- Email: [email protected]
Purification and characterization of neuropathy target esterase (NTE) from rat brain
Life Sciences 78 (2006) 2967 – 2973 www.elsevier.com/locate/lifescie
Purification and characterization of neuropathy target esterase (NTE) from rat brain Pushpinder Kaur a , Geetu Raheja a , Surjit Singh b , K.D. Gill a,⁎ a
b
Department of Biochemistry, Post Graduate Institute of Medical Education and Research, Chandigarh, India Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India Received 8 August 2005; accepted 30 November 2005
Abstract Neuropathy target esterase (NTE) is an integral membrane protein in vertebrate neurons and a member of a novel family of putative serine hydrolases. Neuropathic organophosphates react covalently with the active site serine residue of NTE, causing degeneration of long axons in spinal cord and peripheral nerves which becomes clinically evident 1–3 weeks after exposure to OPs, hence termed as organophosphate induced delayed neuropathy. The present study reports the isolation and characterization of NTE protein from rat brain. Rat brain microsomes were solubilized with phospholipase A2 and they were fractionated by gel filtration chromatography in S-300 column. The sample was eluted in buffer containing polyoxyethylene W1 detergent, which yielded an active fraction of 200 kDa. The most enriched NTE active fraction was further purified by 3-9′-mercaptononylthio-1,1,1-trifluoropropan-2-one bound to sepharose CL4B. The SDS-PAGE confirmed the 155-kDa protein as the most likely candidate for NTE. Database searching of rat N-terminal protein revealed homology with variety of polypeptides from different organisms and suggested that NTE protein has function beyond the nervous system and mediates a biochemical reaction highly conserved through evolution. © 2006 Published by Elsevier Inc. Keywords: Organophosphate; NTE; Organophosphate induced delayed neuropathy; Axonopathies; Serine esterase
Introduction Organophosphorus (OP) compounds represent a vast array of chemicals that are widely used as insecticides, additives, therapeutics agents and notoriously chemical warfare agents (Racke, 1992). Intoxication by OPs represents as much as 80% of pesticide related hospital admissions and deaths (Aldridge, 1993). The manifestations of OP poisoning are clinically divided into three types: the cholinergic syndrome, the intermediate syndrome, and the organophosphate induced delayed neuropathy (Mingxing et al., 2003). In humans, delayed chronic neurotoxic syndromes have been reported in Gulf war veterans, chronically exposed to organophosphates or
⁎ Corresponding author. Tel.: +91 172 2747585x5177; fax: +91 172 2744401, 2745078. E-mail addresses: [email protected], [email protected] (K.D. Gill). 0024-3205/$ - see front matter © 2006 Published by Elsevier Inc. doi:10.1016/j.lfs.2005.11.029
combination of agents (Solberg and Belkin, 1997; Haley and Kurt, 1997, Haley et al., 1999; Enserink, 2001; Hitt, 2002). The most common and best-understood delayed syndrome is organophosphate induced delayed neuropathy (OPIDN), which becomes clinically evident 1–3 weeks after exposure to organophosphates. OPIDN is characterized by paralysis of the lower limb due to degeneration of long axons in the spinal cord and in peripheral nerves, hyperexcitability and flaccid extremities (Lotti, 2002). In addition to the tremendous differences on the clinical manifestations, these syndromes involve distinct molecular targets. While a variety of toxic agents can cause axonopathies, OP mediated neuropathy is unique in that its primary target protein neuropathy target esterase has been identified. The mechanism of action remains to be determined regarding the precise role of NTE in the development of OPIDN. Neuropathy target esterase (NTE), the proposed target protein, is an integral membrane protein present maximally in vertebrate neurons. It is detected in vitro by its capacity to
2968
P. Kaur et al. / Life Sciences 78 (2006) 2967–2973
catalyze the hydrolysis of phenyl valerate although its physiological substrate is unknown (Lotti, 1992; Ray and Richards, 2001). Despite the worldwide use of very large quantities of OP pesticides, those with neuropathic potential have been screened out of the market, why then continue studies of NTE and OPIDN? First, it is now becoming apparent that the normal physiological function of NTE is in neuronal development and involves cell-signaling pathway (Glynn, 1999). Secondly, characterization of NTE and events subsequent to its modification by neuropathic OPs provide a singular opportunity to elucidate the molecular mechanisms of this neuropathy, which in turn may provide insight into mechanisms of normal axonal maintenance and of other neurodegeneration conditions. Thirdly, because NTE is an interesting protein in its own right as it is a member of newly discovered protein family, with a domain conserved through evolution, which may have a second function in addition to its capacity to hydrolyze esters (Glynn, 1999). Elucidation of the structure, subcellular localization and normal functions of NTE are of interest not only from toxicology point of view but also for fundamental neurobiology, since the protein may play a role in normal axonal maintenance (Lotti, 2002). However, subsequent progress in this area had been hampered by difficulty in isolating NTE from brain tissue since it comprises only 0.03% of brain microsomal protein (Johnson, 1974). Moreover NTE has resisted purification and poor recovery from detergent solubilized NTE activity on chromatographic fractionation possibly due to loss of an activating factor (Glynn, 2000). Two forms of NTE have been isolated and characterized, particulate (P-NTE) and soluble (S-NTE) (Escudero and Vilanova, 1997). P-NTE is a membrane bound protein that constitutes the predominant form in chick brain (Glynn et al., 1994). Attempts to purify NTE by gel filtration, sucrose gradient centrifugation, ion exchange chromatography and isoelectric focussing have so far failed to yield significant amount of purified preparation of NTE. Previous methods to isolate active NTE by gel filtration chromatography have yielded a wide range of estimated sizes for the protein ranging from approximately 850 to 1800 kDa (Chemnitius et al., 1984; Pope and Padilla, 1989). Glynn et al. (1994) reported the isolation of P-NTE from chick brain with a molecular mass of 155 kDa using a biotinylated saligenin phosphate analog as an active site ligand (Thomas et al., 1989). Most of these purification studies have been concentrated on hen, which is the habitual model to study OPIDN, but in recent years, objections have been raised on the limited value of this animal in addressing the mechanistic aspects of OP neuropathies. Further rat too has been shown to be a model for OPIDN (Veronesi, 1984). In view of this, easy handling and abundant baseline data available in case of rat prompted us to attempt purification of NTE from rat brain. The purpose of the present line of investigation was to isolate and characterize rat brain NTE using 3′-(9′-mercaptononylthio)-1,1,1-trifluoropropanone-2-one (MNTFP) bound to epoxy activated sepharose CL4B as an affinity ligand.
