Molecular cloning of porcine soluble angiotensin-binding protein

Abstract

Soluble angiotensin-binding protein (sABP) is a 75-kDa cytosolic protein that binds angiotensins and its analogues with high affinity. In this study, the primary structure of porcine sABP is determined by cDNA cloning. Based on the partial amino acid sequences of sABP tryptic fragments, fully degenerate oligonucleotides were synthesized, and used as primers for polymerase chain reactions to amplify the corresponding sABP cDNA fragment from porcine liver first-strand cDNA. By using initially the polymerase chain reaction product and later partial cDNA clones as probes, porcine heart and liver cDNA libraries were screened, and several positive clones were obtained including one covering the entire coding region. From the cDNA sequence, an open reading frame that encodes sABP as a 704-amino acid protein with molecular mass of 80,800 daltons is predicted. No significant homology was seen between sABP and other proteins in GenBank and NBRF data bases, including the angiotensin-related proteins such as angiotensin converting enzyme, renin, and AT1 angiotensin II receptor. Northern blot analysis of poly(A)+ RNA revealed that the mRNA for sABP is expressed as 5.3- and 2.8-3.2-kilobase transcripts. These transcripts are generated by the use of alternative polyadenylation signals. Within the 3'-untranslated region of the cDNA sequence downstream from the polyadenylation signals for smaller transcripts, a porcine short interspersed repetitive element (SINE) was found; only the longer 5.3-kilobase transcript had the SINE sequence.

Full text

THE
JOURNAL
OF
BIOLOGICAL
CHEMISTRY
(CI
1992
by The American Society far Biochemistry
and
Molecular Biology,
Inc.
Vol.
267,
No.
25,
Issue
of September
5,
pp.
18067-18072,1992
Printed
in
1I.S.A.
Molecular Cloning
of
Porcine Soluble Angiotensin-binding Protein*
(Received for publication, May 12, 1992)
Naoaki Sugiura, Hiromi Hagiwara, and Shigehisa Hirose
From the Department of Biological Sciences, Tokyo Institute
of
Technology, Ookayama, Meguro-ku, Tokyo
152,
Japan
Soluble angiotensin-binding protein (sABP) is a
75-
kDa cytosolic protein that binds angiotensins and its
analogues with high affinity. In this study, the primary
structure of porcine sABP is determined by cDNA
cloning. Based on the partial amino acid sequences of
sABP tryptic fragments, fully degenerate oligonucle-
otides were synthesized, and used
as
primers
for
po-
lymerase chain reactions to amplify the corresponding
sABP cDNA fragment
from
porcine liver first-strand
cDNA. By using initially the polymerase chain reaction
product and later partial cDNA clones
as
probes, por-
cine heart and liver cDNA libraries were screened, and
several positive clones were obtained including one
covering the entire coding region. From the cDNA
sequence, an open reading frame that encodes sABP
as
a
704-amino acid protein with molecular mass of
80,800
daltons is predicted. No significant homology
was
seen between sABP and other proteins in GenBank
and NBRF data bases, including the angiotensin-re-
lated proteins such
as
angiotensin converting enzyme,
renin, and ATI angiotensin I1 receptor. Northern blot
analysis of poly(A)+ RNA revealed that the mRNA for
sABP
is
expressed
as
5.3-
and 2.8-3.2-kilobass tran-
scripts. These transcripts are generated by the use of
alternative polyadenylation signals. Within the 3‘-un-
translated region of the cDNA sequence downstream
from the polyadenylation signals
for
smaller tran-
scripts, a porcine short interspersed repetitive element
(SINE) was found; only the longer 5.3-kilobase tran-
script had the SINE sequence.
Angiotensin
11,
an octapeptide, plays a major role in the
maintenance
of
extracellular fluid volume and blood pressure
(1-3).
In addition to this well established role in circulatory
homeostasis, angiotensin
I1
has been linked to
a
variety of
processes including the control of nervous system activity
(1,
4),
cell growth
(5-7),
metabolic processes in the liver (8, 9),
and ovulatory
(10, 11)
and development
(12-14)
processes.
For
establishing the molecular basis
for
these broad physio-
logical effects, angiotensin
I1
receptors have long been a focus
of
intensive research
(15-17).
Following the report claiming
cloning of a neuronal type angiotensin
I1
receptor in 1988
(18),
one of the key receptor subtypes (AT,) that mediates
* This work was supported by a grant-in-aid for Scientific Research
from the Ministry
of
Education, Science and Culture, Japan, and
grants from Chichibu Cement, the Japan Shipbuilding Industry Foun-
dation, the Mitsubishi Foundation, the Mitsukoshi Prize of Medicine
1990, and the Mombusho International Scientific Research Program.
