RESUMO
Recombinant Francisella tularensis universal stress protein with a C-terminal histidine-tag (rUsp/His6) was expressed in Escherichia coli. Endogenous F. tularensis Usp has a predicted molecular mass of 30 kDa, but rUsp/His6 had an apparent molecular weight of 33 kDa based on Western blot analyses. To determine the source of the higher molecular weight for rUsp/His6, post translational modifications were examined. Tryptic peptides of purified rUsp/His6 were subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) and fragmentation spectra were searched for acetylated lysines and polyaminated glutamines. Of the 24 lysines in rUsp/His6, 10 were acetylated (K63, K68, K72, K129, K175, K201, K208, K212, K233, and K238) and three of the four glutamines had putrescine, spermidine and spermine adducts (Q55, Q60 and Q267). The level of post-translational modification was substoichiometric, eliminating the possibility that these modifications were the sole contributor to the 3 kDa extra mass of rUsp/His6. LC-MS/MS revealed that stop codon readthrough had occurred resulting in the unexpected addition of 20 extra amino acids at the C-terminus of rUsp/His6, after the histidine tag. Further, the finding of polyaminated glutamines in rUsp/His6 indicated that E. coli is capable of transglutaminase activity.
Assuntos
Proteínas de Bactérias , Códon de Terminação , Escherichia coli , Francisella tularensis , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Acetilação , Códon de Terminação/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Francisella tularensis/genética , Francisella tularensis/metabolismo , Espectrometria de Massas em Tandem , Histidina/metabolismo , Sequência de AminoácidosRESUMO
Bacterial and mammalian cells are rich in putrescine, spermidine, and spermine. Polyamines are required for optimum fitness, but the biological function of these small aliphatic compounds has only been partially revealed. Known functions of polyamines include interaction with nucleic acids that alters gene expression and with proteins that modulate activity. Although polyamines can be incorporated into proteins, very few naturally occurring polyaminated proteins have been identified, which is due in part to the difficulty in detecting these adducts. In the current study, bovine albumin and the recombinant universal stress protein from Francisella tularensis were used as models for mass spectrometry analysis of polyaminated proteins. The proteins were covalently bound to putrescine, spermidine, or spermine by the action of carbodiimide or microbial transglutaminase. Tryptic peptides, subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS), were identified using Protein Prospector software. We describe the search parameters for identifying polyaminated peptides and show MS/MS spectra for adducts with putrescine, spermidine, and spermine. Manual evaluation led us to recognize signature ions for polyamine adducts on aspartate, glutamate, and glutamine, as well as neutral loss from putrescine, spermidine, and spermine during the fragmentation process. Mechanisms for the formation of signature ions and neutral loss are presented. Manual evaluation identified a false-positive adduct that had formed during trypsinolysis and resulted in peptide sequence rearrangement. Another false positive initially appeared to be a 71 kDa putrescine adduct on a cysteine residue. However, it was an acrylamide adduct on cysteine for a sample extracted from a polyacrylamide gel. The information presented in this report provides guidance and serves as a model for identifying naturally occurring polyaminated proteins.
RESUMO
Butyrylcholinesterase (BChE) is present in plasma and numerous cells and organs. Its physiological function(s) is(are) still unclear. However, this enzyme is of pharmacological and toxicological importance. It displays a broad specificity and is capable of hydrolyzing a wide range of substrates with turnovers differing by several orders of magnitude. Nowaday, these substrates include more than two dozen carboxyl-ester drugs, numerous acetylated prodrugs, and transition state analogues of acetylcholine. In addition, BChE displays a promiscuous hydrolytic activity toward amide bonds of arylacylamides, and slowly hydrolyzes carbamyl- and phosphoryl-esters. Certain pseudo-substrates like carbamates and organophosphates are major drugs of potential medical interest. The existence of a large genetic poly-allelism, affecting the catalytic properties of BChE is at the origin of clinical complications in the use of certain drugs catabolized by BChE. The number of drugs and prodrugs hydrolyzed by BChE is expected to increase in the future. However, very few quantitative data (Km, kcat) are available for most marketed drugs, and except for myorelaxants like succinylcholine and mivacurium, the impact of BChE genetic mutations on catalytic parameters has not been evaluated for most of these drugs.
