BIOCHEMICAL

Vol. 168, No. 2, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Pages 863-870

April 30, 1990

C-TYPE

A NEW MEMBER

OF

NATRIIJRBTIC

SUDOH, Naoto

Tetsuji

NATRIDRBTIC

Department

PEPTIDE

MINAMINO*,

*National

19,

Kenji

(CNP):

IDENTIFIED

Miyazaki Miyazaki

Cardiovascular

Center Suita,

IN

PORCINE

KANGAWA and Hisayuki

Kiyotake,

Fujishirodai,

March

FAMILY

of Biochemistry,

Kihara,

Received

PEPTIDE

Medical 889-16,

565,

MATSUO*#

College,

Japan

Research

Osaka

ESRAIN

Institute, Japan

1990

SUMMARY: Two types of natriuretic peptide, atria1 natriuretic peptide (ANP) and brain natriuretic peptide (BNPI, very similar to each other in structure and in pharmacological effect, are known to be present in mammalian heart and brain. In our present survey for unidentified peptides in porcine brain extracts, we found a new peptide of 22 amino acid residues, eliciting a potent relaxant activity on chick rectum. The amino acid sequence determined for the peptide shows remarkable similarity to those of ANP and BNP, especially in the 17-residue sequences flanked by two cysteine residues. The peptide shows a pharmacological spectrum similar to ANP and BNP. Thus, the peptide was designated "C-type natriuretic peptide (CNP) ", the third member to join the natriuretic peptide family. In contrast to ANP and BNP, CNP terminates in the second cysteine residue, lacking a further C-terminal extension. Q 1990 Academic P?xSS,

1°C.

Atrial atrium,

natriuretic is

natriuretic only

in

hormones homeostatic hormonally

#

peptide

now also peptide

brain of the

but

known (BNP) also

exist

first

in

natriuretic

balance

(ANP),

to

identified

heart peptide

of body

in

fluid,

(4-6). family

originally brain

discovered

(l-3).

in porcine These may

electrolytes

all

facts

brain

is

suggest

participate and

in cardiac

Similarly,

blood

brain present that

not

similar

in regulating pressure

both

and neuronally.

To whom correspondence

should

be addressed.

Abbreviations: ANP, atria1 natriuretic peptide (A-type natriuretic peptide); BNP, brain natriuretic peptide (B-type natriruetic peptide); CNP, C-type BNP-32, pro-BNP[75-106); HPLC, natriuretic peptide; ci-ANP, pro-ANP(99-126); relative molecular mass; RIA, high performance liquid chromatography; Mr, RCM, reduced and S-carboxymethylated; radioimmunoassay; ir, immunoreactive; TFA, trifluoroacetic acid; PTH, phenylthiohydantoin; IgG, immunoglobulin G. 0006-291x/9-8

863

$1.50

Copyright 0 I990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

168,

No.

