- Email: [email protected]
N-Terminally extended form of C-type natriuretic peptide (CNP-53) identified in porcine brain
Vol.
170,
July
31.
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1990
Pages
N-TERMINALLY
EXTENDED
FORM
OF C-TYPE
IDENTIFIED
Naoto
MINAMINO,
National
IN
Kenji
.June 20,
Hisayuki
Institute,
565,
Miyazaki
Kiyot.ake,
(CNP-531
MATSUO
Research Osaka
Biochemistry,
Kihara,
Received
Suita,
PEFTIDE
BRAIN
and
Center
Fujishirodai, of
PORCINE
KANGAWA*
Cardiovascular
*Department
NATRIURETIC
973-979
Japan Medical
Miyazaki
889-16,
College, Japan
1990
SUMMARY: C-type natriuretic peptide of 22 residues (CNP-22) is very recently identified in porcine brain as a third member of the mammalian natriuretic peptide family (1). Using a radioimmunoassay system newly established for CNP-22, we searched for CNP-related peptides in porcine brain. In addition to CNP-22, one major form of immunoreactive CNP was detected in porcine brain extracts, being isolated by immunoaffinity chromatography and reverse phase high performance liquid chromatography. By microsequence analysis, the peptide was deduced to be a 53-amino acid peptide carrying a CNP-22 sequence at the C-terminus, and was designated C-type natriuretic peptide(CNP-53). CNP-53 was found to be a major molecular form of CNP in porcine brain. Q 1990 Academic Press, Inc.
In
a
systematic
relaxant
activity
natriuretic
peptide
designated
similar
to
acid
were
However,
the
these
case three
Abbreviations: peptide natriruetic relative trifluoroacetic
the
of
from
and
BNP,
types
including
effects, in
homologous 17-residue
The
each to
discovery natriuretic
exerted
assay the
of
The
peptide
atria1 natriuretic
peptide spectrum
CNP-22
ANP
CNP
following probably
by and ANP
of
also
units
in
peptide
potencies
formed
found
(B-
hypotensive
relative
bioactive
was
natriuretic
a pharmacological
(11.
for a novel
diuretic-natriuretic,
structure
commonly
(1).
brain
although
ring tail
of
CNP
bioassay
isolated
following
and
a
recently
brain
(CNPI" ANP)
using
very
porcine
(2-4).
different
CNP.
have
peptide
BNPI
C-terminal
peptides
we
peptide,
ANP
highly in
unidentified
residues
relaxant
rather
sequence
especially
the
of
rectum
peptides
22
peptide, those
chick
rectum,
natriuretic
natriuretic
and
for
chick
natriuretic
(~-type
type
survey
of
"C-type
peptide
in
on
has
of a BNP and function
the
ANP
an
amino
and
disulfide is
three
completely BNP
BNP,
linkage. lost
suggests to
that regulate
CNP, C-type natriuretic peptide; ANP, atria1 natriuretic natriuretic peptide); BNP, brain natriuretic peptide (B-type peptide); HPIJC, high performance liquid chromatography; Mr, molecular mass; RIA, radioimmunoassay; ir, immunoreactive; TFA, acid; PTH, phenylthiohydantoin; IgG, immunoglobulin G.
(A-type
0006-291X/90 973
$1.50
Copyright 0 I990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
17‘0,
No.
2,
homeostatic concerted
1990
balance but
an
of body
distinct
In order CNP,
BIOCHEMICAL
to
system
specific
system,
we
isolated
a new
reports
isolation
against
for
volume,
endogenous CNP-22
RESEARCH
electrolytes
molecular
COMMUNICATIONS
and blood
for
53-residue
CNP-related peptide,
and sequence
forms
was prepared,
CNP was established
searched
BIOPHYSICAL
pressure
in a
manner.
elucidate
antiserum
fluid
AND
(to
peptides
MATERIALS
in
porcine
CNP-53. of
system
and a radioimmunoassay
be published).
designated
determination
and processing By using brain The
(RIA) this
extracts present
of RIA and
paper
CNP-53.
