Due to its low ionization efficiency, a Cookson‐type reagent, 1,2,4‐triazoline‐3,5‐dione (TAD), is used to improve the detection/quantification of VD metabolites by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, the high reactivity of TAD makes its solution stability low and inconvenient for practical use. We here describe the development of a novel caged Cookson‐type reagent, and we assess its performances in the quantitative and differential detection of four VD metabolites in serum using LC/MS/MS.Caged 4‐(4′‐dimethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione (DAPTAD) analogues were prepared from 4‐(4′‐dimethylaminophenyl)‐1,2,4‐triazolidine‐3,5‐dione. Their stability and reactivity were examined. The optimized caged DAPTAD (14‐(4‐(dimethylamino)phenyl)‐9‐phenyl‐9,10‐dihydro‐9,10‐epitriazoloanthracene‐13,15‐dione, DAP‐PA) was used for LC/MS/MS analyses of VD metabolites.The solution stability of DAP‐PA in ethyl acetate dramatically improved compared with that of the non‐caged one. We measured the thermal retro‐Diels‐Alder reaction enabling the release of DAPTAD and found that the derivatization reaction was temperature‐dependent. We also determined the detection limit and the lower limit of quantifications for four VD metabolites with DAPTAD derivatization.DAP‐PA was stable enough for mid‐ to long‐term storage in solution. This advantage shall contribute to the detection and quantification of VD in clinical laboratories, and as such to the broader use of clinical mass spectrometry. Because significant levels of the C‐3 epimer of 25(OH)D3, 3‐epi‐25(OH)D3, are present in the serum in both infants and adults,R,25‐dihydroxyvitamin D3 2D3) is essential for the differential diagnosis of infantile hypercalcemia of unknown etiology.Vitamin D (VD) metabolites play an important role in homeostatic maintenance, such as bone metabolism.s‐cis diene. Consequently, VD selective derivatization reagents that take advantage of the reactive dienophile, 1,2,4‐triazoline‐3,5‐dione (TAD), which is known as a Cookson‐type reagent, have been developed for enhancing their detection limits in mass spectrometric analyses.3, 25(OH)D2, 3‐epi‐25(OH)D3, and 24,25(OH)2D3) in serum by LC/MS/MS using DAPTAD derivatization.VD metabolites have a particular structural feature: a conjugated We describe the development of a novel caged Cookson‐type reagent, 14‐(4‐(dimethylamino)phenyl)‐9‐phenyl‐9,10‐dihydro‐9,10‐epitriazoloanthracene‐13,15‐dione (DAP‐PA), which can produce DAPTAD by thermal retro‐Diels‐Alder (RDA) reaction and present preliminary analytical performances of DAP‐PA when applied to the quantitative and differential measurement of the four VD metabolites in serum by LC/MS/MS.22.11H‐NMR (nuclear magnetic resonance) spectra were recorded on a JEOL JNM‐ECA400II and a JEOL JNM‐ECA500 spectrometer  operating at 400 MHz (1H) and 100 MHz (13C), respectively. Chemical shifts are reported in δ units and referenced to the solvent, that is, 7.24 (1H) and 77.0 (13C) for CDCl3. Multiplicities are indicated as br (broadened), s (singlet), d (doublet), t (triplet), or m (multiplet). Coupling constants (J) are reported in Hertz (Hz). High‐resolution mass spectra (HRMS) were recorded on a JEOL T100LP mass spectrometer  under fast atom bombardment (FAB) conditions using m‐nitrobenzyl alcohol as a matrix. IR spectra were recorded on a Spectrum One spectrometer .2.2H‐triazolopyridazine‐1,3(2H)‐dione, DAP‐DP).4‐(4′‐Dimethylaminophenyl)‐1,2,4‐triazolidine‐3,5‐dione2.31H‐NMR (CDCl3) δ 7.85 , 7.59–7.49 , 7.31 , 7.22 , 7.03 , 6.78 , 6.56 , 6.40 , 2.87 ; 13C‐NMR (CDCl3) δ 158.2, 157.3, 150.3, 139.3, 132.5, 129.8, 128.8, 128.1, 128.0, 127.8, 126.6, 124.5, 123.9, 119.4, 112.2, 72.6, 62.6, 40.5; IR  3062, 1774, 1714, 1613, 1526, 1460, 1448, 1417, 1361, 1248, 1230, 1181, 1158, 1039, 1004, 946, 846, 805, 784, 764, 753, 702, 679, 634, 541; HRMS (FAB) calculated for C30H25N4O2 [(M + H)+] 473.19775, found 473.19683.2.41H‐NMR (CDCl3) δ 7.52 , 7.42–7.32 , 7.18 , 6.63 , 5.99 , 5.53 , 2.89 ; 13C‐NMR (CDCl3) δ 152.6, 137.4, 128.8, 128.6, 127.9, 126.4, 125.4, 112.3, 58.3, 40.5; IR  3031, 2941, 2802, 1771, 1701, 1614, 1523, 1493, 1447, 1420, 1352, 1289, 1193, 1158, 1143, 1063, 946, 878, 846, 819, 753, 698, 610, 588, 517; HRMS (FAB) calculated for C26H25N4O2 [(M + H)+] 425.19775, found 425.18956.2.5The chromatographic analysis of the RDA reaction was performed using an Alliance HPLC system equipped with a model 2489 UV–Vis detector . Chromatography was performed using an Inertsil ODS‐3 column  with a binary gradient system with water (A) and acetonitrile (B) and a flow rate of 1.0 mL/min. The detection wavelength was 210 nm, and the elution program was as follows: 0 → 10 min, 50% B; 10 → 20 min, 50 → 5% B; 20 → 50 min, 5% B; 50 → 55 min, 50% B; 55 → 60 min, 50% B.2.6k1 was determined using the following equation describing the irreversible diene tag, 1,4‐diphenyl‐1,3‐buthadiene D2, 3‐epi‐25(OH)D3, and 24,25(OH)2D3); internal standards (25(OH)D3‐, 25(OH)D2‐, 3‐epi‐25(OH)D3‐, and 24,25(OH)2D3‐); quality controls; and the derivatization reagent (DAP‐PA ethyl acetate solution [0.5 mg/mL]) were obtained from JEOL . The calibrator concentrations for 25(OH)D3, 25(OH)D2, 3‐epi‐25(OH)D3, and 24,25(OH)2D3 were as follows: 0.873, 0.0920, 0.0940, and 0.0960 ng/mL for calibrator level 1; 8.80, 0.924, 0.938, and 0.962 ng/mL for calibrator level 2; 44.0, 4.62, 4.69, and 4.81 ng/mL for calibrator level 3; and 88.0, 9.23, 9.37, and 9.61 ng/mL for calibrator level 4. These concentrations were determined by quantitative NMR measurements.The JeoQuant kit for LC/MS/MS analysis of vitamin D metabolites including calibrators (25(OH)D2.8DAP‐PA solution (100 μL) was added to the dried samples, and the resultant mixture was heated at 80°C for 15 min. Ethanol (20 μL) was added to the mixture to terminate the reaction, and the solvent was removed by centrifuged evaporation. The residue was dissolved in 50 μL of 30% aqueous acetonitrile for LC/MS/MS analyses.2.9SRM972a level 2 serum (50 μL) and internal standards (250 μL) were mixed and vortexed for 1 min. Each sample was loaded into a supported liquid extraction column  and eluted three times with 600 μL ethyl acetate/hexane  using a PRESSURE+48 positive pressure manifold (Biotage). The combined eluents were evaporated to dryness using a centrifuged evaporator. DAPTAD derivatization of the obtained samples was carried out as described above.2.1018 (2.7 μm) 2.1 mm I.D. × 100 mm column  equilibrated with 0.1% formic acid 30% acetonitrile with a flow rate of 0.3 mL/min. The injection volume was 20 μL. The elution program was as follows: 0.01 → 0.5 min, 30 → 58% B; 0.5 → 7.0 min, 58% B; 7.0 → 7.01 min, 58 → 90% B; 7.01 → 8.0 min, 90% B; 8.0 → 8.01 min, 90 → 30% B; 8.01 → 9.0 min, 30% B. For analyses of non‐derivatized VD metabolites, an InertSustain PFP HP (3 μm) 2.1 mm I.D. × 150 mm column  was used. The flow rate was 0.4 mL/min, and the elution program was as follows: 0.01 → 0.5 min, 50 → 67% C; 0.5 → 9.0 min, 67% C; 9.0 → 9.01 min, 67 → 90% C; 9.01 → 11.0 min, 90% C; 11.0 → 11.01 min, 90 → 50% C; 11.01 → 13.0 min, 50% C. Ionization was done with the positive ion mode, and selected reaction monitoring (SRM) was used for quantification. The transitions (m/z) and collision energies (CE) for the VD metabolites are summarized in Table LC/MS/MS analyses were performed using an QTRAP4500® triple quadruple mass spectrometer  equipped with a model 1290 Infinity liquid chromatography system . Chromatography was performed using a binary gradient system with 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B) or methanol (C). Samples were dissolved in 50 μL of 30% aqueous acetonitrile and loaded into a CAPCELL CORE C33.1Although TAD is one of the most reactive dienophiles and provides an excellent derivatization tag, its solution instability has been reported.3.2o‐dichlorobenzene) gave better rate constants than that with ethyl acetate , LOD values, and sensitivity increase are summarized in Table Four VD metabolites with and without DAPTAD derivatization in the absence of serum were detected using the previously reported procedure.3.43 2D3) could not be detected quantitatively due to its extremely low concentration and the difficulty in chromatographic separation of 1,25(OH)2D3 and other dihydroxylated VD metabolites such as 4β, 25‐dihydroxyvitamin D.LLOQs of four VD metabolites in SRM972a level 2 serum with DAPTAD derivatization are summarized in Table 4We screened several diene groups to develop the caged Cookson‐type reagent, DAP‐PA, which was stable enough for mid‐ to long‐term storage in solution. Highly stable reagents are essential for data reproducibility in clinical laboratories. The stability of the reagents in a solution is guaranteed by the production of pure products, which is generally achieved by crystallization, and the caged DAPTAD is easy to crystallize, which is a strong advantage in terms of quality control. In addition, from a practical viewpoint, the caged DAPTAD is available in large quantities, and thus market supply is stable and ample. This advantage will contribute to the field of VD detection and quantification in clinical laboratories, and thus to the broader use of clinical mass spectrometry.1H‐NMR spectra of the aromatic region of DAPTAD in ethyl acetate‐d8. DAPTAD signals assigned to aromatic protons are indicated by arrows.Figure S1. 1H‐NMR spectrum of DAP‐PA in CDCl3.Figure S2. 13C‐NMR spectrum of DAP‐PA in CDCl3.Figure S3. Figure S4. IR spectrum of DAP‐PA.1H‐NMR spectrum of DAP‐DP in CDCl3.Figure S5. 13C‐NMR spectrum of DAP‐DP in CDCl3.Figure S6. Figure S7. IR spectrum of DAP‐DP.Figure S8. HPLC chromatograms of the retro‐DA reaction products.Figure S9. Peak area of DAPTAD‐derivatized VD metabolites on SRM chromatograms. The reaction condition was 0.25 mg/mL in ethyl acetate for 60 min (n=2). Data were obtained using a Waters Xevo TQ‐XS mass spectrometer.Figure S10. Peak area of DAPTAD‐derivatized VD metabolites on SRM chromatograms. The reaction condition was 0.25 mg/mL in ethyl acetate at 80°C (n=2). Data were obtained using a Waters Xevo TQ‐XS mass spectrometer.Figure S11. Peak area of DAPTAD‐derivatized VDs on SRM chromatograms. The reaction was performed at 80°C for 15 min (n=2). Data were obtained by using a Waters Xevo TQ‐XS mass spectrometer.Table S1. Kinetics of anthracene analogues in ethyl acetate at 70°C.Table S2. Solvent effect for the retro‐DA reaction of DAP‐PA at 70°C.Click here for additional data file.