The costs of publication of this article were defrayed in part by the
payment
of
page charges. This article must therefore be hereby
marked “aduertisement” in accordance with
18
U.S.C. Section 1734
solely
to
indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted
to
the GenBankl”/EMBL Data Bank with accession number(s)
I)
11336.
the major cardiovascular and hydromineral effects has re-
cently been cloned from vascular smooth muscle cells (19)
and adrenal zona glomerulosa cells
(20)
by expression cloning
strategies. Since the last component of the classical renin-
angiotensin system that eluded molecular cloning was finally
cloned, the stage now seems to be
set
for elucidating the
mechanism of action of angiotensin
I1
at the molecular level.
During the biochemical pursuit of the angiotensin
I1
recep-
tor, Soffer and his co-workers
(21)
have found an angiotensin-
binding protein in rabbit hepatic plasma membranes. The
protein was later shown to be present
at
much higher concen-
trations in cytosols
(22,23).
This binding protein was purified
and well characterized
(23-26);
for example, it has been shown
to have a
M,
of
75,000
by
SDS-PAGE’
and affinity labeling
and to exhibit a wide tissue distribution by Western blotting.
To
obtain information that may help elucidate the physiolog-
ical role, we initiated two approaches: immunohistochemical
localization of the protein and determination
of
the primary
structure through cDNA cloning. Here we report the results
of the later approach.
EXPERIMENTAL PROCEDURES
Purification and Amino Acid Sequencing of sABP-sABP was
purified to apparent homogeneity from porcine liver as described
previously (23). About
150
pg
of
purified protein was denatured in
2.5 ml
of
6
M
urea,
0.6
M
Tris/Cl (pH
8.6),
6
mM EDTA, and
0.5
mM
phenylmethylsulfonyl fluoride, degassed, reduced with 1.9 mM
p-
mercaptoethanol for
1
hat 30 “C, and S-carboxamidomethylated with
3.7
mM
iodoacetoamide for 20 min at
30
“C. The reaction mixture
was dialyzed against
50
mM Tris/Cl (pH
8.0).
After dialysis, CaCl,
was added to 2 mM, and trypsin cleavage of S-carboxamidomethylated
sABP was performed with 2 pg of TPCK-treated trypsin (Sigma) for
12 h at 37 “C. Followed by another 2 pg of TPCK-treated trypsin, t.he
react.ion was continued for 12 h. The digest was subjected directly to
reverse phase HPLC on a Waters pBondasphere Cle column (3.9
X
150
mm) with a linear gradient
of
CHJCN from
5
to
40% in 0.1%
trifluoroacetic acid
for
100
min at
a
flow rate of
0.8
mllmin. Amino
acid sequence analysis of isolated peptides was performed by a gas-
phase sequenator (Applied Biosystems, model 470A) followed by
identification
of
the
phenylthiohydantoin-derivative
by HPLC (Ap-
plied Biosystems, model 120A).
Oligonucleotides-Oligonucleotides were synthesized
on
a Milligen
apparatus and purified using oligonucleotide purification cartridges
(Applied Biosystems).
PCR-mediated Amplification of sABP cDNA Fragment-Porcine
liver total RNA was isolated by the guanidinium isothiocyanate-CsCl
method (27). Poly(A)+ RNA was selected by oligo(dT) chromatogra-
phy using an Amersham mRNA purification kit. First-strand cDNA
synthesis and polymerase chain reaction (PCR) were performed (28,
29) according to the instructions (30) with some modifications. First-
strand cDNA was synthesized using random hexamer primers and
Moloney murine leukemia virus reverse transcriptase (Bethesda Re-
’
The abbreviations used are:
SDS,
sodium dodecyl sulfate; PAGE,
polyacrylamide gel electrophoresis; sABP, soluble angiotensin-bind-
ing protein;
TPCK,~-l-tosylamido-2-phenylethyl
chloromethyl ke-
tone; HPLC, high performance liquid chromatography; PCR, polym-
erase chain reaction; bp, base pair(s); kb, kilobase(s);
SINE(s), short
interspersed repetitive element(s).
18067

18068
cDNA
Cloning
of
Angiotensin-binding Protein
search Laboratories). Subsequent PCR was performed, on a Techne
thermal cycler, with 5 units of Tag DNA polymerase (Perkin-Elmer
Cetus) and 320 pmol each of the fully degenerated oligonucleotide
primers (Fig.
1A)
which were derived from amino acid sequences of
fragments 7 and
8
(see Table
I).
The PCR was run for 30 cycles
(1
min at 94 "C; 2 min at 30 "C; and 3 min at 72 'C) with an additional
extension at 72 "C for 10 min in the last cycle. To obtain sufficient
material for subcloning, the product was diluted 1000-fold and used
as
the template for further synthesis using a Cetus GeneAmp kit.
