Gene, 121 (1992) 295-304

0 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/92/$05.00

GENE 06772

Production and secretion of high acetylcholinesterase in cultured cell lines: subunit

295

levels of recombinant human microheterogeneity of the catalytic

(Eukaryotic vectors; 293 cells; cytomegalovirus promoter; glycosylation; signal processing)

Chanoch Kronman a, Baruch Velan a, Yehoshua Gazes a, Moshe Leitner a, Yehuda Flashner a,

Aryeh Lazarb, Dino Marcus b, Tamar Sery a, Yoel Papierb, Haim Grosfeld a, Sara Cohen a and

Avigdor S htierman a

a Department of Biochemistry, and b Department of Biotechnology, Israel Institute for Biological Research, 70450 Ness-Ziona, Israel

Received by J.K.C. Knowles: 11 February 1992; Revised/Accepted: 24 June/30 June 1992; Received at publishers: 3 August 1992

SUMMARY

To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was

designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell

lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA

was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR)

or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was

achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression

vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting

to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of

6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a

K, value of 120 PM for acetylthiocholine; a k,,, value of 3.9 x 105/min, and selective inhibition by AChE-specific inhibi-

tors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67-

and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by

N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two

alternative cleavage sites for the removal of the signal peptide, in which the ‘mature’ position 1 is either Ala31 or Gly33. Both

of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected process-

ing of signal peptides on a native molecule.

Correspondence to: Dr. A. Shafferman, Israel Institute for Biological Re-

search, P.O.B. 19, 70450 Ness-Ziona, Israel. Tel. (972-8)381408/381518;

Fax (972-8)401404.

antigen H-2L of the d haplotype; hAChE, human AChE; hAChE, gene

(DNA) encoding hAChE; kb, kilobase or 1000 bp; LTR, long termi-

nal repeat; K,, Michaelis-Menten constant; k,,,, catalytic first-order rate

constant; MCS, multiple cloning site; neo, gene encoding neomycin phos-

Abbreviations: aa, amino acid(s); AChE, acetylcholinesterase; bp, base photransferase; nt, nucleotide(s); R, resistance/resistant; re, recombinant;

pair(s); CMV, cytomegalovirus; DMEM, Dulbecco’s modified Eagle’s RSV, Rous sarcoma virus; SDS, sodium dodecyl sulfate; SV40, simian

medium; FBS, fetal bovine serum; H,Ld, mouse major transplantation virus 40; t-PA, tissue-type plasminogen activator; u, units; wt, wild type.

296

E + H + Pol I k

B 3 CMV Promoter; B = RSV LTR; m = SV40 Promoter;

immmmnU t H2Ld Promoler; B I SV40 Poly(A).

Fig. 1. Construction of multicistronic hAChE expression vectors. The genetic elements used for construction of the multicistronic vectors were as follows.

The hAChE cassette: the pL5CA hAChE expression vector has been described previously (Velan et al., 1991a) and was used for construction of vari-

ous derivatives. Plasmids pL5RA and pL5S.A are equivalent to pL5CA and differ only in the promoter controlling hAChE transcription; in pLSRA, the

promoter element is the RSV LTR (Gorman et al., 1983), while in pL5SA the hAChE cDNA is under control of the SV40 early gene promoter (Gorman

et al., 1982). The neo cassette: the HzLd murine histocompatability gene promoter was isolated as an Xbal-BarnHI fragment from pLd (Evans et al., 1983)

and inserted between the XbaI and BarnHI sites of pGEM-7Zf( - ) (P romega, Madison, WI) to give rise to pHP. A BglII-BarnHI fragment encompassing

the neo gene and the downstream SV40 poly(A) signal was isolated from a derivative of pSV2NEO (Southern and Berg, 1982) from which nonessential

sequences residing between the SmaI and HpaI sites have been previously deleted. This fragment was cloned into the unique BarnHI site downstream

from the H,Ld promoter within pHP. The resultant plasmid, pHPNEOSA, contains the neo gene under control of the HzLd promoter. The dhfr cassette:

two different cassettes were constructed: (1) wt dhjr cassette which should allow gene amplification in dizj? cells and (2) a mutant MTXR dhfr cassette

allowing gene amplification in dfhr+ background (Simonsen and Levinson, 1983). The unique Hind111 site within pSV2DHFR (Subramani et al., 1981)

was eliminated (by filling in) and the resulting PvuII-BglII fragment encompassing the SV40 early gene promoter and dhfi gene was cloned into a deriv-

ative of pGEM-9Zf (Promega) in which the original MCS was replaced by a synthetic MCS. A HpaI-BamHI fragment containing the SV40 poly(A) site

was isolated from pSV2NEO and inserted downstream from the dhf gene to generate pSPDHSA-1. The dhjr MTXR variant was generated by replac-

ing the PJIMI-BstXI fragment in pSPDHSA-I, with a synthetic DNA fragment in which the wt codon 22 (CTA, Leu) is mutated (CGA, Arg). The re-

