From the
The aspartyl protease renin, an important modulator of blood
pressure in humans, is present in the circulation not only in its
active form, but also as an inactive precursor, prorenin, in which a
43-amino acid prosegment blocks access of the substrate to the active
site of the enzyme. Site-directed mutagenesis of the prosegment has led
to the following conclusions. 1) Maintenance of the enzymatically
inactive state of prorenin requires a short peptide sequence between
positions 10P and 20P (where P denotes prosegment and numbering is
relative to amino terminus) of the prosegment; and 2) there is an
inverse relationship between the ability of prosegment mutations to
activate and their effect on the secretion of the various prorenins,
suggesting that this same region of the prosegment plays a critical
role in the biosynthesis of human prorenin. Since these results
demonstrated that single amino acid mutations could activate human
prorenin to varying degrees, mutations in this region of the renin gene
could be clinically important in humans. To test this hypothesis,
genomic screening was carried out on the corresponding region of the
human renin gene (exon 2) in a cohort of patients selected for a likely
familial component to their hypertension. While this study identified a
novel polymorphism in exon 2 of the human renin gene, evidence was not
obtained for either the presence of prosegment mutations or the
association of the novel polymorphism with hypertension in the patient
population studied. In conclusion, both structure-function studies and
genetic screening suggest that mutation of the prorenin prosegment is
an unlikely factor in activation of the renin-angiotensin system in
humans.
The renin-angiotensin system (RAS)
Active renin in the circulation is derived almost
exclusively from the juxtaglomerular cells of the kidney(5) .
Within these cells, renin is first synthesized as a precursor,
prorenin, in which a 43-amino acid amino-terminal prosegment maintains
the enzyme in an inactive state, presumably by blocking access to the
active site by steric hindrance (6). A portion of the prorenin made by
juxtaglomerular cells is irreversibly activated by the proteolytic
cleavage of the prosegment before secretion. Nevertheless, both the
kidney and numerous other tissues secrete prorenin into the
circulation, where it is present at 3-5 times the level of active
renin(7) . Speculation abounds regarding the function of
prorenin both in the circulation and in tissues for several reasons.
First, the distribution of extrarenal tissues secreting prorenin in
mammals is largely conserved, including the pituitary, ovaries, testes,
adrenal gland, uterus, and placenta. Prorenin expression in the tissues
is also modulated; during pregnancy, prorenin accumulates in the
amniotic fluid at concentrations approaching 100 times that seen in the
circulation(7) . Second, prorenin-secreting tissues also express
other components of the RAS including angiotensinogen,
angiotensin-converting enzyme, and angiotensin II receptors, leading to
the suggestion that local or ``tissue'' RAS may exist and be
physiologically important(8) . Third, prorenin can be
transiently activated in vitro by either acidic conditions or
extended storage in the cold(9, 10) , raising the
possibility that such activation might occur under certain conditions in vivo.
Hypertension is a multifactorial disease whose
etiology stems from both environmental and genetic factors. Studies
have suggested that multiple genetic loci contribute to inheritable
forms of hypertension in inbred strains of laboratory animals (11, 12, 13) and that as much as 20-40% of
blood pressure variation in humans may be due to genetic
factors(14) . Inappropriate activity of the RAS is both a major
contributor to the pathophysiology of hypertension and an important
target for pharmacologic intervention in its treatment(15) .
Gene mutations could presumably lead to either increased expression or
altered activity of RAS components. Indeed, prior studies have
suggested a linkage between a polymorphism in the gene encoding
angiotensinogen, circulating angiotensinogen levels, and hypertension
in humans(16) . While a similar association has been reported
for the renin gene locus in genetically hypertensive strains of
laboratory rats(17) , no such linkage has been established to
date for the human renin gene.
In this study, we have tested the
hypothesis that some forms of inheritable hypertension might be due to
mutations that cause prorenin to be partially active. Site-directed
mutagenesis was used to identify the region of the human prorenin
prosegment crucial for maintenance of the protease in its inactive
state. This same region of the prosegment was found to play a critical
role in the biosynthesis of prorenin. Genome sequencing of the portion
of the human renin gene encoding the prosegment was subsequently
carried out on a group of hypertensive patients with a likely familial
inheritance of hypertension to test whether mutations in this region of
the prosegment might lead to spurious activation of the
renin-angiotensin system in humans.
