Vol.
167,
March
No.
16,
;!, 1990
BIOCHEMICAL
AND
RESEARCH
COMMUNICATIONS
AND
IDENTIFICATION
OF IN
Jun
HINOl
1
Department
L
TATEYAMA
1,3
of
Fukushima, January
27,
MINAMIN02,
Medical
College, Japan
Research
Osaka
Medicine,
Institute,
565,
Osaka Osaka
PEPTIDES
MATSU02
889-16,
Center Suita,
Geriatric
, Naoto
Hisayuki
Miyazaki
Fujishirodai,
NATRIURETIC
Miyazaki
Kiyotake, Cardiovascular
National
Department
and
Biochemistry,
Kihara,
BRAIN
ATRIDM
KANGAWAl
of
HUMAN
CARDIAC
, Hitone
Kenji
Received
693-700
Pages
1990
ISOLATION
3
BIOPHYSICAL
Japan
University
553,
Medical
School,
Japan
1990
SUMMARY: By using a radioimmunoassay (RIA) system newly established for human brain natriuretic peptide (BNP), a high concentration of immunoreactive (ir-l human BNP (hBNPI has been found in cardiac atrium (1). Two molecular forms of ir-hBNP of 4K and 13-15K were isolated from atria1 extracts by using anti-hBNP IgG immunoaffinity chromatography and reverse phase high performance liquid chromatography (HPLCI. By microsequencing, the peptides were determined to be a pro-hBNP (y-hBNP) and its C-terminal 32-amino acid peptide (hBNP-32). Based on these results, in cardiac atrium, hBNP is in a found to be processed to that pathway similar of porcine BNP (pBNP) but distinct from that of rat BNP, although low MW hBNP-32 is a major form in contrast to pBNP which exists as a high MW y-pBNP. 01990 Academic Press, Inc.
The recently a
amino been
probe
among in
rat
probably major
hBNP
the
of cDNA
Structural
have
revealed
mammalian
BNPs.
cardiac
atrium,
the In
hBNP,
each
tissue.
the
it
rat
method
using
of
porcine,
of
large
was
Thus, to
identify
693
human
of
by
unique
ir-rBNP
processing
been
shown
study
on
molecular
as BNP
differences
identification
further
Abbreviations: BNP, brain natriuretic peptide; porcine; r, rat; y-hBNP, peptide; h,. human; p, hBNP(77-108); RIA, radioimmunoassay; ir, immunoreactive; HPLC, high performance liquid chromatography; IgG, trifluoroacetic acid; PTH, phenylthiohydantoin; carboxymethylated.
cDNA
and
species
endogenous
have
precursor rat
has
for
precursors
pBNP
generated
difference,
(5-71.
(rBNPI
structural and
which species
necessary
BNP
cloning
characterization
form is
and
elucidation presence
structural
molecular
of
hBNP
rBNP-45,
from
endogenous
in
by
.
derived
functions
sequences
deduced
(2-41
precursors
acid
to
be
a
physiologic forms
of
atria1 natriuretic ANP, pro-hBNP; hBNP-32, proMW, molecular weight; immunoglobulin TFA, G; RCM, reduced and S-
0006.291X'90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
167,
No.
Since
an
established, highest forms
we
RIA
of
ir-hBNP Their
present
system
have
concentration
atria. the
BIOCHEMICAL
2, 1990
of isolation
highly
screened of
MW 4K and amino
BIOPHYSICAL
specific ir-hBNP
ir-hBNP
and
AND
in 13-15K
acid
for
in
cardiac were
RESEARCH
human atrium
has
tissue
and Two
(II.
observed
sequence
hBNP
in
determination
COMMUNICATIONS
acid
been
recently have
found
molecular extracts are
the weight
of reported
human in
paper.
MATERIALS
AND
METHODS
Isolation: Heart tissue was collected at autopsy from 3 patients without cardiac disease. Resected cardiac atria mainly comprised of auricles (wet 50 g) were diced and boiled for 10 min in 10 volumes of water tissue weight, to inactivate intrinsic proteases. After cooling, glacial acetic acid and 1M added (final concentration = 1 M and 20 mM, respectively), and the HCl were mixture was homogenized with a Polytron mixer for 4 min. The resulting after centrifugation were supernatants, obtained at 26,000 x g for 30 min, loaded onto a reverse phase C-18 column (90 ml, LC-SORB SPW-C-ODS, Chemcol . adsorbed materials were eluted with 60% After washing with 0.5M acetic acid, CH3CN containing 0.1% trifluoroacetic acid (TFA). The eluate was evaporated in and then loaded onto an SP-Sephadex C-25 column (1.4 x 6.5 cm, vacuum, Pharmacia) , pre-equilibrated with 1M acetic acid. Successive elutions with 1M acetic acid, 2M pyridine and 2M pyridine-acetate (pH 5.0) yielded three respective fractions, SP-I, SP-II and SP-III. After lyophilization, the SPin 5 ml of 1M acetic III fraction was dissolved acid and subjected to gel filtration on a Sephadex G-50 column (1.8 x 134 cm, Pharmacia). An aliquot of each fraction was submitted to RIAs for hBNP and hANP. Pooled fractions #44and then 47 and #30-33 exhibiting ir-hBNP were lyophilized, subjected to (see immunoaffinity chromatography on an anti-hBNP IgG-AFFI-GEL 10 column The below). peptide fraction adsorbed on the immunoaffinity column was purified by reverse phase HPLC on a diphenyl column (219TP54, 4.6 x 250 mm, Vydacl with a linear gradient elution of CH3CN from 10% to 60% in 0.1% TFA. The high MW hBNP was finally purified also by reverse phase HPLC on a C-18 column (u-Bondasphere 300A, 