INTRODUCTION

Acetylcholinesterase (acetylcholine hydrolase, EC

3.1.1.7, AChE), an enzyme of cardinal importance in neu-

rotransmission systems, is responsible for rapid termina-

tion of impulse transmission at cholinergic synapses by

hydrolysis of the neurotransmitter acetylcholine (Rosen-

berry, 1975). Though the complex structure and organiza-

tion of the AChE holoenzyme of various sources has been

extensively studied (Massoulie and Bon, 1982; Gennari

et al., 1987; Chatonnet and Lockridge, 1989; Taylor, 1991)

detailed analysis of the structure and properties of human

AChE has been hampered due to inadequate sources for

sufficient amounts of homogeneous soluble enzyme. The

elucidation of the gene structure of mammalian AChE (Li

et al., 1991), isolation of hAChE cDNA from human brain

(Soreq et al., 1990) and observation that this cDNA codes

for a soluble, secretable enzyme (Velan et al., 1991a) pave

the way for generation of substantial quantities of reh-

AChE.

Various parameters which influence production effi-

ciency of recombinant polypeptides have been established

in optimal systems for high expression of foreign genes.

These include the choice of an appropriate cell line and

method of gene introduction (Sompayrac and Danna, 198 1;

Gorman et al., 1983; Chen and Okayama, 1987), selection

of highly potent promoter sequences which should enable

high transcription levels of the gene of choice (Friedman

et al., 1989; Israel et al., 1989; Sevarino et al., 1989) and

inclusion of accessory genes allowing gene amplification

such as the dhfr gene (Ringold et al., 1981; Kaufman et al.,

1985; Conners et al., 1988). An important feature of opti-

mized cellular production of foreign proteins is that the

production design program must select the correct combi-

nation of host cells and expression elements (Laimins et al.,

1982). A wide range of foreign protein synthesis levels in

various systems has been reported (Whittaker et al., 1987;

Goto et al., 1988; Friedman et al., 1989; Hendricks et al.,

1989; Filbin and Tennekoon, 1990). Generally, a synthesis

level of over 1 pg of foreign protein per lo6 cells/24 h is

considered to be high (Yan et al., 1989) and is sufficient for

extensive biochemical analysis of the product. Very effi-

cient systems have been shown to display synthesis levels

of over 100 pg per lo6 cells/24 h (Cockett et al., 1990).

The biological activity of a complex secreted glycopro-

tein such as AChE would probably depend upon post-

translation modifications specific to eukaryotic cell sys-

297

terns. We have recently reported that transient transfection

of the human 293 cell line with an expression vector con-

taining the hAChE cDNA allowed synthesis and secretion

of functional AChE (Velan et al., 1991a). We set forth to

establish stable homogeneous cell lines which produce and

secrete constitutively high levels of rehAChE, utilizing a

series of expression vectors which contain the hAChE

cDNA under control of various eukaryotic promoters. In

the present study, we describe the isolation of several stable

human 293 cell lines which produce and secrete high lev-

els of hAChE reaching up to 25 pg/106 cells/24 h. Soluble

recombinant enzyme secreted by high producer cell lines

was purified and analyzed for catalytic properties as well

as for post-translational modifications, such as processing

of the N terminus and glycosylation.

RESULTS AND DISCUSSION

(a) Optimization of production of rehAChE in eukaryotic

cells

Several recombinant plasmids which allow expression of

the hAChE gene were designed. The multicistronic expres-

sion vectors contain the hAChE cDNA (coding for the

soluble hAChE form: Soreq et al., 1990) downstream from

various eukaryotic promoters, as well as expression ele-

ments for the selectable markers neo and dhfr. To allow for

maximum flexibility, the various genetic elements compris-

ing the hAChE expression vectors were engineered as por-

table DNA fragments bounded by unique restriction sites.

A schematic representation of various hAChE expression

vectors is given in Fig. 1.

