Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission.Transmission represents a population bottleneck in the   •P. falciparum sexual differentiation is repressed by LysoPC found in human serum•LysoPC is a major building block of parasite phospholipid metabolism•LysoPC acts upstream of the earliest known events of sexual differentiation•Parasite metabolism alters host LysoPC levels during infection Plasmodium falciparum cell fate by repressing parasite sexual differentiation, a key step in malaria transmission.The host-derived lipid lysophosphatidylcholine controls  Plasmodium falciparum gametocytes are the only stage of the malaria parasite able to establish an infection in the Anopheles mosquito vector. Therefore, gametocyte formation and maturation are critical steps for successful parasite dissemination in the human population, and major targets of the ongoing malaria elimination agenda , rescue attempts at 38 ± 2 hpi and later time points were ineffectual (Exposing parasites to spent or “parasite-conditioned” medium (CM) dramatically induces sexual commitment relative to fresh medium complemented with human serum (+SerM) . Based offectual E, indicaTo identify the molecule(s) present in human serum that control parasite differentiation, we separated its components based on polarity A. The reUsing a fluorescent analog of the lipid, we demonstrated that trophozoites and schizonts, but not younger stage asexual parasites or gametocytes, rapidly incorporated LysoPC or metabolites thereof into parasite membranes D and S2A13C palmitate- or 2H choline-labeled LysoPC, parasites were subjected to extraction and liquid chromatography-mass spectrometry (LC-MS) analysis. These experiments revealed that parasites indeed metabolized LysoPC and used it to synthesize choline- and fatty-acid-containing products. The majority of free choline in the parasite (68%) was labeled even in the presence of equimolar amounts of unlabeled choline (21.5 μM) in the medium, suggesting that LysoPC is the major choline source for P. falciparum blood stage parasites , we attempted to block sexual commitment by addition of these lipids to the growth medium. However, parasites failed to efficiently access exogenous pools of these molecules D, explaiP. falciparum strains revealed a dramatic induction of ap2-g transcription in absence of LysoPC, demonstrating that the lipid acts upstream of the earliest known regulator of differentiation. We found a total of 342 genes significantly induced in response to LysoPC depletion, while 45 genes were downregulated (ek), phosphoethanolamine N-methyltransferase (pmt), and genes encoding enzymes required for methyl-donor synthesis , suggests a compensatory utilization of ethanolamine as a Kennedy pathway substrate for PC biosynthesis. Under LysoPC-limiting conditions, elevated ek and pmt activity may act in concert to catalyze production of the Kennedy substrate phosphocholine (P-choline) from ethanolamine via stepwise phosphorylation and SAM-dependent methylation events, ultimately securing cellular PC levels. It has previously been demonstrated that this alternative Kennedy pathway route is indeed active in blood-stage P. falciparum  timecourse experiment A. In briegulated . No signegulated A, demonsegulated B. The prlciparum .Figure 4GFP) in order to mark sexually committed schizonts  induces sexual commitment even in the presence of LysoPC or excess choline and glucose C. This rchizonts  and usedchizonts D. These The events that follow this initial metabolic response are dominated by a stepwise induction of epigenetic regulators, kinases, and factors involved in cell-cycle regulation and differentiation B. The prP. falciparum strains. We noted that a subset of LysoPC-responsive genes is specifically absent in the rodent parasite lineage  and form gametocytes within the same cell cycle  to 3.9%  significantly increased the LysoPC (16:0) concentration required for preventing parasite sexual commitment  located close to the ap2-g locus. Both transcription factors harbor frequent nonsense mutations in culture-adapted P. falciparum strains  and Trypanosoma brucei .Further information and requests for reagents should be directed to and will be fulfilled by the Lead Contact, Matthias Marti . P. berghei infections for serum extraction and metabolic analysis of correlations of LysoPC levels and peripheral parasitemias were performed on female C57BL/6 mice (6-8 weeks). Naïve mice received an initial inoculum of 106 parasites of the P. berghei ANKA line PbmCherryHsp70  were maintained according to Home Office license (60/4443) regulations. rryHsp70 , and werP. falciparum: P. falciparum cell culture and synchronization was performed as described  to select for stable episomes. The Pf2004 strain was transfected with reporter plasmid 164tdTom to generate Pf2004/164tdTom. Consequently, this cell line expresses the fluorescent protein tdTomato under control of the gametocyte-specific promoter of PF3D7_1016900  and gassed with 5 CO2/5% O2 and 90% N2 mixture. For mouse serum extraction and LysoPC quantification experiments, parasitemia was calculated prior to exsanguination. To this end, a drop of blood was obtained by tail vein puncture and the proportion of fluorescent (infected) red blood cells was quantified by flow cytometry.HPLC was performed with an Agilent 1200 series HPLC system (Agilent Technologies) equipped with a photo-diode array detector; all solvents were HPLC grade. Low-resolution Mass Spectrometry data were obtained using the above system and a 6130 quadrupole mass spectrometer; all solvents were LC-MS grade. High-resolution Mass Spectrometry data were obtained using an Agilent 1290 LC system equipped with an Agilent 6530 QTOF mass spectrometer in full scanning positive or negative ion mode in a range of 100-1700 m/z. Instrument conditions were as follows: Gas temperature at 325°C, gas flow at 10 L/min, Nebulizer at 40 psi, Capillary (positive) at 3500 V, Fragmentor at 120 V, and Skimmer at 65 V. Synthetic standards were used to establish retention times and characteristic ionization of lipid classes .2, 1 g) that had been conditioned with 6 mL of hexanes. After loading the lipid extract, the columns were eluted with 5 mL each of (1) hexanes, (2) hexanes/dichloromethane/diethyl ether (89:10:1), (3) hexanes/ethyl acetate (85:15), (4) chloroform/methanol (2:1), (5) methanol, (6) methanol with 1% ammonium hydroxide, (7) methanol with 1% acetic acid, (8) water with 10% ammonium hydroxide. The common fractions from the two columns were combined and concentrated in vacuo.Human serum (45 mL) was split evenly into 4 x 50 mL polypropylene centrifuge tubes. Each tube was extracted with 25 mL of chloroform/methanol (2:1). After collecting and pooling the bottom layers, the top layers were re-extracted with 20 mL chloroform. The bottom layers were combined with the bottom layers from the first extraction, filtered, and vacuum concentrated. This lipid extract was dissolved in 0.5 mL of chloroform and split into two aliquots. Each of these 250 μL aliquots was passed through an aminopropyl solid phase extraction column  fractions 13-16, and (2) fractions 23-24. These active fractions were investigated by high-resolution LC-MS in the positive mode using a Phenomenex Kinetex EVO C18 column. Solvent A was water and solvent B was acetonitrile. A 12-minute gradient of 80% of solvent B up to 100% of solvent B was utilized. The main components in fractions 13-16 and 23-24 were LysoPCs (in fractions 13-16 LysoPC(16:0); in fractions 23-24 LysoPC(18:0), see Activity of each fraction was tested by dissolving the fractions in 4 mL of mixtures of chloroform and methanol. These solutions were added in 2.2 μL aliquots to a glass bottom 96-well plate, the solvent was evaporated, and 220 μL of parasite culture in CM  was addO-palmitoyl-sn-glycero-3-phosphocholine(d9)  in methanol as an internal standard. The samples were randomized and extracted via a modified Bligh and Dyer method   were added in methanol as an internal standard. Extractions and LC-MS analyses were performed as described above. To calculate the concentration of LysoPCs in undiluted bone marrow, each value was multiplied by the dilution factor (200x).Analysis was performed essentially as described above with the following exceptions. One 3 μL aliquot of serum from each of five mice was diluted to 0.2 mL and added to 0.3 mL of 2M NaCl . Cell-free bone marrow dilutions were obtained by extracting bone marrow from the femur and tibia of both lower extremities of each mouse. The bone marrow was collected in 0.5 mL of PBS, and its weight recorded using a high sensitivity scale, prior to disruption using an insulin needle followed by centrifugation at 10,000 g for 5 minutes. The supernatant was recovered and weighed following centrifugation to determine the percentage of the collected material. A 0.2 mL aliquot of the diluted cell-free bone marrow from each of five mice was added to 0.3 mL of 2 M NaCl. To all 10 samples  4 μL of a 25 μM solution of 1-9-choline and 13C2-palmitate) were synthesized following the protocol published by Lindberg et al. (Two heavy-atom labeled LysoPC(16:0) analogs  followed by acidification with p-toluenesulfonic acid and crystallization from acetone.Generally, reactions were carried out in flame-dried glassware under Argon. Anhydrous solvents were purchased from Sigma-Aldrich. N,N-Diisopropylethylamine  were added to 20 mL anhydrous CHCl3. Phosphorus(V) oxychloride  was added dropwise to the solution stirring in an ice-water bath. After stirring at 0°C for 2 hours, unlabeled choline tosylate  was added, followed by pyridine . After pyridine addition, the reaction turned magenta. The flask was removed from the ice-water bath and allowed to stir at room temperature for 5 hours. The reaction was quenched with 1.0 mL water and allowed to stir at room temperature for 1 hour. The mixture was concentrated by rotary evaporation. Almost half of this crude product (6.0 g out of 13.5 g) was purified by silica gel chromatography, eluting with 70% ethanol in water (Rf ∼0.2). Fractions containing the product were combined, concentrated by rotary evaporation, and centrifuged to remove particles. The supernatant was concentrated to yield thick oil (R)-glycidyl phosphocholine .S-glycidol  and 1H NMR  δ 2.68 , 2.81 , 3.19 – 3.27 , 3.62 – 3.67 , 3.67 – 3.77 , 4.18 , 4.23 – 4.34 ; 13C NMR  δ 44.99, 51.96 , 54.66, 54.70, 54.74, 60.42 , 67.37 – 67.59 (m), 67.72 .8H18NO5P: 240.1 [M+H]+; found: 240.2 [M+H]+.MS (single quadrupole): calculated for CN,N-Diisopropylethylamine  were added to 7 mL anhydrous CHCl3. Phosphorus(V) oxychloride  was added dropwise to the solution stirring in an ice-water bath. After stirring at 0°C for 2 hours, d9-labeled choline tosylate  was added, followed by pyridine . After pyridine addition, the reaction turned magenta. The flask was removed from the ice-water bath and allowed to stir at room temperature for 3 hours. The reaction was quenched with 0.3 mL water and allowed to stir at room temperature for 1 hour. The mixture was concentrated by rotary evaporation and purified by silica gel chromatography, eluting with 70% ethanol in water. Fractions containing the product were combined, concentrated