Assuntos
Butirilcolinesterase , Pró-Fármacos , Humanos , Butirilcolinesterase/genética , Succinilcolina/farmacologia , Hidrólise , MutaçãoRESUMO
The acute effects of exposure to organophosphorus toxicants are explained by inhibition of acetylcholinesterase activity. However, the mechanisms that explain long term illness associated with organophosphorus exposure are still under investigation. We find that organophosphorus nerve agents and organophosphorus pesticides make covalent adducts not only on the serine from acetylcholinesterase, but also on tyrosine, lysine, glutamate, serine and threonine from a variety of proteins. Almost any protein can be modified by a high dose of organophosphorus toxicant. A low dose of 10 µM chlorpyrifos oxon added to the serum-free culture medium of human neuroblastoma SH-SY5Y cells resulted in tyrosine adducts on 48 proteins immunopurified from the cell lysate. We identified the adducted proteins by mass spectrometry after immunopurifying modified proteins with a rabbit anti-diethoxyphospho-tyrosine monoclonal antibody which biased this study for tyrosine adducts. In cultured cells, the primary organophosphate targets are abundant proteins. Organophosphate-modified proteins may disrupt physiological processes. In separate experiments we identified organophosphate adducts on lysine. Organophosphylation activates the lysine for protein crosslinking. The activated lysine reacts with glutamic acid or aspartic acid protein side chains to form an isopeptide bond between proteins, resulting in high molecular weight crosslinked proteins. Crosslinked proteins form insoluble aggregates that may lead to neurogenerative disease.
Assuntos
Neuroblastoma , Praguicidas , Animais , Humanos , Coelhos , Acetilcolinesterase/metabolismo , Lisina/química , Compostos Organofosforados/metabolismo , Peso Molecular , Proteínas/química , Tirosina/metabolismo , Ácido Glutâmico/químicaRESUMO
Adductomics is epidemiology at the molecular level. Untargeted adductomics compares levels of chemical adducts on albumin, hemoglobin, and DNA between healthy and exposed individuals. The goal is to determine a cause-and-effect relationship between chemical exposure and illness. Chemical exposures are not necessarily due to synthetic chemicals but are often due to oxidation products of naturally occurring lipids, for example, 4-hydroxynonenal and acrolein produced by lipid peroxidation of arachidonic and linoleic acids. The preferred method used in adductomics is ultra-high pressure liquid chromatography coupled to with nanoelectrospray tandem mass spectrometry. The mass of the adduct indicates its structure and identifies the chemical. The advantages of molecular epidemiology include information about the many toxicants to which a person is exposed over a period of weeks or months and the relative exposure levels. The disadvantage is the absence of information about the mechanism of toxicity. Untargeted adductomics examines albumin and hemoglobin adducts, which serve as biomarkers of exposure but do not identify the proteins and genes responsible for the toxicity. Targeted adductomics is used when the origin of the toxicity is known. This can be either an adducted protein, such as the butyrylcholinesterase protein modified by nerve agents, or a toxicant, such as acetaminophen. Untargeted adductomics methods have identified potential protein adduct biomarkers of breast cancer, colorectal cancer, childhood leukemia, and lung cancer. Adductomics is a new research area that offers structural insights into chemical exposures and a platform for the discovery of disease biomarkers. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.
Assuntos
Butirilcolinesterase , Adutos de DNA , Humanos , Albuminas , Biomarcadores , DNA/químicaRESUMO
Protection from the toxicity of nerve agents is achieved by pretreatment with human butyrylcholinesterase (BChE). Current methods for purifying large quantities of BChE from frozen Cohn fraction IV-4 produce 99% pure enzyme, but the yield is low (21%). Our goal was to simplify the purification procedure and increase the yield. Butyrylcholinesterase was extracted from frozen Cohn fraction IV-4 in 10 volumes of water pH 6. The filtered extract was pumped onto a Hupresin affinity column. The previously utilized anion exchange chromatography step was omitted. Solvent and detergent reagents used to inactivate lipid enveloped virus, bacteria and protozoa did not bind to Hupresin. BChE was eluted with 0.1 M tetramethylammonium bromide in 20 mM sodium phosphate pH 8.0. BChE protein was concentrated on a Pellicon tangential flow filtration system and demonstrated to be highly purified by mass spectrometry. A high pump rate produced protein aggregates, but a low pump rate caused minimal turbidity. Possible contamination by prekallikrein and prekallikrein activator was examined by LC-MS/MS and by a chromogenic substrate assay for kallikrein activity. Prekallikrein and kallikrein were not detected by mass spectrometry in the 99% pure BChE. The chromogenic assay indicated kallikrein activity was less than 9 mU/mL. This new, 1-step chromatography protocol on Hupresin increased the yield of butyrylcholinesterase by 200%. The new method significantly reduces production costs by optimizing yield of 99% pure butyrylcholinesterase.