The reamplified product was electrophoresed on 3% agarose gel, and
the bands were cut out, recovered electrophoretically, digested with
EcoRI and HindIII, and ligated into pBluescript
I1
(Stratagene).
cDNA Library and Plaque Screening-A porcine XgtlO heart cDNA
library and a
XgtlO
liver cDNA library were purchased from Clontech.
The probe used for screening was the PCR-amplified 450-bp sABP
cDNA fragment (Fig. 2) labeled with [a-"'PIdCTP (Amersham) by
the random primer method (31). Duplicate plaque lifts were prehy-
bridized and hybridized at 60 "C in 6
X
SSPE
(1
X
SSPE is 0.15
M
NaCl, 15 mM NaH,PO, (pH 7.0),
1
mM EDTA), 5
X
Denhardt's
solution (0.1% each of bovine serum albumin, polyvinylpyrrolidone,
Ficoll), 100 pg/ml denatured herring sperm DNA, and 0.1% SDS.
The labeled probe was hybridized for 16 h at 2.5
X
lo6
cpm/ml of
hybridization solution. The filters were washed three times at room
temperature with 2
X
SSC
(1
X
SSC is 0.15
M
NaCl, 15 mM sodium
citrate, pH 7.0) and 0.1% SDS (5 min/wash), and then two times at
60
"C with
1
X
SSC and 0.1% SDS (45 min/wash). Following auto-
radiography, double positive plaques were rescreened until pure, and
the phage DNA was purified (30). Thus, two clones, denoted XPABl
and XPAB4, were isolated from the heart library and subcloned into
pBluescript
I1
for further analysis.
To isolate cDNA clones extending further 5', a specific-primer
extended library was constructed in XZAP
I1
(Stratagene). Double-
stranded cDNA was synthesized from porcine heart poly(A)+ RNA
according to Ref. 30. An oligonucleotide complementary to the 5'
portion of the XPAB4 (nucleotides 1349-1365) was used as primer in
the first-strand synthesis. Following adaptor ligation, cDNA greater
than
1
kb was selected by Sepharose CL-4B chromatography (Phar-
macia), ligated to XZAP
I1
arms, and packaged. Using the 705-bp
EcoRI-XbaI fragment of XPAB4 which was "lP-labeled by the random
primer method, screening was performed using the conditions of
hybridization and washing as described above. This screening yielded
clone XPABs2. Phagemid carrying the cloned insert was excised in
oitro from this phage by co-infection of R408 helper phage. XPABs2
contained an unrelated sequence 3' to the sABP cDNA, probably
representing double insert recombinant.
To obtain poly(A)-tailed cDNA clones and confirm alternative use
of multiple polyadenylation signals to generate length polymorphism
of mRNA, an oligo(dT)-primed cDNA library was constructed in
XZAP
I1
starting from porcine liver poly(A)+ RNA. Oligo(dT)l~-l~
(Clontech) was used as primer in the first-strand cDNA synthesis
with Superscript reverse transcriptase (Life Technologies). Before
ligation to
XZAP
I1
arms, cDNA was size-selected through a Chroma-
Spin 1000 column (Clontech). The recombinants from the library
were screened with the 553-bp XbaI fragment of XPABl as described
above.
Similarity Searches of Nucleic Acid and Protein Data Bases-
Comparison of the nucleotide
or
the predicted amino acid sequence
of sABP with the nucleic acid and protein data bases was performed
using the algorithm of Wilber and Lipman (32,33) with a k-tuple size
of
2.
The nucleotide sequence of sABP (including untranslated se-
quences) was compared with nucleic acid sequences in the GenBank
data base using the SEQFN program of the IDEAS package (34).
SEQFP program was employed to search the National Biomedical
Research Foundation protein library for homologous sequences to the
predicted amino acid sequence of sABP.
DNA Sequence Analysis-The nucleotide sequences of plasmids
harboring PCR-amplified product
or
phage inserts were determined
by the chain termination method (35, 36) using Sequenase (United
States Biochemical).
All
sequences in this report were determined
from both strands either after construction of a series of nested
deletions (37)
or
by the use of oligonucleotide primers.
Northern Blot Analysis-Poly(A)+ RNA, 10 pgllane, was fraction-
ated on a 2.2
M
formaldehyde, 1% agarose gel, and transferred to
Genescreen Plus membrane (Du Pont-New England Nuclear). The
blot was prehybridized for 3 h in 50% formamide, 5
X
SSPE, 2
X
Denhardt's, 100 pg/ml denatured herring sperm DNA, and 1% SDS
and then hybridized with 2
X
10' cpm of "P-labeled probe/ml for 24
h. Washing was carried out three times at room temperature in
1
X
SSC and 0.1% SDS, and twice at 65 "C in 0.1
X
SSC and 0.1% SDS.