by rotary evaporation, and centrifuged to remove particles. The supernatant was concentrated to yield thick oil (R)-glycidyl phosphocholine(d9) .S-glycidol  and 1H NMR  δ 2.68 , 2.81 , 3.19 – 3.27 , 3.61 – 3.67 , 3.67 – 3.76 , 4.19 , 4.24 – 4.38 ; 13C NMR  δ 45.02, 51.99 , 53.48 (weak), 53.70 (weak), 53.93 (weak), 60.40 , 67.33 – 67.01 (m), 67.72 .8H9D9NO5P: 249.2 [M+H]+; found: 249.1 [M+H]+.MS (single quadrupole): calculated for C13C2 , cesium carbonate , and the unlabeled (R)-glycidyl phosphocholine (1)  were dissolved in 10 mL dimethylformamide and stirred at 80°C for 6.5 hours. The reaction mixture was cooled on ice and added to 40 mL of 20% methanol in water, which was loaded onto a C18 Seppak cartridge  that had been activated with methanol and equilibrated with 20% methanol in water. The cartridge was eluted with 20 mL each of 20%, 50%, and 80% methanol in water, and finally with 30 mL 100% methanol. The product 1-O-palmitoyl-sn-glycero-3-phosphocholine was present in the 100% methanol elution, which was further purified by reverse phase HPLC using a phenyl-hexyl resin . Solvent A was water, and solvent B was acetonitrile. After a 5 minute equilibration at 50% B, a 15 minute gradient from 50% B to 100% B eluted pure product 1-O-palmitoyl-sn-glycero-3-phosphocholine (3), which was concentrated to yield a white powder Palmitic acid-1,2-1H NMR  δ 0.90 , 1.29 , 1.62 , 2.35 , 3.23 , 3.64 , 3.85–3.93 , 3.93–4.00 , 4.11 , 4.18 , 4.29 ; 13C NMR  δ 14.4, 23.7, 26.0 , 30.2 , 30.4 , 30.5, 30.6, 30.7–30.8 (multiple peaks), 33.1, 34.9 , 54.64, 54.68, 54.72, 60.4 , 66.2 , 67.5 (m), 67.8 , 69.9 ,175.3 .2213C2H50NO7P: 498.3465 [M+H]+; found: 498.3460 [M+H]+.MS (ESI-TOF): calculated for C2CO3, 270 mg, 0.82 mmol), and the (R)-glycidyl phosphocholine (1)  were dissolved in 10 mL dimethylformamide and stirred at 80°C for 6 hours. The reaction mixture was worked up and purified as described above to yield pure product 1-O-palmitoyl-sn-glycero-3-phosphocholine(d9) (compound 4) as a white powder Unlabeled palmitic acid , cesium carbonate  δ 0.90 , 1.29 , 1.62 , 2.35 , 3.63 , 3.85–3.93 , 3.93–4.00 , 4.11 , 4.18 , 4.29 ; 13C NMR  δ 14.4, 23.7, 26.0, 30.2, 30.4, 30.5, 30.6, 30.7–30.8 (multiple peaks), 33.1, 34.9, 60.4 , 66.2, 67.2 (m), 67.8 , 69.9 , 175.4.24H41D9NO7P: 505.3963 [M+H]+; found: 505.3958 [M+H]+.MS (ESI-TOF): calculated for COMe-LysoPC analog (compound 5) was synthesized by modification of the procedure described by Lindberg et al.  and ethyl N,N-diisopropylamine  in dichloromethane (10 mL) with constant stirring. After 2 hours, pyridine  and choline tosylate  were added, and the mixture was allowed to attain room temperature. The reaction mixture was stirred overnight at room temperature. Next, 0.2 mL water were added, and stirred for an additional 1.5 hours. After evaporation, the crude product was purified by silica gel flash column chromatography with a chloroform-methanol gradient  followed by a methanol-water gradient  as eluent. The final 1-O-palmitoyl-2-OMe-LPC (compound 5) was isolated after lyophilization. Yield 105.7 mg (25%). 1H NMR  δ 0.91 , 1.31 , 1.60 , 2.38 , 3.29 , 3.50 , 3.66 , 3.99 , 4.1-4.34 ; 31P NMR (Methanol-d4) δ 1.47. MALDI TOF MS: calculated MW for C25H52NO7P 509.65 Da; found: (M+H)+ m/z 510.5.g et al. . The staescribed . PhosphoP. falciparum parasites were grown for 12 hours (28±2 hpi to 40±2 hpi) in −SerM medium  either in the absence or presence of 20 μM LysoPC(16:0) (Avanti Polar Lipids), 20 μM 13C2-palmitate-labeled-LysoPC(16:0) , or 20 μM d9-choline-labeled-LysoPC(16:0) . Cultures were pelleted by centrifugation for 5 min at 300 g. Erythrocytes were lysed in ice-cold 0.015% saponin (Sigma-Aldrich) in PBS for 10 min. After pelleting , parasites were washed 5 times in 10 mL ice cold PBS. After the final wash, the parasite pellet was snap frozen in liquid nitrogen and stored at -80°C.To test incorporation and metabolism of exogenously added LysoPC, 9-choline chloride (Sigma-Aldrich).The same procedure was followed for characterizing incorporation and metabolism of exogenously added choline. Parasites were grown in −SerM medium either in the absence or presence of 1 mM choline chloride (Sigma-Aldrich) or 1 mM din vacuo, resuspended in 500 μL hexanes, dried, resuspended in 300 μL methanol, dried, and resuspended in 200 μL chloroform for LC-MS analysis (5 μL injections). High-resolution LC-MS was performed in both positive mode and negative mode using a Phenomenex Luna C5 column. Solvent A was 95% water, 5% methanol, 0.1% formic acid, and 5 mM ammonium formate; solvent B was 60% isopropanol, 35% methanol, 5% water, 0.1% formic acid, and 5 mM ammonium formate. After injection, solvent A was pumped at 0.1 mL/min for five minutes, after which the flow rate was increased to 0.4 mL/min, and a 40-minute linear gradient was run from 20% solvent B to 100% solvent B. Peak areas of lysophosphatidylcholines, phosphatidylcholines, phosphatidylethanolamines, sphingomyelins, and ceramides were quantified by integration of [M+H]+ peaks in Agilent MassHunter software. Peak areas of fatty acids, diglycerides, and triglycerides were quantified by integration of [M+NH4]+ peaks, and monoglycerides were quantified by integration of [M+Na]+ peaks in Agilent MassHunter software. Peak areas of phosphatidic acids, phosphatidylserines, phosphatidylglycerols, and phosphatidylinositols were quantified by integration of [M–H]– peaks in Agilent MassHunter software.Pellets were extracted following a previously described protocol . Brieflyin vacuo. The residue was re-dissolved in 90% acetonitrile in water for LC-MS analysis (10 μL injections). High-resolution LC-MS was performed in positive mode using a Phenomenex Kinetex HILIC column. Solvent A was acetonitrile; solvent B was 5 mM ammonium formate in water. After injection, solvent A was pumped at 0.1 mL/min for five minutes, after which the flow rate was increased to 0.4 mL/min, and a 40-minute linear gradient was run from 20% solvent B to 100% solvent B. Peak areas of choline and phosphocholine were quantified by integration of [M+H]+ peaks in Agilent MassHunter software.Diluted pellets from the lipid analysis above were split into 3 aliquots of 75 μL each and their order was randomized. To each 75 μL aliquot in a 1.75 mL centrifuge tube was added 1.0 mL of ice-cold 80% methanol in water. These mixtures were kept on dry ice for 5 minutes, then vortexed one minute, kept on dry ice for 10 minutes, and vortexed again for one minute. The tubes were centrifuged at 4°C for 5 minutes at 18’000 g. The supernatant was collected, and the pellets were re-extracted with 1.0 mL of dry-ice-cooled 80% methanol in water. After pelleting again, the supernatants were combined and concentrated P. falciparum parasites (28±2 hpi) by incubating the cells for 22 hours in CM  or in −SerM as described . Mature schizonts were intravenously (IV) administered to naïve TO mice. Ring stage parasites were isolated at 4 hpi and mature trophozoites and gametocytes were removed by passing through a MACS LD column (Miltenyi Biotec). Infected erythrocytes were incubated in −SerM medium, −SerM medium supplemented with 20 μM LysoPC (−SerM/LysoPC), or serum-complemented medium (+SerM) for 20 hours. Mature schizont stage parasites were then isolated on a 55% Nycodenz (Axis-Shield POC)/RPMI gradient and injected intravenously into 2 or 3 naïve mice. GFP-expressing cells were examined by flow cytometry at 16 hpi to calculate parasitemia, while cells expressing both RFP and GFP (gametocytes) were assessed at 21 hpi. Gametocytemia was calculated as [(RFP+ and GFP+ cells)/GFP+ cells]∗100._1018700  and GFP Reticulocytes were isolated as described . BrieflyP. falciparum NF54 and Pf2004/164tdTom parasite cultures were synchronized to a 4-hour time window using consecutive sorbitol (Sigma-Aldrich) treatments and exposed to conditions that induce (−SerM) or prevent sexual commitment (−SerM/LysoPC (20 μM LysoPC); +SerM) at 30±2 hpi. Total RNA was collected in 1 mL Trizol (Invitrogen) from approximately 5x108 parasites per condition at this time point and in 4-hour intervals thereafter and stored at -80°C. Prior to Trizol treatment, erythrocytes were lysed in 0.015% saponin  and parasites were pelleted  and washed three times in 10 mL PBS at room temperature. RNA was isolated from 500 μL Trizol solution by three extraction steps ; 2x using 100 μL chloroform). After vigorous shaking for 30 seconds, phases were separated by centrifugation . RNA was purified from 200 μL of the aqueous phase using an RNeasy micro kit (Qiagen) and corresponding protocol. RNA was eluted in 32 μL DEPC-treated water, quantified by Qubit (Life Technologies) and stored at -80°C.0.5 μg - 1.0 μg total RNA of each sample was used for library preparation. Libraries were prepared by using TruSeq Stranded mRNA kit (Illumina). Library quantification was conducted by qPCR and TapeStation (Agilent Technologies) measurements. Libraries were subsequently pooled and sequenced using 300 cycle V2 MiSeq reagent kit (Illumina).P. falciparum early trophozoite-infected RBCs were incubated in either −SerM (inducing), −SerM conditions supplemented with 20 μM LysoPC , or serum-complemented medium . Parasites were allowed to develop until the trophozoite stage (26±2 hpi), late trophozoite stage (30±2 hpi), early schizont (36±2 hpi), late schizont (42±2 hpi), or ring stage (4±2 hpi).Prior to imaging, et al. . ConA was added to the dish for 30 minutes at 37°C. It was then washed off twice, using 1x PBS, after which the culture was re-suspended in PBS, added to the glass bottom of the dish, and allowed to settle for 20 minutes at 37°C. Thereafter, non-bound cells were washed off using PBS, leaving a monolayer in the glass bottom, and 2 mL of pre-warmed PBS were added to the dish for imaging. Cells were viewed using a Zeiss Observer Z1 spinning disc confocal microscope equipped with an incubation chamber, a Yokogawa CSU-X1 filter wheel and spinning disc unit, a Photometrics Evolve 512 delta EM-CCD camera and four laser lines: 405, 488, 561, and 642 nm, and an α-Plan-Apochromat 100x 1.46 NA DIC VIS immersion oil lens.Parasites were processed as described by Grüring et al. . BrieflyImmediately prior to imaging, cells were incubated in either 20 μM TopFluor LysoPC (1-(dipyrrometheneboron difluoride) undecanoyl-2-hydroxy-sn-glycero-3-phosphocholine); in 20 μM TopFluor phosphatidylcholine (PC)  undecanoyl]-sn-glycero-3-phosphocholine); or in 20 μM NBD phosphatidic acid (PA) -sn-glycero-3-phosphate ) . Lipids were dissolved in methanol and stored as stocks at a concentration of 1 mM. For use, the lipid solution was added to a dish and the methanol was allowed to evaporate under sterile conditions. The lipids were then re-suspended in PBS to a concentration of 20 μM prior to addition to the iRBCs. For ER colocalization and nuclear staining, ER tracker red  and Hoechst 33342 (ThermoScientific) were used, respectively. Cells were viewed using 40 nm (Hoechst), 488 nm (TopFluor) or 561 nm (ER-tracker and dsRed) laser lines.Image collection parameters for localization at different stages were 512 x 512 d.p.i, 50-72 z-stacks (0.2 μm step size), a zoom level of 1-2 and laser levels of 1-5% for 488 nm, 1-10% for 561 nm, and 10% for 