(Received for publication, July 25, 1995; and in revised form, December 7, 1995)
From the
Common molecular variants of the angiotensinogen gene have been associated with human hypertension. The rare Tyr to Cys change at residue 248 of mature angiotensinogen was identified in one pedigree. Heterozygous individuals (Y248C) had a 40% decrease in plasma angiotensinogen concentration and a 35% reduction of the angiotensin I production rate. Recombinant wild-type (Tyr-248) and mutant (Cys-248) proteins were stably expressed in Chinese hamster ovary cells. Angiotensinogen monoclonal antibodies revealed marked differences in the epitope recognition of the mutant protein and allowed the demonstration of its presence in plasma of Y248C individuals. Similar kinetic constants of angiotensin I production with human renin were observed for both proteins. Western blot analysis showed similar heterogeneities; however, a 3-kDa increase in molecular mass for the Cys-248 protein was observed after immunopurification. Metabolic labeling of the intracellular Cys-248 protein showed a 61-kDa band in addition to the 55.5- and 58-kDa bands observed for the Tyr-248 protein, with all bands being sensitive to endoglycosidase H. In addition, pulse-chase studies revealed a slower intracellular processing for the Cys-248 protein. In conclusion, the Cys-248 mutation alters the structure, glycosylation, and secretion of angiotensinogen in Chinese hamster ovary cells and is accompanied by a decrease in plasma angiotensinogen concentration in Y248C individuals.
Angiotensinogen is the natural and specific substrate of the
renin-angiotensin system that plays a major role in water and salt
homeostasis, vascular tone, and blood pressure regulation. The specific
interaction between renin and angiotensinogen is the initial and
rate-limiting step of this enzymatic cascade that generates the
decapeptide angiotensin I (Ang I), ()which is processed
further to the active octapeptide angiotensin II by angiotensin
I-converting enzyme. Angiotensinogen is a glycoprotein constitutively
secreted by the liver into plasma and extracellular
fluid(1, 2) . In humans, plasma angiotensinogen
concentration is close to the K
for its
interaction with renin, and variations of its concentration might
therefore influence Ang I generation(3) . Pharmacological (4, 5) and genetic (6, 7, 8) studies in rodents and in humans (9, 10) have demonstrated a positive relationship
between plasma angiotensinogen levels and blood pressure.
The role of angiotensinogen in blood pressure regulation has been highlighted by the linkage observed between a polymorphic marker of the angiotensinogen (AGT) gene and essential hypertension in two large series of hypertensive sibships (11) and two others set of hypertensive families(12, 13) . Several molecular variants have been discovered in the AGT gene, and each of these could potentially be responsible for inherited predisposition to human blood pressure variation. Most notably, a common variant (M235T, change from Met to Thr at residue 235) was found to be more frequent in hypertensive probands than in controls(11, 14) . Despite strong ethnic differences in Thr-235 allele frequency, these findings have been replicated in other ethnic groups (15, 16) , although other studies failed to detect any significant difference in the Thr-235 allele frequency between normotensives and hypertensives(12, 17) . Whether Thr-235 directly accounts for a physiological effect or acts as a marker for a causative mutation is as yet unresolved, although its association with a 10-20% increase in plasma angiotensinogen (11, 14, 18) led to the hypothesis that such a common variant could induce a chronic overstimulation of the renin-angiotensin system, which would explain the susceptibility to hypertension.
In addition to this common variant, some rare mutations have been detected in a few hypertensive probands(11) . Their clinical and biochemical significance can only be established by analysis of the rare affected individuals and the biochemical characteristics of the mutant protein. As an example, the replacement of leucine 10 by a phenylalanine corresponding to the site of angiotensinogen cleavage by renin and to the C-terminal amino acid of Ang I was found in a woman who had suffered preeclampsia, which significantly alters the kinetics of the enzymatic reactions of both renin and angiotensin I-converting enzyme(19) .
In this study, we have investigated the clinical and biochemical significance of the change from a tyrosine to a cysteine residue at position 248, present in the heterozygous state (Y248C) in several subjects belonging to the same family.
