Brain natriuretic peptide-32: N-terminal six amino acid extended form of brain natriuretic peptide identified in porcine brain

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 726-732

September 15, 1988

B R A I N NATRI[R~ETIC PEPTIDE-32: B R A I N NATRIURETIC

N-TERMINaL

PEPTIDE

SIX A M I N O A C I D w~TE~DED F O R M OF

IDENTIFIED

IN PORCINE B R A I N

Tetsuji SUDOH #, Naoto MINAMINO*, Kenji KANGAWA & Hisayuki MATSUO

Departments of Biochemistry and Anesthesiology*,

Miyazaki Medical College,

5200 Kihara, Kiyotake, Miyazaki 889-16, Japan #Tokyo Research Institute, Daiichi Pure Chemicals Co. Ltd., Narihira,

Sumida-ku, Tokyo 130, Japan

Received July 28, 1988

SUMMARY: Brain natriuretic peptide (BNP) is a newly identified peptide of 26 residues, which has a remarkable homology to but is distinct from atrial natriuretic peptide. The peptide exerts natriuretic-diuretic activity as well as potent chick rectum relaxant activity. By using radioimmunoassay specific to BNP and immunoaffinity chromatography, we have isolated from porcine brain a novel peptide of 32 residues carrying a BNP structure at the C-terminus. The amino acid sequence of this peptide was determined to be: Ser-Pro-Lys-ThrMet -Arg-Asp- Ser-Gly- Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg- Ile-Gly- Ser-Leu- Ser-GlyLeu-Gly-Cys-Asn-Val-Leu-Arg-Arg-Tyr. This peptide is an N-terminal six amino acid extended form of BNP and henceforth is designated BNP-32~ BNP and BNP-32 are found to be major forms of BNP family in porcine brain. © 1988 Academic Press, Inc.

During the course of our search for undiscovered neuropeptides brain,

we

residues,

very

recently

designated

have

"brain

pharmacological remarkably

high

sequence

in porcine brain suggests maintaining

a novel

peptide

peptide

(BNP)",

spectrum very similar to that of ANP. homology

atrial natriuretic peptide

for

identified natriuretic

to ANP

(ANP) (I).

but

is

of

in porcine

26-amino

which

acid

elicits

a

The peptide also has a

definitely

different

from

The occurrence of BNP along with ANP

that two types of natriuretic peptides may function

homeostatic

developed radioimmunoassay

balance

of

(RIA) for BNP

screened BNP-related peptides

in

body

fluid.

By

using

a recently

(to be reported elsewhere),

porcine brain extracts

we have

and have isolated a

different form consisting of the original

26-residue BNP with an additional 6

amino acids attached at the N-terminus.

This new peptide we have designated

BNP-32.

In this

paper we

report isolation

and structural

determination

of

BNP-32.

MATERIALS AND METHODS Tissue extraction and isolation: Extraction and purification of BNP-32 were performed by the method similar to those used for the isolation of BNP, ~-

