Molecular cloning of a cDNA for rat liver monoamine oxidase B

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 30, 1988

MOLECULAR

Pages 970-976

CLONING

Akio

OF A c D N A FOR RAT L I V E R M O N O A M I N E

Ito .I, T o y o k o Kuwahara***, S h u i c h i r o and Y a s u h i r o S a g a r a * *

OXIDASE

B

Inadome*

* D e p a r t m e n t of Biology, F a c u l t y of S c i e n c e and * * L a b o r a t o r y of M i c r o b i o l o g y , F a c u l t y of P h a r m a c e u t i c a l Sciences, K y u s h u U n i v e r s i t y , F u k u o k a 812, JAPAN ***Department

of B i o l o g y , Sciences,

Daiichi Fukuoka

C o l l e g e of P h a r m a c e u t i c a l 815, JAPAN

Received October 31, 1988

SUMMARY: The c D N A for rat m o n o a m i n e o x i d a s e B m R N A was isolated from liver c D N A l i b r a r y in Xgtll u s i n g specific antibody and o l i g o n u c l e o t i d e p r o b e s d e r i v e d f r o m F A D - c o n t a i n i n g p e p t i d e of the enzyme. The p r i m a r y s t r u c t u r e of the protein, d e d u c e d f r o m the n u c l e o t i d e sequence, c o n s i s t e d of 520 amino acid r e s i d u e s and its molecular w e i g h t was c a l c u l a t e d to be 58.4 kD w h i c h is in good agreement with that of the in vitro-synthesized peptide. F A D - b i n d i n g site is l o c a t e d in the c a r b o x y - t e r m i n a l region. There is no t y p i c a l s t r u c t u r a l f e a t u r e c o m m o n to the t a r g e t i n g signals for m i t o c h o n d r i a , the p e r i o d i c d i s t r i b u t i o n of b a s i c amino acids spaced by several uncharged residues, at its amino-terminal region. This region has an uninterrupted stretch of 14 hydrophobic residues. ©1988AcademicPress,lnc.

Monoamine

oxidase,

oxidative

deamination

important

functions

active

amines

tissues

(i).

in

In the

show

the

existence

and

B.

These

inhibitor chemical contains

I To w h o m

an F A D - c o n t a i n i n g of b i o g e n i c

in the m e t a b o l i s m the

central

last

distinct

are d i s t i n g u i s h e d

specificities, property two

with

correspondence

Abbreviation:

SDS,

sodium

Each

which

970

sulfate.

has vaso-

peripheral

accumulated

of

differ

to

oxidases,

in s u b s t r a t e

type

the

and

and

distribution,

be a d d r e s s e d .

dodecyl

0006-291 X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved,

size,

and

has

of m o n o a m i n e

cell

size.

similar

should

forms

and

and m o l e c u l a r

subunits

system

evidence

by d i f f e r e n c e s

tissue

catalyzes amines

of n e u r o a c t i v e

nervous

two decades,

of two

enzyme,

and x e n o b i o t i c

A

and

immuno-

the in at

enzyme least

Vol. 157, No. 3, 1988

one

regard,

flavin

that

is

intrinsic previous

well protein

on free

in

molecular

size

that

signal

a

present

in

membrane

outer

covalently

the enzyme

attempted

the

targeting

rat

nucleotide

MATERIALS

the

bound

had

the

(2).

to

as in the case (4)

In our

oxidase

same

extension

protein

proteins

typical

synthesized

in the m e m b r a n e .

70 kD p r o t e i n

of

and yeast

B in

apparent

The

results

peptide

and

mitochondria

like A T P / A D P

other

is

outer

porin

(5),

translocator

(7).

difference

and a n c h o r i n g the p r i m a r y

communication

liver m o n o a m i n e and d e d u c e d

a

membrane

peptide

system

enzyme

molecule,

is

that m o n o a m i n e

the

protein

CoA thiolase

to d e t e r m i n e

for

that

has no c l e a v a b l e

yeast

This

reported

and

the m o l e c u l a r

oxidases.

of a c D N A

we

mitochondrial

3-oxoacyl

To c l a r i f y

oxidase

mitochondrial

translation

targeting

proteins,

to l o c a t e

(3),

polysomes

the enzyme

a few inner

(6) and

of the

as the m a t u r e

that

of its

contains

that m o n o a m i n e

a cell-free

suggest

amine

one of t h e m

known

communication

made

vitro

and

only

(i).