The autoradiogram was obtained by exposing the blot to Kodak
X-
Omat AR film with an intensifying screen at -70 "C.
Southern Blot Analysis-Porcine genomic DNA was prepared from
liver by a standard procedure (30). Ten micrograms of DNA were
digested with restriction enzymes to completion, electrophoresed on
a 1.0% agarose gel, and blotted onto Genescreen Plus membrane.
Hybridization was performed using the same conditions as for North-
ern blot hybridization. The blot was washed three times in 2
X
SSC
and 0.1% SDS, and twice at 60 "C in
1
X
SSC and 0.1% SDS.
RESULTS
Amino Acid Sequences
of
Tryptic Fragments
of
sABP-
Since initial attempts to determine the N-terminal sequence
of sABP were unsuccessful, probably due to the blockade of
N terminus, amino acid sequence determination was per-
formed after tryptic digestion. Purified protein was digested
with TPCK-treated trypsin after
S-carboxamidomethylation.
The resulting tryptic peptides were separated by reverse phase
HPLC. The sequences of the major peaks were determined as
shown in Table
I.
cDNA Cloning
of
sABP-cDNA clones encoding sABP were
isolated using a PCR-generated probe. As PCR primers, oli-
gonucleotides were designed on the basis of partial amino acid
sequences of sABP (Fig.
1A
).
PCR, performed using porcine
liver first-strand cDNA as template, gave exclusively a
450-
bp product seen by agarose gel electrophoresis (Fig.
1B).
TABLE
I
Amino acid sequences
of
tryptic fragments
of
sABP
Tryptic fragments of sABP separated by reverse phase HPLC were
subjected to amino acid sequence analysis on a gas-phase sequenator.
X represents unidentifiable residues.
Fragment Sequence Residue"
YIVE
FEYDGK
TEELIAQ
NLNEDDTFLVFSK
STHHVTAFLDDLSQK
VDQSLHTNTSLDAASEYAK
(P/N)AWDLHYYMTQTEELK
XYFHEFGHVMHQICAXXDFAR
FSGTNVETDFVEVPSQMLENWVWDT
99-102
350-355
61-67
205-217
311-325
584-602
357-372
494-514
515-537
Residue numbers of the amino acid sequence predicted from
cloned cDNA (Fig. 3).
A
Sense
primer:
5'
CUTG CAT
TAT
TAT
ATG ACG CAG
cis
Tyr Tyr
Het
Thr
Gln
AA
T
C
Hlndlll
Antisense
primer:
3'
GIG
CAG
TAC GTG GTT TA CC-G
5
ois
Val
mt
nis
Gln
Ile
AA
T
C
''
EcoRl
FIG.
1.
Amplification of sABP cDNA fragment
by
PCR.
A,
oligonucleotide primers used for PCR. Amino acid sequences used to
design the sense and antisense primers were adopted from tryptic
fragments 7 and
8
(Table
I),
respectively. Two different restriction
enzyme sites were introduced in these primers to facilitate the rescue
of the amplified product. B, agarose gel electrophoresis of the PCR
product. The product of PCR amplification, obtained starting from
porcine liver first-strand cDNA and the primers illustrated in A, was
electrophoresed on a 4% agarose gel. The arrow points to a 450-bp
fragment corresponding to sABP cDNA.

cDNA Cloning
of
Angiotensin-binding Protein
18069
Because the relative order of tryptic peptides within sABP
was not known, another possible combination of primers was
employed in a parallel PCR reaction, resulting in amplifica-
tion of numerous nonspecific products (data not shown).
Sequencing of the amplified 450-bp fragment provided the
initial evidence for the amplified fragment
as
sABP cDNA,
since nucleotide sequences flanking each primer encoded ad-
ditional amino acid residues of corresponding sABP tryptic
peptides.
To isolate the cDNA covering the rest of the coding region,
the amplified 450-bp fragment was used to screen two com-
mercially available libraries, porcine liver and heart cDNA
libraries. Two overlapping clones, XPABl and XPAB4, were
isolated from 2
x
lo6
recombinants of the heart library; no
positive clone was found in the liver library. As sequence
analysis of these clones indicated that they lacked the
5'
portion of the open reading frame, a second library was
constructed by 3' extension on heart mRNA of an oligonucle-
otide complementary to the
5'
region of XPAB4 (nucleotides
1349-1365). Screening of this library with a XPAB4 restriction
fragment identified one clone, XPABs2, among 2
x
IO5
recom-
binants.
In addition, a third library was constructed from porcine
liver poly(A)+ RNA using oligo(dT) primers to determine
whether the multiple polyadenylation signals present in the
3'-untranslated region (described below) are really used. From
2
X
lo6
recombinants, 12 clones were isolated when the library
was screened with the 553-bp
XbaI
fragment of XPAB1. Of
these clones, XPAB-L9 and XPAB-L15 terminated in poly(A)
at
their 3' ends, and XPAB-L1 encompassed the entire coding
region (Fig. 2).