405 nm. Image collection parameters for time-lapse imaging of LysoPC uptake and co-localization with ER-tracker were 512 x 512 d.p.i, at an interval of 1 s per frame. Image acquisition was initiated (time zero) prior to the addition of the labeled dye. Without interruption of image acquisition, 20 s after acquisition of the first image, the dissolved labeled dye was added to the culture dish, and image acquisition continued to visualize uptake of the dye and distribution within the infected erythrocyte.To avoid crosstalk between channels images were collected in line sequential mode with z-increments of 0.19 μm. Images were acquired using the Zen 2012 (Zeiss) software. Images were analyzed with Fiji  and ImarP. falciparum trophozoite-infected erythrocytes were incubated in either −SerM medium (inducing), −SerM medium supplemented with 20 μM LysoPC , or serum-complemented media . Following a 4-6 hour incubation period, parasites were incubated in +SerM, and allowed to develop until 32±2 hpi. Parasites were then immobilized in a glass-bottom dish as described above, stained with Hoechst 33342, and 50-72 z-stacks (0.2 μm step size) obtained using bright field and the 405 nm laser line, to determine the number of nuclei in every infected erythrocyte. This procedure was repeated bi-hourly for a total of 18 hours per condition, and 50-100 images quantified per time point and condition.P. berghei ring stage parasites were isolated as described earlier. Infected erythrocytes were incubated in −SerM conditions, −SerM supplemented with 20 μM LysoPC (−SerM/LysoPC), or serum-complemented medium (+SerM). At the indicated time points, infected erythrocytes were fixed in 4% paraformaldehyde/0.075% glutaraldehyde in PBS for 10 minutes, washed twice in PBS, smeared on glass slides and mounted in Vectashield containing DAPI (Vectorlabs). Z-stacks of 4-5 μm (0.1 μm step) were acquired on a DeltaVision epifluorescence microscope (Applied Precision) using the 405 nm and bright field filter sets. Images were deconvoluted using SoftWoRx to count number of merozoites.Synchronized and newly invaded P. berghei-infected mouse LysoPC quantification with published levels of LysoPC in parasite- and bacteria-infected humans. In the case of bacterial sepsis . In addition, we inserted into p_gC a HindIII-SalI-EcoRI-BamHI multi-cloning site for insertion of a drug resistance cassette and replaced the original BtgZI recognition site in the U6 sgRNA expression cassette with a BsaI site for directional insertion of the sgRNA target sequence. We then inserted a hdhfr resistance cassette into BamHI-digested p_gC to obtain the Cas9/sgRNA mother plasmid pH_gC. To engineer marker-free parasites expressing AP2-G-GFP from the endogenous locus (PF3D7_1222600) we generated transfection vectors pH_gC-ap2g-3’ (Cas9/sgRNA suicide plasmid) and pD_ap2g-gfp (donor plasmid)  encoding the sgRNA target sequence sgt_ap2-g-3’ and appropriate single-stranded overhangs were annealed and inserted into the sgRNA expression cassette using BsaI-digested pH_gC and T4 DNA ligase. The sgRNA target sequence sgt_ap2g3’ (AGTTATAGGGAATATTCAAA) is positioned 60bp downstream of the ap2-g coding sequence and was identified using CHOPCHOP (ap2g-gfp was generated using Gibson assembly joining four PCR fragments encoding (1) the plasmid backbone amplified from pUC19 using primers PCRA_F and PCRA_R, (2) the ap2-g 5’ homology box amplified from 3D7 gDNA using primers PCRB_F and PCRB_R, (3) the gfp coding sequence  amplified from pH-GFP (ap2-g 3’ homology box amplified from 3D7 gDNA using primers PCRD_F and PCRD_R. All oligonucleotide sequences are provided in Supplementary Using the previously published transfection vectors pUF1_Cas9 and pL-6_eGFP  as templCHOPCHOP . The donm pH-GFP  using prap2g-3’ (Cas9/sgRNA suicide plasmid) and 50 μg pD_ap2g-gfp (donor plasmid) using electroporation conditions as described (ap2-g locus (NF54/AP2-GGFP), transfected parasites were grown in presence of 4 nM WR99210 for the first six days and then in absence of drug pressure until a stably propagating parasite population was established (approximately four weeks post-transfection). Successful tagging of the ap2-g locus in the NF54/AP2-GGFP population was confirmed by PCR on gDNA , statistical details can be found in the figure and figure legends.P. falciparum reference genome (PlasmoDB version 28). A maximum of one mismatch per read was allowed. The mapped reads from TopHat (IS) to measure transcriptional responses of genes to different culture conditions. For two strains  in different conditions , the transcription level (Exp) of each gene i in 4 time points  can be represented as SerM ) with SerM, −SerM/LysoPC ) and represented as IS can be represented asRNA-seq reads from each sample were aligned to the m TopHat  were usem TopHat . The expTo identify deregulated genes, the group of non-induced parasites can be represented as vectorard test  was usedGSE104114.The accession number for the transcriptional data reported in this paper is GEO: P. berghei experiments. M.D.N. and J.P.G. performed in vivo extraction and LysoPC analysis from P.berghei-infected and non-infected mouse tissues. M.Z. processed RNA samples and performed MiSeq sequencing. C.W. and S.R.A. analyzed RNA-seq data. M.A.C. performed reticulocyte purifications and provided experimental advice, supervised by M.T.D. D.R. supported RNA-sample isolation and processing. E.H., S.D.B., and I.N. generated the NF54/AP2-GGFP line, and T.S.V. supervised these experiments. M.-C.L. assayed the role of BSA in LysoPC activity. C.G. transfected Pf2004 parasites with the 164tdTom reporter plasmid. E.G.-D. synthesized OMeLysoPC. B.R.-R. and A.C.-G. synthesized choline-kinase inhibitor compound BR23. A.D. and S.B. optimized −SerM composition, and D.F.W. supervised these experiments. J.H.A. and R.H.Y.J. supervised RNA-seq data generation and data interpretation. A.P.W. supervised mouse model work. M.M. and J.C. designed the study, interpreted data, and supervised the study. N.M.B.B., J.P.G., J.C., and M.M. wrote the manuscript with input from all co-authors.N.M.B.B. and J.P.G. designed and performed experiments, analyzed and interpreted data, prepared illustrations, and wrote the manuscript. M.D.N. and N.M.B.B. performed and analyzed microscopy experiments. A.B.S., M.-C.L., and N.M.B.B. performed EV and BSA experiments. N.P. and M.D.N. performed and analyzed


Amino acid sequence comparison and homology modeling indicated the close relation between Ldc1E and other putative LDCs. Multiple sequence alignment analysis revealed that Ldc1E contained a highly conserved motif Ser-X-His-Lys (Pxl), and molecular docking results showed that this motif was located in the active site and could combine with the cofactor pyridoxal 5′-phosphate. The ldc1E gene was subcloned into the pET-30a(+) vector and highly expressed in Escherichia coli BL21 (DE3) pLysS. The recombinant protein was purified to homogeneity. The maximum activity of Ldc1E occurred at pH 6.5 and 40°C using L-lysine monohydrochloride as the substrate. Recombinant Ldc1E had apparent Km, kcat, and kcat/Km values of 1.08±0.16 mM, 5.09±0.63 s−1, and 4.73×103 s−1 M−1, respectively. The specific activity of Ldc1E was 1.53±0.06 U mg−1 protein. Identifying a metagenome-derived LDC gene provided a rational reference for further gene modifications in industrial applications.L-lysine decarboxylase  is a key enzyme in the decarboxylation of L-lysine to 1,5-pentanediamine and efficiently contributes significance to biosynthetic capability. Metagenomic technology is a shortcut approach used to obtain new genes from uncultured microorganisms. In this study, a subtropical soil metagenomic library was constructed, and a putative LDC gene named  Selenomonas ruminantium and Anaerovibrio lipolytica [L-Lysine decarboxylase LDC, EC 4.1.1.18) catalyzes the decarboxylation of L-lysine to 1,5-pentanediamine  with pyridoxal 5′-phosphate (PLP) as a coenzyme ,3. 1,5-P.1.1.18 cpolytica –9. ThereEscherichia coli are commonly reported. LdcC is more active in vitro under a broad pH range than CadA, but the expression level of the latter is higher than that of the former [Corynebacterium glutamicum and E. coli are used to construct 1,5-pentanediamine-producing strains, especially C. glutamicum, which is not synthesized LDC by itself but is widely used to produce L-lysine in industrial applications [cadA gene in C. glutamicum produces 1,5-pentanediamine at a concentration of 2.6 g L−1 [ldcC gene combined with the strong promoter tuf is overexpressed in C. glutamicum, and the yield is 300 mM M−1 glucose with the addition of the cofactor PLP [Klebsiella oxytoca [Hafnia alvei, can also produce 1,5-pentanediamine [ldc gene from K. oxytoca was expressed with the tac promoter in E. coli, and the conversion yield of lysine-HCl to 1,5-pentanediamine was 92% without generating side products [ldc gene from H. alvei was expressed in E. coli JM109 and subjected to directed evolution to obtain a mutant strain. The mutant strain produced 1,5-pentanediamine at a concentration of 63.9 g L−1, which was 5.6% higher than that generated by the original strain [K. pneumoniae and H. alvei have been reported. The optimum pH of LDC from K. pneumoniae in vitro is 6.0 [H. alvei has an optimum pH of 6.5, and the specific enzyme activity is higher than that of CadA and LdcC from E. coli [The current production of 1,5-pentanediamine is mainly through traditional chemical synthesis. Although the synthetic process yields 1,5-pentanediamine at a relatively low cost, the method uses non-renewable fossil fuels and produces serious pollutants in the environment . Microbie former . Corynebications –18. Over.6 g L−1 . The ldcctor PLP . A recen oxytoca  and Hafnediamine . The ldcproducts . The ldcl strain . The chao is 6.0 . The LDC E. coli . Hence, In the microbial community, only 1% or fewer microorganisms can be cultured in a medium ,23, whilIn the current study, a plasmid metagenomic library was constructed from subtropical soil microorganisms. The gene encoding of a putative LDC, Ldc1E, was isolated and identified through functional and sequence-based screenings of the metagenomic DNA library. The derived amino acid sequence exhibited moderate similarity to the ornithine decarboxylase family and the aminotransferase (AAT) superfamily. This research will take an insight into the novel L-lysine decarboxylase from uncultured soil microorganisms. Moreover, gene cloning and functional identification of a metagenome-derived LDC provide novel materials for 1,5-pentanediamine biosynthesis.The study was approved by the insititutional review board of the State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, China. Subtropical soil sample collection in this study did not require permission from relevant authorities.E. coli DH5α (Novagen) and pGEM-3Zf(+) (Promega) served as the host and the vector for the construction of a subtropical soil metagenomic library, respectively. Plasmid pET-30a(+) (Novagen) and bacterial strain E. coli BL21 (DE3) pLysS (Novagen) were used as the expression vector and host, respectively. The E. coli strains were grown at 37°C in Luria–Bertani (LB) medium. Restriction enzymes and ligases were purchased from