Genetic engineering of lamelllipodin-enhanced green fluorescent protein

expression vectors: Effect on cholinesterase activity levels and tetramerization

in vitro

William King and Leo Pezzementi

Department of Biology

Birmingham-Southern College

Birmingham, Alabama

In partial fulfillment of the requirements of the degree of Bachelor of Science

Submitted 8 May 2015

1

ABSTRACT

Soluble ChE tetramers in vertebrate sera were found to be associated with lamellipodin (Lpd) and other proline-rich cytoskeletal proteins. In vitro studies found an increase in ChE tetramer formation when ChEs were expressed with Lpd, with a related proline-rich protein, Rap1-GTP interacting adaptor molecule (RIAM), or with poly-L-proline. However, there was a decrease in ChE enzymatic activity levels in the presence of Lpd, which was not seen with RIAM or poly-L-proline. It was thought that the pEGFP plasmid that was used to express the Lpd-EGFP fusion protein was responsible for the decrease in ChE activity via the production of siRNA from the Neo/Kan cassette and from the EGFP. siRNAs are small interfering RNAs that inhibit translation of proteins. Previous studies involving subcloning of the Lpd-EGFP cassette from pEGFP into pcDNA3.1 ruled out the Neo/Kan cassette as the source of ChE down regulation, leaving the possibility of the EGFP as the source. Here, we present evidence that after successful EGFP excision, ChE activity remained downregulated by Lpd.

INTRODUCTION

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are evolutionarily

related cholinesterases (ChEs) (Massoulié et al., 1993; Pezzementi et al., 2011). While

the main function of AChE is the hydrolysis of acetylcholine (ACh) at the neuromuscular

junction, until recently, the function of BChE has remained unclear. BChE is now

thought to be an important detoxifying bioscavenger enzyme in the serum, and BChE

may even act secondarily in synaptic transmission (Johnson and Moore, 2012).

Furthermore, BChE is being studied more due to findings that show the enzyme as a

viable antidote against accidental or intentional poisoning by organophosphate

compounds (OPs) (Masson and Lockridge, 2010). Because of the function of AChE

within the human body, AChE is the target of OP nerve gases like sarin and VX which

can be used as a weapon of mass destruction (WMD) (Evison et al., 2002). Even

though thousands of deaths may occur from an organophosphate WMD, the World

Health Organization lists 200,000 yearly deaths from 3 million accidental OP insecticide

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poisonings (Singh and Sharma, 2000; Walker and Nidiry, 2002; Leibson and Lifshitz,

2008; Eddleston et al., 2008). In the clinical setting, a recent study has shown BChE

expressed in the human plasma to be the treatment of choice for acute OP poisoning

and OP poisoning prophylaxis (Ilyushin et al., 2013).

AChE, through alternative splicing (Massoulié et al., 2008), forms three subunits.

Duval et al. (1992) found that the most important of the subunits, AChET, produces

amphiphilic monomers (G1a) and dimers (G2

a) as well as non-amphiphilic tetramers

(G4na) of the globular protein form. Perrier et al. (2002) found that tailed forms are also

produced when globular AChE subunits associate with two different transmembrane

proteins. These are the Proline-Rich Membrane Anchor (PRiMA) and the triple-helical

collagen Q (ColQ). PRiMA anchors the ChE to the membrane, while ColQ anchors ChE

to the basal lamina. Both PRiMA and ColQ anchor ChE to the synapse. While AChE

tetramers are most likely to be found in the synapse, some fetal mammals also have

AChE soluble tetramers in the plasma (Ralston et al., 1985). BChE protein structure has

both similarities and differences to AChE structure. The BChET soluble tetramer variant

is the only subunit found in mammals, due to the absence of alternative splicing, (Moya

et al., 1991), and the levels of BChE are ten times greater than AChE (Li et al., 2000).

Similarly, BChE has been shown to associate with PRiMA and ColQ in the same

manner as AChE (Blong et al., 1997; Altamarino and Lockridge, 1999; Perrier et al.,

2002).