DNA samples from 356 probands of hypertensive Caucasian families selected in the Broussais Hypertension Clinic (20) and 325 normotensive Caucasians were screened. Heterozygosity for the Cys-248 allele was found in one hypertensive individual and in one normotensive individual. Consequently, we screened all members of the hypertensive families for the presence of the Cys-248 mutation (635 subjects in addition to the 356 hypertensive probands). Two positive hypertensive brothers with mild and late onset of hypertension were unavailable for subsequent studies. The other positive subject was an offspring of a hypertensive individual. We extended clinical and biological data from this pedigree. After informed consent, nine individuals were analyzed. Blood pressure values correspond to the mean of three successive readings in the sitting position without any antihypertensive treatment, except for individual I-1, the grandmother of the proband who was treated by thiazide diuretics for mild and late onset hypertension. For plasma angiotensinogen determination, blood was drawn in the morning, in the fasting state, and in the sitting position. Genotypes were performed as described above. The Cys-248 allele did not come from the hypertensive father's branch, but from the maternal branch, where most subjects were normotensive.
Figure 1:
Pedigree of the family with Y248C
subjects. Nine subjects were studied and genotyped for Cys-248 and T235
variants of the human angiotensinogen gene. M and T are the respective alleles for the Met to Thr change at position
235 of the angiotensinogen gene. Y and C are the
respective alleles for the Tyr to Cys change at position 248. The
maternal branch carried the Cys-248 variant, whereas the common Thr-235
variant was found on both the maternal and paternal sides. ,
Y248Y; &cjs0937;, Y248C
The frequent Thr-235 allele has been previously associated with a 10-20% increase in plasma angiotensinogen concentration(11) . In the present pedigree, seven subjects were found to be M235T heterozygotes, originating from both branches of the pedigree (Fig. 1). When a comparison was made between M235T and M235M individuals, independently of the presence of the Cys-248 mutation, no relationship could be established with plasma angiotensinogen levels (Table 1). The presence of the Cys-248 mutation in the heterozygous state was associated by itself with a decrease in both plasma angiotensinogen and Ang I production rate.
Figure 2: Immunological measurement of recombinant Tyr-248 and Cys-248 angiotensinogens. A, direct RIA versus enzymatic measurement. Shown are correlations between enzymatic angiotensinogen measurement and polyclonal RIA. High correlations were observed for each form of recombinant angiotensinogen (r = 0.982 (Tyr-248) and r = 0.955 (Cys-248)), but with significantly different slopes (Tyr-248 = 12.53 and Cys-248 = 29.07, t = 5.42, df = 13, p < 0.01). B, measurements using polyclonal and monoclonal antibodies. Shown are the ratios calculated between different radioimmunological assays and the enzymatic measurement of angiotensinogen. Values observed for recombinant Tyr-248 angiotensinogen (open bars) are arbitrarily adjusted to 1 and compared with the values observed for recombinant Cys-248 angiotensinogen (closed bars). From left to right are shown the ratios using polyclonal RIA (Cys-248 = 0.60), IRMA using 7B2-4G3 mAbs (Cys-248 = 0.80), and IRMA using 1H8-1C11 mAbs (Cys-248 = 0.11)
To document further the altered immunological recognition of the Cys-248 variant, measurements of both proteins were performed with the angiotensinogen RIAs and IRMAs using different mAbs. The ratio between the RIAs and the enzymatic assay showed important differences according to the mAbs used. IRMA with either the 7C11 or 1C11 mAb as the labeled antibody showed a strong discrimination between the wild-type and mutant proteins (Fig. 2B). The ratio of the angiotensinogen concentration measured by the 1H8-1C11 and 7B2-4G3 antibody pairs was 1.35 ± 0.11 for Tyr-248 and 0.18 ± 0.03 for Cys-248 (7.5-fold difference) (Fig. 3A), whereas ratios between other pairs did not significantly differ (data not shown).