Materials and methods Materials Diethyl p-nitrophenyl phosphate (Paraoxon), sepharose (CL4B) DTT, EDTA, EGTA, polyoxyethylene W1 ether were obtained from Sigma; Sephacryl S-300 was purchased from Amersham Biosciences and 1-9, nonanedithiol, MNTFP, 1-4 butanediol diglycidyl ether was obtained from Fluka. Phenyl valerate was provided as a kind gift from Defence Research and Development Establishment, Gwalior, India. Microsomal preparation Microsomes were prepared from male Wistar rat brain. Rats were decapitated and the whole brain was placed on ice, weighed and washed with 0.33 M sucrose. Twelve grams of fresh brain tissue was homogenized with mechanically driven Teflon fitted potter Elvejhem type homogenizer in a 50 mM Tris–HCl buffer pH 8.0. The homogenate was centrifuged at 10,000×g for 20 min at 4 °C. The supernatant was removed and the pellet resuspended in the same volume of buffer and again centrifuged at 100,000×g for 60 min at 4 °C. NTE solubilization The pellet obtained above was suspended in 50 mm Tris– HCl buffer pH 8.8. To this suspension, the equivalent of 12.5 U/ ml phospholipase A2 (isolated from apis mellifera) was added and microsomal suspension was incubated at 37 °C for 30 min (Richardson et al., 1979; Dessen, 2000). After incubation, the suspension was placed on ice and EDTA, EGTA, DTT were added to yield final concentration of 1 mM, 1 mM and 250 μM respectively. This suspension was again centrifuged at 100,000×g for 60 min at 4 °C and the resulting microsomal supernatant (Seifert and Wilson, 1994) was collected and tested for NTE activity. Gel filtration chromatography Gel filtration chromatography was performed using a 2 × 100 cm S-300 HR column (Amersham Biosciences)—using peristaltic pump at a constant output of 3.5 ml/min (Fig. 1). The column's exclusion volume was determined using dextran blue (avg. mass 2000 kDa) and standardized using known commercial protein standards within a range of 663 kDa to 12.5 kDa (thyroglobulin to cytochrome c). The PLA2 solubilized NTE extracts were combined with gel filtration buffer and incubated at 4 °C for 1 h. An aliquot of 500 μl of PLA2 solubilized sample was retained for NTE activity, protein determination and rest of the sample applied to the column. The sample was eluted in 50 mM Tris–HCl, 1 mM EDTA, 1 mM EGTA, 0.1% (w/v) polyoxyethylene W1, 500 mM NaCl buffer pH 8.2. Eluates were collected in 3.5 ml aliquots. The reproducibility of the chromatograms was controlled by monitoring the absorbance at 280 nm. The total esterase activity, NTE activity and protein were measured in each
P. Kaur et al. / Life Sciences 78 (2006) 2967–2973
2969
2 × Lamelli buffer (Laemmli, 1970). After the addition of the Lamelli buffer, the samples were boiled at 100 °C for 3 min, subjected to 7.5% SDS-PAGE and electrophoresed at a constant voltage of 300 V for 1 h. The gels were then silver stained (Sammons et al., 1981). Molecular equivalent masses were determined by comparing with known standards run concurrently with the samples. NTE activity
Fig. 1. Calibration curve of the gel filtration chromatography column. The column used was 2 × 10 cm S-300 HR column, flow was maintained using a peristaltic pump at a constant output of 3.5 ml/min. The column total exclusion volume was determined to be 189.5 ml using dextran blue (average mass, 2000 kDa). The selective permeation range was standardized using non-commercial protein standards; thyroglobulin (663 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.5 kDa).
subfraction by standard methods. The subfractions containing the highest specific activity (110–135 ml elution volume) were pooled and concentrated to 5 ml by ultrafiltration. Affinity chromatography Affinity chromatography was performed using 3′-(9′mercaptononylthio)-1,1,1-trifluoropropan-2-one (MNTFP) bound to sepharose CL4B that has been shown to be an inhibitor of NTE activity (Thomas et al., 1990). The inhibitor was prepared in our laboratory according to the procedure of Szekacs et al. (1989). Aliquots of 1 ml ultrafiltrated sample from previous step containing approximately 116 nmol/min/mg protein NTE activity in 50 mM Tris–HCl, 1 mM EDTA, 1 mM EGTA, 0.01% polyoxyethylene W1, 500 mM NaCl, 250 mM sucrose buffer pH 8.2 were applied to a 20% (w/v) slurry of prewashed gel in a ratio of 1:1 and incubated with a gentle agitation for 20 h at 4 °C. The supernatant fractions containing non-bound proteins were recovered after centrifuging the suspensions for 2 min at 10,000×g at 4 °C. The pellet affinity gel was then washed with 1 ml of 0.02% (w/v) TritonX-100, 0.5 M NaCl, 1 mM EDTA, 1 mM EGTA, and 20 mM Tris– HCl buffer pH 7.2 and recovered by centrifuging at 10,000×g for 2 min at 4 °C. Protein bound to the affinity gel was eluted for analysis of NTE activity by the addition of 1 volume of 2 × Lamelli buffer followed by incubation at 60 °C for 1 h. SDS-PAGE Samples were desalted when necessary by ultrafiltration using amicon concentrator. These were combined 1:1 with
NTE activity was determined according to the method of Johnson (1977). NTE activity has been operationally defined as that portion of the phenyl valerate hydrolase activity in tissue homogenates or a fraction that is sensitive to the neuropathic OP mipafox and resistant to the non-neuropathic organophosphate Paraoxon. Samples were combined with either 40 μM Paraoxon or a combination of 40 μm Paraoxon and 50 μM mipafox. Samples were incubated at 37 °C for 15 min. After incubation 2 ml of 6.7 mM phenyl valerate was added and the incubation was continued for another 15 min. The reaction was stopped by the addition of 2 ml 10% SDS containing 0.04% 4-Aminoantipyrine. The chromophore was resolved by the addition of 1 ml of 0.4% potassium ferricyanide. The stable red colour was read at 510 nm. Total phenyl valerate activity was determined as enzymatic hydrolysis with no inhibitor added. NTE activity was expressed as nmol/min/mg protein. Estimation of protein Protein concentration was determined by the method of Lowry et al. (1951). N-Terminal sequencing of NTE 15 μg protein from affinity chromatography eluate was subjected to 7.5% SDS-PAGE and electroblotted to PVDF membrane (Towbin et al., 1979). The blotted proteins were analyzed in a Perkin Elmer/Applied Biosystems 476A sequencer. Results Enrichment of NTE activity The results of various studies have suggested that NTE may be an integral membrane protein. Therefore, such proteins have been characterized as requiring detergent for solubilization, losing activity upon solubilization and generally associated with lipids or may require lipid for maximal activity. Incorporation of phospholipase A2 in addition to EDTA, EGTA and DTT for the preparation of brain microsomes has resulted in 95% solubilization of NTE. The levels of NTE activity were higher in phospholipase A2 solubilized NTE extracts and microsomal supernatant (Table 1) as compared to crude brain homogenate and in other samples obtained during the process of preparation of microsomes. The effect of PLA2 on the increased activity of
2970
P. Kaur et al. / Life Sciences 78 (2006) 2967–2973
Table 1 Enrichment of PVase activities Extract
Activity
Activity (nmol/min/mg)
Protein (mg/ml)
Specific activity (nmol/min/mg)
Fold
Yield
Crude brain homogenate
Total PVase NTE Total PVase NTE Total PVase NTE Total PVase NTE
586 114 568 128 712 716 623 116
9.43
62.14 12.08 120.85 27.23 319.2 78.92 254.2 50.43
–
–
–
–
–
–
0.79 0.63
87% 65%
100,000 g supernatant Phospholipase A2 solubilized microsomes Sephacryl S-300 (104–160 ml of elution)
4.70 4.23 2.3
The 10% homogenates was prepared from male Wistar strain rat brain. The 5 ml of 100,000 g supernatant i.e. rat brain microsomes was subjected to Sephacryl S-300 gel filtration chromatography column. ‘A’ represents the total PVase activity NTE as the Paraoxon-resistant mipafox sensitive activity by the differential assay described in Materials and methods. The table shows data corresponding to activities and protein in the most enriched fractions pooled and concentrated for the use in the next step.
NTE activity in the microsomal supernatant is consistent with the results of Seifert and Wilson (1994).
PAGE of gel filtration chromatography showed numerous bands co-eluted with NTE activity (Fig. 3).
Gel filtration chromatography
Affinity chromatography
The 5 ml sample (4.3 mg protein/ml) of phospholipase A2 solubilized NTE extract was combined with gel filtration chromatography buffer and applied to the column. In the Sephacryl S-300 column only one peak of NTE activity was obtained which co-eluted with total PVase activity. A preparation with 0.63 fold purification and 65% yield NTE activity was obtained by the addition of phospholipase A2 and 0.1% W1 detergent in the elution buffer. The majority of the NTE activity eluted at 200-kDa fraction (Fig. 2). The SDS-
Final identification and purification of the 155-kDa band as the source of NTE activity was performed by selective binding to the MNTFP sepharose gel. The eluted fractions from the Sephacryl S-300 column containing the highest specific NTE activity (116 nmol/min/mg protein) were pooled and concentrated by ultrafiltration and subjected to MNTFPsepharose CL4B affinity gel. This triflouroketone has been shown to be a potent inhibitor of NTE as well as being able to selectively bind the 155 kDa NTE protein. The sample was eluted with Lamelli buffer and 155 kDa fragment of NTE was finally identified by running affinity chromatography eluate on 7.5% SDS-PAGE (Fig. 4).