Nucleotide Sequence and Deduced Amino Acid Sequence
of
sABP-A
diagrammatical representation of the sABP cDNA
clones along with their restriction map and sequencing strat-
0
05
1
15
2
25
3
35
kb
~
"
m-0
rn
-rnrnwmm
a-
_~~"
C,-S
+
4"ZBdOb
40
~-
~
$2
y+
f
,?U>
-~
11
1,
-€=
'L.L.II!L-
1-1
APABs2
-
PCR-generated
probe
AATAAA
'INE
GATAAA
"
A
IPABl
A
/"
L
-
.~
"
-&
-
1
L"
"1
-~-
-1
L
~~-3
"A
".
-2
L-
L
/"
L__
k-
i"
L
-~
L-
~
2""
L-
IPAB4
"
"
L
~.
"
L__
L
~"
A
c-
__
A
APAB-L1
d__
L
d
L_u
i
LL
d
1
IPAB-L9
"1
(Ab
?
a
IPAB-L15
,-
.-
~
"
(Ah
IPAB-L3
L
~~~-
"
/
~-
"
FIG.
2.
cDNA clones for
sABP
along with their restriction
map
and
sequencing strategy.
The predicted open reading frame
is shown by an
open bar.
The PCR-amplified cDNA fragment, which
was used to isolate XPABl and XPAB4 from a heart cDNA library, is
represented by a
closed
box;
hPABs2 was isolated from a primer-
specific extended library, and XPAB-L1, -L3, -L9, and -L15 were
from a liver cDNA library.
A
hatched box
represents SINE found in
the 3'-untranslated region. hPABl has a 137-bp deletion indicated
by a
notch.
XPAB-L3, -L9, and -L15 have divergent sequences up-
stream from the common boundary which is denoted by
arrowheads
(N. Sugiura,
H.
Hagiwara, and
S.
Hirose, unpublished data); XPAB-
L9 and XPAB-L15 possess the same sequence as represented by the
same
open boxes,
and XPAB-L3 has a unique sequence represented
by a
dotted
box.
The extent and direction
of
sequence determinations
are indicated by
horizontal arrows.
egy are shown in Fig. 2. By sequencing these clones, an open
reading frame of 2112 nucleotides coding for 704 amino acids
is predicted (Fig. 3) when the first AUG in the open reading
frame is assigned as the translation initiation codon. This
open reading frame
is
preceded by a 157-nucleotide 5'-un-
translated region which includes the in-frame stop codon at
nucleotide 65. A termination codon (TAA) appears at nucleo-
tide 2270, followed by
a
3"untranslated region of 1547 nucle-
otides. Complete assignment of sequences of nine tryptic
peptides (Table
I)
derived from purified sABP to the deduced
amino acid sequence (Fig. 3,
underlined)
verified the authen-
ticity of this open reading frame. The calculated molecular
mass of 80,800 Da is slightly larger than a value of 75 kDa
determined by migration on SDS-PAGE (23, 25). A hydrop-
athy profile of the deduced amino acid sequence
is
shown in
Fig. 4. Although the AUG codon closest to the
5'
end of
eukaryotic mRNAs is usually the initiation site for translation
(38), there is a considerable possibility that the second AUG
at
nucleotide 227 (Fig. 3) is used as the initiating methionine
codon. In fact, the consensus sequence for eukaryotic trans-
lation initiation (39) prefers the second rather than the first
AUG, in that the first AUG does not have a purine in position
-3 (3 nucleotides upstream from the AUG codon) which is
the most critical feature of the consensus sequence, whereas
the first one does not; if this is the case, the encoded protein
would be 681 amino acids having
a
molecular mass of 78,200
Da.
DNA sequence analysis revealed the presence of sequence
variations among cDNA clones: AA at nucleotides 2168-2169
were substituted by a
T
in XPAB4. A 137-nucleotide sequence
(nucleotides 2001-2137) was deleted from the coding region
of XPABl (Fig. 2, denoted by a
notch).
The sequence at the
junction of this 137-bp deletion, AGG, coincides with the
sequence seen at a potential exon junction. Also considering
that the sABP gene is present as a single copy gene (described
below), this deletion of potential exon most likely resulted
either from alternative or aberrant splicing. In XPAB-L3,
-
L9, and -LE, the sequences upstream from the common
boundary (nucleotide 198, denoted by
arrowheads
in Fig.
2)
diverged, suggesting that there appear to be at least three
exons which are spliced alternatively as a mechanism for
regulation of sABP expression. A final conclusion on the
exact nature of these sequence heterogeneities will, however,
have to await analysis of the sABP gene structure. In the case
of the transcript corresponding to XPAB-L3, it is supposed
that translation initiates at the "second AUG" described
above
(at
nucleotide 227 in Fig. 3) because there is no AUG
triplet between the upstream in-frame stop codon and this
second AUG.
