Download Biochemical Similarity of Expressed Human Prorenin

Biochemical Similarity of Expressed Human
Prorenin and Native Inactive Renin
WILLA A. HSUEH, YUNG SHUN D O , TATSUO SHINAGAWA, HELEN TAM, PHYLLIS A. PONTE,
JOHN D. BAXTER, JOHN SHINE, AND LAWRENCE C. FRITZ
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SUMMARY Prorenin is secreted by mammalian cells transfected with a human preprorenin expression construct. The purpose of this investigation was to compare the physicochemical properties of
expressed prorenin in culture medium with the known characteristics of human inactive renin, which
accounts for nearly half the renin in plasma and kidney. We found that expressed human prorenin
strongly resembles human renal and plasma inactive renin. The expressed prorenin was inactive and
could be equally activated by acid (dialysis to pH 3.3) or trypsin. Acid activation was completely
reversible; reexposure to acid could reactivate the expressed inactive renin. Exposure to cold ( —5°C
for 3 days) could also activate expressed renin. The Michaelis-Menten constant of acid-activated
expressed renin with sheep substrate was 0.29 /JLM, and the pH optimum was 7.8. Expressed inactive
renin bound to a cibacron-blue affinity column and could be eluted with 0.5M NaCl. All the above
characteristics resemble those of human renal and plasma inactive renin. In addition, the molecular
weight of expressed prorenin and human chorionic renin was 47,000, as determined by sodium
dodecyl sulfate—polyacrylamide gel electrophoresis, and 46,000, as measured by high-performance
liquid chromatography. These data, taken together with the published observation that native human
inactive renin cross-reacts with antibodies generated against amino acid sequences in the prosegment
of renin, provide strong support for the hypothesis that human inactive renin is prorenin.
(Hypertension 8 [Suppl II]: II-78-II-83, 1986)
KEY WORDS
• expressed human prorenin • inactive renin
I
The question of whether or not human inactive renin
is a biosynthetic precursor of renin (i.e., prorenin) has
been controversial. Several studies provide indirect
evidence to support the prorenin nature of inactive
renin. First, it has been observed that renin-secreting
tumors synthesize and secrete large quantities of inactive renin,5"7 like polypeptide hormone-secreting tumors, which produce large quantities of prohormone.
Second, absolute levels of inactive renin have been
found to change with perturbation of the renin system;
for example, with marked, acute stimulation of renin,
such as that which occurs with inhibition of converting
enzyme, there is a reciprocal drop in inactive renin
while active renin rises.8 Third, antibodies developed
against pure human renal renin cross-react with inactive renin.9 l0 Fourth, pulse-labeling studies in mouse
submandibular gland, which makes large quantities of
renin, indicate that renin is synthesized in two forms
— preprorenin and prorenin, both of which are inactive.""13 Fifth, peptide mapping suggests that human
renal inactive renin is a renin zymogen.14 Finally, antibodies generated against synthetic peptides derived
from the prosegment of human prorenin react with
human inactive renin.15"17
N humans more than half the renin in the circulation exists as an inactive form.1 2 Even higher
levels of circulating inactive renin are found in
some patients who have defects in active renin secretion, such as in the syndrome of hyporeninemic hypoaldosteronism.3 4 Indeed, it has been suggested that
the syndrome may result from an inability to convert
inactive renin to active renin. To understand such defects in renin secretion, it is important to identify the
physiological role of inactive renin and to understand
the relationships between the active and inactive forms
of the enzyme.
From the Section of Endocrinology, Los Angeles County/University of Southern California Medical Center, Los Angeles (W.A.
Hsueh, Y.S. Do, T. Shinagawa, H. Tarn); California Biotechnology, Inc., Mountain View (P.A. Ponte, J. Shine, L.C. Fritz); and
the Metabolic Research Unit, University of California, San Francisco (J.D. Baxter), California.
Supported by Grant AM30254 and Research Career Development Award AM0 1035, both from the National Institutes of Health
(NIH); Biotechnology Research Partners, Ltd.; and NIH Grant
HL34231 under the Small Business Innovation Research Program.
Address for reprints: Willa Hsueh, M.D., LAC/USC Medical
Center, 2025 Zonal Avenue, Unit I 18-632 Los Angeles, CA
90033.