The structures of the dimethylated versions of two natural products found in toad secretions, bufotenine and bufotenidine, are reported, as well as the hydrate of 2,5-dimethylbufotenidine. N,N-trimethyltryptammonium) (5-MeO-2-Me-DMT) fumarate (systematic name: bis{[2-(5-methoxy-2-methyl-1H-indol-3-yl)ethyl]dimethylazanium} (2E)-but-2-enedioate), 2C14H21N2O+·C4H2O42−, the bufotenidine derivative 5-methoxy-2,N,N,N-tetramethyltryptammonium (5-MeO-2-Me-TMT) iodide {systematic name: [2-(5-methoxy-2-methyl-1H-indol-3-yl)ethyl]trimethylazanium iodide}, C15H23N2O+·I−, and the hydrate of the same {systematic name: [2-(5-methoxy-2-methyl-1H-indol-3-yl)ethyl]trimethylazanium iodide monohydrate}, C15H23N2O+·I−·H2O, are reported. The structure of 5-MeO-2-Me-DMT fumarate possesses one tryptammonium cation and a half of a fumarate dianion in the asymmetric unit, linked together by N—H⋯O hydrogen bonds in infinite two-dimensional networks parallel to the (101) plane. The structure of 5-MeO-2-Me-TMT iodide possesses one tryptammonium cation and one iodide anion in the asymmetric unit. The ions are linked via N—H⋯I hydrogen bonds, and indoles are coupled in dimers through π–π interactions. The hydrate of 5-MeO-2-Me-TMT iodide possesses one tryptammonium cation, one iodide anion and one water molecule in the asymmetric unit. It shows N—H⋯I and O—H⋯I hydrogen bonds that couple the tryptammonium cations into dimers.The solid-state structure of the bufotenine derivative bis(5-methoxy-2, N,N-dimethyl analogue of serotonin, and bufotenidine, the N,N,N-trimethyl analogue of serotonin, were both identified in toad secretions in 1934 ]  was first reported in 1955, and crystallized as its picrate salt in two different forms   , along with its structure. Lastly, we report the structure of the first solvate of 5-methoxy-2,N,N,N-tetramethyltryptammonium iodide as its hydrate.Herein we report the structure of 5-methoxy-2,N,N-trimethyltryptammonium) fumarate contains one tryptammonium cation and one half of a fumarate dianion  hydrogen bond with the two oxygens of a carboxylate unit, and the indole nitrogen is involved in an N—H⋯O hydrogen bond with one of the carboxylate oxygens Table 1. This sevia N-–H⋯I hydrogen bonds, between the indole nitrogen and the iodide   and 4-acetoxy-N,N,N-trimethyltryptamine (4-AcO-TMT)  and N,N-dimethyl-N-allyltryptammonium (DMALT) as their iodide salts : δ 7.36 , 7.04 , 6.88 , 3.88 , 3.44–3.40 , 3.21 , 3.16–3.12 , 2.38 . 13C NMR : δ 152.6 (ArC), 135.1 (ArC), 130.3 (ArC), 127.3 (ArC), 111.6 (ArC), 109.9 (ArC), 103.0 (ArC), 99.9 (ArC), 65.2 (AkC), 55.9 (AkC), 52.4 (AkC), 17.2 (AkC), 10.5 (AkC).Crystals of 5-MeO-2-Me-DMT fumarate suitable for diffraction studies were obtained from the evaporation of a methanol solution of a commercial sample (The Indole Shop). 5-MeO-2-Me-TMT iodide was synthesized when 128 mg of 5-MeO-2-Me-DMT fumarate was dissolved in 6 mL of methanol, and 6 mL of methyliodide was added. The mixture was refluxed under an atmosphere of nitrogen for 12 h. The solvent was removed Ueq of the parent nitrogen atom. The hydrogen atoms on the water of the hydrate structure  were found from a difference-Fourier map and were refined isotropically, using a DFIX restraint with an O—H distance of 0.88 (1) Å. Isotropic displacement parameters were set to 1.5Ueq of the parent oxygen atom. All other hydrogen atoms were placed in calculated positions (C—H = 0.93-0.97 Å). Isotropic displacement parameters were set to 1.2Ueq(C) or 1.5Ueq(C-methyl). A certain number of reflections is missing from the data of all three structures. This is likely a beamstop related technical issue which could not be resolved as of yet.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989021000803/yz2004sup1.cifCrystal structure: contains datablock(s) umd1954c_a, umd2018f_a, umd2009b_a. DOI: 10.1107/S2056989021000803/yz2004umd1954c_asup2.hklStructure factors: contains datablock(s) umd1954c_a. DOI: 10.1107/S2056989021000803/yz2004umd2018f_asup3.hklStructure factors: contains datablock(s) umd2018f_a. DOI: 10.1107/S2056989021000803/yz2004umd2009b_asup4.hklStructure factors: contains datablock(s) umd2009b_a. DOI: 2058145, 2058144, 2058143CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Beta-amyloid (Aβ) has been recognized as an early trigger in the pathogenesis of Alzheimer's disease (AD) leading to synaptic and cognitive impairments. Aβ can alter neuronal signaling through interactions with nicotinic acetylcholine receptors (nAChRs), contributing to synaptic dysfunction in AD. The three major nAChR subtypes in the hippocampus are composed of α7-, α4β2-, and α3β4-nAChRs. Aβ selectively affects α7- and α4β2-nAChRs, but not α3β4-nAChRs in hippocampal neurons, resulting in neuronal hyperexcitation. However, how nAChR subtype selectivity for Aβ affects synaptic function in AD is not completely understood. Here, we showed that Aβ associated with α7- and α4β2-nAChRs but not α3β4-nAChRs. Computational modeling suggested that two amino acids in α7-nAChRs, arginine 208 and glutamate 211, were important for the interaction between Aβ and α7-containing nAChRs. These residues are conserved only in the α7 and α4 subunits. We therefore mutated these amino acids in α7-containing nAChRs to mimic the α3 subunit and found that mutant α7-containing receptors were unable to interact with Aβ. In addition, mutant α3-containing nAChRs mimicking the α7 subunit interact with Aβ. This provides direct molecular evidence for how Aβ selectively interacted with α7- and α4β2-nAChRs, but not α3β4-nAChRs. Selective coactivation of α7- and α4β2-nAChRs also sufficiently reversed Aβ-induced AMPA receptor dysfunction, including Aβ-induced reduction of AMPA receptor phosphorylation and surface expression in hippocampal neurons. Moreover, costimulation of α7- and α4β2-nAChRs reversed the Aβ-induced disruption of long-term potentiation. These findings support a novel mechanism for Aβ's impact on synaptic function in AD, namely, the differential regulation of nAChR subtypes. Alzheimer's disease (AD) is the predominant cause of dementia in the elderly, which is characterized by two histopathological hallmarks, beta-amyloid peptide (Aβ)-containing senile plaques and hyperphosphorylated tau-based neurofibrillary tangles . One of i.e., monomers, soluble oligomers, or fibrils), and incubation times . The findings have implications for regulation of nAChRs as therapeutic targets in the hippocampus for neuroprotection in AD.Here, we investigated the Aβ interaction with nAChRs in Aβ-induced Ca1–42 (Aβ42) with nAChRs is important for neuronal hyperactivity by using the Aβcore peptide  mouse hippocampal pyramidal neurons transfected with GCaMP6f  as described previously  , confirming that soluble 250 nM Aβ42 oligomers were sufficient to increase neuronal Ca2+ activity  . Consist; sAβ42) , the totactivity .Figure 12+ hyperexcitation  (min)  (min)  was added in conjunction with 250 nM oAβ42, Aβcore treatment was able to reverse oAβ42-induced Ca = 0.04) . However4) (min) , which ima cells . Next, wΔF/Fmin) . Finallyn) (min) . We thusXenopus oocytes (A), consistent with previous evidence for an interaction between Aβ42 and α7-nAChR in a neuronal cell line . Similar analysis using cells overexpressing human α3-nAChR-GFP receptors with human β4 subunits yielded no Aβ42 as a coimmunoprecipitate (C). As a separate control, we showed that the anti-actin antibody failed to pull down Aβ42 or α7-nAChRs . We next used the inactive Aβcore peptide and found that it had no effect on the association between Aβ42 and α7-nAChRs . This suggests that the Aβcore peptide can inhibit the interaction between Aβ42 and α7-nAChRs, which may underlie, at least in part, the neuroprotective effects of the Aβcore peptide, in addition to its direct action on Aβ-regulated receptors . Among them, Y210 is the ligand-binding residue and conserved in all human and mouse α3, α4, and α7 subunits . Mutation studies have shown that Y210 is essential for acetylcholine binding and Aβ interactions . Of importance, R208 in the human α7 subunit contains a positively charged side chain, and mouse α7 subunits contain lysine (K), a positively charged amino acid, instead of arginine . However, both mouse and human α3 subunits contain hydrophobic isoleucine (I) instead of positively charged arginine in the human α7 subunit or lysine in the mouse α7 subunit . Moreover, E211 in the human α7 subunit contains a negatively charged side chain, which is conserved in both human and mouse α4 and α7 subunits, while both mouse and human α3 subunits include uncharged asparagine (N) . Mutations in R208 and E211 in α7-nAChRs alter the binding affinity of the receptor to acetylcholine (E and F). Co-IP experiments were performed with lysates from HEK293 cells overexpressing human α7-nAChR-R208I-GFP or α7-nAChR-E211N-GFP receptors. We found that Aβ42 was unable to interact with either mutant α7-nAChR, a loss-of-function effect (E and F). Furthermore, we made mutant α3 subunits by substituting I284 for arginine (I284R) or N287 for glutamate (N287E) to mimic the α7 subunit to test whether these mutants would show gain-of-function effects on the interaction between Aβ42 and the receptors. We carried out co-IP experiments with lysates from HEK293 cells overexpressing human α3-nAChR-I284R-GFP or α3-nAChR-N287E-GFP receptors with human β4 subunits. Both mutant receptors were unable to pull down Aβ42 (G and H). We next generated a double mutant receptor that contained both I284R and N287E . In contrast to the single mutations, we found that the double α3 mutant was able to interact with Aβ42 when α3-nAChR-I284R/N287E-GFP and the β4 subunits were expressed in HEK293 cells . These data suggest that the charged arginine and glutamate residues in the loop C in the α4 and α7 subunits play important roles in the interaction between Aβ and nAChRs, providing direct molecular evidence of how Aβ selectively interacts with α7- and α4β2-nAChRs, but not α3β4-nAChRs.Computer-simulated docking studies using the homology model of the human α7-nAChRs derived from the X-ray structure of the acetylcholine-binding protein (AChBP) and human Aβ show that the N terminus of Aβ is predicted to bind to the loop C of the α7 subunit, which is located within the binding interface of two α7 subunits , 54, 55.Fig. 2ractions , 55. Of 2+ hyperexcitation promotes AMPAR endocytosis, which ultimately decreases the surface expression of AMPA receptor subunits GluA1 and GluA2, a cellular mechanism underlying Aβ-induced depression of AMPAR-mediated synaptic transmission  and neuronal Ca2+ activity in Aβ-treated cultured neurons (A). As shown before (p = 0.0424) (A and B). Of significance, Aβ-induced reduction of pGluA1 was reversed by costimulation of α7- and α4β2-nAChRs when we treated neurons with 1 μM PNU-282987 and 2 μM RJR-2403 Oxalate for 1 h  (A and B). However, coactivation of these receptors in control cells had no effect on pGluA1 levels  (A and B). We next treated neurons with a glycine-based medium, well established to induce a form of chemical LTP (cLTP), as shown previously (A). As shown previously (A). However, pGluA1 levels were significantly lower in oAβ42-treated neurons compared with sAβ42-treated control cells after cLTP induction  (A and C), an indication of impaired synaptic plasticity. Of importance, normal pGluA1 levels were restored when we activated both α7- and α4β2-nAChRs and induced cLTP in oAβ42-treated neurons  (A and C). However, coactivation of these receptors in control cells had no effect on pGluA1 levels following cLTP induction  (A and C). Thus, coactivation of α7- and α4β2-nAChRs was sufficient to reverse the Aβ effects on AMPAR phosphorylation and cLTP.Several studies suggest that Aβ-induced Caasticity , 59, 62.asticity , 58, 61.asticity , 65, 66.asticity B and neu neurons , we exam neurons A. As shon before , oAβ42 t 0.0424) , A and B= 0.003) , A and B ± 0.36) , A and Beviously . We treaeviously A. As shoeviously , followieviously A. Howeve 0.0075) , A and C 0.0359) , A and C ± 0.61) , A and CCurrent therapeutic approaches to AD suffer from lack of specificity and poor efficacy. Preclinical approaches based on altering Aβ have failed in clinical trials. Consequently, novel approaches are being explored, including targeting receptors regulated by Aβ. Nicotinic receptors have emerged as potential targets for reversing cognitive deficits in AD , owing tA). This is supported