(Received for publication, November 1, 1995; and in revised form, January 2, 1996)
From the
For facilitating crystallization and structural studies of the
testicular isozyme of angiotensin-converting enzyme (ACE),
we attempted the production of enzymatically active ACE
proteins which are unglycosylated or underglycosylated.
Expression in Escherichia coli of the rabbit ACE
cDNA resulted in the synthesis of an unglycosylated but inactive
protein. Similarly, unglycosylated ACE
synthesized in HeLa
cells, by using a cDNA in which all five potential N-glycosylation sites had been mutated, was inactive and
rapidly degraded. Several ACE
variants carrying mutations
in one or more of the potential N-glycosylation sites were
used to examine the role of glycosylation at specific sites on
ACE
synthesis, transport to the cell surface, cleavage
processing, and enzyme activity. These experiments demonstrated that
allowing glycosylation only at the first or the second site, as counted
from the NH
terminus, was sufficient for normal synthesis
and processing of active ACE
. In contrast,
ACE
g3, which had only the third glycosylation site
available, was unglycosylated, enzymatically inactive and rapidly
degraded. N-Glycosylated ACE
could also be
produced in yeast. Surprisingly, the mutant ACE
g3 was
synthesized, N-glycosylated, and properly transported in
yeast. Wild type and mutant ACE proteins were cleavage-secreted from
yeast and enzymatically active.
Angiotensin-converting enzyme (ACE) ()(EC 3.4.15.1,
dipeptidyl carboxypeptidase) plays a major role in blood pressure
regulation and fluid and electrolyte homeostasis by acting on two major
vasoactive peptides. It converts the inactive precursor angiotensin I
to an active vasopressor peptide angiotensin II and inactivates the
vasodepressor peptide bradykinin(1, 2, 3) .
ACE has two structurally related isozymic forms (4, 5, 6) encoded by two different mRNAs,
which arise from the same gene by tissue-specific choice of the
alternative transcription initiation
sites(7, 8, 9, 10, 11, 12, 13) .
In rabbit, the smaller isozyme, testicular (T) ACE has 737 amino acid
residues and the larger pulmonary (P) isozyme has 1309
residues(7, 9) . The COOH-terminal 665 residues of the
isozymes are identical, whereas their NH
termini are
unique. They carry signal sequences at their NH
termini
which are removed during their biosynthesis. Both isozymes are
extensively glycosylated and expressed as a cell surface Type 1
ectoprotein anchored in the plasma membrane by a 17-residue-long
hydrophobic transmembrane domain near the COOH terminus. There is ample
evidence to suggest that the COOH-terminal 30 residues constitutes the
cytoplasmic tail and the rest of the protein is extracellular. In
tissue
culture(14, 15, 16, 17, 18) ,
as well as in vivo, the extracellular domain of ACE is
released into the culture media or in body fluids, by a regulated
proteolytic cleavage of the membrane anchoring domain. Indeed, a
COOH-terminally truncated, soluble form of enzymatically active ACE is
found in many body fluids, including
serum(16, 18, 19) . Thus, several important
post-translational modifications occur during biosynthesis of ACE.
Rabbit ACE has five potential N-glycosylation
sites as indicated by the presence of the Asn-X-Ser/Thr motif in its
primary structure. In addition, it has a cluster of threonine residues
near the amino terminus that can potentially be O-glycosylated(7) . ACE
isolated from
tissues and transfected cell lines is heavily glycosylated carrying
both N- and O-linked sugars. Using inhibitors of
glycosylation and a mutant cell line defective in protein
glycosylation, we have shown that complete blockage of glycosylation
causes rapid intracellular degradation of ACE
. However,
ACE
synthesized without N-linked complex sugars
and O-linked sugars is transported to the cell surface,
cleavage-secreted, and enzymatically active(20) . Similarly,
Ehlers et al.(21) have shown that a mutant ACE
devoid of most of its O-linked sugars has enzymatic
activity.