Discovery member of the opioid

peptide,

(7-9). member of peptide),

BIOCHEMICAL

2, 1990

of

ANP and

natriuretic

the

peptide

tachykinin

The present

paper

natriuretic

in porcine

BNP in

brain

AND

RESEARCH

mammals raised family

and endothelin describes peptide

BIOPHYSICAL

a possibility

remained

to be

families isolation

family,

COMMUNICATIONS

all and

designated

have

that

a third

identified, three

identification CNP (C-type

since

members of

each a new

natriuretic

extracts. MATERIALS

AND METHODS

Porcine brain (40 kg obtained from 480 pigs) was minced and boiled Isolation: (V/W) of water to inactivate intrinsic proteases. for 10 min in two volumes After cooling, glacial CH3COOH was added to make a final concentration of 1 M, The homogenates were and boiled tissue was homogenized with a Polytron mixer. with Pellicon cassette (PCAC centrifuged, and the supernatant was condensed acetone-precipitation (final concentration = #OOO-05 < Milliporel. After 66%), the supernatant was evaporated in vacuum. One-fourth of the resulting solution was each loaded onto a LC-SORB SPW-C-ODS column (1.5 L, Chemco) in the presence of 0.5M CH COOH, and materials adsorbed on the column were eluted with H20:CH3CN:lO% trif . 4 uoroacetic acid (TFA) at 40:60:1 (V/W. The adsorbed materials were pooled and lyophilized (dry weight; 26 g in total), and were divided into two equal portions, both of which were processed by the same Half of the dried materials was dissolved+in 1M CH COOH and subjected method. 3, 28 cm) in the to batchwise chromatography on SP-Sephadex C-25 (H -form, presence of 1M CH COOH. Successive elutions with 1M CH3COOH, 2M pyridine and 2M pyridine-aceta P e (pH 5.01 yielded three respective fractions of SP-I, SP-II After lyophilization, the SP-III fraction (dry weight; 5.2 g in and SP-III. 7.5 x 145 cm). totall was separated by gel filtration on Sephadex G-50 (fine, Fractions corresponding to relative molecular mass (Mr1 l-SK (dry weight; 2.96 filtration on total) were collected and subjected to a second gel g in Sephadex G-25 (fine, 7.5 x 150 cm). Fraction B of Mr 3K was subjected to CM Whatman) eluting with a ion exchange chromatography (CM-52, 2.4 x 45 cm, linear gradient elution of HCOONH4 (pH 6.6) from 10 mM to 0.5 M in the presence of 10% CH3CN. Peptides (29 mg) in peak 1 of Fig. 1 were then subjected to anti-a-ANP immunoglobulin G (IgGI immunoaffinity chromatography, as described previously for the purification of a-ANP(4-28) and a-ANP[5-28) (10). The adsorbed peptides on the immunoaffinity column were eluted with a solution of 1M CH COOH containing 10% CH CN, and then finally purified by reverse phase HPL 2 on a diphenyl column (2 ?9TP54, 4.6 x 250 mm, Vydac) with a linear gradient elution of CH CN from 10% to 60% in 0.1% TFA for 120 min at a flow rate of 1.0 ml/min. Czick rectum relaxant activity in all fractions of each purification step was monitored, and contents of immunoreactive (ir-1 ANP and BNP were measured in the first several chromatographies. Bioassays: Chick rectum relaxant activity was assayed by the described method, using freshly isolated chick rectum strips (11,121. Natriuretic and diuretic activities were assayed as described previously after injection of (111, peptides into assay rats through the jugular vein in one shot. Systemic blood pressure was measured from the carotid artery in rats, and peptides were administered through a cannula in the femoral vein (11). Radioimmunoassays (RIAs) for ANP and BNP: RIAs for porcine ANP and BNP were carried out as reported previously (13,14). Synthetic CNP showed less than 0.001% crossreaCctivity in each RIA system. Sequence analysis: Reductive S-carboxymethylation of CNP was performed by the described method (4,111, and the resulting reduced and S-carboxymethylated (RCM) CNP (ca. 150 pm011 was sequenced with a gas-phase sequencer coupled with on-line HPLC identification system of the resulting phenylthiohydantoin (PTH) amino acids (Applied Biosystems 470A/l20A). PTH-amino acids were detectable down to 0.2 pmol. Amino acid analysis was performed with Hitachi L-8500 amino acid analyser after acid hydrolysis of RCM-CNP (ca. 150 pmol) in 6N HCl containing 0.1% phenol and 0.02% 2-mercaptoethanol at llO°C for 24 h.

864

Vol.

168,

No.

BIOCHEMICAL

2, 1990

AND

Synthesis: Synthetic CNP was prepared phenylacetamidomethyl resin using a 430A). An intramolecular disulfide K3(Fe2(SCN) 1. Synthetic peptide was exchange HP2 C, and correct synthesis sequencing.

