Characterization of recombinant bovine phenylethanolamine N-methyltransferase expressed in a mouse C127 cell line
Dong H. Park 1, Thomas Wessel 1, Harriet Baker 1, Tong H. Joh 1 and Himadri Samanta 2
~Laboratory of Molecular Neurobiology, Cornell University Medical College, The Burke Rehabilitation Center, White Plains, NY 10605 (U.S.A.) and 2Eugene Tech International, Inc., Allendale, NJ 07401 (U.S.A.)
(Accepted 8 January 1991)
Key words: Recombinant phenylethanolamine N-methyltransferase; cDNA; mRNA; In situ hybridization; Charge isozyme; Immunostaining; Mouse cell line
Bovine phenylethanolamine N-methyltransferase (PNMT) cDNA was inserted into a bovine papilloma virus-based expression vector and used to transfect a mouse C127 cell line. The resultant recombinant bovine PNMT was characterized biochemically and immunochemically. Recombinant bovine PNMT activity, like the native bovine enzyme, was enhanced by phosphate ion in a concentration-dependent manner. Their molecular weights were shown to be identical by Western blot analysis. Antibodies raised against native bovine adrenal PNMT equally immunoprecipitated the activity of the recombinant and native enzymes. In addition, double immunodiffusion analysis showed a single precipitin line of confluence with both enzyme preparations, indicating immunological identity of native and recombinant bovine PNMT. These antibodies immunostained the recombinant enzyme protein in transfected cells and in their neurite-like processes. In addition, in situ hybridization with the bovine PNMT cDNA probe resulted in a labelling pattern similar to the immunostaining. The recombinant bovine PNMT as the native bovine enzyme exist in multiple-charge forms, but only one form is predominant. Taken together, our results suggest that recombinant bovine PNMT, expressed from bovine PNMT cDNA in a mouse cell line is enzymatically active and shares many common features with native bovine adrenal PNMT.
INTRODUCTION
Phenyle thanolamine N-methyltransferase (PNMT) is
the enzyme that catalyzes the conversion of norepineph-
rine to epinephrine and is a specific marker for adrener-
gic neurons. Glucocorticoids appear to play an important
role in the normal maintenance of PNMT activity 3'19. The
enzyme has been purified from bovine, rat and rabbit
adrenal glands and has been well characterized 4'5'9'13'1a.
PNMT in rabbit and bovine adrenal glands and bovine
brain exists in multiple-charge forms. In contrast, rat
PNMT exhibits a single-charge form in both the adrenal gland and the CNS 5'9'12'13'15'18. Recently, full-length
cDNAs encoding PNMT from various species have been cloned and sequenced 2"7A1'17.
To ascertain whether the translation product from
bovine PNMT cDNA is enzymatically active and has the
same physiochemical and immunochemical properties as
the native PNMT, bovine PNMT cDNA was inserted into
an expression vector and used to transfect a mouse C127
cell line. The resultant recombinant bovine PNMT was
characterized biochemically and immunochemically as
well as by immunocytochemistry and in situ hybridiza-
tion. In addition, to further extend the existence of the
multiple-charge isozymes of PNMT among other species,
we determined the isoelectric point of mouse and human
adrenal as well as recombinant bovine PNMT by chro-
matofocusing and compared them with those for the
native bovine PNMTs previously published 15. Our find-
ings indicate that both native and recombinant bovine
PNMT share many common features.
MATERIALS AND METHODS
To express the PNMT gene at high levels we used a bovine papilloma-based vector. A fragment containing the coding region of bovine PNMT (bPNMT) was obtained from the clone PNMT-172 and was inserted into the expression vector pBMT3X at the XhoI site, so that bPNMT transcription was under the control of the mouse metallothionein promoter. The expression plasmid pBMT3X-bPNMT with the correct orientation of the bPNMT insert was purified and transfected into mouse C127 cells. A SV40 polyadenylation site was provided at the 3' end of the insert. Transfection of the C127 cells and selection of the transformants were carried out according to the published procedures 16. Clones were tested for PNMT activity after induction by CdCI 2.