by our previous work in which stimulation of GABAA receptors is sufficient to reverse Aβ42-induced Ca2+ hyperactivity in cultured neurons  and other Ca2+ signaling pathways. This promotes the dephosphorylation of the AMPAR subunit, GluA1, which allows for AMPAR endocytosis, resulting in an overall decrease of AMPAR surface expression . This ultimately contributes to disruption of LTP  and may lead to cognitive decline. As Aβ42 inhibits both α7- and α4β2-nAChRs but not α3β4-nAChRs, costimulation of α7- and α4β2-nAChRs by using selective agonists can reverse the Aβ effects on synapses by restoring normal activity of both hippocampal inhibitory and excitatory cells . With restoration of normal Ca2+ activity, calcineurin activity decreases, leading to AMPAR phosphorylation and decreased AMPAR endocytosis, ultimately restoring normal LTP . Given that Aβ42 inhibits both α7- and α4β2-nAChRs, stimulation of each receptor by themselves has no neuroprotective effect (A).Based on the present findings, we propose the following model . Given tFig. 5 neurons . Excitatcineurin   and glutamate 185 (E185), located in the loop C region of the α7-nAChR-AChBP chimera , do not have direct contact to agonists but impact the affinity of α7-nAChRs for ligands . Cholinergic signaling in GABAergic inhibitory networks is generally stronger than direct actions on glutamatergic neurons in the hippocampus, as nAChRs are more densely expressed on inhibitory interneurons than on excitatory cells (C).Of interest, costimulation of α4β2-and α7-nAChRs can prevent the Aβ effects in cultured neurons, whereas coadministration of agonists for three nAChR subtypes has no effect C. Cholinry cells , 77, 78.ry cells , 87, 88 ry cells . Thus, cry cells , reductiry cells C.C), pointing to confounding effects of activating α3β4-nAChRs, which may result in unexpected side effects, including imbalance of excitation and inhibition in hippocampal circuits. Therefore, acetylcholinesterase inhibitors are not very effective in slowing AD progression due to nonselective stimulation of acetylcholine receptors . By contrast, selective coactivation of α7- and α4β2-nAChRs was sufficient to reverse Aβ-induced neuronal hyperexcitation  and imaged on DIV 12 to 14. For biotinylation assays and cLTP, hippocampal neurons were plated in 6-cm dishes (3 × 106 cells) and used on DIV 14. Cells were grown in Neurobasal medium (Life Technologies) with B27 supplement (Life Technologies), 0.5 mM Glutamax (Life Technologies), and 1% penicillin/streptomycin (Life Technologies). Colorado State University's Institutional Animal Care and Use Committee reviewed and approved the animal care and protocol (978).Mouse hippocampal neuron cultures were prepared as described , 103. HiSoluble Aβ42 oligomers or soluble scrambled Aβ42 oligomers were prepared as described , 104. OnHEK293 cells were grown in Dulbecco's modified Eagle's medium with fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin (Life Technologies) and transfected by jetPRIME DNA and siRNA transfection reagent (Polyplus) according to the manufacturer's protocol. Cells used for each experiment were from more than three independently prepared cultures. Two micrograms of human α7-nAChR-GFP plasmid was transfected into HEK293 cells to express the homopentameric form of α7-nAChRs. Both α3β4- and α4β2-nAChRs expressed by 1:1 α:β transfection ratio formed functional channels in HEK293 cells , 108. Thhttp://n2t.net/addgene:62629; RRID:Addgene_62629)  . β4-nAChR (DPM negative control) was a gift from Jaime Modiano  (ATA TTC TAT GAG TGC TGC-3′ and 5′-CCT TAG GGG CCG TTC TCC TCA CTT TAT AAG ATA CTC ACG ACG-3′), E211N (5′-AGG TTC TAT AAC TGC TGC AAA GAG CCC TAC CCC GAT GTC-3′ and 5′-TCC AAG ATA TTG ACG ACG TTT CTC GGG ATG GGG CTA CAG-3′), I208R (5′-CCA GGC TAC AAA CAC GAC CGC AAG TAC AAC TGC TGC-3′ and 5′-GCA GCA GTT GTA CTT GCG GTC GTG TTT GTA GCC TGG-3′) and N211E (5′-GGC TAC AAA CAC GAC ATC AAG TAC GAG TGC TGC GAG GAG-3′ and 5′-CTC CTC GCA GCA CTC GTA CTT GAT GTC GTG TTT GTA GCC-3′); bold and italic nucleotides indicate mutations introduced.Human pcDNA3.1-CHRNA7-mGFP was a gift from Henry Lester  . Mouse ne_15244) . pCI-neoe_86651) . Human αCo-IP experiments were performed using a Co-IP kit (Pierce) following the manufacturer's protocol with samples from three independently prepared cultures as carried out previously . Two micComputational peptide docking of the N terminus of Aβ into the α7-nAChR-AChBP (the acetylcholine-binding protein) chimera  was perf2+ imaging was carried out by the previously reported method   /Fmin in each image, and values of ΔF/Fmin < 0.1 were rejected due to bleaching. Ten to 20 neurons were used for imaging in each individual experiment, and one individual neuron was assayed in an image.GCaMP Cad method , 47. DIVe_40755)  for image_83899)  for imag2 and 0.5 mM MgCl2 and incubated with 1 mg/ml Sulfo-NHS-SS-biotin (Thermo Scientific) for 15 min on ice. Following biotin incubation, neurons were washed with 20 mM glycine to remove the excess of biotin, and cells were lysed in 300 μl radioimmunoprecipitation assay buffer for 1 h. Ten percent of the total protein was separated as an input sample, and protein lysates were incubated overnight with streptavidin-coated beads (Thermo Scientific) at 4 °C under constant rocking. The beads containing surface biotinylated proteins were separated by centrifugation. Biotinylated proteins were eluted from streptavidin beads with SDS loading buffer. Surface protein fractions and their corresponding total protein samples were analyzed by immunoblots.Surface biotinylation was performed according to the previous studies , 103. Ce2+-free buffer , treated with 200 μM glycine in Mg2+-free buffer for 5 min at 37 °C, and returned to Mg2+ buffer (Mg2+-free buffer and 2 mM MgCl2) for 20 min at 37 °C. Cells were then lysed with 100 μl radioimmunoprecipitation assay buffer, and cell lysates was used for immunoblots.cLTP was performed by a modification of the previously described method , 67. Celhttps://imagej.nih.gov/ij/). Immunoblots were at least duplicated for quantitative analysis.Protein samples for biotinylation and cLTP were loaded on 10% glycine-SDS-PAGE gel. Co-IP samples were loaded on 16% Tricine-SDS-PAGE gel as described . SDS-PAGt tests were used in single comparisons. For multiple comparisons, one-way ANOVA followed by Fisher's least significant difference (LSD) test was used to determine statistical significance. Results are represented as mean ± standard deviation (SD), and p < 0.05 was considered the minimum for statistical difference.Statistical comparisons were analyzed with the GraphPad Prism 9 software. Unpaired two-tailed Student All data are contained within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The Neisseria meningitidis protein FrpC contains a self-processing module (SPM) undergoing autoproteolysis via an aspartic anhydride. Herein, we establish NeissLock, using a binding protein genetically fused to SPM. Upon calcium triggering of SPM, the anhydride at the C-terminus of the binding protein allows nucleophilic attack by its target protein, ligating the complex. We establish a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We optimize NeissLock using the Ornithine Decarboxylase/Antizyme complex. Various sites on the target (α-amine or ε-amines) react with the anhydride, but reaction is blocked if the partner does not dock. Ligation is efficient at pH 7.0, with half-time less than 2 min. We arm Transforming Growth Factor-α with SPM, enabling specific covalent coupling to Epidermal Growth Factor Receptor at the cell-surface. NeissLock harnesses distinctive protein chemistry for high-yield covalent targeting of endogenous proteins, advancing the possibilities for molecular engineering. 2+, the authors here develop a system that enables the covalent capture of endogenous binding partners.Covalent conjugation of endogenous protein complexes offers many opportunities for fundamental and clinical research. Based on a bacterial protein domain that forms a reactive anhydride in the presence of Ca Much attention has focused on peptide tags able to react with protein partners6, e.g., SpyTag, split inteins, sortase or OaAEP1, or reactions through click chemistry pairs7. However, distinct strategies are required to react with unmodified endogenous proteins, which has greater relevance for therapeutic settings. For this challenge, proximity-directed ligation has been an important approach, employing either small molecules or protein binders8. Small molecules with affinity for a target protein may be equipped with reactive functionalities, favoring covalent reaction with nearby nucleophiles in the binding site 4. This approach has been successful for certain proteins, particularly those with deep and unique pockets facilitating specific ligand binding4. To generalize this approach to a wider range of protein targets, one would ideally harness natural or engineered proteins and confer on them covalent reactivity with their specific protein partners. We previously applied this strategy through chemical attachment of the weak electrophile acrylamide, for covalent ligation by an affibody to an apposed nucleophile on its cognate partner9. Since then, various electrophilic unnatural amino acids have been genetically encoded for coupling to endogenous proteins14. However, it would be preferable to obtain such reactivity using standard amino acids and with inducible reaction.Covalent conjugation to proteins presents unique opportunities. Compared to typical non-covalent coupling approaches, decoration through a stable covalent bond can enhance long-term imaging, biomaterial strength, therapeutic or vaccine efficacy, and diagnostic sensitivityNeisseria meningitidis is a secretory protein containing a self-processing module (SPM), which displays calcium-dependent autoproteolytic activity at an aspartate-proline dipeptide16. Moving from the low calcium environment inside the cell (Ca2+ ~ 0.1 µM) to the extracellular medium (Ca2+ 1–2 mM)17, calcium-dependent conformational change in SPM mediates FrpC processing20. Autoproteolysis follows protonation of the main-chain nitrogen of proline, leading to formation of an aspartic anhydride at the newly generated C-terminus21  , ahead of SDS-PAGE with Coomassie staining  Fig. . Proteining Fig. . When X ing Fig. . H, Y, aing Fig. . Therefot to lysine ε-amine <10 Å  and Ornithine Decarboxylase Antizyme (OAZ) (PDB 4zgy)24  a short distance between the C-terminal anhydride of the binding protein and the nearest accessible nucleophile on the target protein, (2) SPM should not sterically disrupt docking between the binding protein and target protein, and (3) avoidance of an own-goal (self-reaction where a nucleophile on the binding protein rather than on the target protein reacts with the anhydride) Fig. . Therefo0 Å Fig.   vary from amino acid residues 414–65724. We predicted this short distance to be favorable for conjugation , is 3.5 Å from ODC K92′s ε-amine (atom Nε)ion Fig. . Followiion Fig. , E219 ap2+, an efficient reaction occurred between OAZ and ODC, with little free OAZ remaining 17 are sufficient for the NeissLock approach.Previously, it was shown that the cleavage rate of SPM in fusion proteins was enhanced with higher calcium concentrations but still highly efficient at 2 mM CaWe then tested NeissLock’s compatibility with different buffers by incubating OAZ-GSY-SPM and ODC in HEPES-buffered