Assuntos
Butirilcolinesterase , Pré-Calicreína , Humanos , Butirilcolinesterase/química , Cromatografia Líquida , Cromatografia de Afinidade/métodos , Espectrometria de Massas em TandemRESUMO
Chronic low-dose exposure to organophosphorus (OP) toxicants is correlated with an increase in the risk of impaired cognition and neurodegenerative diseases. A mechanism to explain this relationship is needed. We suggest that the formation of organophosphate-induced high-molecular-weight protein aggregates that disrupt cell function may be the missing link. It has been demonstrated that such aggregation can be promoted by OP-labeled lysine. Alternatively, OP-labeled glutamate may be the initiator. To test this hypothesis, we treated MAP-rich tubulin Sus scrofa and human transglutaminase with chlorpyrifos oxon. Trypsin-digested proteins were subjected to liquid chromatography-tandem mass spectrometry followed by Protein Prospector searches to identify diethyl phosphate adducts and cross-linked peptides. We report the presence of diethyl phosphate adducts on the side chains of glutamate, lysine, and tyrosine, as well as cross-links between glutamate and lysine. Glutamate-lysine cross-linking could be initiated either by diethyl phosphate-activated glutamate or by diethyl phosphate-activated lysine to form stable isopeptide bonds between and within proteins. It was concluded that organophosphate-induced high-molecular-weight protein aggregates could promote brain dysfunction.
Assuntos
Clorpirifos , Lisina , Humanos , Lisina/química , Ácido Glutâmico , Agregados Proteicos , Proteínas/química , Clorpirifos/químicaRESUMO
Exposure to organophosphorus pesticides (OP) can have chronic adverse effects that are independent of inhibition of acetylcholinesterase, the classic target for acute OP toxicity. In pure proteins, the organophosphorus pesticide chlorpyrifos oxon induces a cross-link between lysine and glutamate (or aspartate) with loss of water. Tubulin is particularly sensitive to OP-induced cross-linking. Our goal was to explore OP-induced cross-linking in a complex protein sample, MAP-rich tubulin from Sus scrofa and to test 8 OP for their capacity to promote isopeptide cross-linking. We treated 100 µg of MAP-rich tubulin with 100 µM chlorpyrifos, chlorpyrifos oxon, methamidophos, paraoxon, diazinon, diazoxon, monocrotophos, or dichlorvos. Each sample was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue. Five gel slices (at about 30, 50, 150, and 300 kDa, and the top of the separating gel) were removed from the lanes for each of the eight OP samples and from untreated control lanes. These gel slices were subjected to in-gel trypsin digestion. MSMS fragmentation spectra of the tryptic peptides were examined for isopeptide cross-links. Sixteen spectra yielded convincing evidence for isopeptide cross-linked peptides. Ten were from the chlorpyrifos oxon reaction, 1 from dichlorvos, 1 from paraoxon, 1 from diazinon, and 3 from diazoxon. It was concluded that catalysis of protein cross-linking is a general property of organophosphorus pesticides and pesticide metabolites. Data are available via ProteomeXchange with identifier PXD034529.