2.1 x 150 nun, Waters) with the same solvent system as described above. The column effluents of HPLC were monitored by measuring absorbance at 210 and 280 nm. Aliquots of all the fractions from immunoaffinity chromatography and reverse phase HPLC were submitted to RIAs for hBNP and hANP. RIAs for hBNP and hANP: Details on RIA for hBNP were reported in a separate paper (1). Antiserum #189-6, which was raised in rabbit by immunizing hBNP26-thyroglobulin conjugate, recognized hBNP-26 and hBNP-32 at the same affinity. RIA was performed as reported (1,8), using hBNP-32 as a standard, and free and bound tracers were separated by the double antibody method. When the antiserum was used at a final dilution of 1:420,000, peptides were measurable in a range of 0.2-30 fmol/tube, and cc-hANP did not show any crossreactivity. RIA for a-hANP was performed as reported previously (9). This RIA system showed 0.04% crossreactivity with hBNP-32. Immunoaffinity chromatography: Anti-hBNP immunoaffinity column was prepared mainly as reported for the isolation of pBNP-32 (101. In brief, IgG fraction of antiserum #189-6, prepared with Protein A-Agarose (Bio-Rad1, was coupled with AFFI-GEL 10 (Bio-Rad). Samples dissolved in O.lM sodium phosphate buffer (pH 7.4) containing 0.001% Triton X-100 were loaded onto the immunoaffinity column (total bed volume: 2.5 ml). After washing the column, the adsorbed materials were eluted with 1M acetic acid containing 10% CH CN. Sequence analyses: Half of the purified hBNP-32 (ca. 6Od pmol) was reduced with 20mM dithiothreitol in 0.5M Tris-HCl (pH 8.51 at 37OC for 4 hr, and then was carboxymethylated with sodium monoiodoacetic acid. The reduced and Scarboxymethylated (RCM-) hBNP-32 was purified by reverse phase HPLC on a w Bondasphere C-18 column (300 A, 3.9 x 150 mm) using a linear gradient elution
694
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
of CH CN in 0.1% TFA solution. Sequence analysis of RCM-hBNP-32 was performed using3a gas-phase sequencer equipped with a phenylthiohydantoin (PTHI-amino acid analyzing HPLC system (Model 470A/120A, Applied Biosystems) . Amino acid analysis of RCM-hBNP-32 was carried out after acid hydrolysis with a Hitachi L-8500 amino acid analyzer by the post o-phthalaldehyde-labeling method. All the purifield high MW y-hBNPs were directly submitted to sequence analyses with the gas-phase sequencer. Biological ,activity: Chick rectum relaxant activity was assayed by the described method (11). RESULTS AND DISCUSSION By utilizing have
a newly
measured
about
100
(12).
pmol/g
Thus,
scale
tissue
desalted
wt
we began
the
to that
extraction, with
of ir-hBNP
(l),
which
present used
acid
a reverse
phase
C-18
were
then
separated
SP-III
by an SP-Sephadex
ion
exchange
peptides G-50
containing gel
filtration.
more
than AS
Fraction Figure
from
into
gel
atria
column.
shown
in
total Fig.
in
porcine
atrium
condensed
was
SP-II
and and
fraction
of
subjected
to
peaks
of ir-hBNP
number
1. Sephadex G-50 gel filtration of human cardiac atria1 extracts. Sample: SP-III fraction of acid extracts of human cardiac atria (50 9) (see details in text). Flow rate: 7 ml/hr. Column: Sephadex G-50 (1.8 x 134 cm, Pharmacial. 1M acetic acid. Fraction size: 5 ml/tube. Solvent: Arrows indicate elution positions of II bovine serum albumin, 2) y-hANP, 31 hBNP-32, 4) cx-hANP and 51 NaCl.
695
on a
materials
The SP-III two
to be
atria
of SP-I,
ir-hBNP
we
(13).
The adsorbed
1,
hBNP,
atrium
were
fractions
chromatography. 90% of the
cardiac
of y-pBNP
three
for
50 g of cardiac
purification silica
specific to that
of human cardiac
C-18
basic
was comparable
the
extracts
highly in human
purification
for
on the
Sephadex
column
RIA system
concentration wet
comparable After
established
Vol.
167,
No.
corresponding MW form
to MW 4K and 13-15K
comprised
5/6
Fractions pooled
of
total
#30-33
column
were
shown
in
then Fig.
chromatography compatible impurity ir-hBNP
separated 2,
the
used.
HPLC system
High on a diphenyl in all
column.
of the
was then
five
not
to
the
10
0
from
A)
also
was
were
by immunoaffinity min was
derived
subjected
t.0
reverse state,
from
20
phase
by reverse
HPLC on
30
in the
40
Purification
2.
Sample:
Anti-hBNP of fraction
of hBNP-32 by reverse phase IgG immunoaffinity chromatography B (#44-471 in Fig. 1.
phase
Column: 219TP54 diphenyl Solvent: H20:CH3CN:10%
a C-18
column
50
Linear Flow
rate:
1.0
gradient ml/min.
HPLC. fraction
(4.6 x 250 mm, Vydacl. TFA = (11 9O:lO:l (V/V,, elution from (I1 to (III Temperature: ambient.
696
(II) for
40:60:1 80 min.
HPLC
Each peak
case of peak
adsorbed
MW
was observed
Time (mln) Figure
low
atria.
immunoaffinity
30 and 38 min.
as shown
from
confirmed
of the peptide
between
As
BNP immunoreactivity
50 g of human cardiac purified
an
affinity
column.