AND METHODS
Starting materials used in the present purification was a side Isolation: (1). Porcine brain fraction of our previous purification of porcine CNP-22 prepared from 40 kg (480 pigs) of fresh porcine brain. A extracts were peptide fraction was prepared from crude brain extracts by Pellicon cassette condensation, acetone precipitation and batch-wise reverse phase C-18 chromatography. From the peptide fraction, an SP-III fraction containing basic peptides was obtained by SP-Sephadex ion exchange chromatography. The SP-III fraction was then separated by gel filtration on a Sephadex G-50 column (fine, 7.5 x 145 cm, Pharmacial. Fractions corresponding to relative molecular mass (Mr) lK-5K were collected and subjected to a second gel filtration on a Sephadex ~-25 column (fine, 7.5 x 146 cm). An aliquot of each fraction in gel filtration was submitted to RIAs for CNP, BNP and ANP. Two peaks of immunoreactive (ir-l CNP were observed on the chromatogram, and CNP22 was isolated from the low Mr ir-CNP peak in fraction C in the present fractionation. The present purification concerned the high Mr ir-CNP peak in fraction B of Fig. 1. Fraction B of Mr 3K-5K was subjected to CM ion exchange chromatography (CM-52, 2.4 x 52.5 cm, Whatmanl eluting with a linear gradient elution of HCOONH (pH 6.6) from 10 mM to 0.6 M in the presence of 10% CH CN (Fig. 2). Frac&ons #ill-113 in Fig. 2 were then subjected to anti-cc- a NP immunoglobulin G (IgGI immunoaffinity chromatography, as described previously for the purification of a-ANP(4-28) and CX-ANP(5-28) (5). The adsorbed peptides on the immunoaffinity column were eluted with a solution of 1M CH3COOH containing 10% CH3CN, and then separated by reverse phase high performance liquid chromatography (HPLC) on a C-18 column (Hi-Pore RP-318, 4.6 x 250 mm, Bio-Rad) with a linear gradient elution of CH3CN from 10% to 60% in 0.1% trifluoroacetic acid (TFA) for 120 min at a flow rate of 1.5 ml/min. Final purification of CNP-53 was also performed by reverse phase HPLC on a diphenyl column (219TP54, 4.6 x 250 mm, Vydac) under the same solvent system as described above at a flow rate of 1.0 ml/min. Aliquots of all fractions were submitted to RIA for CNP, and column effluents of HPLC were monitored by measuring absorbance at 210 nm and 280 nm. RIAs for CNP, BNP and ANP: Details on preparation of an antiserum against CNP and characterization of RIA for CNP will be reported in a separate paper. In brief, antiserum #171-4 was usable at a final dilution of 1:60,000, and peptides were measureable in a range of l-100 fmol/tube. In this RIA system, CL-ANP and BNP-26 showed 0.015% and 0.46% crossreactivity, respectively. RIAs for porcine ANP and BNP were carried out as reported previously (6,7\. Synthetic CNP-22 showed less than 0.001% crossreactivity in each RIA system. Sequence analysis: Three-fourths of the finally purified CNP-53 were directly submitted to microsequence analysis with a gas-phase sequencer coupled with on-line HPLC identification system of the resulting phenylthiohydantoin (PTHJ amino acids (Applied Biosystems 47OA/l2OA). PTH-amino acids were detectable down to 0.2 pmol. Bioassay: Chick rectum relaxant activity was assayed by the described method, using freshly isolated chick rectum strips (8,91. 974
Vol.
170,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS AND DISCUSSION
from of
CNP-53
was
a side
fraction
porcine
isolated
brain
weight:
5.2
ion was
subjected
exchange
in
all
and ir-ANP
ir-BNP Mr
lK-5K
Sephadex
the
G,-25
separated
into
isolated
from
(1).
emerged
lyophilized gel
two peaks
respectively,
as reported
740
the
chromatogram,
peak
comprising
about
region
of
As
and
fraction of
were
previous B of
ee
1.
Fig.
peaks
of
the was
of Mr 2K-5K,
while
from
eluted
in
isolation
of
fraction
separated
ir-CNP
by a ratio
crude first ir-CNP
The peptide 1,
and
of
and further
CNP-22
subjected
ir-CNP
were
Mr 4K-SK to
as shown
in
Fig.
75% of
the
total
20
ion
3:4.
fraction
by
was clearly CNP-53
was
C of Mr 2K-3K
fractions
C
of BNP-26,
30 Fraction
2.
was pooled,
exchange
observed
1 23 t tt ----ABCDE
10
in
and 4K-5K
ANP
in the
gl
by Pellicon
ir-CNP fraction
Mr 2K-4K.
dry
chromatography
A portion
major
2.96
shown while
of the SP-III
CNP
extracts
fraction,
extracts C-18
column.
with
acid
and
D,
BNP-32,
CL-
(4,5,10).
and then
mg1
Two peaks
column.
G-50
weight:
of Mr 2K-3K
and a-ANP[5-28)
CNP-immunoreactive weight:
the
(dry
BNP
crude
for
system
from
(SP-III
phase
Most The
along
B of Mr 4K-5K,
Immunoreactive
ANP(4-28)
in
filtration.
fraction
from
fraction.
of Mr SK-6K
RIA
CNP-22
fraction
reverse
on a Sephadex
region
was
peptide
prepared
this
established of
chromatography. in
filtration
was eluted of
The basic precipitation,
recovered
to gel
a newly purification
was efficiently
gl
acetone
SP-Sephadex
utilizing previous
(40 kgl .
ultrafiltration, extracts
by
in our
chromatography
in tubes CNP-53
lyophilized
#99-102
was purified
ir-CNP.
After
(dry
on a CM-52
and
#ill-113
from
the
on latter
lyophilization,
CNP-
45 tt t_-
40 number
Sephadex G-25 gel filtration
of Porcine
F
50
brain
60
extracts.