saline (HBS) or Tris-buffered saline (TBS). The rate of conjugation to ODC was similar in HBS and TBS  compared to the untreated control  after 2 h  and no observable conjugation to sfGFP  or superfolder green fluorescent protein (sfGFP). At 10 µM each of OAZ-GSY-SPM and added protein, there was only trace conjugation to MBP . Therefore, we incorporated charge inversion mutations at R188 and K153 of OAZ , we introduced an AviTag to OAZ-GSY-SPM for site-specific biotinylation, allowing anchoring to the streptavidin-chip. We also introduced C175A in OAZ to reduce aggregation. We tested two mutations reported to reduce binding based on the mouse ODC/OAZ complexSPM Fig. . In summ6-tags at the N-termini of OAZ and ODC 29 suggested that the N-terminus of ODC might extend back towards the OAZ interface. Based on PDB 1d7k, we, therefore, removed the N-terminal His6-tag as well as 9 flexible residues from ODC 4KR, to generate ODC ΔN 4KR. Incubation of OAZ-GSY-SPM with ODC ΔN 4KR yielded only small amounts of conjugation . There are multiple nucleophilic residues within the unresolved C-terminus of ODC that are potential reactive sites. Incubation of OAZ-GSY-SPM with ODC ΔN ΔC 4KR now made conjugation barely detectable  in ODC ΔN 4KR, we introduced a further truncation of the C-terminus of ODC that was unresolved in PDB 4zgyble Fig. . Re-intrw for a monomeric protein . This analysis gave a close correspondence between the predicted and observed M30. AZI has a well-positioned lysine for reaction with OAZ-GSY-SPM  . AZI binds with high affinity to OAZ, so reducing OAZ’s inhibition of ODC and leading to increased cellular polyamine levels which may favor tumor progression 3) Fig. . The cov 3) Fig. .In the OAZ/ODC model system, we established coupling via NeissLock to the α-amine or ε-amines on ODC. To further understand the reactivity of the SPM-generated aspartic anhydride, we developed a plate-based assay to compare a panel of biotin-linked nucleophiles, including those mimicking cysteine, tyrosine, α-amine, and ε-amine Fig. . SDS-PAG31. First, we validated that this complex was suitable by testing conjugation of TGFα-GSY-SPM to the soluble ectodomain of EGFR (sEGFR)32 in vitro. Glycosylation of sEGFR in Expi293 cells led to heterogeneous gel mobility. Therefore, we expressed sEGFR in the presence of the mannosidase inhibitor kifunensine and deglycosylated sEGFR with Peptide:N-glycosidase (PNGase) F, resulting in a single sharp band for sEGFR  and Epidermal Growth Factor Receptor (EGFR) as a promising candidate Fig. . TGFα/EGGFR Fig. . Incubatone Fig. . Using tone Fig. . This reone Fig.  and the 2+ 20. To test that SPM does not promote promiscuous cellular interaction, we tested the binding of TGFα-GSY-SPM at the mammalian cell surface. Here, we used A431, a human epidermoid carcinoma cell-line expressing EGFR33. TGFα-GSY-SPM bound strongly to the cell-surface, as did the non-reactive TGFα-GSY-[DA]SPM control   was mutated out, K121 at 14 Å from OAZ’s C-terminal E219 still reacted efficiently. Therefore, given the abundance of lysines on protein surfaces, many protein complexes should have a suitable lysine in range, particularly when including a GSY-linker before SPM. In fact, we found thousands of such instances, promising broad applicability  or too high reactivity (leading to non-specific coupling and spontaneous inactivation upon storage)41. NeissLock gives a system with intrinsic low reactivity  until high reactivity is induced by the mild conditions of typical extracellular calcium concentration. Since anhydrides have high reactivity, they have been little explored for directed protein modification. In neutral conditions, succinic anhydride or glutaric anhydride hydrolyzes with t½ ~4 min at 25 °C42. Compared to constitutive electrophiles, the anhydride is generated in situ, offering high reactivity to allow efficient coupling, although raising concerns about non-specificity. We found minimal non-specific reaction of OAZ-GSY-SPM with irrelevant non-binding proteins. Fine tuning of reactivity could include modifying accessibility of the reactive C-terminus, change of pH, removal/addition of competing self-reacting residues, or addition of free nucleophile to modulate anhydride life-time.Previous approaches for proximity ligation have either used UV induction of highly reactive free radicals or weak electrophiles43, so future work will investigate NeissLock in different cellular locations. Since even peptide tags of 6 residues can induce immune responses44, it is important that the protein retains minimal foreign sequence for potential use in vivo. NeissLock leaves a small scar, i.e., just D, although a short spacer may improve performance. In summary, NeissLock should allow generation of covalent probes for a broad range of protein assemblies, increasing their resilience to time, force, or harsh conditions, with future potential for diagnostic and therapeutic applications41.We demonstrated NeissLock conjugation of TGFα to EGFR on live cells. We observed changes in STAT1 signaling post conjugation of TGFα to EGFR. More study will be required to further understand the changes in signaling pathways from covalent coupling to cell surface receptors. EGFR can be rapidly internalized into acidic compartments upon ligand bindingN. meningitidis serogroup B (strain MC58) (UniProt Q9JYV5). The SPM sequence was based on residues 414–657 of FrpC21. When testing C-terminal truncations of SPM, SPM635 was based on residues 414–635, SPM613 on 414–613 and SPM591 on 414–591. pET28a-His6-SpyTag-A-SPM (GenBank MW364947) has the following organization: (M)GSS-linker, His6–tag, SSG-linker, thrombin cleavage site, NdeI restriction site, G-spacer, SpyTag, A, SPM, GSG-linker, C-tag. pET28a-His6-SpyTag-X-SPM has the alanine replaced by each of the other 19 amino acids. Residue numbers for OAZ and ODC were based on the crystal structure of the human OAZ/ODC complex (PDB 4zgy)24. pET28a-His6-OAZ-Y-SPM-Ctag has the following organization: N-terminal (M)GSS-linker, His6–tag, residues 95–219 of human OAZ1 (UniProt P54368), Y-spacer, SPM, GSG-linker, C-tag. pET28a-His6-OAZ-GSY-SPM-Ctag  has the same organization, except for the addition of a GS-spacer before Y. C175A was introduced into OAZ in pET28a-AviTag-His6-OAZ-GSY-SPM-Ctag by site-directed mutagenesis using Gibson assembly, to minimize any disulfide-mediated aggregation. The mutations K153A, K153E, V198A, A215R, and R188E were introduced into OAZ by site-directed mutagenesis using Gibson assembly to disrupt binding to ODC. D414A was introduced into SPM of OAZ-GSY-SPM by site-directed mutagenesis using Gibson assembly to generate a non-cleaving SPM variant. Human ODC1 (UniProt P11926) was cloned as pET28a-His6-ODC-Ctag  to give the following organization: (M)GSS-linker, His6–tag, SSG-linker, ODC1, GSG-linker, C-tag. The ΔN version of ODC had the organization ODC1(10–460), GSG-linker, C-tag. The ΔN ΔC version of ODC had the organization ODC1(10–421) with K92R, K121R, K74R, and K78R mutations, GSG-linker, C-tag (GenBank MW364945). Human AZI1 (UniProt O14977) was cloned as pET28a-His6-AZI-Ctag (GenBank MW364946) to give the following organization: (M)GSS-linker, His6–tag, SSG-linker, AZI1, GSG-linker, C- tag. pET28a-TGFα-GSY-SPM  includes mature TGFα sequence that was taken from residues 40–89 of human protransforming growth factor alpha (UniProt P01135). pET28a-TGFα-GSY-SPM has the following organization: (M), TGFα, GSY-linker, SPM, GSS-linker, His6-tag, SSG-linker, C-tag. DNA primers and gene fragments codon-optimized for E. coli expression were ordered from Integrated DNA Technologies before cloning into the pET28a backbone. pET28a-SpyTag003-sfGFP (Addgene plasmid #133454) and pET28a-SpyCatcher003-sfGFP (Addgene plasmid #133449) have been described45. pET28a-AviTag-DogTag-MBP has the following organization: (M)GSS-linker, His6–tag, SSG-linker, Thrombin site, AviTag, GSG-linker, DogTag, GSGESG-linker, MBP46. pENTR4-sEGFR-His6 has the organization: tissue plasminogen activator leader sequence, soluble fragment of extracellular domain of human EGFR 47, GSGESG, His6. All constructs were validated by Sanger sequencing. pGEX-2T encoding GST-BirA was a gift from Chris O’Callaghan, University of Oxford, and purified by glutathione-affinity chromatography48. Oligonucleotides used in this study are listed in Supplementary Data For cloning of constructs, Q5 High-Fidelity Polymerase (NEB) or KOD polymerase (EMD Millipore) was used for PCR, followed by Gibson assembly. Residue numbers for SPM derive from FrpC of 49. Biological assemblies were generated using code provided by PDBx/mmCIF Dictionary Resources (mmcif.wwpdb.org/docs/sw-examples/python/html/assemblies.html). To preserve computational resources, the file-size of biological assemblies was limited prior to analysis to < 10 MB . For structures with multiple models, only the first 10 were analyzed. For each structure, initial analysis was performed using Python (Python Software Foundation) with the code we have provided (https://github.com/arnescheu/NeissDist). Herein, the Biopython PDB module was used to interpret structural data and parse dictionary information51. As parsed by the Biopython module, the last resolved standard amino acid for each polypeptide chain was defined as the ‘C-terminal’ residue for that chain. The first resolved standard amino acid residue in each polypeptide chain as well as all lysines were defined as ‘target’ residues. If applicable atoms were resolved in the structure, distances were calculated from atoms C (main-chain carbonyl carbon), CA  and N (main-chain nitrogen) in the backbone of C-terminal residues to atoms CA and N in the backbone of the target residues as well as Nε in the side-chain of target lysines. For lysine, ‘primary distance’ was chosen by the first available distance in order of C-terminal to target residue: C to Nε > CA to Nε > N to Nε > C to CA > CA to CA > N to CA > C to N > CA to N > N to N. For N-terminal residues, ‘primary distance’ was chosen in order of C-terminal to target residue: C to αN > CA to αN > N to αN > C to CA > CA to CA > N to CA. If the N-terminal residue was also Lys, αN or Nε was selected, if available, according to which resulted in a shorter distance .To identify candidates for covalent fusion by C-terminal activation, protein structures were screened for the distance between the C-terminal resolved residue and the N-terminal resolved residue or lysine residues. Protein structures were retrieved from the worldwide protein data bank (wwPDB)t atom C to lysine atom Nε were considered . For all biological assemblies generated from a single asymmetric unit, one distance was retrieved per category (which can be across assemblies). Using the SQLalchemy module in Python, this dataset was interrogated for all available structures of a given category in 0.1 Å intervals from 0 to 50 Å. Increments were binned into 1 Å intervals. In Fig. For Fig. 6-SpyTag-X-SPM and related plasmids, plasmids were transformed into chemically-competent E. coli BL21 (DE3) RIPL (Agilent Technologies). Cells were plated on LB agar with 50 μg/mL kanamycin and 34 µg/mL chloramphenicol and incubated for 16 h at 37 °C. Single colonies were picked to inoculate 60 mL of Auto-induction medium LB  with 50 μg/mL kanamycin and 34 µg/mL chloramphenicol in a 200 mL round-bottom flask. Cells were incubated for 24 h at 30 °C with 200 rpm shaking. Cells were harvested by centrifugation