Figure 3: Presence of Cys-248 angiotensinogen in plasma of heterozygous Y248C individuals. A, values of the 1H8-1C11/7B2-4G3 IRMA ratio from wild-type (Tyr-248 = 1.38 ± 0.11; open bar) and mutant (Cys-248 = 0.18 ± 0.03; closed bar) recombinant angiotensinogens. B, values of the 1H8-1C11/7B2-4G3 IRMA ratio observed in plasma of heterozygous Y248C individuals compared with those observed in plasma of individuals not bearing the mutation (Y248Y). Taking into account the influence of the M235T variant on this immunological ratio and the presence of this variant in the family, separate comparisons were performed in M235M and M235T individuals. In M235M individuals, values observed in five unrelated Y248Y controls (open bar) were 1.58 ± 0.06 compared with 0.90 and 0.92 observed in the II-2 and II-4 heterozygous Y248C subjects (hatched bar). In M235T individuals, values observed in five unrelated Y248Y controls (0.95 ± 0.03; open bar) were comparable to those observed in the II-1, II-5, II-6, and III-2 Y248Y subjects (0.85, range of 0.81-0.88), but 290% higher than those obtained in the I-1, II-3, and III-1 Y248C subjects (0.33, 0.30, and 0.31, respectively; hatched bar).
Ratios observed in five unrelated M235M control subjects not bearing the Cys-248 variant (1.58 ± 0.06) were on average 73% higher than those observed in the II-2 and II-4 heterozygous Y248C subjects (Fig. 3B). Values obtained in five controls (0.95 ± 0.03) and in the II-1, II-5, II-6, and III-2 individuals (0.85, range of 0.81-0.88), all being M235T heterozygous and Y248Y homozygous, were almost 3-fold higher than those obtained in the I-1, II-3, and III-1 individuals (0.33, 0.30, and 0.31, respectively; p < 0.01), heterozygous for both M235T and Y248C mutations. Taking into account the difference in the IRMA ratio observed for recombinant Tyr-248 and Cys-248 angiotensinogens, these results strongly suggest that Cys-248 angiotensinogen was present in plasma of Y248C individuals of our family.
Figure 4: Characterization and immunopurification of recombinant Tyr-248 and Cys-248 angiotensinogens. A, Western blot analysis of Tyr-248 and Cys-248 angiotensinogens secreted by CHO cells. Samples were denatured with reducing agent (5 mM dithiothreitol). Western blotting was performed using the anti-angiotensinogen polyclonal antibody HCL(22) . Antigen-antibody complexes were revealed by alkaline phosphatase activity after biotin-streptavidin enhancement. B, elution profiles of Tyr-248 and Cys-248 angiotensinogens. CHO cell media containing 196 µg of Tyr-248 protein or 290 µg of Cys-248 protein were loaded onto mAb 1B1-Sepharose. Bound proteins were eluted with 0.1 M sodium acetate buffer, pH 3.5. The angiotensinogen amount was measured in each fraction by angiotensinogen assays. C, Coomassie Blue staining of immunopurified Tyr-248 and Cys-248 angiotensinogens. Each protein (2 µg) was denatured with 50 mM dithiothreitol. Proteins were separated on an SDS-9% acrylamide gel and visualized by Coomassie Blue staining. D, Western blot analysis of immunopurified Tyr-248 and Cys-248 angiotensinogens. Each protein (10 ng) was denatured with 10 mM dithiothreitol. Western blot analysis was performed as indicated above.
The single-step
immunopurification technique led to similar purification yields of
Tyr-248 and Cys-248 angiotensinogens (78 and 65%, respectively). Purity
of both angiotensinogens was 95% as judged from Coomassie Blue
staining. N-terminal sequence analysis performed by the Edman
degradation cycle method showed that the sequence corresponded to that
of Ang I, which constitutes the N-terminal part of angiotensinogen. The
angiotensinogen concentration in the immunopurified fractions was
determined by direct RIA and enzymatic assay. For the wild-type
protein, concentrations obtained with both methods were equivalent,
indicating that the capacity of cleavage of the immunopurified
angiotensinogen by renin was not altered by the immunopurification
step.
In vitro cleavage of both substrates by pure human
recombinant renin did not significantly differ as measured by the
determination of K (0.81 and 0.77 µM for Tyr-248 and Cys-248 angiotensinogens, respectively) and k
(0.95 and 0.89 s
,
respectively), leading to similar k
/K
ratios (1.17 versus 1.15 µM
s
,
respectively)
Figure 5:
Metabolic labeling of Tyr-248 and Cys-248
angiotensinogens. CHO cells stably transfected with the cDNA encoding
Tyr-248 angiotensinogen (clone Y248-7) and three different clones of
CHO cells expressing the Cys-248 mutant (C248-17, C248-15, and C248-14)
were metabolically labeled for 10 min with 50 µCi/ml
[S]methionine and
[
S]cysteine in methionine/cysteine-free medium.