Gle: Half of basic peptide fraction of Mr lK-5K (dry weight: Column: Sephadex G-25 (fine, 7.5 x 146 cm, Pharmacia). Fraction size: 100 ml/tube. Flow rate: 100 ml/h. Arrows indicate elution positions of II bovine serum albumin, 3) BNP-26, 4) c(-ANP(4-28) and 51 neurotensin.
975
1.48
g).
21 BNP-32,
Vol.
170,
No.
2,
1990
BIOCHEMICAL
AND
Fraction
Figure
fraction
immunoaffinity
chromatography
which
RESEARCH
COMMUNICATIONS
number
Cation exchange chromatography of basic peptide fraction 2. of Mr 4K-5K. Sample: Fraction B (tubes #29-31) in Fig. 1 (dry weight: 740 Column: CM-52 (NH:-form, 2.4 x 52.5 cm, Flow rate: 40 ml/h. Fraction size: 20 ml/tube. Solvent system: Linear gradient elution from (Al to (B). (A) 1OmM HCOONH4 (pH 6.6):CH3CN = 9O:lO (V/VI, (Bi 0.6M HCOONH4 (pH 6.6):CH3CN = 9O:lO (V/V). Fraction marked with black bar was used for further purification.
immunoreactive
ANP,
BIOPHYSICAL
was
(tubes
#ill-113, using
more
effective
30
40
for
dry an
IgG
weight:
8.5
fraction
of
immunoaffinity
mg)
antiserum
chromatography
was
mgl. Whatmanl.
subjected
than
a
i
50 Time
Figure
60
70 20
(min)
30
40 Time
50
60
(mlnl
3. Purification of CNP-53 by repeated reverse phase HPLC. Sample: (a) Anti-or-ANP IgG immunoaffinity chromatography-adsorbed fraction of tubes #ill-113 (black bar region1 in Fig. 2. (b) CNP-immunoreactive fractions eluted around 48 min in (a). Column: (a) Hi-Pore RP-318 (4.6 x 250 mm, Bio-Rad), (bl 219TP54 diphenyl (4.6 x 250 mm, Vydac). Flow rate: (a) 1.5 ml/min, (b) 1.0 ml/nun. Solvent system: Linear gradient elution from (A) to (B) for 120 min. H20:CH3CN:10%TFA = (Al 90:10:1, (B) 40:60:1 (V/VI.
976
to
against that
cIof
Vol.
170,
No.
;!,
1990
BIOCHEMICAL
0
AND
BIOPHYSICAL
L 10
30
20
Cycle
antiserum column AS
4. Yield of P'TH-amino CNP-53. one letter amino cycles #30-45 under 4-fold
#171-4 were
against
eluted
shown
in
indicating
Fig.
that
phase
highly
immunoreactivity
peptide kg
of
estimated
porcine
brain.
Because fourths
of
of
sequencer. identified After
up the
45th
PTH-amino
acid
i
45th
step
step
of
analysis,
peptide
except
pmol
the
were
on
at
each
for
the
subsequence
peak
of
as
well of from
of
was
in but
Fig. were
peptide
15
Aso~Leu-Arg-Val-Asa-Thr-Lys-Ser-Arg-Ala-Ala-Tr~-Ala-Arg-Leu-Leu-H~sFI~-H~s~~~~-As~-Ala-Arg-Lys-~~r-Lys-G;y-6:y-Asnf~~~~y-L~~-~~r-L~sGIy-Cys-Phe-G1y-L4eO-Lys-Leu-Asp-Arg-14;e-Gly-SeT-Met-Ser-dPy-Leu-Giy-CS:s I
I
: CNP-53
&LP,"-SWLW G;y-Cys-Phe-Giy-Leu-;yOs-leu-Arg-,,~-~~y-~~r-~~~-~~~-~,y-~~~-~,y-C~s 1
--Figure
Amino acid sequences of porcine 5. Intramolecular disulfide linkage is in each peptide. Lys-Lys sequence, qenerate CNP-22, is boxed.
977
I
CNP-53 and CNP-22. formed between two cysteine probably a processing signal
40
a gas-phase
degradation
this
as the
three-
shown
detected
(32-45)
10
to
as
were
reverse
purified,
Edman
step,
affinity by
yield
submitted
acids
the
starting
thus
cycle
fraction,
height
ngl,
(700
peptide
37th
by
isolation
directly
PTH-amino The
5
of
amount
peak
homogeneity
the 130
liberated
the
identifiable.
definitely
Based
column.
a c-18
on
a main
t-o
of of
immunoaffinity
performed
was
3b).
HPLC
at
purification,
limited purified
phase
purified
approximately
finally
to
(Fig.
the
on
eluted
Was
finally
final
be
reverse
purification was
the to
a very
the
ir-CNP
column in
waci
peptides
by
efficient peptide
a diphenyl
on
adsorbed
separated
most.