at 4000 g for 10 min at 4 °C. Cells were resuspended with 1.5 mL Ni-NTA buffer  supplemented with cOmplete mini EDTA-free protease inhibitor cocktail (Roche), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/mL lysozyme (Merck) and 2 U/mL benzonase and samples were transferred to microcentrifuge tubes. For lysis, cells were incubated at 25 °C with end-over-end rotation for 30 min and subjected to 6 rounds of freeze-thawing (−80 °C freezer to 23 °C thermomixer). After thawing, the total lysate was spun twice at 16,100–16,900 g for 30 min at 4 °C to separate the cleared lysate. Cleared lysate was incubated with 100 µL 1:1 slurry of pre-equilibrated Ni-NTA beads in Ni-NTA buffer at 4 °C before transfer to a 96-well filter plate (AcroPrep). Flow-through was collected by centrifugation at 300 g for 30 s. Samples were washed twice with 300 µL Ni-NTA binding buffer supplemented with 10 mM imidazole  and then washed twice with 300 µL Ni-NTA buffer supplemented with 30 mM imidazole . Finally, the protein was eluted with 300 µL Ni-NTA buffer supplemented with 200 mM imidazole. Eluates were concentrated using Vivaspin 500 5 kDa spin concentrators. The samples were buffer-exchanged into HEPES-buffered saline  using the same concentrators. Samples were flash-frozen in an ethanol/dry ice bath and stored at −80 °C until further use.For pET28a-His6-OAZ-SPM-Ctag, pET28-His6-ODC1-Ctag, pET28-His6-AZI1-Ctag or related plasmids as well as pET28a-TGFα-GSY-SPM-Ctag, the plasmids were transformed into chemically-competent E. coli BL21 (DE3) RIPL. pET28a-TGFα-GSY-SPM-Ctag was transformed into E. coli BL21 (DE3) RIPL or E. coli T7 Express (NEB). Cells were then plated on LB agar with 50 μg/mL kanamycin and incubated for 16 h at 37 °C. Single colonies were picked to inoculate 11 mL of LB with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol, before 16–19 h incubation at 37 °C with shaking at 200 rpm. 10 mL of the overnight culture was used to inoculate 1 L of LB with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol in a baffled flask. Cultures were incubated at 37 °C with shaking at 200 rpm until A600 reached 0.6. Then the cultures were induced using 0.42 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Fluorochem) and incubated at 25 °C with shaking at 200 rpm for 16–18 h. For constructs containing TGFα, induction was at 18 °C. Cell pellets were immediately processed or stored at −80 °C until further use.For pET28a-HisBacterial cells were harvested and lysed by sonication in lysis buffer  supplemented with cOmplete mini EDTA-free protease inhibitor cocktail, 1 mM PMSF, 1 mg/mL lysozyme (Merck) and 2 U/mL benzonase (Merck). While kept on ice, the lysate was sonicated 3–4 times for 1 min at 50% duty cycle with 1 min rest periods. The cell lysate was then centrifuged in microcentrifuge tubes in a benchtop centrifuge at 16,900 g for 10–30 min at 4 °C or with a JA25−50 rotor (Beckman) at 30,000–35,000 g for 30–40 min at 4 °C.6-tag purification, the clarified lysate was then added to pre-equilibrated Ni-NTA resin (Qiagen). After addition to a Poly-Prep gravity column, the Ni-NTA resin was washed with Ni-NTA buffer with 10 mM imidazole, followed by Ni-NTA buffer with 30 mM imidazole. The protein was eluted from the resin using Ni-NTA buffer with 200 mM imidazole. For variants of ODC, OAZ, and AZI, the lysis buffer, wash buffers and elution buffer were supplemented with 5 mM 2-mercaptoethanol.For His2, pH 7.8.For ODC variants without an N-terminal His-tag, the clarified lysate was added to CaptureSelect™ C-tagXL Affinity Matrix (Thermo Fisher). After addition to a Poly-Prep gravity column, the resin was washed with 20 mM Tris-HCl, 5 mM 2-mercaptoethanol, pH 7.4. The protein was eluted using 50 mM HEPES, 5 mM 2-mercaptoethanol, and 2 M MgCl280 peak and verified by SDS-PAGE, before another round of concentration using pre-rinsed Vivaspin centrifugal concentrator with 5, 10, or 30 kDa cut-off  and then storage at −80 °C.After Ni-NTA or C-tag purification, variants of TGFα-GSY-SPM, ODC, OAZ-SPM, and AZI were concentrated using a Vivaspin centrifugal concentrator with 5, 10, or 30 kDa cut-off  and loaded onto a pre-equilibrated HiLoad 16/600 Superdex 200 pg size exclusion chromatography (SEC) column  connected to an ÄKTA Pure 25  fast protein liquid chromatography (FPLC) machine at 4 °C. 50 mM HEPES, 150 mM NaCl pH 7.4 buffer was used for SEC of TGFα-GSY-SPM. SEC buffer was supplemented with 2 mM tris(2-carboxyethyl)phosphine (TCEP) for OAZ-SPM and AZI variants and with 2 mM TCEP as well as an additional 0.02 mM pyridoxal phosphate (PLP) as cofactor for ODC variants. Fractions were collected according to the A6 cells/mL in 25 mL volume. 80 µL ExpiFectamine (Thermo Fisher) in 1.4 mL Expi293 Expression Medium was incubated for 5 min and then combined with 25 µg of 0.22 µm filter-sterilized pENTR4-sEGFR-His6 diluted with 1.5 mL Expi293 Expression Medium. After 20 min incubation, the mixture was slowly added to the cell suspension. Where indicated, 30 µL of 5 mM kifunensine (Sigma-Aldrich) was added to the suspension. Cells were incubated at 37 °C, 8% (v/v) CO2 and 125 rpm orbital shaking. After 20 h, enhancer 1 (150 µL) and enhancer 2 (1.5 mL), as well as 150  µL of 100× Penicillin/Streptomycin , were added. Cells were grown for an additional 4 days, after which sEGFR was recovered from the cell supernatant using Ni-NTA affinity purification. Cell supernatant was supplemented with 5 mL Ni-NTA buffer to adjust pH, as well as mixed protease inhibitors  and 1 mM PMSF. The supernatant was incubated with 5 mL of 1:10 Ni-NTA bead slurry (0.5 mL beads equilibrated to 5 mL in Ni-NTA buffer) at 4 °C with horizontal rolling at 33 rpm (Stuart roller mixer SRT6). Samples were applied to a gravity-flow column and washed twice with 10 mL Ni-NTA buffer supplemented with 10 mM imidazole and once with 2 mL Ni-NTA buffer supplemented with 30 mM imidazole. Samples were eluted with Ni-NTA buffer supplemented with 200 mM imidazole. Eluates were combined before buffer exchange and spin concentration using 30 kDa Vivaspin centrifugal concentrators. The purification yielded 0.21 mg sEGFR (weight estimated without considering glycosylation).Expi293 cells were seeded in Expi293 Expression Medium (Thermo Fisher) at 2.5–3 × 10280, with extinction coefficients estimated using the ExPASy server52. SDS-PAGE employed 10%, 16%, or 18% polyacrylamide gels in an XCell SureLock system (Thermo Fisher) run at 180 V or 200 V. Optimized conditions for OAZ-Y-SPM or OAZ-GSY-SPM conjugation to ODC refers to 18% SDS-PAGE at 180 V for 110 min. Gels were stained using InstantBlue (Expedeon) and destained with MilliQ water before imaging with a ChemiDoc XRS imager. Quantification was carried out using Image Lab software Protein concentrations were measured from AReactions were carried out at 37 °C in HBS supplemented with 2 mM TCEP. When measuring pH-dependence, an additional 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) was added for proper buffering over the pH range tested. For reactions analyzing the effect of the −1 position on cleavage rate, 4–6 μM SpyTag-X-SPM was used with 10 mM cysteine in 50 mM HEPES, 150 mM NaCl, pH 7.4. For reactions analyzing the speed and pH-dependence of coupling, OAZ-SPM was reacted with ODC with each protein at 10 μM or at the indicated concentrations. When investigating buffer-dependence, TBS consisted of 20 mM Tris-HCl, 150 mM NaCl, pH 7.4 supplemented with 2 mM TCEP. OAZ-GSY-SPM was reacted with AZI with each protein at 2.5 μM for both the overnight reaction as well as the time course of conjugation. The cleavage of SPM was induced by addition of the HEPES reaction buffer, pre-equilibrated to 37 °C, containing calcium chloride at a final 10 mM. When investigating the calcium-dependence of the reaction, calcium chloride diluted in HEPES reaction buffer was added to give final concentrations of 2 mM, 1 mM or 0.5 mM calcium chloride. After the indicated time, the reaction was stopped by addition of 5× SDS-loading buffer  containing EDTA added for a final 15 mM. The reaction was stopped with only EDTA to allow subsequent MS. SDS-loading buffer was added only for subsequent SDS-PAGE. Protein samples were then heated on a Bio-Rad C1000 thermal cycler at 95 °C for 6 min or 99 °C for 3 min. For time-courses, the 0 h time point was taken by addition of the stop buffer to the reaction before addition of the start buffer. Replicates represent multiple measurements derived from the same protein preparation. The fraction of uncleaved SPM was determined by dividing the intensity of the band for SpyTag-X-SPM or OAZ-SPM at each time point by the intensity of the SpyTag-X-SPM or OAZ-SPM band at the 0 h time point, i.e.,:The fraction of conjugated ODC was determined by dividing the decrease in intensity of the band for ODC by the intensity of the ODC band at the 0 h time point, i.e.,:The fraction of conjugated AZI was determined by dividing the decrease in intensity of the band for AZI by the intensity of the AZI band at the 0 h time point, i.e.,:The relative conjugation to ODC in the pH/buffer/calcium-dependence time-course was determined by dividing the intensity of the band for OAZ:ODC by the intensity of the OAZ:ODC band after 2 h for the most efficient reaction, i.e.,:The relative conjugation to MBP compared to ODC was determined by dividing the intensity of the band for OAZ:MBP by the intensity of the OAZ:ODC band (adjusted for the relative molecular weights of OAZ:MBP and OAZ:ODC), i.e.,:The relative conjugation to each ODC variant was determined, for example, by dividing the intensity of the band for OAZ: ΔN 4KR ODC by the intensity of the OAZ:ODC (wt) band (adjusted for the relative molecular weights of OAZ: ΔN 4KR ODC and OAZ:ODC (wt), i.e.,7 cells in 1 mL lysis buffer consisting of 20 mM Tris-HCl, 150 mM NaCl, 1% (v/v) Triton-X-100, pH 7.4, supplemented with mixed protease inhibitors and 1 mM PMSF. The cell lysate was incubated on ice for 20 min before centrifugation at 12,000 g for 10 min at 4 °C. 1 μM biotinylated AviTag-OAZ-GSY-SPM was added to the clear lysate, with or without 1 μM ODC. Cleavage of SPM was induced by addition of clear lysate containing calcium chloride at a final concentration of 2 mM. The reaction was carried out at 25 °C for 10 min, before the reaction was stopped by addition of 5× SDS-loading buffer containing EDTA at a final concentration of 15 mM. Samples were heated at 99 °C for 3 min before resolving by SDS-PAGE. Protein samples were transferred to a nitrocellulose membrane at 25 V for 10 min using the iBlot2 Dry Blotting System (Thermo Fisher). The membrane was blocked for 1 h at 25 °C with 5% (w/v) bovine serum albumin (BSA) in PBS with 0.05% (v/v) Tween-20 (Merck) (PBS-T) before incubation for 20 h at 4 °C with mouse anti-His  at 1:1,000 dilution in 5% (w/v) BSA in PBS-T. The membrane was then washed 6 times with PBS-T. Subsequently, the membrane was incubated with goat anti-mouse horseradish peroxidase (HRP)  at 1:1,000 in 5% (w/v) BSA in PBS-T for 1 h at 25 °C. The membrane was washed 6 times with PBS-T and incubated with SuperSignal West Pico PLUS Chemiluminescent Substrate. Chemiluminescence was measured using ChemiDoc XRS imager using Image Lab Software version 5.2.1 (Bio-Rad).A431 cell lysate was prepared by first washing A431 cells once with PBS before resuspending 2 × 10OAZ-GSY-SPM at 2 mg/mL in 100 μL 50 mM HEPES, 150 mM NaCl, 2 mM TCEP, 0.02 mM PLP, pH 7.4 was injected at 25 °C into a Superdex 200 HR 10/30 column , connected to a Shimadzu HPLC system with a Wyatt Dawn HELEOS-II 8-angle light scattering detector and Wyatt Optilab rEX refractive index monitor. The running buffer was HBS with 2 mM TCEP and 0.02 mM PLP. ASTRA 6 software (Wyatt Technology) was used for analysis of the light scattering, refractive index, and UV absorbance data. Error bars in the observed molecular weight represent the uncertainty in fitting to the molar mass curve.48. 