To determine the optimal host for expression of reh-

AChE, we examined intracellular and extracellular levels of

AChE following transient transfection of several estab-

lished cell lines with the pL5CAN expression vector in

which the hAChE-coding sequences are under control of

the CMV immediate-early promoter. Three human cell lines

were employed: 293 (transformed primary human embry-

onal kidney) cells, HeLa cells and HeLaS3 cells. The 293

cells allow high production levels of foreign protein follow-

ing transfection and are able to cope with intricate post-

translation processing requirements (Yan et al., 1989). The

HeLaS3 cells differ from HeLa cells in their ability to adapt

to growth in suspension and may thus prove to be benefi-

cial for large-scale growth. Rodent cells tested include Rat2,

BHK and CHOdhfr- cells. Determination of secreted and

sultant plasmid is named pSPDHmSA-1. The final constructs pLSCAN, pACHEl0 and pACHE4 contain the hAChE cDNA under control of the CMV

promoter. In pACHE20 and pLSSAN, hAChE transcription is controlled by the RSV-LTR and SV40 promoters, respectively. Plasmids pACHEl0 and

pACHE20 contain a mutated version of the dhfi gene (Simonsen and Levinson, 1983). In contrast to pLSCAN, pACHEl0 and pACHE20 which include

a neo gene under control of the murine HzLd promoter, in pACHE4 the neo gene is controlled by the SV40 promoter. Restriction enzyme and recogni-

tion site designations: BamHI - Bm; BclI - Bc; BglII - Bg; BspMII - Bs; BstXI - Bt; &I - C; EcoRI - E; Hind111 - H; KpnI - K; PflMI - P; Sac1

- S; .SpeI - Sp; X&I - X. PolIk, Klenow (large) fragment of E. co/i DNA polymerase I.

298

intracellular levels of AChE activity in each of these cell

lines prior to transfection revealed negligible amounts of

AChE activity (less than 10d3 units/ lo6 cells/24 h). 24 h

following transfection, the human cell lines 293, HeLa and

HeLaS3 displayed higher transient levels of rehAChE than

the rodent cell lines BHK, Rat2 or CHOdhfr~ (Table I).

(Intracellular levels of AChE did not increase significantly

above background level in all cell lines.)

To determine the optimal promoter for high-level expres-

sion of the hAChE gene, various plasmids in which the

hAChE cDNA is under control of either the CMV (pLSCA,

pLSCAN), RSVLTR (pLSRA), or SV40 (pL5SA) promot-

ers were used to transfect the human cell lines. These pro-

moters have been found efficient in various eukaryotic ex-

pression systems (Sompayrac and Danna, 1981; Foecking

and Hofstetter, 1986). Enzymatic activity secreted into the

cell growth medium was determined (Table II). As ex-

pected, the CMV promoter proved to be more efficient than

the RSV or SV40 promoters in HeLa cells. However, in

293 cells, the CMV and RSV promoters were apparently

equally efficient in driving expression of the hAChE cDNA

while the SV40 promoter was less efficient. Comparison of

the two cell lines showed that the ratio of rehAChE expres-

sion under the RSV promoter is approximately 7: 1 in favor

of 293 cells, while the ratio of SV40-driven expression is

inversed (> 1:5). These results demonstrate the complex

relationships between gene expression, various promoters

and different cell lines. This observation substantiates the

need for experimental studies prior to selection of an op-

TABLE I

Expression of the hAChE gene under the CMV promoter in various cell

lines transiently transfected with pL5CAN

Cells:* CHOdhfi- BHK Rat2 HeLa HeLaS3 293

Exp.

No. hAChE activity (lo-’ u/IO6 cells)b

1 < 1.0 11.0 9.0 120 80 170

2 13.0 11.0 120 _ 380

3 < 1.0 7.0 - _ 100

4 4.0 - _ 50

5 70 - 160

’ 293(ATCC CRL1573), HeLa(ATCC CCLZ), HeLaS3 (ATCC

CCL2.2), BHK-21(ATCC CCLlO) RAT-2(ATCC CRL1764) and CHO-

dhfr- (ATCC CRL9096) were all obtained from the American Type Cul-

ture Collection and cultivated as recommended by ATCC.

’ Cells (1.5 x IO6 in lOO-mm plates) were transfected by the Ca,phosphate

coprecipitation method as modified by Wigler et al. (1977). 24 h after

transfection, cells were rinsed and refed. Secreted AChE levels were de-

termined 24 h post medium change by the Ellman method (1961), using

the procedure described earlier (Velan et al., 1991b). Background AChE

levels determined immediately after refeeding (l-3 x 10 3 u/ml, depend-

ing on the cell line) were substracted from the 24-h values. AChE values

are an average of results obtained in three independent transfections

comprising each experiment.