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This evidence, however, cannot exclude the alternative hypothesis that inactive renin represents active
renin bound to an inhibitor protein. Indeed, these "latent" forms of renin, as labeled by Inagaki and Inagami,18 have been extensively described in animal
plasma and tissue.2
Recently, human kidney renin complementary DNA
(cDNA) and the human renin gene have been cloned
and sequenced.19"22 This has allowed deduction of the
amino acid sequence of preprorenin. Using cloned
cDNA, Fritz et al. 23 have constructed a eukaryotic
expression plasmid that directs the synthesis of human
preprorenin. Chinese hamster ovary (CHO) cells transfected with this plasmid were shown to secrete an
inactive prorenin into the culture medium, which could
subsequently be activated with trypsin. Using this material, we have been able to make a direct comparison
between prorenin and inactive renin, and we report
herein that they demonstrate a striking biochemical
similarity.
Methods
Active renin was measured by radioimmunoassay of
angiotensin I after incubation of samples with sheep
angiotensinogen at 37°C, pH 7.5, in the presence of
angiotensinase inhibitors.24 Total renin was measured
by two different methods: one using low pH,24 and the
other using trypsin. Briefly, the supernatant was dialyzed against 0.05 M glycine buffer, pH 3.3, at 4°C for
18 hours, quickly neutralized, and incubated with
sheep angiotensinogen as above for 0, 10, 20, and 30
minutes. The slope of angiotensin I concentration versus time was determined by linear regression analysis
(r > 0 . % was considered acceptable). Trypsin (Sigma
Chemical, St. Louis, MO, USA) was used according
to a modification of the method of Atlas et al.23 in a
concentration of 500 /Ag/ml at 0°C in the presence of
200 mM benzamidine.
Fritz et al.23 cloned a cDNA sequence coding for
human preprorenin from a human kidney cDNA library. The cDNA was expressed in CHO cells in culture with a mammalian cell expression system. This
resulted in the secretion of prorenin into the culture
medium, which consisted of Dulbecco's minimal essential medium 21/Coon's F12 (1:1), 10% fetal calf
serum, and antibiotics. Characterization of prorenin
secreted into the culture medium included 1) comparison of trypsin, acid, and cold activation; 2) confirmation of the presence of reversible acid activation; 3)
determination of the pH optimum of activity with
sheep angiotensinogen as substrate; 4) determination
of the Michaelis-Menten constant, Km, with sheep angiotensinogen at pH 7.5; and 5) a test of the ability to
bind to cibacron-blue. These methods have previously
been described in detail.24 Cryoactivation was performed by incubating the culture supernatant at — 5°C
for 4 days. To reverse acid activation, culture supernatant that had been dialyzed to a low pH was incubated
at 37°C, pH 7.5, for 4 hours. Reactivation occurred
with redialysis against a low pH. Binding to cibacron
blue was tested by applying 1.5 ml of supernatant to a
0.5 x 6-cm cibacron blue affinity column. After application of the sample, the column was washed with
0.025 M phosphate buffer, pH 7.0, and eluted with a
gradient of 0 to 1.0 M NaCl.
In addition, the molecular weight of the expressed
prorenin was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting and immunoanalysis, and
gel filtration high-performance liquid chromatography
(HPLC). SDS slab gels containing 12.5% acrylamide
were made according to the method of Laemmli, 26 and
stained with Coomassie blue.27 Protein standards
(BioRad, Concord, CA, USA) included phosphorylase
B (92,500), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean
trypsin inhibitor (21,500), and lysozyme (14,400). For
Western blotting, proteins were separated on SDSpolyacrylamide gels and blotted onto nitrocellulose paper, using the sandwich technique of Burnette.28 The
nitrocellulose paper was incubated at 25 °C for 1 hour
with rabbit polyclonal antibody generated against the
last 12 amino acids of the prosegment of prorenin; this
antiserum is known to cross-react with human inactive
renin.16 Immunoglobulin bound to the nitrocellulose
was visualized with a horseradish peroxidase system
(BioRad). CHO supernatant (50 ml) was applied directly to SDS. Native inactive renin was extracted
from human chorionic homogenate.29
Gel filtration was performed on a BioRad HPLC
system with aTSK 3000 (Altech, Deerfield, IL, USA)
column. One hundred microliters of either CHO supernatant or amniotic fluid was applied to the column and
eluted with 50 mM NaHPO4 and 0.5M NaCl, pH 6.5,
containing 2% C2H6OS. This concentration of C2H6OS
had no effect on the renin assay. The flow rate was
0.5 ml/min. Protein standards (BioRad) were bovine
thyroglobulin (670,000), immunoglobulin (Ig) G
(158,000), ovalbumin (45,000), myoglobin (17,000),
and B l2 (1350). Eluates were activated by acid dialysis
and assayed for total renin.