40
number
acid at each cycle of Edman degradation (Inset) Data acid notation is used. vertical magnification.
The
then
3a,
The
HPLC
CNP
CNP.
and
chromatography.
COMMUNICATIONS
-
0
Figure
RESEARCH
: CNP-22
residues to
4. not was
Vol.
170,
No.
2,
completely
identical
peptide both
to
showed
chick
which
require
of
cysteine
residues,
molecular
weight
peptide
BIOCHEMICAL
1990
was
sequence
and was the
natriuretic recently
of a CNP precursor
CNP, a termination CNP-22
codon
or CNP-53, In the
molecule
processing
the
presumably
relative
content
G-25
filtration
step
gel
filtration
step.
isolation due to
is its
suggest
relatively
that
addition,
basic
CNP-53 our
is
that imply By the present
CNP-53
brain. from
forms These
those
2K-3K
ir-CNP
the
preceding
of
whole itself
identification different
facts
indicate
yield that
RIA data
molecular brain also
that
of
of
CNP-53
in
which
from
gel
is
be
more
CNP-22
Thus,
CNP-22
in Fig.
CNP-22
G-50
the is
CNP has a unique
probably
filtrations In
brain. predominant
These in brain.
to exist
and CNP-53,
processing
gel
present
published).
shown
1,
in Sephadex
as a neuropeptide CNP is
two
the
Sephadex
CNP in porcine (to
functions
In these
was 4:3
CNP-53
extract
weights,
5).
of CNP-22,
that
of CNP-53, molecular
of
obtained
form
indicate
residue
As indicated
in
RIA data
for
proteolytic
CNP-22.
to
a major
results
into
brain.
to
structure
preceding
higher
the
porcine
(Fig. just
ir-CNP
as compared
sequence
putative
4K-5K
low
in the
uncut
CNP-53
recovery
CNP-22
hereafter
cysteine
and was
nature,
CNP-22
precusor
the
30-31,
converting
a final
preliminary
remain
of CNP in porcine of
than
endogenous
for
Although
highly
of
at positions
this
conclusion.
sets
two
a
its
peptide-
CNP is
a C-terminal
present
and Lys-Lys,
form
however,
the
CNP-53,
signal
a mature
In the
follows
Thus,
nucleotide
and
two
when
natriuretic
cloning
in
between
basis,
22-residue
be published).
signal
as the
(CNP-22)".
supports
Arg-Lys
Lys-Lys
may serve
also of
signals,
signals,
peptide-
TAG directly
which
"C-type
isolated
(to
formed
on a molar
originally
the
as CNP immunoreactivity,
acid peptide. peptide carrying
designated
succeeded
COMMUNICATIONS
Furthermore,
structure
of CNP-22
as a 53-amino to be a 53-residue
deduced
very
as well
ring
to those
RESEARCH
of CNP-22.
activity
17-residue
C-terminus
"C-type
analysis
is
the
BIOPHYSICAL
sequence
relaxant
comparable
Therefore,
We have
unit,
rectum
finally
designated
N-terminal
was calculated
in the
(CNP-53)".
the
AND
as two in porcine
system
distinct
of ANP and BNP.
Acknowledgments: This Ministry of Education, and Welfare of Japan.
work was supported in Science and Culture,
part by research grants and from the Ministry
from the of Health
REFERENCES 1. Sudoh, T., Minamino, N., Kangawa, K. & Matsuo, H. (1990) Res . Commun., 168, 863-870. 2. de Bold, A. J. (19851 Science, 230, 767-770. 3. Matsuo, H. & Nakazato, H. (1987)ndocrinol. Metab. Clin. 16, 43-61. 4. Sudoh, T., Kangawa, K., Minamino, N. & Matsuo, H. (1988) 78-81. 978
Biochem. North Nature,
Biophys. Am., 332,
Vol.
170,
No.
;!,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
5. Ueda, S., Sudoh, T., Fukuda, K., Kangawa, K., Minamino, N. & Matsuo, H. (1987) Biochem. Biophys. Res. Commun., 149, 1055-1062. 6. Miyata, A., Kangawa, K., Toshimori, T.,xtoh, T. & Matsuo, H. (19851 Biochem. Biophys. Res. Commun., 129, 248-255. 7. Ueda, S.,, Minamino, N., Sudoh, T., Kangawa, K. & Matsuo, H. (1988) Biochem. Biophys. Res. Commun., 155, 733-739. 8. Kangawa, K. & Matsuo, H. (1984) Biochem. Biophys. Res. Commun., 118, 131-139. 9. Currie, M-G., Geller, D.M., Cole, B-R., Siegel, N.R., Fok, K-F., Adams, Eulanks, S.R., Galluppi, G.R. & Needleman, P. (19831 Science, 221, S-P., 71-73. 10. Sudoh, T., Minamino, N., Kangawa, K. & Matsuo, H. (1988) Biochem. Biophys. Res. Commun., 155, 726-732.