TABLE II

Efficiencies of CMV, RSV and SV40 promoters in transient expression of

hAChE in human cell lines

Promoter: CMV RSV sv40

Cell line”

hAChE activity (10 3 u/lo6 cells)b

293 110 160 < 1.0

160 120 8.0

250 _ 5.0

HeLa 120 20 20

120 34

rl See Table I, footnote a.

b Cells were transiently transfected with expression vectors in which the

hAChE cDNA is under control of the CMV promoter (pLSCA), the

RSV-LTR (pL5RA) or the SV40 early promoter (pL5SA). (For details on

transfection and AChE assay, see Table I, footnote b.)

timal configuration of an expression system for a given

gene. This is true in particular for 293 cells in which the

endogenously expressed adenovirus ElA protein was

shown to activate some promoters while repressing others

(Lewis and Manley, 1985). In light of these results, we

decided to examine both CMV-AChE and RSV-AChE ex-

pression elements as possible candidates for optimal ex-

pression of hAChE.

(b) Establishment of cell lines secreting rehAChE

Of the various mammalian cell lines examined as can-

didates for AChE production and secretion, the human cell

lines 293, HeLa and HeLaS3 displayed significantly higher

transient levels of rehAChE than the rodent cell lines (see

Table I). Selection for stable integration was accompanied,

however, by a complete loss of AChE expression in the

HeLa cells but not in 293 cells. The 293 cells were there-

fore chosen as host cells for stable transfection with hAChE

expression controlled by the most promising promoters,

CMV and RSV LTR (Table II). Cells were transfected with

the hAChE expression vectors pACHE10, pACHE20 or

pLSCAN, which contain a copy of the neo gene under

control of the H2Ld promoter. While hAChE expression is

controlled by the CMV promoter in pACHE10 and

pLSCAN, in pACHE20 hAChE expression is driven by the

RSV-LTR promoter unit. pL5CAN differs from pACHEl0

and pACHE20 by not containing a copy of the dhfr gene.

Each experiment was comprised of four to five independent

transfections. Cells in which stable integration of the plas-

mid has occurred were selected by G418. As shown in

Table III, secreted AChE activity of the stably transfected

cell pools, was mostly within a range of 0.5-4 u/lo6 cells

during 24 h. In two cases, AChE activity was considerably

higher: in one instance a pACHEl0 (Exp. 1) transfection

gave rise to 60 AChE u/lo6 cells during a 24-h period.

299

TABLE III

Establishment of high-hAChE-producer 293 cell lines

Step” Procedure Vecto& pACHEl0 (exp. 1) pAChEl0 (exp. 2) pAChE20 pL5CAN

I Selection by G418

Pool AChE activity’ 0.6-1.5; 60 0.08-l 0.08- 1 2-4; 60

II First cloning cycle

Number of cell clones established 287 259 180 45*

Number of clones expanded 45 26 12 10

Range of AChE activity 5-26 30-80 0.06-16 5-100

in expanded clones’

III Subcloning

AChE activity in highest

producer subclones

Subclone Activity Subclone Activity Subclone Activity Subclone Activity

lo-2FlO G9 105 ClObl-D2-E6 50 20 AD5 E8 28 C33-20-B4-2C4 150

IO-2FlO El0 40 ClObl-D4-E2 45 20ADS C3 7 C33-20-B4-lC2 85

lo-18B9 D4 20 ClObl-D4-F7 35 C33-20-B4-1C5 73

lo-3C2 D8 18 ClObl-C7-E5 20

‘I Each experiment consists of four to five independent transfections. 48-h post-tranfection cells were refed with growth medium (DMEM + 10% FBS)

containing 0.8 mg/ml of the neomycin analogue, G418 (Geneticin, Sigma). Growth in the presence of the drug was continued for approx. three weeks.

AChE levels of the stable pool cells were determined and individual cells were cloned by limiting dilution in 96 well microtiter plates (cell concentration = 0.3

cells/well volume), in the presence of DMEM containing 20”/, FBS. Relative AChE levels were determined for the various clones and high AChE pro-

ducers were expanded. To ascertain clonality, cells were subcloned by limiting dilution as above. Once again, relative high AChE producers were expanded.

b See Fig. 1.

’ AChE activity values are given in units per lo6 cells during 24 h. Extensive clumping of 293 cells following trypsinization, renders cell counting inac-

curate and therefore values of AChE activity/cell should be considered correct within a f 5O”/b range.

d In this experiment, cloning was performed only to cells comprising the high level pool exhibiting 60 u/IO6 cells.

Likewise, a single transfection with pL5CAN resulted in a

similarly high enzymatic activity (Table III).

Individual clones of the transfected pACHE10,

pACHE20 and pL5CAN pools were isolated by limiting

dilution followed by seeding onto 96-well microtiter plates.