Fig. 2. Gel filtration profile of NTE, total PVase eluted in polyoxyethylene W1 buffer on S-300 column. This figure shows the fractionation of total PVase (▴) and NTE activities (■) by molecular exclusion chromatography in S-300 column. 5 ml of phospholipase A2 solubilized rat brain microsomes were combined with buffer containing 1 M NaCl and 0.2% (w/v) polyoxyethylene W1 ether. The sample was applied to a 2 × 100 cm S-300 column and eluted with the same buffer and subfractions of 3.5 ml were collected. Total PVase, NTE activities and protein were measured in each fraction as described in Materials and methods. (♦) Represent absorbance at 280 nm as measured by UV diode array detector.
Fig. 3. SDS-PAGE of crude rat brain homogenate, microsomes and gel filtration exclusion chromatography eluted fractions. The molecular weight (kDa) marker position is shown at right. The figure shows the Coomassie blue stained 7.5% gel of samples from different enrichment PVase activity steps. Lane 1: pooled fractions (104–160 ml of elution) of the S-300 gel filtration exclusion chromatography (30 μg protein). Lane 2: 10% rat brain homogenate sample (30 μg protein). Lane 3: 10,000 g pellet (30 μg protein). Lane 4: 100,000 g pellet (30 μg protein). Lane 5: phospholipase A2 solubilized sample (30 μg protein). Lane 6: brain microsomal sample (30 μg protein). Lane 7: high molecular weight markers.
P. Kaur et al. / Life Sciences 78 (2006) 2967–2973
2971
Table 3 Rat brain NTE N-terminal sequence shares similarity with other proteins Source
Protein sequence
Protein component
Rat
MGSSNLHELNT TSV S N L EL NT
Neuropathy target esterase
Lipotes vexillifer Homo sapiens
NLHELN
NADH dehydrogenase subunit 5 Patatin-like phospholipase domain containing 5
Rat brain NTE N-terminal sequence found few hits on running a BLAST search. BLAST search revealed that NTE protein shares protein homology (shown in bold amino acid symbol) with variety of polypeptides from different organisms. Fig. 4. SDS-PAGE of MNTFP affinity chromatography eluted NTE protein. Lane 1: silver stained 7.5% gel of the NTE affinity chromatography sample eluted in Lamelli buffer. Highly prominent band of 155-kDa molecular weight confirms the NTE protein. Lane 2: high molecular weight markers.
N-Terminal amino acid analysis The first 14 N-Terminal amino acid residues of purified NTE protein were identified as M, G, S, S, N, L, H, E, L, N, T, T, S, V, and the amino acids were matched by BLAST and database searching suggests rat NTE protein shares similarity with many known proteins (Tables 2 and 3). Discussion Neuropathy target esterase is inhibited by several organophosphorus (OP) pesticides, chemical warfare agents, lubricants and plasticizers, leading to organophosphorus induced delayed neurotoxicity (OPIDN). Brain NTE is considered a membrane bound protein, mainly obtained in the microsomal fraction. The low abundance and apparent requirement for membrane lipids to maintain NTE activity impeded its isolation for several years. Advances in determining NTE structures and functions have come from studies with several species and various levels of organization. The adult hen is the habitual model to study OPIDN (Lotti, 2002) but of lately rat has also been used as an animal of choice by many workers since preferential damage to Table 2 N-terminal amino acid sequence of purified rat brain NTE protein S. no.
Amino acid
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Met Gly Ser Ser Asn Leu His Glu Leu Asn Thr Thr Ser Val
15 μg protein from affinity chromatography eluate was subjected to 7.5% SDSPAGE and electroblotted to PVDF membrane. The blotted proteins were analyzed in a Perkin Elmer/Applied Biosystems 476A sequencer.
the spinal cord's long ascending and descending tract is shared by chicken as well as rats which emphasizes the dying back nature of neuropathy in both species (Veronesi, 1984). Moreover, the Km value of the rat brain NTE, i.e. 1.36 × 10− 3 M, is in line with the reported Km value of hen brain NTE, i.e. 1–2 mM (Sarin and Gill, 2000). Thus, the present paper describes the isolation and characterization of NTE protein from rat brain microsomes. Chemnitius et al. (1984) observed a molecular weight of about 1800 kDa for brain P-NTE solubilized with n-octylglucoside on S-300 gel filtration column; it probably reflected micelle containing membrane lipids and proteins. Although, it had been difficult to isolate NTE in its active form for so many years but as reports of Ishikaa et al. (1983) and Thomas et al. (1989) suggested that if the ambient conditions for the isolation of NTE protein are provided, it became possible to isolate NTE in active form with a mass of 155–178 kDa. Earlier, all the chaotropic agents tested by Mackay et al. (1992) have resulted in the inactivation of NTE. Some serine hydrolases that catalyze the hydrolysis of ester bonds show maximal catalytic activity only when the enzyme is adsorbed at an oil/water interface. NTE seems to require interfacial activation, therefore the addition of phospholipase A2 for the NTE solubilization in our study had resulted in 95% increased activity of NTE. A similar effect has been observed in the previous study carried out by Davis and Richardson (1987) which suggested that it was necessary to add exogenous phosphatidylcholine in order to maintain the activity in the solution, as phosphatidyl being amphipathic protected NTE activity in aqueous solution by providing lipophillic cocoon around it. Moreover the presence of polyoxyethylene W1 detergent in the elution buffer of gel filtration chromatography further permitted NTE protein to remain in its active form thus allowing it to migrate in the 200-kDa fraction. As NTE is an integral membrane protein its firm association with membrane supports the possibility that addition of phospholipids completely restores the catalytic activity of purified NTE. The membrane association of NTE likely points to the importance of the cellular functions, particularly in the genesis and remodeling of membranes. The specific activity of the starting material i.e. PLA2 solubilized microsomal NTE was 78.92 nmol/min/mg protein. The enrichment of PVase activities was made possible by using molecular exclusion chromatography in S-300 and showed 65% yield of NTE protein. Our results suggest that although incorporation of PhospholipaseA2
2972
P. Kaur et al. / Life Sciences 78 (2006) 2967–2973
has resulted in increased solubilization as well as increased activity of NTE the specific activity of gel filtration NTE fractions was found to be 50.43 nmol/min/mg, which indicated that there was no substantial increase in purity. The loss of lipid cofactor during chromatographic separation may underlie some of the difficulties encountered in isolation of active NTE. Further, SDS-PAGE of most enriched NTE gel filtration fractions at 200 kDa peak showed a predominant band with a molecular weight of 155 kDa (the proposed NTE protein) and 2 bands with a molecular weight of 66 and 55 kDa, which suggested that there may be more than one source of NTE activity or they may be subfragments of 155 kDa larger fragment. When the ultrafiltrated fractions of gel filtration with 116 nmol/min/mg NTE activity were subjected to MNTFP sepharose CL4B affinity chromatography, the 155 kDa bind specifically to MNTFP which was confirmed after eluting the protein from affinity column and running on SDS-PAGE. The SDS-PAGE confirmed single 155 kDa band that has been proposed as an NTE protein. This also suggested that NTE specifically binds the MNTFP affinity ligand at its active site. Nevertheless the application of MNTFP bound sepharose CL4B affinity purification method led to much important progress in the purification of NTE. Therefore, inactive NTE can now be obtained in one simple step after gel filtration chromatography followed by preparative SDS-PAGE. Although, Glynn et al. (1994) have also reported the use of S9B (biotinylated organophosphate ester) for the affinity purification of NTE from chicken brain microsomes with a molecular weight of 155 kDa. Mackay et al. (1992, 1996) had successfully employed