A control patient
population was obtained from healthy, predominantly French Canadian
volunteers at Hydro-Quebec (Montreal, Quebec, Canada) who had
participated in a previously reported study(22) . Criteria for
participation were normal blood pressure at interview and age of >40
years.
The results of this study suggest that the region of the
human prorenin prosegment from positions 10P to 20P plays an important
role in the maintenance of the inactive state of the zymogen. As CHO
cells do not cleave the prosegment of prorenin(24) , the most
likely explanation for this activation would be that certain of these
mutations result in a disruption of both electrostatic and hydrophobic
interactions, which are important for the binding of the prosegment to
the body of renin. This would presumably have the effect of causing an
unfolding of the prosegment, thereby exposing the active site of the
enzyme to create enzymatically active prorenin. Analysis of the acid
activation kinetics of recombinant prorenin whose prosegment had been
subjected to limited proteolysis has likewise led to the suggestion
that amino acids 10P-14P contribute to the binding of the
prosegment to the body of renin(29) . Since the activation of
prorenin by prosegment mutation is not an ``all or none''
phenomenon, but rather follows a continuum (), it is
likely that the induced mutations shift the equilibrium of prosegment
binding in the active site. That prorenin exists in an equilibrium
between a closed and open conformation in vitro has previously
been suggested on the basis of its activa-tion kinetics (9) and
active-site ``trapping'' experiments in which purified
prorenin was incubated with active site-directed
inhibitors(29, 30) .
The degree of mutation-induced
prorenin activation also exhibits an inverse relationship with the
amount of total renin (prorenin + renin) detected in culture
supernatants (Fig. 2). Thus, as has been described in other
protease families(27) , the prosegment of prorenin (and perhaps
other aspartyl proteases) may play a dual role as both an inhibitor and
a folding ``catalyst'' during biosynthesis of the active
enzyme. These results also suggest that even though prorenin can be
activated to varying degrees by single amino acid mutations in the
prosegment, the effect of these mutations on the overall physiological
activity of the RAS might be offset by the decreased secretion of such
mutant proteins. Indeed, the combined effect of these two factors in vitro is a normalization of active (pro)renin levels (, corrected activity). In addition to these
structure-function studies, our inability to detect coding mutations in
a limited survey of patients selected for a likely familial component
to their hypertension suggests that prosegment mutations resulting in
activated prorenin are an unlikely explanation for inappropriately high
renin activity such as has been reported in some hypertensive
patients(31) .
To date, there has been only one published
report of a protein-altering mutation in human prorenin(32) . In
the family identified, affected individuals were heterozygous for a
mutation inducing a premature stop codon within the prorenin coding
sequence. Interestingly, these individuals had compensated for their
prorenin hemizygosity by increasing the expression level from their
unaffected allele. In addition, no homozygotes for this or any other
protein-altering mutation in prorenin have been reported, and although
several variants of angiotensinogen (the renin substrate) have been
described(16) , none affect the region of the protein that codes
for the angiotensin I peptide released by renin. Taken together, these
findings may suggest an additional role for the RAS in development such
that mutations that would inactivate renin (or the RAS) in humans would
be lethal.
This study also defines a previously undescribed
polymorphism in exon 2 of the human renin gene. While this sequence
alteration leads to the appearance of a novel StyI restriction
site in exon 2 (data not shown), there are numerous additional StyI sites in the human renin gene, making the detection of
this polymorphism difficult in the context of the whole gene. However,
by using direct DNA sequencing, a selective polymerase chain reaction
amplification strategy, or StyI digestion of the polymerase
chain reaction-amplified exon 2 region (data not shown), it has been
possible to catalogue the frequency of this polymorphism in populations
of hypertensive and normotensive patients (I). While we
have not found any obvious correlation with hypertension within the
limitations of this study, two points merit further comment. First, we
detected the StyI polymorphism in only 1 of 35 (2.9%)
ethnically heterogeneous normotensive volunteers drawn from laboratory
workers of our institution (data not shown). It is possible, therefore,
that the StyI polymorphism will display some degree of ethnic
segregation, being more prevalent in the French Canadian population.