The nucleotide and amino acid sequence of sABP was
compared to the nucleotide sequence data of GenBank and
protein sequence data of the National Biomedical Research
Foundation, respectively.
No
significant homology was ob-
served with any proteins in the data bases including type-1
(AT,) angiotensin receptor,
mas
oncogene, angiotensin-con-
verting enzymes of somatic and testicular types, and renin.
Therefore, sABP does not belong to the known protein fam-
ilies.
Expression
of
sABP
mRNA-Northern blot analysis of
poly(A)+ RNA using a
PstI
970-bp restriction fragment of
XPABs2 demonstrated expression of transcripts of 5.3 and
2.8-3.2 kb in both liver and heart (Fig. 5). The absence of
polyadenylation signal around the 3' end of the cloned 3.8-kb
cDNA sequence indicates that the corresponding message is
longer than
3.8
kb, meaning that it should be the 5.3-kb
transcript. Accordingly, Northern hybridization using a

18070
cDNA Cloning
of
Angiotensin-binding Protein
FIG.
3.
Nucleotide and deduced
amino acid sequence
of
sABP.
The
deduced amino acid sequences that cor-
respond to the tryptic peptide sequences
are
underlined.
The polyadenylation sig-
nals which are used by the 2.8-3.0-kb
sABP transcript (Fig.
5)
are in
black
boxes.
The SINE (PRE-1) sequence is
shaded,
and the flanking direct repeats
are
boxed.
Two AUG triplets, namely
putative translation initiation codon and
second AUG, which is also a possible
candidate for translation initiation site,
are in
gray boxes.
-3
0
L","
100
200
300
400
500
600
700
Amino Acid Number
FIG.
4.
Hydropathy profile
of
sABP.
Hydropathy profile of the
deduced amino acid sequence is obtained according to the method of
Kyte and Doolittle
(40)
with a window size of
13.
cDNA probe located near the
3'
end of XPABl detected only
a
5.3-kb transcript (data not shown). A common polyadenyl-
ation signal (AATAAA, nucleotides 2989-2994) and its var-
iant (GATAAA, nucleotides 2676-2681) occur in the middle
of
the 3"untranslated sequence. In fact, it has been demon-
strated
in
vitro
that the GATAAA sequence, which is
a
very
rare variant among natural mRNA, can serve as a signal for
addition .of poly(A) even though the efficiency of processing
is greatly reduced (41); by the use of these signals, the 2.8-
3.2-kb species would be produced as indicated by the presence
of
poly(A)-containing short cDNA clones PAB-L15 and -L9,
respectively. Indeed, with close examination of the autoradi-
kb
-7.4
-
-
5.3
-
-
2.8
-
-
1.9
-
-1.6-
157
37
277
40
397
80
517
120
637
160
757
200
871
240
991
280
1117
320
I237
360
1357
4DD
1477
440
1591
480
1717
520
1837
560
1957
600
2077
640
2197
680
2317
704
2437
2557
2611
2191
2911
3037
3151
3271
3391
3511
3631
3751
3819
A
B
exposure
2-week exposure
2-days
FIG.
5.
Northern blot analysis
of
porcine liver and heart
mRNA.
Poly(A)+ RNA
(10
pg/lane) from porcine liver and heart
was fractionated on a formaldehyde/agarose gel, blotted
onto
nylon
membrane, and hybridized with a '"P-labeled
5'
0.96-kb
PstI
fragment
of sABP cDNA. RNA molecular weight markers (Boehringer Mann-
heim) were used to estimate the size
of
transcripts. Autographic
exposure was done for
2
weeks
(A)
or
2
days
(R).

cDNA Cloning
of
Angiotensin-binding Protein
18071
ogram shown in Fig. 5A, the hybridization signal of 2.8-3.2-
kb heart transcripts appears to be
a
doublet. This minor
heterogeneity of 2.8-3.2-kb transcripts seems to reflect the
alternative choice of these polyadenylation signals located
about 300 bp apart. The different use of at least three poly-
adenylation signals, therefore, contributes to the heterogene-
ity
of
sABP mRNA. The abundant expression of the sABP
messages in liver, rather than heart, correlates with that
expected from the contents of the sABP protein observed by
Western blotting (26) and ligand binding study (23). Concern-
ing the faint hybridization to small RNAs of liver at longer
exposures (Fig. 5A), it is not clear whether they represent
sABP-related species or nonspecific hybridization signals.
Genomic DNA Blot Analysis-Copy number of the sABP
gene in the porcine genome was examined by Southern blot
analysis of porcine genomic DNA under conditions of mod-
erate stringency. As shown in Fig.