Results
Effect of Acid and Trypsin
The active renin concentration in the untreated culture supernatant was 370 ng/ml/hr. Treatment with
acid resulted in a total renin concentration of 25,425
ng/ml/hr (Figure 1), and treatment with trypsin yielded
a similar value: 22,770 ng/ml/hr. After overnight dialysis of the supernatant against pH 3.3 buffer, neutralization, and incubation at 37°C for 3 hours, the renin
concentration decreased to 6% of the acid-activated
level. Redialysis of the sample against the pH 3.3
buffer completely reactivated the sample. Incubation
at - 5 ° C for 4 days increased the concentration to
18,120 ng/ml/hr, or 7 1 % of the level determined by
acid. Details of reversible acid activation are shown in
Figure 2. Reversible acid activation and reactivation
occurred in the presence or absence of aprotinin, 200
1985 BLOOD PRESSURE COUNCIL
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•tmtrolpH,
37"C
80 -
topH3J
««'C
8, No 6,
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1986
• — • WITHOUT APTOT1NIN
o — o WITH APR0T1NIN
100
80
60 -
60
40 a?
40
20 20
UnfrMtvd
Acid
dialysis
Add
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Add
Incubation
dialysis
at
+
-3*C
+
Incubation Incubattan
at nsutral at nsutral
2
TrypsJn
dialysis
PH
PH
3
TIME (hra)
FIGURE 2. Reversible acid activation of expressed prorenin
(see text for details).
add dialysis
FIGURE I. Effect of acid, cold, and trypsin on expressed protein. Chinese hamster ovary supernatant was treated as indicated beneath each bar (see text for details).
2-O-,
11,000
IOOO0
IU/ml. Reversal at 37 °C occurred within 1 hour. Reactivation by low pH at 4°C required 5 to 8 hours.
Kinetic Studies
Prorenin from the culture medium was activated by
dialysis against pH 3.3 buffer and human renal kallikrein, as previously described. 24 The optimum pH for
enzyme activity with sheep angiotensinogen was 7.8.
The Km with sheep substrate was determined to be 0.29
Clbacron Blue Binding
When the supernatant was applied to cibacron blue,
active renin did not bind, whereas prorenin did (Figure
3). The bound prorenin eluted at a concentration of 0.5
M NaCl, as demonstrated by acid activation of the
eluates. Recovery of prorenin from the column was
71%.
Determination of Molecular Weight
SDS-PAGE and jmmunoblot analysis are shown in
Figure 4. Prosegment antibody cross-reacted with both
expressed prorenin and human chorionic renin. The
molecular weight for both renins, determined by a plot
of log of molecular weight versus retardation factor
was 47,000. HPLC yielded a similar value of molecular weight — 46,000 — for expressed prorenin and
inactive renin in amniotic fluid (Figure 5), as did
SDS-PAGE.
Discussion
Expressed human prorenin and native human inactive renin are similar in their activation characteristics,
molecular weight, kinetic activity, and binding to a
A - - A T 0 T A L RENIN
•
• ACTIVE RENtN
9,000
•4
. tpoo
£
2.OO0
1,000
3
4
9
6 7 8 9 1 0
FRACTION NUMBER
II
I 2 B I 4
FIGURE 3. Binding of expressed prorenin to cibacron blue.
Small amounts of active renin did not bind, whereas prorenin
bound to cibacron blue and eluted at 0.5 M NaCl. Recovery of
prorenin from the column was 71%.
cibacron blue affinity column (Table 1). These data,
taken together with the observation that native human
inactive renin cross-reacts with antibodies generated
against amino acid sequences in the prosegment of
prorenin, provide strong support for the hypothesis
that human inactive renin is prorenin.
