Moringa oleifera is a plant found in many tropical and subtropical countries. Many different uses and properties have been attributed to this plant, mainly as a nutritional supplement and as a water purifier. Its antibacterial activity against different pathogens has been described in different in vitro settings. However the potential effect of this plant leaf as a hand washing product has never been studied. The aim of this study is to test the efficacy of this product using an in vivo design with healthy volunteers.Escherichia coli. Moringa oleifera leaf powder was tested as a hand washing product and was compared with reference non-medicated liquid soap using a cross over design following an adaptation of the European Committee for Standardization protocol (EN 1499). In a second part of tests, the efficacy of the established amount of Moringa oleifera leaf powder was compared with an inert powder using the same protocol.The hands of fifteen volunteers were artificially contaminated with Moringa oleifera leaf powder  was significantly less effective than the reference soap . Application of the same amounts of Moringa oleifera (2 and 3 g) but using a wet preparation, was also significantly less effective than reference soap . However there was no significant difference when using 4 g of Moringa oleifera powder in dried or wet preparation  compared with reference soap (2.97 ± 0.28). Application of calcium sulphate inert powder was significantly less effective than the 4 g of Moringa oleifera powder (p < 0.01).Application of 2 and 3 g of dried Moringa oleifera powder in dried and wet application had the same effect as non-medicated soap when used for hand washing. Efficacious and available hand washing products could be useful in developing countries in controlling pathogenic organisms that are transmitted through contaminated hands.Four grams of  Diarrhoeal diseases are a common cause of morbidity and the leading cause of death among children under five, accounting for 19% of mortality in this age group . The vasMoringa oleifera is a plant that can grow to 5–10 meters in a year and it is cultivated in many countries from South Asia to West and East Africa  wThe study was performed in the Medical Microbiology Laboratory of the London School of Hygiene and Tropical Medicine (LSHTM) from June to July 2012, and was approved by LSHTM Ethics Committee on 19th April 2012.Fifteen adult volunteers from LSHTM were selected for the study, and a formal written consent was received from all of them. The volunteers were physically examined to ensure they were healthy with healthy skin. None had skin disorders like eczema, paronychia, scabies, abrasions, lacerations or skin allergy. They all had short fingernails with no artificial nails. They had no history of drug allergy and had not taken any systemic antibiotic in the two weeks prior to the study, which could otherwise impair the efficacy of the product being tested. All forms of jewellery were removed from their hands prior to hand washing, since it had the potential of retaining some bacteria, which could affect the recovery pre and post values.Moringa oleifera leaf powder  was obtained from LUTOR Ltd. (UK). The aim of this study was to test the efficacy of the leaves as a hand washing product in dried and wet preparation and also to test the effect of using different amounts .Moringa oleifera powder as a hand washing product. The other four groups of volunteers repeated the same procedure on days 2, 3, 4 and 5.Because we wanted to test more than one preparation, we used a cross-over design with 5 groups of 3 subjects each. For day one, all the three volunteers in the first group used 5 ml of non–medicated liquid soap, 2 g of dry and wet Moringa oleifera powder; 3 and 4 g respectively. However, the quantity of non -medicated liquid soap was maintained at 5 ml. At the end of the whole series of runs every subject had used each hand-washing product once (Table During the second and third experiments (which were carried out the same day for each group), the aforementioned procedure was repeated with increasing amounts of ce Table .Escherichia coli (ACTC 25922) 8.3×108 cfu/ml. Excess of fluid was drained off and hands were air-dried for 3 min.The hands of each volunteer were washed with a non-medicated soap, dried and immersed for 5 seconds in a contamination fluid which contained non-pathogenic et al. technique [Bacteria were recovered for the initial pre value by kneading the fingertips of each hand separately for 60 seconds in 10 ml of tryptone soya broth (TSB) without neutralizers. The pre values were estimated using the Miles echnique .Moringa oleifera for one minute; the hands of the other third were washed with the reference solution (5 ml of non-medicated liquid soap) for one minute. Hands treated with dried Moringa oleifera received one of the 3 different amounts  whilst hands treated with wet Moringa oleifera received 2, 3 or 4 g of the herb powder plus 20 ml of tap water which was added on the top of the Moringa powder deposited previously into each pair of hands. In both cases the participants then rubbed their hands with the product for 1 minute. Hands were rinsed under running water for 15 seconds and were allowed to dry for 3 minutes.The hands of two thirds of the volunteers were treated with the et al. [After drying of the hands, the thumbs and fingers of both left and right hands were rubbed in separate petri dishes containing 10 ml of TSB for 60 seconds. Post values were determined using the method of Miles et al. . After tMoringa oleifera powder efficacy could be caused by mechanical friction with the hands, we compared the efficacy of 4 g of Moringa oleifera powder with an inert dried powder . The same protocol described before was used.In order to test whether 10 reduction values were calculated. Statistical analysis was performed with the Statistical Package STATA version 11.0. First it was checked whether the data were normally distributed using Kurtosis and Skewness test. Since the data did not follow a normal distribution, the Wilcoxon matched-pair signed ranks test was used to test for differences between each Moringa oleifera preparation and the reference soap and also between Moringa oleifera and the inert powder. The new product (Moringa oleifera) was considered to have the same efficacy as the reference product (soap) if the mean log10 reduction factor was not significantly smaller for the former than for the latter. Because of the confirmative nature of the test on this application, the level of significance is set at p = 0.1. The test is to be used one-sided. The discrimination efficiency of the test procedure described has been set to detect a difference between the two mean log reduction factors of approximately 0.6 log at a power of 95%.For both reference and test products, log counts from the left and right hands of each subject were averaged separately, for both pre and post values. The arithmetic means of all individual logMoringa oleifera leaf powder resulted in a mean log10-reduction of 2.44 ± 0.41 and 2.58 ± 0.34, respectively. The reduction observed was significantly less effective than with the reference soap  (Table Moringa oleifera (2 and 3 g) but using a wet preparation was also significantly less effective than reference soap . However, when using 4 grams of Moringa oleifera powder in dried or wet preparation there was no significant difference  compared with reference soap .Application of 2 and 3 g of dried Moringa oleifera, our results showed that Moringa oleifera was more efficacious in reducing bacterial contamination when used in wet preparation in all the 3 doses, but the difference was significant  only when 4 g of Moringa oleifera was used . 4 g of wet Moringa oleifera was more efficacious than 2 and 3 g of wet preparation but the reduction was only significantly higher when compared with 2 g of wet Moringa oleifera (0.21 log10 reduction (p = 0.01).When we compared the efficacy of 2, 3 and 4 g of dried vs. wet Moringa oleifera bacterial reduction effect was due to the mechanical friction of hands rubbed when using dried powder. Therefore we selected calcium sulphate as an inert and sterile powder and compared its efficacy with 4 g of Moringa oleifera in dried and wet preparation. The results are shown in Table 10-reduction of 2.33 ± 0.35 less than those obtained with 4 g of dried and wet Moringa oleifera . The reduction observed was also significantly less than with the reference soap; p < 0.001).We wanted to test whether the Moringa oleifera can reduce bacterial load on artificially contaminated hands when used as a dried or wet powder formulation. A dose of 4 g of Moringa oleifera in wet or dried formulation presented the same efficacy as non-medicated soap.The main finding of this study is that in vitro antibacterial effect from Moringa oleifera against different bacteria. Peixoto et al. [Moringa oleifera leaf extracts on the growth of gram-positive and negative bacteria, and they found that some strains of E. coli, P. aeruginosa and S. Enteritidis were resistant to all the Moringa oleifera preparations but the same extracts were most effective against S.aureus, V. parahaemolyticus, E. faecalis and A. caviae. Other authors found that aqueous and ethanolic Moringa oleifera leaf extracts were effective against Salmonella[. The authors attributed the antibacterial effect to the presence of saponins, tannic, phenolic and alkaloid phytoconstituents [Moringa oleifera leaves had been found in other studies to present an important amount of saponins (80 g/kg) [Moringa oleifera as a hand washing product.Different studies have shown an o et al.  evaluatealmonella. The auttituents . Moringa80 g/kg) . Saponin80 g/kg)  and thisMoringa oleifera, with a dose of 4 g associated with a greater bacterial reduction, comparable with the efficacy of non-medicated soap. These results suggest that when using Moringa oleifera, the hands have to be completely full of the powder in order to have the best results. The use of a wet preparation also presented better results than when dried powder was used. This could be explained by the water’s effect [In this study we also showed a dose–response reduction effect of s effect  in extras effect . Thus ths effect -22.Moringa oleifera leaf powder could be due only to the mechanical friction exerted when rubbing the hands together. For this purpose we compared the effect of 4 g of Moringa oleifera leaf powder with inert calcium sulphate powder, and observed that although calcium sulphate could reduce bacterial load on hands, the reduction was significantly smaller than obtained using 4 g of Moringa oleifera or non-medicated soap. These results suggest that Moringa oleifera has an extra effect due to its active components that cannot be attributed only to the mechanical action of rubbing hands together.Different studies have found that mechanical friction applied during hand washing plays a role in removing microorganisms adhering to hands . Therefoin vivo methods to test antiseptics are those of the European Committee for Standardization (CEN) [Moringa oleifera did not pass this criterion, but it met the EN1500 requirement for a hand-rubbing agent, which said that the mean reduction in the numbers of viable bacteria shall not be significantly inferior to that with the reference product [Moringa oleifera powder has the same effect as non-medicated soap, as both can significantly reduce bacteria counts in artificially contaminated hands. However we suggest this product is more suitable for domestic settings, rather than healthcare settings, where other products have already been demonstrated to be effective and available, and where the use of this powder would be less convenient.Many regulatory agencies require hand-wash (except for non-medicated soap) and hand-rub agents used in health care settings to meet certain performance standards when assessed using standardized test protocols ,25. In Eon (CEN) ,27. EN 1on (CEN)  and EN 1on (CEN)  belong ton (CEN) . 