Assuntos
Clorpirifos , Monocrotofós , Praguicidas , Acetilcolinesterase/metabolismo , Ácido Aspártico , Clorpirifos/análogos & derivados , Clorpirifos/química , Diazinon , Diclorvós , Glutamatos , Lisina/química , Compostos Organofosforados/química , Paraoxon/metabolismo , Peptídeos/química , Praguicidas/toxicidade , Dodecilsulfato de Sódio , Tripsina , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , ÁguaRESUMO
Chlorpyrifos oxon catalyzes the crosslinking of proteins via an isopeptide bond between lysine and glutamic acid or aspartic acid in studies with purified proteins. Our goal was to determine the crosslinking activity of the organophosphorus pesticide, dichlorvos. We developed a protocol for examining crosslinks in a complex protein mixture consisting of human SH-SY5Y cells exposed to 10 µM dichlorvos. The steps in our protocol included immunopurification of crosslinked peptides by binding to anti-isopeptide antibody 81D1C2, stringent washing of the immobilized complex, release of bound peptides from Protein G agarose with 50% acetonitrile 1% formic acid, liquid chromatography tandem mass spectrometry on an Orbitrap Fusion Lumos mass spectrometer, Protein Prospector searches of mass spectrometry data, and manual evaluation of candidate crosslinked dipeptides. We report a low quantity of dichlorvos-induced KD and KE crosslinked proteins in human SH-SY5Y cells exposed to dichlorvos. Cells not treated with dichlorvos had no detectable KD and KE crosslinked proteins. Proteins in the crosslink were low abundance proteins. In conclusion, we provide a protocol for testing complex protein mixtures for the presence of crosslinked proteins. Our protocol could be useful for testing the association between neurodegenerative disease and exposure to organophosphorus pesticides.
Assuntos
Neuroblastoma , Doenças Neurodegenerativas , Praguicidas , Diclorvós/química , Diclorvós/metabolismo , Humanos , Compostos Organofosforados , Peptídeos/químicaRESUMO
Zero-length isopeptide crosslinks between the side chains of glutamine and lysine are the product of transglutaminase activity. It is generally accepted that transglutaminase activity is dormant under physiological conditions because the calcium concentration inside cells is too low to activate transglutaminase to an open conformation with access to the catalytic triad. Traditional assays for transglutaminase activity measure incorporation of biotin pentylamine or of radiolabeled putrescine in the presence of added calcium. In this report, we identified naturally occurring isopeptide crosslinked proteins using the following steps: immunopurification of tryptic peptides by binding to anti-isopeptide antibody 81D1C2, separation of immunopurified peptides by liquid chromatography-tandem mass spectrometry, Protein Prospector database searches of mass spectrometry data for isopeptide crosslinked peptides, and manual evaluation of candidate crosslinked peptide pairs. The most labor intense step was manual evaluation. We developed criteria for accepting and rejecting candidate crosslinked peptides and showed examples of MS/MS spectra that confirm or invalidate a possible crosslink. The SH-SY5Y cells that we examined for crosslinked proteins had not been exposed to calcium and had been lysed in the presence of ethylenediaminetetraacetic acid. This precaution allows us to claim that the crosslinks we found inside the cells occurred naturally under physiological conditions. The quantity of crosslinks was very low, and the crosslinked proteins were mostly low abundance proteins. In conclusion, intracellular transglutaminase crosslinking/transamidase activity is very low but detectable. The low level of intracellular crosslinked proteins is consistent with tight regulation of transglutaminase activity.
RESUMO
Cultured SH-SY5Y human neuroblastoma cells are used in neurotoxicity assays. These cells express markers of the cholinergic and dopaminergic systems. Acetylcholinesterase (AChE) activity has been reported in these cells. Neurotoxic organophosphate compounds that inhibit AChE, also inhibit butyrylcholinesterase (BChE). We confirmed the presence of AChE in the cell lysate by activity assays, Western blot, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of immunopurified AChE. A nondenaturing gel stained for AChE activity identified the catalytically active AChE in SH-SY5Y cells as the unstable monomer. We also identified immature BChE in the cell lysate. The concentration of active BChE protein was similar to that of active AChE protein. The rate of substrate hydrolysis by AChE was 10-fold higher than substrate hydrolysis by BChE. The higher rate was due to the 10-fold higher specific activity of AChE over BChE (5000 units/mg for AChE; 500 units/mg for BChE). Neither cholinesterase was secreted. Tryptic peptides of immunopurified AChE and BChE were identified by LC-MS/MS on an Orbitrap Lumos Fusion mass spectrometer. The unfolded protein chaperone, binding immunoglobulin protein BiP/GRP78, was identified in the mass spectral data from all cholinesterase samples, suggesting that BiP was co-extracted with cholinesterase. This suggests that the cytoplasmic cholinesterases are immature forms of AChE and BChE that bind to BiP. It was concluded that SH-SY5Y cells express active AChE and active BChE, but the proteins do not mature to glycosylated tetramers.