In the T subunits of AChE and BChE, at both C-termini, a tryptophan (W)

amphiphilic tetramerization (WAT) domain is present. This WAT domain allows the

enzyme to associate with proline-rich areas in PRiMA and ColQ called the proline-rich

3

attachment domains (PRADs; Simon et al., 1998). Vertebrate and invertebrate ChEs

can be assembled into tetramers through the WAT domains when treated with synthetic

poly-L-proline (Bon and Massoulié, 1997; Altamirano and Lockridge, 1999; Pezzementi

et al., 2012). PRiMA and ColQ have been identified only in vertebrates, but invertebrate

ChEs are still able to assemble tetramers (Johnson and Russell, 1983; Arpagaus et al.,

1992; Combes et al., 2000). In vitro expression of an invertebrate ChE with PRiMA and

ColQ was reported by both Frederick et al. (2008) and Pezzementi et al. (2012), and

those studies show invertebrate ChE readily participating in WAT-PRAD interactions.

Interpretations of these data began to raise questions about invertebrate ChE

organization by PRADs from an unknown protein, although the identity of the

invertebrate PRAD is unknown.

Li et al. (2008) found that soluble tetrameric BChE from both equine and human

plasma was associated with a PRAD from a proline-rich protein called lamellipodin

(Lpd). Li et al. (2008) also found that cells expressing BChE, when treated in vitro with a

proline-rich peptide derivative from Lpd, favored tetrameric assemblage of the BChE,

suggesting that the PRAD-containing PRAD-fragments of Lpd assembled soluble

plasma BChE. These data reporting the existence of the organizing protein Lpd and its

ability to assemble soluble tetramers via WAT-PRAD interactions was novel in the field

of ChE study. These data suggest that all tetrameric ChEs, whether they are anchored

to synapses in the central of peripheral nervous system or soluble in the blood of

hemolymph, have some form of organization by PRADs (Biberoglu et al., 2013).

Lpd, as reported by Li et al. (2008), was the first identified PRAD-containing

protein associated with soluble plasma BChE. Our laboratory found that Lpd had the

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highest amount of proline-rich sequences of any of

its related proteins, indicating that Lpd and its two

closest relatives could serve as a model system

for studying proline-rich regions of proteins

involved in ChE tetrameric assemblage. Due to

the fact that the presence of ChE tetramers

increases ChE activity (Simon and Massoulié,

1997; Chitlaru et al., 2001; Liang et al., 2009), it

was hypothesized that Lpd, through the proline-

rich sequences, would have a positive effect on

ChE activity. It was also predicted that co-

expression of Lpd with ChE would show an

increase in the amount of tetramers. The

results obtained in our lab are partially contrary

to what was predicted. The percentage of

tetramers across the two conditions relative to the control increased somewhat, but

there was a steep decline in ChE activity compared to the control (Figure 1). These data

show that ChE enzymatic activity is being down regulated by an unknown source

(Pezzementi, pers. comm.).

The Lpd used in our laboratory is part of an expression vector described by

Krause et al. (2004). The Lpd is fused to an enhanced green fluorescent protein (EGFP)

in the expression vector pEGFP-N1 (Figure 2). Nejepinska et al. (2012) asserted that

Control

Lpd-EGFP-pEGFP

Lpd-EGFP-pcDNA

%G

4 o

n G

rad

ient

0

20

40

60

80

D. viviparus AChE +

Lpd-EGFP-pEGFP or Lpd-EGFP-pcDNA

Control

Lpd-EGFP-pEGFP

Lpd-EGFP-pcDNA

%C

ontr

ol A

ChE

Activi

ty

0

20

40

60

80

100

120

Figure 1. ChE activity and tetrameric forms in the presence of lamellipodin. Top panel,

Percent ChE activity relative total control activity. Bottom panel, % G4 (tetramer) on gradient, which is reflective of the ChE found in the tetramers. Mean ± SE.