BIOPHYSICAL

RESEARCH

COMMUNlCATiONS

by solid phase techniques conducted on a peptide synthesizer (Applied Biosystems linkage was formed by the action of purified by reverse phase HPLC and ion was confirmed by amino acid analysis and

RESULTS AND DISCUSSION The porcine

present brain

rectum,

survey

extract

which

for

provided

previous

isolations

chick

rectum

relaxant

activity

condensed

3K

(fraction

isolation

ion

exchange The

G-50).

immunoreactive rectum

column

relaxant

(Fig.

isolation

1).

of CNP.

induced

the

basic

peptide

fraction

activity

by

Among them, Peaks

20

cation peak

60

80 Fraction

Fisure

Mr

which

yielded

120

was used were

140

mass

for SP-

G-25

contained

about

5 major

chromatography

#51-531

was

reported

(Sephadex

3K,

ir-BNP,

100 number

of potent

chromatography,

filtrations

exchange

1 (tube

in a manner

The bulk

manner

C-18

ir-ANP,

3 and 5, exhibiting

40

the

phase

of from

on chick

of porcine brain of relative molecular

in

and gel

was free

activity

in

extracts

41,

reverse

peptide

activity

(4,111.

fraction

of ref.

chromatography

relaxant

of natriuretic

peptide

la

BNP but

natriuretic

and BNP by acid

successive

basic (ir-1

assessment of ANP

in Fig.

of BNP including

Sephadex

of

in

B

unidentified

out by monitoring

a rapid

to

(Mr)

so far

was carried

similar efficiently

a

on for

identified

most

peaks a CM-52

the

present

as BNP-26

160

1. CM ion exchange chromatography of basic peptide fraction of Mr 3K. Sample: Basic peptide fraction of Mr 3K obtained from SP-III fraction by Sephadex G-50 and G-25 gel filtrations (dry weight; 440 mg). Flow rate: 35 ml/h. Column: CM-52 (2.4 x 45 cm, Whatmanl. Fraction size: 20 ml/tube. Solvent system: Linear gradient elution from (A) to (BI. (A) 1OmM HCOONH4 (pH 6.61:CH3CN = 9O:lO W/VI, (B) 0.5M HCOONH4 (pH 6.61:CH3CN = 9O:IO (V/V,. Relaxant activity on chick rectum is indicated by black columns and BNP-like inununoreactivity by white columns. 865

and

Vol.

BIOCHEMICAL

168, No. 2, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0.06 3 2 I! i3 0.04 cm f :: s 0.02 I 0 20

10

30

40

50

Time

Figure

respectively.

In addition,

related

calcitonin-gene

peptide

respectively. on an anti-cc-ANP

homology

to ANP.

immunoaffinity-adsorbed

fraction, of

carboxymethylated, preliminary carboxymethylated (1) i

Ser ,

2.78

(3);

Phe,

0.96

(11;

acid

analysis

pg (400 pmol), definitely of the

GUY,

data,

purified

were

of a cystine acid

(61;

(II;

1.14

(II,

yield

up to the

and

(Fig. column.

first

reduced

Ile,

was deduced (2)

1.00

(Fig.

disulfide

linkage.

acid

Thus, the as indicated

established, sequence

sequences

determined

of porcine

for

866

4.01

residues.

CNP was Based

on 1.0

of RCM-CNP was sequenced

by

acids 3).

were

able

Positive

complete in Fig. CNP shows

ANP and HNP (Fig.

Leu,

that

4).

amino 4. high These

to

be

confirmation

above was provided by chromatographic an identically sequenced synthetic peptide

CNP was unambiguously

1.23

to be about

PTH-amino

22nd residue

from. and S-

; Asp,

(1);

indicating

of CNP was estimated Half

21,

and S-

of reduced

two cysteine

40 kg of brain.

of the

homogeneity

CmCys, 1.67

0.88

including

column

residue

Met,

structural

on a C-18

composition

to be:

completely

its

determined

CNP with

The amino

to

HPLC system

2.10 (2); Arg,

sequencer,

identified

corresponding

6.03

isolation from

automated

intramolecular

thus Amino

LYS I acid residues

starting

structure

of natural

purified

as peptide

1 was

suggesting

HPLC on a diphenyl

phase

presence

analysis.