979
170,
July
31.
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
1990
Pages
N-TERMINALLY
EXTENDED
FORM
OF C-TYPE
IDENTIFIED
Naoto
MINAMINO,
National
IN
Kenji
.June 20,
Hisayuki
Institute,
565,
Miyazaki
Kiyot.ake,
(CNP-531
MATSUO
Research Osaka
Biochemistry,
Kihara,
Received
Suita,
PEFTIDE
BRAIN
and
Center
Fujishirodai, of
PORCINE
KANGAWA*
Cardiovascular
*Department
NATRIURETIC
973-979
Japan Medical
Miyazaki
889-16,
College, Japan
1990
SUMMARY: C-type natriuretic peptide of 22 residues (CNP-22) is very recently identified in porcine brain as a third member of the mammalian natriuretic peptide family (1). Using a radioimmunoassay system newly established for CNP-22, we searched for CNP-related peptides in porcine brain. In addition to CNP-22, one major form of immunoreactive CNP was detected in porcine brain extracts, being isolated by immunoaffinity chromatography and reverse phase high performance liquid chromatography. By microsequence analysis, the peptide was deduced to be a 53-amino acid peptide carrying a CNP-22 sequence at the C-terminus, and was designated C-type natriuretic peptide(CNP-53). CNP-53 was found to be a major molecular form of CNP in porcine brain. Q 1990 Academic Press, Inc.
In
a
systematic
relaxant
activity
natriuretic
peptide
designated
similar
to
acid
were
However,
the
these
case three
Abbreviations: peptide natriruetic relative trifluoroacetic
the
of
from
and
BNP,
types
including
effects, in
homologous 17-residue
The
each to
discovery natriuretic
exerted
assay the
of
The
peptide
atria1 natriuretic
peptide spectrum
CNP-22
ANP
CNP
following probably
by and ANP
of
also
units
in
peptide
potencies
formed
found
(B-
hypotensive
relative
bioactive
was
natriuretic
a pharmacological
(11.
for a novel
diuretic-natriuretic,
structure
commonly
(1).
brain
although
ring tail
of
CNP
bioassay
isolated
following
and
a
recently
brain
(CNPI" ANP)
using
very
porcine
(2-4).
different
CNP.
have
peptide
BNPI
C-terminal
peptides
we
peptide,
ANP
highly in
unidentified
residues
relaxant
rather
sequence
especially
the
of
rectum
peptides
22
peptide, those
chick
rectum,
natriuretic
natriuretic
and
for
chick
natriuretic
(~-type
type
survey
of
"C-type
peptide
in
on
has
of a BNP and function
the
ANP
an
amino
and
disulfide is
three
completely BNP
BNP,
linkage. lost
suggests to
that regulate
CNP, C-type natriuretic peptide; ANP, atria1 natriuretic natriuretic peptide); BNP, brain natriuretic peptide (B-type peptide); HPIJC, high performance liquid chromatography; Mr, molecular mass; RIA, radioimmunoassay; ir, immunoreactive; TFA, acid; PTH, phenylthiohydantoin; IgG, immunoglobulin G.
(A-type
0006-291X/90 973
$1.50
Copyright 0 I990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
17‘0,
No.
2,
homeostatic concerted
1990
balance but
an
of body
distinct
In order CNP,
BIOCHEMICAL
to
system
specific
system,
we
isolated
a new
reports
isolation
against
for
volume,
endogenous CNP-22
RESEARCH
electrolytes
molecular
COMMUNICATIONS
and blood
for
53-residue
CNP-related peptide,
and sequence
forms
was prepared,
CNP was established
searched
BIOPHYSICAL
pressure
in a
manner.
elucidate
antiserum
fluid
AND
(to
peptides
MATERIALS
in
porcine
CNP-53. of
system
and a radioimmunoassay
be published).
designated
determination
and processing By using brain The
(RIA) this
extracts present
of RIA and
paper
CNP-53.