4 g of  product . It can  product . Thus, a product . For exa product , various product ,32. In tMoringa oleifera powder and spoons to dispense it, close to crucial places for hand hygiene, for example next to the cooking place and the latrine.The challenges of using this product in a domestic setting should be explored. How would users accept using a dried plant to clean their hands? How could the material be made available in specific domestic points where hand hygiene takes place? One possibility would be to place baskets with dried Moringa oleifera leaves in reducing bacteria on hands. It would be very interesting to test the bacterial reduction effect of fresh leaves. The possibility of using fresh leaves could facilitate its use as preparation process would not be necessary, opening up new avenues for its use, for example in open defecation places where Moringa trees are available.In this study we have demonstrated the effect of dried Moringa oleifera powder in dried and wet application had the same effect as non-medicated soap when used for hand washing. Moringa oleifera could be very useful in places where soap or water is not available, and where this tree grows naturally. It could also be a cheap and healthy hand-washing optional product. Our data have been obtained in a laboratory setting, so the next step will be to try this product in real conditions and study its acceptability and convenience for potential users. Moringa oleifera formulations should be investigated further in randomized controlled trials.Four grams of The authors declare that they have no competing interests.Idea for the research question: SC, BB, BT. Design of the experiments: BT, DO. Performed the experiments: DO. Analyzed the data: BT, DO. Contributed reagents/materials/techniques: EC. Wrote the paper: BT. Comments and review of paper: SC, DO, EC. All authors’ read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/57/prepub


Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa), the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein's digestibility is not affected by such processing.  Fish allergies are common in North America, Europe, and Asia and can potentially be fatal . Several2+  and with at least 1 mL of volume were pooled equivolumetric quantities. This pool was used in the immunoblotting studies.Deidentified and discarded sera from 21 individuals with a positive history of immediate-type hypersensitivity reactions to fish were obtained from the Johns Hopkins University Dermatology, Allergy and Clinical Immunology Reference Laboratory, Baltimore, Maryland, USA. At the time of collection, all individuals provided general consent for use of their serum in research studies related to food allergy. All sera  were initially analyzed for cod-specific IgE by Immuno-CAP . Sera , and the data were corrected for blank gelsections on the same gel. Gates were set such that the target protein bands  were not overlapping. Values of intensity of each band were compared with the summed density in the whole lane and reported as percentages (relative quantity). The sum of the shown intensities of the 3, 4, and 10 kDa bands is therefore not necessarily 100%. For IgE-immunoblotting, gels were electroblotted onto PVDF membranes. Protein transfer was confirmed using Ponceau Red . Membranes were blocked for 2 h using 5% (W/V) nonfat dry milk in wash buffer consisting of 0.01 M sodium phosphate buffered saline, pH 7.4 containing 0.05% Tween. The serum pool was diluted 1 : 10 in wash buffer +2.5% (W/V) nonfat dry milk and preincubated at room temperature for 1 hr before adding to the washed, blocked membranes for overnight incubation. After washing 4 times with wash buffer, the membranes were incubated with mouse anti-human IgE conjugated with horseradish peroxidase  diluted 1 : 1000 (v/v) in wash buffer +2.5% (W/V) nonfat dry milk for 1 hr at room temperature. The blots were washed (4X with wash buffer), and substrate solution was added . Emitted light was detected using a Kodak Gel Logic 440 image station, and the resulting images were stored digitally.SDS-PAGE was performed as described previously for the characterization of peanut allergen digests , in thisGiven the high degree of Lys modifications in the Maillard-treated parvalbumin, it was speculated that its isoelectric point (IEP) would be substantially lowered. Isoelectric focusing showed, however, only a minor shift in IEP of 0.1 pH units towards being more acidic as a result of the glycosylation (data not shown). Similar observations were made for beta-lactoglobulin  and are Addition of a single glucose to a Lys residue results in a mass addition of 162 Da. The average degrees of modification of 9 and 11 reached at 5 and 12 hours of treatment, respectively correspond with an average mass increase of 1,458 Da for the 5-hour-treated sample, and 1,782 Da for the 12-hour-treated sample. Mass spectroscopy on the 12-hour-treated sample show mass increase in line with this calculation, while for the 5-hour-treated sample, the average mass shift seems less than the expected . OverallThe far UV CD spectrum of native parvalbumin  resembleBecause we intend to investigate the digestibility at low pH, we investigated whether decreasing the pH alters the overall secondary structure of the different forms of parvalbumin. Far UV-CD spectra were recorded from 190 to 260 nm at various pHs. At pH 1.2, the spectra could not be recorded properly at the low wavelengths because of the high absorbance by chloride required to reach this pH. Also at pH 2, a relative high noise was observed in the spectra below 195 nm (not shown). The spectra of all samples at the various pHs were comparable, though some differences were observed at the extreme low pH values. To facilitate comparison of spectra, the wavelength at which the signal crosses 0 ellipticity (zero-crossing) is investigated. Zero-crossing is considered a relevant marker for conformational changes of proteins rich in alpha-helix and beta structures. A shift of zero-crossing to lower wavelength indicates loss of alpha-helix content . Zero-crInformation on tertiary protein folding was obtained by intrinsic fluorescence spectroscopy. At neutral pH a single peak around 330 nm was observed, representing fluorescence of tryptophans that are readily shielded from the aqueous solvent, for native and glycosylated parvalbumins (not shown). At low pH (2.0 and 1.2), the emission maximum for native parvalbumin is found at 328 nm, whereas for glycosylated parvalbumin, a distinct red-shifted emission maximum (340 nm) was observed, indicating a slightly more water-exposed environment for tryptophan. A fully exposed tryptophan would exhibit a maximum around 355 nm. A moderate red-shift could thus reflect a slightly destabilized tertiary fold, without necessarily a loss of secondary structure. Summarizing, analysis indicates that native parvalbumin is structurally sensitive to low pH conditions and around its IEP, while glycosylation provides some protection against such sensitivity.in vitro study using plant proteins showed that allergenic food proteins from peanut and soy were stable toward digestion [igestion . In contigestion  which waigestion . We usedigestion . One stuigestion . Peanut The use of acid-suppression medication has become more common in western countries in the last decades. Symptoms of gastritis, ulcer, erosions, and reflux syndromes can be treated with medication that are available over-the-counter, that is, without prescription by a physician. Such drugs can increase the pH of the stomach up to pH 5, thereby limiting the individual's digestive capacity. It has been hypothesized that the more common use of acid-suppressing drugs is associated with the increasing prevalence of food allergies , but no MALDI-ToF MS was applied to further characterize the peptides. However, due to the complexity of the degradation pattern of the multiple isoforms together with variable glycosylations, made an unambiguous assignment was not feasible. This latter was, for example, illustrated in the mass spectrum of the digested sample of the 5-hour glycosylated parvalbumin where a series of peaks separated 162 Da from each other could be observed (not shown), which indicates the same peptide with different degrees of modification. The rather stable peptides that are obtained after digestion are of sufficient size to comprise IgE epitopes, because the molecular weights of 3 and 4 kDa  correspoThe observation that the 3 and 4 kDa peptides do not bind IgE is consistent with the earlier work of Untersmayr et al. who reported that the binding of IgE to codfish extract was virtually absent upon digestion with pepsin for a short time. Although not exactly comparable with our experimental set-up, they also found peptides in the low molecular weight area (estimated at 5 kDa) on electrophoresis that had no IgE-binding . For othLimited information exists in the literature with regard to the effects of food processing on the allergenicity of fish. One report described that canning reduced the allergenicity of tuna substantially . AnotherThe amount of dimers and multimers of parvalbumin in glycosylated material is low as judged by the intensities of the SDS-PAGE Figures . Howeverin vivo. It can be speculated that the dimers and multimers of parvalbumin have a more pronounced allergenicity when tested in a biological system, because such systems require the binding of at least two IgE molecules, and this can be facilitated by larger size proteins and multiplication of IgE epitopes. A similar observation was recently made by Vissers et al. [The stronger IgE-binding suggests that the dimers (and higher order multimers) are potentially more allergenic than the monomeric form of parvalbumin; however, this needs to be investigated using biological assays, such as basophil histamine-release assays or s et al.  who obseOur research deals with isolated codfish parvalbumin. It is unknown whether dimers and multimers of parvalbumin will occur as the effect of the Maillard reaction in fish muscle tissue too. Whole tissue extracts are difficult to investigate because the presence of the excess of other (muscle) proteins may interfere in the analytical processes. The western blot applying IgE could be considered because it resolves the monomeric (approximately 12 kDa) and various multimeric forms . However, the sera from fish-allergic patients commonly also contain IgE to other fish muscle proteins rather than parvalbumin. These proteins have typical molecular weights between 20 and 80 kDa, thereby increasing the complexity of the band pattern on the western blot. Tailor-made analytical methods should be developed to answer the question if glycosylation-induced multimerization of parvalbumin occurs in whole fish tissue too.We have prepared different forms of glycosylated cod parvalbumin and characterized them biochemically at different pH mimicking gastric conditions. While some minor differences were observed in the change in secondary and tertiary structures as function of pH between native and glycosylated parvalbumins, the pepsin digestion was comparable for both forms. Primarily peptides of 3 and 4 kDa were formed, and glycosylation had no significant impact on the generation of these peptides. The peptides were no longer able to bind IgE and are therefore considered less allergenic than the intact parvalbumin. In contrast, the glycosylation resulted in small amounts of dimers and higher order multimers with more pronounced IgE-binding. We conclude that food-processing conditions applied to fish allergen can potentially lead to an increase in allergenicity, even while the protein's digestibility is not affected by such processing. Therefore, the allergenicity of fish products should be monitored with great care when food-processing steps are used that may induce glycosylation.


