Expression of Choline Acetyltransferase mRNA and Protein in T ...

No. 71

Expression

Proc.

of Choline

Japan

Acetyltransf

Acad.,

erase

71, Ser.

231

B (1995)

mRNA

and

Protein

in T-Lymphocytes

By Takeshi FUJII,*) Shin YAMADA,*)Hidemi MISAWA,**)Sonoko TAJIMA,*)Kazuko FUJIM0T0,*) Takeshi SUzuKI,*) and Koichiro KAWASHIMA*),t) (Communicatedby Setsuro EBASxi,M.J. A., Sept. 12, 1995)

Abstract : Acetylcholine (ACh) is present in human blood and localized in T-lymphocytes which exhibit ACh-synthesizing activity. As the final step towards clarifying the origin of ACh present in the blood, we elucidated the enzyme involved in ACh synthesis in T-lymphocytes by detecting the mRNA for choline acetyltransferase (ChAT), which is known to catalyze ACh synthesis in the nervous system. The ChAT mRNA in the MOLT-3 human leukemic T-cell line was amplified by reverse transcriptionpolymerase chain reaction (RT-PCR) using specific order and reverse primers. A single specific RT-PCR product was observed on agarose gel. The sequence of the RT-PCR product of MOLT-3 was completely identical with nucleotide positions 322-973 of human brain ChAT cDNA. Western blot analysis with an antibody to ChAT confirmed the presence of ChAT protein in MOLT-3. These findings demonstrate that mRNA for the same ChAT as that in the nervous system is expressed in T-lymphocytes, and indicate that ACh synthesized by ChAT in T-lymphocytes is the origin of ACh in the blood. Key words : Acetylcholine; chain reaction; T-lymphocyte.

choline acetyltransferase;

Introduction. Acetylcholine (ACh), a classical neurotransmitter in both the central and peripheral nervous system, is synthesized by choline acetyltransferase (ChAT, E.C.2.3.1.6) from acetyl coenzyme A and choline.') Both muscarinic and nicotinic ACh receptors have been shown to be present in various blood cells, including lymphocytes2)-7> and granulocytesg) using selective cholinergic ligands. In addition, muscarinic and nicotinic agonists modulate the functions and metabolism of lymphocytes.9)-12) These findings suggest that ACh in the blood is also capable of modulating immune function by interacting with cholinergic receptors on leukocytes. 13),14) It has been considered unlikely that a sufficient concentration of ACh is available in the blood since there is a high level of cholinesterase (ChE) which rapidly degrades ACh in the blood. However, using a sensitive and specific radioimmunoassay, we previously measured ACh concentration in human plasma and *)

Department

of Pharmacology

, Kyoritsu College of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105, Japan. **) Department of Neurology , Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu-city, Tokyo 183, Japan. t) Correspondence to: K . Kawashima.

MOLT-3; reverse

transcription-polymerase

whole blood at 456±53 (n=32) and 1264± 149 pg/ml (n=30) (mean± S.E . M.), respectively. 15),16) In addition, about 60% of ACh in whole blood was found to be localized in circulating mononuclear leukocytes (MNL).16) More recently, we found that ACh content in the human leukemic T-cell lines MOLT-3, HSB-2 and CEM was about 9- to 70-fold higher than those in other human leukemic cell lines including Daudi (B-cell) and U-937 (NK-cells).17) In addition, AChsynthesizing activity was found in the MOLT-3, HSB-2 and CEM T-cell lines.17)Rinner and Schauenstein also recently reported the presence of ChAT activity in lymphocytes. 18)These findings suggest that the major portion of blood ACh originates from T-lymphocytes. However, there is no direct evidence that mRNAs for ACh-synthesizing enzyme(s), probably including that for ChAT, are present in T-lymphocytes. In the present study, we detected ChAT mRNA in the MOLT-3 human leukemic T-cell line using reverse transcription-polymerase chain reaction (RTPCR) in order to confirm the presence and origin of ACh in the blood. We have identified the partial sequence of MOLT-3 ChAT cDNA and found it was identical at positions 322-973 of human brain ChAT

232

T.

