inhibitory activity were then fractionated by reverse phase high performance liquid chromatography (RPHPLC). Based on the results reported by Lau et al.,[Protein extraction from us study. Briefly et al.,, the actThe protein content of the SEC fractions was estimated using the Pierce® Bicinchoninic Acid (BCA) Protein Assay Kit  according to the protocol provided by the manufacturer. The absorbance values were measured using a Sunrise™ ELISA microplate reader  at 562 nm. The protein content was determined by comparing the absorbance value of the samples with a standard curve of bovine serum albumin.50.In the current study, ACE inhibitory activity was determined using an ACE inhibitory assay kit . The assay was carried out according to the protocol provided by the manufacturer. Absorbances of the reactions were measured using a Sunrise® ELISA microplate reader  at 450 nm. The ACE inhibitory activity of the samples was calculated using the formula given in the protocol. The concentration of the ACE inhibitor required to inhibit 50% of ACE activity under the above assay conditions was defined as the ICIdentification of the peptide sequences present in SEC fraction 1 was carried out by LC-MS/MS at Proteomics International Pty Ltd, WA, Australia. Briefly, the SEC fraction was digested with trypsin and the peptides extracted were analysed by electrospray ionisation mass spectrometry using an Ultimate 3000 nano HPLC system  coupled to a 4000 QTRAP mass spectrometer . Peptides were loaded onto a C18 PepMap100, 3 μm (LC Packings) column and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). Protein identification was carried out using Mascot sequence matching software (Matrix Science) with the Ludwig NR database.The two identified potential ACE inhibitory peptides, AHEPVK and GPSMR were chemically synthesised by Peptron, Inc., Republic of Korea. The purity of the synthesised peptides was >98% measured by RPHPLC and MS analysis.in vitro by the method of Wu and Ding[The stability of the synthesised peptides against gastrointestinal proteases was assessed and Ding. The pepThe inhibition pattern of peptide AHEPVK on ACE activity was determined spectrophotometrically using HHL as substrate. Basically, 20 μl of the ACE solution (0.1 UN/ml) and 50 μl of peptide were incubated with 200 μl of various HHL concentrations . The enzymatic reaction was terminated by the addition of 250 μl of 1.0 M HCl. The liberated hippuric acid was extracted with ethyl acetate and evaporated under vacuum condition. The hippuric acid residue was re-dissolved in 1.0 ml of distilled water and the absorbance was determined at 228 nm using a spectrophotometer . The enzyme activities were measured in the presence (0.05 and 0.50 mg/ml) and absence (control) of peptide. The kinetic of ACE inhibition was determined by Lineweaver-Burk plots.The analysis of ACE inhibitory activity was carried out in triplicate and result was reported as mean ± standard deviation. Mean differences of ACE inhibitory activity in SEC fractions was analyzed using one-way ANOVA in Statgraphics Plus 3.0 at p < 0.05.The RPHPLC fraction of E5PcF3 was further fractionated by SEC into seven fractions C1 to C7), as shown in Figure  to C7, aThe amino acid sequences of the peptides in C1 were determined by LC-MS/MS. Two potential ACE inhibitory peptides were identified. The LC-MS/MS spectra of these peptides are shown in Figure 50 value of 62.8 μM. This is lower than the IC50 value of ACE inhibitory peptides isolated from other edible mushrooms, i.e. G. frondosa (129.7 μM), P. adiposa (254 μM) and P. cornucopiae (277.3 μM)[50 values of P. adiposa and P. cornucopiae[In the current study, peptide AHEPVK exhibited potentially high ACE inhibitory activity with an IC77.3 μM),20,21. Ornucopiae,20.50 value of 2.4 μM[50 value with the current study. Peptides GTEKC and GPCSR from sweet potato exhibited IC50 values of 61.67 and 275.8 μM, respectively[The peptides in the current study have lower ACE inhibitory activity than proteins from other food sources. Hexapeptide from milk (PYVRYL) and pentapeptide from bonita fish (LKPNM) have exhibited ICf 2.4 μM,28. Peptf 2.4 μM. Howeverectively,30.in vivo studies of peptides from mushrooms, P. adiposa and P. cornucopiae have shown similar antihypertensive effect with the commercial drug, captopril[There are several ACE inhibitory peptides from food sources that have been sold in Japan and Canada. Examples are Vasotensin®, PeptACE™ and Valtyron®. These paptopril,20. Addiin vitro provided an easy method to evaluate the fate of these peptides after oral administration.Proteins or peptides delivered by the oral route have to be able to maintain their biological activity throughout the digestion process in the gastrointestinal tract before they reach their target site inside the body. The most important sites for the digestion of proteins and peptides are the stomach and small intestine. They contain gastrointestinal enzymes such as pepsin (stomach), trypsin and chymotrypsin . PrelimiReferring to Figure http://www.uwm.edu.pl/biochemia/index.php/en/biopep) is an online program that can serve as a tool to predict possible proteolysis products by gastrointestinal enzymes and define the possible biological activity of the proteolysis fragments[In order to verify the stability of these peptides, the changes without and following gastrointestinal digestion were analysed by SEC. The chromatograms are illustrated in Figures ragments. TherefoAccording to BIOPEP, AHEPVK was not hydrolysed by the three proteolytic enzymes. It was predicted to remain stable throughout the digestion process. Referring to Figure 50 value of 252.63 μM[Referring to Figure 52.63 μM. Therefo50 62.8 μM) and it shows stability against gastrointestinal digestion. Therefore, it was selected to determine its inhibition pattern against the ACE enzyme. According to the Lineweaver-Burk plot in Figure Peptide AHEPVK exhibited the most potent ACE inhibitory activity IC50 2.8 μM anG. frondosa, P. cornucopiae, P. adiposa and T. giganteum[The competitive inhibition pattern exhibited by AHEPVK is similar to ACE inhibitory peptides purified from the edible mushrooms giganteum-21. In agiganteum.P. cystidiosus were shown to be potential ACE inhibitors. Peptide AHEPVK exhibited a high IC50 value (62.8 μM) and its peptide sequence remained stable following gastrointestinal digestion. It exhibited a competitive inhibition pattern against ACE. Peptide GPSMR was predicted to release a dipeptide ACE inhibitor, GP, from its precursor after gastrointestinal digestion. Although these peptides had lower ACE inhibitory activity compared to commercial antihypertensive drugs, they are derived from food sources and should have no side effects.In the current study, peptides isolated from ACE: Angiotensin I-converting enzyme; RPHPLC: Reverse phase high performance liquid chromatography; SEC: Size exclusion chromatography; LC-MS/MS: Liquid chromatography mass spectrometry.The authors declare that they have no competing interests.CCL carried out all the experimentation, analysis of data and drafting of the manuscript. NA involved in monitoring and coordinating the work on mushroom biology and antihypertensive activity. ASS involved in coordinating the work on isolation and purification of peptides; and proteomic analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/313/prepub