Modified gangliosides may be overexpressed in certain types of cancer, thus, they are considered a valuable target in cancer immunotherapy. Structural knowledge of their interaction with antibodies is currently limited, due to the large size and high flexibility of these ligands. In this study, we apply our previously developed site mapping technique to investigate the recognition of cancer-related gangliosides by anti-ganglioside antibodies. The results reveal a potential ganglioside-binding motif in the four antibodies studied, suggesting the possibility of structural convergence in the anti-ganglioside immune response. The structural basis of the recognition of ganglioside-mimetic peptides is also investigated using site mapping and compared to ganglioside recognition. The peptides are shown to act as structural mimics of gangliosides by interacting with many of the same binding site residues as the cognate carbohydrate epitopes. These studies provide important clues as to the structural basis of immunological mimicry of carbohydrates. Campylobacter jejuniGangliosides are glycosphingolipids which feature one or more sialic acid residues. They are most often associated with nervous system function, where they play a crucial role in maintaining the stability of myelin and axons N-glycolylneuraminic acid (Neu5Gc), such as N-glycolyl GM3 (Neu5Gc-GM3), are also useful targets for cancer treatment N-acetylneuraminic acid, Neu5Gc cannot be synthesized by humans, due to the lack of a functional sialic acid hydroxylase Gangliosides have been identified as tumor-associated carbohydrate antigens (TACAs), a group which includes Lewis Y, Lewis X, Thomsen-Friedenreich and Thomsen-nouvelle Carbohydrates are normally considered T cell-independent antigens, typically incapable of inducing a strong immune response Although prior structural studies into the recognition of gangliosides and their mimics by antibodies have been performed For method validation, high resolution complexes (<2.5 Å) of the antichlamydial antibodies S25-2, S73-2 and S25-39 with poly-Kdo (ketodeoxyoctulosonic acid) antigens were obtained from the Protein Data Bank (PDB) . The tesGlide 5.6 The site mapping procedure was applied to the test systems as described previously The metrics of reproduction and correctness were used in the assessment of site map quality. Reproduction is calculated as the number of crystallographic interactions identified by the site map divided by the number of crystallographic interactions observed. Correctness is calculated as the number of crystallographic interactions identified by the site map divided by the total number of mapped interactions. Reproduction and correctness values close to one indicate that the generated site maps accurately identify the crystallographic interactions, without the inclusion of erroneous contacts. The product of reproduction and correctness was used to assess the quality of mapping; larger values indicate optimal reproduction and correctness. The cumulative sum cutoff was optimized for the validation systems by assessing the average product of reproduction and correctness at 10% cutoff intervals from 0–100%. The cutoff which provided the greatest average product for the series of validation cases  was appla is the cutoff for selection of important hydrogen bonding contacts (as a fraction), b is the cutoff for selection of important van der Waals contacts (as a fraction), 2HBr is the correlation coefficient calculated between a particular conformer and the ensemble average for hydrogen bonding contacts, and 2vdWr is the correlation coefficient calculated between a particular conformer and the ensemble average for van der Waals contacts. The conformer exhibiting the highest similarity to the ensemble average maps was selected as the most likely conformer.Three residues of the HCDR3 loop are missing from the structure of chP3 (PDB 3IU4) Ganglioside-mimetic peptides were separated into overlapping hexapeptide fragments and docked to the antibody targets using GOLD. The site mapping technique (see above) was applied to the resulting ensembles of poses. The interaction data for the set of hexapeptides was pooled to give one set of site maps for the complete peptides, as described earlier d) was computed as described previously d values indicate a greater number of interactions by that residue with the mimic, while negative d values indicate more interactions with the ganglioside. Residues with d greater than an absolute value of 3.00 were considered to vary significantly from the equivalence line.To compare the recognition of gangliosides and their peptide-based mimics, scatter plots comparing the interaction contributions of antibody residues in ganglioside recognition and mimic recognition were generated. The distance between each point and the line representing equivalence of ganglioside and mimic recognition  is missing three residues from the HCDR3 loop From site-directed mutagenesis studies, it is known that Arg111.2H (Arg100AH in Kabat numbering) is critical for ganglioside recognition In all cases, van der Waals contacts were dominated by Trp57H and Trp116L . The HCDIn analyzing the generated site maps, it was determined that the chP3 conformer most representative of the average state was the tenth-ranked conformer , Table 5We had investigated ganglioside recognition by the four anti-ganglioside antibodies  using idGanglioside recognition by the antibodies was generally dominated by interactions with the heavy chain  and 5. IDespite the differences in binding site topographies, there are key similarities between the antibodies which become apparent upon structural examination of the site maps. Four residues, arranged in a relatively similar “spiral” around each antibody binding site, are largely responsible for hydrogen bonding interactions with the gangliosides. Proceeding clockwise, the likely ganglioside-binding motif of the antiganglioside antibodies comprises two polar residues , an aromatic residue  and a basic residue (Arg). Not all of the antibodies strictly conform to this motif; for example, R24 features a threonine residue where an arginine would be expected, and chP3 features an alanine residue where a serine would be expected.The peptide mimic of GD3 – RHAYRSMAEWGFLYS – could replicate almost all of the van der Waals interactions made by the ganglioside with R24, but some marked differences in the hydrogen bonding profile occurred . The mapThe peptide mimic of GD2 could more closely mimic carbohydrate binding to ME36.1 compared to the peptide mimic of GD3 binding to R24 . SimilarCarbohydrate-protein recognition is particularly challenging for molecular docking to predict accurately, due to the multitude of chemically equivalent hydroxyl groups in carbohydrates and their potential to form many specific interactions, including CH-π interactions In this study, the site mapping methodology is extended to study the recognition of acidic sugars, particularly gangliosides, by antibodies. Furthermore, to study ganglioside recognition by chP3, a “dynamic” version of the site mapping technique was developed, whereby multiple antibody conformers are considered to select one with the most likely ligand-binding conformation. Ganglioside mimicry is investigated by comparing the site maps describing the recognition of gangliosides and their peptide-based mimics.All of the antibodies examined in the validation set  prominenvia site mapping In our previous site mapping studies, identical cutoffs of hydrogen bonding and van der Waals interactions were used to select residues involved in ligand recognition The dynamic mapping study of chP3 demonstrates that the site mapping technique has applications not just in identifying antibody residues important for ligand interactions, but also in identifying the likely bioactive conformation of CDR loops, particularly HCDR3, which is often crucial for antigen recognition and for which no canonical structures exist Although the four anti-ganglioside antibodies studied have quite distinct binding site topographies and varying specificities , enough In comparing ganglioside and peptide recognition, we were able to determine that peptides known to act as immunological mimics of gangliosides could also act at least as partial structural mimics of the gangliosides. Therefore, it may be possible to design peptides which are capable of inducing an anti-ganglioside immune response using the antibody site maps generated in this study. Such a “design by mapping” procedure was proposed in our earlier work in investigating peptide mimicry of αGal-terminating carbohydrates While the site mapping technique solely evaluates ligand recognition from the protein's “point-of-view”, it can be combined with ligand-based mapping techniques to provide the complete picture of ligand-protein recognition To summarize our findings, the anti-ganglioside antibodies studied here largely utilize a motif of four residues to recognize gangliosides . These residues are arranged within the binding site of each antibody studied here in a relatively similar fashion. Peptides which bind to anti-ganglioside antibodies and elicit anti-ganglioside immune responses were found to act as structural mimics of the gangliosides, providing a case where structural mimicry translates into immunological mimicry. Our findings provide structural details invaluable for the future development of ganglioside-targeting cancer vaccines or optimizing therapeutic antibodies, as well as demonstrating the potential role of the site mapping technique in structure-based vaccine design.Table S1Initial optimization of site mapping cutoff using validation systems.(DOC)Click here for additional data file.Table S2Optimization of site mapping cutoff for hydrogen bonding using validation systems.(DOC)Click here for additional data file.Table S3Optimization of site mapping cutoff for van der Waals interactions using validation systems.(DOC)Click here for additional data file.Table S4Hydrogen bonding interactions in top ranked HCDR3 conformers of chP3.(DOC)Click here for additional data file.Table S5van der Waals interactions in top ranked HCDR3 conformers of chP3.(DOC)Click here for additional data file.Table S6Comparison of carbohydrate and peptide recognition by R24.(DOC)Click here for additional data file.Table S7Comparison of carbohydrate and peptide recognition by ME36.1.(DOC)Click here for additional data file.