50 µM of each AviTagged protein was incubated with 5 mM MgCl2, 1 mM ATP, 0.75 mM biotin, and 3.3 µM GST-BirA in PBS for 1 h at 25 °C. The same amount of GST-BirA was added again, before incubation for another for 1 h at 25 °C. GST-BirA was removed by incubation with Glutathione HiCap matrix (Qiagen) for 1 h at 4 °C, before spinning the resin down. Excess biotin was removed subsequently by gel filtration using the HiLoad 16/600 Superdex 200 pg size exclusion chromatography (SEC) column  into HBS with 2 mM TCEP. Biotinylated AviTag-OAZ-GSY-SPM, AviTag-OAZ-GSY-SPM, AviTag-OAZ-GSY-SPM or AviTag-OAZ-GSY-SPM was immobilized onto the sensor chip using the Biotin CAPture Kit, Series S . C175A was introduced into OAZ to minimize any disulfide-mediated aggregation. The flow buffer was HBS with 2 mM TCEP and 0.02 mM PLP. Measurements were made at 25 °C. Following each run, the chip was regenerated following the manufacturer’s protocol. Biacore T200 Software v3.0  was used for data analysis and a 1:1 binding model between OAZ and ODC was used for fitting and to derive the kinetic binding constants. Replicates represent multiple measurements derived from the same protein preparation.SPR was carried out using a Biacore T200 . AviTag-OAZ-GSY-SPM, AviTag-OAZ-GSY-SPM, AviTag-OAZ-GSY-SPM and AviTag-OAZ-GSY-SPM were biotinylated using GST-BirA2 for 16 h at 37 °C in HBS with 2 mM TCEP. Processing was stopped by addition of EDTA to 15 mM (1/5 volume). Samples were diluted 1:1 with water and acidified to 0.9% (v/v) formic acid before aspiration under vacuum for 0.3 s and loading onto a C4 solid-phase extraction cartridge. Washes using 0.1% (v/v) formic acid in water were carried out for 5.5 s, before the sample was eluted onto the Q-TOF detector for 5.5 s. Data were analyzed using MassHunter Qualitative Analysis B.07.00 (Agilent), with the following deconvolution settings: Deconvolute (protein), maximum entropy deconvolution algorithm, mass range 10,000.00–80,000.00 Da, mass step 1,0000 Da, m/z range limited to 600,0000–5,000,0000 m/z, with baseline subtraction at baseline factor 3.00, proton adduct and automatic isotope width. Expected Mw was calculated using the ExPASy ProtParam tool. For ODC and OAZ-Y-SPM, we considered full-length protein with fMet removed. For post-cleavage OAZ-Y-D or SPM, the expected molecular weight of a corresponding protein was considered . For the expected molecular weight of an ODC:OAZ-Y-D conjugate, masses for each protein were added and the mass of water (−18.01 Da) was subtracted.For intact protein MS, we used a RapidFire 365 platform (Agilent) comprising a jet-stream electrospray ionization source coupled to a 6550 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) (Agilent) detector. 30 µM ODC was incubated with 10 µM OAZ-Y-SPM and 10 mM CaClConjugated protein samples were resolved on SDS-PAGE to separate different conjugate species. Bands were cut from the gel. For in-gel tryptic digest, gel slices were fragmented and transferred to LoBind tubes (Eppendorf). Gel pieces were covered with 50% (v/v) acetonitrile (ACN) + 50% (v/v) 100 mM ammonium bicarbonate and destained at 37 °C. Supernatant was removed and samples were reduced with 10 mM TCEP in 100 mM ammonium bicarbonate for 30 min at 25 °C, before drying the gel slices with ACN. Supernatant was removed and samples were incubated for 30 min at 25 °C with 50 mM 2-chloroacetamide in 100 mM ammonium bicarbonate in the dark (leading to carbamidomethylation of cysteines). Supernatant was removed, gel slices were washed twice with ACN and rehydrated with Sequencing Grade Modified Trypsin  at 100 ng trypsin per band. After overnight digestion, supernatant was collected in LoBind tubes and tryptic digestion inhibited upon wash of gel pieces with 10% (v/v) formic acid. The supernatant was combined with the previous supernatant. Finally, remaining (crosslinked) peptides were extracted from the gel pieces with ACN. After transfer to a new LoBind tube, the solvent was removed by vacuum evaporation. Extracted peptides were resuspended with 5% (v/v) formic acid and 5% (v/v) dimethyl sulfoxide (DMSO) in water and combined with the previous supernatant. Samples were stored at 4 °C or frozen at −20 °C until analysis.53 1000 ultra-high-performance liquid chromatography (UHPLC) system (Proxeon) and electrosprayed directly into a Q Exactive mass spectrometer (Thermo Fisher). Peptides were trapped on a C18 PepMap100 pre-column  using solvent A [0.1% (v/v) formic acid in water] at 500 bar and then separated on an in-house packed analytical column  with a linear gradient from 10 to 55% (v/v) solvent B [0.1% (v/v) formic acid in ACN] in 45 min at 200 nL/min. Full scan MS spectra were acquired in the Orbitrap . After the MS scans, the 10 most intense peaks were selected for Higher-energy collisional dissociation (HCD) fragmentation at 30% of normalized collision energy. HCD spectra were also acquired in the Orbitrap  with first fixed mass at 100 m/z. Charge states 1+ and 2+ were excluded from HCD fragmentation.Peptides were separated on an EASY-nLC54. The database contained the target proteins and common contaminants. Search parameters were as follows: maximum number of missed cleavages = 2, fixed modification = carbamidomethyl-Cys, variable modification 1 = Oxidation-Met, variable modification 2 = Glu to pyro-Glu. Crosslinking from D to K, S, T, or N-terminus was considered. Data were initially filtered to a False-discovery rate (FDR) of 1%. Crosslinks were further filtered/inspected with specific emphasis on fragmentation patterns. Data were deposited in the ProteomeXchange Consortium via the PRIDE55 partner repository with the dataset identifier PXD023073.MS data were converted into Mascot generic format (mgf) using pParse and searched using the pLink softwarew 3,000 Da)  were added to each well at varying final concentrations, dissolved in 10% (v/v) DMSO in HBS with 10 mM CaCl2. The buffer control consisted of 10% (v/v) DMSO in HBS with 10 mM CaCl2. The biotinylated ligands were incubated at 37 °C for 0.5 h, shaking at 300 rpm. Excess unbound ligands were removed by washing the plate 6 times with 400 µL PBS-T. 100 µL Pierce High Sensitivity Streptavidin-HRP (Thermo Fisher) dissolved 1:5,000 in 3% (w/v) BSA in PBS-T was added to each well and incubated at 25 °C for 1 h. The ELISA plate was washed 6 times with 400 µL PBS-T. 100 µL 1-Step Ultra 3,3’,5,5’-Tetramethylbenzidine (TMB) ELISA substrate (Thermo Fisher) was added to each well for 1 min, before the reaction was stopped by addition of 100 µL 1 M HCl. Absorbance at 450 nm was read using the FLUOstar Omega microplate plate reader (BMG Labtech).A Nunc MaxiSorp 96-well flat-bottom ELISA plate (Thermo Fisher) was coated with 100 µL per well of SpyCatcher003-sfGFP at 2 µg/mL in PBS pH 7.4  and incubated at 4 °C for 16–18 h. Excess unbound SpyCatcher003-sfGFP was removed by washing the plate 6 times with 400 µL PBS-T each time. Blocking was done by incubating each well with 200 µL 3% (w/v) BSA in PBS-T at 25 °C for 1 h. After another wash in PBS-T, 100 µL SpyTag-Y-SPM at 2 µg/ml in HBS was added to each well and incubated at 25 °C for 30 min. Unbound SpyTag-Y-SPM was removed by washing the plate 6 times with 400 µL PBS-T. Biotin pentyldiamine , biocytinamide (Merck), biotin-PEG-alkoxyamine , biotin tyramide (Merck) and biotin-PEG-SH  for 20 h at 0.14 mbar and −72.5 °C. The lyophilized OAZ-GSY-SPM was then reconstituted in 100 μL MilliQ water and centrifuged at 16,900 g for 10 min. After the removal of aggregates, the supernatant was used to test for coupling to ODC by SDS-PAGE with Coomassie staining.2 in 50 mM HEPES, 150 mM NaCl, pH 7.4 at 37 °C. After 5 h, samples were incubated with 1× Glycoprotein Denaturing Buffer . Samples were heated for 10 min at 100 °C. Subsequently, Glycoprotein Buffer 2 (NEB) was added to 1×, NP-10 (NEB) to 10% (v/v), and PNGase F (NEB) to 25,000 U/mL. Samples were digested for 1 h at 37 °C. Finally, SDS-loading buffer was added. Samples were heated to 95 °C for 6 min and resolved on SDS-PAGE, before Coomassie staining.2.5 µM sEGFR was incubated with 12.5 µM TGFα-GSY-SPM and 2 mM CaCl2. For cell staining, sub-confluent A431 were seeded at 2 × 104 cells/cm2 onto glass-bottom petri dishes (MatTek). Cells were grown for 24 h, washed twice with serum-free medium (DMEM supplemented with 1× penicillin/1× streptomycin and 1× GlutaMAX) and then maintained for an additional 16–18 h at 37 °C with 5% (v/v) CO2 The dishes were transferred to 4 °C to prevent receptor internalization and cells were washed twice with cold 1 mL HBS-Mg . Cells were incubated at 4 °C with 5 µM TGFα-GSY-SPM  in HBS-Mg supplemented with 1% (w/v) BSA  or only HBS-Mg with BSA as indicated. After 1 h, cells were washed twice with HBS-Mg. Subsequently, samples were incubated in the dark with 1 mL Anti-His-Phycoerythrin (BioLegend 362603) at 1:100 in HBS-Mg for 1 h at 4 °C. Cells were washed thrice and then covered with 1 mL HBS-Mg. Samples were imaged with a DeltaVision core inverted wide-field microscope with softWoRx software (Applied Precision and Micron Oxford), using brightfield or an mCherry filter for fluorescence imaging of PE . All fluorescence images were collected and analyzed using the same settings.A431 cells were from Cancer Research UK, Lincoln’s Inn Fields. Cells were cultured in complete media  at 37 °C, with 5% (v/v) CO5 A431 cells per well were seeded into 6-well plates, grown at 37 °C with 5% (v/v) CO2 in complete media for 24 h, washed twice in 4 mL serum-free medium, and subsequently starved with serum-free medium for an additional 24 h at 37 °C with 5% (v/v) CO2. Prior to cell conjugation, cells were washed twice with 2 mL HBS-Mg. 150 µL TGFα-GSY-SPM (wt or DA) diluted to 2 µM in HBS-Mg with or without 5 mM hydroxylamine (Sigma-Aldrich) [final concentration 2% (v/v) DMSO] in HBS-Mg was added to cells. Rapidly thereafter, 150 µL HBS-Mg with 4 mM CaCl2 with or without 5 mM hydroxylamine [final concentration 2% (v/v) DMSO] was added to the cells, without prior removal of the protein solution. Cells were incubated for an additional 10 min, during which time they were placed in an incubator at 37 °C with 5% (v/v) CO2. The supernatant was removed and cells were incubated for the indicated time with serum-free medium with or without 5 mM hydroxylamine [final concentration 2% (v/v) DMSO] at 37 °C with 5% (v/v) CO2. Cells were washed twice with HBS-Mg. For cell lysis, 150 µL ice-cold modified RIPA buffer , supplemented with cOmplete mini EDTA-free protease inhibitor cocktail, 1 mM PMSF and 1 mM sodium orthovanadate was added to each well. Lysates were incubated on ice for 20 min, before centrifuging at 12,000 g at 4 °C for 20 min. The supernatant was mixed with 6× SDS loading buffer containing 120 mM dithiothreitol and heated for 6 min at 95 °C, before resolving on SDS-PAGE.In total 5 × 10For Western blotting with anti-TGFα and anti-EGFR, proteins were transferred from SDS-PAGE to nitrocellulose