22 amino

a gas-phase

CNP the

phase

identified

intestinal

in peak

column,

CNP was reverse

2 and 4 were

activity

(RCM-1 CNP was determined

(4); amino

the

since sequence

of

relaxant reverse

by another

Three-fourths

composed

80

vasoactive

and

IgG immunoaffinty

By subsequent

was confirmed

peaks

(CGRPI

Rectum

adsorbed

which

70

2. Final purification of CNP by reverse phase HPLC. Sample: Anti-a-ANP IgG immunoaffinity chromatography adsorbed fraction of Peak 1 in Fig. 1. Flow rate: 1.0 ml/min. Column: 219TP54 diphenyl (4.6 x 250 mm, Vydac). Solvent system: Linear gradient elution from (A1 to (BI for 120 min. H 0:CH CN:IO%TFA = (A) 90:10:1, (B) 40:60:1 (V/VI. Rectum re?axanz activity was observed in black bar region.

and BNP-32, (VIP),

60

(min)

comparison containing

acid

an

sequence

of

homology

to the

peptides

share

Vol.

BIOCHEMICAL

168, No. 2, 1990

AND BIOPHYSICAL

1 5

a 17-residue is

ring

ring

structure

characteristic

sequence

identical,

except

that to the

ANP and BNF in the

20

only

sequence,

CNP ends at the from

precursor

demonstrated

ring

with

par-tion,

second

structure. that

residue

peptide

CNP is

a

observed,

cDNA

codon

be published).

This

in

the

further

analysis

for

fact

directly

S-L-R-R-S-S-C~~.~'G-R-M~~-~~!-~~A-D-S-G'C-G~C-N-S-F-R-

C-terminal a porcine

a C-

that

the C-

: a-ANP

B-type

:BNP-S2

C-type

:m HZN

b

a-ANP

Figure

ENP-32

CNP

4. (a) IImino acid sequences of porcine CNP, BNP-32 and a-ANP. Intramolecular disulfide linkage is formed between two cysteine residues in each peptide. Identical residues among three peptides are shaded. (b) Schematic representation of porcine wANP, BNP-32 and CNP structures. Identical residues among three peptides are indicated by open circles.

867

CNP

followed

confirmed

-y

from

C-terminal

.~., .~ A-type

exactly

different

and lacks TAG

is

respectively.

quite

Especially

recent

a termination (to

were

residue

Our

linkage,

The 17-residue

of ANF and BNF,

structurally

regions. cysteine

disulfide family.

those

5 and 3 replacements

ring

N- and C-terminal

the

an intramolecular

by

natriuretic

when compared

extension

cysteine

formed

of the

of CNP,

In contrast

terminal

15

number

3. Yield of ETH-amino acid at each cycle of Edman degradation of amino acid notation is used, except CinC for RCM-CNP. One letter carboxymethyl-cysteine.

Figure

which

10 Cycle

RESEARCH COMMUNICATIONS

Vol.

168,

No.

BIOCHEMICAL

2, 1990

Diuretic-natriuretic

Table 1.

Dose (nmol) % Increase in urine output Na+ excretion K+ excretion Cl- excretion Decrease in mean blood pressure (mmHg)

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

and hypotensive responses induced by CNP and a -ANP CNP 16

4.8

a-ANP 0.4

32

229 f 38 192 + 28 2OOf43 228 rf: 32

251+ 286 + 167 f 3Olk

62 125 29 83

6.7 + 1.7

16.7 + 4.2

42lk 371 k 238 + 400 f

40 69 46 45

545 725 270 575

14.2 k 5.2

+ iz zk f

43 74 21 36

13.3 zk 1.7

Diuretic-natriuretic responses are expressed as % change (mean + s.e.m.) in urine output, and in excretions of Na+, K+ and Cl- from 15-min urine samples collected before and after intravenous injection into anesthetized rats. Hypotensive responses are expressed as decrease (mean + s.e.m.) in mean arterial blood pressure. Six rats were used for each sample.

terminal

truncated

terminal

tail

In

the

is

CNP is

of

BNP.

an

is

important

N-terminal

portion and

form

These

intrinsic

for

exerting

region,

the

clearly

different

facts

demonstrate

two members

expected

from

structural

resemblance

to

be essential

found

to exert

Table

1 of ref.