3a, hBNP immunoreactivity
eluted
onto
on the
peak was further
The yield
peptides
second
at 34
of the main
in Fig.
to a homogenous
eluted
B) were
loaded
peptides
efficiently
1.2 nmol
peaks
was first
HPLC on a diphenyl
purified
shown).
adsorbed
As shown absorbance
subjected purified
and low
(fraction
#44-47
at 24 min exhibited
The peak
(fraction
and the
chromatography,
was
to be about
MW ir-hBNP
COMMUNICATIONS
chromatogram,
adsorbed
phase
Homogeneity
(data
on the
low MW ir-hBNP
peak eluted
of reagents
RESEARCH
fractions
and the
by reverse
peak height.
was estimated
and
B of
peptide
and a main
BIOPHYSICAL
observed
Al
column,
to its
by another
were
Fraction
IgG immunoaffinity
AND
ir-hBNP.
(fraction
and lyophilized.
anti-hBNP
finally
BIOCHEMICAL
2, 1990
(V/VI.
and
5 (Fig.
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
b
Time
Figure
3bl.
3. Successive purification of y-hBNP by reverse phase HPLC. Sample: a) Anti-hBNP IgG immunoaffinity chromatography adsorbed fraction of fraction A (#30-331 in Fig. 1. bl Human BNP-immunoreactive peak 5 in (al. Column: a) 219TP54 diphenyl (4.6 x 250 mm, Vydacl. bj u-Bondasphere C-18 (300A, 2.1 x 150 mm, Waters). Solvent: H20:CH3CN:10% TFA = (Ii 9O:lO:l (V/VI, (III 40:60:1 (V/V). Llnesr gradient elution from (II to (III for 80 min in (ai and 40 min in (b1. Flow rate: (a1 1.0 ml/min and (bl 0.3 ml/min. Temperature: ambient,
The yield
peak
5 and An
finally
of high
3-10
pmol acid
amino
purified
by reverse analysis, (Fig.
(2, ; that
HPLC.
Half
acid
1.05 (2); Asp, 5.13 (51; Val, 0.75
(1);
LYS I
molar
methionine in this
were
low,
analysis
peptides.
these low
of cardiac
was definitely
(11; (21;
2.81
(3); acid
His,
on the
atria,
comprising was finally
32-residue
peptide activity
hBNP-32
showed
chick
rectum
relaxant
hBNP-32,
which
also
supported
its
(11;
chemical 697
Arg,
up to the
1.21 0.86 3.70
(Numbers
recovery
yields
be read
analysis
about
85"s of
total
structure.
Pro, Leu,
(41,
indicating
of
CM-Cys and equivalents of
ir-hBNP
as hBNP-32, to that
1.00 1.96
in parentheses
data
of hBNP precursor. comparable
32nd
to amino
(11; (1);
as 2 molar
acid identified
to
MW hBNP was determined Glu, Ile,
peptide.
should amino
of the and
was submitted
(6); (21;
1.06
Although
MW hBNP,
to a C-terminal
6.04 1.46
residue
two residues
identified
of RCM-low
Ser, Met,
Half
MW hBNP was submitted
MW hBNP
composition
1.12 1.81
based
of
carboxymethylated,
RCM-low
RCM-low
numbers.)
system
Thus,
extracts
identical
residue
in the case
determined.
was reduced, of the
acid
of the
low MW hBNP was a 32-amino
indicate
acid
600 pmoll
One-third
20 pmol
4 peaks.
and PTH-amino
4b).
to be about
of low MW hBNP was first
The amino
to be CM-Cys, Gly, Phe,
other (cd.
phase
analysis.
(1) ;
in the
peptide
sequence acid
MW hBNP was deduced
sequence
purified
residue
30
20 Time (min)
(mln)
synthetic in
which This
the
was
natural
of synthetic
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
10 a I Hls-Pro-Leu-Gly-Ser-Pro-Glv-Ser-A*a-Ser-Aso-Le”-G,“-Thr-ser-G,Y-Le”-G,“-G~“-~~“-Arg-As”-H*s-Le” -7-----7--ir----7---77---T--25
I
40
30
Gln-Gly-Lys-Leu-Ser-Glu-Leu-Gln-Val-Glu-G~n-Thr-Ser-Leu-Glu-Pro-Leu-Gln-Glu-Ser-Pro-Arg-Pro-Thr -7----T-7
100 Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-HIS
b
108
I
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-~~s-~h~-G~y-Arg-Lys-Me~-As~-Ar~-lle-Ser-~~r-Ser-Ser-GIv-Leu ------------T-----T-------;r* G?Y-Cys-Lys-Val-Leu-A%Arg-t?Fs --z------
Figure
4. Amino acid sequences of human (a) y-BNP and (bl BNP-32. Amino acid sequence analyses were performed with purified y-hBNPs (after re-chromatography of hBNP-immunoreactive peaks 1-5 in Fig. 3a) and reduced and S-carboxymethylated natural hBNP-32. Arrows indicate amino acid residues identified by stepwise Edman degradation. Two cysteines in each molecule form a disulfide linkage. *I identified as a carboxymethylcysteine.
All
the
submitted
peptides
t-o
sequence
available. terminus
in
other
4
acids
the
of
in
acids,
no
analysis
(21.
comparable unit
be
HPLC
be
due also
been
atrium
(5).
cardiac
By
identification by
indicating amino
acid
peptide
to
of
BNP
sequence sequencing.
a
and
was gene of
is y-hBNP
deduced As
shown
698
was in
Fig.
the
cDNA
as
predicted
a
deduced
in on
our
reverse since
of
hBNP
by
a
y-rBNP
precursor
a peptide
mammals.
processing
carry
directly
peptides,
confirmed 5,
was was
atrium,
in
also
to
hBNP
purification
exist
expressed
the
5 peptides
cardiac
to
generally
the of
in
5
completely
thought
which
previous
y-hBNP
verified
PTH-amino
from
as
of
differences during
22nd
these
were
of
peptide
times
were the
among
were
5al,
signal
elution
observed
hBNP-32
analysis
the
in
conformational
of
cDNA that
removal
the
hBNP-immunoreactivity
MW form
(Fig.
to as
deduced
high
residues
up
N-
In
sequences
observed
they
the
the
4aJ.
and
sequences
basis,
molar
(Fig. acid
elicited
MW hBNPs
a
108
was
were
from
identified
acid
directly
peptides step
5
amino
pro-hBNP
Thus,
Differences
has
rat
deduced
by
(2).
may
fact
on
of
pre-hBNP
paper
similar
hBNP-32 C-termini.
peak
residue
were
of
32nd
4,
amino
high
the
residues
of
MW BNP
13th
sequence
sequence
(pro-hBNPI
from
previuos
of
their
the
acid
these
to the
Among
to from
1
least
determined
N-terminal
that in
peaks
amino the
Since
y-hBNP
generated
direct
the
to
hBNP-32
the
in
high amounts
up
purified
at
4.