A 30-min chase period in complete medium was performed. Cells were then
solubilized and immunoprecipitated with the anti-angiotensinogen
polyclonal antibody HCL. The immunoprecipitated material was analyzed
by SDS-PAGE. Autoradiography was revealed after an overnight
exposure.
Figure 6:
Effect of glycosidases on intracellular
and secreted forms of recombinant Tyr-248 and Cys-248 angiotensinogens.
CHO cells expressing wild-type Tyr-248 and mutant Cys-248
angiotensinogens were metabolically labeled with
[S]methionine and
[
S]cysteine. Cellular (A) and secreted (B) proteins were immunoprecipitated with the HCL polyclonal
antibody. Immunoprecipitated materials were then subjected to mock
treatment, or endoglycosidase H, or N-glycosidase F, or N-glycosidase F followed by neuraminidase and O-glycosidase digestions. The samples were analyzed by
SDS-PAGE and autoradiography.
Figure 7:
Pulse-chase experiments on recombinant
Tyr-248 and Cys-248 angiotensinogens. CHO cells expressing Tyr-248 or
Cys-248 angiotensinogen were metabolically labeled with
[S]methionine and
[
S]cysteine for 10 min (pulse period). Different
times of chase were performed: 0, 1.5, 2, 2.5, 3, 3.5, 24, and 48 h. At
each time, cells were lysed, and the supernatant was immunoprecipitated
with the anti-angiotensinogen polyclonal antibody HCL. The samples were
analyzed by SDS-PAGE and autoradiography.
The presence of natural mutations of the AGT gene provides a
unique opportunity to obtain further insight into the
pathophysiological impact of angiotensinogen on the renin-angiotensin
system and blood pressure regulation. Among the different genetic
variations discovered in the coding sequence of the AGT gene, the
change from Tyr to Cys at position 248 of angiotensinogen could
represent a mutation with a negative impact on the renin system
activity. In the family studied here, the presence of the Cys-248
variant in the heterozygous state was associated with a 42% decrease in
plasma angiotensinogen concentration. Since the K of the renin reaction (1 µM) is close to the average
plasma angiotensinogen concentration(3) , it was interesting to
test whether the difference in plasma angiotensinogen concentration
observed in individuals bearing the Cys-248 mutation could affect the
generation of Ang I. In this pedigree, the presence of the Cys-248
variant in the heterozygous state was associated with a 35% decrease in
Ang I production rate. Whether this decrease can promote a lower blood
pressure level is as yet undetermined and will require an extensive
family study of a large number of affected and unaffected individuals.
It is interesting to note that a direct causal relationship was
established between plasma angiotensinogen levels and blood pressure in
transgenic mice carrying one to four copies of the AGT
gene(8) . However, pathophysiological adaptations of other
systems regulating blood pressure and especially feedback regulation by
renin should play a major role in the final blood pressure
level(34, 35) .
To investigate the mechanism
responsible for this reduction of angiotensinogen concentration in
Y248C individuals, the mutant angiotensinogen was expressed in
eucaryotic cells. Its structure, function, and biosynthesis were
compared with those of the wild-type form of angiotensinogen. Human
angiotensinogen amino acid sequence contains four potential N-linked glycosylation sites (Asn-X-Ser/Thr) that
represent 14% of the molecular mass of the
protein(1, 2) . Using different multiple-step
procedures for the purification of angiotensinogen, several authors
demonstrated a heterogeneity of the protein varying from 55 to 65 kDa,
which corresponds mainly to multiple glycosylated forms of
angiotensinogen (1, 36) . For both wild-type and
mutant proteins, a similar heterogeneous pattern and molecular mass
were identified by Western blot analysis (Fig. 4A) and
by metabolic labeling (Fig. 6B) in CHO cell medium.