Fu

cDNA.19) In addition, using a polyclonal antibody against C-terminal peptide of human brain ChAT, we showed that ChAT protein was present in MOLT-3 cells. Materials and methods. RNA extraction kit, p(dN)6 and DEAE-Sephadex A25 were from Pharmacia (Uppsala, Sweden). The Poly(A) Quik`KmRNA isolation kit was from Stratagene (La Jolla, CA, U.S.A.). Fetal calf serum (FCS), penicillin and streptomycin were from Grand Island Biological Co. (Grand Island, NY, U. S.A. ). Glutamine and RPMI 1640 medium were from Nissui Seiyaku Co. (Tokyo, Japan). NuSieve GTG agarose was from FMC Bioproducts (Rockland, ME, U.S.A). SuperScriptTM II was from GIBCO BRL (Gaithersburg, MD, U.S.A. ). 0X1'7'4/ Hinc II digest was from Toyobo (Tokyo, Japan). Human placental ChAT and phenylmethylsulfonyl fluoride were from Sigma (St. Louis, MO, U.S.A. ). Biotinylated molecular marker was from Bio-Rad Laboratories (Hercules, CA, U.S.A.). The ABC-kit was from Vector Laboratories (Burlingame, CA, U.S.A.). Oligonucleotides were synthesized using Gene Assembler Plus (Pharmacia). Other chemicals used were of reagent grade. Cell culture and preparation of poly(A)RNA from MOLT-d. The MOLT-3 human leukemic T-cell line was cultured in a culture flask (Corning 2511-75) in RPMI 1640 medium supplemented with 7% heatinactivated FCS at 37°C in a humidified atmosphere of 5% CO2 in air. The cell line (5x 10~ cells) was transferred to a centrifuge tube (Corning 25311) and pelleted at 300 x g for 8 min at 4°C. The pellet was washed three times with 30 ml of phosphate-buffered saline (PBS). The resulting pellet was used to isolate total RNA using RNA extraction kit as described by manufacturer's protocol. In addition, poly(A)+RNA was purified using Poly(A) Quik° mRNA isolation kit. First strand cDNA synthesis and PCR amplification. First strand cDNA was synthesized from 20 yg of MOLT-3 poly(A)+RNA with 50 pmol of p(dN)6 and Superscript reverse transcriptase as described elsewhere.2o>,2I)As a negative control, reverse transcriptase was omitted from the reaction mixture. Human brain poly(A)RNA was also used to synthesize first strand cDNA as a positive control template. One-twentieth of the cDNA was amplified for 40 cycles with 25 pmol of both order and reverse primers using Ampli Taq DNA polymerase and Thermal Cycler (Perkin-Elmer Cetus, Norwalk, CT, U.S.A.) as described elsewhere. 21),22) The amplification reaction was

ii et

al.

[Vol.

71(B),

performed using a step program (94°C, 1 min; 62°C, 1 min; and 72°C, 2 min), followed by 15-min final extension at 72°C. The specific primers used were as follows: 5'-AAGACGCCCATCCTGGAAAAG-3' for the order primer; and 5'-TGAGACGGCGGAAATTAATGAC-3' for the reverse primer, which correspond to positions 322-342 and 952-973 of human brain ChAT cDNA,19) respectively. Sequence analysis o f RT-PCR product. RTPCR products (10 ul) were separated on 2.5% agarose gel containing ethidium bromide (0.6 yg/ml). The RT-PCR product of MOLT-3 was extracted from agarose gel and subcloned in pT7Blue(R) T-vector (Novagen Inc, Madison, WI, U.S.A.). Plasmid DNA was purified using the Wizard Miniprep DNA purification system (Promega, Madison, WI, U.S.A.) and sequenced with an automated sequencer (Model 373A, Applied Biosystems, Foster City, CA, U.S.A. ). Western blot analysis. MOLT-3 Cells (6x107) were homogenized in 10 ml of 50 mM Tris/HC1(pH7.4) containing 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (buffer A) using a UD-201 ultrasonic disruptor (Tomy, Tokyo, Japan). The homogenate was centrifuged at 100,000 x g for 60 min, and the supernatant was partially purified using a DEAESephadex A25 column equilibrated with buffer A. The fraction was pelleted with 60% ammonium sulfate and re.dissolved in a minimal volume of buffer A. An aliquot containing 100 ,yg protein was electrophoresed on a 10% polyacrylamide-sodium dodecyl sulfate gel and electrophoretically transferred to a nitrocellulose filter (BA85, Schleicher & Schull, Keene, NH, U.S.A. ) using the Trans-Blot' system (Bio-Rad). The filter was incubated overnight at 4°C with a rabbit polyclonal antibody against C-terminal peptide of human brain ChAT in PBS, washed with PBS three times, and visualized with ABC-Kit as described by manufacturer's protocol. The sizes of MOLT-3 ChAT and human placental ChAT as a positive control were compared. Results. RT-PCR analysis of the MOLT-3 poly(A)RNA sample was performed to confirm the expression of ChAT mRNA using specific primers corresponding to positions 322-342 and 952-973 of human brain ChAT cDNA. A single transcript of RT-PCR products was observed on agarose gel (Fig. 1, lane 3). The position of RT-PCR product obtained from poly(A)+RNA of MOLT-3 was identical with that of the human brain sample used as a positive control (Fig. 1, lane 4), and was equivalent

to the calculated

Expression

No. 7]

of ChAT

Fig. 1. The expression of choline acetyltransferase mRNA in MOLT-3. Ethidium bromide-stained PCR products. Lane 1 contains the DNA size standards,

with size listed in base pairs

(bp); lane 2, MOLT-3 mRNA sample, with reverse transcriptase omitted from PCR mixture as a negative control; lane 3, MOLT-3 mRNA sample; lane 4, human brain mRNA sample as a positive

control.

size of 652 base pairs on agarose gel. However, when reverse transcriptase was omitted from the reaction mixture, as a negative control, no DNA band was observed on the agarose gel (Fig. 1, lane 2). In addition, we analyzed the ChAT transcript expressed in MOLT-3 cells using another 2 sets of primers

Fig.