In our current study, we evaluated the contributions of
each of the five potential N-glycosylation sites of ACE toward its synthesis, glycosylation, intracellular transport,
cleavage secretion, and enzymatic activity. This was accomplished by
site-directed mutagenesis of these sites individually or in
combinations. Our studies demonstrated interesting differences among
the contributions made by the different sites. Moreover, we attempted
the production of wild type and mutant ACE
proteins in
bacteria and yeast so that large quantities of these proteins can be
easily produced for structural studies. In the process, we revealed an
unexpected difference between yeasts and mammalian cells in the
utilization of a specific glycosylation site in ACE
.
Figure 8:
Glycosylation status of ACE produced in P. pastoris. Culture media from P.
pastoris expressing ACE
WT (lanes 1-4)
or ACE
g3 (lanes 5-8) were immunoprecipitated
with anti-ACE antibody 2 days after methanol induction. The
immunoprecipitates were boiled with SDS and left untreated(-) or
treated with (+) deglycosylating enzymes as
indicated.
Figure 1:
Expression of ACE in E.
coli. Total cellular protein isolated from E. coli BL21(DE3) containing either pET3a vector alone (Control)
or pET3a-ACE
clone was analyzed by SDS-PAGE. The proteins
were transferred onto a nitrocellulose membrane and subjected to
Western blot analysis with anti-ACE-antibody. The arrow indicates the expressed
ACE
.
Figure 2:
Expression of unglycosylated ACE mutants in HeLa cells. The figure shows synthesis, intracellular
processing, and secretion of wild type ACE
(ACE
WT) and the mutant
ACE
g0 in HeLa cells. HeLa cells, infected with the
recombinant vaccinia virus expressing T7 RNA polymerase, were
transiently transfected with ACE cDNA (left and middle
panels) and ACE
g0 cDNA (right panel). The
cells were pulse-labeled with [
S]methionine for
30 min followed by incubation without the labels for different periods
of time as indicated by Chase (h). Labeled detergent lysates
of cells (C) and medium (M) were immunoprecipitated
and analyzed by SDS-PAGE. The middle panel had 5 µg/ml
tunicamycin present in the culture medium at all times. Positions of
molecular mass markers in kilodaltons are shown on the left.
Figure 3:
Expression of underglycosylated ACE mutants. HeLa cells were transfected with wild type or mutant
ACE
cDNAs, pulse-labeled with
[
S]methionine, and the label was chased for 15
h. Detergent lysates of cells (C) and the culture medium (M) were immunoprecipitated and analyzed. A,
single-site mutants. B, double-site mutants. C,
triple-site mutants.
Figure 4:
Expression of ACE mutants with
single N-glycosylation sites. A, HeLa cells were
transfected with wild type or mutant cDNAs, and pulse-chase (15 h)
analysis was performed as described in the legend of Fig. 3.
Immunoprecipitated cell lysates (C) and media (M)
from cells transfected with ACE
WT (lanes 1 and 5), ACE
g2 (lanes 2 and 6),
ACE
g3 (lanes 3 and 7), and
ACE
g1 (lanes 4 and 8) were analyzed. B, kinetics of biosynthesis: transfected HeLa cells were
pulse-labeled and chased for 0, 2, 4, 8, and 15 h as indicated.
Immunoprecipitated lysates of cells transfected with ACE
WT (lanes 1-5), ACE
g2 (lanes
6-10), ACE
g1 (lanes 11-15), and
ACE
g3 (lanes 16-20) cDNA were
analyzed.
The
glycosylation status of these mutant proteins was directly tested by
measuring their sensitivity to glycosidases (Fig. 5). As
expected, ACEWT was both N- and O-glycosylated. The same was true for both ACE
g2
and ACE
g1, although the extent of their N-glycosylation was less than that of ACE
WT. The
76-kDa ACE
g3 protein, on the other hand, was neither O-glycosylated nor N-glycosylated. It should be noted
that for this analysis of ACE
g3, we used a relatively
larger quantity of an extract of cells that had been pulse-labeled but
not chased. Enough of the 76-kDa protein was available for analysis
only under these conditions. The lack of glycosylation of
ACE
g3 was further confirmed by the failure to react with an
anti-sugar antibody (data not shown). Thus, ACE
g3, although
synthesized, remained unglycosylated in the HeLa cells. As a
consequence, it was rapidly degraded and not transported to the cell
surface. The above conclusions drawn from the metabolic labeling
experiments ( Fig. 4and Fig. 5) were confirmed by
immunofluorescence studies (Fig. 6). ACE
WT was
present both on the surface and inside of the transformed cells. In
contrast, a small amount of ACE
g3 was present inside the
cells, but none was displayed on the cell surface. The pattern of
intracellular distribution of ACE
g3 suggests that the
protein is arrested in the endoplasmic reticulum. Taken together, these
experiments demonstrate that all glycosylation sites in ACE
are not equivalent. Site 3, by itself, is not sufficient for
glycosylation of the protein, but sites 1 and 2 are.