Assuntos
Neuroblastoma , Síndromes Neurotóxicas , Acetilcolinesterase/metabolismo , Butirilcolinesterase , Inibidores da Colinesterase/toxicidade , Cromatografia Líquida , Humanos , Espectrometria de Massas em TandemRESUMO
Chronic low-dose exposure to organophosphorus pesticides is associated with the risk of neurodegenerative disease. The mechanism of neurotoxicity is independent of acetylcholinesterase inhibition. Adducts on tyrosine, lysine, threonine, and serine can occur after exposure to organophosphorus pesticides, the most stable being adducts on tyrosine. Rabbit monoclonal 1C6 to diethoxyphosphate-modified tyrosine (depY) was created by single B cell cloning. The amino acid sequence and binding constant (Kd 3.2 × 10-8 M) were determined. Cultured human neuroblastoma SH-SY5Y and mouse neuroblastoma N2a cells incubated with a subcytotoxic dose of 10 µM chlorpyrifos oxon contained depY-modified proteins detected by monoclonal 1C6 on Western blots. depY-labeled peptides from tryptic digests of cell lysates were immunopurified by binding to immobilized 1C6. Peptides released with 50% acetonitrile and 1% formic acid were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Lumos mass spectrometer. Protein Prospector database searches identified 51 peptides modified on tyrosine by diethoxyphosphate in SH-SY5Y cell lysate and 73 diethoxyphosphate-modified peptides in N2a cell lysate. Adducts appeared most frequently on the cytoskeleton proteins tubulin, actin, and vimentin. It was concluded that rabbit monoclonal 1C6 can be useful for studies that aim to understand the mechanism of neurotoxicity resulting from low-dose exposure to organophosphorus pesticides.
Assuntos
Doenças Neurodegenerativas , Praguicidas , Acetilcolinesterase , Animais , Linfócitos B , Células Cultivadas , Clorpirifos/análogos & derivados , Cromatografia Líquida , Clonagem Molecular , Camundongos , Compostos Organofosforados , Peptídeos , Praguicidas/toxicidade , Espectrometria de Massas em TandemRESUMO
Isopeptide crosslinked proteins can be the product of transglutaminase or of exposure to organophosphorus toxicants (OP). Transglutaminase links glutamine to lysine with loss of ammonia. OP toxicants induce a link between glutamic acid and lysine with loss of water. Our goal was to establish criteria to distinguish real from false isopeptide crosslinks reported by software searches of mass spectrometry data. We used fragmentation spectra of tryptic peptides from MAP-rich tubulin Sus scrofa as a test system for detection of naturally-occurring isopeptide crosslinks. Data were analyzed with Protein Prospector. Criteria for the assignments included the presence of at least 1 crosslink specific product ion, fragment ions from both peptides, Protein Prospector scores ≥20, and best fit of the MS/MS data to the crosslinked peptide as opposed to a linear peptide. Out of 301,364 spectra, 15 potential transglutaminase-type crosslinked peptide candidates were identified. Manual evaluation of these MS/MS spectra reduced the number to 1 valid crosslink between Q112 of NFH and K368 of Tau. Immunopurification with anti-isopeptide 81D1C2 confirmed that MAP-rich tubulin contained only one isopeptide. Support for this isopeptide bond was obtained by showing that transglutaminase was capable of incorporating dansyl-aminohexyl -QQIV into K368. A model of the KIETHK-QLEAHNR isopeptide was synthesized with the aid of transglutaminase. MS/MS spectra of the model validated our interpretation of the native isopeptide. An OP-induced isopeptide bond between K163 of tubulin alpha-1A and E158 of tubulin beta-4B was induced by treating MAP-rich tubulin with 100 µM chlorpyrifos oxon. This crosslink was supported by the criteria described above and by the presence of diethoxyphospho-lysine 163 in the tubulin alpha-1A peptide. The information obtained in this work is valuable for future studies that aim to understand why exposure to OP is associated with increased risk of neurodegenerative disease.
Assuntos
Peptídeos/química , Espectrometria de Massas em Tandem/métodos , Doenças Neurodegenerativas/metabolismoRESUMO
Bacterial transglutaminase was used to label human plasma proteins with fluorescent tags. Protein lysines were modified with dansyl-epsilon-aminohexyl-Gln-Gln-Ile-Val-OH (dansylQQIV), while protein glutamines were modified with dansyl cadaverine. Labeled proteins included human butyrylcholinesterase, apolipoprotein A-1, haptoglobin, haptoglobin-related protein, immunoglobulin heavy chain, and hemopexin. Tryptic peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Modified residues were identified in Protein Prospector and Proteome Discoverer searches of mass spectrometry data. The MS/MS fragmentation spectra from dansylQQIV-modified peptides gave intense peaks at 475.2015, 364.1691, 347.1426, 234.0585, and 170.0965 m/z. These signature ions are useful markers for identifying modified peptides. Human butyrylcholinesterase retained full activity following modification by dansylQQIV or dansyl cadaverine.