It

was

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of m o n o a m i n e

signals

structure reports

oxidase

amino

acid

oxidases

in the m o l e c u l e , of rat

liver

the m o l e c u l a r

B and

and we

monocloning

the d e t e r m i n a t i o n

sequences.

AND METHODS

Preparation of Rabbit Antibody against the Bovine Liver M o n o a m i n e 0 x i d a s e B: M o n o a m i n e o x i d a s e B was p u r i f i e d f r o m b o v i n e liver mitochondria according to the m e t h o d of Minamiura and Yasunobu (8). Antibody a g a i n s t the enzyme was prepared by immunizing a rabbit w i t h the p u r i f i e d b o v i n e enzyme. The antibody could react w i t h the m o n o a m i n e o x i d a s e B of rat liver m i t o c h o n d r i a (3). Construction of the Rat L i v e r c D N A L i b r a r y and Screening: Igtl0 and Igtll l i b r a r i e s w e r e c o n s t r u c t e d from p o l y (A) + RNA isolated from rat liver. The Igtll l i b r a r y was subjected to screening for the m o n o a m i n e o x i d a s e B by immunological method using the s p e c i f i c a n t i b o d y (9). Positive clones were further screened by in situ p l a q u e h y b r i d i z a t i o n w i t h 32P-labeled synthetic o l i g o n u c l e o t i d e s (I0). The o l i g o n u c l e o t i d e probes were designed from the amino acid sequence of FAD-containing pentapeptide, Ser-Gly-Gly-Cys-Tyr (ii). Two sets of mixed oligonucleotides, 5'(A/G)TA(A/G)CA(G/C)CC(G/C)CC(G/T)GA3' and 5'(A/G)TA(A/G)CA(G/C)CC(G/C)CC(A/G)CT3', w e r e s y n t h e s i z e d on an Applied B i o s y s t e m s D N A S y n t h e s i z e r m o d e l 380B and used for the hybridization. A p o s i t i v e clone o b t a i n e d , rMAOBI6, w a s about 1.3 kbp long, which was m u c h s m a l l e r t h a n that expected for the full-length cDNA. F u l l - l e n g t h c D N A for rat liver monoamine oxidase B was screened again from several cDNA libraries constructed w i t h Igtl0 and Igtll u s i n g the i n s e r t of r M A O B I 6 labeled with ~ [ 3 2 P ] d C T P as a probe. A c D N A clone, rMAOBF3, having the

971

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

longest insert of a b o u t 2.4 kbp was s e q u e n c e d . Sequencing was c a r r i e d out by the d i d e o x y n u c l e o t i d e m e t h o d (12). R N A Gel Blot A n a l y s i s : T o t a l and p o l y (A) + R N A p r e p a r e d f r o m rat liver w e r e s u b j e c t e d to e l e c t r o p h o r e s i s on a 1.2% agarose/ formaldehyde gel and t r a n s f e r e d to a n i t r o c e l l u l o s e filter as d e s c r i b e d (i0). The R N A blot was h y b r i d i z e d with 32p-labeled r M A O B I 6 insert.

RESULTS A from

AND D I S C U S S I O N c D N A clone

a

shows

for m o n o a m i n e

cDNA library

restriction maps

rMAOBF3. sequence

The

nucleotides

long and the

size

3kb,

s h o w n by R N A blot

peptide

of the m R N A

from

ence, (13). this the

CC(A/G)CC, There

is,

codon.

of

of

detected

for rat

and

(Fig.