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

726

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ANP[4-28] and ~--ANP[5-28] (i,2). Brain tissue (ca. 21o4 kg wet weight) excluding cerebellum was collected from 250 pigs soon after killing. Diced tissue was heated at 95-I00°C for 10 min in two volumes of water to inactivate proteases. After addition of glacial acetic acid (IM final concentration), the boiled tissue was homogenized by a Polytron mixer. The resulting homogenates were centrifuged at 20,000 x g for 30 min. The supernatant was then concentrated by filtration with a Pellicon PCAC membrane (#000-05, Millipore), which retained materials larger than IK daltons. The concentrate was precipitated by addition of acetone (final concentration=66%) and centrifuged. The supernatant thus obtained was then evaporated under reduced pressure. The residual materials were dissolved in 0.5M acetic acid and pumped onto a C-18 silica gel column (1.4 L, Chemco LC-SORB ODS); the materials adsorbed on the column were eluted with 60% CH_CN containing 0.1% 3 trifluoroacetic acid (TFA) % The eluates were evaporated and loaded onto a SP-Sephadex C-25 column (H -form, 3 x 28cm, Pharmacia) in 1M acetic acid. Successive elutions with IM acetic acid, 2M pyridine, and 2M pyridine-acetate (pH 5.0) yielded three fractions: SP-I, SP-II and SP-III, respectively. The SP-III fraction containing basic peptides was then subjected to successive gel filtrations on a Sephadex G-50 fine column (7.5 x 140 cm, Pharmacia) and on a Sephadex G-25 fine column (7.5 x 135 cm, Pharmacia) using IM acetic acid as an elution solvent. An aliquot of each fraction was submitted to RIA for BNP. A fraction containing immunoreactive BNP was + further separated by cation exchange chromat(~raphy on a CM-52 column (NH 4 -form, 2.4 x 45 cm, Whatman). Two major peaks of ir-BNP were each subjected to immunoaffinity chromatography on an anti-BNP IgG-AFFI-GEL column (see Delow). The peptide fraction adsorbed on the immunoaffinity column was then subjected to reverse phase high performance liquid chromatography (HPLC) on a 219TP54 diphenyl column (4.6 x 250 mm, Vydac) with a linear gradient elution of CH3CN in the solvent system of H20-CH3CN-10%TFA at a flow rate of 1.0 ml/min. The column effluents of HPLC were monitored by measuring absorbance at 210 nm and 280 nm. Structural analyses: After reductive S-carboxymethylation (RCM) of cystine with dithiothreitol and monoiodoacetic acid, the purified RCM-BNP-32 (ca. 150 pmol) and RCM-BNP (ca. 200 pmol) were each sequenced with a gas-phase sequencer, equipped with reverse phase HPLC for identification of the resulting phenylthiohydantoin (PTH) amino acids (Applied Biosystems, 470A/120A). Amino acid analyses were carried out with IRICA A-5550 amino acid analyzer, after acid hydrolysis of RCM-peptides (ca. 200 pmol) in 6M HCI containing 0.1% phenol and 0.02% 2-mercaptoethanol at II0°C for 24hr. Syntheses: BNP-32 was synthesized by solid phase techniques conducted on a chloromethylated polystyrene resin in a manner similar to those used for BNP (i). Correct synthesis was confirmed by amino acid analysis and sequencing. RIA for BNP: Details on preparation of an antiserum against BNP and characterization of RIA for BNP will be reported in a separate paper. In brief, an antiserum (#158-4) mainly recognizes the portion flanked by a disulfi~inkage, and BNP-32 has an affinity with the antiserum comparable to BNP. [~-~I]-BNP was prepared by the lactoperoxidase method and purified by reverse phase HPLC. Half-maximum inhibition of binding was observed at 9 fmol/tube of BNP, and peptides were measurable in a range of i-i00 fmol/tube. Immunoaffinity chromatography: Immunoglobulin G (IgG) fraction was purified by Protein A-Sepharose CL-4B (Pharmacia) from 2 ml of antiserum #158-5. IgG fraction was then coupled with 2 ml of AFFI-GEL I0 (Bio-Rad) in 0.1M sodium phosphate buffer (pH 7.4). Samples were d i s s o l v e d i n 0.1M sodium phosphate buffer (pH 7.4), loaded onto the column (gel volume, 1.0 ml), and washed with the same buffer; the adsorbed materials were then eluted with a solution of IM acetic acid containing 10% CH_CNo Bioassa~: Chick rectum relaxant effect, natriuretic-diuretic activity, and hypotensive activity of BNP-32 were assayed as reported (3,4). RESULTS AND DISCUSSION BNP-32 was

isolated

from porcine

brain by immunoaffinity

chromatography

and reverse phase HPLC coupled with RIA specific to BNP in a manner similar to

727

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1,o! 2

I° o

2;

30

4'0

5'o

Frectlon number

60

Figure i. Sephadex G-25 gel filtration of the basic peptide fraction obtained from 21.4 kg of porcine brain. Sample: Basic peptide fraction of molecular weight IK-5K daltons (dry weight, 1.48 g). Column: Sephadex G-25, fine (7.5 x 135 cm). Flow rate: 120 ml/hr. Fraction size: 100 ml/tube. Solvent: 1M acetic acid. The arrows indicate the elution positions of bovine serum albumin (BSA), gastrin releasing peptide (GRP) and neurotensin (NT), respectively. The fraction shown with a hatched bar was submitted to CM ion exchange chromatography.