4).

rats

resulted

in

electrolytes

into

is

in

mean

structurally

Thus,

urine,

blood

(atria11

peptide

the natriuretic

peptides

but

As was

portion,

which

that

excretion

elicited

by

caused it

natriuretic

CNP was anesthetized

and in

rats

is

(ANPI W and "B-type

of ct-ANP.

a significant evident

that

peptide

peptide

is

1) and BNP (see

a member of natriuretic "C-type

peptide

so

isolation

far

C-type,

based

on their

while

mammalian

third

to be

member, widely

according with

of CNP in porcine

identified

A-type,

along

to

family.

family.

(CNP)", (brain)

CNP

following natriuretic

(BNP) ". The present

the

peptide

to

Accordingly,

pressure.

similar

CNP into

of urine

ring

ANP

to CL-ANP (Table

similar

the

activity,

anesthetized

and pharmacologically

we designated

"A-type

into

(151.

of

peptide ring

C-

sequences

structure

of synthetic

a manner

of CNP

arterial

similar

the

from

natriuretic-diuretic

in excretion

in

injection

has a

17-residue

injection

increase

extending

corresponding

natriuretic

of the

effects

Intravenous an

of the

exerting

pharmacological

Furthermore, decrease

for

the

CNP

that

the other

al though activity

sequence from

from

CNP,

of

natriuretic-diuretic

5-residue

distinct

thought

structure

are

chemical

structure.

CNP, has only preliminary

ANP and

into

BNP and chicken

distributed,

to our

divided

not

been only

screening

BNP strongly

brain

suggests

three

B-type and A-type, and frog ANPs constitute

Mammalian

to B-type

(3,16,17).

in

pig,

but

in mammals but

also

in other

(to be published).

suggests

868

the

natriuretic

types;

NP belong identified

that

probable

ir-CNP

is

thought

vertebrates,

Occurrence presence

So far

of CNP

of a complex

Vol.

168, No. 2, 1990

system

for

by these

BIOCHEMICAL

regulating three

the homeostatic

members

of the

Identification

of

characterizing cloned

their

in respect

isolation

of

rise

present.

In this

hypotensive

effects

and BNP,

while

that

of

CNP

dissociated

ANP and

natriuretic in

the

the

third

as

to whether

context,

it

K

rectum-relaxant

receptors

the

to

physiological controlled CNP and their

ANP

family although

The present family

gives

specific

for

CNP is

that

natriuretic-diuretic as those

on

P,

by ANP

higher

than

features

of

multiplicity

ligands,

of substance studies

a

and

induced

Pharmacological suggest

to multiple

further

been well

peptide

BNP

composed

been within

natriuretic

of BNP.

corresponding

tachykinin

has not

of CNP was 3-4 times

and

so far

domain

(18-21).

as potent

activity of

receptor,

for

a receptor

be noted l/100

to that

those

(22-241,

remain

With

should

have

cyclase

significance

about

pressure

studies

of receptors

clearance

or not

and blood

extensive

a guanylate

member of the

was comparable

case of the

receptors shown

physiological

from

types

fluid

family.

induced

include

of its

peptide

neuromedin

Three

of CNP were

the

BNP

a so-called

question

of body

peptide

and

another,

CNP as

to the

ANP

two of these

while

evaluated

balance

natriuretic

receptors.

and sequenced;

the molecule,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

as observed substance

natriuretic

K and peptide

to be done.

present

comprise

identification at

functions by a combination respective

Acknowledgments: This Ministry of Education, and Welfare of Japan.

least so far

of CNP, the three

natriuretic

different

attributed

of a series

mainly of natriuretic

peptide

peptides, to ANP and its peptides,

family

implying receptor

is that are

ANP, BNP and

receptors.