Figure 5:
N- and O-glycosylation
status of mutants with single N-glycosylation sites. HeLa
cells were transfected with ACEWT (lanes
1-4), ACE
g2 (lanes 5-8),
ACE
g1 (lanes 9-12), and ACE
g3 (lanes 13-16) cDNAs. After pulse-labeling, the label was
chased for 0 h (ACE
g3-transfected cells) or 15 h (all other
cells). Cell lysates were immunoprecipitated, boiled with SDS, and left
untreated(-) or treated with (+) deglycosylating enzymes as
indicated. For the analysis of ACE
g3 (lanes
13-16), five times more extract was used than all other
lanes.
Figure 6:
Absence of ACEg3 on the cell
surface: detection of ACE
WT and ACE
g3 proteins
by indirect immunofluorescence. HeLa cells grown on coverslips were
transfected with ACE
WT or ACE
g3 cDNA.
Transfected cells were processed for indirect immunofluorescence as
described under ``Experimental Procedures'' using anti-ACE
antibody and fluorescence-conjugated rabbit anti-goat IgG, to detect
ACE proteins expressed intracellularly (Internal) or on the
cell surface (Surface).
Figure 7:
Expression of ACEWT,
ACE
13, ACE
g2, and ACE
g3 in
methylotropic yeast, P. pastoris. ACE
WT and three
other mutant proteins were expressed in P. pastoris as
described under ``Experimental Procedures.'' Two days after
methanol induction, 40 µl of the clarified culture media were
analyzed by Western blot analysis using anti-ACE antibody. Lane
1, ACE
WT; lane 2, ACE
g13; lane 3, ACE
g2; and lane 4,
ACE
g3. Numbers on the left indicate the
molecular masses of the expressed proteins in
kilodaltons.
Finally, the mode of secretion of
ACEWT and ACE
g3 from yeast was examined. We
have shown previously that secretion of ACE
from mammalian
cell surface is accomplished by proteolytic cleavage of the ectodomain.
As a result, the secreted form of ACE
does not contain the
membrane-anchoring domain and the intracellular domain present in the
cell-bound form of ACE
. In the experiment shown in Fig. 9, we examined if the ACE
secreted by yeast
also lacks these domains. For this purpose, we used a
COOH-terminal-specific antibody, which reacts with HeLa cell-bound
ACE
, but not with secreted ACE
(23) .
This antibody also failed to react with ACE
WT and
ACE
g3 secreted by yeast (Fig. 9), thus indicating
that ACE
is also cleavage-secreted by P. pastoris.
Figure 9:
Cleavage secretion of ACE in P. pastoris. Culture media of P. pastoris expressing
ACE
WT (lanes 1 and 3, 20 µl each) and
ACE
g3 (lanes 2 and 4, 60 µl each)
were analyzed by Western blot analysis using anti-ACE (lanes 1 and 2) or anti COOH-terminal peptide antibody (lanes
3 and 4).