Ethnic heterogeneity has been reported for two other restriction
fragment length polymorphisms in the human renin 5`-flanking DNA in
Afro-Caribbeans(33) , reinforcing the importance of selecting an
ethnically comparable control population in these studies. Second, the
multifactorial nature of hypertension and the implied difficulty in
assigning a sufficiently precise clinical subclassification, the
contribution of environmental factors, and the potential variation in
penetrance of contributing genes will likely require efforts to
identify genetic causes in individual families with approaches such as
combinations of polymorphisms to increase the power of the statistical
analysis. In this effort, the StyI polymorphism described
herein may be of greater use in combination with other previously
described restriction fragment length polymorphisms in the human renin
gene(33, 34, 35) .
Percent active renin values are from
Table I. Percent expression is calculated by dividing the average level
of total renin (prorenin + renin) obtained for each mutant
prorenin by the average level of expression of native prorenin in the
same experiment times 100. Corrected activity is calculated by
multiplying percent activity by percent expression.
French Canadian origin was determined by interview.
We thank Micheline Vachon for expert technical
assistance; Michelle LaPointe for patient interviews and blood
sampling; Richard Larivière and Anne Minnick for assistance in
sample procurement; W. N. Chu for assistance with some of the plasmid
constructions; Ken Morgan, Michael N. G. James, and John D. Baxter for
helpful advice; and Christian F. Deschepper for critical reading of the
manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)plays
a critical role in the maintenance of blood pressure, and its activity
has been implicated in the pathogenesis of a number of cardiovascular
disorders including hypertension, cardiac hypertrophy, and vascular
pathologies associated with diabetes and renal disease (1-4). The
rate-limiting step in the RAS is the cleavage of angiotensinogen by the
circulating aspartyl protease renin to release the decapeptide
angiotensin I. Angiotensin-converting enzyme subsequently cleaves two
amino acids from the carboxyl terminus of angiotensin I to release
angiotensin II, a potent vasoconstrictor and regulator of aldosterone
release.
Recombinant Plasmid Construction
The mammalian
expression vector pRhR1100, which expresses the human prorenin cDNA
under the control of the Rous sarcoma virus promoter/enhancer, has been
described elsewhere(18) . Site-directed mutagenesis of amino
acids in the prosegment of human prorenin was carried out by overlap
extension polymerase chain reaction(19) . Amplified regions of
the prorenin cDNA were verified by direct DNA sequencing(20) .
Cell Culture and Transfection
Chinese hamster
ovary (CHO) cells were grown in Dulbecco's modified Eagle's
medium supplemented with 200 µg/ml proline and 10% fetal calf
serum. For expression studies, 5 10
cells were
plated in a 35-mm dish, and transfection was carried out 24 h later by
the calcium phosphate method (21) using 6 µg of the appropriate
prorenin expression vector. Twenty-four hours after transfection, the
medium was removed, and fresh medium was added. Sixteen hours later,
the medium was collected and used immediately to determine renin and
prorenin.
Measurement of Renin and Prorenin
Prorenin and
renin content in tissue culture supernatants was determined by the
angiotensin I generation assay as described previously(21) .
Briefly, 16-h culture supernatants were incubated in the presence of
excess semipurified sheep angiotensinogen either directly (active renin
content) or after incubation with trypsin (total renin content
(prorenin + renin)). Percent renin activity was calculated as
(active renin content/total renin content) 100. Relative
percent activity of the various mutant prorenins was compared with that
of native human prorenin (see ). Statistical analysis was
carried out by one-way analysis of variance with a posthoc Dunnet multiple comparison test. describes total renin content obtained by transfection of
CHO cells with the various prosegment mutant prorenins as a percentage
of the total renin content obtained with native prorenin (relative
expression), representing the mean of three or more individual
transfection experiments. To minimize for variation in transfection
efficiency, total renin levels obtained with the various mutant
prorenins were normalized to the levels exhibited by non-mutant
(native) human prorenin obtained within the same experiment.
Labeling and Immunoprecipitation
Forty-eight hours
after transfection of CHO cells as described above, cells were washed
twice in prewarmed methionine-free Dulbecco's medium (Life
Technologies, Inc.) and incubated in 500 µl of the same medium for
2 h at 37 °C. [S]Methionine (300 µC;
>1000 µCi mmol; DuPont NEN) was added to each well, and the
cells were incubated for 1 h, followed by the addition of 500 µl of
complete medium. After a further 5 h of incubation, the culture
supernatants were collected, and prorenin/renin was immunoprecipitated
and displayed by gel fluorography as described previously(18) .