Reversible acid activation is a hallmark of human
inactive renin. It occurs in inactive renin extracted
from plasma, kidney, amniotic fluid, and chorion.24 N
The process appears to be unimolecular11 and is not
dependent on other proteases in the system. Acid exposure induces a conformational change in inactive renin
such that the active site is exposed.32 The critical pH at
4°C is 3.3; incomplete activation occurs at a higher
pH. With neutralization and an increase in the temperature to 37°C, a time-dependent loss of enzyme
n-8i
EXPRESSED HUMAN PRORENlN/«5«e/i et al.
1
2
3
(4
5
6
7
1
2 3
•»
5
6
7
so
-i
r
032
70
30
[
r
40
0.16 g
i
30
20
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FIGURE 4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of expressed prorenin and human chorionic inactive
renin. Lanes in the left panel are as follows: lane 1, protein
standards (see text); lane 2, Chinese hamster ovary supernatant
containing prorenin; lane 3, cibacron blue eluates containing
prorenin; lane 4, pure human renal renin and its subunits30;
lane 5, blank; lane 6, human chorionic renin29; lane 7, protein
standards. The right panel shows the results of nitrocellulose
blot developed with prosegment antibody; the lanes are the
same as those in the left panel. Note that expressed prorenin
(lanes 2 and 3) and chorionic inactive renin (lane 6) bind to the
antibody, whereas pure human renal renin (lane 4) does not.
The estimated molecular weight of both expressed prorenin and
inactive renin is 47,000.
activity occurs, consistent with a refolding of the molecule to its original conformation. With reexposure to
pH 3.3, the enzyme becomes reactivated. In plasma
and kidney, the presence of aprotinin is necessary to
demonstrate reversible acid activation, since kallikrein
in plasma and kidney irreversibly activates renin. The
reversible acid activation observed with expressed
prorenin was independent of aprotinin, reflecting the
relative absence of kallikrein-like activating proteases
in the supernatant. As with inactive renin,33 trypsin
treatment and acid dialysis were roughly equivalent in
their ability to activate prorenin. Cold incubation activated prorenin to 7 1 % of the concentration with acid
dialysis, whereas cryoactivation of plasma inactive
renin yields about 30% activation. This discrepancy
could reflect differences in inactive renin preparations
and culture supernatants. Enzymes in fetal calf serum
may be responsible for the increased cryoactivation of
expressed prorenin. The mechanism of cryoactivation
remains unknown.
Binding to a cibacron blue affinity column is also a
unique feature of human inactive renin.24 The prosegment is required, since active renin does not bind. This
technique is thus useful in separating active from inactive renin. The nature of the binding is unknown. Expressed human prorenin binds readily to cibacron blue
and is eluted at 0.5 M NaCl (when a gradient of NaCl is
10
0
K)
IS
20
FRACTION NUMBER
25
30
B
800
1P.M
r
- 032
10
IS
20
FRACTION NUMBER
30
FIGURE 5. Gelfiltration high-performance liquid chromatography of expressed prorenin (A) and human amniotic fluid inactive renin (B). Arrows denote protein standards in order of
decreasing molecular weight (see text). The estimated molecular weight of both expressed prorenin and inactive renin is
46,000.
employed), which is similar to the elution pattern of
human inactive renin.
The molecular weight of human inactive renin has
been reported to range from 40,000 to 60,000, depending on the fractionation technique used and the method
of subsequent activation of renin.2 Use of Sephadex
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1986
TABLE 1. Comparison of Physicochemical Properties of Expressed Human Prorenin and Native Inactive Renin
Inactive renin
Expressed
prorenin
Kidney
Plasma
+*
+•
+
Reversible acid activation
Activity with sheep angiotensinogen
pH optimum
Binding to cibacron blue
7.8
0.29 jiM
+
Expressed
prorenin
7.8*
0.34 + 0.05 MM*
+*
7.8*
0.29±0.05/xM*
+*
Native inactive renin
(chorion)
Apparent molecular weight
47,000
47,000
SDS-PAGE
46,000
46,000
HPLC
Km = Michaelis-Menten constant; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis;
HPLC = high-performance liquid chromatography.
•Taken from Hsueh et al.24
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G100 generally yields values of 48,000 to 55,000,
whereas use of other molecular sieving gels and
SDS-PAGE yields lower values of molecular weight.