However, several lines of evidence suggest structural differences
between Tyr-248 and Cys-248 angiotensinogens. After immunopurification,
Cys-248 angiotensinogen displayed a slower migration on SDS-PAGE
(
3 kDa) as compared with Tyr-248 angiotensinogen (Fig. 4, C and D), indicating that the acidic denaturation and
renaturation used during the immunopurification procedure modified the
two angiotensinogens differently. In addition, we were able to
demonstrate that the Tyr to Cys change at position 248 of
angiotensinogen markedly affects the immunological recognition of the
protein. Among the four monoclonal antibodies directed against human
angiotensinogen that had been previously generated(23) , two
(4G3 and 1C11) that belong to the same antigenic cluster
allowed a clear distinction between the mutant and wild-type
forms of the protein and the detection of Cys-248 angiotensinogen in
plasma of Y248C individuals.
Whether the altered immunological
recognition of Cys-248 angiotensinogen is due to the modification of
the carbohydrate content or to a more profound structural modification
due to the change of amino acids cannot be determined from this study.
It is interesting to note that the Tyr residue at position 248 is part
of a region of six residues that are conserved between proteins of the
serine protease inhibitor family(37, 38) . From the
three-dimensional structures of -antitrypsin and
antithrombin III, this conserved sequence corresponds to the s3 segment
of the large
-sheet A, which is not exposed at the surface of the
molecule. Since comparison between wild-type and Cys-248
angiotensinogens shows a significant alteration of the predicted
hydropathy profile(39) , it is tempting to speculate that the
mutation disrupts the association with the following antiparallel
strand of the
-sheet A, thus allowing the Cys residue at position
248 to be exposed at the surface and to modify the corresponding
epitope site of angiotensinogen. However, this interpretation should be
cautiously accepted since the entire
-antitrypsin and
antithrombin III molecules share only 21 and 18% similarity at the
amino acid level with angiotensinogen. A potential impact of these
structural modifications either on plasma angiotensinogen half-life or
on its interaction with other plasma proteins (40) was not
tested in this study. However, we did not observe any modification of
the functional properties of the protein as a renin substrate, but only
a decrease in plasma angiotensinogen in individuals bearing the
mutation.
The other major difference between Cys-248 and Tyr-248 angiotensinogens was observed during the biosynthesis and secretion of the corresponding recombinant proteins. In all clones analyzed, the Cys-248 mutant presented a 61-kDa intracellular form in addition to the two bands of 55.5 and 58 kDa observed with the wild-type protein. Since treatment with endoglycosidase H or F suppressed this difference, it was interpreted as a modification in the glycosylation process. In addition to this qualitative change of glycosylation, pulse-chase studies demonstrated that the intracellular processing of the different forms of angiotensinogen was not similar. While the intracellular 55.5-kDa form was similarly secreted for both proteins, the 58- and 61-kDa forms of Cys-248 angiotensinogen were significantly retained in CHO cells, suggesting an abnormal intracellular processing of the mutant protein.
These experimental observations may be differently interpreted. One explanation is that the structural modifications induced by the Cys-248 mutation markedly affect the processing of the protein, explaining the accumulation of different high mannose forms of angiotensinogen. Therefore, the observed 61-kDa band could represent an initial transient and rapidly processed glycosylated form of angiotensinogen synthesized for both the Tyr-248 and Cys-248 proteins, which is not detectable unless a slower intracellular processing occurs. Another more speculative but also attractive hypothesis is that the change from Tyr to Cys at residue 248 creates an unusual site of N-glycosylation (Asn-X-Cys)(41) . According to this hypothesis, the 61-kDa form of Cys-248 angiotensinogen would present an additional N-linked carbohydrate side chain that would be deleterious for the efficient secretion of angiotensinogen. Indeed, N-glycans play a major role in protein folding and intracellular processing of secretory protein(42) . Both hypotheses could explain the abnormal glycosylation and secretion that were observed in CHO cells, but cannot be directly extrapolated to other cellular systems. In both cases, the observed decreased plasma angiotensinogen concentration in subjects bearing the Cys-248 mutation may be explained by altered intracellular processing and secretion of angiotensinogen in hepatocytes.
In conclusion, this study demonstrates that the presence of the rare Cys-248 variant in the heterozygous state is associated with a significantly lower plasma angiotensinogen concentration. Recombinant Cys-248 angiotensinogen exhibits an altered immunological profile and is abnormally glycosylated and secreted in CHO cells. Taken together, these results strongly suggest that the Cys-248 variant of angiotensinogen alters the structure of the protein in addition to decreasing its concentration in individuals heterozygous for this mutation.