5

the reduction in ChE levels may be due to a problem with the expression vector. It was

also found that difficulties

with the pEGFP vectors

may arise from the

Kan/Neo antibiotic

resistance cassette. This

Kan/Neo cassette

produces small interfering

RNA (siRNA) that inhibits

translation of the vector’s

proteins. This problem was

overcome by Nejepinska

et al. (2012), who excised

the Kan/Neo cassette and replaced it with an ampicillin resistant gene. However, the

study still noted decreased enzymatic activity. The EGFP is also known to have

associated siRNA that inhibits translation of vector proteins, therefore it was

hypothesized that the EGFP may be causing the decrease of ChE activity (Baens et al.,

2006; Koelsch et al., 2013).

The purpose of this study is excise EGFP in order to determine if EGFP has a

causal relationship with ChE down regulation. This will be the first study in our

laboratory whose sole purpose is to excise EGFP, as previous projects have examined

the the Kan/Neo cassette theory of ChE down regulation. Previously in our laboratory,

the Lpd-EGFP fusion protein was subcloned into another plasmid expression vector,

Figure 2. Expression vector pEGFP-N1. Expression vector showing Lpd, EGFP, and Kan/Neo constructs

6

pcDNA3.1, in order to avoid translation and transcription of the Kan/Neo cassette, but

the suppression of ChE activity was still present, as shown in Figure 1. This down

regulation after subcloning effectively eliminated the vector and the Kan/Neo cassette

as the possible sources of ChE down regulation. Now with the Lpd or the EGFP of the

Lpd-EGFP fusion cassette identified as the cause of the ChE activity suppression,

previous projects in our laboratory have also attempted to engineer a STOP codon and

a NotI restriction site into the vector between Lpd and EGFP to complement an

endogenous NotI site downstream of EGFP in order to excise the EGFP by NotI

digestion (see Figure 3).

Figure 3. Proposed genetically engineered pEGFP expression vector. pEGFP expression vector showing the proposed new NotI restriction site indicated by the arrow.

The STOP codon was successfully engineered, tetramers still increased, but the second

NotI

7

NotI site was not successful, and the ChE down regulation was still observed (see

Figures 4 and 5). Because these projects were not entirely successful, their data

demonstrate the need to completely excise EGFP from the expression vector in order to

determine Lpd’s role in ChE enzymatic activity suggesting that the transcription of the

EGFP RNA, perhaps as siRNA, could be responsible for the decrease in ChE activity

(Pezzementi, pers. comm.). If transcription of EGFP causes ChE down regulation, then

EGFP’s successful excision may increase ChE activity levels and tetramerization.

MATERIALS AND METHODS

1. Site-Directed Mutagenesis

In order to genetically engineer the second NotI restriction site to complement the

endogenous NotI site, site-directed mutagenesis was used to mutate the DNA

sequences of both pEGFP and pcDNA3.1 expression vectors. A QuikChange

kit was obtained along with the necessary sense and antisense primers from

Invitrogen. Step-wise methods were followed according to the QuikChange

kit, and then a PCR was performed. After PCR, the mutants were confirmed with

0.8% agarose gel electrophoresis. An E. coli transformation followed with amp

and kan LB selection plates.

2. NotI Restriction Digestion

Following DNA mutation, restriction digestion was employed to excise EGFP

from the Lpd-EGFP fusion protein. Having added the second NotI restriction site

and confirmed mutants with gel electrophoresis, restriction digestion was

performed. A Qiagen Spin Miniprep kit was obtained, and the DNA was

amplified and quantified. Digestion reactions with 10x buffer 3.1, NotI, milliQ

8

water, and the plasmid were prepared and allowed to incubate at 37C for one

hour. Following incubation, the digests were loaded onto a 0.8% agarose gel for

electrophoresis to confirm digestion. For EGFP excision, a second set of digests

were prepared in the same manner and loaded onto a 0.5% agarose gel. After 1

hour of electrophoresis, the gel was transferred to an ultraviolet light box, and the

portion of the gel containing the Lpd was cut out. The DNA was purified from the

gel, and religated with 10x ligation buffer, milliQ water, and ligase. A subsequent

E. coli (Invitrogen Subcloning Efficiency DH5) transformation was

performed with amp and kan selection plates.