ThermoFisher. FastPfu DNA polymerase and related reagents were purchased from TransGen Biotech, Beijing, China, and 1,5-pentanediamine, acetonitrile, acetone, and dansyl chloride were obtained from Sigma–Aldrich. A BCA Protein Assay Kit was purchased from Solarbio, China. The rest of the chemicals were analytical grade.PstI and HindIII, and the DNA fragments were examined in 0.8% agarose gel. Fractions containing 2.0–10.0 kb DNA were ligated into the pGEM-3Zf(+) vector, and the ligated products were transformed to competent E. coli DH5α. The constructed metagenomic library was collected in 96-well plates and then stored at −80°C.Soil samples were collected from the ground surface in mountainous areas located in Guangxi, South China . The collected soil samples were maintained at 30°C and pH 6.5 and covered with rotten leaves for several years. Metagenomic DNA was extracted from subtropical soil samples following the method described by Bürgmann with modifications . Purifie−1 ampicillin at 37°C for less than 24 h. The appearance of a purple clearance zone characterized the clones with LDC activity [Metagenomic library clones were cultured onto screening agar plates  supplemented with 50 μg mLactivity . Positivhttp://web.expasy.org/translate/). Sequence similarities and conserved domain search were performed in the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Multiple sequence alignment was conducted with Align X in Vector NTI . A phylogenetic tree was generated with Molecular Evolutionary Genetics Analysis (MEGA 6.0) using the neighbor-joining method . The secondary structure was predicted using the PSIPRED Server [http://bioinf.cs.ucl.ac.uk/psipred/). Protein homology modeling structure was constructed with the GalaxyWEB server [http://galaxy.seoklab.org/). The protein model quality was precisely evaluated using MOLPROBITY [http://molprobity.biochem.duke.edu/). The protein model was used for docking with PLP and L-lysine in Autodock 4.2  with default parameters. The protein structure model was displayed using PyMOL .Protein translation was performed using the translation tool from the Expasy homepage  (http://D Server  (http://B server  (http://LPROBITY  (http://ldc1E nucleotide sequence was amplified from the plasmid of a positive clone. Restriction enzyme sites (underlined) for NcoI and HindIII were designed in the forward/reverse primer (CCATGGCTAACATCATTGCCATCCTCAACC-3′/ 5′-CCCAAGCTTACGCAGCACCTTGACGGTGT-3′5′-CTA). Polymerase chain reaction (PCR) was performed in a 50 μL reaction consisting of 0.2 μM forward primer, 0.2 μM reverse primer, 0.2 mM dNTP, 1×TransStart FastPfu buffer, 2.5 U TransStart FastPfu DNA polymerase, and 20 ng plasmid. The PCR program was as follows: 95°C for 2 min; 30 cycles of 95°C for 20 s, 62°C for 20 s, and 72°C for 1 min; final extension step at 72°C for 5 min. The PCR product was purified and digested with NcoI and HindIII at 37°C for 2 h and then directly ligated into the NcoI-HindIII site of expression vector pET-30a(+). The confirmed recombinant plasmid pET-30a-ldc1E was transformed into competent E. coli BL21 (DE3) pLysS.The −1) and chloramphenicol (50 μg mL−1). When the cell density at 600 nm reached 0.6, the cells were induced with 0.8 mM isopropyl-β-D-galactopyranoside (IPTG) and further grown at 28°C for 12 h. The cells were harvested by centrifugation at 4,000 rpm for 20 min at 4°C and then washed twice with pre-cooled phosphate buffer saline (pH 7.6). The cells were lyzed by sonication, and the lysate was centrifuged at 12,000 rpm for 20 min at 4°C. Recombinant Ldc1E was purified with nickel-nitrilotriacetic acid (Ni-NTA) agarose resin and analyzed by denaturing discontinuous sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The protein concentration was determined using the BCA Protein Assay Kit.The transformed bacterial cell was cultivated at 37°C in LB medium with kanamycin . The HPLC system was equipped with a Waters 2475 detector and a chromatographic column . Isocratic elution with 65% acetonitrile as the mobile phase was used to separate the product, and the product was detected at 340 nm. The column temperature was maintained at 25°C. The injection volume of the sample was 10 μL, and the flow rate was 1 mL min−1, 200 μL) was added to the mixture and then incubated at 60°C for 30 min. 25% ammonium hydroxide (100 μL) was added to remove the residual dansyl chloride, and the volume of the mixture was adjusted with acetonitrile to 2 mL [The sample (0.1 mL) was mixed with 2 M sodium hydroxide (20 μL) and saturated sodium bicarbonate solution (30 μL). Dansyl chloride solution . The reaction mixture was incubated at 40°C for 15 min and terminated by adding 1 M K2CO3 (250 μL). The mixture was added with 9.6 mM (200 μL) TNBS and then incubated at 40°C for 5 min. Each sample was thoroughly mixed with toluene (1 mL) at top speed for 1 min. The toluene layers were separated through centrifugation at 4,000 rpm for 15 min. Heat-inactivated recombinant Ldc1E served as a negative control. The absorbance was determined at 340 nm [LDC activity was determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS). This method was based on the differential solubility of lysine and 1,5-pentanediamine reaction products in toluene . The rea2HPO4/0.1 M citric acid buffer (pH 6.5). The enzyme activity obtained at the optimum temperature was used to calculate the relative percentage of activity at other temperature values. To evaluate thermostability, Ldc1E was incubated in 0.2 M Na2HPO4/0.1 M citric acid (pH 6.5) for up to 1 h at the temperature range of 20–60°C.Temperature-dependent activity assays were performed at various temperatures from 20°C to 60°C with 5°C intervals in 0.2 M Na2HPO4/0.1 M citric acid pH 4.0–8.0 and 0.1 M glycine–NaOH pH 8.0–10.0 at 40°C. To estimate pH stability, Ldc1E was pre-incubated at the pH range of 4.0–9.0 at 4°C for 60 h. All reactions were performed in three independent experiments.pH-dependent activity assays were measured in 0.2 M NaTo investigate the effect of different PLP concentrations on Ldc1E activity, we measured the enzyme activity supplemented with 0, 0.05, 0.1, 0.5, and 1.0 mM PLP. All reactions were performed in three independent experiments.2, ZnCl2, CuCl2, CaCl2, CoCl2, BaCl2, SrCl2, CrCl2, MnCl2, NiCl2, FeCl3, AlCl3, ethylenediamine tetraacetic acid (EDTA), dimethyl sulfoxide (DMSO), Triton-100, Tween-20, Tween-80, or SDS. The control group did not contain a metal compound or a chemical reagent. All reactions were performed in three independent experiments.The sensitivity of Ldc1E to metal compounds and chemical reagents was analyzed by measuring the enzyme activity supplemented with different concentrations of MgCl2HPO4/0.1 M citric acid (pH 6.5). One unit was defined as the enzyme consumption of 10 μg L-lysine-HCl/L-arginine-HCl/L-ornithine-HCl per minute under the standard assay conditions.The substrate specificity of Ldc1E was measured using the standard assay. The reaction mixture contained 2 mM L-lysine-HCl/L-arginine-HCl/L-ornithine-HCl, 0.1 mM PLP, 3 μg recombinant protein, and 0.2 M NaThe kinetic parameters of purified Ldc1E were assayed by linear regression from Lineweaver–Burk plots with L-lysine HCl as the substrate at pH 6.5 and 40°C . Substraldc1E nucleotide sequence was deposited in the GenBank database with the accession number KX463450.The PstI and HindIII were used to digest randomly selected clones. DNA inserts were obtained in the range of 2.5–6.0 kb, with an average of 4.0 kb  [E. coli O157:H7  [Salmonella enterica subsp. LT2  [H. alvei  [E. coli K-12  [3+ of Lys could form a covalent bond with PLP [Sequence analysis showed that the ilarity) , which iilarity) , Salmoneilarity) , H. alveilarity) , and E. with PLP . The seqwith PLP .E. coli, S. enterica, H. alvei and A. hydrophila) have evolved from a common ancestor. Among them, Ldc1E was closely related to A. hydrophila AL06-06 LDC. In addition, Ldc1E belonged to the same cluster as the LDC of the AAT superfamily, and other similar decarboxylases belonged to the Orn/Lys/Arg decarboxylase class-I family.A phylogenetic tree was constructed using the neighbor-joining method to show Ldc1E having a distinct clade from the LDCs of other microorganisms . AccordiE. coli K12, and its quaternary structure was a homo-decamer [The 3D structure model of Ldc1E was constructed by using the GalaxyWEB server, and a template (PDB: 3n75) was found by searching the amino acid sequence in Protein Data Bank . 3n75 wa-decamer . The ide-decamer . The pre-decamer ,45.Docking of the Ldc1E monomer with PLP from the Autodock 4.2 software showed that the binding sites probably contained the residues Trp333, Ser364, His366, and Lys367 . The Lysldc1E was subcloned into the expression vector pET30a(+) and transformed into E. coli BL21 (DE3) pLysS. Intracellular Ldc1E was expressed without any modification. The recombinant Ldc1E was expressed by induction with 0.8 mM IPTG. Crude Ldc1E was purified by Ni-NTA magnetic agarose chromatography and analyzed using SDS–PAGE. A single ~80 kDa band corresponded to the predicted size of Ldc1E  at pH 6.5 and 40°C. Lineweaver–Burk plots of the initial velocities determined x values . Ldc1E h sources . The Km  present . The Km eumoniae ,21. Thusectively ,21. The A metagenome-derived LDC gene was cloned and characterized from subtropical soil microorganisms. Sequence analysis showed that the identified gene product had moderate similarities to known LDCs. The detailed biochemical characterization of the Ldc1E protein was performed, including the temperature–activity profile, pH–activity profile, metal ion–activity profile, chemical cheating activity profile, and enzyme kinetics analysis. These results indicated that Ldc1E could be a good candidate for industrial and chemical applications. Furthermore, this research also revealed the potential of using metagenomics to discover novel LDCs for converting L-lysine into 1,5-pentanediamine.S1 FigHind III Marker; Lane 2: metagenomic DNA from subtropical soil microorganisms; (B) Agarose gel electrophoresis profile of the metagenomic DNA double digested with PstI and HindIII. Lane 1: λ DNA/HindIII Marker; Lane 2: metagenomic DNA digested with PstI and HindIII; (C) Agarose gel electrophoresis profile of random positive plasmid DNA from the metagenomic library. Lane 1: 1 kb DNA Marker; Lanes 2–9: recombinant plasmid digested with PstI and HindIII; Lane 10: plasmid pGEM-3Zf(+) digested with PstI and HindIII.(A) Agarose gel electrophoresis profile of the metagenomic DNA. Lane 1: λ DNA/(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigIsolation of potential positive clones with functional screening strategy of LDC from the metagenomic library of subtropical soil microorganisms.(TIF)Click here for additional data file.S4 Fig−1.The concentrations of PLP varied at 0, 0.05, 0.1, 0.5, and 1.0 mM. LDC activity assay was performed as described in materials and methods. The specific activity of Ldc1E at 0.1 mM PLP was 1.53±0.05 U mg(TIF)Click here for additional data file.S5 FigThe kinetic parameters of the purified enzyme Ldc1E were assayed by linear regression from Lineweaver–Burk plots with L-lysine-HCl substrate at pH 6.5 and 40°C.(TIF)Click here for additional data file.