Mclntyre et al. 34 reported that the molecular weight of
pure human renal inactive renin was 48,000, as determined by SDS-PAGE, and 51,000, as determined by
gel filtration on Sephadex G100 with trypsin treatment
of the eluates. Human prorenin expressed in CHO cells
is a doublet of 49,000 and 50,000, as demonstrated by
immunoprecipitation of prorenin from biosynthetically
radiolabeled supernatants followed by SDS gel electrophoresis.23 Our estimation of molecular weight by
SDS-PAGE is in agreement with the estimation using
HPLC. Expressed human prorenin and native inactive
renin have a molecular weight of 47,000 by S D S PAGE and 46,000 by HPLC.
The Km and pH optimum for expressed human prorenin with sheep substrate are also similar to those
reported for human renal and plasma inactive renin and
for human renal active renin.24 Despite the fact that
renin is an acid protease, the pH optimum for renin
with sheep angiotensinogen is near neutrality. The Km
of human renin with sheep substrate is one third the Km
of human renin with human substrate.
The biosynthetic processing of renin has been studied in mouse submaxillary gland, one of the richest
known sources of renin. Cell-free translation and pulse
labeling indicate that the primary translation product of
renin messenger RNA is preprorenin.""13 During translocation into the rough endoplasmic reticulum, preprorenin is cleaved to prorenin, which is then converted to renin in the Golgi complex. 12 Studies of human
renin-secreting tumor cells demonstrate that tumoral
renin is synthesized as a 55,000-molecular-weight inactive form, which is then converted to a 44,000-molecular-weight active renin.6 However, when cells
from this tumor were cultured, the 55,000 form was
exclusively released into the medium, and no conversion to the smaller form occurred. Galen et al. 6 postulated the existence of two pathways for the secretion of
human renin: in one prorenin is packaged into secre-
tory granules where it is processed into active renin,
and in the other prorenin is nof stored or processed but
is released directly by the cell. These pathways are
analogous to the "regulated" and "constitutive" pathway described in pituitary tumor cells.33 In the latter
pathway, preforms are not stored but released by the
cell. If the constitutive pathway is normally operative
in human juxtaglomerular cells, it may explain the
large quantities of inactive prorenin in human plasma.
Cloning and sequencing of the human renin gene
indicate that the prosegment of human renin consists of
a 46-amino acid peptide. In an attempt to determine
whether inactive renin is prorenin, several investigators developed antibodies to fragments of the prosegment and demonstrated that they bind human inactive
renin. Bouhnik et a l . " synthesized a 13-amino acid
segment of this peptide (Glu 28 —• Trp 40) and generated polyclonal antibodies to the fragment. When the
antibodies were purified and coupled to an affinity gel,
they appeared to bind inactive renin from human kidney and plasma. However, elution with 6 M urea destroyed the activity of the bound renin, which could
then be demonstrated only by direct radioimmunoassay using an enzyme-linked immunosorbent assay.
Presumably, inactive renin was measured in the
eluates containing bound protein. Atlas and colleagues16 developed a polyclonal antibody to the last
12 amino acids of the prosegment (Arg 35 —* Arg 46).
Antibody binding to plasma and renal inactive renin
could be demonstrated by using protein A to precipitate the antibody and by finding no activatable renin
activity in the supernatant. Kim et a l . " developed an
antibody to a 15-residue peptide in the prosegment
(Asp 32 —* Lys 45) and demonstrated that it crossreacted with both human plasma inactive renin (binding to an immunoaffinity column) and human preprorenin obtained from cell-free translation of poly(A) +
RNA, isolated from ischemic human kidney in a rabbit
reticulocyte lysate system.
The present studies strongly support the findings of
these previous investigations. Ultimate identification
EXPRESSED HUMAN PRORENIN///w/i et al.
of human inactive renin lies in its complete purification
and amino acid sequencing. However, the lability of
inactive renin and its susceptibility to activation render
purification in large quantities a difficult task. Nevertheless, the identification of human inactive renin as
prorenin is a major step in determining the physiological importance of human inactive renin and in defining
defects in renin secretion in humans.
Acknowledgments
The authors wish to thank Ms. Susan Bitolas for preparation of
the manuscript and Ms. Anne Santo for preparation of the figures.
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Hypertension. 1986;8:II78
doi: 10.1161/01.HYP.8.6_Pt_2.II78
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