Angiotensin I-converting enzyme (ACE) has an important function in blood pressure regulation. ACE-inhibitory peptides can lower blood pressure by inhibiting ACE activity. Based on the sequence of an ACE-inhibitory hexapeptide (TPTQQS) purified from yeast, enzyme kinetics experiments, isothermal titration calorimetry (ITC), and a docking simulation were performed. The hexapeptide was found to inhibit ACE in a non-competitive manner, as supported by the structural model. The hexapeptide bound to ACE via interactions of the N-terminal Thr1, Thr3, and Gln4 residues with the residues on the lid structure of ACE, and the C-terminal Ser6 attracted the zinc ion, which is vital for ACE catalysis. The displacement of the zinc ion from the active site resulted in the inhibition of ACE activity. The structural model based on the docking simulation was supported by experiments in which the peptide was modified. This study provides a new inhibitory mechanism of ACE by a peptide which broads our knowledge for drug designing against enzyme targets. Angiotensin I-converting enzyme  is a zinc-dependent dipeptidyl carboxypeptidase that plays an important role in regulating blood pressure by catalyzing the conversion of the inactive form of a decapeptide (angiotensin I) into a potent vasoconstrictor, an octapeptide (angiotensin II) ACE-inhibitory peptides derived from food proteins have attracted particular attention for their ability to prevent hypertension. Compared with chemosynthetic drugs, peptides derived from food proteins may have reduced toxic effects in humans; therefore, these food-derived peptides could be used as potent functional food additives and represent a healthier and more natural alternative to ACE inhibitor drugs. There is evidence that dietary ACE-inhibitory peptides may be bioavailable. Some ACE-inhibitory peptides have resistance to digestion, ability of intestinal absorption and stability in the blood Because the primary activity of ACE is to cleave the C-terminal dipeptide of oligopeptide substrates with a wide specificity, the inhibitory activity of ACE-inhibitory peptides is strongly influenced by their C-terminal tripeptide sequence. The most potent ACE inhibitors contain hydrophobic amino acid residues at each of the three C-terminal positions that interact with the subsites S1, S1′, and S2′ at the ACE active site. Many studies have shown that peptides with high ACE-inhibitory activities have tryptophan, phenylalanine, tyrosine, or proline at their C-terminus and branched aliphatic amino acids at the N-terminus, and ACE is known to have little affinity for inhibitors with C-terminal dicarboxylic amino acids  However, many ACE-inhibitory peptides are not competitive inhibitors of ACE, which may be the reason why these peptides are not in accordance with the results of the previous structure-activity correlation studies on ACE-inhibitory peptides. The precise inhibition mechanisms of ACE-inhibitory peptides are not yet clear, and the relationship between the inhibition kinetics and the crystal structure of ACE has not been fully researched. Further investigations are necessary to elucidate the relationship between the inhibition mechanism and the structure of these peptides.In a previous study To determine the inhibitory mechanism, Lineweaver-Burk plots were determined for the ACE-inhibitory peptide from yeast (TPTQQS). As shown in Non-competitive inhibition is a system in which the inhibitor and the substrate may both be bound to the enzyme at any given time. When both the substrate and the inhibitor are bound, the enzyme-substrate-inhibitor complex cannot form a product but can only be converted back into the enzyme-substrate complex or the enzyme-inhibitor complex To investigate the non-competitive inhibition mechanism of ACE by TPTQQS further, DS2.1 (a suitable a program for performing automated docking simulations of ligands to their macromolecular receptors) was used to carry out the docking simulation. Because tACE can completely perform the blood pressure regulation function of sACE, the three-dimensional structure of tACE (1O8A) was used as the macromolecular receptor in the docking simulation to investigate the inhibition mechanism of ACE by TPTQQS According to the results of the docking of TPTQQS onto ACE, TPTQQS forming coordination bonds with the zinc in the active site and the H-bonds with other amino acids outside of the active site played a key role in the inhibitory effect of TPTQQS. We chemically modified the key amino acids and changed the length of TPTQQS to investigate the non-competitive inhibition mechanism of TPTQQS further. First, we confirmed the effect of Ser6 by deleting the residue and changing it to an alanine in the peptide. The results showed that the inhibition rate of the peptide significantly decreased from 72.55% to 26.36% when the Ser was deleted as a result of the lack of interaction between the Ser and the active site of ACE. When Ser6 was replaced with Ala6, the interaction force decreased because Ala contains only two oxygen ions that can interact with the active site of ACE; the inhibition rate decreased to 48.61% .Second, to investigate the effect of the other amino acids in TPTQQS on the inhibitory activity, we deleted one or three residues from the N-terminus and changed Gln4 to Leu4 to weaken the interaction force between TPTQQS and amino acid residues outside of the active site of ACE. When the strength of the interaction between TPTQQS and helix α1 of tACE was decreased, the inhibition rate decreased by more than 40% . Thus, iTPTQQS has been shown to be an effective ACE-inhibitory peptide that was isolated from hydrolyzed yeast protein in our previous study The active site of ACE is composed of a zinc ion and a HEXXH…E motif, which includes His383, Glu384, His387 and Glu411. ACE accommodates two substrate residues that are C-terminal to the zinc ion (in the S1′ and S2′ subsites) and, thus, has dipeptidase activity. The S1′ and S2′ subsites are conserved and important subsites in ACE and are occupied by competitive inhibitors. According to the proposed mechanism of HHL hydrolysis catalyzed by ACE The docking simulation of TPTQQS binding with ACE showed that Thr1, Thr3 and Gln4 are the key amino acids for the non-competitive inhibition: all of these amino acids interacted with residues in helix α1 of ACE. The three N-terminal helices (helices α1 to α3) form a lid-like structure (referred to as the lid) that has significant flexibility according to the B-factor The zinc ion is the key ion in the active site of tACE, and this ion can be moved away from the opposite wall of the activesite cleft by residues 569–578, thus opening up the catalytic site These results suggest that the inhibition of ACE by TPTQQS is non-competitive, and a non-competitive inhibition model of TPTQQS to ACE can be built . When ACThe determination of ACE-inhibitory activity was performed using a High Performance Liquid Chromatography (HPLC) method, modified from a spectrophotometric method Inhibition kinetics of TPTQQS- Various substrate (HHL) concentrations  were incubated with the ACE solution in the presence or absence of 100 µg/mL, of 200 µg/mL TPTQQS at 37°C. The ACE-inhibitory activity was determined as described as described in the “Assay of ACE-inhibitory activity". The kinetics of ACE in the presence of the inhibitory peptide was determined using Lineweaver-Burk plots The molecular modeling package, ChemOffice 2004 , was used to construct and view the two- and three-dimensional structures of the hexapeptides. The structures of all of the ligands were optimized using “Prepare Ligands" in Discovery Studio (DS) 2.1  The isothermal titration calorimetry (ITC) experiments were carried out using a Nano ITC Low Volume System  with a 190 µL cell. All of the experiments were performed at 37°C. ACE was prepared by diluting the stock solution in a buffer of the appropriate pH; the substrate was dissolved in the same buffer as the enzyme. In a control experiment, the ITC cell was filled with 0.024 µmol/L ACE (250 µL), and the ACE solution was titrated with 1.55 mmol/L HHL. The injection volume was 50 µL, and the time between two injections was 300 s during which the substrate was completely hydrolyzed. In the inhibition experiment, the ITC cell was filled with 0.024 µmol/L ACE and 0.13 µmol/L TPTQQS (250 µL), and the solution was titrated with 1.55 mmol/L HHL. The titration conditions were the same as those for the control experiment.


In silicio docking studies support the view that EGCG binds directly to the active site of 11β-HSD1 by forming a hydrogen bond with Lys187 of the catalytic triade. Our study is the first to provide evidence that the health benefits of green tea and its polyphenolic compounds may be attributed to an inhibition of the cortisol producing enzyme 11β-HSD1.The microsomal enzyme 11β-hydroxysteroid deydrogenase type 1 (11β-HSD1) catalyzes the interconversion of glucocorticoid receptor-inert cortisone to receptor- active cortisol, thereby acting as an intracellular switch for regulating the access of glucocorticoid hormones to the glucocorticoid receptor. There is strong evidence for an important aetiological role of 11β-HSD1 in various metabolic disorders including insulin resistance, diabetes type 2, hypertension, dyslipidemia and obesity. Hence, modulation of 11β-HSD1 activity with selective inhibitors is being pursued as a new therapeutic approach for the treatment of the metabolic syndrome. Since tea has been associated with health benefits for thousands of years, we sought to elucidate the active principle in tea with regard to diabetes type 2 prevention. Several teas and tea specific polyphenolic compounds were tested for their possible inhibition of cortisone reduction with human liver microsomes and purified human 11β-HSD1. Indeed we found that tea extracts inhibited 11β-HSD1 mediated cortisone reduction, where green tea exhibited the highest inhibitory potency with an IC50 value of 3.749 mg dried tea leaves per ml. Consequently, major polyphenolic compounds from green tea, in particular catechins were tested with the same systems. (−)-Epigallocatechin gallate (EGCG) revealed the highest inhibition of 11β-HSD1 activity . Detailed kinetic studies indicate a direct competition mode of EGCG, with substrate and/or cofactor binding. Inhibition constants of EGCG on cortisone reduction were Ki = 22.68 µM for microsomes and Ki = 18.74 µM for purified 11β-HSD1.  