MNTFP sepharose for its ability to isolate sufficient quantities of NTE for amino terminal sequencing. Therefore, the incorporation of phospholipase A2 for the NTE solubilization, polyoxyethylene W1 ether detergent in the elution buffer, use of MNTFP sepharose for affinity purification of NTE and preparative SDS-PAGE demonstrates the feasibility of isolating NTE for amino acid analysis. Furthermore, the purified NTE protein was characterized by N-terminal sequence of the excised peptide from the electroblot. Amino acid analysis confirmed that 15 μg of 155 kDa protein were recovered from 7.5% SDS-PAGE on to a PVDF membrane. Our Wistar strain rat NTE 14 amino acids Nterminal sequence shares similarity to number of polypeptides predicted from the sequencing of bacteria, yeast, nematodes, Drosophila, patatin-like phospholipase domain containing 5 [Homo sapiens]. As reported earlier by Marianne et al. (2002) the recombinant esterase domain of NTE i.e. NEST liberates fatty acid from phospholipids, monoacylglycerol and lysophospholipids. Rat NTE sequence similarity with patatin domain provide structural basis that NTE belong to superfamily of serine enzyme and its catalytic activity is being regulated by signaling events. Thus, our data and earlier reports on NTE demonstrate that NTE might be a putative lipase, and provides new clues to its possible cellular functions and its function beyond the nervous system. The sequence similarity with so many polypeptides on the database suggest that rat NTE protein is not only involved in the etiopathogenesis of OPIDN but may be involved in many other physiological process. Therefore further studies
are needed to know the exact function of NTE, which can open new avenues of investigation into the characteristics of NTE and the mechanism causing OPIDN. Acknowledgement We thank E. Vilanova for kindly providing mipafox for carrying out NTE assays. We also thank Dr. Pandey (DRDE, Gwalior, India) for his valuable suggestions regarding the preparation of mipafox. This study was supported by the Department of Biotechnology, Ministry of Science and Technology, India. References Aldridge, W.N., 1993. The esterases: perspectives and problems. ChemicoBiological Interactions 87, 5–13. Chemnitius, J.M., Haselmeyer, K.H., Zech, R., 1984. Neurotoxic esterase: gel filtration and isoelectric focussing of carboxylesterases solubilized from hen brain. Life Sciences 34, 1119–1125. Davis, C.S., Richardson, R.J., 1987. Neurotoxic esterase: characterization of the solubilized enzyme and the conditions for its solubilization form chicken brain microsomal membranes with ionic, zwitterionic, or nonionic detergents. Biochemical Pharmacology 36, 1393–1399. Dessen, A., 2000. Structure and mechanism of human cytosolic phospholipase A2. Biochimica et Biophysica Acta 1488, 40–47. Enserink, M., 2001. Gulf war illness. The battle continues. Science 291, 812–817. Escudero, M.A., Vilanova, E., 1997. purification and characterization of naturally soluble neuropathy target esterase from chicken sciatic nerve by HPLC and Western Blot. Journal of Neurochemistry 69, 1975–1982. Glynn, P., 1999. Neuropathy target esterase. Biochemical Journal 344, 625–631. Glynn, P., 2000. Neural development and neurodegeneration: two faces of neuropathy target esterase. Progress in Neurobiology 61, 61–74. Glynn, P., Read, D., Guo, R., Wylie, S., Johnson, M.K., 1994. Synthesis and characterization of a biotinylated Organophosphorus ester for the detection and affinity purification of a brain serine esterase: neuropathy target esterase. Biochemical Journal 301, 551–556. Haley, R.W., Kurt, T.L., 1997. Self reported exposure to neurotoxic chemical combinations in the Gulf war. A cross sectional epidemiological study. JAM 277, 231–237. Haley, R.W., et al., 1999. Evaluation of neurological function in Gulf war veterans. A blinded case-control study. JAM 277, 223–230. Hitt, E., 2002. New investigations into Gulf War syndrome. Nature Medicine 8, 198. Ishikaa, Y., Chow, E., McNamee, M.G., McCheune, M., Wilson, B.W., 1983. Separation of Paraoxon and mipafox sensitive esterase by sucrose density gradient sedimentation. Toxicology Letters 17, 315–320. Johnson, M.K., 1974. The primary biochemical lesion leading to the delayed neurotoxic effects of some organophosphorus esters. Journal of Neurochemistry 23, 785–789. Johnson, M.K., 1977. Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential. Archives of Toxicology 37, 113–115. Laemmli, U.K., 1970. cleavage of structural proteins during the assembly of bacteriophage T4. Nature 227, 680–685. Lotti, M., 1992. The pathogenesis of organophosphate polyneuropathy. Critical Reviews in Toxicology 21, 465–488. Lotti, M., 2002. Low-level exposure to organophosphorus esters and peripheral nerve function. Muscle & Nerve 25, 492–504. Lowry, O.H., Rose borough, J.N., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. Journal of Biochemistry 193, 265–275. Mackay, C.E., Thomas, T.C., Szekacs, A., Hang, T., Hammock, B.D., McNamee, M.G., Wilson, B.W., 1992. Isolation and characterization of neuropathy target esterase. The Toxicologist (Abstr. Soc. Toxicol.) 12, 39.
P. Kaur et al. / Life Sciences 78 (2006) 2967–2973 Mackay, C.E., Hammock, B.D., Wilson, B.W., 1996. Identification and isolation of a 155-kDa protein with neuropathy target esterase activity. Fundamental and Applied Toxicology 30, 23–30. Marianne, van Tienhoven, Atkins, Jane, Li, Yang, Glynn, Paul, 2002. Human neuropathy target esterase catalyze hydrolysis of membrane lipids. Journal of Biological Chemistry 277 (23), 20942–20948. Mingxing, Xie, Dongfang, Yang, Lynna, Mtoney, Bingfang, Yan, 2003. Rat NTE-related esterase is a membrane-associated protein, hydrolyzes phenyl valerate, and interacts with diisopropylfluorophosphate through serine catalytic machinery. Archives of Biochemistry and Biophysics 416, 137–146. Pope, C.N., Padilla, S.C., 1989. Chromatographic characterization of NTE. Toxicology and Applied Pharmacology 97, 272–278. Racke, K.D., 1992. Degradation of organophosphorus insecticides in environmental matrices. In: Chambers, J.E., Levi, P.E. (Eds.), Organophosphates: Chemistry, Fate and Effects. Academic Press, San Diego, CA, pp. 47–72. Ray, D.E., Richards, P.G., 2001. The potential for toxic effects of chronic low dose exposure to organophosphates. Toxicology Letters 120, 343–351. Richardson, R.J., Davis, C.S., Johnson, M.K., 1979. Subcellular distribution of marker enzymes and of neurotoxic esterase in adult hen brain. Journal of Neurochemistry 32, 607–615. Sammons, D.W., Adams, L.D., Nishizawa, E.E., 1981. Ultrasensitive silver based color staining of polypeptides in polyacrylamide gels. Electrophoresis 2, 135–141. Sarin, S., Gill, K.D., 2000. Biochemical characterization of dichlorvos induced delayed neurotoxicity in rat. IUBMB Life 49, 125–130.
2973
Seifert, J., Wilson, B.W., 1994. Solubilization of neuropathy target esterase and other phenyl valerate carboxylesterases from chicken embryonic brain by phospholipase A2. Computational Biochemistry and Physiology C 108, 337–341. Solberg, Y., Belkin, M., 1997. The role of excitotoxicity in organophosphorus nerve agents central poisoning. Trends in Pharmacological Sciences 18, 183–185. Szekacs, A., Hammock, B.D., Abdel-Aal, Y.A.I., Halarnkar, P.P., Philpott, M., Matolesy, G., 1989. New trifluoropropanone sulfides as highly active selective inhibitors of insect juvenile hormone esterase. Pesticide Biochemistry and Physiology 33, 112–124. Thomas, T.C., Szekacs, A., Rojas, S., Hammock, B.D., Wilson, B.W., 1989. Correlation of neuropathy target esterase activity with specific tritiated DFP labeled proteins. Biochemical Journal 257, 109–116. Thomas, T.C., Szekacs, A., Hammock, B.D., Wilson, B.W., McNamee, M.G., 1990. Affinity chromatography of neuropathy target esterase. ChemicoBiological Interactions 87, 347–360. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76, 4350–4354. Veronesi, B., 1984. A rodent model of organophosphorus induced delayed neuropathy: distribution of central (spinal cord) and peripheral nerve damage. Neuropathology and Applied Neurobiology 10, 357–362.