6,
only one band was seen
with three out of six genomic DNAs digested with restriction
enzymes when the blot was hybridized with a 290-bp cDNA
from the coding region
of
XPABs2. This result suggests that
the sABP gene exists
as
a single copy gene per haploid
genome. Also, the presence of a single human sABP gene was
indicated by Southern analysis of human genomic DNA (data
not shown).
SINE
in the 3'-Untranslated Region
of
the 5.3-kb Tran-
script-Within the 3"untranslated region of cDNA there was
a
stretch of 250 nucleotides highly homologous to PRE-1
sequence (Figs. 3 and 7). PRE-1 was first described by Singer
et
al.
(42) as the repetitive DNA element in the genome of
miniature swine, and recent characterization of this sequence
concluded it to be
a
member of the SINE family (Ref. 43; for
review of SINEs, see Refs. 45 and 46). The PRE-1 sequence
in sABP cDNA has characteristics of SINEs, that is, it has
an A-rich sequence in its 3' end and is flanked by direct
repeats of
8
bp (Fig. 3). In addition to these features, PRE-1
appears to have been derived from tRNA like most SINEs
(44); it showed strong homology (-64%) in its 5' third with
arginine tRNA (Fig. 7). This sequence similarity was not
pointed out in earlier reports on PRE-1.
Since this SINE is located downstream from sequences
AATAAA and GATAAA that serve
as
polyadenylation signals
for the 2.8-3.2-kb transcripts, it follows that only the 5.3-kb
transcript has SINE.
$~,O.@GK
$*$$\,
8
8
cc,
$'
Qq3
kb
-
21
-
9.4
-
6.7
-
5.0
-
3.5
-2.0
-
1.4
-
0.8
FIG.
6.
Southern
blot
analysis
of
porcine genomic DNA.
Porcine genomic DNA was digested with restriction enzymes indi-
cated
aboue
each
lane,
fractionated on a 1% agarose gel, and blotted
onto nylon membrane. The blot was probed with a radiolabeled 293-
bp
RsaIIPstI
fragment of the coding region.
PRI-I
ACCCAGIICC CAIC-CTGGC ICAGIGG-IA ACCAACCICA CIA-ClAlCC
AIGACCA-IC
ARC-tRNl
CCCC.1
.....
C..A...A..
...
CCI
.......
C.C.:. .-..A..T.C
rA8P
mRNA
1CA
........
G.
.A-
.......
TCT.-..
.
1
.....
ClG
... -.
c
.....
.....
A
box
PRI-I CACAllCAAl CCClCCCCll NCTCACIGGC IIAACCAICC NCCAIICClC ICACNTGI-C
ARC-tRNA
...
C...
6.
C
I
......
-.C
C...
GCCA
rA8P
mRNA
.
C.C..
IC.
C
..........
C...
...............
1
6..
0
.........
AC
...
A.
QRI-l
ClClAG----
-GICNCAGAI CCGCCICCCA ICCCGICllG ClCTCCClCl GGlClACGCC
I
box
rA8Q
nRNA
A...C.
CIAC
A.
CTC
.....
A
...........
AT.CA
......................
1
PRl-l
GGCAGCIACA CCICCCAIIN GACCCCTAGC CICGCAACCT CCATAICCCG CCCCIGNGCC
rA8P
"RNA
.......
6..
.........
G
..................
1.
..
1
.....
1.
..
A...
Cl.
1
QRt-l
CCI
Illn
tA8P
-RNA
,
..AAAlACC AACCAAACAA
CAAAIAIATA
TAlAlAlAlA
TAlAlAlAlA
IA1AIAT711
FIG.
7.
Sequence alignment of PRE-I, its equivalent
of
sABP mRNA and arginine tRNA.
PRE-1 and mouse arginine
tRNA sequences are taken from Refs.
42
and
47,
respectively. The
sABP mRNA sequence shown is nucleotides 3466-3761
of
cDNA.
Dots
indicate identical nucleotides. Gaps
(-)
were introduced to
maximize alignment. The sequences corresponding to the internal
promotor sequences for DNA polymerase
111
(the
A
and
B
boxes)
are
shaded.
DISCUSSION
In this study cDNA clones for porcine sABP were isolated,
and the amino acid sequence of sABP was deduced from their
nucleotide sequences. sABP is
a
cytosolic protein that binds
angiotensins and its peptide analogues with high affinity
comparable to that of the membrane-bound angiotensin
receptors (23, 25). The order of affinities of the binding
protein for peptide analogues, as well as sensitivity of its
binding activity to the reducing agent dithiothreitol, are sim-
ilar to those of the receptor (23, 25), implying their possible
structural resemblance. The deduced amino acid sequence of
sABP, however, shares no homology with the angiotensin
receptor which has recently been cloned by expression clon-
ing, shown to be
a
member of the heptahelical receptor family,
and classified as the subtype ATI (19, 20) based on its selec-
tivity for non-peptide angiotensin antagonist losartan (DuP
753; Ref. 48). The absence of homology is not totally unex-
pected since sABP exhibited no binding ability for losartan
potassium or EXP655, another non-peptide antagonist selec-
tive for the
AT2
angiotensin receptor subtype.* The fact that
no significant sequence homology to other proteins
was
de-
tected in the GenBank and NBRF data bases indicates that
the sABP cloned and sequenced here belongs to a new protein
family.