The title compound is the product of a new synthetic route towards tetraaryl/hetaryl-substituted ethenes that reduces the occurrence of side-products. In the crystal, the molecule is centrosymmetric and the cyclopentadienyl rings are nearly coplanar and aligned slightly closer to a staggered conformation than to an eclipsed one. 2(C5H5)2(C20H14O2)], is the product of a new synthetic route towards tetraaryl/hetaryl-substituted ethenes that reduces the occurrence of side-products. In the crystal, the molecule is centrosymmetric and the cyclopentadienyl (Cp) rings are nearly coplanar and aligned slightly closer to a staggered conformation than to an eclipsed one. The ethene plane is tilted by 32.40 (18)° with respect to that of the substituted Cp ring and by 63.19 (19)° with respect to that of the furan ring. C—H⋯π interactions link the molecules into a three-dimensional supramolecular framework.The title compound, [Fe The same method was applied for the preparation of some ferrocenyl/hetaryl-substituted ethenes -1et al., 1990E)-configuration to the obtained compound. In our case, single crystals of (E)-1 were grown from hexane/CH2Cl2 and used for an X-ray diffraction analysis, from which the previous tentatively postulated structure of the obtained isomer could be confirmed.Here we report the analogous synthesis and crystal structure of the known title compound, (E)-1The molecule of -isomers. The archetypal structure is (E)-1,2-diferrocenylethene -configuration display Cp arrangements that are much closer to eclipsed than observed for (E)-1 -configured examples have a bulky substituent on one of the distal Cp rings; those with a less bulky Cp substituent have the eclipsed arrangement. Interestingly, (E)-1 has no Cp substituents yet the Cp ring arrangement deviates significantly from eclipsed. The degree of eclipsing of the Cp conformations found among the molecules with the (Z)-configuration, two of which have a cyclopropene ring as the ethene bridge  was added in small portions. Stirring was continued for 20 min, and after warming to room temperature, the solvent was evaporated under vacuum. The crude product was analyzed by 1H NMR spectroscopy, which revealed the presence of two isomeric ethenes in a ratio of ca 10:1. After column chromatography , the major product was isolated, contaminated with a small admixture of the minor one, as an analytically pure sample (78% yield). After additional crystallization from a hexane/CH2Cl2 mixture, 285 mg (54%) of pure (E)-1 were isolated as orange crystals with m.p. 485–487 K. From this material, crystals suitable for the X-ray diffraction measurements were separated without additional recrystallization.The title compound was prepared according to the reaction sequence presented in the scheme below1H NMR : 3.63–3.65 , 4.13–4.15 , 4.16 , 6.40 , 6.54 , 7.58 . 13C NMR : 68.5, 68.9 [2 signals for 8CH(Fc)], 69.6 [10CH(Fc)], 85.4 [2C(Fc)], 109.1, 111.1, 140.8 [3 signals for 6CH(Fur)], 129.1 (C=C), 153.3 [2C(Fur)]. ESI–MS (mixture of isomers): 528 , 529 . Elemental analysis calculated for C30H24Fe2O2 (528.20): C 68.22, H 4.58%; found: C 68.38, H 4.61%.Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018005078/sj5552sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989018005078/sj5552Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018005078/sj5552Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018005078/sj5552Isup4.cdxSupporting information file. DOI: 1833400CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the methods section “Characterization by HPLC and LC-FTMS”, the identities of solvents A and B were switched. Solvent A should be listed as 0.1% formic acid in water, not in 0.1% formic acid in acetonitrile; and vice versa for solvent B. The corrected text is indicated in bold and underlined below:0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 1 mL/min. Initial conditions were 98:2 (A:B) changing to 70:30 (A:B) at 50 min, to 2:98 (A:B) at 70 min and held until 85min. Samples were prepared in DMSO and 10 μL injections were made. Chromatograms were monitored at 254 nm and 314 nm.An analytical HPLC-method was developed for the purposes of characterization of 224 and fractions. The analysis was performed on an Agilent 1260 Infinity system running OpenLab CDS ChemStation  with an Agilent ZORBAX Eclipse XDB-C18  column with compatible guard column at a column temperature of 40°C. Mobile phase reagents were HPLC-grade and purchased from Fisher Scientific, except for the Type 1 water, which was obtained from an EMD Millipore MILLI-Q water system . Mobile phase consisted of a linear gradient elution 0.1% formic acid in water (A) and 0.1% formic acid in Optima LC/MS acetonitrile (Fisher Scientific) (B) at a flow rate of 1 mL/min. Initial conditions were 98:2 (A:B) changing to 64:36 (A:B) at 12 min, to 52:48 (A:B) at 86 min, 2:98 (A:B) at 102.6 min and held until 117.6 min before returning to initial conditions to equilibrate the column. The data was acquired in MS1 mode scanning from a m/z of 150–1500 on a Thermo Scientific LTQ-FT Ultra MS in negative ESI mode and processed with Thermo Scientific Xcalibur 2.2 SP1.48 software . The capillary temperature was 275.0°C, sheath gas of 60, source voltage and current 5.0 kV and 100.0 μA, and the capillary voltage -49.0 V.Liquid chromatography-Fourier transform mass spectrometry (LC-FTMS) was performed on 224C-F2 using a Shimadzu SIL-ACHT and Dionex 3600SD HPLC pump with a modification of the previous chromatographic conditions. A 20 μL injection at ambient temperature with


Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation. Mapping protein-ligand interactions is fundamental to advance the understanding of cellular processes and to develop drug discovery strategies. Here, the authors present a photo-activated probe that allows highly efficient labelling and identification of protein binding sites using mass spectrometry. Interactions between proteins, and between proteins and small molecules are at the heart of practically all biochemical processes3101113141 and 4,4-azipentanoic acid for diazirine-based protein carbene footprinting. The former has been used to map the calmodulin-M13 interaction site1 to be highly protein dependent, and that labelled peptides exhibited, in some cases, sub-optimal behaviour during MS/MS fragmentation. Despite this, we believed that the carbene-labelling strategy offered considerable promise owing to its use of benign, near-ultraviolet photoactivation by compact low-cost laser systems, and clear mass shifts upon protein labelling. To provide a platform for high-resolution mapping of protein interactions by MS, we have designed and synthesized a bespoke diazirine-based footprinting agent for differential protein labelling experiments. This probe labels proteins with much higher efficiency than photoleucine at both lower concentrations, and lower irradiation times. We have successfully used the probe to map the lysozyme substrate-binding site accurately at high resolution, and identify changes in intramolecular interactions associated with binding. In a second example of the technology we have identified the substrate-binding sites of a large, multi-domain deubiquitinating protease and used the results to clarify uncertainties raised by the X-ray crystal structure.Diazirine-based footprinting agents, in contrast, offer the potential to use near-ultraviolet wavelengths (≈350 nm)–outside the absorbance window of amino acids–to generate highly reactive carbenes, which rapidly label the surface of a protein202, based on a sodium benzoate core was considered an appropriate choice, where the presence of the –CF3 group has the benefit of reducing side reactions associated with the diazoisomer2 has been synthesized some years previously, for incorporation into photoaffinity probes, but not for use as a footprinting tool2 was achieved following a modified literature method , enzyme activity assays were performed in the presence and absence of the probe. No change in enzyme activity was detected upon addition of 10 mM 2 to HEWL, indicating that the enzyme's structure was unaffected by the diazirine (N-acetylchitopentaose (NAG5) substrate to highlight regions of HEWL masked by NAG5. Interactions between enzyme and substrate in this system have been studied in detail, and a large number of high-resolution structures are available2 as part of a carbene labelling strategy.An important consideration when using probes such as iazirine . These e2 was performed in the presence and absence of NAG5 (see the ‘Methods' section). Irradiation of the sample following flash freezing had the dual benefit of halting enzyme turn-over and reducing diffusion of the probe. Indeed, we found freezing a necessary step for efficient labelling. Following proteolysis of HEWL with either AspN or trypsin, the resulting peptides (representing 79% sequence coverage) were analysed by liquid chromatography-tandem MS (LC-MS/MS) to quantify the degree of labelling at each position  reduced in the presence of the NAG5 ligand , of current interest in our laboratory, provides an excellent example of such a protein. The enzyme is responsible for the disassembly of unanchored poly-ubiquitin (poly-Ub) chainsde novo assembly. Recent work has identified important physiological roles for unanchored poly-Ub in regulating the 26S proteasome2 was performed on the catalytically inactive C335A USP5, both in the presence and absence of one equivalent of K48-linked di-Ub. Following irradiation and subsequent trypsin digestion, the differential labelling pattern of USP5 was quantified by LC-MS , and its structural features make it a highly efficient labelling reagent. It was not designed to be an accurate measure of solvent accessibility of a protein, but rather for use in a differential, ±binding partner assay. Once formed, the carbene has a lifetime of a few ns, reacting with either protein, buffer or water molecules. By conducting labelling experiments on flash frozen samples we were able to reduce diffusion effects232-amino acid sidechain interactions, rather than the inherent reactivity profile of the carbene. This notion was supported by the amino acid-labelling distribution of the hydrophobic and anionic probe 2 (on both HEWL and USP5), which generally favoured hydrophobic and basic (cationic) residues. The spin state of aryltrifluoromethyl carbenes, such as 3, is relatively complicated; p-toluyl(trifluoromethyl)carbene, for example, has a triplet ground state, but it principally reacts through its low-lying singlet stateMethods that permit high-resolution mapping of protein interaction sites with improved throughput and sensitivity are highly sought after. We have discovered and demonstrated that differential carbene footprinting with diazirine n oligomers, to HEWL has been studied in detail over the last 50 years, and the precise protein–ligand interactions are well described. The importance of W62, for example, in making van der Waals contacts with a sugar ring in the oligomeric substrate has been demonstrated by X-ray and NMR studiesThe binding of carbohydrate substrates, such as NAGIn contrast, the interactions between USP5 and its poly-Ub substrates are more complex and less well studied than those of lysozyme. USP5 is a large, dynamic enzyme with specific Ub-binding regions in addition to the active-site domain itself. The ZnF-UBP domain has a particularly high affinity for the C-terminus of Ub ref. , and thi2 in the presence and absence of small molecule or protein-binding partners allows high-quality mapping of interaction sites.In summary, we have shown that differential carbene labelling of proteins with diazirine 2 under a nitrogen atmosphere. NEt3 was distilled from CaH2 and stored over 4 Å molecular sieves. All other reagents were obtained from commercial sources and used without further purification. Reactions were monitored using thin layer chromatography, and visualized using ultraviolet light and stained using a basic KMnO4 (potassium permanganate) solution. Column chromatography was performed using Merck silica gel 60 as the stationary phase. Petrol refers to petroleum spirit (b.p. 40–60 °C).Unless otherwise stated, reactions were carried out in flame-dried glassware, under an atmosphere of argon with magnetic stirring. Tetrahydrofuran (THF) was freshly distilled over sodium and benzophenone; dichloromethane (DCM) was freshly distilled over CaH1H, 13C and 19F) were recorded on Bruker AV400 (400 MHz), DPX400 (400 MHz), AV3400 (400 MHz) and DPX300 (300 MHz) spectrometers, in CDCl3, DMSO-D6 or CD3OD. Infrared spectra were obtained using a Bruker Tensor 27 FT-IR spectrophotometer as a solution in CHCl3, with the peaks recorded as υmax (cm−1) or using an attenuated total reflectance (ATR) adaptor for the Nicolet IsT Spectrometer. High-resolution mass spectra (HRMS) were obtained using a Bruker MicroTOF time of flight mass spectrometer operating in electrospray ionization mode, or a Fisons AutoSpec magnetic sector mass spectrometer in electron impact ionization (EI) mode. Melting point data was collected using a Stuart SMP3. Spectral data showed agreement with the literatureNMR spectra  and the solution stirred for 15 min. Ethyl trifluoroacetate  was then added dropwise over 5 min, the solution slowly warmed to room temperature over 2 h before addition of 2 M HCl(aq) (10 ml). Volatiles were removed under reduced pressure, before the product was extracted into the organic layer of a work up between EtOAc (50 ml) and 2 M HCl(aq) (50 ml). The organic layer was dried over MgSO4, concentrated under reduced pressure to yield a colourless oil, which was used without further purification. A separate reaction was purified for analytical data.To a solution of 4-bromotoluene  in anhydrous THF (20 ml) at −78 °C was added H : 7.97–8.00 , 7.34–7.38 , 2.47 ; δC: 180.1 , 147.0, 130.3, 129.8, 127.5, 116.8 , 21.9; δF : −71.3; υmax (cm−1): 1,714, 1,608, 1,176, 939.