Purification and characterization of neuropathy target esterase (NTE) from rat brain Pushpinder Kaur a , Geetu Raheja a , Surjit Singh b , K.D. Gill a,⁎ a
b
Department of Biochemistry, Post Graduate Institute of Medical Education and Research, Chandigarh, India Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India Received 8 August 2005; accepted 30 November 2005
Abstract Neuropathy target esterase (NTE) is an integral membrane protein in vertebrate neurons and a member of a novel family of putative serine hydrolases. Neuropathic organophosphates react covalently with the active site serine residue of NTE, causing degeneration of long axons in spinal cord and peripheral nerves which becomes clinically evident 1–3 weeks after exposure to OPs, hence termed as organophosphate induced delayed neuropathy. The present study reports the isolation and characterization of NTE protein from rat brain. Rat brain microsomes were solubilized with phospholipase A2 and they were fractionated by gel filtration chromatography in S-300 column. The sample was eluted in buffer containing polyoxyethylene W1 detergent, which yielded an active fraction of 200 kDa. The most enriched NTE active fraction was further purified by 3-9′-mercaptononylthio-1,1,1-trifluoropropan-2-one bound to sepharose CL4B. The SDS-PAGE confirmed the 155-kDa protein as the most likely candidate for NTE. Database searching of rat N-terminal protein revealed homology with variety of polypeptides from different organisms and suggested that NTE protein has function beyond the nervous system and mediates a biochemical reaction highly conserved through evolution. © 2006 Published by Elsevier Inc. Keywords: Organophosphate; NTE; Organophosphate induced delayed neuropathy; Axonopathies; Serine esterase
Introduction Organophosphorus (OP) compounds represent a vast array of chemicals that are widely used as insecticides, additives, therapeutics agents and notoriously chemical warfare agents (Racke, 1992). Intoxication by OPs represents as much as 80% of pesticide related hospital admissions and deaths (Aldridge, 1993). The manifestations of OP poisoning are clinically divided into three types: the cholinergic syndrome, the intermediate syndrome, and the organophosphate induced delayed neuropathy (Mingxing et al., 2003). In humans, delayed chronic neurotoxic syndromes have been reported in Gulf war veterans, chronically exposed to organophosphates or
⁎ Corresponding author. Tel.: +91 172 2747585x5177; fax: +91 172 2744401, 2745078. E-mail addresses: [email protected], [email protected] (K.D. Gill). 0024-3205/$ - see front matter © 2006 Published by Elsevier Inc. doi:10.1016/j.lfs.2005.11.029
combination of agents (Solberg and Belkin, 1997; Haley and Kurt, 1997, Haley et al., 1999; Enserink, 2001; Hitt, 2002). The most common and best-understood delayed syndrome is organophosphate induced delayed neuropathy (OPIDN), which becomes clinically evident 1–3 weeks after exposure to organophosphates. OPIDN is characterized by paralysis of the lower limb due to degeneration of long axons in the spinal cord and in peripheral nerves, hyperexcitability and flaccid extremities (Lotti, 2002). In addition to the tremendous differences on the clinical manifestations, these syndromes involve distinct molecular targets. While a variety of toxic agents can cause axonopathies, OP mediated neuropathy is unique in that its primary target protein neuropathy target esterase has been identified. The mechanism of action remains to be determined regarding the precise role of NTE in the development of OPIDN. Neuropathy target esterase (NTE), the proposed target protein, is an integral membrane protein present maximally in vertebrate neurons. It is detected in vitro by its capacity to
2968
P. Kaur et al. / Life Sciences 78 (2006) 2967–2973
catalyze the hydrolysis of phenyl valerate although its physiological substrate is unknown (Lotti, 1992; Ray and Richards, 2001). Despite the worldwide use of very large quantities of OP pesticides, those with neuropathic potential have been screened out of the market, why then continue studies of NTE and OPIDN? First, it is now becoming apparent that the normal physiological function of NTE is in neuronal development and involves cell-signaling pathway (Glynn, 1999). Secondly, characterization of NTE and events subsequent to its modification by neuropathic OPs provide a singular opportunity to elucidate the molecular mechanisms of this neuropathy, which in turn may provide insight into mechanisms of normal axonal maintenance and of other neurodegeneration conditions. Thirdly, because NTE is an interesting protein in its own right as it is a member of newly discovered protein family, with a domain conserved through evolution, which may have a second function in addition to its capacity to hydrolyze esters (Glynn, 1999). Elucidation of the structure, subcellular localization and normal functions of NTE are of interest not only from toxicology point of view but also for fundamental neurobiology, since the protein may play a role in normal axonal maintenance (Lotti, 2002). However, subsequent progress in this area had been hampered by difficulty in isolating NTE from brain tissue since it comprises only 0.03% of brain microsomal protein (Johnson, 1974). Moreover NTE has resisted purification and poor recovery from detergent solubilized NTE activity on chromatographic fractionation possibly due to loss of an activating factor (Glynn, 2000). Two forms of NTE have been isolated and characterized, particulate (P-NTE) and soluble (S-NTE) (Escudero and Vilanova, 1997). P-NTE is a membrane bound protein that constitutes the predominant form in chick brain (Glynn et al., 1994). Attempts to purify NTE by gel filtration, sucrose gradient centrifugation, ion exchange chromatography and isoelectric focussing have so far failed to yield significant amount of purified preparation of NTE. Previous methods to isolate active NTE by gel filtration chromatography have yielded a wide range of estimated sizes for the protein ranging from approximately 850 to 1800 kDa (Chemnitius et al., 1984; Pope and Padilla, 1989). Glynn et al. (1994) reported the isolation of P-NTE from chick brain with a molecular mass of 155 kDa using a biotinylated saligenin phosphate analog as an active site ligand (Thomas et al., 1989). Most of these purification studies have been concentrated on hen, which is the habitual model to study OPIDN, but in recent years, objections have been raised on the limited value of this animal in addressing the mechanistic aspects of OP neuropathies. Further rat too has been shown to be a model for OPIDN (Veronesi, 1984). In view of this, easy handling and abundant baseline data available in case of rat prompted us to attempt purification of NTE from rat brain. The purpose of the present line of investigation was to isolate and characterize rat brain NTE using 3′-(9′-mercaptononylthio)-1,1,1-trifluoropropanone-2-one (MNTFP) bound to epoxy activated sepharose CL4B as an affinity ligand.