In general, tea can be divided into three types (percentage of worlds tea production): non-fermented green tea (20%), semi-fermented tea (e.g. oolong tea and white tea) (2%) and fermented black tea (78%) The most popular form of tea consumed in the world is black tea, whereas green tea is mainly consumed in China and Japan. Recently, plenty of commercial beverages came to market that contain tea extracts or catechins from tea. Nowadays, tea or beverages containing tea extracts, if consumed daily, belong to a life-style that might support healthiness and long life, which is underpined by several laboratory, epidemiological and human intervention studies In particular, consumption of green tea has been associated with a reduction of the risk of cardiovascular disease, some forms of cancer, as well as with the promotion of oral health and other physiological functions such as antibacterial and antiviral activity, neuroprotective properties, anti-hypertensive effects, body weight control and diabetes type 2 prevention The latter diseases are risk factors of the metabolic syndrome  against which the therapeutical potential of tea has been shown in humans and model organisms in numerous studies In most cases the beneficial effects have been attributed to the polyphenolic compounds, especially catechins, although a large number of potentially bioactive chemicals are present in tea as well Unlike semi-fermented and fermented teas (black and white teas) unfermented green tea contains more catechins Other catechins are present in smaller amounts: (−)-epigallocatechin (EGC)>(−)-epicatechin gallate (ECG)>(−)-epicatechin (EC)> gallocatechin gallate (GCG)> gallocatechin (GC)> catechin gallate (CG)> catechin (C)> epigallocatechin digallate > epicatechin digallate Many other components have been identified in tea that might have an effect on human health: theaflavins, thearubigins, theasinesins, gallic acid, quinic acid, theogallin, coumaryl-quinic acid, caffeine, theobromine and theophylline, L-theanine (unique to tea), kaempferol, myricetin and quercetin It has been estimated that up to one-third of patients with diabetes type 2, hypertension and dyslipidaemia consume some form of complementary and alternative medicine, involving the use of herbs and other dietary supplements, like plant infusions, as alternatives to mainstream Western medical treatment In humans high levels of intracellular cortisol can increase glucose output in the liver, induce fat accumulation and weaken insulin sensitivity in adipose tissues, resulting in an increased risk for the metabolic syndrome Multiple evidence argues for an important aetiological role of 11β-HSD1 in various metabolic disorders including insulin resistance or rather diabetes type 2, hypertension, dyslipidaemia and obesity Collectively, these findings emphasize the potential benefits of a specific inhibitor of human 11β-HSD1 in the treatment of obesity and type 2 diabetes. In the present investigation, we tested several teas and some major tea-specific polyphenolic compounds, catechins, for their capability to inhibit the cortisol producing enzyme 11β-HSD1. Inhibition of 11β-HSD1 would shed light on – at least some of – the mechanisms by which green tea performs its health effect.Work on pluripotent carbonyl reductases/hydroxysteroid dehydrogenases, including 11β-HSD1, was approved by the ethics commission of the Medical Faculty, University of Kiel. Human liver tissue was obtained with written informed consent and approval by ethics commission of the Medical Faculty, University of Kiel. All experimental studies were performed in accordance with German legislation.Cortisol, cortisone and EGCG were purchased from Sigma-Aldrich . The following chemicals were procured from Carl Roth GmbH + Co. KG : Ethylacetate, NADP, NADPH, non-fat dry milkpowder, monosodium phosphate and potassium chloride, HPLC column LiChrospher® 100 RP-18, methanol, sodium chloride and monopotassium phosphate were obtained from Merck . (−)-Epicatechin gallate, (−)-catechin and (−)-gallocatechin were purchased from Biomol GmbH , Cayman Chemical  and LKT Laboratories Inc. , respectively. The different types of commercially available teas consisting of green, oolong and black teas were bought from supermarkets in Kiel, Germany. The anti-HSD11B1 antibody (ab83522) was obtained from Abcam .Dried tea leaves were reduced to small pieces and 1 g of the crushed material was suspended in 10 mL water without stirring for 90 min at room temperature. Insoluble material was removed by centrifugation  and the resulting supernatant was used in enzyme inhibition experiments.Human liver microsomes and purified human 11β-HSD1 were prepared as described previously In terms of 11β-HSD1 reductase activity, inhibition analysis was performed as follows: 50 µl of tea extract were added to a mixture containing 400 µM cortisone, 0.8 mM NADPH solution in 100 mM phosphate buffer pH 7.4. The reaction was started by adding 5 µl of human liver microsomes (218 µg protein) and the mixture was incubated for 3 h at 37°C. The total assay volume was 250 µl.The reaction was stopped by adding 250 µl ice-cold ethylacetate and vortexing, followed by a 30 sec centrifugation at 13,000 RPM in a microcentrifuge. The organic phase was separated and the aqueous phase extracted two more times with 250 µl ethylacetate. The ethylacetate phases were combined and the solvent was removed in a SpeedVac (ThermoSavant). The resulting residue was dissolved in 150 µl methanol-water  and used for metabolite quantification by HPLC.To analyze inhibition of 11β-HSD1 dehydrogenase activity, the oxidation of cortisol to cortisone was measured essentially as described above, except for the substitution of NADPH with 0.8 mM NADP, and the use of 400 µM cortisol instead of cortisone. Here, the incubation time was 60 min at 37°C in a 100 mM phosphate buffer pH 9.0.After enzymatic conversion the glucocorticoids were resolved as published previously The type of inhibition by EGCG (dissolved in buffer solution) was determined with four inhibitor  and substrate  concentrations. The data obtained were plotted as substrate-velocity curves and analyzed by nonlinear-regression. The type of inhibition was tested with both human liver microsomes (218 µg) and purified 11β-HSD1 (16.4 µg).To exclude EGCG-induced 11β-HSD1 aggregate formation, purified 11β-HSD1 (2.14 µg) was preincubated with 25–100 µM EGCG (3 h at 37°C) and then prepared for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the manufacturer's protocol. As a control, the same volume of untreated purified protein was loaded onto the parallel lane.The resolved proteins were transferred to nitrocellulose membranes. After blocking the membrane for 1 h at RT with 5% non-fat dry milkpowder in PBS-buffer, the blocking solution was replaced by the primary antibody at a 1∶2,000 dilution and the membrane was incubated at 4°C for about 16 h. Primary and secondary antibodies were diluted in PBS containing 2.5% non-fat dry milkpowder. The membrane was washed three times and then the corresponding secondary antibody was added at a dilution of 1∶10,000. After a 1.5 h incubation at RT the membrane was washed three times and the specific Western blot signals of 11β-HSD1 were detected by the ECL Western Blotting System according to the manufacturer's instructions.According to Halliwell Inhibition of 11β-HSD1 activity was assessed by determining cortisol yields after 3 h incubation at 37°C with equal volumes of green, black and white tea infusions . Quantification was performed by integration of the well-resolved product peaks in the above discribed HPLC assay. As shown in Inhibition of cortisone reduction in human liver microsomes was assessed with several green tea constituents. To determine possible differences in the potential of EGCG to inhibit reductase and dehydrogenase activity of 11β-HSD1, the influence of EGCG on the oxidation of cortisol to cortisone was monitored. To determine the mechanism of 11β-HSD1 inhibition by EGCG various inhibitor and substrate concentrations were used. Non-linear regression analysis yielded Ki -values of Ki = 22.68±10.10 µM and Ki = 18.74±8.00 µM for microsomes and the purified enzyme, respectively Insets . The datAs shown in When 200 µM EGCG was incubated for 3 h at 37°C in buffer only (same buffer as used in our assays above), the formation of 220 µM hydrogen peroxide was determined by the xylenol orange assay. However, when human liver microsomes, NADPH and cortisone was added (in the same concentrations as in the enzyme assays), only 12 µM hydrogen peroxide could be detected. Further, we tested the influence of up to 300 µM hydrogen peroxide on cortisol production with both human liver microsomes and purified human 11β-HSD1, respectively. No 11β-HSD1 inhibition by hydrogen peroxide could be detected (data not shown).Tea infusions have not only been a popular beverage for thousands of years but also have been traditionally used for the treatment of the metabolic syndrome and other chronic diseases. The metabolic syndrome is a complex metabolic disorder that may lead to diabetes type 2 and is one of the fastest growing diseases within the western civilisation. The role of glucocorticoid hormones in maintaining the blood glucose level by mobilization of energy sources is essential for homeostasis: Cortisol deficiency results in low blood glucose levels, dizziness, generalized weakness and stress sensitivity while cortisol overproduction, hypercortisolism, leads to high blood pressure, increased abdominal fat deposition, insulin resistance etc. Due to its pivotal role in cortisol production, inhibition of 11β-HSD1 maybe of therapeutic value in the treatment of the metabolic syndrome and diabetes.For these reasons our laboratory has investigated the inhibitory effects of tea, in particular green tea and some of its constitutents, on microsomal and purified 11β-HSD1 mediated cortisone reductase (and cortisol dehydrogenase) activity. The present study demonstrates that tea and green tea, respectively, and some of their polyphenolic constituents, EGCG and GC, have the capability to inhibit human 11β-HSD1.Initially, we could show that non-fermented green tea, semi-fermented tea (e.g. oolong tea and white tea) and fermented black tea have different potencies to inhibit human 11β-HSD1 mediated cortisone reduction. Non-fermented green tea showed the highest inhibitory potential , whereas a weaker inhibition potency was observed by semi-fermented white and fermented black tea.A reason for higher 11β-HSD1 inhibition by green tea compared to other teas might be a higher concentration of phenolic compounds in the former. During the fermentation process phenolic compounds, like catechins, are enzymatically oxidized. To protect the polyphenolic compounds from enzymatic oxidation, tea leaves that are destined to become green tea are withered by air drying prior to heat inactivation of polyphenol oxidases.Consequently, we tested several polyphenolic compounds known from green tea, in particular catechins, for their inhibition of 11β-HSD1 cortisone reduction. Interestingly, we observed big differences in the inhibition potency among the structurally related catechins see . For exaFrom cell culture experiments it is known that polyphenols like EGCG and EGC trigger a rapid hydrogen peroxide generation in various buffer systems. Concentrations up to 220 µM of hydrogen peroxide generated by 200 µM EGCG (incubated for 3 h at 37°C) in the buffer systems used could be detected. In contrast, addition of human liver microsomes, NADPH and cortisone to the reaction mixture resulted in about 12 µM hydrogen peroxide. A possible explanation for this hydrogen peroxide preventing effect might be the presence of peroxisomes in the microsomal fraction which contain several peroxidases known to catalyze the reduction of hydrogen peroxide to water. Further, due to the possibility that hydrogen peroxide generated by catechins could be responsible for the 11β-HSD1 inhibition observed in our study, we tested the inhibitory effects of up to 300 µM hydrogen peroxide on cortisone reduction. However, no inhibition of cortisol formation by both human liver microsomes and purified human 11β-HSD1, respectively, could be detected.vice versa by human liver microsomes. The results indicate that the dehydrogenase activity seems to be less affected by EGCG than the reducing activity  being responsible for the EGCG dependent effect As reported by others (Camellia sinensis) are able to inhibit cortisol formation by the enzyme 11β-HSD1. From several abundant constitutents of tea, the major phenolic compound EGCG could be identified as the most potent inhibitor of human 11β-HSD1, with inhibition constants of Ki = 22.68 µM in microsomes and Ki = 18.74 µM for the purified enzyme, respectively. The mechanism of EGCG inhibition is most likely a direct binding to the active site of 11β-HSD1, which is supported by enzyme kinetic studies and a computer aided docking model. Our results decipher the mechanism by which catechins such as EGCG, or green tea in general, have been successfully consumed for thousand of years for general health benefits. These polyphenolic compounds may serve as model structures for the development of novel agents to treat the metabolic syndrome and related diseases.In conclusion, we provide evidence that aqueous extracts of tea