For the enzyme assays and determination of protein concentra- tion, cells were harvested by trypsinization. Human and mouse adrenal glands were homogenized in 5 mM potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100 and centrifuged at
Correspondence: D.H. Park, Laboratory of Molecular Neurobiology, Cornell University Medical College, The Burke Rehabilitation Center, 785 Mamaroneck Avenue, White Plains, NY 10605, U.S.A.
214
10,000 g for 10 min. Protein concentration was determined by the method of Lowry et a1.1°, using bovine serum albumin as a standard. However, when monitoring the eluate from the chromatofocusing column, protein was measured by A280nm reading. PNMT activity was measured by using S-[methyl-3H]adenosyl-L-methionine as a methyl donor and phenylethanolamine as a substrate, as described by Park 12. The effects of phosphate ion at various concentrations was studied as previously described TM. The isoelectric point was determined by chromatofocusing as described by Park and Joh ]5. The production of antibodies to PNMT and immunochemical titration experiments were performed as described by Park et al.'3. Sodium dodecyl sulfate (SDS)-slab gel electrophoresis was per- formed according to Laemmli s. Western immunoblot was per- formed as described by Albert et al. 1.
For immunocytochemical staining and in situ hybridization, mouse C127 cultures were grown to confluence on double cham- bered glass tissue culture slides. For immunocytochemistry, the cells were washed briefly with 0.1 M phosphate buffer, pH 7.2. The cultures were fixed for 10 min in 4% buffered (0.1 M phosphate buffer, pH 7.2) formaldehyde generated from paraformaldehyde. The cultures were rinsed in phosphate buffer, pretreated with 0.2% Triton X-100 and 1% bovine serum albumin (BSA) in 0.1 M phosphate buffered saline (PBS). Following 2 washes in PBS containing 0.5% BSA, the cultures were incubated overnight with specific antiserum to bovine PNMT (diluted 1/20,000). Following another 2 washes in PBS-BSA, the cultures were incubated 1 h with biotinylated goat anti-rabbit IgG, washed and incubated 1 h with avidin-biotinylated horseradish peroxidase from the Vectastain 'Elite' kit (Vector Labs, Burlingame, CA) according to the manufacturer's protocol. The antigen was visualized with 3,3'° diaminobenzidine-HC1 (50 mg/100 ml) and 0.003% H202. Cultures were dehydrated through graded alcohols, cleared in xylene and cover-slipped with Permount. As controls for non-specific staining, parallel cultures were incubated with antisera to TH (diluted 1/10,000), neuron-specific enolase (diluted 1/10,000) and normal rabbit serum (diluted 1/10,000).
For in situ hybridization, slides were fixed as described above, and rinsed twice in phosphate buffer, and 200/~l of prehybridization buffer [50% formamide, 10% dextran sulfate, 2x SSC (1× SSC = 0.15 M sodium chloride-0.015 M sodium citrate), l x Denhart's, 50 mM dithiothreitol, 0.5 mg/ml yeast tRNA and 0.5 mg/ml sonicated and denatured salmon sperm DNA] was added to completely cover the floor of each chamber, Prehybridization was carried out for 30 min at 55 °C. The denatured rat PNMT cDNA probe was labeled with [3sS]dCTP by the random-priming method. The labeled probe (2 X 10 6 cpm/chamber) was then added to the prehybridization buffer in an additional 50/~1. Hybridization was performed overnight at 55 °C. Five consecutive washes were carried out the next morning, starting with 2× SSC and ending with 0.125x SSC (1:1 dilution with each step) at 50 °C. Cells were then dehydrated through an ethanol series (70%, 95%, 100%). Slides were dipped in Kodak NTB-2 emulsion and stored in a light-tight box for 10 days at 4 °C. After developing in Kodak D-19, sections were counter- stained with Cresyl violet and cover-slipped with Permount (Fisher Scientific).