AND METHODS
Starting materials used in the present purification was a side Isolation: (1). Porcine brain fraction of our previous purification of porcine CNP-22 prepared from 40 kg (480 pigs) of fresh porcine brain. A extracts were peptide fraction was prepared from crude brain extracts by Pellicon cassette condensation, acetone precipitation and batch-wise reverse phase C-18 chromatography. From the peptide fraction, an SP-III fraction containing basic peptides was obtained by SP-Sephadex ion exchange chromatography. The SP-III fraction was then separated by gel filtration on a Sephadex G-50 column (fine, 7.5 x 145 cm, Pharmacial. Fractions corresponding to relative molecular mass (Mr) lK-5K were collected and subjected to a second gel filtration on a Sephadex ~-25 column (fine, 7.5 x 146 cm). An aliquot of each fraction in gel filtration was submitted to RIAs for CNP, BNP and ANP. Two peaks of immunoreactive (ir-l CNP were observed on the chromatogram, and CNP22 was isolated from the low Mr ir-CNP peak in fraction C in the present fractionation. The present purification concerned the high Mr ir-CNP peak in fraction B of Fig. 1. Fraction B of Mr 3K-5K was subjected to CM ion exchange chromatography (CM-52, 2.4 x 52.5 cm, Whatmanl eluting with a linear gradient elution of HCOONH (pH 6.6) from 10 mM to 0.6 M in the presence of 10% CH CN (Fig. 2). Frac&ons #ill-113 in Fig. 2 were then subjected to anti-cc- a NP immunoglobulin G (IgGI immunoaffinity chromatography, as described previously for the purification of a-ANP(4-28) and CX-ANP(5-28) (5). The adsorbed peptides on the immunoaffinity column were eluted with a solution of 1M CH3COOH containing 10% CH3CN, and then separated by reverse phase high performance liquid chromatography (HPLC) on a C-18 column (Hi-Pore RP-318, 4.6 x 250 mm, Bio-Rad) with a linear gradient elution of CH3CN from 10% to 60% in 0.1% trifluoroacetic acid (TFA) for 120 min at a flow rate of 1.5 ml/min. Final purification of CNP-53 was also performed by reverse phase HPLC on a diphenyl column (219TP54, 4.6 x 250 mm, Vydac) under the same solvent system as described above at a flow rate of 1.0 ml/min. Aliquots of all fractions were submitted to RIA for CNP, and column effluents of HPLC were monitored by measuring absorbance at 210 nm and 280 nm. RIAs for CNP, BNP and ANP: Details on preparation of an antiserum against CNP and characterization of RIA for CNP will be reported in a separate paper. In brief, antiserum #171-4 was usable at a final dilution of 1:60,000, and peptides were measureable in a range of l-100 fmol/tube. In this RIA system, CL-ANP and BNP-26 showed 0.015% and 0.46% crossreactivity, respectively. RIAs for porcine ANP and BNP were carried out as reported previously (6,7\. Synthetic CNP-22 showed less than 0.001% crossreactivity in each RIA system. Sequence analysis: Three-fourths of the finally purified CNP-53 were directly submitted to microsequence analysis with a gas-phase sequencer coupled with on-line HPLC identification system of the resulting phenylthiohydantoin (PTHJ amino acids (Applied Biosystems 47OA/l2OA). PTH-amino acids were detectable down to 0.2 pmol. Bioassay: Chick rectum relaxant activity was assayed by the described method, using freshly isolated chick rectum strips (8,91. 974
Vol.
170,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
RESULTS AND DISCUSSION
from of
CNP-53
was
a side
fraction
porcine
isolated
brain
weight:
5.2
ion was
subjected
exchange
in
all
and ir-ANP
ir-BNP Mr
lK-5K
Sephadex
the
G,-25
separated
into
isolated
from
(1).
emerged
lyophilized gel
two peaks
respectively,
as reported
740
the
chromatogram,
peak
comprising
about
region
of
As
and
fraction of
were
previous B of
ee
1.
Fig.
peaks
of
the was
of Mr 2K-5K,
while
from
eluted
in
isolation
of
fraction
separated
ir-CNP
by a ratio
crude first ir-CNP
The peptide 1,
and
of
and further
CNP-22
subjected
ir-CNP
were
Mr 4K-SK to
as shown
in
Fig.
75% of
the
total
20
ion
3:4.
fraction
by
was clearly CNP-53
was
C of Mr 2K-3K
fractions
C
of BNP-26,
30 Fraction
2.
was pooled,
exchange
observed
1 23 t tt ----ABCDE
10
in
and 4K-5K
ANP
in the
gl
by Pellicon
ir-CNP fraction
Mr 2K-4K.
dry
chromatography
A portion
major
2.96
shown while
of the SP-III
CNP
extracts
fraction,
extracts C-18
column.
with
acid
and
D,
BNP-32,
CL-
(4,5,10).
and then
mg1
Two peaks
column.
G-50
weight:
of Mr 2K-3K
and a-ANP[5-28)
CNP-immunoreactive weight:
the
(dry
BNP
crude
for
system
from
(SP-III
phase
Most The
along
B of Mr 4K-5K,
Immunoreactive
ANP(4-28)
in
filtration.
fraction
from
fraction.
of Mr SK-6K
RIA
CNP-22
fraction
reverse
on a Sephadex
region
was
peptide
prepared
this
established of
chromatography. in
filtration
was eluted of
The basic precipitation,
recovered
to gel
a newly purification
was efficiently
gl
acetone
SP-Sephadex
utilizing previous
(40 kgl .
ultrafiltration, extracts
by
in our
chromatography
in tubes CNP-53
lyophilized
#99-102
was purified
ir-CNP.
After
(dry
on a CM-52
and
#ill-113
from
the
on latter
lyophilization,
CNP-
45 tt t_-
40 number
Sephadex G-25 gel filtration
of Porcine
F
50
brain
60
extracts.