3. DNA Sequencing

To confirm the successful mutation and religation of the expression vector, DNA

samples were quantified (diluted as needed), and sequenced. Sequencing was

performed by the University of Alabama at Birmingham – Genomics Core

Laboratory in Birmingham, Alabama.

4. DNA Amplification

After a glycerol stock was prepared, a Qiagen Maxiprep DNA amplification was

performed. Step-wise methods were followed according to Qiagen protocols.

DNA was subsequently quantified.

5. Cell Transfection and Expression

Cells were trypsinized, counted, and plated as described previously (Pezzementi

et al., 2012). ChE- and Lpd-expression vectors were combined with Fugene 6 in

Opti-MEM media for transfection of COS-7 cells in vitro as described previously

(Pezzementi et al., 2012). In a multi-well plate, pure medium served as the

9

control in one well, and the varying transfections (0.1 g/well, 1 g/well) with the

Lpd protein were added to the other wells. After a 48 hour incubation time, the

media and cell extracts were harvested. Cell extract was prepared in HIS buffer

(high ionic strength buffer; 10 mM NaHPO4, pH 7, 1 M NaCl, 1% Triton X-100, 1

mM EDTA) as described previously (Pezzementi et al., 2012).

6. Velocity Sedimentation Centrifugation on Sucrose Gradients

5-25% sucrose gradients were prepared in a gradient maker as described

previously (Pezzementi et al., 2012). Mixtures were combined into a

sedimentation gradient by using a gradient maker. Each week for four weeks, six

gradients will be made and stored at 4C. Three media samples and three extract

samples with the most enzymatic activity as determined by a preliminary enzyme

screen were added to the gradients with catalase. The gradients were then

centrifuged overnight on the Ultracentrifuge at 34-38,000 rpm. After

centrifugation, a fraction collector was used to obtain 35-40 equal fractions of

each gradient.

7. Ellman’s Esterase Assay

Ellman’s solution (100 mM NaHPO4, pH 7, 0.3 mM DTNB, 167 mM NaCl, 258 μM

Triton X-100, and 1 mM ATCh) was prepared during the fractionation as

previously described (Pezzementi et al., 2012). The contents of each fraction

tube was added to a 96-well micro-titer plate, combined with Ellman’s solution,

and then analyzed by spectrophotometry. Data was fit to Michaelis–Menten

kinetics equations using SigmaPlot. Integration was performed (Pezzementi et

al., 2012).

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RESULTS

QuikChange site-directed mutagenesis was successful in expression vectors

pEGFP and pcDNA3.1 in inserting a second NotI restriction site. This successful

mutation was confirmed by the subsequent agarose gel electrophoresis. Once these

mutants were transformed to chemically competent E. coli, it was realized that the

mutation may not have been successful for pEGFP as no growth had occurred on the

selection plates. It was demonstrated that pcDNA3.1 has been successfully mutated

into pcDNA3.1-Lpd-STOP-NotI. After multiple attempts to genetically engineer pEGFP

into pEGFP-Lpd-STOP-NotI with no success, that expression vector was abandoned,

and the study continued with pcDNA3.1-Lpd-STOP-NotI (figure 4).

Figure 4. Final genetically engineered pcDNA3.1 expression vector. pcDNA3.1 expression vector was genetically engineered to add a second NotI restriction site

upstream from the endogenous NotI site. Agarose gel electrophoresis confirmed NotI restriction digestion in pcDNA3.1-

Amp

pcDNA3.1

NotI

11

Lpd-STOP-NotI. As noted, the digests were excised from the gel, and the final

[pcDNA3.1-Lpd-STOP-NotI] was 18.4 ng/L in the most concentrated sample. Newly

ligated pcDNA3.1-Lpd-pcDNA3.1 showed effective transformation with Invitrogen

DH5 chemically competent E. coli, and the final [pcDNA3.1-Lpd-pcDNA3.1] after

Qiagen spin miniprep was 356.6 ng/L in the most concentrated sample. Post-ligation

DNA sequence analysis of the vector confirms previous engineering of the STOP

codon, 5’TAA3’ at base pair 336, as well as the region of interest, the newly engineered

NotI restriction site. This new NotI site, 5’GCGGCCGC3’, was found to begin at base

pair 371, upstream from the endogenous NotI site (Figure 4). A Qiagen maxiprep DNA

amplification of the pcDNA3.1-LpdX gave a [pcDNA3.1-LpdX] of 1533.0 ng/L.