Materials and methods Materials Diethyl p-nitrophenyl phosphate (Paraoxon), sepharose (CL4B) DTT, EDTA, EGTA, polyoxyethylene W1 ether were obtained from Sigma; Sephacryl S-300 was purchased from Amersham Biosciences and 1-9, nonanedithiol, MNTFP, 1-4 butanediol diglycidyl ether was obtained from Fluka. Phenyl valerate was provided as a kind gift from Defence Research and Development Establishment, Gwalior, India. Microsomal preparation Microsomes were prepared from male Wistar rat brain. Rats were decapitated and the whole brain was placed on ice, weighed and washed with 0.33 M sucrose. Twelve grams of fresh brain tissue was homogenized with mechanically driven Teflon fitted potter Elvejhem type homogenizer in a 50 mM Tris–HCl buffer pH 8.0. The homogenate was centrifuged at 10,000×g for 20 min at 4 °C. The supernatant was removed and the pellet resuspended in the same volume of buffer and again centrifuged at 100,000×g for 60 min at 4 °C. NTE solubilization The pellet obtained above was suspended in 50 mm Tris– HCl buffer pH 8.8. To this suspension, the equivalent of 12.5 U/ ml phospholipase A2 (isolated from apis mellifera) was added and microsomal suspension was incubated at 37 °C for 30 min (Richardson et al., 1979; Dessen, 2000). After incubation, the suspension was placed on ice and EDTA, EGTA, DTT were added to yield final concentration of 1 mM, 1 mM and 250 μM respectively. This suspension was again centrifuged at 100,000×g for 60 min at 4 °C and the resulting microsomal supernatant (Seifert and Wilson, 1994) was collected and tested for NTE activity. Gel filtration chromatography Gel filtration chromatography was performed using a 2 × 100 cm S-300 HR column (Amersham Biosciences)—using peristaltic pump at a constant output of 3.5 ml/min (Fig. 1). The column's exclusion volume was determined using dextran blue (avg. mass 2000 kDa) and standardized using known commercial protein standards within a range of 663 kDa to 12.5 kDa (thyroglobulin to cytochrome c). The PLA2 solubilized NTE extracts were combined with gel filtration buffer and incubated at 4 °C for 1 h. An aliquot of 500 μl of PLA2 solubilized sample was retained for NTE activity, protein determination and rest of the sample applied to the column. The sample was eluted in 50 mM Tris–HCl, 1 mM EDTA, 1 mM EGTA, 0.1% (w/v) polyoxyethylene W1, 500 mM NaCl buffer pH 8.2. Eluates were collected in 3.5 ml aliquots. The reproducibility of the chromatograms was controlled by monitoring the absorbance at 280 nm. The total esterase activity, NTE activity and protein were measured in each
P. Kaur et al. / Life Sciences 78 (2006) 2967–2973
2969
2 × Lamelli buffer (Laemmli, 1970). After the addition of the Lamelli buffer, the samples were boiled at 100 °C for 3 min, subjected to 7.5% SDS-PAGE and electrophoresed at a constant voltage of 300 V for 1 h. The gels were then silver stained (Sammons et al., 1981). Molecular equivalent masses were determined by comparing with known standards run concurrently with the samples. NTE activity
Fig. 1. Calibration curve of the gel filtration chromatography column. The column used was 2 × 10 cm S-300 HR column, flow was maintained using a peristaltic pump at a constant output of 3.5 ml/min. The column total exclusion volume was determined to be 189.5 ml using dextran blue (average mass, 2000 kDa). The selective permeation range was standardized using non-commercial protein standards; thyroglobulin (663 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), carbonic anhydrase (29 kDa) and cytochrome c (12.5 kDa).
subfraction by standard methods. The subfractions containing the highest specific activity (110–135 ml elution volume) were pooled and concentrated to 5 ml by ultrafiltration. Affinity chromatography Affinity chromatography was performed using 3′-(9′mercaptononylthio)-1,1,1-trifluoropropan-2-one (MNTFP) bound to sepharose CL4B that has been shown to be an inhibitor of NTE activity (Thomas et al., 1990). The inhibitor was prepared in our laboratory according to the procedure of Szekacs et al. (1989). Aliquots of 1 ml ultrafiltrated sample from previous step containing approximately 116 nmol/min/mg protein NTE activity in 50 mM Tris–HCl, 1 mM EDTA, 1 mM EGTA, 0.01% polyoxyethylene W1, 500 mM NaCl, 250 mM sucrose buffer pH 8.2 were applied to a 20% (w/v) slurry of prewashed gel in a ratio of 1:1 and incubated with a gentle agitation for 20 h at 4 °C. The supernatant fractions containing non-bound proteins were recovered after centrifuging the suspensions for 2 min at 10,000×g at 4 °C. The pellet affinity gel was then washed with 1 ml of 0.02% (w/v) TritonX-100, 0.5 M NaCl, 1 mM EDTA, 1 mM EGTA, and 20 mM Tris– HCl buffer pH 7.2 and recovered by centrifuging at 10,000×g for 2 min at 4 °C. Protein bound to the affinity gel was eluted for analysis of NTE activity by the addition of 1 volume of 2 × Lamelli buffer followed by incubation at 60 °C for 1 h. SDS-PAGE Samples were desalted when necessary by ultrafiltration using amicon concentrator. These were combined 1:1 with
NTE activity was determined according to the method of Johnson (1977). NTE activity has been operationally defined as that portion of the phenyl valerate hydrolase activity in tissue homogenates or a fraction that is sensitive to the neuropathic OP mipafox and resistant to the non-neuropathic organophosphate Paraoxon. Samples were combined with either 40 μM Paraoxon or a combination of 40 μm Paraoxon and 50 μM mipafox. Samples were incubated at 37 °C for 15 min. After incubation 2 ml of 6.7 mM phenyl valerate was added and the incubation was continued for another 15 min. The reaction was stopped by the addition of 2 ml 10% SDS containing 0.04% 4-Aminoantipyrine. The chromophore was resolved by the addition of 1 ml of 0.4% potassium ferricyanide. The stable red colour was read at 510 nm. Total phenyl valerate activity was determined as enzymatic hydrolysis with no inhibitor added. NTE activity was expressed as nmol/min/mg protein. Estimation of protein Protein concentration was determined by the method of Lowry et al. (1951). N-Terminal sequencing of NTE 15 μg protein from affinity chromatography eluate was subjected to 7.5% SDS-PAGE and electroblotted to PVDF membrane (Towbin et al., 1979). The blotted proteins were analyzed in a Perkin Elmer/Applied Biosystems 476A sequencer. Results Enrichment of NTE activity The results of various studies have suggested that NTE may be an integral membrane protein. Therefore, such proteins have been characterized as requiring detergent for solubilization, losing activity upon solubilization and generally associated with lipids or may require lipid for maximal activity. Incorporation of phospholipase A2 in addition to EDTA, EGTA and DTT for the preparation of brain microsomes has resulted in 95% solubilization of NTE. The levels of NTE activity were higher in phospholipase A2 solubilized NTE extracts and microsomal supernatant (Table 1) as compared to crude brain homogenate and in other samples obtained during the process of preparation of microsomes. The effect of PLA2 on the increased activity of
2970
P. Kaur et al. / Life Sciences 78 (2006) 2967–2973
Table 1 Enrichment of PVase activities Extract
Activity
Activity (nmol/min/mg)
Protein (mg/ml)
Specific activity (nmol/min/mg)
Fold
Yield
Crude brain homogenate
Total PVase NTE Total PVase NTE Total PVase NTE Total PVase NTE
586 114 568 128 712 716 623 116
9.43
62.14 12.08 120.85 27.23 319.2 78.92 254.2 50.43
–
–
–
–
–
–
0.79 0.63
87% 65%
100,000 g supernatant Phospholipase A2 solubilized microsomes Sephacryl S-300 (104–160 ml of elution)
4.70 4.23 2.3
The 10% homogenates was prepared from male Wistar strain rat brain. The 5 ml of 100,000 g supernatant i.e. rat brain microsomes was subjected to Sephacryl S-300 gel filtration chromatography column. ‘A’ represents the total PVase activity NTE as the Paraoxon-resistant mipafox sensitive activity by the differential assay described in Materials and methods. The table shows data corresponding to activities and protein in the most enriched fractions pooled and concentrated for the use in the next step.
NTE activity in the microsomal supernatant is consistent with the results of Seifert and Wilson (1994).
PAGE of gel filtration chromatography showed numerous bands co-eluted with NTE activity (Fig. 3).
Gel filtration chromatography
Affinity chromatography
The 5 ml sample (4.3 mg protein/ml) of phospholipase A2 solubilized NTE extract was combined with gel filtration chromatography buffer and applied to the column. In the Sephacryl S-300 column only one peak of NTE activity was obtained which co-eluted with total PVase activity. A preparation with 0.63 fold purification and 65% yield NTE activity was obtained by the addition of phospholipase A2 and 0.1% W1 detergent in the elution buffer. The majority of the NTE activity eluted at 200-kDa fraction (Fig. 2). The SDS-
Final identification and purification of the 155-kDa band as the source of NTE activity was performed by selective binding to the MNTFP sepharose gel. The eluted fractions from the Sephacryl S-300 column containing the highest specific NTE activity (116 nmol/min/mg protein) were pooled and concentrated by ultrafiltration and subjected to MNTFPsepharose CL4B affinity gel. This triflouroketone has been shown to be a potent inhibitor of NTE as well as being able to selectively bind the 155 kDa NTE protein. The sample was eluted with Lamelli buffer and 155 kDa fragment of NTE was finally identified by running affinity chromatography eluate on 7.5% SDS-PAGE (Fig. 4).