(In the case of pLSCAN-transfected cells, only the cell pool

exhibiting 60 u of AChE/ lo6 cells during 24 h was subjected

to clone isolation.) Cell clones producing high levels of

AChE were expanded, and AChE values for each expanded

clone was determined. The range of activity exhibited by

the various clones, upon reaching confluence (Table III),

suggests that subclones harboring pACHEl0 or pL5CAN

vectors may reach higher AChE levels than cells contain-

ing pACHE20. To ensure homogeneity of the cell clones,

individual cells comprising the cell clone population were

subcloned. The AChE values demonstrated by high- pro-

ducer subclones are presented in Table III. Subclones har-

boring pACHEl0 or pL5CAN exhibit enzymatic values

which may reach up to 150 u/lo6 cells during a 24-h period.

(c) High level of expression of rehAChE Our considerations in design of an optimal expression

vector included the choice of a potent promoter controlling

AChE transcription (Table II) and a selector gene under a

weak [murine histocompatibility (H2Ld)] promoter. Usu-

ally, selector cassettes in eukaryotic vectors rely on the use

of a strong promoter such as SV40 early promoter for ex-

pression of the selection gene product. In contrast to the

highly potent SV40 early promoter we chose the murine

H,Ld promoter which was shown to function inefficiently

in murine cells (Vogel et al., 1986). Furthermore, histocom-

patibility genes were shown to be repressed in 293 cells

(Lewis and Manley, 1985). We reasoned that the bacterial

neo gene under the control of the weak H,Ld promoter will

confer G418 resistance only upon cells which have inte-

grated the plasmid into sites of active transcription within

the chromosome which will allow high level of transcription

of the neo gene. This in turn may lead to a concomitant high

transcription levels of the proximal hAChE cDNA.

The contribution of plasmid copy number to the produc-

tion levels of hAChE, was assessed by Southern blot anal-

ysis performed on high-producer cell lines (not shown).

Only one to three copies of the hAChE cDNA were found

in cells producing as high as 20-150 u of hAChE/106 cells

per day (lo-18-B9-D4 and C33-20-B4-2C4; Table III).

These results suggest that the most pertinent factor deter-

mining production levels in this system is the specific site

of integration into the host genome rather than a high num-

ber of integrated cDNA copies. Indeed, a comparative

study revealed that the number of productive chromosomal

integrations giving rise to G418R colonies was at least ten-

fold lower with the vector containing the H,Ld-neo con-

struct (pACHE1) than with that containing the analagous

SV40-neo construct (pACHE4). This observation would

300

be consistent with the notion that the weak H,Ld promoter

restricts cell survival in the presence of the neomycin an-

alogue, G418, to cells in which integration occurred into

transcriptionally active sites in the chromosome. Using this

strategy we were able to isolate several high-hAChE-

producer 293 cell clones when using either a CMV or RSV-

LTR promoters to drive AAChE transcription. Some clones

displayed stable levels of secreted hAChE which reach up

to 150 u/10’ cells during a 24-h period (Table III). The

highest amount of protein synthesized and secreted is ap-

proximately equivalent to 25 pg/cell per day, which corre-

sponds to 10% of the total cellular protein synthesis under

optimal growth conditions (Griffiths, 1990). Following

more than 50 cell passages in the absence of G418, all

tested high producer subclones retained a stable produc-

tion level of rehAChE (within a 10% deviation). A similar

method to attain cell lines producing high levels of recom-

binant protein was pursued by Conners et al. (1988), by

linking the t-PA gene to a neo gene under the control of a

weak murine /I-globin promoter. However, in their system

high production levels of t-PA were obtained only after dhfr

coamplification, resulting in cell lines containing approxi-

mately ten integrated copies of the t-PA gene.

A clear advantage of our expression system is that prop-

agation of producer cell lines does not require the presence

of a selective drug for preservation of high levels of reh-

AChE synthesis. Inclusion of the dhfr gene in several of the

hAChE expression vectors will allow us to determine

whether the high levels of hAChE synthesized in the 293

established clones represent the upper limit of foreign pro-

tein synthesis potential, or whether higher rates may be

approached following gene amplification.

(d) Biochemical characterization of secreted rehAChE

The rehAChE secreted by the established cloned cell

lines (Table III) C33-20-B4-2C4 and ClO-bl-C7-E5 was

purified, utilizing procainamide affinity columns (Fig. 2).

The purification profile of C33-20-B4-2C4 is given in Table

IV. Specific activity of the purified enzyme was approx.

6000 u/mg protein. The polypeptide eluted from the first

and second procainamide columns appears mainly as a

discrete band of 130 kDa accompanied by two weaker

bands of 70 kDa and 67 kDa (lanes 4,5). Upon reduction

with P-mercaptoethanol, the 130-kDa band virtually dis-

appears while the two bands of 70 kDa and 67 kDa are

intensified (lane 6). It therefore appears that the secreted

hAChE consists mostly of disulfide-bonded oligomeric

forms as suggested previously (Velan et al., 1991b). Immu-

noblots developed with sequence-specific anti hAChE an-

tibodies, indicate that all protein bands in the purified prep-

aration are AChE-related molecules (Fig. 2, lanes 7-9).