Assuntos
Butirilcolinesterase/química , Cadaverina/química , Compostos de Dansil/química , Glutamina/química , Lisina/química , Espectrometria de Massas em Tandem/métodos , HumanosRESUMO
Glutamine residues susceptible to transglutaminase-catalyzed crosslinking can be identified by incorporation of dansyl cadaverine or biotin cadaverine. Bacterial transglutaminase and human transglutaminase 2 were used to modify residues in beta-casein with dansyl cadaverine. Bacterial transglutaminase was used to modify residues in human butyrylcholinesterase with biotin cadaverine. Tryptic peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Modified residues were identified in Protein Prospector searches of mass spectrometry data. The MS/MS spectra from modified casein included intense peaks at 336.2, 402.2, and 447.2 for fragments of dansyl cadaverine adducts on glutamine. The MS/MS spectra from modified butyrylcholinesterase included intense peaks at 329.2, 395.2, and 440.2 for fragments of biotin cadaverine adducts on glutamine. No evidence for transglutaminase-catalyzed adducts on glutamic acid, aspartic acid, or asparagine was found. Consistent with expectation, it was concluded that bacterial transglutaminase and human transglutaminase 2 specifically modify glutamine. The characteristic ions associated with dansyl cadaverine and biotin cadaverine adducts on glutamine are useful markers for modified peptides.
Assuntos
Biotina/química , Cadaverina/química , Glutamina/química , Biotina/metabolismo , Butirilcolinesterase/metabolismo , Cadaverina/metabolismo , Glutamina/metabolismo , Humanos , Íons/química , Íons/metabolismo , Streptomyces/enzimologia , Transglutaminases/metabolismoRESUMO
Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger that protects from the toxicity of nerve agents. Non-human primates are suitable models for toxicity studies that cannot be performed in humans. We evaluated the biochemical properties of native macaque (MaBChE) tetramers, compared to recombinant MaBChE monomers, PEGylated recombinant MaBChE tetramers and monomers, and native HuBChE tetramers. Km and kcat values for butyrylthiocholine were independent of subunit assembly status. The Km for all forms of MaBChE was about 70 µM, compared to 13 µM for HuBChE. The kcat was about 100,000 min-1 for MaBChE and 30,000 min-1 for HuBChE. The reversible inhibitor ethopropazine had similar Ki values of 0.05 µM for all MaBChE forms and HuBChE. The bimolecular rate constant, ki, for inhibition by diisopropylfluorophosphate (DFP), an analog of sarin, was 2.2 to 2.5 × 107 M-1 min-1 for all MaBChE forms and for HuBChE. A major difference between MaBChE and HuBChE was the rate of reactivation by 2-PAM. The second order rate constant for reactivation of DFP-inhibited MaBChE by 2-PAM was 1.4 M-1 min-1, but was 380 fold faster for DFP-inhibited HuBChE (kr 531 M-1 min-1). The acyl pocket of MaBChE has Leu285 in place of Pro285 in HuBChE. The reactivation rate of DFP-inhibited HuBChE mutant P285L by 2-PAM was reduced 5.8-fold (kr 92 M-1 min-1) indicating that P285 determines whether 2-PAM binds in an orientation that favors release of diisopropylphosphate. DFP-inhibited MaBChE treated with 0.2 M 2-PAM recovered 10% of its original activity, whereas DFP-inhibited HuBChE recovered 80% activity. It was concluded that the biochemical properties of MaBChE are similar to those of HuBChE except for the reactivation of DFP-inhibited BChE.