Patient Selection
Hypertensive patients were
recruited from the Hypertension Clinic at the Clinical Research
Institute of Montreal after provision of an informed consent to
participate in the study and in adherence to a protocol approved by the
institutional human ethics committee. In answer to a questionnaire, all
participants reported having at least one hypertensive parent or
sibling and either were receiving antihypertensive medication or had a
diastolic blood pressure of >90 mm Hg or a systolic pressure of
>140 mm Hg on at least two occasions. Blood was collected on EDTA
and frozen at -20 °C until assayed.
Genome Sequencing
Genomic DNA was purified from
100 µl of whole blood using the DNA microextraction kit from
Stratagene (La Jolla, CA). A 312-base pair DNA fragment corresponding
to exon 2 of the human renin gene was selectively amplified by
polymerase chain reaction with the following oligonucleotides: forward
primer, CGGG-ATCCACGTTAAAGGTGGTTGTACTA; and reverse primer,
CGGAAT-TCGCCTAGGTCCATGAGGGAAA. The amplified DNA fragment was
subsequently digested with the restriction enzymes BamHI and EcoRI (underlined in the forward and reverse primer sequences,
respectively) and cloned into the Bluescript II plasmid vector
(Stratagene). Direct DNA sequencing was carried out on plasmids
prepared by the alkaline lysis method (23) from random pools of
bacterial colonies using the method of Sanger and Coulson (20) and the sequencing primer GGCTGAGCCAAGCACTC, which
corresponds to a reverse primer in exon 2 of the human renin cDNA. All
polymorphisms identified by this method were confirmed by a second
round of genome amplification and sequencing.
Prosegment Sequences Involved in Renin
Inactivation
As previously reported, expression of recombinant
human prorenin in CHO cells leads to secretion of mostly active
prorenin (, native). Recombinant human prorenin expressed
in CHO cells migrates on SDS-polyacrylamide gel electrophoresis at the
expected molecular weight for full-length prorenin (see Fig. 3)(24) . By contrast, expression of a human prorenin
mutant in which the prosegment is deleted results in the secretion of
constitutively active renin ()(18) . Transfection of
CHO cells with expression vectors encoding human prorenin in which
individual amino acids in the prosegment (Fig. 1) have been
mutated results in the secretion of prorenins with varying amounts of
renin activity (). The prosegment mutations tested can be
subdivided into two categories. The first category of mutations
involves charge reversals: Lys and Arg residues were converted to Asp,
and Glu and Asp residues were converted to Arg. These results indicate
that the effect of charge reversal on prorenin activation was not
uniform over the prosegment, but rather clustered in the amino-terminal
region from Arg-10P (where P denotes prosegment and numbering is
relative to amino terminus) to Arg-20P. Within this region, the effect
of charge reversal was most pronounced with Arg-10P (59% activation),
Lys-14P (45% activation), and Arg-15P (53% activation) and least
effective with reversal of Arg-20P (29% activation). The critical
region for charge interaction is well defined since the reversal of
Lys-9P and Glu-21P had no significant effect on prorenin activity. As
well, charge reversals outside this region, at Lys-24P, Arg-26P, and
Asp-29P, did not activate prorenin. Thus, it appears that charged amino
acids in region 10P-20P in the prosegment contribute to the
maintenance of the inactive state of human prorenin.
Figure 3:
Immunoprecipitation of radiolabeled native
and mutant prorenins from transfected CHO cell supernatants. Lane1, native prorenin (7.6% activity, 100% expression); lane2, Arg-10P Ala (15.9% activity, 34.4%
expression); lane3, Leu-13P
Ala (91.2%
activity, 3.8% expression); lane4, control
supernatant from nontransfected cells. Shown at left is the relative
migration of size markers. Data for activity and expression are derived
from Table II.
Figure 1:
Schematic diagram representing the
prosegment of human prorenin. The amino acid sequence is depicted using
the one-letter code. Solidarrows denote those amino
acids that, when mutated, led to a significant increase in prorenin
activity (see Table I for details). Also denoted is the region of the
protein encoded by exon 2 of the human renin gene (drawing not to
scale).