Specific activity of the purified enzyme was also deter-

mined by active site titration. The hAChE preparations

12345 6 kDa

140-

94-

67-

rpro ..

43-

78 9

Fig. 2. SDS-PAGE and Western blot analyses of hAChE. Samples from

the various purification steps (Table IV) were loaded on a 0.1% SDS-

10% polyacrylamide gel (Laemmli, 1970). 4.5 pg (lanes l-6) or 0.15 pg

(lanes 7-9) was loaded on an SDS polyacrylamide gel in absence (lanes

1-5, 7,8) or presence (lane 6 and 9) of /%mercaptoethanol. The gel was

either stained with Coomassie blue (lanes 1-6) or electrotransferred to

nitrocellulose paper and labeled with alkaline phosphatase-conjugated

anti-hAChE antibodies (lanes 7-9) as described elsewhere (Velan et al.,

1991b). The purification method for rehAChE is described in Table IV,

footnote a. Lanes: 1, loaded material; 2, flow-through of the first procain-

amide column; 3, protein released in presence of 0.4 M NaCl (see legend

to Table IV); 4, protein eluted from first procainamide column with deca-

methonium (0.15 M); 5, elution from second procainamide column with

decamethonium (0.15 M); 6, same as 5, but sample was boiled in the

presence of ,!?-mercaptoethanol (0.7 M) prior to loading; 7, Western blot

of 5; 8, Western blot of 293 mock-transfected purification product; 9,

Western blot of 6.

were incubated in the presence of varying amounts of the

inhibitors MEPQ [ 7-(methylethoxy-phosphinyloxy)-l-me-

thylquinolinium iodide] (Levy and Ashani, 1986), or with

Soman (methylpinacolylphosphono-fluoride). Following

incubation at 25 “C for 2 h, residual activity of enzyme-

inhibitor mixture was determined. With both inhibitors, 0.4

u of rehAChE was found to be equivalent to 1 pmol of

active sites. Based on this result and using the apparent

value of 70 kDa for hAChE (Fig. 2, lane 6) a specific ac-

tivity value of approx. 5650 u/mg protein was determined

confirming the purity and integrity of the protein prepara-

tion. A summary of kinetic parameters determined for the

purified recombinant enzyme is given in Table V. All pa-

rameters determined, including the extremely high turnover

number (k,,& are similar to those reported for other eu-

karyotic AChE (Rosenberry and Scoggin, 1984; Ralston

et al., 1985).

(e) Glycosylation heterogeneity of rehAChE subunits ex-

pressed in 293 cells

To examine the possibility that differential glycosylation

of the enzyme molecule is responsible for the 70-kDa and

67-kDa polypeptides (Fig. 2, lane 6), purified rehAChE was

treated with N-glycanase, which catalyzes the hydrolysis of

Asn-linked oligosaccharides (Elder and Alexander, 1982).

Following incubation with N-glycanase (Fig. 3, lane 3), a

discrete band of 62 kDa was discerned while the 67-kDa

301

TABLE IV

Purification of rehAChE

Purification steps” Activity (u/ml) Protein” (mg/ml) Specific activity (u/mg) Purification factor Accumulated yield (%)

Cells supernatant

Ultr~ltration

Procainamide

60 3 20 1 100

160 4 40 2 90

chromatography I

Procainamide

3 000 1.2 2500 125 65-70

chromatography II 21000 3.5 6000 300 55-60

a Recombinant human AChE was purified from the high AChE producer cell line C33-20-B4-2C4 (200 ml growth medium) on proc~n~ide-Sepharose

aIIinity columns (Ralston et al., 1985). Cell culture supernatants were concentrated and dialyzed by ultratiltration (Minitan System, 100 K cutoff mem-

branes). Dialysis buffer was 10 mM Na.phosphate buffer pH 8.0. The concentrated enzyme solution was adsorbed to the procainamide-Sepharose 4B

column (3000 u/ml resin) which was then rinsed with 50 mM Na.phosphate buffer pH 8.0/l mM EDTA, and again with 50 mM Na*phosphate buffer

pH S.OjO.4 M NaCl/l mM EDTA. Enzyme elution was performed with decamethonium (0.15 M) in 50 mM Na.phosphate buffer pH 8.0/l mM EDTA.

Further purification was achieved on a second procainamide column as above. Purified enzyme was dialyzed against 50 mM Na.phosphate buffer pH

8.0 and concentrated by ultr~ltration (Minit~, 30 kDa cutoff membrane).