Gle: Half of basic peptide fraction of Mr lK-5K (dry weight: Column: Sephadex G-25 (fine, 7.5 x 146 cm, Pharmacia). Fraction size: 100 ml/tube. Flow rate: 100 ml/h. Arrows indicate elution positions of II bovine serum albumin, 3) BNP-26, 4) c(-ANP(4-28) and 51 neurotensin.
975
1.48
g).
21 BNP-32,
Vol.
170,
No.
2,
1990
BIOCHEMICAL
AND
Fraction
Figure
fraction
immunoaffinity
chromatography
which
RESEARCH
COMMUNICATIONS
number
Cation exchange chromatography of basic peptide fraction 2. of Mr 4K-5K. Sample: Fraction B (tubes #29-31) in Fig. 1 (dry weight: 740 Column: CM-52 (NH:-form, 2.4 x 52.5 cm, Flow rate: 40 ml/h. Fraction size: 20 ml/tube. Solvent system: Linear gradient elution from (Al to (B). (A) 1OmM HCOONH4 (pH 6.6):CH3CN = 9O:lO (V/VI, (Bi 0.6M HCOONH4 (pH 6.6):CH3CN = 9O:lO (V/V). Fraction marked with black bar was used for further purification.
immunoreactive
ANP,
BIOPHYSICAL
was
(tubes
#ill-113, using
more
effective
30
40
for
dry an
IgG
weight:
8.5
fraction
of
immunoaffinity
mg)
antiserum
chromatography
was
mgl. Whatmanl.
subjected
than
a
i
50 Time
Figure
60
70 20
(min)
30
40 Time
50
60
(mlnl
3. Purification of CNP-53 by repeated reverse phase HPLC. Sample: (a) Anti-or-ANP IgG immunoaffinity chromatography-adsorbed fraction of tubes #ill-113 (black bar region1 in Fig. 2. (b) CNP-immunoreactive fractions eluted around 48 min in (a). Column: (a) Hi-Pore RP-318 (4.6 x 250 mm, Bio-Rad), (bl 219TP54 diphenyl (4.6 x 250 mm, Vydac). Flow rate: (a) 1.5 ml/min, (b) 1.0 ml/nun. Solvent system: Linear gradient elution from (A) to (B) for 120 min. H20:CH3CN:10%TFA = (Al 90:10:1, (B) 40:60:1 (V/VI.
976
to
against that
cIof
Vol.
170,
No.
;!,
1990
BIOCHEMICAL
0
AND
BIOPHYSICAL
L 10
30
20
Cycle
antiserum column AS
4. Yield of P'TH-amino CNP-53. one letter amino cycles #30-45 under 4-fold
#171-4 were
against
eluted
shown
in
indicating
Fig.
that
phase
highly
immunoreactivity
peptide kg
of
estimated
porcine
brain.
Because fourths
of
of
sequencer. identified After
up the
45th
PTH-amino
acid
i
45th
step
step
of
analysis,
peptide
except
pmol
the
were
on
at
each
for
the
subsequence
peak
of
as
well of from
of
was
in but
Fig. were
peptide
15
Aso~Leu-Arg-Val-Asa-Thr-Lys-Ser-Arg-Ala-Ala-Tr~-Ala-Arg-Leu-Leu-H~sFI~-H~s~~~~-As~-Ala-Arg-Lys-~~r-Lys-G;y-6:y-Asnf~~~~y-L~~-~~r-L~sGIy-Cys-Phe-G1y-L4eO-Lys-Leu-Asp-Arg-14;e-Gly-SeT-Met-Ser-dPy-Leu-Giy-CS:s I
I
: CNP-53
&LP,"-SWLW G;y-Cys-Phe-Giy-Leu-;yOs-leu-Arg-,,~-~~y-~~r-~~~-~~~-~,y-~~~-~,y-C~s 1
--Figure
Amino acid sequences of porcine 5. Intramolecular disulfide linkage is in each peptide. Lys-Lys sequence, qenerate CNP-22, is boxed.
977
I
CNP-53 and CNP-22. formed between two cysteine probably a processing signal
40
a gas-phase
degradation
this
as the
three-
shown
detected
(32-45)
10
to
as
were
reverse
purified,
Edman
step,
affinity by
yield
submitted
acids
the
starting
thus
cycle
fraction,
height
ngl,
(700
peptide
37th
by
isolation
directly
PTH-amino The
5
of
amount
peak
homogeneity
the 130
liberated
the
identifiable.
definitely
Based
column.
a c-18
on
a main
t-o
of of
immunoaffinity
performed
was
3b).
HPLC
at
purification,
limited purified
phase
purified
approximately
finally
to
(Fig.
the
on
eluted
Was
finally
final
be
reverse
purification was
the to
a very
the
ir-CNP
column in
waci
peptides
by
efficient peptide
a diphenyl
on
adsorbed
separated
most.