membrane (Bio-Rad) in transfer buffer  for 16 h at 30 V at 4 °C. Membranes were blocked for 1 h at 25 °C with 5% (w/v) skim milk (Sigma-Aldrich) in PBS-T. Subsequently, membranes were incubated for 20 h at 4 °C with primary antibodies at 1:1,000 in 5% (w/v) skim milk in PBS-T, i.e., 0.2 µg/mL mouse anti-TGFα  or mouse anti-EGFR . Membranes were washed 6 times with PBS-T before addition of goat anti-mouse horseradish peroxidase (HRP)  at 1:1,000 in 5% (w/v) skim milk with PBS-T. After incubation at 25 °C for 1 h, membranes were washed 6 times with PBS-T.For Western blotting with anti-pSTAT1 and anti-GAPDH , proteins were transferred from SDS-PAGE to nitrocellulose (Bio-Rad) in transfer buffer for 3 h at 30 V at 25 °C. Membranes were blocked for 1 h at 25 °C with 5% (w/v) BSA (Sigma-Aldrich) in PBS-T. Subsequently, membranes were incubated for 20 h at 4 °C with primary antibodies at 1:1,000 in 5% (w/v) BSA in PBS-T, i.e., rabbit anti-pSTAT1(Y701)  or mouse anti-GAPDH . Membranes were washed 6 times with PBS-T before addition of goat anti-rabbit horseradish peroxidase (HRP)  at 1:1,000 in 5% (w/v) BSA with PBS-T for anti-pSTAT1(Y701) or goat anti-mouse horseradish peroxidase (HRP)  at 1:1,000 in 5% (w/v) BSA with PBS-T for anti-GAPDH. After incubation at 25 °C for 1 h, membranes were washed 6 times with PBS-T. Membranes were incubated with SuperSignal West Pico PLUS Chemiluminescent Substrate before measuring chemiluminescence on a ChemiDoc XRS imager using Image Lab Software version 5.2.1 (Bio-Rad).24, OAZ/AZI from PDB 4zgz24, and TGFα/EGFR from PDB 1mox47. Structures were visualized using PyMOL version 2.2.0 (Schrödinger). Distances shown were drawn from atom C of the C-terminal resolved residue to atom Nε of lysine. Figures were prepared using the FIJI distribution of ImageJ56 and the open-source graphics editor Inkscape (inkscape.org). In Supplementary Fig. 20 and the secondary structure of SPM591-657 was predicted by the Jpred4 server26. FrpC414–657 was subject to Ginzu Domain Prediction using the Robetta server25. Figure www.flaticon.com. Chemical structures were prepared in ChemDraw.The structure of OAZ/ODC was obtained from PDB 4zgya values were predicted using the ROSIE Rosetta server38 based on the ODC/OAZ crystal structure (PDB 4zgy). Values were derived from a single run using the pKa app with re-packing of neighbor residues at a pack radius of 6.0 Å and no pre-packing.pKd values are representative of two separate experiments. For Supplementary Fig. For kinetic analysis of protein conjugation or processing, replicate samples refer to multiple analyses from the same protein preparation, after which gels were processed and resolved in parallel. For representative SDS-PAGE Fig. , observaFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupp Data 1Supp Data 2Reporting Summary


In the crystal, C—H⋯O interactions link the molecules into infinite columns along the b-axis direction. Parallel columns interact by additional C—H⋯O hydrogen bonds.In the title compound, C 18H16N2O3S, was synthesized by reaction of 2-mercapto-3-phenylquinazolin-4(3H)-one with ethyl chloroacetate. The quinazoline ring forms a dihedral angle of 86.83 (5)° with the phenyl ring. The terminal methyl group is disordered by a rotation of about 60° in a 0.531 (13): 0.469 (13) ratio. In the crystal, C—H⋯O hydrogen-bonding interactions result in the formation of columns running in the [010] direction. Two parallel columns further interact by C—H⋯O hydrogen bonds. The most important contributions to the surface contacts are from H⋯H (48.4%), C⋯H/H⋯C (21.5%) and O⋯H/H⋯O (18.7%) interactions, as concluded from a Hirshfeld analysis.The title compound, C In the IR spectrum of (4), the disappearance of the NH stretching and the presence of a strong C=O absorption at 1732 cm−1 indicate the existence of an ester compound. In the 1H NMR spectrum of (4), the signal at δ 13.05 ppm disappears and three new signals in the aliphatic area  are consistent with the presence of the –CH2COOCH2CH3 moiety in (4).As shown in Fig. 1As no X-ray crystallographic information is available for this ester, we have determined the crystal structure by single-crystal X-ray diffraction and a Hirshfeld surface analysis has been performed to gain further insight into the intermolecular interactions.P21/n with four molecules in the unit cell. The asymmetric unit of the title compound is illustrated in Fig. 2B⋯O14 contact is observed .The title compound crystallizes in the space group 3) may exist in the thione form, namely 3-phenyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one. Therefore, it could react with ethyl chloroacetate to give ethyl 2--yl)acetate as illustrated in Fig. 34), which proves that in the alkaline environment, (3) converts into the thiolate form and then reacts with ethyl chloroacetate to yield the title compound (4).Theoretically, compound  x, y + 1, z]. Two parallel columns interact via C7—H7⋯O23i hydrogen-bonding interactions [symmetry code: (i) −x + y − z + The crystal packing is mainly characterized by C—H⋯O hydrogen-bonding interactions Table 1 and 5 ▸.CrystalExplorer  Å]. Small faint-red spots appear near atoms H8 and H17E are due to a short H8⋯H17E contact (2.352 Å). The S11⋯H23 contact mentioned is only visible as a white spot, while a white region above the C18–C23 phenyl ring is present because of the proximity of atom H20. The distance between H20 and the centroid of this phenyl ring of 3.204 Å, however, is too long for a C—H⋯π interaction. The fingerprint plots  and MUDNAC  were recorded on FTIR-8400S-SHIMADZU spectrometer using KBr pellets. The NMR spectra were recorded on a Bruker Avance III spectrometer (500 MHz for 1H NMR) using residual solvent DMSO-d6 signals as internal reference. The spin–spin coupling constants (J) are given in Hz. Peak multiplicity is reported as s (singlet), d (doublet), dd (doublet-doublet), t (triplet), q (quartet), m (multiplet). The synthetic protocol for title compound (4) is shown in Fig. 1et al., 2019Anthranilic acid, phenyl isothiocyanate and ethyl chloroacetate were purchased from Acros and used without purification. Melting points were measured in open capillary tubes on a Gallenkamp melting point apparatus. IR spectra :2) (0.1 mol) was added to the solution of anthranilic acid (1) (0.1 mol) and triethylamine (3.0 mL) in absolute ethanol (200 mL). The reaction mixture then was refluxed for 4 h. After cooling to room temperature, the reaction mixture was poured into cold water. The resulting solid was filtered and recrystallized from a mixture of DMF and water, then washed with cold ethanol to give the product (3). M.p. 569 K; yield 80%. IR : 3217, 3134 (N—H), 3028 (C—H aromatic), 1659 (C=O), 1618, 1524, 1485 . 1H NMR : 13.05 , 7.96 , 7.80 , 7.50–7.40 , 7.42 , 7.36 , 7.29 .Phenyl isothiocyanate  sulfanyl]acetate (4):3) (20 mmol) and anhydrous potassium carbonate (20 mmol) in dry DMF (30 mL) was stirred for 30 min, ethyl chloroacetate (20 mmol) was then added. After refluxing for 5 h, the reaction mixture was cooled to room temperature and poured into ice-cold water. The white precipitate was filtered off and recrystallized from ethanol to afford crystals of (4). Colourless crystals, m.p. 485 K, yield 65%. IR : 3059 (C—H aromatic), 2976, 2906 , 1732 (C=O ester), 1680 (C=O ketone), 1607, 1598, 1468 . 1H NMR : 8.09 , 7.84 , 7.61-7.48 , 4.15 , 3.99 , 1.23 .A mixture of (Uiso(H) values of 1.2Ueq or 1.5Ueq of the parent atoms, with C—H distances of 0.93 (aromatic), 0.97 (CH2) and 0.96 Å (CH3). In the final cycles of refinement, two outliers were omitted.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020005071/dj2002sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020005071/dj2002Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020005071/dj2002Isup3.cmlSupporting information file. DOI: 1996127CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Splenic red pulp macrophages (RPMs) contribute to erythrocyte homeostasis and are required for iron recycling. Heme induces the expression of SPIC transcription factor in monocyte-derived macrophages and promotes their differentiation into RPM precursors, pre-RPMs. However, the requirements for differentiation into mature RPMs remain unknown. Here, we have demonstrated that interleukin (IL)-33 associated with erythrocytes and co-cooperated with heme to promote the generation of mature RPMs through activation of the MyD88 adaptor protein and ERK1/2 kinases downstream of the IL-33 receptor, IL1RL1. IL-33- and IL1RL1-deficient mice showed defective iron recycling and increased splenic iron deposition. Gene expression and chromatin accessibility studies revealed a role for GATA transcription factors downstream of IL-33 signaling during the development of pre-RPMs that retained full potential to differentiate into RPMs. Thus, IL-33 instructs the development of RPMs as a response to physiological erythrocyte damage with important implications to iron recycling and iron homeostasis.  •IL-33 signaling promotes development of monocyte-derived red pulp macrophages (RPMs)•Il33−/− and Il1rl1−/− mice have decreased RPMs and splenic erythrophagocytosis•ERK activation is required for RPM development and is potentiated by hemin and IL-33•Il1rl1−/− RPM precursorsGATA2 instructs RPM development and is aberrant in  Splenic red pulp macrophages (RPMs) are critical for iron recycling. Here, Lu et al. implicate a role for IL-33 signaling in the regulation of GATA2, which controls the transition of monocytes to produce pre-RPMs that are competent to terminally differentiate into mature RPMs. P. Chabaudi) and are able to cross-prime early effector T cell responses against viruses, and may therefore play a role in the defense against infections  are Spic-EGFPhi, whereas splenic pre-RPMs (CD11bhi F4/80lo) are Spic-EGFPlo/int (hi F4/80−) express little SPIC (not shown). This was further confirmed by gene expression analysis on sorted populations of splenic monocytes, pre-RPMs, and RPMs, which revealed intermediate expression of RPM-associated genes in pre-RPMs compared to monocytes and RPMs  in comparison with mice that received murine IgG1  f IL1RL1 A and S4Bf IL1RL1 A, we detf IL1RL1 B and a 3f IL1RL1 C. Substaf IL1RL1 D. In casf IL1RL1 . We founf IL1RL1 E, suggesf IL1RL1 C. Howevef IL1RL1 C. Taken Myd88−/− mice displayed reduced numbers of RPMs  at steady state and did not increase their RPMs after in vivo administration of IL-33 and hemin  number compared to vehicle-treated mice  compared to control animals  generated by the same source, to fulfil its homeostatic function. This pathway is essential for the normal regulation of iron recycling. Similarly to its role as an alarmin in initiating immune responses during tissue damage, IL-33 appears to act as an alarmin during physiological damage of senescent erythrocytes, thereby instructing the differentiation of a specific subset of tissue-resident macrophages involved in the clearance of senescent erythrocytes and the recycling of their iron content. Beyond its role in iron homeostasis and the response to erythrocyte damage, IL-33 might also be involved in other immunological properties of RPMs, including the response to infections .zm255@medschl.cam.ac.uk. Methods, including statements of data availability and any associated additional references are available in the online version of the paper.Lead author: Ziad Mallat, Spicigfp/igfp reporter mice were kindly provided by Kenneth M. Murphy , BALB/c background of Il1rl1−/− and Il33−/− were from Andrew McKenzie and C57/Bl6 background of Il1rl1−/− were from Padraic Fallon, C57/Bl6 Myd88−/− mice were from Bernhard Ryffel and C57/Bl6 CD45.1 mice were originally from Jackson Labs. For the hemin (500 μg/200 μl) and IL-33 (1 μg/200 μl) in vivo experiment, the mice were injected intra-peritoneally once a day for 3 days. Control mice received PBS (200 μl) injections. For the ERK1/2 in vivo experiment, C57/Bl6 Spicigfp/igfp reporter mice were treated with the MEK inhibitor U0126 or DMSO control 3 times per week for 6 weeks. U0126 solution was prepared in DMSO as previously described  of 2x109 erythrocytes from either WT or Il33−/− mice.To reconstitute 4OH and stored at −20°C. Hemin was used at a final concentration of 40uM for cell culture experiments and 500 μg/200 μL sterile 0.15M NaCl for i.p. injections in vivo, as previously described , and a stock solution was prepared at 25mg/mL in 0.15M NaCl containing 10% NHescribed .7 bone marrow cells obtained from CD45.2 WT or Il1rl1−/− mice. Donor-derived cells in spleen or liver (CD45.2+) were analyzed 10 weeks after transfer using flow cytometry. In other experiments, CD45.2 mice were lethally irradiated and injected intravenously (i.v.) with 107 bone marrow cells containing 50% of CD45.2 WT or Il1rl1−/− mice and 50% of CD45.1 mice.CD45.1 mice were maintained overnight with Baytril before irradiation with two doses of 5.5 Gy (separated by 4 h) followed by reconstitution with 10Samples of the spleen (20-60 mg) and liver (40-90 mg) were digested (1 in 10) in 16% nitric acid, prepared by diluting a stock solution of high purity nitric acid  with ultra-high purity water, in acid-cleaned 15 mL PTFE vials in an UltraWave Single Reaction Chamber Microwave Digestion System . The following digestion conditions were followed: 5 min ramp to 120°C, then 10 min ramp from 120°C to 230°C and maintained at 230°C for 15 min. Blank samples, containing just the acid mixture, were similarly prepared to check for background Fe contributions.Digested samples were analyzed for total Fe by inductively coupled plasma optical emission spectrometry , equipped with a concentric micro-nebulizer and cyclonic spray chamber. A sample introduction pump speed of 10 rates/min, nebulizer flow rate of 0.72 mL/min and a plasma gas flow rate of 10 L/min was used with low-flow sample tubing. The 259.940 nm analytical line for Fe was used. All samples, including blank samples, were analyzed in a single batch with acid-based Fe standards (0-40 mg/L). Each sample was analyzed in triplicate and the average value was used. For serum measurements, 20 μl of serum was incubated with 20 μl of acid reagent for 5 min. Supernatant was mixed with 40 μl chromagen reagent, and absorbance at 535 nm was measured as described above.For Perl’s Prussian blue stain, tissues were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0), embedded in paraffin, and stained with Perl’s Prussian blue and pararosaniline (Sigma).2 atmosphere. Four days after seeding, an extra 10 mL of fresh differentiation media were added per plate and incubated for an additional 3 days.Fresh bone marrow cells were used to generate BMDM. Cells were resuspended in 10 mL bone marrow differentiation media , 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine and 20ng/mL of M-CSF(BioLegend)). Cells were seeded in Petri dishes (Corning Brand) and incubated at 37°C in a 5% COSingle-cell suspensions of bone marrow, spleen and liver were incubated with Fc block solution , dilution of 1:200 in flow buffer  for 10 min at 4°C. Cells were then stained with fluorophore-conjugated antibodies, dilution of 1:200 in flow buffer for 30 min at 4°C, prior to extensive wash and analyzed using an LSRII Fortessa (BD) flow cytometer. The following antibodies were used in the experiments, Zombie Aqua Fixable Viability Kit, Alexa Flour 700 anti-mouse CD11b (BD Bioscience MI170), PE/Cy7 anti-mouse CD11c (BioLegend N418), Brilliant Violet 421 anti-mouse F4/80 (BioLegend BM8), APC-eFluor 780 anti-mouse Ly6C (eBioscience HK1.4), PerCP/Cy5.5 anti-mouse Ly-6G (BioLegend 1A8), Brilliant Violet 650 anti-mouse NK-1.1 (BioLegend PK136), Brilliant Violet 605 anti-mouse CD335 (BioLegend 29A1.4), FITC anti-mouse CD169 (BioLegend 3D6.112), APC anti-mouse CD209b (eBioscience eBio22D1), FITC anti-mouse T1/IL1RL1 (ST2) (mdbioproducts DJ8), FITC anti-mouse Ter119 (BioLegend Ter119), PE anti-mouse CD71 (BioLegend RI7217), APC anti-mouse DYKDDDDK (BioLegend L5), Brilliant Violet 570 anti-mouse CD45.1 (BioLegend A20) and Brilliant Violet 785 anti-mouse CD45.2 (BioLegend 104), APC anti-mouse CD45 (BioLegend 30-F11). Cell analysis was done using BD FACSDiva v8.01 Software and figure displayed dot plots and histograms were obtained using FlowJo v10.5 software .We used MesoScale Discovery IL-33 V-Plex assay kit (product code K152XBD-1) and human IL-33 Duoset ELISA kit  according to manufacturer’s instructions. Measurements were performed blindly by the Core Biochemical Assay lab at Addenbrookes Hospital, the University of Cambridge, Cambridge, UK.SPIC forward 5′-ACGGTAATTAACAGTGCTGCG-3′ and reverse 5′-GCTGGAGAAGAGTGGGTTGT-3′, human HMOX1 forward 5′-TAGAAGAGGCCAAGACTGCG-3′ and reverse 5′- GGGCAGAATCTTGCACTTTGTT-3′, human TREML4 forward 5′- AGACCAGGAAATCAAGAGCCC-3′ and reverse 5′-AAACCTCGTCACTGCTGTCC-3′, human VCAM1 forward 5′-GTTTGCAGCTTCTCAAGCTTTT-3′ and reverse 5′-AGATGTGGTCCCCTCATTCG −3′, human LCN2 forward 5′-GCCCTGAAATCATGCCCCTA-3′ and reverse 5′-TCCCCTGGAATTGGTTGTCC-3′, human BACH1 forward 5′-TCGCGTAAGAAAAGCCGAG-3′ and reverse 5′-CATCAACCATATTGTGCGAGGC-3′, human IL1RL1 forward 5′- CCCTCTGTCTTTCAGTTTGGTTGA-3′ and reverse 5′- ACGACAGTGAAGGTCACCAC-3′. Mouse Spic forward 5′-ACTGGAGAGGTGTAACAAATGGT-3′ and reverse 5′-CAAACAGCCGAAGCTTTCTCC-3′, mouse Treml4 forward 5′-AAGCACAGCCACCATCTTTATG-3′ and reverse 5′-GCACACAGAAAACTGACAGCA-3′, mouse Bach1 forward 5′-CTCTGAGACGGACACGGAAG-3′ and reverse 5′-CCTTCTGCGGATGTCATGGA-3′, mouse Itgam (Cd11b) forward 5′-GTGAGGTCTAAGACAGAGACCAA-3′ and reverse 5′-TGCCGCTTGAAAAAGCCAAG-3′, mouse Hmox1 forward, 5′-GCTAGCCTGGTGCAAGATACT-3′ and reverse 5′-TGGGGGCCAGTATTGCATTT-3′, mouse Il1rl1 forward 5′-CCAGCCCTTCATCTGGGTATC-3′ and reverse 5′-TGGCAATGGCACAGGATAGT-3′, mouse Lcn2 forward 5′-CTGTCCCAATCGACCAGTGT-3′ and reverse 5′-CCAGCTCCCTCAATGGTGTT-3′, mouse Adgre1 (F4/80) forward 5′-AATCGCTGCTGGTTGAATACAG-3′ and reverse 5′-CCAGGCAAGGAGGACAGAGTT-3′, mouse Vcam1 forward 5′-CCGGCATATACGAGTGTGAA-3′ and reverse 5′-GATGCGCAGTAGAGTGCAAG-3′.For gene expression analysis, RNA from sorted RPM, Pre-RPM and Monocytes were isolated using an RNAeasy mini kit (QIAGEN). RT-PCR was performed using a QuantiTect Reverse Transcription kit (QIAGEN). Real-time PCR was performed on 5 μL cDNA product (diluted 10 to 20 times) using SYBR Green qPCR mix (Eurogentec) on a Roche Lightcycler. Primer sequences are: human These experiments were independently repeated three times and each treatment consisted of triplicate samples.10/200 μL for intravenous injection. 24 h after i.v. injection, spleens were collected and single-cell suspensions were prepared by mechanical disruption. Following incubation in Fc block, splenocytes were labeled with antibodies against Ly6G, CD11b and F4/80 and analyzed by flow cytometry (Fortessa) for PKH26+ cells in splenic monocytes (CD11bhi F4/80-) or pre-RPM (CD11bhi F4/80lo) or RPM (CD11blo/- F4/80hi) or neutrophil (Ly6GhiCD11b+) population.Anti-mouse CD47 Fab antibody treated RBCs were stained with PKH26 (Sigma) according to the manufacturer’s protocol. Briefly, 250 μL of RBCs were mixed with 2.25 mL diluent C from the PKH26 cell linker kit and incubated with 2 μM PKH26 dye for 5 min at room temperature. The staining reaction was stopped by the addition of 2.5 mL FBS for 1 min, followed by further dilution with 5 mL PBS. Cells were then washed three times with PBS and resuspended at 1x10Human or mouse whole blood was collected in EDTA-treated blood collection tubes, followed by centrifugation at 400 g for 10 min. The RBC were transferred to new tubes and washed twice in PBS. The samples were incubated with RBC lysis buffer at room temperature for 10 min. After centrifugation at 13,000 g for 15 min at 4°C, the supernatants were passed through Amicon ultra (50 kDa) cutoff filter to remove haemoglobin (64 kDa), followed by a centrifugation at 7,000 g for 20 min at 4°C.The positive selection of human erythrocytes from fresh human whole blood was performed using anti-CD235a (Glycophorin A)-conjugated microbeads and MACS LS columns (Miltenyi Biotec) according to manufacturer’s instructions. Enriched erythrocyte preparations (2e8 cells/mL) were lysed in PBS/0.1% Triton X-100 at 4°C and lysates snap frozen on dry ice.Samples were analyzed by SDS-PAGE on NuPAGE Novex 4%–12% or 12% Bis-Tris mini gels (Invitrogen) with MOPS running buffer (Invitrogen) according to manufacturer’s instructions under reducing conditions. All samples were reduced by heating to 95°C for 3 min in SDS-PAGE buffer containing 2% beta-mercaptoethanol. Proteins were transferred to nitrocellulose membranes (Invitrogen) and detected by western blotting. IL-33 was detected with an anti-mouse IL-33 antibody (R&D systems AF3626) and anti-human IL-33 antibodies . Immunoreactive proteins were identified with HRP-conjugated secondary antibodies  and Supersignal West Femto substrate (Pierce 34095) and visualized on LI-COR cDigit.RNA was extracted from freshly sorted splenic monocytes, pre-RPM and RPM, using the RNEasy plus micro kit (QIAGEN). RNA libraries were prepared using the SMARTer stranded total RNA-Seq v2 kit (Takara) and sequenced using Illumina NovaSeq (2x50bp), obtaining approximately 20 million reads per sample.4 freshly sorted splenic monocytes, pre-RPM and RPM. Transposition reactions were performed in 50 μL using Tn5 transposase (Illumina) for 30 min at 37°C. Libraries were prepared using the recommended Nextera barcodes  with default parameters, trimmed with cutadapt 2.5 (https://pypi.org/project/cutadapt/2.5/) under Python 3.6.2 (parameters:–nextseq-trim = 20 -m 15 for the R1 reads and–nextseq-trim = 20 -m 15 -u 3 for the R2 reads) and counted using Subread-Feature Counts (v1.6.2) (https://sourceforge.net/projects/subread/files/subread-1.6.2/). DGE analysis was performed using DESeq2 (v1.25.9) (http://bioconductor.org/packages/release/bioc/html/DESeq2.html). Heatmaps were generated using the heatmap.2 function from the R Package gplots version 3.0.1.1 (https://cran.r-project.org/src/contrib/Archive/gplots/). Lists of differentially expressed genes were submitted to Pathway analysis using Ingenuity Systems Pathway analysis software (QIAGEN).Reads were mapped to Ensembl/GRCm38.95 using STAR (v2.5.1)  with a quality Phred score cutoff of 20 and a minimum required sequence length for both reads before a sequence pair gets removed set to 20 bp. Trimmed reads were mapped to Ensembl/GRCm38.95 using Bowtie2 (https://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.3.5.1/) with standard parameters. Peaks were called using Genrich (https://github.com/jsh58/Genrich#quick) and excluding mitochondrial reads. Differential peak analysis was performed using BEDtools 2.29.0 (https://github.com/arq5x/bedtools2/releases/tag/v2.29.0). Motif enrichment was performed using Meme-Chip . The accession number for the sequencing data reported in this paper is GEO: GSE146782. All other relevant data and scripts are available on request.