δp-tolyl)ethanone (from the previous step) in pyridine (10 ml) and ethanol (5 ml) was added hydroxylamine hydrochloride . The reaction mixture was heated at 50 °C for 16 h, cooled and diluted with EtOAc (100 ml), and the organic phase washed with 2 M HCl(aq) (50 ml), dried over MgSO4, and concentrated under reduced pressure. Purification by column chromatography (5% EtOAc/Petrol) yielded the title compound as a white solid . m.p. 77–80 °C (ethanol).To a solution of crude 2,2,2-trifluoro-1-: 9.61 , 7.50–7.55 , 7.32–7.36 , 2.45 ; δC : 147.5 , 141.2, 129.3, 128.6, 122.8, 120.7 , 21.4; δF : −66.6; υmax (cm−1): 3,561, 1,612, 1,516, 1,381; HRMS (EI): 203.0551 [M+] found, C9H8F3NO2 requires 203.0558.δp-tolyl)ethanone oxime  and 4-(dimethylamino)pyridine (DMAP)  in anhydrous dichloromethane  was added triethylamine  followed by tosyl chloride  at 0 °C. The reaction mixture was stirred at room temperature for 1 h before the organic layer was washed with H2O, dried over MgSO4 and concentrated under reduced pressure. Purification by column chromatography (5% EtOAc/Petrol) yielded the title compound as a white solid . m.p. 101–103 °C (Et2O/hexane).To a solution of 2,2,2-trifluoro-1-: 7.87–7.93 , 7.20–7.42 , 2.49 , 2.41 ; δC: 154.0 , 146.1, 142.5, 131.3, 129.9, 129.5, 129.3, 128.5, 121.7, 119.7 , 21.8, 21.6; δF: −66.6; υmax (cm−1): 3,043, 2,926, 1,598, 1,389; HRMS (electrospray ionization): 380.0546 found, C16H14F3NNaO3S (M+Na) requires 380.0539.δp-tolyl)ethanone oxime  and DMAP  in anhydrous dichloromethane (30 ml) was added triethylamine  followed by tosyl chloride  at 0 °C. The reaction mixture was stirred at room temperature for 1 h, then the organic layer was washed with H2O (50 ml), dried over MgSO4 and concentrated under reduced pressure. The crude tosylate 6 was then dissolved in anhydrous dichloromethane (3 ml), added to liquid nitrogen (3 ml) at −78 °C in a microwave vial, sealed and stirred for 16 h.To a solution of 2,2,2-trifluoro-1-(2O (50 ml), dried over MgSO4 and concentrated under reduced pressure. Purification by column chromatography (20% EtOAc/Petrol) yielded the title compound as a white solid . m.p. 42–44 °C (hexane).The microwave vial was de-capped, excess ammonia evaporated, and the residue diluted in dichloromethane (50 ml). The organic phase was washed with HH: 7.48–7.53 , 7.20–7.26 , 2.77 , 2.39 , 2.19 ; δC: 140.3, 129.4, 129.1, 128.0, 123.6 , 57.9 , 21.3; δF: −75.7; υmax (cm−1): 3,282, 3,009, 1,731, 1,602, 1,394; HRMS (EI): 202.0719 [M+·] found, C9H9F3N2 requires 202.0718.δp-tolyl)-3-(trifluoromethyl)-3H-diaziridine  in pyridine (10 ml) and H2O (10 ml) was added KMnO4  and the reaction stirred at 50 °C for 16 h. The solution was cooled, diluted with H2O (10 ml) and acidified with 2 M HCl(aq) (50 ml) before the addition of 10% sodium bisulfite(aq) (20 ml). The product was extracted into Et2O (50 ml), before the organic layer was separated, basified with 2 M NaOH(aq) (60 ml), extracted into aqueous layer before being acidified once more and extracted into Et2O (100 ml). The product was dried over MgSO4 and concentrated under reduced pressure to yield the title compound 8 as a white solid . m.p. 121–122 °C (Et2O/hexane).To a homogenous solution of 3-: 8.02–8.07 , 7.38–7.42 ; δC : 166.3, 132.4, 131.9, 130.1, 126.6, 121.7 , 28.1 ; δF : −64.4; υmax (cm−1): 3,513, 3,061, 1,737, 1,700 and 1,611.δ2O (1 ml) was added 4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoic acid , and the suspension stirred for 5 min. The suspension was filtered, and the filtrate concentrated under reduced pressure to yield the title compound as a white solid .To a solution of sodium hydroxide  in HH : 7.98–8.02 , 7.20–7.25 ; δC : 174.0, 141.1, 131.7, 131.0, 127.0, 123.8 , 29.7 ; δF : −66.9; υmax (cm−1): 1,592, 1,546, 1,418, 1,343, 1,144, 942 and 777.δN-acetylchitopentaose (NAG5), dithiotreithol (DTT), iodoacetamide, calmodulin, equine cytochrome c, horse heart myoglobin, melittin and ubiquitin were purchased from Sigma-Aldrich . Trypsin and AspN were obtained from Promega . Lys48 poly-ubiquitin ladder, wild-type USP5 and Lys48 diubiquitin were purchased from Boston Biochem . C335A USP5 was expressed and purified as previously described1 was purchased from Thermo Fisher Scientific, Loughborough, UK, and aryldiazirine 2 synthesized as described above.HEWL, penta-1 or 2) in 20 mM Tris/150 mM NaCl, to give a final label concentration of 10 mM. The mixture was left equilibrating for 5 min at room temperature. Aliquots (6 μl) of this solution were placed in ‘11 mm PP vial crimp/snap 250 ul' sample vials (Thermo Fisher Scientific). To avoid enzymatic cleavage of the pentasaccharide substrate, NAG5 was added to the mixture, to a final concentration of 80 μM, just before snap-freezing the samples in liquid nitrogen (77 K). Freezing was found to be important for efficient and reproducible labelling. For lysozyme footprinting in the absence of substrate, the NAG5 solution was substituted by an equal volume of buffer. The labelling reaction was initiated by irradiation (16 s) of the mixture using a Spectra Physics Explorer 349 laser . The laser beam was directed into the open top of the sample vial using a small 45° mirror.An aqueous solution of lysozyme (160 μM) in 25 mM ammonium acetate or 20 mM Tris/150 mM NaCl was mixed with an equal volume of a 20 mM solution of the diazirine labelling agent  and diazirine 2  was snap-frozen and irradiated at 349 nm as described for lysozyme.Labelling efficiency. Solutions of calmodulin, myoglobin, cytochrome c, ubiquitin or melittin (70 μM in 20 mM Tris/150 mM NaCl) and photoleucine 1 (100 mM in 20 mM Tris/150 mM NaCl) or diazrine 2 (10 mM in 20 mM Tris/150 NaCl) were snap-frozen and irradiated at 349 nm for 4 s in the case of diazirine 2 , and 15 s in the case of photoleucine 1. Samples were diluted to 5 μM with mobile phase A (H2O/acetonitrile 95/5+0.1% formic acid) and analysed directly by LC-MS.Protein digestion. Following separation by SDS–polyacrylamide gel electrophoresis , protein bands were excised, reduced , alkylated  and digested at 37 °C with either trypsin (1:15 protease/protein ratio in 10 mM ammonium bicarbonate) or AspN (1:70 protease/protein ratio in 5 mM Tris-HCl) for 16 h  system using a commercially available C4 trapping column (Dionex) and a custom packed WP C4 column  with a Picofrit tip . The mobile phases A and B consisted of 95/5 water/acetonitrile (v/v) and 5/95 water/acetonitrile (v/v), respectively, and both contained 0.1% formic acid. The samples (1 μl) were injected in load-trapping mode. Peptides were eluted using a 30 min linear gradient of mobile phase B from 0–70% at a flow rate of 0.3 μl min−1 followed by column equilibration. The high-performance liquid chromatography system was coupled to a Thermo Scientific  LTQ FT Ultra mass spectrometer equipped with a commercial nanoelectrospray ionization source. A 1.7 kV voltage was applied to a coated PicoTip emitter (New Objective). The capillary temperature was set at 275 °C, with inner capillary voltage value set on 37 V and tube lens value of 145 V. Spectra were acquired in positive ion mode over a 400–2,000 m/z range at a nominal resolution of 100,000 (at m/z=400). The instrument was controlled by Xcalibur software (Thermo Fisher). Identification of USP5 peptides was conducted using an automated data-dependent acquisition mode, followed by manual examination of the raw data. For data-dependent acquisition the four most intense ions for each scan were isolated within a window of 8 Th and subjected to collision induced dissociation (CID) using a nominal energy of 35.0. Signals with +1 charge state were rejected. The data were searched against a custom database including the C335A USP5 sequence using Bioworks software (Thermo Fisher Scientific).Q-value of 0.250. The scans for each labelled peptide were combined to give an average spectrum containing labelled and unlabelled fragments.The proportion of labelling at the peptide level was determined by integrating the signals for each labelled and unlabelled peptide ion. Partial, or even complete chromatographic separation was seen for some labelling isomers , and spectra were combined across the entire peak or set of peaks to ensure that the quantities of all labelled forms were included in the subsequent calculations. CID experiments for residue-level labelling identification/quantitation were performed at a nominal energy of 15.0. Each manually selected labelled precursor ion was isolated within a window of 10.0 Th  and the activation time was set at 30 ms with an activation et al.Data analysis: the fractional modification per peptide, and the average number of labels per residue were determined using the approach described by Jumper Lysozyme activity assay. Cell suspensions of Micrococcus lysodeikticus ATCC  were equilibrated in a Multiskan Go microplate spectrophotometer (Thermo Scientific) at 25 °C for 5 min with or without the presence of 10 mM diazirine 2. A solution of lysozyme  was added to each suspension, and the decrease in absorbance at 450 nm was measured every 60 s for a total of 10 min. As a control, a cell suspension without the addition of either lysozyme or 2 was similarly monitored.USP5 activity assay. The deubiquitination assay was performed under conditions (1:4 enzyme: substrate molar ratio) previously used by Komander et al.−1 in 10 × deubiquitinating buffer (500 mM Tris (pH 7.5), 1500, mM NaCl, 100 mM DTT), to yield a 1 × deubiquitinating buffer and pre-incubated at room temperature for 10 min. 10 μl of the pre-incubated USP5, in the presence or absence of 3 μl of aryldiazirine 2 (100 mM), was then mixed with 3 μg of K63-linked tetraubiquitin , 3 μl of 10 × deubiquitinating buffer and made up to 30 μl with water. 6 μl aliquots were removed at the indicated time points and quenched via the addition of 9 μl of SDS–PAGE gel application buffer  SDS, 20% v/v glycerol, 10% (w/v) 2-mercaptoethanol, 3% (w/v) DTT, 0.1% (w/v) bromophenol blue, pH 6.8 (HCl)). 50% of each sample was resolved on a 5–20% SDS–PAGE, western blotted and visualized with anti-ubiquitin  for the presence of poly-Ub disassembly. Densitometric analysis of the indicated tetraubiquitin band  via the PRIDE partner repository with the data set identifier PXD004971.Data supporting the findings of this study are available within the article, and its How to cite this article: Manzi, L. et al. Carbene footprinting accurately maps binding sites in protein–ligand and protein–protein interactions. Nat. Commun.7, 13288 doi: 10.1038/ncomms13288 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Methods, Supplementary References