Of interest is the presence of SINE in sABP mRNA. SINEs
are short (70-300 bp), interspersed, and highly repeated se-
quences that are first identified by classical interspersion
analysis and later shown to have the following common prop-
erties: SINEs appear to be derived from class I11 genes that
encode small cytoplasmic RNAs, such as tRNAs and 7SL
RNA, and therefore their sequences are homologous to those
of such RNAs; SINEs are usually surrounded by direct repeats
(7-21 bp) that indicate a target site duplication and have a
3'-terminal A-rich stretch; and the sequences of SINEs are
diverse and each species has its own typical set of SINEs. The
porcine version of SINE, PRE-1, found in the 3"untranslated
region of the sABP mRNA had the above common properties
of SINEs. The sequence homology, described under "Results,"
between the PRE-1 and its possible parental arginine tRNA
gene includes the internal promoter elements for RNA polym-
erase 111, the A and B boxes (Fig. 7). PRE-1 is also present in
the 3"untranslated region of inhibin &subunit (49) and cal-
pastatin (50) mRNA. Although the significance of SINES is
still elusive, some roles have been attributed
1)
an evolution-
ary force contributing fluidity
of
genome (45, 51); 2) origins
of DNA replication (52, 53); and 3) regulatory elements
of
gene expression (46, 54-57). The third hypothesis includes
*
N. Sugiura, H. Hagiwara, and
S.
Hirose, unpublished observa-
tions.

18072
cDNA
Cloning
of
Angiotensin-binding Protein
coordinate regulation of a certain set of genes associated with
a
common regulatory element (SINE) during critical cellular
events, such as development, differentiation, cell prolifera-
tion, oncogenesis, and viral infection (58-62). In this context,
the report by Glaichenhaus and Cuzin (61)
is
interesting that,
in rat fibroblast cells, growth-dependent accumulation of a
set of mRNAs, which also increase following transformation
by
polyoma virus and oncogenes, is under the regulation of
SINE present in these transcripts. Likewise, expression of
sABP transcripts might be regulated by the SINE in the 3'-
untranslated region. Then, the difference in the regulation
would occur between two species of sABP transcripts revealed
by
Northern analysis (Fig.
5),
because the 2.8-3.2-kb tran-
script has a relatively short 3"untranslated region and there-
fore lacks SINE.
Although the physiological function of sABP remains an
enigma, it seems to be involved in the regulation of basic cell
physiology as suggested by its widespread tissue distribution
(23, 24). Some investigators have proposed an intracellular
action of angiotensins based on preferential perinuclear lo-
calization of radiolabel following exposure to labeled angio-
tensin (63), formation of angiotensins by an intracellular
renin-angiotensin system (64, 65), and specific nuclear bind-
ing of angiotensin
I1
in an isolated nuclei (66, 67). Several
peptide hormones and growth factors have also been reported
to associate with intracellular sites, especially nuclei (68-70).
sABP may mediate the intracellular transport of angiotensins
and/or their actions yet to be determined. Once such intra-
cellular actions of angiotensins are defined, sABP may become
an important target of therapeutic drugs, like angiotensin-
converting enzyme, renin, and angiotensin I1 receptors. Re-
cently, Sharma
et
al.
(71) have demonstrated an inhibition of
the 60-kDa bovine brain calmodulin-dependent cGMP phos-
phatase by angiotensins and non-peptide antagonists, and
suggested that the modulation of this intracellular enzyme is
mediated through a site recognizing these ligand in a similar
manner as angiotensin receptors. This finding of a cytosolic
angiotensin-sensitive protein lends a support to the functional
significance of intracellular angiotensins. The availability of
cDNA clones and the determination of the primary structure
of sABP will open an avenue towards elucidating physiological
role(s) of sABP.
Acknowledgments-We thank Yoshihiro Fukumori and Taketomo
Fujiwara for help with peptide sequencing, Nobuo Tanaka and Hi-
deaki Moriyama for computer sequence analysis, Takeo Kishimoto,
Naohiro Hashimoto, and Kazunori Tachibana for expert advice, and
Norihiro Okada
for
helpful discussions.
We
also thank Setsuko Satoh
and Tomohito Hombe for secretarial and technical assistance.
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