Organophosphorus nerve agents - PowerPoint Presentation

Slide1

Organophosphorus nerve agents covalently bind to and inhibit acetylcholinesterase (AChE), preventing AChE from hydrolyzing the neurotransmitter acetylcholine. This inhibition causes muscle spasms, organ failure, and possibly death. Enzyme bioscavengers can bind to nerve agents, thereby preventing the inhibition of AChE. Atypical butyrylcholinesterase (aBuChE) from the Medaka Oryzias latipes has been identified as a bioscavenger candidate. Research by Shah and Otto found that aBuChE binds to G-type and V-type nerve agents. Interestingly, VR-inhibited aBuChE can spontaneously reactivate and may be able to serve as a pseudo-catalytic bioscavenger (2013). The amino acid residue tyrosine 337 (Y337) may play a role in the mechanism of reactivation (Neuman & Otto, 2014). Understanding the role of this residue in spontaneous reactivation may lead to improvements in the effectiveness of aBuChE as a catalytic bioscavenger.The purpose of this study was to determine if Y337 is involved in the spontaneous reactivation of VR-inhibited aBuChE. Y337 was mutated to six other amino acids, and whole cell lysates expressing each variant were examined for the capacity to spontaneously reactivate after inhibition with VR. If Y337 is critical to reactivation, then substituting any other amino acid residue at this site will result in no observed reactivation. Y337 may not be vital for reactivation, however, and another amino acid substitution may improve rates of reactivation.

DNA sequencing confirmed the presence of each mutation, resulting in variants Y337D/F/G/K/V/M. The KM for PtCh was derived for each variant. The values were 1.6 to 2.75-fold greater than the KM value of wild type aBuChE (Chart 1). PtCh hydrolysis by variant Y337K was too low to determine the KM. Each variant was examined for the capacity to undergo spontaneous reactivation after inhibition by VR. All variants were able to reach 100% reactivation following VR inhibition. Variants Y337F, Y337G and Y337V had reactivation half-times similar to wild type aBuChE, whereas variant Y337M had a t1/2 that was 1.4 fold longer than wild type. Interestingly, VR-inhibited Y337D was able to spontaneously reactivate with a t1/2 of 4 hours, which was approximately 1.6-fold faster than the reactivation rate for wild type aBuChE (Chart 1). The reactivation rate for variant Y337K could not be determined.

Assessing the importance of tyrosine-337 in the spontaneous reactivation of

VR-inhibited atypical butyrylcholinesterase from Oryzias latipesPatrick HealyMentored by Dr. Tamara Otto

Introduction

Results (continued)

Thanks to Dr. Tamara Otto and Melanie

Kirkpatrick for their guidance.

Mutation of Y337

. Gene splicing by overlap extension (SOE) (Horton, Cai, Ho, & Pease, 1990) was conducted in six separate procedures to mutate Y337 to either valine (V), phenylalanine (F), glycine (G), lysine (K), aspartic acid (D), or methionine (M). The products were cloned into pcDNA 3.1 using restriction enzymes HindIII and ApaI. The encoded amino acid substitutions were confirmed by DNA sequencing.Expression of aBuChE variants. Plasmid DNA was transfected into human embryonic kidney 293T (HEK293T) cells with Lipofectamine™ 2000 transfection reagent according to the manufacturer’s instructions. HEK293T cells were grown in a 37 ºC CO2 incubator in Dulbecco’s Minimal Essential Media. Media was exchanged in a biosafety cabinet to avoid contamination. Forty-eight hours post transfection, cells were lysed in 100 mM KPO4, 1 M NaCl, 1% Triton X-100, pH 7.4 for 5 minutes at room temperature. The lysates were clarified by centrifugation at 20,000 g for 20 minutes and stored at -20 ºC.

Measuring propionylthiocholine (PtCh) turnover rate. The Michaelis constant (KM) for the substrate PtCh was derived for each variant of aBuChE. Clarified lysates were incubated with 50 mM KPO4 at pH 7.4, 2 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and PtCh (7.8 mM – 1 mM). The hydrolysis of PtCh was followed in a spectrophotometer at A412 for five minutes. Measuring reactivation of aBuChE variants. Modified Ellman assays (Ellman, Courtney, Andres, & Featherstone, 1961) were conducted to determine reactivation half-times (t1/2) for each variant after VR inhibition. In these assays, 50 μl of clarified lysate was incubated with a molar excess of VR for ten minutes at room temperature. The total volume was spun through hydrated spin columns to remove any unbound nerve agent. Lysate incubated with 1 μl of 50 mM KPO4 was treated in the same way to serve as the uninhibited control. Both samples were assayed for hydrolysis of PtCh using 10 μl of diluted lysate in 100 μl of substrate solution containing 1 mM DTNB, 2 mM PtCh and 50 mM KPO4, pH 7.4. The rate of hydrolysis of PtCh was recorded with a spectrophotometer at A412. Readings were taken at 15, 30, 45, 160, 1140 and 1260 minutes. The reaction rate of the VR-inhibited sample was compared to the reaction rate of the uninhibited sample, determining a rate of reactivation of VR-inhibited sample over time.

Materials and Methods (continued)

Ellman, G., Courtney, K., Andres, V., & Featherstone, R. (1961). A new and rapid colormetric determination of acetylcholinesterase activity. Elsevier Biochemical Pharmacology, 7(2), 88-95. Horton, R. M., Cai, Z., Ho, S. N., & Pease, L. R. (1990). Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. BioTechniques, 8(5), 528-535.Neuman, A., & Otto, T. (2014). Probing the active site of atypical butyrylcholinesterase with V-agents and V-agent analogs. Retrieved from www.scienceandmathacademy.com/academics/srt4/student_work/2014/neuman.pdfShah, N., & Otto, T. (2013). Evaluation of atypical butyrylcholinesterase from Oryzias latipes as a scavenger of nerve agents. Retrieved from www.scienceandmathacademy.com/academics/srt/student_work/2013/shah.pdf

Chart 1: (right) This chart displays KM and t1/2 values with standard error for each variant of aBuChE. The KM and t1/2 values with standard error for wild type aBuChE was taken from data published by Shah and Otto (2013) and Neuman and Otto (2014), respectively.

Six variants at site Y337 in aBuChE were examined for the capacity to spontaneously reactivate after VR inhibition. Changes at this site affected the rate of the enzyme to reactivate and suggest that this residue may play a role in this mechanism. Y337D had the best reactivation half-time at 4 hours. However, this rate is still too low to be physiologically relevant. The decreased ability of Y337K to hydrolyze PtCh indicates that Y337 may also be involved in hydrolyzing this substrate. Future studies will explore if another negatively charged amino acid (Y337E) produces a similar result as observed for Y337D. These studies will aid in the understanding of the reactivation of aBuChE.

Materials and Methods

Acknowledgments

References

Graph 1: (above) This graph shows averaged Michaelis-Menten assay data with standard error for Y337G with labeled Vmax and KM.

KM and t1/2 of aBuChE variantsVariantKMt1/2 (hr)Wild Type0.12 ± 0.0036.5 ± 0.07Y337D0.19 ± 0.0094.0 ± 0.35Y337F0.24 ± 0.0106.0 ± 0.27Y337G0.31 ± 0.0276.5 ± 0.51Y337M0.33 ± 0.0349.0 ± 0.28Y337V0.33 ± 0.0186.9 ± 0.26

Conclusions

Figure

1: (above) This is a gel of reverse primer PCRs for each variant specified. Each sample contains 722 base pairs.

Ladder

D

F

G

K

V

650

1650

2000

400

850

100

3’ SOE PCR

V

max

K

M

V

max

Results