The second
category of mutations tested involved alanine substitution. Only
substitution of alanine for either Leu-13P or Arg-15P resulted in a
significant activation of prorenin (). These residues lie
in the same region as identified using charge reversal (see above). In
addition, with the exception of the substitution of Arg-15P, which
leads to a minor activation of prorenin (21%), alanine substitution of
charged amino acids within region 10P-20P resulted in only a
slight, but nonsignificant activation of prorenin. By contrast, the
rather conservative alanine substitution of Leu-13P led to a major
activation of prorenin (91%), whereas alanine substitution on either
side of Leu-13P (for Phe-12P and Lys-14P) had only a slight, but
nonsignificant activating potential on prorenin. Substitution of a
charged amino acid (Asp) for Leu-31P did not lead to further activation
of prorenin. Taken together, these results suggest that prorenin
inactivation involves charged amino acids between positions 10P and 20P
on the prosegment and is critically dependent on a leucine residue at
position 13P.
Role of the Prosegment in the Biosynthesis of Human
Prorenin
Several groups have reported the expression of
recombinant active renin in tissue culture cells transfected with a
prorenin-encoding expression vector in which the prosegment is
selectively deleted(18, 25, 26) , suggesting
that the prosegment is not absolutely required for biosynthesis of the
active enzyme. Nevertheless, in AtT-20 cells transfected with a similar
construction, we have previously reported that expression of the
resulting active renin was severely suppressed(18) . Comparison
of the overall production of total renin (prorenin + renin) from
native and prosegment-deleted prorenins in transfected CHO cells
confirms this observation: removal of the prosegment results in an
apparent 20-fold decrease in biosynthetic capacity (). One
possible explanation of this finding is that the prosegment, while not
essential, nevertheless plays a role in the biosynthesis or stability
of the renin molecule as has been suggested with other
proteases(27) . Plotting total renin production achieved by
transfection of the various mutant prorenin expression vectors () against their corresponding percent activities () reveals an inverse relationship (Fig. 2), i.e. the more a prorenin molecule is activated by a given mutation, the
lower are its levels of secretion in the culture supernatant of
transfected cells. Immunoprecipitation and SDS-polyacrylamide gel
electrophoresis analysis of selected mutant prorenins demonstrated a
protein with the expected molecular weight for intact prorenin (M
49,000)(24) . In addition, the
quantity of immunoprecipitable prorenin secreted into the supernatant
of transfected cells correlated well with the total renin production as
determined by the enzymatic assay (Fig. 3). Thus, while these
data should only be considered semiquantitative due to the inherent
variation in efficiencies of transfection assays, they identify an
inverse relationship between prosegment inactivation and prorenin
secreted.
Figure 2:
Relationship between mutation-induced
activation and secretion of the various recombinant prorenins studied. Arrows denote the positions of native prorenin and prorenin in
which the prosegment has been removed by site-directed mutagenesis (ProDel.). Data are from Table II.
Sequence Analysis of Renin Gene Exon 2 in Human
Hypertensive Patients
The short region of the prorenin
prosegment responsible for the maintenance of the inactive state of
human renin is located within exon 2 of the human renin gene (Fig. 1)(28) . To test whether mutations of this region
might account for some forms of hypertension due to partial activation
of prorenin, a method was developed to allow the direct sequencing of
this portion of the genome. Specific oligonucleotides designed to flank
exon 2 were used to amplify and clone this region of the genome into
bacterial plasmids. Subsequent DNA sequencing of the cloned insert DNA
allowed the determination of the complete sequence of exon 2 in the
patients tested. Sixty-five patients were selected according to the
following criteria: hypertension as defined by either a diastolic
pressure of >90 mm Hg or a systolic pressure of >140 mm Hg or a
history of hypertension requiring at least two medications for control
and at least one parent or sibling with hypertension. In the 65
patients analyzed, no mutations were detected that would lead to a
change in the peptide sequence encoded by exon 2. However, a novel
polymorphism (CAA to CAC) resulting in the creation of a StyI
restriction site (data not shown) at the position in the exon
corresponding to Thr-2 of the active renin protein was detected in 14
of the 65 subjects analyzed (I). Preliminary tests to
determine whether this polymorphism might serve as a marker for
hypertension were negative in a French Canadian normotensive population (I). In summary, there is no evidence for mutation of
prosegment sequences responsible for maintaining the inactive state of
human renin in the hypertensive patient population analyzed in this
study.
Table: Relationship between activity and expression in
prorenin prosegment mutations
Table: Characteristics of
hypertensive/normotensive patient populations used in this study
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.