After four trials, it was found that with increasing concentration of Lpd,

tetramerization of DvAChE increased, however only slightly. It was found

observationally that there was slightly more G4 in the extract than in the media on

average, but this did not correlate with enzymatic activity. Overall, the data show no

drastic change in AChE enzymatic activity from the control (no Lpd) to 0.1 g/well, but

there was a noticeable decline in ChE enzymatic activity with increasing activity to 1.0

g/well. Across the trials (n=4), cellular enzymatic activity as a percent of the total

control activity was equal to 25.99±1.79% (control; mean±SE), 27.51±5.91% (0.1

g/well; mean±SE), and 13.32±1.08% (1.0 g/well; mean±SE). The drop in activity was

also found to be in the media as shown by the secreted enzymatic activity as a percent

of the total control activity. Again with n=4, secreted activity was 74.00±1.79% (control;

mean±SE), 70.75±11.86% (0.1 g/well; mean±SE), and 44.51±2.55% (1.0 g/well;

12

mean±SE). These data are displayed in Figure 5.

DvAChE + pcDNA-LpdX

[Lpd] g/culture

Control 0.1 1

% G

4 o

n G

rad

ient

-60

-40

-20

0

20

40

60

80

DvAChE + pcDNA-LpdX

[Lpd] g/culture

Control 0.1 1

% A

ctivi

ty a

s P

erc

ent o

f T

ota

l Co

ntr

ol A

ctivi

ty

-40

-20

0

20

40

60

80

Figure 5. G4 increased with increasing Lpd but enzymatic activity still down regulated. After co-transfection with DvAChE and various [pcDNA3.1-LpdX],

Michaelis–Menten kinetics equations were fit to data from the Ellman’s esterase assay. Integration was performed, and percent G4 on the gradient as well as percent activity was calculated. (A) With increasing [Lpd], tetramerizaiton only marginally increased,

even in the media. (B) DvAChE enzymatic activity decreased in response to increasing [Lpd]. Data shown as mean ± standard error. N=4.

DISCUSSION

The novel finding of Lpd to be associated with ChE tetramer formation through

the WAT-PRAD interaction has been widely demonstrated since first reported by Li et

al. (2008). This finding has been especially significant due to the fact that the ChE

tetramer is the only functional form of the enzyme (Dvir et al., 2004). It is thought that

Lpd may increase production of tetramerization due to the poly-proline nature of the

PRAD near the carboxy-terminus (Biberoglu et al., 2012; Biberoglu et al., 2013; Li et al.,

2008; Pezzementi et al., 2012). Again, because of the finding by Dvir et al. (2008),

Extract

B A

Media Media

Extract

13

among others, as to the tetramer being the only functional cholinesterase form, an

increase in tetrameric forms should increase the enzymatic activity. The results

presented here do not support this hypothesis (Figure 5).

One of the Lpd expression vectors used in our study was previously described by

Krause et al. (2004). As shown in Figure 2, the Lpd construct is genetically fused to an

enhanced green fluorescent protein (EGFP), as Krause et al. (2012) performed

fluorescence experiments in their study. Data analyzed before this current study

showed that AChE enzymatic activity was decreasing in response to an increase of

[Lpd] (Figure 1). It was hypothesized that transcription of the Kan/Neo antibiotic

resistance cassette could be producing small-interfering RNA (siRNA) which may serve

in an inhibitory capacity for protein translation (Baens et al., 2006; Koelsch et al., 2013;

Nejepinska et al., 2012).