Furthermore, to
peptide
to
5 of
limited
identified
from
peak
difference
identical
from
the
up
of
1 to
since
purified
case
peaks
were
N-termini
the
peptides.
phase
case
from
residue
from
analyses,
PTH-amino
peptides
determined
to
isolated
in
heart,
About
60%
the
present
pathways
of
and
Vol.
167,
No.
BIOCHEMICAL
2, 1990
human
BNP
AND
pre
heart
RESEARCH
COMMUNICATIONS
BNP
Porcine
pre -- BNP
BIOPHYSICAL
rat
- BNP
BNP
pre - BNP
-r - BNP BNP
- 32
BNP-46
c
blood BNP-32 BNP - 26 (BNP - 29)
Figure
5. human,
endoqenous
in in
to
between
these
the nothing
to
be in
candidate
a for
whether
or
not
is
the
similar,
of
rBNP-45
from
manner
similar
(Fig.
form
structural
differences meaning.
and to
pBNP,
of
rBNP
function
to
order
molecular
while
of
hBNP,
together
If rBNP-45
to
hBNP
rBNP-45
(5-7).
identify
form
of
pBNP
are
different rBNP,
its
and
pBNP,
as
but
hBNP-32
is
with
hANP
(5-7). form
between In
and
5)
and
of
each system
are
case
those
a circulating
heart
a circulating
from
hBNP
rates the
in
processing
of
processing In
of
different
a uniform
pathways
distinct
pathways
rather
have
but
about
secreted
concentration
which
(8,10,13,15).
known
biosynthetic
were
Processing
apparently
is
BNPs
ANPs
(14).
precursors
physiological
hormone,
mammalian
presence
far
hormone
to
of processing and heart and blood.
mammalian
fundamentally two
by
of
species
pathway
indicated
have
contrast
be
processing
assumed
forms
different
thought
So
representation and rat BNPs in
molecular
species, even
Schematic porcine
of
is
so,
it and hBNP
hBNP
in
considered should
be
hBNP-32 as
blood
or a
as
a
as
a
examined pBNP-32
circulating must
first
be
part by a Grant-in-Aid from Human heart tissue Japan. (Department and M. Koono
the was of
investigated.
Acknowledgments: Ministry of Education, presented courtesy Miyazaki Pathology,
This
work was supported Science and Culture of Profs. A. Sumiyoshi Medical College).
699
in of
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES 1. 2. 3. 4.
5. 6.
7. 8. 9. 10. 11. 12. 13. 14. 15.
Tateyama, H., Hino, J., Minamino, N., Kangawa, K., Ogihara, T. & Matsuo, H. Biochem. Biophys. Res. Commun., in press. Sudoh, T., Maekawa, K., Kojima, M., Minamino, N., Kangawa, K. & Matsuo, H. (19891 Biochem. Biophys. Res. Commun., E, 1427-1434. Kojima, M., Minamino, N., Kangawa, K. & Matsuo, H. (19891 Biochem. Biophys. Res. Commun., 159, 1420-1426. Maekawa, K., Sudoh, T., Furusawa, M., Minamino, N., Kangawa, K., Ohkubo, H ., Nakanishi, S. & Matsuo, H. (1988) Biochem. Biophys. Res. Commun., 157, 410-416. Aburaya, M., Hino, J., Minamino, N., Kangawa, K. & Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 163, 226-232. Kambayashi, Y., Nakao, K., Itoh, H., Hosoda, K., Saito, Y., Yamada, T., Mukoyama, M., Arai, H., Shirakami, G., Suga, S., Ogawa, Y., Jougasaki, M., Minamino, N., Kangawa, K., Matsuo, H., Inouye, K. & Imura, H. (1989) Biochem. Biophys. Res. Commun., 163, 233-240. M. Aburaya, N. Minamino, J. Hino, K. Kangawa, & H. Matsuo. (19891 Biochem. Biophys. Res. Commun., 165, 880-887. Ueda, S., Minamino, N., Sudoh, T., Kangawa, K. & Matsuo, H. (19881 Biochem. Biophys. Res. Commun., 122, 733-739. Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T. & Matsuo, H. (19851 Biochem. Biophys. Res. Commun., 2, 248-255. Sudoh, T. Minamino, N., Kangawa, K. & Matsuo, H. (19881 Biochem. Biophys. Res. Commun., 155, 726-732. Kangawa, K. & Matsuo, H. (19841 Biochem. Biophys. Res. Commun., 2, 131-139. Minamino, N., Aburaya, M., Ueda, S., Kangawa, K. & Matsuo, H. (1988) Biochem. Biophys. Res Commun., 155,, 740-746. Minamino, N., Kangawa, K. & Matsuo, H. (1988) Biochem. Biophys. Res. Commun., 151, 402-409. Matsuo, H. & Nakazato, H. (19871 Endocrinol. Metab. Clin. North Am., 5, 43-62. Aburaya, M., Minamino, N., Kangawa, K., Tanaka, K. & Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 165, 872-879.
700
167,
March
No.