work was supported Science and Culture,

in part by research grants and from the Ministry

from the of Health

REFERENCES 1. de Bold, A. J. (1985) Science, 230, 767-770. 2. Nakao, K., Morii, N., Itoh, H., Yamada, T., Shiono, S., Sugawara, A., Saito, Y., Mukoyama, M., Arai, H., Sakamoto, M. & Imura, H. (1986) J. Hyperten., 4 (Suppl. 61, S492-S496. 3. Matsuo, H. & Nakazato, H. (1987) Endocrinol. Metab. Clin. North Am., 16, 43-61. 4. Sudoh, T., Kangawa, K., Minamino, N. & Matsuo, H. (19881 Nature, 332, 78-81. 5. Minamino, N., Aburaya, M., Ueda, S., Kangawa, K. & Matsuo, H. (19881 Biochem. Biophys. Res Commun., 155, 740-746. 6. Aburaya, M., Minamino, N., Kangawa, K., Tanaka, K. & Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 165, 872-879. 7. Hollt, V. (1983) Trend NeuroSci., 6, 24-26. 8. Nakanishi, S. (1987) Physiol. Review, 67, 1117-1142. 9. Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K. & Masaki, T. (1989) Proc. Natl. Acad. Sci. U.S.A., 86, 2863-2867. 10. Ueda, S., Sudoh, T., Fukuda, K., Kangawa, K., Minamxo, N. & Matsuo, H. (1987) Biochem. Biophys. Res. Commun., 149, 1055-1062. 869

Vol.

11. 12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

168,

No.

Kangawa, 131-139.

2, 1990

K. & Matsuo

BIOCHEMICAL

H.

(1984)

AND

Biochem.

BIOPHYSICAL

Biophys.

RESEARCH

Res.

COMMUNICATIONS

Commun.,

118,

M.G., Geller, D.M., Cole, B.R., Siegel, N.R., Fok, K.F., Adams, S.P., Eulanks, S.R., Galluppi, G.R. & Needleman, P. (1983) Science, 221, 71-73. Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T. & Matsuo, H. (19851 Biochem. Biophys. Res. Commun., 129, 248-255. Ueda, S., Minamino, N., Sudoh, T., Kangawa, K. & Matsuo, H. (1988) Biochem. Biophys. Res. Commun., l';s, 733-739. Watanabe, T.X., Noda, Y., Chino, N., Nishiuchi, Y., Kimura, T., Sakakibara, S. & Imai, M. (1988) Eur. J. Pharmacol., 147, 49-57. Sakata, J., Kangawa, K. & Matsuo, H. (19881 Biochem. zphys. Res. Commun., 155, 1338-1345. Miyata, A., Minamino, N., Kangawa, K. & Matsuo, H. (1988) Biochem. Biophys. Res. Commun., 155, 1330-1337. Chinkers, M., Garbers, D.L., Chang, M.S., Lowe, D.G., Chin, H., Goeddel, D.V. & Schulz, S. (19891 Nature, 338, 78-83. Chang, M.S., Lowe, D-G., Lewis, M., Hellmiss, R., Chen, E. & Goeddel, D.V. (1989) Nature, 341, 68-72. Schulz, S., Singh, S., Bellet, R.A., Singh, G., Tubb, D-J., Chin, H. & Garbers, D.L. (1989) Cell, F, 1155-1162. Fuller, F., Porter, J.G., Arfsten, A.E., Miller, J., Schilling, J.W., Scarborough, R.M., Lewicki, J.A. & Schenk, D.B. (19881 J. Biol. Chem., 263, 9395-9401. Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M. & Nakanishi, S. (1988) Nature, 329, 836-838. Yokota, Y., Sasai, Y., Tanaka, K., Fujiwara, T., Tsuchida, K., Shigemoto, R ., Kakizuka, A., Ohkubo, H. & Nakanishi, S. (1989) J. Biol. &em., 264, 17649-17652. Shigemoto, R., Yokata, Y., Tsuchida, K. & Nakanishi, S. (1990) J. Biol. Chem., 265, 623-628. Currie,

870