those used

in our previous purification

of ~-ANP[4-28]

From the acid extracts of porcine brain was prepared

by Pellicon

(21.4 kg),

ultrafiltration,

chromatographies on a reverse phase C-18 25 ion exchange column.

acetone

and ~-ANP[5-28]

the basic peptide fraction precipitation,

shown),

(ir-)

The resulting SP-III

fraction

(dry weight,

Most of the

BNPs were eluted in a region of 2K-4K daltons

(data not The bulk

subjected

to ion exchange

combined i ~ u n o r e a c t i v i t y

ir-BNP on the chromatogram

(Fig. I),

chromatography

peaks of ir-BNP emerged at fractions A),

2.61 g)

and were further separated by Sephadex G-25 gel filtration.

of ir-BNP was eluted in fractions #32-36 then

step-wise

silica gel column and SP-Sephadex C-

was first subjected to gel filtration on a Sephadex G-50 column. immunoreactive

(2).

#68-71

which were lyophilized and

on a CM-52

column.

Two major

(Fr B) and fractions

#98-101

(Fr

of which corresponded to more than 70% of total

(Fig. 2).

After desalting with a C-18 cartridge,

Fr A and Fr B were each loaded onto an anti-BNP IgG in~nunoaffinity column, and the adsorbed materials were eluted with 1M acetic acid containing The peptides

related to BNP were

thus effectively

reverse phase HPLC on a diphenyl column,

purified.

The

peptides

peptides.

thus

isolated

were

into their respective

Amino acid analysis data

peptide B were

By subsequent

peptide A and peptide B were finally

purified to a homogeneous state from Fr A and Fr B, respectively

after conversion

10% CH3CN.

each

submitted

to

(Fig. 3).

structural

analyses

reduced and S-carboxymethylated (Table i) indicated

composed of 32 and 26 amino acid residues

728

(RCM-)

that peptide A and respectively,

each

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

i00 v

o

1

0.2

o 1.o .~ 0,5~

20

0

40

60

80 i00 Fraction number

120

140

160

Figure 2. CM ion exchange chromatography of ir-BNP fraction in Sephadex G-25 gel filtration. Sample: Fractions #32-36 from Sephadex G-25 gel fil~ration (hatched area in Fig. I) (dry weight, 440 rag). ColLmun: CM-52 (NH 4 -form, 2.4 x 45 cm), pre-equilibrated with solvent (I). Flow rate: 35 ml/hr. Fraction size: 20 ml/tube. Solvent system: Started with solvent (I), and changed at fraction #6 to a linear gradient elution from (I) (1.5 L) to (If) (1.5 L). (I) i0 mM HCOONH 4 (pH 6.6) : CH_CN = 90 : i 0 (v/v). (If) 0.5 M HCOONH 4 (pH 6.6) CH3CN 90 l0 (v/v).

with

2 cysteine

to that of BNP, residues yields peptide

in were

residues. while

addition

peptide to

estimated

B, s t a r t i n g

Peptide

to

B had

an amino

A consisted

those

of

be

about

BNP.

of 32 amino Based

560

acid composition

pmol

from 21.4 kg of p o r c i n e

acids

on these

for

peptide

containing

data, A

identical

and

the 750

6 more

isolation ix~ol for

brain.

a

60~ 40

0.1

2O >

o(

8

o

b 0,ii

ol

10

20

30

40 50 T t m e (mln)

60

70

80

Figure 3. Reverse phase HPLC of anti-BNP inununoaffinity purified fractions. Sample: Anti-BNP immunoaffinifty purified fractions of (a) Fr A and (b) Fr B in CM ion exchange chromatography (Fig. 2). Column: 219TP54 diphenyl (4.6 x 250 ram, Vydac). Flow rate: 1.0 ml/min. solvent system: H_O:CH_CN:I0%TFA = (I) 90:10:1, (II) 40:60:1 (v/v). Linear gradient e~utioJn from (1) to (If) for 120 rain. BNP irmuunoreaetivity was observed only at the black bar regions.

729

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table i.