ATG

520 a m i n o

the

3).

initiation

the

analysis,

codon

nucleotide

of

in

frame for

sequence sequ-

preceding

c o d o n is

enzyme.

including

for

A

two

3'poly-

c D N A is s m a l l e r

true

mRNA

than

species

adenylation

ii

NcoI B g l ~

I

NcoI

I

Pstl

•

,

I

AcclHindIKAccI

100bp Fig. I. Restriction Cleavage Maps and Sequencing Strategy for Rat Liver Monoamine Oxidase B cDNAs (rMAOBI6 and rMAOBF3). Thick bar in rMAOBF3 represents the coding region. Arrows indicate the direction and extent of sequencing. Restriction enzymes used for sequence analysis are shown.

972

was

signal

rMAOB16 rMAOBF3

a

eukaryotes

codon

the

single

another

about

the c o n s e n s u s

long,

and

from

B,

codes

this ATG

size of the

acid 2,389

open r e a d i n g

stop

that

amino

oxidase

1

and

expected

codon

an i n - f r a m e

766 n u c l e o t i d e s Since

that

The

The

residue

rMAOBI6

The c D N A is

than

with

indicate

for

deduced 2.

initiation

c o r r e s p o n d i n g mRNA,

in R N A blot

Fig.

acids.

methionine is

isolated

mRNA.

is c o n s i s t e n t

results

signals.

was

liver

liver m o n o a m i n e

analysis

furthermore,

sequence

rMAOBF3,

strategy

is s m a l l e r

of the a c t i v e

These

the

sequence

size

codon

amino-terminal

adenylation

sequencing

putative

this ATG

noncoding

that

the

consisting

preceding

B,

f r o m rat

c D N A is s h o w n in Fig.

the

starting

and

nucleotide

of r M A O B F 3

oxidase

constructed

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-i GTCTCAGOCAOAGGTCCAGACTCAGTGGAAGCAGAGOAGAOAGCCTGAAACCTGGCGAGCACC

ATGA~AA~AAATG~GA~TGAT~TG~T~G~G~G~CAT~AG~A~cAG~AGCCAAACTTT~GCATGAC~G~CcTCAG~GT~ V

90 30

GTGGTTCTGGAAGcACGAGACTGTGTGGGAGGCA~GACTTAcACAA~TAGGAA~AAAAATG~TAAATATGTGGACCTTGGAGGATCT~AT V V L E A R D C V G G R T Y T I R N K N V K Y V D L G G S Y

180 6O

GTTGGGCCGAcCCAGAATCGTATCTTACGATTGGCCAAAGAGCTAGGATTGGAGAcCTA~AAAGTGAAT~AAGTTGAGCGGC~GA~CCAC V G P T Q N R I L R L A K E L 0 L E T Y K V N E V E R L I H

270 30

TTTGTAAAGG~AAAATCATATGCCTTCA~GG~CcCA~TCCCACCAGTGTGGAATCCAATcACTTACCTAGATTATAAcAACcTCTGGAGA F V K G K S Y A F R G P F P P V W N P I T Y L D Y N N L W R

390 120

A~AATGGAT~AGATG~GC~AAGAGAT~CCCAGTGATGCTC~AT~AAGGCACCCCTT~T~AAGAGT~GGAc~A~ATGACAAT~AAAGAG

45O 150

M

S

N

K

C

D

V

I

V

V

G

G

G

I

S

G

M

A

A

A

K

L

L

H

D

C

G

L

S

T M D E M G Q E I P S D A P W K A P L A E E W D Y M T M K E

TTGCTAGATAAGATCTGCT~GACCAACTCTACAAAGCAGATTGCCACACTCTTTGTGAACCTATGTGTAA~TGCGGAGACcCATGA~GTT L L D K I C W T N S T K Q I A T L F V N L C V T A E T H E V

TCTGCACT~TGGTTCCT~TGGTATGTGAAGCAGTGTGGGG~TACAACCAGAATCATATCAACAACCAATGGA~GAcAGGA~AG~AAATTT 8 A L W F L W Y V K Q C G G T T R I I S T T N G G Q E R K F