16,
;!, 1990
BIOCHEMICAL
AND
RESEARCH
COMMUNICATIONS
AND
IDENTIFICATION
OF IN
Jun
HINOl
1
Department
L
TATEYAMA
1,3
of
Fukushima, January
27,
MINAMIN02,
Medical
College, Japan
Research
Osaka
Medicine,
Institute,
565,
Osaka Osaka
PEPTIDES
MATSU02
889-16,
Center Suita,
Geriatric
, Naoto
Hisayuki
Miyazaki
Fujishirodai,
NATRIURETIC
Miyazaki
Kiyotake, Cardiovascular
National
Department
and
Biochemistry,
Kihara,
BRAIN
ATRIDM
KANGAWAl
of
HUMAN
CARDIAC
, Hitone
Kenji
Received
693-700
Pages
1990
ISOLATION
3
BIOPHYSICAL
Japan
University
553,
Medical
School,
Japan
1990
SUMMARY: By using a radioimmunoassay (RIA) system newly established for human brain natriuretic peptide (BNP), a high concentration of immunoreactive (ir-l human BNP (hBNPI has been found in cardiac atrium (1). Two molecular forms of ir-hBNP of 4K and 13-15K were isolated from atria1 extracts by using anti-hBNP IgG immunoaffinity chromatography and reverse phase high performance liquid chromatography (HPLCI. By microsequencing, the peptides were determined to be a pro-hBNP (y-hBNP) and its C-terminal 32-amino acid peptide (hBNP-32). Based on these results, in cardiac atrium, hBNP is in a found to be processed to that pathway similar of porcine BNP (pBNP) but distinct from that of rat BNP, although low MW hBNP-32 is a major form in contrast to pBNP which exists as a high MW y-pBNP. 01990 Academic Press, Inc.
The recently a
amino been
probe
among in
rat
probably major
hBNP
the
of cDNA
Structural
have
revealed
mammalian
BNPs.
cardiac
atrium,
the In
hBNP,
each
tissue.
the
it
rat
method
using
of
porcine,
of
large
was
Thus, to
identify
693
human
of
by
unique
ir-rBNP
processing
been
shown
study
on
molecular
as BNP
differences
identification
further
Abbreviations: BNP, brain natriuretic peptide; porcine; r, rat; y-hBNP, peptide; h,. human; p, hBNP(77-108); RIA, radioimmunoassay; ir, immunoreactive; HPLC, high performance liquid chromatography; IgG, trifluoroacetic acid; PTH, phenylthiohydantoin; carboxymethylated.
cDNA
and
species
endogenous
have
precursor rat
has
for
precursors
pBNP
generated
difference,
(5-71.
(rBNPI
structural and
which species
necessary
BNP
cloning
characterization
form is
and
elucidation presence
structural
molecular
of
hBNP
rBNP-45,
from
endogenous
in
by
.
derived
functions
sequences
deduced
(2-41
precursors
acid
to
be
a
physiologic forms
of
atria1 natriuretic ANP, pro-hBNP; hBNP-32, proMW, molecular weight; immunoglobulin TFA, G; RCM, reduced and S-
0006.291X'90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
167,
No.
Since
an
established, highest forms
we
RIA
of
ir-hBNP Their
present
system
have
concentration
atria. the
BIOCHEMICAL
2, 1990
of isolation
highly
screened of
MW 4K and amino
BIOPHYSICAL
specific ir-hBNP
ir-hBNP
and
AND
in 13-15K
acid
for
in
cardiac were
RESEARCH
human atrium
has
tissue
and Two
(II.
observed
sequence
hBNP
in
determination
COMMUNICATIONS
acid
been
recently have
found
molecular extracts are
the weight
of reported
human in
paper.
MATERIALS
AND
METHODS
Isolation: Heart tissue was collected at autopsy from 3 patients without cardiac disease. Resected cardiac atria mainly comprised of auricles (wet 50 g) were diced and boiled for 10 min in 10 volumes of water tissue weight, to inactivate intrinsic proteases. After cooling, glacial acetic acid and 1M added (final concentration = 1 M and 20 mM, respectively), and the HCl were mixture was homogenized with a Polytron mixer for 4 min. The resulting after centrifugation were supernatants, obtained at 26,000 x g for 30 min, loaded onto a reverse phase C-18 column (90 ml, LC-SORB SPW-C-ODS, Chemcol . adsorbed materials were eluted with 60% After washing with 0.5M acetic acid, CH3CN containing 0.1% trifluoroacetic acid (TFA). The eluate was evaporated in and then loaded onto an SP-Sephadex C-25 column (1.4 x 6.5 cm, vacuum, Pharmacia) , pre-equilibrated with 1M acetic acid. Successive elutions with 1M acetic acid, 2M pyridine and 2M pyridine-acetate (pH 5.0) yielded three respective fractions, SP-I, SP-II and SP-III. After lyophilization, the SPin 5 ml of 1M acetic III fraction was dissolved acid and subjected to gel filtration on a Sephadex G-50 column (1.8 x 134 cm, Pharmacia). An aliquot of each fraction was submitted to RIAs for hBNP and hANP. Pooled fractions #44and then 47 and #30-33 exhibiting ir-hBNP were lyophilized, subjected to (see immunoaffinity chromatography on an anti-hBNP IgG-AFFI-GEL 10 column The below). peptide fraction adsorbed on the immunoaffinity column was purified by reverse phase HPLC on a diphenyl column (219TP54, 4.6 x 250 mm, Vydacl with a linear gradient elution of CH3CN from 10% to 60% in 0.1% TFA. The high MW hBNP was finally purified also by reverse phase HPLC on a C-18 column (u-Bondasphere 300A, 2.1 x 150 nun, Waters) with the same solvent system as described above. The column effluents of HPLC were monitored by measuring absorbance at 210 and 280 nm. Aliquots of all the fractions from immunoaffinity chromatography and reverse phase HPLC were submitted to RIAs for hBNP and hANP. RIAs for hBNP and hANP: Details on RIA for hBNP were reported in a