Further genetic engineering with expression vector pEGFP was abandoned after

agarose gel confirmed that the mutation of the NotI site had occurred. Even though the

gel demonstrated a successful mutation, pEGFP-Lpd-EFGP was unable to be

transformed with chemically competent E. coli. It is possible that the cells used in both

pEGFP transformations were not viable, even though aliquots had been properly stored

at -80C. Due to the fact that both pcDNA3.1-Lpd-EGFP transformations were

successful after site-directed mutagenesis, it is highly unlikely and would represent a

coincidental anomaly for both pEGFP mutant samples to be unable to be transformed.

A more likely explanation is an error with site-directed mutagenesis. Our study was able

to proceed as we had a viable vector with Lpd present and EGFP excised, pcDNA3.1-

LpdX.

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Contrary to our hypothesis that EGFP was the cause of ChE down regulation, the

mean enzymatic activity declined when expressed with just Lpd. As seen in figure 5, the

decrease appears to be in a dose-dependent manner as well. Tetramerization was

unaffected by dosing of Lpd. Of all the MRL proteins studied, Lpd is the only protein to

cause a decrease in ChE enzymatic activity. Concurrent studies by Babi (unpublished,

2015), Cain (unpublished, 2015), and Mayfield (unpublished, 2015), all show that

expression of either DvAChE or hBChE with RIAM, poly-L-proline, or MIG-10 increases

tetramerization but does not affect ChE enzymatic activity. These data present a new

question regarding possible structural differences between Lpd and others of the MRL

protein family.

The amino acid sequences of MIG-10, RIAM, and Lpd share some distinctive

features, indicating evolutionary conservation (Legg and Machesky, 2004). While these

MRL proteins are not present in the lower invertebrates, soluble tetrameric assemblage

is possibly achieved by a WAT-PRAD interaction of some other poly-proline intracellular

protein (Pezzementi et al., 2015). The MRL proteins do share similar regions in their

polypeptides, and these homologous domains have been named RA, PH, and PRI II by

Legg and Machesky (2004). Because this study was the first in our laboratory to

express Lpd with any ChE, albeit AChE or BChE, our study of Lpd is somewhat behind

RIAM. This genetic engineering project was first necessary to isolate Lpd from the Lpd-

EGFP fusion protein in the expression vectors. After the positive excision of EGFP, the

study of the entire Lpd protein and its effects on ChE tetramerization and activity levels

began. Now that it is known that EGFP was not the cause of the ChE down regulation,

Lpd needs to be studied segmentally like studies of RIAM (Babi, unpublished, 2015;

15

Cain, unpublished, 2015). In its current form in our laboratory, however, Lpd exists as

the complete protein, so future projects would need to engineer truncated clones of the

Lpd genome for expression.

CONCLUSION

We hypothesized that EGFP, a fluorescence protein fused to Lpd in the

expression vector pcDNA3.1, was the cause of previously studied ChE enzymatic

activity down regulation. After successful excision of EGFP by NotI restriction digestion,

we expressed pcDNA3.1-LpdX with DvAChE. Our data demonstrate that while

tetramerization was slightly increased, AChE enzymatic activity decreased substantially.

Therefore, we are able to conclude that Lpd, not the hypothesized EGFP, is the cause

of ChE enzymatic down regulation. Future studies should examine domains of Lpd in

order to determine which region(s) are the decrease in enzymatic activity.

ACKNOWLEDGMENTS

This research was supported through the BI 470/472 senior capstone experience

program in the Department of Biology at Birmingham-Southern College. Dr. Leo

Pezzementi was of invaluable assistance throughout the entire project as the project

sponsor and second author of this paper. Dr. Megan Gibbons’s help is also greatly

appreciated through contributions during Summer 2014 and through the BI 470

proposal process. Kyle Cain and Mohamad Babi assisted greatly in the laboratory.

Heather Daniel and Jacob Mayfield helped with data analysis. Isa Delgado assisted with

lab maintenance, and the Genomics Core Laboratory at the University of Alabama at

Birmingham did DNA sequencing.

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