ATTG~TGGATCTGGTCAAGTGA~TGAGCGGATAAA~GATATCCTTGG~ACAGA~TGAAGCTGGAGA~GCCGGTGATCcACATTGACCAG I

O

G

S

G

Q

V

S

E

R

I

K

D

I

L

G

~

V

K

L

E

R

P

V

I

H

I

D

540 180 630 210 720 240

Q

ACAG~A~AAAATGTTGTTG~GAAAA~C~TAAACCAT~AAATATATGAGGCTAAA~ATGTGATTA~GCCATCC~AC~TGTTTTGGGCAT~ T O E N V V V K T L N H E I Y E K Y V I S A { P P V L G M

810 270

AAGATTCACCATAGTCCTCCTCTGCCCATTCTAAGAAACcAGCTGATTACTCGTGTGCCTTTGGGTTCAGTTATTAAGTG~A~GGTTTAT K I H H S P P L P I L R N Q L [ R V P L G S V I K C M V Y

9OO 3OO

TATAAAGAA~CCTTCT~GAGGAAAAAGGATTTCTGTGGAA~CATG~TTATTGAAGGAGAG~AAGCTCCAATTGCGTACACATTGGATGAT Y

K

E

P

F

W

R

K

K

D

F

C

G

T

M

V

I

E

G

E

E

A

P

I

A

Y

T

L

D

D

ACCAAGCCAGATGCAGOCTGTGCTGCTATAATGGGATTTATCCTTGCTCACAAAGCTAGAAAA

CTGGTACGCCTTACTAAAGAAGAAAGA

T

L

990 330

R

1080 380

CTGAGGAAG CTCTG TGAGCTATACGCGAAAGTTCTGAACTCTCAAGAAGCTCTGCAGCCAGTCCATTATGAAGAOAAGAACTGGTGTGAG L R K L C E L Y A K V L N S Q E A L 0 P V H Y E E K N W C E