RESULTS
T h e b o v i n e p a p i l l o m a v i r u s - b a s e d e x p r e s s i o n v e c t o r
p B M T 3 x - b P N M T p l a c e d t he b o v i n e P N M T inse r t u n d e r
t h e c o n t r o l of an i n d u c i b l e m o u s e m e t a l l o t h i o n e i n p ro -
m o t e r . A s t he e x p r e s s i o n p l a s m i d c o n t a i n e d a h u m a n
m e t a l l o t h i o n e i n g e n e , t h e r ec ip i en t cells cou ld easi ly b e
s e l ec t ed in t he p r e s e n c e of CdCI 2. T h e t r a n s c r i p t i o n of
t h e b P N M T inse r t was u n d e r t h e con t r o l of a m o u s e
m e t a l l o t h i o n e i n p r o m o t e r . Th i s p r o m o t e r cou ld be in-
_-g E
(3 2 0 - o
z
I0-
40
0
Pho4phote conc. (mM)
Fig. 1. Increase of recombinant bovine PNMT activity by phosphate ion. PNMT was assayed in absence and presence of various amounts of phosphate ion as described in the Materials and Methods. Note that recombinant bovine PNMT activity was enhanced by phosphate ion in a concentration-dependent fashion.
d u c e d in t he p r e s e n c e o f CdC12. W e f o u n d t h a t in t he
p r e s e n c e of 1 0 / ~ M CdCI2, o n e of t he e x p r e s s i o n c lones
p r o d u c e d P N M T act iv i ty at t he leve l o f 4 n m o l / m g
50
40-
E ~n 3 0 -
E o_
"] 20 -
F-
Z 0_
10-
ot I I I I I I
0 2 4 6 B 10 12
A n t i s e r u m (p l )
Fig. 2. Immunochemical titration curve for recombinant bovine PNMT using antibodies to bovine adrenal PNMT. Immunotitration was carried out as described in the Materials and Methods. Note that 4 pl of antiserum to native bovine adrenal PNMT almost completely immunoprecipitates recombinant PNMT activity.
2
215
92.5 K
66 .2 K
45.0 K
Fig. 3. Ouchterlony double immunodiffusion. The plate was developed at 37 °C for 1 h and subsequently at 4 °C overnight. The center well contained 10/~l of rabbit antiserum to bovine adrenal PNMT. The outer wells contained clockwise: 12 o'clock, 500/tg of protein from bovine adrenal supernatant; 2 o'clock, 260 /~g of bovine adrenal PNMT from Sepharose 4B column fraction; 5 o'clock, 54 /~g of bovine adrenal PNMT from chromatofocusing colums; 7 and 10 o'clock, 143 /~g of protein from recombinant bovine PNMT supernatant. The different enzyme preparations used were prepared as described in ref. 15 and in the Materials and Methods. Note that precipitin lines of native and recombinant bovine PNMT do not exhibit spurs but are confluent, indicating immunological identity of native and recombinant bovine PNMT.
protein/15 min. The concentration was comparable to
that of native PNMT in the 100,000 g supernatant of bovine adrenal medulla (3.6 nmol/mg protein/15 min).
Enhancement of bovine PNMT activity by phosphate
ion was studied, using cell supernatant containing either 4.1 /~g (Fig. 1) or 8.2 /zg (data not shown) protein.
Phosphate ion addition produced comparable increases in
enzyme activity at both protein concentrations employed. PNMT activity was gradually increased by addition of phosphate ion in a concentrat ion-dependent manner
(Fig. 1). Phosphate at a 150 mM concentration maximally elevated PNMT activity 4.7-fold over control.
Immunoti t rat ion, Western blot analysis and double immunodiffusion analysis were performed to demon- strate the immunochemical identity of the native and
recombinant PNMT proteins. All experiments used a specific rabbit polyclonal antiserum directed against bovine adrenal PNMT. For immunochemical titration,
increasing amounts of antiserum were added to cell supernatant containing 7.1/~g protein. As illustrated in Fig. 2, the activity of recombinant PNMT was markedly reduced by 1 /zl of antiserum. Furthermore, 4 /~1 of antiserum resulted in more than a 95% reduction of the enzyme activity. Double immunodiffusion analysis on Ouchter lony plates showed a single precipitin line of
31.0 K,
21.5 K,
14.4 K •
Fig. 4. Western blot analysis of native and recombinant bovine PNMT. Lane 1, crude preparation (100,000 g supernatant) of PNMT from bovine adrenal medulla. Lane 2, crude preparation (10,000 g supernatant of recombinant bovine PNMT). Each lane of the 12% SDS-polyacrylamide slab gels contains 10/~g of protein and are run and electroblotted and stained with PNMT antiserum (1:40,000 dilution) as described under Materials and Methods. Molecular weights of standard protein markers are indicated. Note that a single 31,000 Da band is detected with native and recombinant bovine PNMT preparations.