Amino acid compositions (residues per mole) of RCM-BNP-32 and RCM-BNP

amino acid

BNP-32*

CmCys Asp Thr Ser Pro Gly Val Met

1.85 3.00 i.I0 3.73 0.90 5.21 1.01 0.95

Ile

Leu Tyr Phe Lys Arg

BNP #

(2) (3) (i) (4) (i) (5) (i)

1.81 3.02 2.84 5.04 1.18

1.07

(1) (1)

0.97

(1)

3.98 0.98 0.97 1.12 5.95

(4) (i) (i) (i) (6)

3.93 1.05 1.09 4.91

(4) (i) (i)

total

(2) (3) (3) (5) (i)

(5)

(32)

(26)

*) purified from Fr A. #) purified from Fr B. Numbers in parentheses represent the nearest integers.

Sequence gas-phase degradation peptides,

analysis

was p e r f o r m e d

sequencer.

PTH-amino

was s u c c e s s f u l l y as

shown

in

on 150-200

acid

identified

Fig.

4.

pmol of each R C M - p e p t i d e

liberated

at

each

up to the C - t e r m i n a l

Peptide

B was

cycle

of

residues

determined

to

be

of both exactly

a 60 <

40

D G F~ R

o 2o vo.

I

S

G k

l/

~

DR

~

\^GLG \/\ /"-'k N

"

S

L R

Y

o TD

100

L >-

50

L

~

GLG

i

Cycle number

Figure 4. Yield of PTH-amino acid at each cycle of Edman degradation. Sample: (a) RCM-BNP-32 and (b) RCM-BNP, prepared by reductive carboxymethylation of isolated peptides (Figs. 3a and 3b) followed by reverse phase HPLC purification. One letter amino acid notation is used. CmC; carboxymethyl cysteine.

730

by a Edman

Vol. 155, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

10

20

30

D-S-G-C-F-G-R-R-L-D-R-I-G-S-L-S-G-L-G-C-N-V-L-R-R-Y i

BNP

I

S~K-T-M~D~G[C-F-GIRR LD-R-I-GS-L.S-G-L-G-C-NIV-L-R ~

BNP-32

R-S-S-C-F-G-G-R-M-D-R-I-G-A-Q-S-G-L-G-C-N-S-F--R-Y t,.

a-hANP[4-28]

i

Figure 5. The amino acid sequences of BNP, BNP-32, y-ANP (human) and ~-ANP[4-28]. Identical residues of BNP-32 and y-ANP are boxed with solid lines. One letter amino acid notation is used.

identical

to BNP,

determined

to

which

the

of

deduced

natural

containing

been

an

isolated

peptide-32

(1).

an additional

at the N-terminus.

"brain natriuretic

structure

HPLC

previously

be a form of BNP carrying

(Ser-Pro-Lys-Thr--Met-Arg-) designated

had

6 amino

Hereafter,

(BNP-32)".

with

intramolecular

an

identically

disulfide

acid sequence

Positive confirmation

sequenced

linkage.

on reverse synthetic

Synthetic

BNP-32

and

identified,

relaxant

activity.

Thus,

thus

identified

was

found

to

elicit

at its N-terminus observed

intrinsic bioactivity

of BNP.

In the

~-hANP

and

isolate

definitely

biological

activity,

present

isolation,

we

employed

unfavorable

degradation

neuromedins

BNP and BNP-32

(5-7).

heat

in comparable

from 21.4 kg of porcine

amounts

fact

(750 pmol

that

we

intrinsic

confirmed in

in our previous

the

respectively)

of

isolation were

able

of to

and 560 pmol starting

in the present

study indicates

that BNP and BNP-32 are most likely to be two major endogenous

entities of BNP

family in porcine brain.

brain,

inactivation

which had been

of peptides Therefore,

This indicates

in BNP-32 may not change any

at the earliest stage of purification,

minimizing

diuretic-

was

to that induced by BNP of 26 amino acid residues.

that the six residues

proteases

BNP-32

peptide

as shown in Fig. 5.