separate paper (1). Antiserum #189-6, which was raised in rabbit by immunizing hBNP26-thyroglobulin conjugate, recognized hBNP-26 and hBNP-32 at the same affinity. RIA was performed as reported (1,8), using hBNP-32 as a standard, and free and bound tracers were separated by the double antibody method. When the antiserum was used at a final dilution of 1:420,000, peptides were measurable in a range of 0.2-30 fmol/tube, and cc-hANP did not show any crossreactivity. RIA for a-hANP was performed as reported previously (9). This RIA system showed 0.04% crossreactivity with hBNP-32. Immunoaffinity chromatography: Anti-hBNP immunoaffinity column was prepared mainly as reported for the isolation of pBNP-32 (101. In brief, IgG fraction of antiserum #189-6, prepared with Protein A-Agarose (Bio-Rad1, was coupled with AFFI-GEL 10 (Bio-Rad). Samples dissolved in O.lM sodium phosphate buffer (pH 7.4) containing 0.001% Triton X-100 were loaded onto the immunoaffinity column (total bed volume: 2.5 ml). After washing the column, the adsorbed materials were eluted with 1M acetic acid containing 10% CH CN. Sequence analyses: Half of the purified hBNP-32 (ca. 6Od pmol) was reduced with 20mM dithiothreitol in 0.5M Tris-HCl (pH 8.51 at 37OC for 4 hr, and then was carboxymethylated with sodium monoiodoacetic acid. The reduced and Scarboxymethylated (RCM-) hBNP-32 was purified by reverse phase HPLC on a w Bondasphere C-18 column (300 A, 3.9 x 150 mm) using a linear gradient elution
694
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
of CH CN in 0.1% TFA solution. Sequence analysis of RCM-hBNP-32 was performed using3a gas-phase sequencer equipped with a phenylthiohydantoin (PTHI-amino acid analyzing HPLC system (Model 470A/120A, Applied Biosystems) . Amino acid analysis of RCM-hBNP-32 was carried out after acid hydrolysis with a Hitachi L-8500 amino acid analyzer by the post o-phthalaldehyde-labeling method. All the purifield high MW y-hBNPs were directly submitted to sequence analyses with the gas-phase sequencer. Biological ,activity: Chick rectum relaxant activity was assayed by the described method (11). RESULTS AND DISCUSSION By utilizing have
a newly
measured
about
100
(12).
pmol/g
Thus,
scale
tissue
desalted
wt
we began
the
to that
extraction, with
of ir-hBNP
(l),
which
present used
acid
a reverse
phase
C-18
were
then
separated
SP-III
by an SP-Sephadex
ion
exchange
peptides G-50
containing gel
filtration.
more
than AS
Fraction Figure
from
into
gel
atria
column.
shown
in
total Fig.
in
porcine
atrium
condensed
was
SP-II
and and
fraction
of
subjected
to
peaks
of ir-hBNP
number
1. Sephadex G-50 gel filtration of human cardiac atria1 extracts. Sample: SP-III fraction of acid extracts of human cardiac atria (50 9) (see details in text). Flow rate: 7 ml/hr. Column: Sephadex G-50 (1.8 x 134 cm, Pharmacial. 1M acetic acid. Fraction size: 5 ml/tube. Solvent: Arrows indicate elution positions of II bovine serum albumin, 2) y-hANP, 31 hBNP-32, 4) cx-hANP and 51 NaCl.
695
on a
materials
The SP-III two
to be
atria
of SP-I,
ir-hBNP
we
(13).
The adsorbed
1,
hBNP,
atrium
were
fractions
chromatography. 90% of the
cardiac
of y-pBNP
three
for
50 g of cardiac
purification silica
specific to that
of human cardiac
C-18
basic
was comparable
the
extracts
highly in human
purification
for
on the
Sephadex
column
RIA system
concentration wet
comparable After
established
Vol.
167,
No.
corresponding MW form
to MW 4K and 13-15K
comprised
5/6
Fractions pooled
of
total
#30-33
column
were
shown
in
then Fig.
chromatography compatible impurity ir-hBNP
separated 2,
the
used.
HPLC system
High on a diphenyl in all
column.
of the
was then
five
not
to
the
10
0
from
A)
also
was
were
by immunoaffinity min was
derived
subjected
t.0
reverse state,
from
20
phase
by reverse
HPLC on
30
in the
40
Purification
2.
Sample:
Anti-hBNP of fraction
of hBNP-32 by reverse phase IgG immunoaffinity chromatography B (#44-471 in Fig. 1.
phase
Column: 219TP54 diphenyl Solvent: H20:CH3CN:10%
a C-18
column
50
Linear Flow
rate:
1.0
gradient ml/min.
HPLC. fraction
(4.6 x 250 mm, Vydacl. TFA = (11 9O:lO:l (V/V,, elution from (I1 to (III Temperature: ambient.
696
(II) for
40:60:1 80 min.
HPLC
Each peak
case of peak
adsorbed
MW
was observed
Time (mln) Figure
low
atria.
immunoaffinity
30 and 38 min.
as shown
from
confirmed
of the peptide
between
As
BNP immunoreactivity
50 g of human cardiac purified
an
affinity
column.
3a, hBNP immunoreactivity
eluted
onto
on the
peak was further
The yield
peptides
second
at 34
of the main
in Fig.
to a homogenous
eluted
B) were
loaded
peptides
efficiently
1.2 nmol
peaks
was first
HPLC on a diphenyl
purified
shown).
adsorbed
As shown absorbance
subjected purified
and low
(fraction
#44-47
at 24 min exhibited
The peak
(fraction
and the
chromatography,
was
to be about
MW ir-hBNP
COMMUNICATIONS
chromatogram,
adsorbed
phase
Homogeneity
(data
on the
low MW ir-hBNP
peak eluted
of reagents
RESEARCH
fractions
and the
by reverse
peak height.
was estimated
and
B of
peptide
and a main
BIOPHYSICAL
observed
Al
column,
to its
by another
were
Fraction
IgG immunoaffinity
AND
ir-hBNP.