We have established transfected mammalian cell lines that are
high producers of enzymatically active ACE(14) . We
purified large quantities of ACE
from the culture medium of
these cells and attempted its crystallization without any success. We
reasoned that the failure to crystallize could be due to an inherent
heterogeneity of the purified protein which, in turn, is caused by its
high level of glycosylation. We, therefore, sought out means to produce
unglycosylated and underglycosylated ACE
. There are reports
in the literature that the sugars may not be required for the activity
of ACE
, since purified ACE
can be
deglycosylated without losing activity(26) . These studies,
however, provided no evidence that the deglycosylation was complete,
nor do they address the issue of whether newly synthesized
unglycosylated ACE can fold into an active conformation. We observed
that unglycosylated ACE
produced in tunicamycin-treated
HeLa cells is inactive and rapidly degraded(20) . In the
present study, we, therefore, produced unglycosylated ACE
in E. coli. This protein was not degraded in the
bacterium, and therefore, it is possible to produce and purify
bacterially produced ACE
in large quantities. It, however,
is devoid of enzyme activity and not suitable for crystallization and
structural studies. This result was not entirely unexpected since many
mammalian glycoproteins, when produced in bacteria, are biochemically
inactive. This lack of activity is ascribed to misfolding of the
proteins in bacteria. Consequently, careful denaturation and
renaturation of some of these bacterially produced proteins regenerate
activity(27) . This, however, could not be accomplished for
ACE
. We, therefore, resorted to alternative methods for
producing active ACE
with minimum sugar modifications. For
this purpose, the glycosylation sites were mutated and the
corresponding proteins were produced in HeLa cells and P.
pastoris. Our results showed that allowing N-glycosylation only at one out of five sites was sufficient
for producing active ACE in both systems. The proteins produced in
yeast had, however, consistently lower molecular weights (Table 2), thus suggesting less post-translational modifications
of ACE
in yeast. In the future, ACE
g3 produced
in yeast will probably be the best candidate for crystallization.
Our studies with the glycosylation site mutants revealed interesting
differences in the nature of contributions of each of the five sites.
The sizes of all of the single mutants (Fig. 3A) or all
of the double mutants (Fig. 3B) are not the same. This
suggests that either the complexity of sugar chains at each of these
sites is different or that N-glycosylation at each site has
different effects on other modifications of the protein such as O-glycosylation. These mutations also affected the rate of
synthesis and the rate of cleavage secretion differently. It would be
interesting to investigate in the future why ACEg134 was
secreted much more efficiently than ACE
g135. Like
ACE
WT, ACE
g2 and ACE
g1 produced in
HeLa cells were both N- and O-glycosylated (Fig. 5). These proteins probably carry additional modifications
whose nature remains to be determined. Note that completely
deglycosylated ACE
WT, g2 and g1 (lanes 4, 8, and 12, Fig. 5) had a molecular mass much higher than 76
kDa, which was the molecular mass of unglycosylated ACE
g3 (lane 13, Fig. 5). All of these proteins, when produced
in yeast, had molecular weights lower than the corresponding species
produced in HeLa cells (Table 2). This could be due to either a
lack of O-glycosylation of ACE
produced in yeast,
or it could be that the other putative additional modifications, be it
phosphorylation, sulfation, or others, are missing in yeast. Completely
deglycosylated secreted form of ACE
WT produced in yeast had
a molecular mass of about 70 kDa (lane 4, Fig. 8),
whereas the corresponding molecular mass of secreted, deglycosylated
ACE
WT produced by HeLa cells is about 84 kDa (data not
shown). Since ACE
produced in yeast was enzymatically
active, the additional modifications are obviously not needed for the
formation of active ACE. These modifications of ACE
probably occur in the Golgi or plasma membrane of HeLa cells,
since ACE
g3 arrested in the endoplasmic reticulum seems to
be devoid of them.
ACE produced in P. pastoris was cleavage-secreted (Fig. 9), since the secreted
ACE
protein was devoid of the cytoplasmic tail. The exact
cleavage site has not yet been determined and compared with the site
used in mammalian cells. The cleavage secretion process in the
mammalian cells is regulated by phorbol esters and the responsible
plasma membrane-associated proteolytic activity has the characteristics
of a specific class of metalloprotease(28) . It remains to be
determined if the cleavage secretion process in yeast has similar
characteristics. If so, it can serve as a model for the mammalian
activity, and the responsible protease may be more easily identifiable
in the yeast system.
The observed differential behaviors of
ACEg3 in HeLa cells and P. pastoris were
unexpected. We are unaware of any other examples of a mammalian
glycoprotein that fails to be glycosylated in the endoplasmic reticulum
of a mammalian cell, but not in yeast. ACE
g3 can, thus,
serve as a useful tool for identifying key differences in the
glycosylation apparatus of the two cell types. Since yeasts are widely
used for studying the process involved in eukaryotic protein
trafficking and post-translational modifications, the differences noted
here may serve as a warning that all the steps may not be equivalent in
yeast and higher eukaryotes.