BNP-32 comparable

rectum

of

phase

was also

found to have an identical potency to native BNP-32 in hypotensive, natriuretic,

A was

peptide A will be

above was made by co-chromatographies

BNP-32

Peptide

It is of interest that the ANP family found in brain

also consists of two molecular These

two

central

related

control

data obtained porcine

brain

ir-ANP

(0.03

families

forms,

of

i.e.,

natriuretic

of water-electrolyte

~-ANP[4-28] peptides

balance

may

and ~-ANP[5-28] participate

and blood pressure

in the present study reveal that the concentration (0.3 pmol/g wet tissue) pmol/g

wet

tissue)

is about ten times

(2).

This

also

in

(8-12).

(2). the RIA

of ir-BNP in

higher than that of

suggests

the

physiologic

importance of BNP family in the neural control of body fluid homeostasis. At present,

the entire

processing

compared with that of the ANP family.

pattern

However,

731

of the

BNP family

identification

cannot be

of BNP-32 along

Vol. 155, No. 2, 1988

with BNP family. precursor, is

may help

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

us elucidate

the processing

ANP of higher mammals

is initially

events involved synthesized as

from which y-ANP of 126 residues is processed.

cleaved in

response to

the Pro-Arg

generate the 28-residue ~-ANP

(13).

signal preceding

in the BNP

a 151-residue

The y-ANP in turn the e-ANP

In the BNP-32 molecule,

unit to

however,

the

Pro-Lys sequence replaces the Pro-Arg signal found in a similar region of the ANP family and remains uncut.

Moreover,

the ANP family in brain undergoes

further processing at the site of the Arg-Arg pair (corresponding to position 3-4 in e-ANP) to yield yet shorter ~-ANP[4-28] of the Arg-Arg sequence found in ~-ANP, These

varying

amino

acid

and e-ANP[5-28]

( 2 ) . Instead

BNP-32 has only one Arg at this site.

substitutions

in

the

BNP

family

may

reflect

a

different processing pattern than that for the ANP family. ACKNOWLEDGEMENTS: The authors are grateful to Dro S. Ueda and Miss T. Hatoh for RIA and synthesis. This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.

REFERENCES i. sudoh, T., Kangawa, K., Minamino, N. & Matsuo, H. (1988) Nature, 332, 78-81. 2. Ueda, S., Sudoh, T. Fukuda, K°, Kangawa, K., Minamino, N. & Matsuo, H. (1987) Biochem. Biophys. Res. Commun., 149, 1055-1062. 3. Kangawa, K. & Matsuo, H. (1984) Biochem. Eiophys. Res. Commun., 118, 131-139. 4. Currie, M.C., Geller, D.M~, Cole, B.R., Boylar, J.G., YuSheng, W., Holmberg, S.W. & Needleman, P. (1983) Science, 221, 71-73. 5. Miyata, Ao, Kangawa, K., Toshimori, T., Hatoh, T. & Matsuo, H. (1985) Biochem. Biophys. ReSo Commun. 129, 248-255. 6. Minamino, N., Masuda, H., Kangawa, K. & Matsuo, H. (1984) Biochem. Biophys. Res. Commun. 124, 731-738. 7. Minamino, N°, Kangawa, K. & Matsuo, H. (1984) Biochem. Biophys. Res. Commun. 124, 925-932. 8. Quirion, R., Daple, M., de Lean, A., Gutkowska, J., Cantin, M. & Genest, J. (1984) Peptides, 5, 1167-1172. 9. Saper, C.B., Standaert, D.G., Currie, M.G., Schwartz, D., Geller, D.M. & Needleman, P. (1985) Science, 227, 1047-1049. i0. Kawata, M., Nakao, K., Morii, N., Kiso, Y., yamashita, H., Imura, H. & Sano, Y. (1985) Neuroscience, 16, 521-546. ii. Jacobowitz, D.M., Skofitsch, G., Keiser, H.R., Eskay, R.L. & Zamir, N. (1985) Neuroendocrinology, 40, 92-94. 12. Nakao, K., Morii, N., Itoh, H., Yamada, T., Shiono, S., Sugawara, A., saito, Y., Mukoyama, M., Arai, H., Sakamoto, M. Imura, H. (1986) J. Hypertension 4 (Suppl. 6), S492-496. 13. Matsuo, H. & Nakazato, H. (1987) Endocrin. Metab. Clin. North America, 16, 43-61.

732