1170 330

•AGCAGTACTCCG•GGG•TGCTA•ACA•CCTACTTCCCTCCT•GCATCTTGACCCA•TA•GGAAGGGTTCTACGCCAGCCA•TG•GCAAG

1260 420

E

K

P

Q

Y

D

A

.~., G

G

C

~

A

y

A

T

I

A

M

Y

G

F

F

P

I

P

L

G

A

I

H

L

K

T

A

Q

R

Y

K

G

R

V

V

R

L

L

R

T

O

K

P

E

V

E

G

K

ATTTTCTTTGCAGG•AC••AGACAGCT•CACATTGGAGT•GCTACAT•GAGGG•G•TG•A•AGGCTGGAGAGAGA•CTGCcAGAGAGATT I

F

F

A

G

T

E

T

A

S

H

W

S

G

Y

M

E

O

A

V

E

A

G

E

R

A

A

R

E

I

CTTCATGCCA•T••GAAGATT•CAGAGGAT•AAATTTGGCAGCCAGAAC•AGAATCT•TG•ATGTC••AG•AAGACCCATTAc•AACACC L

H

A

I

G

K

I

P

E

D

E

l

W

Q

P

E

P

E

S

V

D

V

P

A

R

P

I

T

N

T

1440 480

L

1530 510

TTCCTGGAGAGA~ACTTGCCTT~TGTACCAGGTCTACTAAAGCTGCTTGGATTGAC~ACCATCTT~TCAG~AACA~TCTTGGT~TCCTG F

L

E

R

H

L

P

S

V

P

G

L

L

K

L

L

G

L

T

T

I

L

S

A

T

A

L

G

F

•C••ACAAAAAGGGTCTGTTTG•ACGTTTCTAAAGATGGG•TTTAGGACCATATCCA•A.GGTTT•T•ATTCAGTGTGT••ACAAAAG•T•T A

H

K

K

G

L

F

V

R

F

1350 450

*

1620 820

T~GAAGGAGTTGGGATAAAAAT~TGA~AAAGGTGCAGAGATTA~GGAGTGAGAAAGCACAGTAACTTGG~CT~CATTTTGGCTATCTTTT

1710

AGCATCGCTGTGGTC•ACTCATTTTCAACTTTCCTGCACTCTGAATATT•AGAA•AGATACACAGGCTCTCTCACAACCTACCTGCCCTA

1800

TGCACATAGTTGTTTTTCAAAACCCTATGCCTTTGTGCTTGTCTTTCTT•TGGTGT•TTAGGTC•TCACCTATATCAA•TT•TT•ATcAT

1890

TGTACCTAG AATCCTGTCTTGTTAGAACCAGAAGGCATTAGACACTGTAGCTTATTGTCTACTTTAGAGTTAAA

1980

TAAACCAAATG CAACA

GAAGTGAAAT•TAACCACACAAGGCCTACACAAAGCTACTGGTATT•GGGTGACTGGAACACAA•CTGATGCTTTTcTCACCTC•CAAGG

2070

TTCATTCCCCTGTGATCCTCCTCCACCTTATGTCATAGT~ATTCACGGATCATTGTTCTTGTGGATTTACTCTGTATTAA~T~GTATTGT

2160

GTTACTCAGTAGATTCTTCTAGGCTTGCTA TTTTGTGTAGTGTTGCCAGCTGATTCTAATTTTTCTTGAGAATGGGAGTCTTGTCTTTGT

2250

CATTTCTTTTTTGCATCTTCCAOTA TGCTTCCACTCATAGATTTAAGACATGCTTAAATAATTAAAAATAAAOCTO

2326

Fig. 2. Nucleotide Sequence of Rat Monoamine Oxidase B cDNA and the Deduced Amino Acid Sequence. Nucleotide sequence that corresponds to the oligonucleotide probe derived from FADcontaining peptide is indicated by the double underline. Consensus sequences for polyadenylation signal are underlined. The single letter amino acid code is used.

973

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

28S

18S " - "

Fig. 3. Northern Blot Analysis of Rat Liver mRNA. Approximately 0.8 ~g of poly (A) I RNA was applied on formaldehyde agarose gel. The membrane was hybridized with 32p-labeled rMAOBI6 insert. The hybridization was carried out in IxS$C at 65°C, and blot was washed in 0.5xSSC at 65°C.

might

exist

of r M A O B F 3 region

at 500 to 600 n u e l e o t i d e s eDNA,

and

a poly A tail

FAD-binding clone,

or the m R N A m i g h t

site,

is l o c a t e d

The

eDNA

reported

rat

human

have

about

sequence

the

When

The

for

the

our p r e v i o u s

synthesized

and

to

their signal

amino of

residues,

to m o r e

than

calculated

showing

that

into

(5),

termini.

in

rat

there

et al.

without

(4)

is l o c a t e d

974

have

and

they

amino the are

of

acid entire

included,

in

amino

the v a l u e

acid

determined

SDS-polyacrylamide

yeast

to

be

for

of the

the

that

outer

post-

maturation membrane

the

at

targeting

amino-terminal

distribution

B

protein

inserted

the

with

oxidase

70 kD

proteolytic

showed

gel

is c o n s i s t e n t

Proteins

signals

is p e r i o d i c

of over

liver m o n o a m i n e

shown

targeting

Hase

70 kD p r o t e i n in w h i c h

with

protein.

the m e m b r a n e

uncleavable

similar

The r e s u l t

were

B

95%.

so far c h a r a c t e r i z e d ,

porin

2).

and

for the d e d u c e d

its m o b i l i t y (3).

eDNA

sequences

substituitons

agreement

as a m a t u r e - s i z e

yeast

have

acid

A

acid

The r e g i o n s

amino

sequence. of the

(Fig.

oxidase Amino

distributed

59 kD)

membrane

translationally and

from

(about

region

3'-end

5'-untranslated

AATAAA

are q u i t e

identity.