Fig. 2. Gel filtration profile of NTE, total PVase eluted in polyoxyethylene W1 buffer on S-300 column. This figure shows the fractionation of total PVase (▴) and NTE activities (■) by molecular exclusion chromatography in S-300 column. 5 ml of phospholipase A2 solubilized rat brain microsomes were combined with buffer containing 1 M NaCl and 0.2% (w/v) polyoxyethylene W1 ether. The sample was applied to a 2 × 100 cm S-300 column and eluted with the same buffer and subfractions of 3.5 ml were collected. Total PVase, NTE activities and protein were measured in each fraction as described in Materials and methods. (♦) Represent absorbance at 280 nm as measured by UV diode array detector.
Fig. 3. SDS-PAGE of crude rat brain homogenate, microsomes and gel filtration exclusion chromatography eluted fractions. The molecular weight (kDa) marker position is shown at right. The figure shows the Coomassie blue stained 7.5% gel of samples from different enrichment PVase activity steps. Lane 1: pooled fractions (104–160 ml of elution) of the S-300 gel filtration exclusion chromatography (30 μg protein). Lane 2: 10% rat brain homogenate sample (30 μg protein). Lane 3: 10,000 g pellet (30 μg protein). Lane 4: 100,000 g pellet (30 μg protein). Lane 5: phospholipase A2 solubilized sample (30 μg protein). Lane 6: brain microsomal sample (30 μg protein). Lane 7: high molecular weight markers.
P. Kaur et al. / Life Sciences 78 (2006) 2967–2973
2971
Table 3 Rat brain NTE N-terminal sequence shares similarity with other proteins Source
Protein sequence
Protein component
Rat
MGSSNLHELNT TSV S N L EL NT
Neuropathy target esterase
Lipotes vexillifer Homo sapiens
NLHELN
NADH dehydrogenase subunit 5 Patatin-like phospholipase domain containing 5
Rat brain NTE N-terminal sequence found few hits on running a BLAST search. BLAST search revealed that NTE protein shares protein homology (shown in bold amino acid symbol) with variety of polypeptides from different organisms. Fig. 4. SDS-PAGE of MNTFP affinity chromatography eluted NTE protein. Lane 1: silver stained 7.5% gel of the NTE affinity chromatography sample eluted in Lamelli buffer. Highly prominent band of 155-kDa molecular weight confirms the NTE protein. Lane 2: high molecular weight markers.
N-Terminal amino acid analysis The first 14 N-Terminal amino acid residues of purified NTE protein were identified as M, G, S, S, N, L, H, E, L, N, T, T, S, V, and the amino acids were matched by BLAST and database searching suggests rat NTE protein shares similarity with many known proteins (Tables 2 and 3). Discussion Neuropathy target esterase is inhibited by several organophosphorus (OP) pesticides, chemical warfare agents, lubricants and plasticizers, leading to organophosphorus induced delayed neurotoxicity (OPIDN). Brain NTE is considered a membrane bound protein, mainly obtained in the microsomal fraction. The low abundance and apparent requirement for membrane lipids to maintain NTE activity impeded its isolation for several years. Advances in determining NTE structures and functions have come from studies with several species and various levels of organization. The adult hen is the habitual model to study OPIDN (Lotti, 2002) but of lately rat has also been used as an animal of choice by many workers since preferential damage to Table 2 N-terminal amino acid sequence of purified rat brain NTE protein S. no.
Amino acid
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Met Gly Ser Ser Asn Leu His Glu Leu Asn Thr Thr Ser Val
15 μg protein from affinity chromatography eluate was subjected to 7.5% SDSPAGE and electroblotted to PVDF membrane. The blotted proteins were analyzed in a Perkin Elmer/Applied Biosystems 476A sequencer.
the spinal cord's long ascending and descending tract is shared by chicken as well as rats which emphasizes the dying back nature of neuropathy in both species (Veronesi, 1984). Moreover, the Km value of the rat brain NTE, i.e. 1.36 × 10− 3 M, is in line with the reported Km value of hen brain NTE, i.e. 1–2 mM (Sarin and Gill, 2000). Thus, the present paper describes the isolation and characterization of NTE protein from rat brain microsomes. Chemnitius et al. (1984) observed a molecular weight of about 1800 kDa for brain P-NTE solubilized with n-octylglucoside on S-300 gel filtration column; it probably reflected micelle containing membrane lipids and proteins. Although, it had been difficult to isolate NTE in its active form for so many years but as reports of Ishikaa et al. (1983) and Thomas et al. (1989) suggested that if the ambient conditions for the isolation of NTE protein are provided, it became possible to isolate NTE in active form with a mass of 155–178 kDa. Earlier, all the chaotropic agents tested by Mackay et al. (1992) have resulted in the inactivation of NTE. Some serine hydrolases that catalyze the hydrolysis of ester bonds show maximal catalytic activity only when the enzyme is adsorbed at an oil/water interface. NTE seems to require interfacial activation, therefore the addition of phospholipase A2 for the NTE solubilization in our study had resulted in 95% increased activity of NTE. A similar effect has been observed in the previous study carried out by Davis and Richardson (1987) which suggested that it was necessary to add exogenous phosphatidylcholine in order to maintain the activity in the solution, as phosphatidyl being amphipathic protected NTE activity in aqueous solution by providing lipophillic cocoon around it. Moreover the presence of polyoxyethylene W1 detergent in the elution buffer of gel filtration chromatography further permitted NTE protein to remain in its active form thus allowing it to migrate in the 200-kDa fraction. As NTE is an integral membrane protein its firm association with membrane supports the possibility that addition of phospholipids completely restores the catalytic activity of purified NTE. The membrane association of NTE likely points to the importance of the cellular functions, particularly in the genesis and remodeling of membranes. The specific activity of the starting material i.e. PLA2 solubilized microsomal NTE was 78.92 nmol/min/mg protein. The enrichment of PVase activities was made possible by using molecular exclusion chromatography in S-300 and showed 65% yield of NTE protein. Our results suggest that although incorporation of PhospholipaseA2
2972
P. Kaur et al. / Life Sciences 78 (2006) 2967–2973
has resulted in increased solubilization as well as increased activity of NTE the specific activity of gel filtration NTE fractions was found to be 50.43 nmol/min/mg, which indicated that there was no substantial increase in purity. The loss of lipid cofactor during chromatographic separation may underlie some of the difficulties encountered in isolation of active NTE. Further, SDS-PAGE of most enriched NTE gel filtration fractions at 200 kDa peak showed a predominant band with a molecular weight of 155 kDa (the proposed NTE protein) and 2 bands with a molecular weight of 66 and 55 kDa, which suggested that there may be more than one source of NTE activity or they may be subfragments of 155 kDa larger fragment. When the ultrafiltrated fractions of gel filtration with 116 nmol/min/mg NTE activity were subjected to MNTFP sepharose CL4B affinity chromatography, the 155 kDa bind specifically to MNTFP which was confirmed after eluting the protein from affinity column and running on SDS-PAGE. The SDS-PAGE confirmed single 155 kDa band that has been proposed as an NTE protein. This also suggested that NTE specifically binds the MNTFP affinity ligand at its active site. Nevertheless the application of MNTFP bound sepharose CL4B affinity purification method led to much important progress in the purification of NTE. Therefore, inactive NTE can now be obtained in one simple step after gel filtration chromatography followed by preparative SDS-PAGE. Although, Glynn et al. (1994) have also reported the use of S9B (biotinylated organophosphate ester) for the affinity purification of NTE from chicken brain microsomes with a molecular weight of 155 kDa. Mackay et al. (1992, 1996) had successfully employed MNTFP sepharose for its