confluence with native and recombinant bovine PNMT
preparations (Fig. 3). These data indicate the immuno- logical identity of native and recombinant bovine PNMT and suggest that only one protein is produced by the
transfected cells. Western immunoblot analysis was used to compare the subunit molecular weight of native and recombinant bovine PNMT. As shown in Fig. 4, the antibody recognizes an identical molecular weight band (mol.wt. = 31,000) in both native and recombinant bovine PNMT preparations.
The transfected cultures also were analyzed for the
216
A
" f¢
B C
f
J \ \
i
I<
i
D
d~
I
E 1' -,- t~, , F
~.L~~ ~.:~, i.~!~¢: ~7,. ,~
Fig. 5. Detection of recombinant PNMT and its mRNA in the transfected cell cultures by immunostaining and in situ hybridization. PNMT protein was immunostained using specific antibodies against bovine protein (B,C). Bovine recombinant PNMT mRNA (E,F) was detected in the transfected cells by in situ hybridization with a random primed (full-length) bovine PNMT cDNA probe. Large arrows indicate similar regions of the cultures illustrated at low and high magnification. Note the neurite-like processes containing both protein and message (small arrows). Cells transfected with the plasmid lacking the PNMT insert express neither PNMT message (D) nor protein (A). Bar = 25/~m (A,B,D,E); 50 ~m (C,F).
presence of PNMT protein and m R N A by immunocyto-
chemistry and in situ hybridization, respectively. Trans- fected cultures were stained with the same polyclonal
antiserum mentioned above. Over a broad range of antiserum dilutions (1/10,000 to 1/50,000), distinct cyto- plasmic staining was apparent in the transfected cells (Fig. 5B,C). Staining occurred not only in the cytoplasm,
but in the nucleus and in processes as well. These processes had a neuritic appearance and formed an extensive network of contacts with other cells. Processes are present in untransfected and vector transformed cells but were not apparent since these cell lines did not stain with any of the antisera used. Thus, the visualization of these processes were enhanced by PNMT immunostain- ing. The cells did not express other catecholamine biosynthetic enzymes, such as tyrosine hydroxylase, the first enzyme in the catecholamine biosynthetic pathway (data not shown). In addition, staining was not observed with an antiserum directed against neuron specific eno- lase, a marker often used to demonstrate cells of
neuronal origin (data not shown). They also did not
exhibit staining with preimmune serum. Non-transfected cells and cells transfected with the transforming vector lacking the PNMT insert served as additional controls (Fig. 5A).
The in situ hybridization analysis produced similar
results. PNMT message was detected over the cell bodies and processes in the transfected cell lines (Fig. 5E,F). The presence of message in the processes is supportive of the concept that these structures represent cytoplasmic extensions. In agreement with the immunocytochemical data, non-transfected cells and those tranfected without the PNMT coding fragment (Fig. 5D) did not contain PNMT message.
Typical enzyme elution profile of mouse adrenal, recombinant bovine and human adrenal PNMT from chromatofocusing columns are illustrated in Fig. 6. Mouse adrenal PNMT (Fig. 6, left top) had a single peak of activity in the elution pattern and a mean isoelectric point corresponding to 4.7. Multiple-charge forms of
217
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.15000
.12500
E I :1
.10000 13 .c_
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Fig. 6. Typical elution profile of recombinant bovine (left bottom), human adrenal (right bottom) and mouse adrenal (left top) PNMT from chromatofocusing columns, pH and PNMT activity in column fractions (3 ml/fraction) were measured as described in Materials and Methods. Note that human adrenal and recombinant bovine PNMT have multiple-charge forms, while mouse adrenal enzyme has only one charge form. The difference in scale between graphs is produced primarily because less protein was loaded on the columns for the mouse and human adrenal glands than for the recombinant PNMT. Furthermore, both human and mouse adrenal gland PNMT exhibit lower specific activity when compared to recombinant or native bovine PNMT.