40
number
acid at each cycle of Edman degradation (Inset) Data acid notation is used. vertical magnification.
The
then
3a,
The
HPLC
CNP
CNP.
and
chromatography.
COMMUNICATIONS
-
0
Figure
RESEARCH
: CNP-22
residues to
4. not was
Vol.
170,
No.
2,
completely
identical
peptide both
to
showed
chick
which
require
of
cysteine
residues,
molecular
weight
peptide
BIOCHEMICAL
1990
was
sequence
and was the
natriuretic recently
of a CNP precursor
CNP, a termination CNP-22
codon
or CNP-53, In the
molecule
processing
the
presumably
relative
content
G-25
filtration
step
gel
filtration
step.
isolation due to
is its
suggest
relatively
that
addition,
basic
CNP-53 our
is
that imply By the present
CNP-53
brain. from
forms These
those
2K-3K
ir-CNP
the
preceding
of
whole itself
identification different
facts
indicate
yield that
RIA data
molecular brain also
that
of
of
CNP-53
in
which
from
gel
is
be
more
CNP-22
Thus,
CNP-22
in Fig.
CNP-22
G-50
the is
CNP has a unique
probably
filtrations In
brain. predominant
These in brain.
to exist
and CNP-53,
processing
gel
present
published).
shown
1,
in Sephadex
as a neuropeptide CNP is
two
the
Sephadex
CNP in porcine (to
functions
In these
was 4:3
CNP-53
extract
weights,
5).
of CNP-22,
that
of CNP-53, molecular
of
obtained
form
indicate
residue
As indicated
in
RIA data
for
proteolytic
CNP-22.
to
a major
results
into
brain.
to
structure
preceding
higher
the
porcine
(Fig. just
ir-CNP
as compared
sequence
putative
4K-5K
low
in the
uncut
CNP-53
recovery
CNP-22
hereafter
cysteine
and was
nature,
CNP-22
precusor
the
30-31,
converting
a final
preliminary
remain
of CNP in porcine of
than
endogenous
for
Although
highly
of
at positions
this
conclusion.
sets
two
a
its
peptide-
CNP is
a C-terminal
present
and Lys-Lys,
form
however,
the
CNP-53,
signal
a mature
In the
follows
Thus,
nucleotide
and
two
when
natriuretic
cloning
in
between
basis,
22-residue
be published).
signal
as the
(CNP-22)".
supports
Arg-Lys
Lys-Lys
may serve
also of
signals,
signals,
peptide-
TAG directly
which
"C-type
isolated
(to
formed
on a molar
originally
the
as CNP immunoreactivity,
acid peptide. peptide carrying
designated
succeeded
COMMUNICATIONS
Furthermore,
structure
of CNP-22
as a 53-amino to be a 53-residue
deduced
very
as well
ring
to those
RESEARCH
of CNP-22.
activity
17-residue
C-terminus
"C-type
analysis
is
the
BIOPHYSICAL
sequence
relaxant
comparable
Therefore,
We have
unit,
rectum
finally
designated
N-terminal
was calculated
in the
(CNP-53)".
the
AND
as two in porcine
system
distinct
of ANP and BNP.
Acknowledgments: This Ministry of Education, and Welfare of Japan.
work was supported in Science and Culture,
part by research grants and from the Ministry
from the of Health
REFERENCES 1. Sudoh, T., Minamino, N., Kangawa, K. & Matsuo, H. (1990) Res . Commun., 168, 863-870. 2. de Bold, A. J. (19851 Science, 230, 767-770. 3. Matsuo, H. & Nakazato, H. (1987)ndocrinol. Metab. Clin. 16, 43-61. 4. Sudoh, T., Kangawa, K., Minamino, N. & Matsuo, H. (1988) 78-81. 978
Biochem. North Nature,
Biophys. Am., 332,
Vol.
170,
No.
;!,
1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
5. Ueda, S., Sudoh, T., Fukuda, K., Kangawa, K., Minamino, N. & Matsuo, H. (1987) Biochem. Biophys. Res. Commun., 149, 1055-1062. 6. Miyata, A., Kangawa, K., Toshimori, T.,xtoh, T. & Matsuo, H. (19851 Biochem. Biophys. Res. Commun., 129, 248-255. 7. Ueda, S.,, Minamino, N., Sudoh, T., Kangawa, K. & Matsuo, H. (1988) Biochem. Biophys. Res. Commun., 155, 733-739. 8. Kangawa, K. & Matsuo, H. (1984) Biochem. Biophys. Res. Commun., 118, 131-139. 9. Currie, M-G., Geller, D.M., Cole, B-R., Siegel, N.R., Fok, K-F., Adams, Eulanks, S.R., Galluppi, G.R. & Needleman, P. (19831 Science, 221, S-P., 71-73. 10. Sudoh, T., Minamino, N., Kangawa, K. & Matsuo, H. (1988) Biochem. Biophys. Res. Commun., 155, 726-732.
979