(fraction
and lyophilized.
anti-hBNP
finally
BIOCHEMICAL
2, 1990
(V/VI.
and
5 (Fig.
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
b
Time
Figure
3bl.
3. Successive purification of y-hBNP by reverse phase HPLC. Sample: a) Anti-hBNP IgG immunoaffinity chromatography adsorbed fraction of fraction A (#30-331 in Fig. 1. bl Human BNP-immunoreactive peak 5 in (al. Column: a) 219TP54 diphenyl (4.6 x 250 mm, Vydacl. bj u-Bondasphere C-18 (300A, 2.1 x 150 mm, Waters). Solvent: H20:CH3CN:10% TFA = (Ii 9O:lO:l (V/VI, (III 40:60:1 (V/V). Llnesr gradient elution from (II to (III for 80 min in (ai and 40 min in (b1. Flow rate: (a1 1.0 ml/min and (bl 0.3 ml/min. Temperature: ambient,
The yield
peak
5 and An
finally
of high
3-10
pmol acid
amino
purified
by reverse analysis, (Fig.
(2, ; that
HPLC.
Half
acid
1.05 (2); Asp, 5.13 (51; Val, 0.75
(1);
LYS I
molar
methionine in this
were
low,
analysis
peptides.
these low
of cardiac
was definitely
(11; (21;
2.81
(3); acid
His,
on the
atria,
comprising was finally
32-residue
peptide activity
hBNP-32
showed
chick
rectum
relaxant
hBNP-32,
which
also
supported
its
(11;
chemical 697
Arg,
up to the
1.21 0.86 3.70
(Numbers
recovery
yields
be read
analysis
about
85"s of
total
structure.
Pro, Leu,
(41,
indicating
of
CM-Cys and equivalents of
ir-hBNP
as hBNP-32, to that
1.00 1.96
in parentheses
data
of hBNP precursor. comparable
32nd
to amino
(11; (1);
as 2 molar
acid identified
to
MW hBNP was determined Glu, Ile,
peptide.
should amino
of the and
was submitted
(6); (21;
1.06
Although
MW hBNP,
to a C-terminal
6.04 1.46
residue
two residues
identified
of RCM-low
Ser, Met,
Half
MW hBNP was submitted
MW hBNP
composition
1.12 1.81
based
of
carboxymethylated,
RCM-low
RCM-low
numbers.)
system
Thus,
extracts
identical
residue
in the case
determined.
was reduced, of the
acid
of the
low MW hBNP was a 32-amino
indicate
acid
600 pmoll
One-third
20 pmol
4 peaks.
and PTH-amino
4b).
to be about
of low MW hBNP was first
The amino
to be CM-Cys, Gly, Phe,
other (cd.
phase
analysis.
(1) ;
in the
peptide
sequence acid
MW hBNP was deduced
sequence
purified
residue
30
20 Time (min)
(mln)
synthetic in
which This
the
was
natural
of synthetic
Vol.
167,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
10 a I Hls-Pro-Leu-Gly-Ser-Pro-Glv-Ser-A*a-Ser-Aso-Le”-G,“-Thr-ser-G,Y-Le”-G,“-G~“-~~“-Arg-As”-H*s-Le” -7-----7--ir----7---77---T--25
I
40
30
Gln-Gly-Lys-Leu-Ser-Glu-Leu-Gln-Val-Glu-G~n-Thr-Ser-Leu-Glu-Pro-Leu-Gln-Glu-Ser-Pro-Arg-Pro-Thr -7----T-7
100 Ser-Ser-Gly-Leu-Gly-Cys-Lys-Val-Leu-Arg-Arg-HIS
b
108
I
Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-~~s-~h~-G~y-Arg-Lys-Me~-As~-Ar~-lle-Ser-~~r-Ser-Ser-GIv-Leu ------------T-----T-------;r* G?Y-Cys-Lys-Val-Leu-A%Arg-t?Fs --z------
Figure
4. Amino acid sequences of human (a) y-BNP and (bl BNP-32. Amino acid sequence analyses were performed with purified y-hBNPs (after re-chromatography of hBNP-immunoreactive peaks 1-5 in Fig. 3a) and reduced and S-carboxymethylated natural hBNP-32. Arrows indicate amino acid residues identified by stepwise Edman degradation. Two cysteines in each molecule form a disulfide linkage. *I identified as a carboxymethylcysteine.
All
the
submitted
peptides
t-o
sequence
available. terminus
in
other
4
acids
the
of
in
acids,
no
analysis
(21.
comparable unit
be
HPLC
be
due also
been
atrium
(5).
cardiac
By
identification by
indicating amino
acid
peptide
to
of
BNP
sequence sequencing.
a
and
was gene of
is y-hBNP
deduced As
shown
698
was in
Fig.
the
cDNA
as
predicted
a
deduced
in on
our
reverse since
of
hBNP
by
a
y-rBNP
precursor
a peptide
mammals.
processing
carry
directly
peptides,
confirmed 5,
was was
atrium,
in
also
to
hBNP
purification
exist
expressed
the
5 peptides
cardiac
to
generally
the of
in
5
completely
thought
which
previous
y-hBNP
verified
PTH-amino
from
as
of
differences during
22nd
these
were
of
peptide
times
were the
among
were
5al,
signal
elution
observed
hBNP-32
analysis
the
in
conformational
of
cDNA that
removal
the
hBNP-immunoreactivity
MW form
(Fig.
to as
deduced
high
residues
up
N-
In
sequences
observed
they
the
the
4aJ.
and
sequences
basis,
molar
(Fig. acid
elicited
MW hBNPs
a
108
was
were
from
identified
acid
directly
peptides step
5
amino
pro-hBNP
Thus,
Differences
has
rat
deduced
by
(2).
may
fact
on
of
pre-hBNP
paper
similar
hBNP-32 C-termini.
peak
residue
were
of
32nd
4,
amino
high
the
residues
of
MW BNP
13th
sequence
sequence
(pro-hBNPI
from
previuos
of
their
the
acid
these
to the
Among
to from
1
least
determined
N-terminal
that in
peaks
amino the
Since
y-hBNP
generated
direct
the
to
hBNP-32
the
in
high amounts
up
purified
at
4.