recombinant bovine PNMT (Fig. 6, left bottom) were separated by chromatofocusing. Their mean isoelectric points were 5.27, 5.06, 5.51 and 6.21 in decreasing order of abundance. The isozyme at 6.21 is not shown in the figure as it exists in a relatively small amount. The pls of recombinant bovine PNMT partially correspond to those of the native PNMT. Human adrenal PNMT (Fig. 6, right bottom) also exhibited multiple-charge forms by chro- matofocusing. The mean isoelectric points were 5.57, 5.79, 5.33 and 4.95 in decreasing order of abundance.
DISCUSSION
Recombinant bovine PNMT expressed in this trans- fected mouse C127 cell line was not only enzymatically active but also shared many common features with the
native bovine adrenal PNMT protein. Previously, we demonstrated that the activity of native bovine adrenal PNMT was enhanced by phosphate in a concentration- dependent fashion and not dependent on the cation or simply solute concentration TM. The increase in activity of recombinant bovine PNMT was comparable to that of the native enzyme. The mechanisms underlying this increase in activity by phosphate are unknown.
Immunochemically, there seems to be little difference between the native and recombinant bovine PNMT proteins. First, the patterns of the double immunodiffu- sion, immunotitration curves, and the Western blot for the native and recombinant enzymes were similar. These data indicate that a single protein is expressed by the transfected cells. Second, the enzyme in the transfected mouse cells was demonstrable immunocytochemically.
218
PNMT-Iike immunoreact ivi ty was observed in both the
cell body and the neuri te- l ike processes. Other neuronal
markers , including neuron specific enolase and tyrosine
hydroxylase, were not expressed. PNMT message and
protein exhibi ted identical cytoplasmic distributions. The
presence of the neuri te- l ike processes was not l imited to
the PNMT-transfec ted cells as they were also observed,
but with difficulty, in non-transfected and vector trans-
fected cells. However , as their presence was enhanced by
the PNMT immunostaining, the resemblance to neuri tes
was striking and p rompted the immunostaining with
o ther neuronal markers. The lack of tyrosine hydroxylase
or neuron-specific enolase staining confirmed that no
neuronal t ransformat ion had occurred. Thus, the pres-
ence of neuri te- l ike processes is not an indicator of a
neuronal phenotype.
Charge isozymes of PNMT have been detected in some species 5'9'12A3'15"18. Mult iple-charge forms of bovine ad-
renal and brain PNMT have been demonst ra ted by us
and other investigators 5'12'13'~5"18. In contrast , rat adrenal
and brain PNMT have only a singe-charge form 12A3'15.
Lee et al. observed that rabbi t adrenal PNMT also was
present as mult iple-charge forms 9. To further investigate
mult iple-charge forms in other species, we de termined
the isoelectric points of native mouse and human adrenal
as well as recombinant bovine PNMT. Mouse adrenal
PNMT has a single-charge form, while human and
recombinant bovine PNMT have mult iple-charge forms.
Al though the recombinant PNMT prote in exhibits mul-
t iple-charge forms, the relat ive abundance and the pls of
the isozymes are not identical to those observed in vivo
and may be a reflection of the fact that the prote in is
expressed in mouse, not bovine cells.
The mechanisms that genera te the PNMT isozymes are
present ly not known. Judging from the fact that native
and recombinant bovine PNMTs are identical in molec-
ular weight as well as immunochemical ly and biochemi-
cally, the comparable multiplici ty of isozymes could be
due to a post- t ransla t ional event. The amino acid
sequence for PNMT, as deduced from the nucleotide
sequences, does not indicate any obvious sites for
post- t ranslat ional modificat ions such as glycosylation or
phosphoryla t ion. However , the existence of as yet un-
character ized sites for o ther forms of post- t ranslat ional
modification cannot be ruled out. To fully elucidate the
biochemical mechanisms that p roduce charge isozymes
requires further investigation of PNMT enzyme structure
and modulat ion.
Acknowledgements. This work was supported by NIH Grants MH 24285 and MH 44043. We would like to extend our appreciation to Mrs. Maureen McCrum for her excellent preparation of our manuscript and Mr. Charles Carver for his outstanding work in preparing the photographs.