Assuntos
Butirilcolinesterase/química , Reativadores da Colinesterase/química , Compostos de Pralidoxima/química , Prolina/química , Sequência de Aminoácidos , Animais , Inibidores da Colinesterase/farmacologia , Humanos , Cinética , Macaca , Macaca mulatta , Fenotiazinas/farmacologia , Alinhamento de SequênciaRESUMO
Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by hydrolyzing acetylcholine. The collagen-tailed AChE tetramer is a product of 2 genes, ACHE and ColQ. The AChE tetramer consists of 4 identical AChE subunits and one polyproline-rich peptide, whose function is to hold the 4 AChE subunits together. Our goal was to determine the amino acid sequence of the polyproline-rich peptide(s) in Torpedo californica AChE (TcAChE) tetramers to aid in the analysis of images that will be acquired by cryo-EM. Collagen-tailed AChE was solubilized from Torpedo californica electric organ, converted to 300 kDa tetramers by digestion with trypsin, and purified by affinity chromatography. Polyproline-rich peptides were released by denaturing the TcAChE tetramers in a boiling water bath, and reducing disulfide bonds with dithiothreitol. Carbamidomethylated peptides were separated from TcAChE protein on a spin filter before they were analyzed by liquid chromatography tandem mass spectrometry on a high resolution Orbitrap Fusion Lumos mass spectrometer. Of the 64 identified collagen-tail (ColQ) peptides, 60 were from the polyproline-rich region near the N-terminus of ColQ. The most abundant proline-rich peptides were SVNKCCLLTPPPPPMFPPPFFTETNILQE, at 40% of total mass-spectral signal intensity, and SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK, at 27% of total intensity. The high abundance of these 2 peptides makes them candidates for the principal form of the polyproline-rich peptide in the trypsin-treated TcAChE tetramers.
Assuntos
Acetilcolinesterase/metabolismo , Peptídeos/metabolismo , Torpedo/metabolismo , Sequência de Aminoácidos , Animais , Colágeno/metabolismoRESUMO
A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslinked following treatment with 1.5 mM chlorpyrifos oxon. High molecular weight bands were visualized on an SDS gel. Proteins in the gel bands were digested with trypsin, separated by liquid chromatography and analyzed in an Orbitrap mass spectrometer. MSMS files were searched for crosslinked peptides using the Batch-Tag program in Protein Prospector. MSMS spectra were manually evaluated for the presence of ions that supported the crosslinks. The crosslink between Lys544 in VLEMTGNIDEAEWEWK544AGFHR and Glu542 in VLEMTGNIDEAEWE542WK satisfied our criteria including that of spatial proximity. Distances between Lys544 and Glu542 were 7.4 and 9.5 Å, calculated from the cryo-EM (electron microscopy) structure of the HuBChE tetramer. Paraoxon ethyl, diazoxon, and dichlorvos had less pronounced effects as visualized on SDS gels. Our proof-of-principle study provides evidence that OP have the ability to crosslink proteins. If OP-induced protein crosslinking occurs in the brain, OP exposure could be responsible for some cases of neurodegenerative disease.
Assuntos
Butirilcolinesterase/química , Clorpirifos/análogos & derivados , Peptídeos/química , Sítios de Ligação , Butirilcolinesterase/metabolismo , Catálise , Clorpirifos/química , Clorpirifos/metabolismo , Humanos , Isomerismo , Modelos Moleculares , Conformação Molecular , Agregados Proteicos , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Aqueous solutions of chlorpyrifos oxon are used to study the ability of chlorpyrifos oxon to catalyze protein crosslinking. Assays for protein crosslinking can avoid artifacts by using information on the stability of chlorpyrifos oxon in solution. We undertook to determine the half-life of chlorpyrifos oxon in aqueous solution because literature values do not exist. The rate of conversion of chlorpyrifos oxon to 3,5,6-trichloro-2-pyridinol was measured at 23⯰C in 20â¯mM TrisCl pH 8 and pH 9 by recording loss of absorbance at 290â¯nm for chlorpyrifos oxon and increase in absorbance at 320â¯nm for 3,5,6-trichloro-2-pyridinol. The half-life of chlorpyrifos oxon was 20.9 days at pH 8 and 6.7 days at pH 9. Literature reports for the stability of other organophosphorus toxicants were summarized because our current studies suggest that other organophosphorus toxicants are also crosslinking agents.
Assuntos
Clorpirifos/análogos & derivados , Ésteres/química , Organofosfatos/química , Água/química , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Clorpirifos/química , Clorpirifos/metabolismo , Meia-Vida , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Organofosfatos/metabolismo , Hidróxido de Sódio/química , EspectrofotometriaRESUMO
A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 µs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293â¯cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293â¯cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.