’ AChE activity was assayed according to Ellman et al. (1961).

’ Protein contents was determined according to Lowry et al. (1951).

TABLE V

Summary of enzymatic properties of rehAChE kDa

K,” (ATC) I20 pM

kc,, 3.9 x lO’/min

Activity ratiob g

ATC Activity ratiob PTC

BW284C5 1 IC,,” 0.008 frM

iso-OMPA IC,,” 200 nM

Substrate inhibition” >lmMATC

Specific activityd 6000 u/mg

a Determinations were carried out as described previously (Velan et al.,

199Ib).

b In substrate specificity experiments, acetylthiocholine (ATC was re-

placed by 0.5 mM of butyrylthiocholine (BTC) or propionylthiocholine

(PTC).

c IC,,, Inhibition concentration 50 (concentration which causes 50%

inhibition). Inhibition by BW284C51 (a specific inhibitor of AChE) and

iso-ompa (a specific BChE inhibitor) was carried out as described previ-

ously (Velan et al., 1991a).

d See Table IV.

and 70-kDa bands disappeared. (Extensive incubation with

N-glycanase led to the appearance of faster migrating bands

related to AChE degradation, lane 4.) The 62-kDa band

represents a genuine AChE form as it was identified by

rabbit anti-hAChE antibody when subjected to Western

blot analysis (Fig. 3, lanes 3’,4’). This result suggests that

both the 67-kDa and 70-kDa bands represent differentially

I?-glycosylated versions of the rehAChE monomer. Indeed,

the 62-kDa band is a sharply defined band and differs from

the fuzzy wide bands which characterize microheteroge-

neous glycosylated polypeptides. This apparent molecular

size of the nonglycosylated monomer is in good agreement

with a calculated value of 64.5 kDa based on aa compo-

1 2 3 4 1' 2’ 3’ 4’

94+

Fig. 3. N-Glycanase digestion of rehAChE. Denatured purified hAChE

samples were treated with N-glycanase. Denatured AChE (30 ng) was

subjected to digestion with 0.625 u of N-glycanase [peptide-N”-(N-acetyl-

b-glucosaminyl) asparagine amidase; Genzyme, USA], at 37°C. Diges-

tion buffer included 200 mM Na.phosphate buffer pH 8.0/0.17x SDS/

1.25% NP-40/30 mM ~-mercaptoethanol. Samples (4.5 ng protein each)

removed at various times were rapidly frozen, then boiled (5 min) in the

presence of B-mercaptoethanol, resolved by 0.1% SDS-IO% PAGE and

stained with Coomassie blue (lanes l-4). In parallel, samples (0.15 pg

protein each) were treated and resolved by SDS-PAGE, as above, trans-

ferred to nitrocellulose filters and incubated in the presence of rabbit

polyclonal anti-AChE antibodies which were then iabeled with goat anti-

rabbit ~ti~dies conjugated to alkaline phosphatase (lanes 1’ -4’). Lanes:

1 and l’, mock reaction without N-glycanase; 2 and 2’, T = 0 h; 3 and 3’.

T = 4 h; 4 and 4’, T = 24 h. Arrows denote position of standard molec-

ular weight marker proteins.

sition. Variation in ~ycosylation of a given protein has been

reported in various systems (Kobata, 1985). Sequence

analysis of the hAChE cDNA reveals three potential sites

for N-glycosylation (Asn-Xaa-Thr/Ser). Some of these sites

may be more available to glycosylation than others, so that

302

TABLE VI

N-terminal aa sequence of mature rehAChE

Sequencing

cycle No.”

1 2 3 4 5 6 7 8 9 10 11 12

aa of

sequence No. 1

(pmoBb

aa of

sequence No. 2

(pmol)’

GIU GUY Arg Glu Asp Ala Glu Leu Leu Val Thr Val

(45.8) (65.5) (21.1) (59.5) (39.3) (111.2) (44.2) (56.9) (73.5) (138.2) (14.2) (27.5)

Arg Glu Asp Ala Glu Leu Leu Val Thr Val Arg GUY

(1.8) (20.4) (26.9) (81.3) (25.6) (94.7) (100.5) (20.1) (10.0) (138.2) (5.0) (16.3)

” The N terminus of recombinant hAChE (17.0 pg; 200 pmol) was sequenced by subjection to twelve cycles of Edman degradation in a gas-phase se-

quencer (Applied Biosystems, Model 475A).

’ Peaks corresponding to the major AChE species.

’ Peaks corresponding to the minor AChE species.

differentially glycosylated AChE molecules may comprise

the steady-state extracellular pool.