In Hedgehog (Hh) signaling, the seven-transmembrane protein Smoothened (Smo) acts as a signal transducer that is regulated by phosphorylation, ubiquitination, and cell surface accumulation. However, it is not clear how Smo cell surface accumulation and intracellular trafficking are regulated. Here, we demonstrate that inactivation of Hrs by deletion or RNAi accumulates Smo in the late endosome that is marked by late endosome markers. Inactivation of Hrs enhances the wing defects caused by dominant-negative Smo. We show that Hrs promotes Smo ubiquitination, deleting the ubiquitin-interacting-motif (UIM) in Hrs abolishes the ability of Hrs to regulate Smo ubiquitination. However, the UIM domain neither recognizes the ubiquitinated Smo nor directly interacts with Smo. Hrs lacking UIM domain still downregulates Smo activity even though to a less extent. We have characterized that the N-terminus of Hrs directly interacts with the PKA/CK1 phosphorylation clusters to prevent Smo phosphorylation and activation, indicating an ubiquitin-independent regulation of Smo by Hrs. Finally, we found that knockdown of Tsg101 accumulates Smo that is co-localized with Hrs and other late endosome markers. Taken together, our data indicate that Hrs mediates Smo trafficking in the late endosome by not only promoting Smo ubiquitination but also blocking Smo phosphorylation. The Hedgehog (Hh) morphogen controls such development processes as cell proliferation, embryonic patterning, and cell growth Drosophila, the Hh signal is transduced through a reception system at the plasma membrane, which includes the receptor complexes Ptc-Ihog and the signal transducer Smo decapentaplegic (dpp), ptc, and enDrosophila imaginal discs indicated that Smo is directed primarily to the lysosomes of A compartment cells, but is enriched at the plasma membrane of P compartment cells Drosophila, the endosomal sorting of Smo is likely regulated by the Drosophila homolog of HGF-regulated tyrosine kinase substrate (Hrs), as Smo accumulation has been observed in cells mutating hrsIn Drosophila homolog of the dynamin GTPase).In this study, we provide genetic and biochemical evidence that the ubiquitination and intracellular trafficking of Smo are regulated by Hrs through direct interaction that inhibits the phosphorylation of Smo at the carboxyl-terminal domain. We further provide evidence that both Hrs and tumor susceptibility gene 101 (Tsg101) mediate Smo trafficking in the late endosomes, likely downstream of Shibire  compartment cells express and secrete Hh proteins that act upon neighboring anterior (A) compartment cells located adjacent to the A/P boundary to induce the expression of Hh target genes. P-compartment cells as well as A-compartment cells near the A/P boundary exhibit high levels of Smo cell surface accumulation , by C765-Gal4 caused a reproducible wing phenotype with partial fusion between Vein 3 and 4 , suggesting that Smo and Hrs exist in the same protein complex. Furthermore, we found that the physical interaction between Smo and Hrs was inhibited by Hh-treatment (nd panel), indicating that the downregulation of Smo ubiquitination correlates with the disassociation of Hrs. Consistently, Smo pulled down much less endogenous Hrs in the presence of Hh treatment (nd panel). It is possible that Hh downregulates Smo ubiquitination by inhibiting the physical interaction between Smo and Hrs.In the above experiment examining Smo ubiquitination regulated by Hrs, we immediately detected the Smo-bound Hrs in the immunoprecipitation assay -tagged Hrs truncations  and examrd panel), a deubiquitinase that has been characterized to downregulate Smo ubiquitination ΔU) or Hrs lacking the N-terminal VHS and FYVE domains but containing the UIM domain (HrsNT1) were not regulated by USP8 either  interacted with Smo aa661-678 (not shown). These data suggest that the UIM and N-terminal domains of Hrs interact with different region of Smo  but not transmembrane domain interacted with Hrs. To further map Smo domain that interacts with Hrs, we tested various SmoCT truncations generated previously Phosphorylation promotes Smo cell surface accumulation, whereas dephosphorylation leads Smo endocytosis SD123 that has phospho-mimetic mutations in the residues of three phosphorylation clusters interacted with Hrs weakly and such interaction was completely blocked by the treatment with OA  reporter assay to monitor the activity of Smo in Hh signling. As shown in ptc-luc activity  that are required for membrane receptors to be sequentially targeted to the MVBs. Although some studies showed that the ESCRT-0 consisting of Hrs can be used as early endosome marker In this study, we found that the ubiquitination is not required for the interaction between Smo and Hrs. Hrs normally recognizes the ubiquitinated cargos through its UIM domain, but the UIM domain obviously does not specifically recognize the ubiquitinated membrane receptor. It has been shown that Hrs, Tsg101, and other component protein from the ESCRTs undergo ubiquitination We showed that Hrs promotes the ubiquitination of Smo . It is pIt is also possible that Hrs regulates Smo ubiquitination by facilitating the ubiquitin ligase(s). However, the ubiquitin conjugating enzyme(s) and ubiquitin ligase(s) are unknown although it has been shown that mutating the ubiquitin activating enzyme increases the levels of Smo in wing discs Not only the ubiquitination machinery but also the degradation pathways for Smo have not been clearly addressed, although a recent study has demonstrated VPS36-mediated trafficking of Smo ΔU with the UIM  domain deletion was constructed by two-steps PCR. GST-Smo656−678 was generated by fusing Smo aa656-678 (corresponding nucleotide 1966–2034) to the GST backbone. His-HrsCT1 and His-HrsUIM were generated by fusing Hrs aa1-240 (corresponding nucleotide 1–720) and aa242-300 (corresponding nucleotide 724–900) to the pET-30a-His backbone, respectively. Fly mutants used: D28hrsMS1096 Gal4, ap-Gal4, C765-Gal4, and UAS-GFP-Smo have been described ΔU transgenic lines were generated at the VK5 attP locus to ensure the proteins are expressed at the same levels without positional effects Myc-Smo, Flag-USP8 and a series of Smo truncations including Myc-SmoNT1, NT2, NT3, NT4, and NT5 have been previously described 6 cells were harvested and lysed in 450 µl lysate buffer. 50 µl was saved for direct western blots, out of which 4 µl was used for each load. The remaining 400 µl was used for IP assay, which resulted in 30 µl IP samples. 5 µl IP sample was loaded for each run. For immunoprecipitation, cell lysate was added with beads of protein A ultralink resin (Thermo) after adding the proper primary antibody for 2 hours. Then the samples were resolved by SDS-PAGE and transferred onto PVDF membranes (Millipore) for Western blot. About 16 times more of the immunoprecipitation sample was analyzed compared with the corresponding lysate. Western blot analysis was performed using the indicated antibodies and the enhanced chemiluminescence (ECL) protocol. The use of HhN-conditioned medium has been previously described 4Cl (Sigma-Aldrich), a lysosome inhibitor, to block Smo degradation has been previously described ptc-luciferase reporter assay has been described with S2 cells transfected with tub-Ci and 3 repeats were analyzed S2 cells were cultured as previously described For the in vitro kinase assay, GST-Smo fusion proteins were expressed in bacteria, which is harvested, washed with PBS, and suspended with lysis buffer (PBS supplied with 1% Triton X100 and protease inhibitor) at the ratio of 50 µl buffer/1 ml culture. After sonication and centrifugation, glutathione beads were added to the supernatant aliquots (15 µl beads/500 µl lysate), followed by incubation for one hour and washing with PBS for three times at 4°C. The GST fusion protein was then subjected to a kinase assay with commercial PKA and CK1 (New England Biolabs) according to the supplier’s protocols. Phosphorylation of Smo was detected by western blot with the phospho-SmoP antibody that recognizes the phosphorylated forms of Smo Wing discs from third instar larvae were dissected in PBS then fixed with 4% formaldehyde in PBS for 20 min. After permeabilization with PBT (PBS supplemented with 1% Triton X100), discs were incubated with the indicated primary antibodies for three hours and the corresponding second antibodies for one hour sequentially, and washed with PBT for three times, 20 min per wash, following incubations. Primary antibodies used in this study were as follows: mouse anti-SmoN ; rabbit anti-β-Gal , anti-Rab5 , anti-Rab7 , and anti-Rab11 ; guinea pig anti-Hrs (gift from Dr. Hugo Bellen). Secondary antibodies were from Jackson ImmunoResearch Laboratories Inc., affinity-purified for multiple labeling (1∶500). Samples were mounted on slides in 80% glycerol. Fluorescence signals were acquired with the 20 x objective on an Olympus confocal microscope and images were processed with Olympus Fluoview Ver.1.7c. About 15 imaginal discs were screened and 3–5 disc images were taken for each genotype.


Reports on anaphylactic reactions to exotic fruits, e.g. kiwi and dragon fruit, are increasing. Therefore, the allergenic risk assessment and the identification of allergenic components are important. 9 cases of allergic reactions to pomegranate have already been reported, but the responsible allergens have so far not been characterized in detail.A 26 year old female patient developed erythema, swelling of the ears and pruritus within 10 min after ingestion of pomegranate. She is allergic to birch pollen and apple. Prick and IgE tests revealed a positive reaction to mites, tree and grass pollen, ambrosia, mugwort and apple. Scratch test was positive for pomegranate, the seeds showing a stronger reaction than the juice. Oral provocation test with kiwi, peach and cherry was negative. The serum was analysed for IgE reactivity by Western blotting. As extracts served different sections of pomegranate . IgE-reactive protein bands were analyzed and identified by protein sequencing and homology screening.The IgE-binding patterns differed between juice with one band of 9 kDa and the seeds with three IgE-reactive components of about 21, 16 and 6 kDa. The 9 kDa allergen was identified as a lipid transfer protein (LTP), which revealed a 77% sequence identity to the LTP from peach. While the 21 kDa protein of the seeds was N-terminally blocked, the 16 kDa component showed sequence similarity to Bet v 1-homologous proteins, which is in line with the patient’s reactivity to birch and apple. The 6 kDa protein revealed no significant sequence similarities to proteins in the databases. Tryptic mass fingerprinting is in progress to identify the allergens in more detail.Pomegranate is a relevant allergen source which shows different IgE-reactive components in its compartments. The structural characterization of these components is necessary to define their allergenicity and the underlying IgE-binding epitopes for improving in vivo and in vitro diagnosis and to estimate the potency of novel allergens in the future.