in good

findings

mitochondrial (4)

was

enzyme

electrophoresis

is

weight

a long

second

(14).

enzymes

from the

identification

monoamine

randomly

increases

58,389, rat

B type

are

molecular

sequence,

the

for

et al.

conservative

similarity

used

of h u m a n

sequence

identity

protein.

following was

by B a c h

liver

88%

contain

in the e a r b o x y - t e r m i n a l

cloning

recently and

which

downstream

of three

llbasic

Vol. 157, No. 3, 1988

amino is

acids

known

peptides signal

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

spaced to

by a few uncharged

be a common

of mitochondrial of

residues

into

outer membrane.

the

sion

peptides,

residues

flanked

acid

sequence

in five

485-510)

them

could

the

long

stretch

(residues

in addition

2,

monoamine

rich

amino

165-190,

not

the

in

amino

amino

acids

shown)

shows

260-270,

to the amino-terminal for anchoring

acids the

mitochondria,

in basic

(data

exten-

uncharged

to locate B to

28

protein

to the

uninterrupted

plot

of

this

common

oxidase

sequence

extension

The targeting

of

by a few charged

monoamine

be responsible

outer mitochondrial

feature

of 14

The hydropathy

regions

by the

It is now hard

is a potential 42-52.

hydrophobic

335-350, of

sides

region.

for targeting

there

residues

of (15).

As can be seen in Fig.

structural

on both

amino-terminal

Such a sequence

for the insertion

but has a stretch

the

although

required

B has no typical

feature

precursors

is followed

uncharged

oxidase

structural protein

70 kD protein

residues.

280-300,

region.

enzyme

to

Some the

membrane.

ACKNOWLEDGMENT The authors for

kindly

wish

providing

to thank Dr. one of rat

Y. Misumi, liver

%gtll

Fukuoka

University,

cDNA libraries

used

for screening.

REFERENCES (1) Singer, T.P. (1987) J. Neural Transm.(suppl) 23, 1-23 (2) Greenawalt, J.W. and Schnaitman, C. (1970) J. Cell Biol. 46, 273-179 (3) Sagara, Y. and Ito, A. (1982) Biochem. Biophys. Res. Commun. 109, 1102-1107 (4) Hase, T., MUller, U., Riezman, H. and Schatz, G. (1983) EMBO J. ~, 3157-3164 (5) Mihara, K. and Sato, R. (1985) EMBO J. ~, 769-774 (6) Zimmermann, R., Paluch, U., Sprinzel, M. and Neupert, W. (1979) Eur. J. Biochem. 99, 247-252 (7) Arakawa, H., Takiguchi, M., Amaya, Y., Nagato, S., Hayashi, H. and Mori, M. (1987) EMBO J. 6, 1361-1366 (8) Minamiura, N. and Yasunobu, K.T. (1978) Arch. Biochem. Biophys. 189, 481-489 (9) Huynh, T.V., Young, R.A., and Davis, R.W. (1985) in DNA Cloning: A Practical Approach (Glover, D.M. ed.) Vol. I, pp. 49-78, IRL Press, Oxford (I0) Maniatis, T., Pritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Mannual, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(ii) Kearney, E.B., Salach, J.T., Walker, W.H., Seung, R.L., Kenney, W, Zeszotek, E. and Singer, T.P. (1971) Eur. J. Biochem. 24, 321-327 (12) Sanger F., Coulson, A.R., Barrell, B.G., Smith, A.J.H. and Roe, B.A. (1980) J. Mol. Biol. 143~ 161-178 (13) Kozak, M., (1984) Nucleic Acids Res. 12, 857-872 (14) Bach, A.J.W., Lan, N.C., Johnson, D.L., Abell, C.W., Bembenek, M.E., Kwan, S.-W., Seeburg, P.H. and Shih, J.C. (1988) Proc. Natl. Acad. Sci. USA 85, 4935-4938 (15) Hurt, E.C. and van Loon, A.P.G.M. (1986) Trends Biochem. Sci. ii, 204-207

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