(I) The N-terminal sequence of mature rehAChE secreted

by 293 producer cell lines

The hAChE cDNA employed in these studies contains

the entire coding region for the soluble form of the enzyme

subunit (Soreq et al., 1990). To determine the actual N

terminus of the mature secreted rehAChE, aa sequencing

of the N end of the purified protein was performed. A

major sequence determined by this analysis was found to

be Glu-Gly-Arg-Glu-Asp-Ala-Glu-Leu-Leu-Val-Thr-Val

(Table VI). However, the major peaks de termined for each

sequence cycle were accompanied by additional minor

peaks. The aa sequence represented by the minor peaks is

Arg-Glu-Asp-Ala-Glu-Leu-Leu-Val-Thr-Val-Arg-Gly. (It

should be noted, however, that only a weak Arg peak was

found in the first sequence cycle.) Thus, the purified pro-

tein is comprised of two molecular species differing at their

N end. The terminal 12 aa of the major species, correspond

to aa 32-43 of the hAChE coding region (Fig. 4). Align-

ment of this hAChE sequence with that of the highly ho-

mologous FBS AChE (Doctor et al., 1990), shows that the

terminal Glu residue of the mature hAChE sequence co-

incides with the terminal Glu residue at the N terminus of

the FB S AChE molecule. The second N-terminal sequence

originates at a distance of two aa from this major sequence.

An Arg residue occupies the + 1 site of this species, while

Ala and Gly residues are located at -3 and -1, respec-

tively, relative to the peptidase cleavage site (Fig. 4). Thus,

the putative minor cleavage site also conforms with known

N terminal processing sites of secreted proteins (Fig. 4,

Perlman and Halvorson, 1983). However, the minor

hAChE cleavage site is preceded by 33 aa, whereas in most

cases, the signal peptide is of 15-30 aa (Nothwehr et al.,

1990). This may cause the second sequence cleavage site

to be less favored. Reductive methylation of the N-terminal

Pre AChE MRPWCWTPSLASPULV~ffiREDAELLVTV

Mature AChE #I EGREDAEUVIV

Mature AChE #2 AEDAELLVTV

-3 1 Consensus Hydrophobic core . A .AR signal G GK

peptide S SE V D L

I

Fig. 4. Alignment of the mature rehAChE N termini with the deduced aa

sequence of the hAChE primary translation product and with consensus

cleavage sites. Various examined signal peptides were found to contain a

hydrophobic core preceded by 4-17 aa residues. Signal peptide cleavage

occurs at a distance of six or more aa following the hydrophobic core.

Frequently found aa residues are noted for positions + 1, -1 and -3

relative to the peptidase cleavage site (Perlman and Halvorson, 1983; von

Heijne, 1983).

aa identified Glu as well as Arg at the N terminus of human

erythrocyte AChE (Haas and Rosenberry, 1985). The ex-

istence of a similar mixed population of hAChE molecules

in a native system, strongly suggests that heterogeneity at

the N terminus reflects authentic alternative processing and

not partial degradation of the N terminus. Cleavage within

the FBS AChE primary translation product to form a sec-

ond species parallel to that found in hAChE would have

generated at position + 1, a Pro residue. Such a residue

appears to be excluded from this position (von Heijne,

1983), consistent with the single uniform N-terminal se-

quence reported for FBS AChE (Doctor et al., 1990).

Previous mutagenesis studies have shown that multiple

potential sites of cleavage may compete for recognition by

signal peptidase (Folz et al., 1988), but to the best of our

knowledge, this is the first case of a native protein exhib-

iting cofunctional processing signals. Generation of mature

hAChE molecules differing at the N end in a biological

system may reflect an in vivo process whose significance

awaits clarification.

303

(g) Conclusions

(I) High levels of AChE protein production could be

reached at low copy number of integrated plasmid without

DNA amplification using a stable transfection system uti-

lizing a selectable marker gene under control of a weak

promoter.

(2) Establishment of a cell line producing high levels of

recombinant human AChE requires empirical determina-

tion of an optimal set of genetic elements and host cells.

(3) The high-level expression system allows production

of authentic human AChE molecules as judged by various

kinetic parameters, correct subunit oligomerization and

specific activity .

(4) Two functional signal peptidase cleavage sites reside

within the single hAChE precursor polypeptide giving rise

to mature molecules differing at their N terminus.

ACKNOWLEDGEMENTS

We would like to thank Gila Friedman, Rachel Monzain

and Yitzhak Inbar for their excellent technical assistance.

We thank Dr. E. Elchanati for helpful discussions con-

cerning N-terminal aa analysis of rehAChE. This work was

supported by the U.S. Army Research and Development

Command, Contract DAMD17-89-C-9117 to A.S.

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