All agonists activated Rho-kinase and LIMK2, and activation was eliminated by inhibition of Rho-kinase. Microcystin LF (20 nM) potentiated the angiotensin II, but not the 5-hydroxytryptamine (1 μM)-mediated increase of P-CF/CF. In conclusion, all tested agonists activate the Rho-kinase-LIMK pathway and increase P-CF/CF. Angiotensin II activates PP2A and counteracts the LIMK-mediated CF phosphorylation. CF phosphorylation stabilizes peripheral actin structures and may contribute to the maximal contraction of PA.We hypothesized that agonist-induced contraction correlates with the phospho-cofilin/cofilin (P-CF/CF) ratio in pulmonary artery (PA) rings and cultured smooth muscle cells (PASMCs). PA rings were used for isometric contractions and along with PASMCs for assay of P-CF/CF by isoelectric focusing and immunoblotting. The P-CF/CF measured 22.5% in PA and differentiated PASMCs, but only 14.8% in undifferentiated PASMCs. With comparable contraction responses in PA, endothelin-1 (100 nM) and norepinephrine (1  The pulmonary circulation facilitates gas exchange between inhaled air and blood, and serves as a blood reservoir and a filter. Smooth muscle cell layers of pulmonary arteries (PA) play a primary role in these functions by regulating vascular myogenic tone and pulmonary blood flow. The majority of smooth muscle cells (SMCs) in healthy pulmonary blood vessels is differentiated, and their primary functions are to contract and relax. These functions are carried out by actin and myosin, which provide the driving force of the contraction response , 2. HoweCofilin (CF) is an actin binding protein which functions in the cell periphery as the principal depolymerizing protein of subcortical actin complexes , 7. CF lPhosphorylation of CF is mediated by two protein kinase families: the testicular protein kinase (TESK) and LIM kinase (LIMK). However, TESK is activated upon cell attachment onto fibronectin and transduces signals from integrin receptors to peripheral actin structures . LIMK isThe goal of this study is to evaluate how contraction agonists alter the phosphorylation state of cofilin in freshly dissected canine PA rings and in cultured PASMCs, and to assess signal transduction events involved in the cofilin phosphorylation at rest and during contraction of pulmonary artery smooth muscle.Guide for the Care and Use of Laboratory Animal published by the US National Institutes of Health , and with specific protocols approved by the Animal Care and Use Committee at the University of Nevada Reno. Canine lungs were obtained from adult mongrel dogs of either sex euthanized by barbiturate overdose. Lungs were dissected and second-order pulmonary artery branches were isolated, which were then used to cut rings for isometric contraction experiments, or to disperse and culture vascular myocytes, as described in our previous publications ATP at 30°C for 15 minutes. Protein was resolved by SDS-PAGE (15% acrylamide), gels were stained with Coomassie brilliant blue (CBB) and after distaining were exposed onto phosphorimaging screens. CF radioactive spots were visualized with a Personal Molecular Imager FX  and normalized to the CBB-stained bands. Stimulus-specific LIMK2 activation was expressed relative to the kinase activity of untreated controls.Total protein extracts from control and contraction agonist treatedcanine PASMCs were precleared with protein A/G agarose plus beads and then LIMK2 was immunoprecipitated with a goat polyclonal antibody . Immunoprecipitates were washed first with Ripa buffer and then with phosphorylation buffer  compared to the total CF , we used canine freshly dissected pulmonary artery rings and cultured PASMCs differentiated for three days in serum-free culture medium . AverageCultured PASMCs have been used in previous studies of our and other laboratories. To test how cell culturing affects the fraction of P-CF, we compared the P-CF/CF ratio of differentiated (0% NCS for three days) and in proliferating PASMCs maintained in complete culture medium (10% NCS). Results from three parallel experiments demonstrated that while the P-CF content changed insignificantly, the total CF content increased in proliferating PASMCs . As a reTo test whether smooth muscle contraction is associated with changes of the P-CF, we incubated freshly dissected pulmonary artery strips with NE and at the maximal amplitude we snap-froze tissues. Then we extracted and resolved total protein by SDS-PAGE, and assayed CF and P-CF by simultaneous immunoblotting using CF and P-CF antibodies, respectively. The NE-induced contraction was associated with increased phosphorylation of CF Figures . IncubatNE is the major contraction agonist in healthy blood vessels; however PASMCs are also exposed to other endogenous contraction agonists. To test how other contraction agonists affect P-CF, we incubated PA rings with ET-1, AngII, 5HT, and thrombin and then assayed the P-CF/CF ratio by immunoblotting. Similar to NE, these agonists induced contraction responses and the maximal contractions were partially inhibited by Y-27632 (not shown). However, these agonists had distinct effects on the P-CF/CF ratio: the ET-1 and thrombin-mediated contractions were associated with substantial about 2-fold increase of the P-CF/CF ratio, 5-HT stimulated an increase of 1.7-fold, while AngII caused no change of the CF phosphorylation .We also assayed the P-CF/CF ratio in PASMCs cultured in serum-free growth medium for 4 days prior to incubation with ET-1, AngII, 5-HT, and thrombin. Overall, the pattern of the P-CF/CF ratio in cultured PASMCs was reminiscent of the pattern in PA rings . Togetheγ-32P]ATP. All contraction agonists activated LIMK2, with ET-1 being the strongest (261 ± 52%) and AngII being the weakest activator (125 ± 35%) at these concentrations  indicating that PP2A activity modulates the phosphorylation level of CF. Moreover, MCLF potentiated the AngII-stimulated CF phosphorylation from 104 ± 13%  to 198 ± 42%   . In the , n = 3) . These rThis study demonstrates that exposure of pulmonary artery smooth muscle to various contraction agonists is associated with stimulus-specific changes of the phosphorylation state of CF. CF functions primarily in cell subcortical areas and modulates the remodeling of peripheral actin structures and cellular responses to extracellular stimuli. During smooth muscle contraction, for example, phosphorylation and inactivation of CF could reduce dissociation of peripheral actin filaments and hence stabilize stress fiber attachment to focal adhesion complexes , 31. UltThe role of AngII in the activation of Rho GTPase has been controversial. Sakurada et al., for example, have reported lack of activation of Rho in rabbit aortic smooth muscle , howeverWith respect to the functional role of cofilin in contracting vascular smooth muscle, our results are consistent with the following speculations. Firstly, CF phosphorylation and inactivation plays a minor, if any, role for the contraction response to NE, ET-1, thrombin, and 5-HT, and particularly to AngII. This conclusion does not exclude the possibility that inhibition of the CF phosphorylation may decrease the maximal contraction to NE, ET-1, thrombin, and 5-HT, or that inhibition of CF phosphatases may increase the maximal contraction to AngII. However, addressing these possibilities will require the development of specific pharmacological activators and inhibitors of CF kinases  or CF phosphatases  for use in whole tissue preparations. Secondly, the P-CF content increases from a resting level of about 23% to 42% in thrombin-treated, and to about 50% in NE or ET-1-trated PA rings. Such accumulation of inactive P-CF has been shown to induce the formation of stress fibers and focal adhesions in HeLa cells . Similar


The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) plays a critical scaffolding role in T cell receptor (TCR) signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs) Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway. TCR signaling plays an essential role in the regulation of the adaptive immune response, and it has been intensively investigated and described . TCR eng−CD8− double-negative stage and the lack of peripheral T cells 145 and short motifs within the proline-rich domain of SLP-76 3) and diacylglcycerol (DAG) SLP-76 consists of three domains capable of mediating intermolecular interactions: an N-terminal acidic region containing three tyrosine phosphorylation sites, a central proline-rich region, and a C-terminal SH2 domain T cells must discriminate foreign peptide-MHC agonists from a large variety of self peptide-MHC antagonists to appropriately trigger the TCR activation pathway only in the proper context 2 at 37°C. In addition, growth media for J14-76-11, the stable transfectant of J14, was supplemented with 2 mg/ml G418, which was washed out 2 days prior to SILAC labeling of the cells. For SILAC labeling, all cell lines were washed twice with RPMI 1640 medium without Arginine, Lysine, and Leucine (Sigma), and reconstituted in RPMI 1640 medium containing either 12C6, 14N4 L-Arginine, and 12C6, 14N2 L-Lysine (Sigma) or 13C6, 15N4 L-Arginine, 13C6, 15N2 L-Lysine , supplemented with 10% dialyzed fetal bovine serum (Sigma), 0.381 mM L-leucine (Sigma), glutamine, penicillin, and streptomycin for 7 cell doublings. The concentration of L-Arginine and L-Lysine chosen for SILAC labeling of Jurkat cells in experiments described here was 0.383 mM and 0.219 mM, respectively (The SLP-76 deficient Jurkat-derived cell line J14 and its wild-type (WT) SLP-76 reconstituted derivative J14-76-11 ectively .8 cells/ml in PBS. For each timepoint, 1×108 cells were treated with OKT3 and OKT4 primary antibodies, at a concentration of 2.5 µg/ml of each antibody, for 10 min at 4°C. Cells were then crosslinked with 22 µg/ml of goat anti-mouse IgG  at 37°C for 0, 1, 1.5, 2, 3, 5, or 10 minutes.Anti-CD3 and anti-CD4  stimulation was performed as described 8 cells and eluted peptides were filtered through a 0.22 µM filter . 