Significantly, fluorescent supramolecular ensemble was fabricated from the supramolecular complexation of MSP5 and a neutral guest with tetraphenylethene core. Enhanced fluorescent emission of this system can be detected both in dilute solution and the solid state, and its temperature and competitive guest multi-responsive properties suggest its promising application as a chemical sensor towards alcohol analogs, ethylenediamine, and temperature variations.A pillar[5]arene derivative with a hydrophilic sulfonic group,  Considering their superior properties, special structures and typical functions in supramolecular chemistry, remarkable attentions have been focused on the exploitation of their potentials and progress in molecular machines7, molecular recognition11, nanomaterials16, supramolecular polymers21, chemical sensors and detectors25, biological medicine30 and so on32. As a relatively new class of supramolecular macrocyclic hosts, pillar[n]arenes have attracted extensive attention owning to its unique properties, rigid structures, easy functionalization, etc.35. Monofunctionalized pillar[n]arenes, as one typical type of useful pillar[n]arene derivatives with unique substituent groups, possess variety of abilities such as molecular recognition and fluorescent detection, depending on their single modified functional groups and the different host-guest interactions40.Supramolecular macrocyclic compounds have been largely developed since the first generation of crown ethers till the fifth generation of pillar[n]arenes during the past decades44, was first reported by Tang and coworkers in 200145, which paves a new way for the efficiency of fluorescent dyes in the solid state and the concentrated solution and breaks the limited applications of traditional fluorophores with ACQ properties. Restriction of intramolecular rotation (RIR) of AIE molecules was proven to be the well-known mechanism of their fluorescent enhancement in the aggregated state. Non-radiation energy dissipation channel from the excited state to ground state was blocked because of the hindrance of intramolecular steric interaction resulted from the impeded intramolecular rotations when the AIE molecules are assembled48. Tetraphenylethene (TPE) is a typical AIE molecule that has been widely investigated during the past two decades50. Several studies combining supramolecular approaches with typical fluorescent molecules with AIE properties to construct controllable fluorescent detectors or stimuli-responsive supramolecular materials have been reported53, especially using some pillar[n]arene derivatives as building blocks. This indicates that the combination of pillararenes and TPE is indeed an efficient way for the fabrication of novel smart optical devices to be applied in chemical sensors51, biological imaging55, and detection of pollutants and explosives57, among which the immediately selective detection of alcohol analogs is urgently required in the field of ecological environment and industrial development. The detection of alcohol analogs possesses special significance in traffic safety and medical emergency.Aggregation-induced emission (AIE), entirely opposite to the aggregation-caused quenching (ACQ) effect of traditional fluorescent dyes4, has also been designed and prepared via host-guest complexation, giving the credit to the hydrophilic group of MSP5 was not affected by pH of the solution. MSP5 also exhibited effective binding affinity towards alcohols via hydrogen bonds between hydroxyl of alcohols and sulfonic group of MSP5, making it possible to selectively detect alcohol analogs by destroying the complex of TPE-(Br)4⊂MSP5. Furthermore, the complex of MSP5 and TPE-(Br)4 can also serve as a temperature sensor and fluorescence probe for ethylenediamine.Herein, for the first time, we successfully synthesized sulfonic group-substituent monofunctionalized pillar[5]arene, i.e., monosulfonicpillar[5]arene (MSP5), Fig.  in a goovia Williamson ether-type synthetic method. The structure of MSP5 was confirmed by 1H NMR, 13C NMR, HRMS, and FT-IR spectroscopy  between those alcohols and MSP5, respectively. MSP5 has the strongest binding affinity toward butanediol among other alcohols , indicating its ability of selective recognition toward alcohol analogs particularly with an enlarged selectivity.MSP5 was synthesized through the installation of a 1-butanesulfonic acid sodium onto pillar[5]arene ols Fig. . Molar rols Fig. . Interesols Fig.  as compavia host-guest interaction between MSP5 and TPE-(Br)4 for molecular sensing and detection. We synthesized the TPE derivative with four binding arms that can be included in the cavity of MSP5, which can serve as a fluorescent indicator. A novel binary supramolecular-assembled fluorescent ensemble was constructed from MSP5 and TPE-(Br)4 -4-methoxybenzene (G1) possessing the same binding site as TPE-(Br)4 was synthesized as a model compound. As shown in the Supplementary Fig. 1 and H2 on the alkyl chain shifted upfield, because these protons were located in the cavity of MSP5 and suffered from shielding effect. This provided a strong evidence for the interactions between MSP5 and G1. MSP5 forms a 1:1 complex with G1 as assessed by 1H NMR titration, and the Ka of G1⊂MSP5 was calculated to be (1.08 ± 0.22) × 102 M−1 in chloroform using nonlinear curve-fitting analysis 4 in d-chloroform solution was also obtained and similar complexation-induced chemical shift changes were detected 4 Fig. . In orde4⊂MSP5, while non-fluorescent emission was surveyed from individual MSP5 and TPE-(Br)4 at the same concentration 4. Besides, the fluorescence enhancement of TPE-(Br)4 induced by addition of MSP5 was clearly perceived by naked eyes ion Fig. . Their hyes Fig. , and str4 upon addition of MSP5 confirmed the synergetic importance of host-guest interaction of pillararene and TPE guests and sulfonic functional group on pillararenes by a series of controlled experiments. The addition of MSP5 with a monosulfonic arm to form host-guest inclusion complex was proven to be the necessary condition for the fluorescent enhancement, consistent to RIR mechanism. TPE-(Br)4 and other two different host molecules, i.e., DMP5 and monocarboxylatopillar[5]arene (MCP5), were selected to investigate whether host-guest interaction itself will produce fluorescent enhancement of TPE-(Br)4. 1H NMR titration experiments provided Ka between MCP5 and G1 4 was almost unchanged 4⊂MSP5 was similar to that of TPE-(Br)4⊂MSP5 4 ged Fig. , illustrent Fig. , while s. 4 Fig. , for theSP5 Fig. , indicatr)4 Fig. , indicat4⊂MSP5 to maintain stable under different pH conditions, which was different from that of carboxylic acid group as the hydrophobic group in MCP5. We deduced that the hydrophobicity of the functional groups affected the fluorescence behaviors. Thus, three kinds of anionic monofunctional pillar[5]arenes and their sodium salts  have been synthesized to investigate the role of sulfonic group in the fluorescent ensembles. The enhanced fluorescence of TPE-(Br)4⊂MSP5 can be detected under low concentration, and upon addition of MSP5, fluorescence enhanced gradually. Furthermore, the fluorescence of other five kinds of supramolecular ensembles has also been studied at the same conditions, and the results are shown in Supplementary Fig. 4⊂MCP5 was detected, while the fluorescence was largely enhanced in TPE-(Br)4⊂MSP5 and TPE-(Br)4⊂monosulfonatepillar[5]arene. Monophosphoricpillar[5]arene also induced weaker fluorescence enhancement. On the contrary, monophosphatepillar[5]arene induced remarkable fluorescent enhancement, same as MSP5 and monosulfonatepillar[5]arene. From the above results, we can further concluded that the water-soluble groups-substituents monofunctionalized pillar[5]arene can enhance the fluorescence of TPE-(Br)4via host-guest inclusion. There was an obvious difference in pKa of the substituent groups in pillar[5]arene derivatives: R-SO3H (1.6 in DMSO) < R-PO3H2 (2.59 and 8.19 in water/ethanol) < R-COOH (12.3 in DMSO)61, which indicates that sulfonic group exists in the form of acidic anion in chloroform solution. However, phosphoric has two pKa, the strong acidic hydrogen will be ionized in chloroform, resulting in slight fluorescence enhancement of TPE-(Br)4⊂MSP5. The carboxylic acid group maintains un-ionized form in chloroform owning to the weak acidity, causing no fluorescence enhancement.Sulfonic group, as a hydrophilic entity, plays a key role in the complex system of TPE-(Br)4 have the same diffusion coefficient (2.6 × 10−9 m2s−1), suggesting the host-guest interaction of TPE-(Br)4⊂MSP5. The solution of the host-guest complex exhibited obvious Tyndall effect 4⊂MSP5 can self-assemble into nanoparticles with the average diameter of 16 nm , dynamic laser scattering (DLS), and DOSY NMR spectrum have been used to further investigate the fluorescence and self-assembled behaviors. The DOSY NMR spectrum showed that all the peaks correlated to the signals in the chemical shift dimensions are in a horizontal line 4 and blocked the nonradiative emission, leading to a strong fluorescence emission; (ii) The solubility of the host-guest complex (fluorescent nanoparticles) in chloroform was reduced due to the hydrophilic group, leading to the aggregation state; (iii) MSP5 can self-assemble with TPE-(Br)4 to construct organic fluorescent nanoparticles, reaching aggregation state and exhibiting strong emission.We thus ascribed the fluorescence enhancement to the following reasons: (i) The host-guest interaction of TPE-(Br)4 4⊂MSP5 can be used to detect ethylenediamine sensitively and rapidly 4 can be used to fabricate supramolecular fluorescence composite through supramolecular self-assembly. This new fluorescence complex system possesses multi-stimuli responsive properties, and can selectively recognize butanediol among several similar alcohols. It also can act as a fluorescence probe to detect toxic substance ethanediamine and act as a temperature sensor. We envision that combining the AIE effect of TPE with the host-guest property of functional macrocycles may lead to many potential applications of pseudorotaxanes in sensors, cell imaging, controlled optical materials and smart materials.In summary, we synthesized MSP5 for the first time and employed it to construct a stable AIE-active binary complex system with TPE core via host-guest interaction and supramolecular self-assembly. Upon the formation of host-guest complex, the fluorescence emission of the complex was enhanced dramatically. The resulting pseudorotaxane-type structure restricted the intramolecular rotation of phenyl rings of the TPE-(Br)4, G1, M1 and MCP5 were synthesized according to a published literature procedure (See the Supporting Information for details)62. 1H NMR spectra were collected on a Bruker AVANCE III 300 MHz NMR spectrometer. 13C NMR, 2D NOESY NMR and DOSY NMR spectra were recorded on a Bruker AVANCE III 500 MHz NMR spectrometer. High-resolution electrospray ionization mass spectra (HRESI-MS) were obtained on a Bruker 7-Tesla FT-ICR mass spectrometer equipped with an electrospray source. Mass spectra were recorded on Bruker Daltonics Autoflex Speed Series: High-Performance MALDI-TOF Systems. FT-IR spectra were recorded on a Vertex 80 V spectrometer. Scanning electron microscope (SEM) images were obtained on a HITACHI-SU8082 instrument. The fluorescence experiments were conducted on a RF-5301 spectrofluorophotometer . To determine the stoichiometry and association constants of alcohols⊂MSP5, 1H NMR titration was performed. By a nonlinear curve-fitting method, the association constants between the guests and host were calculated. Through a molar ratio plot, the stoichiometry was determined (see supporting information for details).All reagents were commercially available and used without further purification. TPE-(Br)1H NMR , δ (ppm): 6.73 , 3.46~3.77 , 3.02 , 1.79~1.96 . 13C NMR  δ (ppm): 150.73, 149.82, 128.22, 114.11, 68.31, 55.80, 50.97, 29.76, 28.82, 21.35. HRESIMS is shown in Fig. m/z 871.3468 [M−Na]− (100%). Then the sodium salt (200 mg) was dispersed in water and stirred with hydrochloric acid at room temperature for 12 h. After the solvent was removed, the obtain solid was purified by column chromatograph with dichloromethane/methanol (1:10 v/v) to get the final product of MSP5. Yellow powder: 140 mg, 72%. 1H NMR , δ (ppm): 6.72 , 3.55~3.76 , 3.03 , 1.80~1.99 . 13C NMR  δ (ppm): 150.77, 128.30, 114.17, 68.28, 55.82, 51.01, 29.72, 28.72, 21.38. HRESIMS is shown in Fig. m/z 871.3373 [M−H]− (100%).MonohydroxyDMP[5]  and NaOH  were added into 25 mL THF in a 50 mL flask, the mixture was stirred at room temperature for 1 h. Then, 1,4-butylenesulfone (0.15 mL) was added into the mixture. The mixture was stirred at 40 °C for 24 h. The crude product was recrystallized with dichloromethane/n-hexane and washed with water. After dryness, monosulfonatepillar[5]arene sodium salt was obtained as a yellow powder . 2CO3 (144 mg) were added into 20 mL CH3CN. The mixture was stirred at room temperature for 30 min. Then 5-bromovaleronitrile was added into the above solution, reacted under reflux for 24 h. After the solvent was removed, the obtain solid was purified by column chromatograph with petroleum ether/dichloromethane/ethyl acetate (15:15:1 v/v) to get the final product. Pale yellow powder, 75 mg, 34%. 1H NMR , δ (ppm): 6.90 , 6.60 , 3.93 , 2.44 , 1.90 13C NMR  δ (ppm): 157.04, 137.11, 132.66, 119.62, 113.62, 66.56, 28.31, 22.59, 17.13. MALDI-TOF MS is shown in Fig. m/z 720.2358 [M] (100%).Tetra-hydroxyl-TPE (120 mg) and K1H NMR , δ (ppm): 6.67~6.74 , 4.18 , 3.59~3.76 . 13C NMR  δ (ppm): 175.86, 150.88, 128.45, 114.13, 71.74, 55.95, 29.81. HRESIMS is shown in Fig. m/z 871.3468 [M−Na]− (100%).Monoesterpillar[5]arene  was added into sodium hydroxide aqueous solution  and THF (4.2 mL). The mixture was stirred at 85 °C for 24 h. After solvent was removed, the obtained solid was recrystallized with dichloromethane/n-hexane. White powder: 40 mg, 82%. 1H NMR , δ (ppm): 6.72~6.77 , 4.09 , 3.85 , 3.77 , 3.64 , 1.85~1.89 , 1.70 , 1.29 . 13C NMR  δ (ppm): 150.75, 149.87, 128.32, 114.95, 114.00, 67.74, 61.54, 55.87, 30.68, 29.82, 19.57, 16.45. MALDI-TOF MS is shown in Fig. m/z 930.4110 [M + H]+, 953.2957 [M + H + Na]2+, 969.3292 [M + H + K]2+.Copillar[5]arene 1  was added into triethyl phosphite (1.2 mL). The mixture was stirred at 150 °C for 24 h. The crude product was concentrated and subjected to column chromatograph with petroleum ether/ethyl acetate (1:20 v/v) to get the final product. Pale yellow powder, 260 mg, 78%. 2Cl2. Then TMSBr (0.4 mL) was added into the above solution. The mixture was stirred at room temperature for 24 h. The crude product was concentrated and recrystallized to give the final product. White powder: 204 mg, 78%. 1H NMR , δ (ppm): 6.75 , 3.62~3.76 , 1.67 , 1.02 . 13C NMR  δ (ppm): 150.74, 128.26, 113.97, 67.80, 55.78, 52.55, 30.61, 29.60, 19.30. HRESIMS is shown in Fig. m/z 871.3453 [M−H]− (100%).Monophosphite-DMP[5]  was added into 10 mL CH1H NMR , δ (ppm): 6.69 , 3.53~3.75 , 1.74 , 1.27 . 13C NMR  δ (ppm): 150.78, 128.20, 114.21, 67.97, 61.54, 55.76, 29.71, 25.60, 14.11. HRESIMS is shown in Fig. m/z 871.3604 [M−H]− (100%).Monophosphoricpillar[5]arene (50 mg) was dissolved in 5 mL THF, then added into 5 mL NaOH aqueous (2 M/L) dropwise, which was allowed to react at room temperature for 12 h. Then THF solvent was evaporated and the solid was collected as a pale yellow powder: 30 mg, 59%. Supplementary Information