2.

primers

Partial used

nucleotide

sequence

are underlined.

mRNA

233

in T-cells

corresponding to positions 908-930 and 1597-1616, and 1574-1594 and 2225-2244 of human brain ChAT cDNA. Each primer set yielded an RT-PCR product which agreed in length with that of human brain ChAT (data not shown). The RT-PCR product of MOLT-3 (Fig. 1, lane 3) was subcloned in a plasmid vector and both strands were sequenced. The nucleotide sequence of the RT-PCR product (Fig. 2) was completely identical with the sequence of positions 322-973 of human brain ChAT cDNA,19)with the exception that the nucleotide sequence at positions 781-784 (CCGG) was GGCC in our PCR product (positions 460-463 in Fig. 2). We sequenced the original ChAT cDNA clone obtained from human spinal cord (clone H635)19>and found GGCC in this location. This CCGG to GGCC change resulted in conversions of amino acids from Pro and Glu to Gly and Gln, respectively. The resulting amino acids are identical with those resulting from translation of ChAT cDNAs from rat,23>,24)mouse24~and pig2J~ at the corresponding position. The presence of ChAT protein in MOLT-3 was confirmed by Western blotting using a polyclonal ChAT antibody. The major band in MOLT-3 (Fig. 3, lane 3) was observed at the same molecular weight (70 kDa) as human placental ChAT protein (Fig. 3, lane 2).

of MOLT-3

ChAT cDNA.

The order

and reverse

T. FUJii

234

et al.

that

[Vol. 71(B),

the same

ChAT

mRNA

expressed in MOLT-3 In a previous synthesizing including BrACh,

activity

in

MOLT-3,17) a selective

ACh-synthesizing

activity

of ChAT leukemic determined Fig.

3.

Western

blot analysis

of MOLT-3

ChAT protein.

Lane

1, contains molecular size markers with sizes listed in kDa; lane 2, human placental ChAT; lane 3, MOLT-3 sample.

of the present

T-cell

lines

of MOLT-3 study,

to

ACh-

examined,

by about by that

50% at of ChAT

immunoblot at least part

in MOLT-3

synthesize that

is

of Fonnum.27) inhibited the

the presence

we have shown

for ChAT

in MOLT-3

of the corresponding

is due to

ACh

in the

the expression

region

cells is the same of human

brain

as

ChAT

cDNA. These findings should facilitate further studies of the role of ACh in the blood in the modulation of of local blood

The findings

all

brain

mRNA takes place in the MOLT-3 human T-cell line and that the partial sequence

T-cell-dependent

Discussion.

detected

activity

which is known system.

In conclusion,

that

we

in MOLT-3 was confirmed These findings demonstrate

of the ACh-synthesizing ChAT, nervous

of human

using the method ChAT inhibitor,

100 µM.17) In the present protein analysis.

as that

cells. study,

immune

response

and the regulation

flow.

study

provide the first direct evidence for the presence of ChAT mRNA and protein in T-lymphocytes. Findings of the present and previous studies16>"7) demonstrating that ACh is synthesized by ChAT and stored in T-lymphocyte, suggest that ACh in the blood originating from T-lymphocytes may modulate immune system function,l3),14> and regulate local blood flow via its effects on vascular endothelial cells.26~ Since Tlymphocytes can directly contact the above-mentioned targets, even a small amount of ACh released from T-lymphocytes should be able to interact with the receptors prior to its hydrolysis by ChE. We attempted to determine whether mRNA for ChAT is expressed in the MOLT-3 human leukemic cell line using RT-PCR amplification, since AChsynthesizing activity in MOLT-3 cells was the highest among the T-cell lines tested. 17) A single band of RT-PCR product from MOLT-3 poly(A)RNA was observed on agarose gel, and the length of the band was the same as that of human brain ChAT. In addition, when first strand cDNA was synthesized from MOLT-3 poly(A)+RNA without reverse transcriptase, no PCR product DNA band was observed on agarose gel. The partial sequence of MOLT-3 ChAT cDNA identified by RT-PCR was completely identical with the corresponding region (322-973) of human brain ChAT cDNA.19) These findings strongly suggest

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