ability to isolate sufficient quantities of NTE for amino terminal sequencing. Therefore, the incorporation of phospholipase A2 for the NTE solubilization, polyoxyethylene W1 ether detergent in the elution buffer, use of MNTFP sepharose for affinity purification of NTE and preparative SDS-PAGE demonstrates the feasibility of isolating NTE for amino acid analysis. Furthermore, the purified NTE protein was characterized by N-terminal sequence of the excised peptide from the electroblot. Amino acid analysis confirmed that 15 μg of 155 kDa protein were recovered from 7.5% SDS-PAGE on to a PVDF membrane. Our Wistar strain rat NTE 14 amino acids Nterminal sequence shares similarity to number of polypeptides predicted from the sequencing of bacteria, yeast, nematodes, Drosophila, patatin-like phospholipase domain containing 5 [Homo sapiens]. As reported earlier by Marianne et al. (2002) the recombinant esterase domain of NTE i.e. NEST liberates fatty acid from phospholipids, monoacylglycerol and lysophospholipids. Rat NTE sequence similarity with patatin domain provide structural basis that NTE belong to superfamily of serine enzyme and its catalytic activity is being regulated by signaling events. Thus, our data and earlier reports on NTE demonstrate that NTE might be a putative lipase, and provides new clues to its possible cellular functions and its function beyond the nervous system. The sequence similarity with so many polypeptides on the database suggest that rat NTE protein is not only involved in the etiopathogenesis of OPIDN but may be involved in many other physiological process. Therefore further studies
are needed to know the exact function of NTE, which can open new avenues of investigation into the characteristics of NTE and the mechanism causing OPIDN. Acknowledgement We thank E. Vilanova for kindly providing mipafox for carrying out NTE assays. We also thank Dr. Pandey (DRDE, Gwalior, India) for his valuable suggestions regarding the preparation of mipafox. This study was supported by the Department of Biotechnology, Ministry of Science and Technology, India. References Aldridge, W.N., 1993. The esterases: perspectives and problems. ChemicoBiological Interactions 87, 5–13. Chemnitius, J.M., Haselmeyer, K.H., Zech, R., 1984. Neurotoxic esterase: gel filtration and isoelectric focussing of carboxylesterases solubilized from hen brain. Life Sciences 34, 1119–1125. Davis, C.S., Richardson, R.J., 1987. Neurotoxic esterase: characterization of the solubilized enzyme and the conditions for its solubilization form chicken brain microsomal membranes with ionic, zwitterionic, or nonionic detergents. Biochemical Pharmacology 36, 1393–1399. Dessen, A., 2000. Structure and mechanism of human cytosolic phospholipase A2. Biochimica et Biophysica Acta 1488, 40–47. Enserink, M., 2001. Gulf war illness. The battle continues. Science 291, 812–817. Escudero, M.A., Vilanova, E., 1997. purification and characterization of naturally soluble neuropathy target esterase from chicken sciatic nerve by HPLC and Western Blot. Journal of Neurochemistry 69, 1975–1982. Glynn, P., 1999. Neuropathy target esterase. Biochemical Journal 344, 625–631. Glynn, P., 2000. Neural development and neurodegeneration: two faces of neuropathy target esterase. Progress in Neurobiology 61, 61–74. Glynn, P., Read, D., Guo, R., Wylie, S., Johnson, M.K., 1994. Synthesis and characterization of a biotinylated Organophosphorus ester for the detection and affinity purification of a brain serine esterase: neuropathy target esterase. Biochemical Journal 301, 551–556. Haley, R.W., Kurt, T.L., 1997. Self reported exposure to neurotoxic chemical combinations in the Gulf war. A cross sectional epidemiological study. JAM 277, 231–237. Haley, R.W., et al., 1999. Evaluation of neurological function in Gulf war veterans. A blinded case-control study. JAM 277, 223–230. Hitt, E., 2002. New investigations into Gulf War syndrome. Nature Medicine 8, 198. Ishikaa, Y., Chow, E., McNamee, M.G., McCheune, M., Wilson, B.W., 1983. Separation of Paraoxon and mipafox sensitive esterase by sucrose density gradient sedimentation. Toxicology Letters 17, 315–320. Johnson, M.K., 1974. The primary biochemical lesion leading to the delayed neurotoxic effects of some organophosphorus esters. Journal of Neurochemistry 23, 785–789. Johnson, M.K., 1977. Improved assay of neurotoxic esterase for screening organophosphates for delayed neurotoxicity potential. Archives of Toxicology 37, 113–115. Laemmli, U.K., 1970. cleavage of structural proteins during the assembly of bacteriophage T4. Nature 227, 680–685. Lotti, M., 1992. The pathogenesis of organophosphate polyneuropathy. Critical Reviews in Toxicology 21, 465–488. Lotti, M., 2002. Low-level exposure to organophosphorus esters and peripheral nerve function. Muscle & Nerve 25, 492–504. Lowry, O.H., Rose borough, J.N., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. Journal of Biochemistry 193, 265–275. Mackay, C.E., Thomas, T.C., Szekacs, A., Hang, T., Hammock, B.D., McNamee, M.G., Wilson, B.W., 1992. Isolation and characterization of neuropathy target esterase. The Toxicologist (Abstr. Soc. Toxicol.) 12, 39.
P. Kaur et al. / Life Sciences 78 (2006) 2967–2973 Mackay, C.E., Hammock, B.D., Wilson, B.W., 1996. Identification and isolation of a 155-kDa protein with neuropathy target esterase activity. Fundamental and Applied Toxicology 30, 23–30. Marianne, van Tienhoven, Atkins, Jane, Li, Yang, Glynn, Paul, 2002. Human neuropathy target esterase catalyze hydrolysis of membrane lipids. Journal of Biological Chemistry 277 (23), 20942–20948. Mingxing, Xie, Dongfang, Yang, Lynna, Mtoney, Bingfang, Yan, 2003. Rat NTE-related esterase is a membrane-associated protein, hydrolyzes phenyl valerate, and interacts with diisopropylfluorophosphate through serine catalytic machinery. Archives of Biochemistry and Biophysics 416, 137–146. Pope, C.N., Padilla, S.C., 1989. Chromatographic characterization of NTE. Toxicology and Applied Pharmacology 97, 272–278. Racke, K.D., 1992. Degradation of organophosphorus insecticides in environmental matrices. In: Chambers, J.E., Levi, P.E. (Eds.), Organophosphates: Chemistry, Fate and Effects. Academic Press, San Diego, CA, pp. 47–72. Ray, D.E., Richards, P.G., 2001. The potential for toxic effects of chronic low dose exposure to organophosphates. Toxicology Letters 120, 343–351. Richardson, R.J., Davis, C.S., Johnson, M.K., 1979. Subcellular distribution of marker enzymes and of neurotoxic esterase in adult hen brain. Journal of Neurochemistry 32, 607–615. Sammons, D.W., Adams, L.D., Nishizawa, E.E., 1981. Ultrasensitive silver based color staining of polypeptides in polyacrylamide gels. Electrophoresis 2, 135–141. Sarin, S., Gill, K.D., 2000. Biochemical characterization of dichlorvos induced delayed neurotoxicity in rat. IUBMB Life 49, 125–130.
2973
Seifert, J., Wilson, B.W., 1994. Solubilization of neuropathy target esterase and other phenyl valerate carboxylesterases from chicken embryonic brain by phospholipase A2. Computational Biochemistry and Physiology C 108, 337–341. Solberg, Y., Belkin, M., 1997. The role of excitotoxicity in organophosphorus nerve agents central poisoning. Trends in Pharmacological Sciences 18, 183–185. Szekacs, A., Hammock, B.D., Abdel-Aal, Y.A.I., Halarnkar, P.P., Philpott, M., Matolesy, G., 1989. New trifluoropropanone sulfides as highly active selective inhibitors of insect juvenile hormone esterase. Pesticide Biochemistry and Physiology 33, 112–124. Thomas, T.C., Szekacs, A., Rojas, S., Hammock, B.D., Wilson, B.W., 1989. Correlation of neuropathy target esterase activity with specific tritiated DFP labeled proteins. Biochemical Journal 257, 109–116. Thomas, T.C., Szekacs, A., Hammock, B.D., Wilson, B.W., McNamee, M.G., 1990. Affinity chromatography of neuropathy target esterase. ChemicoBiological Interactions 87, 347–360. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 76, 4350–4354. Veronesi, B., 1984. A rodent model of organophosphorus induced delayed neuropathy: distribution of central (spinal cord) and peripheral nerve damage. Neuropathology and Applied Neurobiology 10, 357–362.