Alteration of PD-1/PD-L1 pathway may be involved in poorer prognosis, as patients with PGN are characterized by higher frequencies of PD-1-positive and PD-L1-positive T and B lymphocytes than patients with NPGN. Our results suggest that deregulation of PD-1/PD-L1 axis may contribute to the PGN and NPGN pathogenesis. High percentages of lymphocytes with PD-1 and PD-L1 expression may be related to the continuous T-cell activation and development of glomerular inflammation and injury.The pathogenesis of primary proliferative and non-proliferative glomerulonephritides (PGN and NPGN) is still not fully understood, however, current evidence suggests that most cases of PGN and NPGN are the results of immunologic response to different etiologic agents that activates various biological processes leading to glomerular inflammation and injury. Programmed cell death protein 1 (PD-1) is the major inhibitory receptor regulating T cell exhaustion. The aim of this study was to evaluate the frequencies of PD-1-positive and PD-ligand 1 (PD-L1)-positive T and B lymphocytes in patients with NPGN and PGN in relation to clinical parameters for the first time. The study included peripheral blood (PB) samples from 20 newly diagnosed PGN and NPGN patients. The control group comprised of 20 healthy age- and sex-matched subjects. The viable PB lymphocytes underwent labelling with fluorochrome-conjugated monoclonal antibodies anti-PD-1 and anti-PD-L1, and were analyzed using a flow cytometer. The frequencies of CD4 The prevalence of chronic kidney disease (CKD) in the United States in 2012  was estimated at 13.6%  samples from 20 newly diagnosed, previously untreated patients with primary glomerulonephritis (11 men and 9 women). Ten patients were diagnosed with PGN , and ten patients were diagnosed with NPGN . The mean age of patients from the study groups was 42.4 ± 12.2 years [median 42.5 years (21–62 years)] in PGN patients, and 45.7 ± 13.4 years [median 50.0 years (23–63 years)] in NPGN patients. The control group comprised 20 healthy subjects (12 men and 8 women), aged 44.4 ± 12.2 years [median 45.0 years (20–61 years)].Neither the patients nor the controls used immunomodulating agents or hormonal preparations, showed signs of infection within at least three months prior to the study, underwent blood transfusion, or presented with autoimmune condition or allergy. Moreover, none of the controls had a history of oncological therapy or prior treatment for tuberculosis or other chronic conditions that could be associated with impaired cellular or humoral immunity.The diagnosis of primary glomerulonephritides was established on the basis of standard diagnostic criteria, with special emphasis on kidney biopsy . Written informed consent was obtained from all patients with respect to the use of their blood for scientific purposes.Venous blood samples in an amount of 5 ml were collected from the study patients and controls by venipuncture using sterile, lithium heparin-treated tubes .+ cells stained with antibody. The percentage of positive cells was calculated by comparing with the control. Background fluorescence was determined using isotype-matched directly conjugated FITC Mouse IgG1 κ Isotype Control and PE Mouse IgG1 κ Isotype Control monoclonal antibodies. The samples were gated on forward scatter vs. side scatter to exclude debris and cell aggregates. Example of cytometric analysis is presented on the Fig. Peripheral blood mononuclear cells were aseptically separated by a standard density gradient centrifugation 1 h after obtaining from the patients and controls . The percentages of cells expressing surface markers were analyzed. The cells were phenotypically characterized by incubation (20 min in the dark at room temperature) with combination of relevant fluorescein isothiocyanate (FITC)—phycoerythrin (PE)—and CyChrome-labelled monoclonal antibodies (mAbs). Immunofluorescence studies were performed using a combination of the following mAbs: CD45 FITC/CD14 PE, CyChrome Mouse Anti-Human CD3, FITC Mouse Anti-Human CD19, FITC Mouse Anti-Human CD4, FITC Mouse Anti-Human CD8, PE Mouse Anti-Human CD279 (PD-1) and PE Mouse Anti-Human CD274 (PD-L1), purchased from BD Biosciences (USA). Three-colour immunofluorescence analyses were performed using a FACSCalibur flow cytometer (Becton–Dickinson) equipped with 488 nm argon laser. A minimum of 10,000 events was acquired and analyzed using CellQuest Software. The results were presented as percentage of CD45t test was used for independent variables, and the Mann–Whitney U test was used for intergroup comparisons. The power and direction of relationships between pairs of continuous variables were determined on the basis of the values of Spearman’s coefficient of rank correlation. The distributions of discrete variables in the studied groups were compared with the Pearson’s Chi-square test or the Fisher’s exact test. Receiver operating characteristic (ROC) curves was generated for significant predictor variables of PGN patients. Areas under the ROC curves (AUCs) were calculated for each parameter and compared. All the calculations were carried out with Statistica 10  package and Graphpad Prism 5 . Differences were considered statistically significant with a p value <0.05.Normal distribution of continuous variables was tested using the Shapiro–Wilk test. Statistical characteristics of the continuous variables were presented as medians, minimum and maximum values, as well as arithmetic means and their standard deviations (SD). The Student Complete blood count parameters of patients from study groups and controls are presented in Table +/PD-1+ T cells were higher among patients suffering from PGN (36.8 ± 8.6%) [median 36.0% (25.2–50.1%)] and NPGN (23.2 ± 4.5%) [median 22.3% (17.8–32.6%)] than in the control group (5.4 ± 1.5%) [median 5.3% (2.7–7.7%)]  [median 24.2% (18.0–64.8%)] exceeded both the NPGN group (15.4 ± 3.3%) [median 14.8% (10.2–20.3%)] and the control group (3.6 ± 1.5%) [median: 3.7% (1.4–6.2%)]  [median 18.8% (15.6–45.6%)] and NPGN (11.6 ± 2.4%) [median 12.0% (5.7–14.0%)] than in healthy subjects (1.67 ± 0.84%) [median 1.8% (0.4–3.0%)]  [median 32.2% (18.2–49.8%)] and with NPGN (20.4 ± 4.3%) [median 20.0% (14.8–28.7%)] were statistically significantly higher than in the control group (1.86 ± 0.70%) [median 1.7% (1.0–3.5%)]  [median 20.2% (14.0–40.7%)] and NPGN group (13.7 ± 2.6%) [median 12.8% (10.7–19.0%)] than in healthy individuals (0.45 ± 0.11%) [median 0.43% (0.31–0.67%)]  [median 17.8% (14.1–43.0%)] than in patients with NPGN (15.6 ± 2.3%) [median 16.2% (11.5–19.1%)] .Figure +/PD-1+ and CD4+/PD-L1+ T cells . We have also found a positive correlation between the frequencies of CD4+/PD-1+ and CD8+/PD-L1+ T cells . Moreover, a positive correlation was stated between the frequencies of CD8+/PD-1+ T cells and the frequencies of CD8+/PD-L1+ T cells in those patients . Finally, the frequencies of CD19+/PD-1+ B cells positively correlated with the frequencies of CD19+/PD-L1+ .In the group of patients with PGN, we have found a positive correlation between the frequencies of CD4+/PD-1+ T cells and CD4+/PD-L1+ T cells  was found. A positive correlation was also stated between the frequencies of CD8+/PD-1+ and CD8+PD-L1+ T cells . Moreover, we have found a positive correlation between the frequencies of CD19+/PD-1+ B cells and the frequencies of CD19+/PD-L1+ B cells in those patients .Next, the group of patients with NPGN was checked, and a positive correlation between frequencies of CD4No statistically significant correlations were found in the healthy subjects.+/PD-1+ B cells , and also with the frequencies of CD19+/PD-L1+ B cells . Moreover, a statistically significant positive correlation was found between the level of serum beta globulins and the frequencies of CD4+/PD-1+ T cells , CD19+/PD-1+ B cells , and CD19+/PD-L1+ B cells .In patients with PGN, we have found a positive correlation between the platelet (PLT) count and the frequencies of CD19+/PD-L1+ T cells . Moreover, the concentration of serum IgM showed a significant positive correlation with the frequencies of CD4+/PD-1+ T cells  and the frequencies of CD4+/PD-L1+ T cells .In patients with NPGN, a statistically significant negative correlation was stated between the blood urea nitrogen (BUN) and the frequencies of CD8No statistically significant correlations were found in the healthy subjects.PD-1 is a member of the CD28/cytotoxic T-lymphocyte-associated protein-4 superfamily, which plays an important role in the regulation of activated T cells  cells was significantly increased in BXD2 mice, which spontaneously develop autoantibodies and subsequent glomerulonephritis, offering a useful animal model to study autoimmune lupus, compared with wild-type mice  production and enhanced levels of T-cell activation marker CD69  has been described in patients with lupus glomerulonephritis and cytopenias , and serum beta globulins level. In the pathogenesis of PGN, therefore, cytotoxic reactions are probably less impaired than antigen recognition and antibody secretion.Our team also found a statistically significant positive correlation between the level of serum beta globulins and the frequencies of CD4+/PD-1+ B cells, and also with the frequencies of CD19+/PD-L1+ B cells. It was proven that CKD patients have significant PLT activation and endothelial dysfunction, which was involved in CKD’s occurrence and development  tubulointerstitial nephritis and immune complex glomerulonephritis as a result of treatment of several malignancies with ipilimumab and tremelimumab (anti-CTLA-4-blocking antibodies), and pembrolizumab and nivolumab (antibodies targeting PD-1 receptors) (Izzedine et al. In conclusion, our results suggest that deregulation of PD-1/PD-L1 axis may contribute the PGN and NPGN pathogenesis. Alteration of PD-1/PD-L1 pathway may be involved in poorer prognosis, as patients with PGN are characterized by higher frequencies of PD-1-positive and PD-L1-positive T and B lymphocytes than patients with NPGN. High percentages of lymphocytes with PD-1 and PD-L1 expression may contribute to the continuous T-cell activation and development of glomerular inflammation and injury. Further studies, currently under investigation by our team, are needed to explain if similar pathologies are found in the glomeruli of PGN and NPGN patients, and if PD-1/PD-L1 blockade is able to induce long-term remission of PGN and NPGN.In this work, we presented findings in a relatively small study group. Further research should include assessment of PD-1 and PD-L1 expression on more cell populations, including antigen-presenting cells in PB and on glomeruli, as well as glomerular cell infiltrates. The study should be continued and the expression of PD-1 and PD-L1 has to be evaluated during the treatment of GN, and after obtaining remission.