Furthermore, to
peptide
to
5 of
limited
identified
from
peak
difference
identical
from
the
up
of
1 to
since
purified
case
peaks
were
N-termini
the
peptides.
phase
case
from
residue
from
analyses,
PTH-amino
peptides
determined
to
isolated
in
heart,
About
60%
the
present
pathways
of
and
Vol.
167,
No.
BIOCHEMICAL
2, 1990
human
BNP
AND
pre
heart
RESEARCH
COMMUNICATIONS
BNP
Porcine
pre -- BNP
BIOPHYSICAL
rat
- BNP
BNP
pre - BNP
-r - BNP BNP
- 32
BNP-46
c
blood BNP-32 BNP - 26 (BNP - 29)
Figure
5. human,
endoqenous
in in
to
between
these
the nothing
to
be in
candidate
a for
whether
or
not
is
the
similar,
of
rBNP-45
from
manner
similar
(Fig.
form
structural
differences meaning.
and to
pBNP,
of
rBNP
function
to
order
molecular
while
of
hBNP,
together
If rBNP-45
to
hBNP
rBNP-45
(5-7).
identify
form
of
pBNP
are
different rBNP,
its
and
pBNP,
as
but
hBNP-32
is
with
hANP
(5-7). form
between In
and
5)
and
of
each system
are
case
those
a circulating
heart
a circulating
from
hBNP
rates the
in
processing
of
processing In
of
different
a uniform
pathways
distinct
pathways
rather
have
but
about
secreted
concentration
which
(8,10,13,15).
known
biosynthetic
were
Processing
apparently
is
BNPs
ANPs
(14).
precursors
physiological
hormone,
mammalian
presence
far
hormone
to
of processing and heart and blood.
mammalian
fundamentally two
by
of
species
pathway
indicated
have
contrast
be
processing
assumed
forms
different
thought
So
representation and rat BNPs in
molecular
species, even
Schematic porcine
of
is
so,
it and hBNP
hBNP
in
considered should
be
hBNP-32 as
blood
or a
as
a
as
a
examined pBNP-32
circulating must
first
be
part by a Grant-in-Aid from Human heart tissue Japan. (Department and M. Koono
the was of
investigated.
Acknowledgments: Ministry of Education, presented courtesy Miyazaki Pathology,
This
work was supported Science and Culture of Profs. A. Sumiyoshi Medical College).
699
in of
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES 1. 2. 3. 4.
5. 6.
7. 8. 9. 10. 11. 12. 13. 14. 15.
Tateyama, H., Hino, J., Minamino, N., Kangawa, K., Ogihara, T. & Matsuo, H. Biochem. Biophys. Res. Commun., in press. Sudoh, T., Maekawa, K., Kojima, M., Minamino, N., Kangawa, K. & Matsuo, H. (19891 Biochem. Biophys. Res. Commun., E, 1427-1434. Kojima, M., Minamino, N., Kangawa, K. & Matsuo, H. (19891 Biochem. Biophys. Res. Commun., 159, 1420-1426. Maekawa, K., Sudoh, T., Furusawa, M., Minamino, N., Kangawa, K., Ohkubo, H ., Nakanishi, S. & Matsuo, H. (1988) Biochem. Biophys. Res. Commun., 157, 410-416. Aburaya, M., Hino, J., Minamino, N., Kangawa, K. & Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 163, 226-232. Kambayashi, Y., Nakao, K., Itoh, H., Hosoda, K., Saito, Y., Yamada, T., Mukoyama, M., Arai, H., Shirakami, G., Suga, S., Ogawa, Y., Jougasaki, M., Minamino, N., Kangawa, K., Matsuo, H., Inouye, K. & Imura, H. (1989) Biochem. Biophys. Res. Commun., 163, 233-240. M. Aburaya, N. Minamino, J. Hino, K. Kangawa, & H. Matsuo. (19891 Biochem. Biophys. Res. Commun., 165, 880-887. Ueda, S., Minamino, N., Sudoh, T., Kangawa, K. & Matsuo, H. (19881 Biochem. Biophys. Res. Commun., 122, 733-739. Miyata, A., Kangawa, K., Toshimori, T., Hatoh, T. & Matsuo, H. (19851 Biochem. Biophys. Res. Commun., 2, 248-255. Sudoh, T. Minamino, N., Kangawa, K. & Matsuo, H. (19881 Biochem. Biophys. Res. Commun., 155, 726-732. Kangawa, K. & Matsuo, H. (19841 Biochem. Biophys. Res. Commun., 2, 131-139. Minamino, N., Aburaya, M., Ueda, S., Kangawa, K. & Matsuo, H. (1988) Biochem. Biophys. Res Commun., 155,, 740-746. Minamino, N., Kangawa, K. & Matsuo, H. (1988) Biochem. Biophys. Res. Commun., 151, 402-409. Matsuo, H. & Nakazato, H. (19871 Endocrinol. Metab. Clin. North Am., 5, 43-62. Aburaya, M., Minamino, N., Kangawa, K., Tanaka, K. & Matsuo, H. (1989) Biochem. Biophys. Res. Commun., 165, 872-879.
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