REFERENCES
1 Albert, V.R., Allen, J.M. and Joh, T.H., A single gene codes for aromatic L-amino acid decarboxylase in both neuronal and non-neuronal tissues, J. Biol. Chem., 262 (1987) 9404-9411.
2 Baetge, E.E., Suh, Y.H. and Joh, T.H., Complete nucleotide and deduced amino acid sequence of bovine phenylethanolamine N-methyltransferase: partial amino acid homology with rat tyrosine hydroxylase, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 5454-5458.
3 Bohn, M.C., Role of glucocorticoids in expression and devel- opment of phenylethanolamine N-methyltransferase (PNMT) in cells derived from the neural crest: a review, Psychoneuroen- docrinology, 8 (1983) 381-390.
4 Hurst, J.H., Guchhait, R.B., Billingsley, M.L., Stolk, J.M. and Lovenberg, W., Phenylethanolamine N-methyltransferase: notes on its purification from bovine adrenal medulla and separation from protein carboxymethyltransferase, Biochem. Biophys. Res. Commun., 112 (1983) 1061-1068.
5 Joh, T.H. and Goldstein, M., Isolation and characterization of multiple forms of phenylethanolamine N-methyltransferase, Mol. Pharmacol., 9 (1973) 117-129.
6 Kajihara, J., Enomoto, M., Seya, K., Sukenaga, Y. and Katoh, K., Physiochemicai properties of charge isomers of recombinant human superoxide dismutase, J. Biochem., 104 (1988) 638-642.
7 Kaneda, N., Ichinose, H., Kobayashi, K., Oka, K., Kishi, F., Nakazawa, A., Kurosawa, Y., Fujita, K. and Nagatsu, T., Molecular cloning of cDNA and chromosomal assignment of the gene for human phenylethanolamine N-methyltransferase, the enzyme for epinephrine biosynthesis, J, Biol. Chem., 263 (1988) 7672-7677.
8 Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685.
9 Lee, H.S., Schulz, A.R. and Fuller, R.W., Isolation and
10 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275.
11 Mezey, E., Cloning of the rat adrenal medullary phenylethano- lamine N-methyltransferase, Nucl. Acid Res., 17 (1989) 2125.
12 Park, D.H., Monoamine Methyltransferases. In A.A. Boulton, G.B. Baker and P.H. Yu (Eds.), Neuromethods, Vol. 5, Neurotransmitter Enzymes, Humana, New Jersey, 1986, pp. 117-145.
13 Park, D.H., Baetge, E.E., Kaplan, B.B., Albert, V.R., Reis, D.J. and Joh, T.H., Different forms of adrenal phenylethano- lamine N-methyltransferase: species-specific posttranslational modification, J. Neurochem., 38 (1982) 410-414.
14 Park, D.H. and Joh, T.H., Activation of adrenal phenyletha- nolamine N-methyltransferase by phosphate, Biochem. Pharma- col., 33 (1984) 2911-2913.
15 Park, D.H. and Joh, T.H., Species-specific charge forms of phenylethanolamine N-methyltransferase, Brain Res., 344 (1985) 402-404.
16 Samanta, H. and Youn, B.W., Expression of hepatitis B virus surface antigen containing the pre-S region in mammalian cell culture system, Vaccine, 7 (1989) 69-76.
17 Weisberg, E.P., Baruchin, A., Stachowiak, M.K., Stricker, E.M., Zigmond, M.J. and Kaplan, B.B., Isolation of a rat adrenal cDNA clone encoding phenylethanolamine N-methyl- transferase and cold-induced alterations in adrenal PNMT mRNA and protein, Mol. Brain Res., 6 (1989) 159-166.
18 Wong, D.L., Yamasaki, L. and Ciaranello, R.D., Characteriza- tion of the isozymes of bovine adrenal medullary phenyletha- nolamine N-methyltransferase, Brain Res., 410 (1987) 32-44.
19 Wurtman, R.J. and Axelrod, J., Control of enzymatic synthesis of adrenaline in the adrenal medulla by adrenal cortical steroids, J. Biol. Chem., 241 (1966) 2301-2305.