10 pmol synthetic phosphopeptide LIEDAEpYTAK was added to each timepoint prior to peptide immunoprecipitation as a control for label-free quantitation. Results presented in this manuscript represent the average of three total replicate analyses.To stop the stimulation, cells were lysed with lysis buffer  and incubated for 20 min at 4°C. Lysates were then cleared at 14,000×g for 15 min at 4°C, and protein concentration was measured by the DC Protein Assay . Cell lysates from J14 and J14-76-11 were combined at a 1∶1 protein concentration ratio and reduced with 10 mM DTT for 1 hr at 56°C, followed by alkylation with 55 mM iodoacetamide for 1 hr at room temperature in the dark. Cell lysates were then diluted 5 fold with 100 mM ammonium bicarbonate, pH 8.9 and digested with sequencing grade modified trypsin  at 1∶100 (w/w) trypsin∶protein ratio overnight at room temperature. Tryptic peptides were desalted using C18 Sep-Pak plus cartridges , as described 783) and PLCγ1 expression were detected by western blotting analysis using Odyssey Infrared Imaging System . Generally, membranes were blocked in Odyssey Blocking Buffer (LI-COR Biosciences) for 1 hour and then incubated overnight at 4°C with rabbit anti-phospho-PLCγ1 (Tyr783) pAb and rabbit anti-PLCγ1 pAb  respectively in Odyssey Blocking Buffer. After rinsing, membranes were incubated with IRDye 800CW Donkey Anti-Rabbit IgG (H+L) (LI-COR Biosciences) in Odyssey Blocking Buffer for 45 minute in the dark at room temperature. Membranes were then extensively rinsed and bands were visualized using Odyssey infrared imaging system.Total cellular protein from 8 M urea cell lysates was diluted 1∶1 with gel loading buffer containing 4% SDS, 125 mM Tris-HCl (pH 6.8), 20% v/v glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue, pH 6.8 from each proteomic sample. Equal amounts of protein  were separated by 4–20% gradient SDS-polyacrylamide gel electrophoresis (Thermo Fisher Scientific), and electroblotted onto an Immobilon transfer membrane (Millipore). SLP-76 expressions in J14-76-11 and J14 Jurkat T cells were probed using rabbit anti-SLP-76 pAb and rabbit anti-β-Actin mAb . Phosphorylation of PLCγ1  Relative quantification of peptide abundance was performed via calculation of selected ion chromatogram (SIC) peak areas of heavy and light SILAC labeled phosphopeptides. For label free comparison of phosphopeptide abundance in the SLP-76 reconstituted Jurkat cells between different timepoints of TCR stimulation, individual SIC peak areas were normalized to the SIC peak area of the copurified synthetic peptide LIEDAEpYTAK in the same timepoint. The exogenous peptide LIEDAEpYTAK was added to each timepoint at 10 pmol. This exogenous peptide accompanied cellular phosphopeptides through the peptide immunoprecipitation, desalt, IMAC, and reversed-phase elution into the mass spectrometer. Peak areas were calculated by inspection of SICs using recently developed 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 peptide using the ICIS algorithm available in the Xcalibur XDK with the following parameters: multiple resolution of 8, noise tolerance of 0.1, noise window of 40, scans in baseline of 5, include of RefExc peaks False. A minimum SIC peak area equivalent to the typical spectral noise level of 500 was required of all data reported for label free and SILAC quantitation. SIC peak areas for all reported phosphopeptides were then manually validated.Temporal changes in phosphorylation abundance are represented as heatmaps from 3 replicate experiments. A label free heatmap that represents the change of abundance of phosphopeptides in J14-76-11 across 10 minutes of TCR stimulation and a SILAC heatmap that represents the ratios of abundance of phosphopeptides between J14 and J14-76-11 at each of the TCR stimulation timepoints were generated as previously described P if identified using phosphoproteomic method alone or * if identified using traditional approaches such as site-directed mutagenesis. For label free heatmap representation, P-values were calculated for each timepoint compared to the timepoint with the minimal average peak area for that phosphopeptide using unpaired, 2- sided student t-test. Q value has been defined as the measure of the minimum positive false discovery rate (pFDR) at which the test can be called significant In both label free and SILAC heatmaps, blanks indicate timepoints without a clearly defined SIC peak in any of the replicate analyses. The coefficient of variation (CV) for each heapmap square amongst the three replicate observations was calculated  and reprA quantitative phosphoproteomic analysis of TCR signaling comparing SLP-76 deficient Jurkat T cell line J14 and SLP-76 reconstituted Jurkat T cell line J14-76-11 was performed . A recepRelative quantification of peptide abundance via calculation of SIC peak areas was performed for each light or heavy SILAC labeled phosphopeptide in each timepoint. SILAC ratios were calculated by comparison of the SIC peak areas of phosphopeptides from SLP-76 deficient cells to their SLP-76 reconstituted counterparts. Label free fold changes were also calculated by comparison of the SIC peak areas of phosphopeptides in SLP-76 reconstituted cells between different timepoints of TCR stimulation. A total of 3 replicate experiments were performed and SIC peak areas were generated from the average values. The complete list of quantitative replicate data, calculated CV, p-values and q-values are available . SIC pea1, R2, R3) at 2 min after TCR stimulation (2 (R2/R1), Log2 (R3/R1), and Log2 (R3/R2) for each identified phosphopeptide. In good agreement of the expected value of 0, the measured average value of Log2 (R2/R1), Log2 (R3/R1), and Log2 (R3/R2) was 0.08±0.27, 0.01±0.18, and 0.01±0.46, respectively. Additionally, histogram plots showed that 85.2% (2 (R2/R1), Log2 (R3/R1), and Log2 (R3/R2) values respectively in the range of −0.6 to 0.6.The reproducibility of SILAC analysis among 3 replicate experiments was assessed by comparisons of three SILAC ratios , Fyn phosphorylation could not be quantified.The data revealed changes in phosphorylation abundance when comparing cells with or without SLP-76 expression through a timecourse of TCR-induced tyrosine phosphorylation on hundreds of proteins. Consistent with previous studies in J14 (SLP-76 deficient) Jurkat T cells, significantly reduced phosphorylation of Itk, PLCγ, Erk was observed . As expeed cells . Reducedern blot  [6], , [46Constitutively reduced phosphorylation on PAG in SLP-76 deficient cells would be expected to lead to a reduction in recruitment of the negative feedback regulator CSK, leading to constitutive increased phosphorylation of Lck within its activation loop as well as pathway substrates of Lck such as CD3, and ξ-chain ITAMs, and the ZAP70. Before Lck is substantially activated at 3 min, the SLP-76 mediated regulation of PAG and its associated negative regulator CSK could explain the observed significant increased phosphorylation of Lck and its pathway substrates. After 3 min, SLP-76 mediated regulation of ERK positive feedback could predominantly regulate the activity level of Lck. An intricate balance of positive and negative feedback pathways could lead to a fine regulation of the precise levels of activation of the T cell signaling pathway. From this data, the newly appreciated role of SLP-76 in the regulation of feedback pathways provides a new glimpse into the pivotal role of this protein.571) and SKAP55 (Tyr142), and several negative regulators of TCR signaling, such as the E3 ubiquitin ligase CBL  and the CBL-interacting protein STS-1 (Tyr19), membrane glycoprotein CD31 (ITIM motif residue Tyr713) and CD5 (ITAM-like motif residue Tyr453), and transmembrane adaptor protein PZR (ITIM motif residue Tyr263) and SIT (Tyr199).Our analysis also revealed that the removal of SLP-76 led to increased phosphorylation of multiple signaling proteins in J14 (SLP-76 deficient) cells, including the SLP-76 interacting adaptor proteins ADAP (Tyr690 and Tyr713) and PZR (Tyr263), as well as the ITAM-like motif tyrosine residue (Tyr453) in CD5 and the tyrosine-based signaling motif Y199ASV in SIT, were significantly elevated in SLP-76 deficient cells. The Src family kinase Lck and/or Fyn have been identified as the putative kinases that phosphorylate these tyrosine residues in T cells This data also revealed previously undescribed SLP-76 regulation of T cell signaling inhibitory proteins. The phosphorylation of immunoreceptor tyrosine-based inhibitory motif (ITIM) tyrosine residues in the cytoplasmic domain of CD31 (Tyr409) and DOK2 (Tyr299) 93) 313) 789) 579) 427) 62 with a strong scansite motif for Src PTKs, have been observed with SILAC profiles very similar to Lck Tyr349 (slightly elevated phosphorylation at the early timepoints and significantly reduced phosphorylation at the later timepoints in the SLP-76 deficient Jurkat T cells compared to reconstituted), supporting the hypothesis that Lck is involved in regulating these sites, either directly or indirectly.Phosphorylation of many other signaling proteins previously known to be phosphorylated by Src PTKs, including adaptor proteins DOK1  in both SLP-76 reconstituted and deficient Jurkat T cells.(XLS)Click here for additional data file.Dataset S2Assigned MS/MS spectra for all identified phosphopeptides in this analysis.(PDF)Click here for additional data file.Dataset S3Phosphorylation kinetics of all identified phosphopeptides in SLP-76 reconstituted and deficient Jurkat T cells. The differences of phosphorylations between SLP-76 reconstituted (WT) and deficient (ΔSLP76) cells are also presented as plots of ΔSLP76–WT across 8 timepoints to show the trend of phosphorylation changes. Results represent the means of three replicate experiments (error bars indicate standard deviation). “*” represents timepoints with false discovery rate less than 2% for significant changes in phosphorylation abundance between WT and ΔSLP76 cells.(PDF)Click here for additional data file.Figure S11, R2, R3) at 2 min after TCR stimulation. Difference between SILAC ratios were represented using a scatter plot of observed phosphopeptides  and a histogram  of Log2 (R2/R1), Log2 (R3/R1), and Log2 (R3/R2) for each identified phosphopeptide. In good agreement of the expected value of 0, the measured average value of Log2 (R2/R1), Log2 (R3/R1), and Log2 (R3/R2) was 0.08±0.27, 0.01±0.18, and 0.01±0.46, respectively. Additionally, histogram plots showed that D) 85.2%, E) 82.2%, and F) 86.8% identified phosphopeptides displayed Log2 (R2/R1), Log2 (R3/R1), and Log2 (R3/R2) values respectively in the range of −0.6∼0.6.Variation assessment of SILAC ratios among three replicate experiments. The reproducibility of SILAC analysis among 3 replicate experiments was assessed by comparisons of three SILAC ratios (R(TIF)Click here for additional data file.Figure S2A) SLP-76 expression in Jurkat T cells. Protein lysates from J14-76-11 (SLP-76 reconstituted) and J14 (SLP-76 deficient) were separated by SDS-PAGE and immunoblotted with SLP-76 specific antibodies. B) Effect of SLP-76 on PLCγ1 phosphorylation in OKT3/OKT4 stimulated Jurkat T cells. Lysates were prepared from J14-76-11 (SLP-76 reconstituted) and J14 (SLP-76 deficient) cells following TCR stimulation for the indicated time periods. Total cell lysates were probed by protein immunoblotting with anti-phospho-PLCγ1 (Y783) (top) and anti-PLCγ1 (bottom).Disruption of SLP-76 from Jurkat T cells and phosphorylation of PLCγ1. (TIF)Click here for additional data file.Figure S3Canonical TCR signaling pathway. Established signaling cascades in activated T cells with quantitative SILAC ratios (SLP-76 deficient in relative to SLP-76 reconstituted) represented as SILAC heatmaps beside individual proteins. SILAC ratios between J14 and J14-76-11 at each of the TCR stimulation timepoints were represented as SILAC heatmaps as described in methods.(PDF)Click here for additional data file.Method S1Selection of SILAC labeling conditions. SILAC labeling conditions were tested for both Jurkat T cell lines J14-76-11 (SLP-76 reconstituted) and J14 (SLP-76 deficient) to make sure cells grow and function normally.(PDF)Click here for additional data file.