From the
Recent structural studies
(9, 10) suggest a related but more complex process may be involved
in the loop-sheet polymerization of the serpins. The structure of a
dimer of antithrombin shows one molecule in the latent form in which
the reactive loop is totally incorporated into the A sheet of the
molecule. This releases a strand from the C sheet and it is this strand
that is replaced by the reactive loop of the second molecule in the
dimer. This alternative mechanism of C sheet polymerization is not
incompatible with the A sheet model, since the common first step in
either polymerization process would be increased rate of refolding of
the loop into the A sheet.
Here we examine the comparative secretion
of M, Z, and S
Although the structural and
other evidence strongly indicates that the underlying defect in
S
Z and S
Although the effect of such mutations on the thermal stability and
inhibitory activity of normal serpins are well established, their
influence on polymerization and secretion have yet to be explored. The
secretion of Z
Alanine to
valine substitutions at P
-Antitrypsin plasma deficiency variants which
form hepatic inclusion bodies within the endoplasmic pathway include
the common Z variant (Glu
Lys) and the rarer
-antitrypsin S
(Ser
Phe). It has been proposed that retention of both abnormal
proteins is accompanied by a common mechanism of loop-sheet
polymerization with the insertion of the reactive center loop of one
molecule into a
-pleated sheet of another. We have compared the
biosynthesis, glycosylation, and secretion of normal, Z and
S
variants of
-antitrypsin using
Xenopus oocytes. S
and Z
-antitrypsin both duplicated the secretory defect seen
in hepatocytes that results in decreased plasma
-antitrypsin levels. Digestion with endoglycosidase H
localized both variants to a pre-Golgi compartment. The mutation
Phe
Leu abolished completely the intracellular
blockage of S
-antitrypsin and
reduced significantly the retention of Z
-antitrypsin.
The secretory properties of M and Z
-antitrypsin
variants containing amino acid substitutions designed to decrease loop
mobility and sheet insertion were investigated. A reduction in
intracellular levels of Z
-antitrypsin was achieved
with the replacement of P
alanines by valines. Thus a
decrease in Z and S
-antitrypsin
retention was observed with mutations which either closed the A sheet
or decreased loop mobility at the loop hinge region.
-Antitrypsin variants Z
(1, 2) and S
(3) are associated with a
deficiency of the inhibitor in the blood and its partial retention
within the endoplasmic reticulum of synthesizing hepatocytes. Recent
evidence indicates that this retention is accompanied by polymerization
of the abnormal protein by the process of loop-sheet linkage in which
the reactive center loop of one molecule inserts into a
-pleated
sheet of another
(4, 5) . The mutation in Z
-antitrypsin, Glu
Lys, occurs at
the junction of strand 5 of the six-membered sheet A of the molecule
(6) with the base of the mobile loop. This alteration in the
hinge region is thought to interfere with normal refolding of the
reactive center into the A sheet, thus favoring intermolecular loop
insertion with the sequential formation of loop-sheet polymers
(5) . The A sheet polymerization model is also compatible with
structural studies on
-antitrypsin S
in which a Ser
Phe mutation is predicted to
cause an opening of the A sheet between strands s3A and s5A
(7, 8) .
-antitrypsins from
Xenopus oocytes to further explore the link between loop-sheet
interactions and defects in secretion. Yu and colleagues
(11) have identified a mutation, Phe
Leu,
which stabilizes
-antitrypsin against polymerization,
predictably by locking its A sheet in the closed position. In
particular we examine the secretion of this mutant
-antitrypsin and of its chimer with Z
-antitrypsin (Phe
Leu/Glu
Lys) and S
-antitrypsin
(Phe
Leu/Ser
Phe) to establish
whether, as predicted, stabilization of the A sheet will prevent the
polymerization that otherwise occurs in these variants with a
consequent failure in protein export.
is due to an opening of the A sheet allowing the
ready formation of polymers, the structural deductions with respect to
the Z mutant are more ambivalent. The Z mutation is at residue
P
, at the base of the hinge region on which the reactive
center loop pivots, and may result in either an opening of the A sheet
or an impedance of loop insertion into the sheet. An inhibition of loop
folding would be expected to favor polymerization, but if this is so,
the mechanism would be that of A sheet rather than C sheet
polymerization. We test this possibility here using constructs of M and
Z antitrypsin containing replacements in the hinge region designed to
constrain loop mobility but which should not effect the mechanism of A
sheet opening. The secretory properties of these M and Z chimeric
mutants provide evidence as to the mechanism underlying the
intracellular polymerization of Z antitrypsin.
Chemicals and Reagents
DNA and RNA modifying
enzymes were from Promega Corp. or Boehringer Mannheim.
-
S-dATP (specific activity > 1000
Ci
mmol
) and
L-[
S]methionine (specific activity >
1000 Ci
mmol
) were supplied by Amersham
International plc. Oligonucleotides were synthesized by Oswel DNA
Service, Edinburgh, United Kingdom. Endoglycosidase H (cloned from
Streptomyces plicatus) was from Boehringer Mannheim. All other
reagents were analytical grade or better and provided by Sigma.
Construction of
Mutants were generated from full-length
-Antitrypsin
Mutants
-antitrypsin cDNA
(12) using the polymerase
chain reaction-based procedure for site-directed mutagenesis described
by Landt et al. (13) . PstI-compatible ends
were added to the 5`- and 3`-flanking primers to facilitate cloning of
the mutated CDNA into the PstI site of SP64T-RCF, a modified
SP64T transcription vector
(14) . Clones were sequenced using
Sequenase reagents (Amersham International plc.) to ensure that the
desired mutation was in place. cDNAs were transcribed in vitro as described previously
(14) using the Promega Ribomax
transcription system.
Secretion from Xenopus Oocytes
The preparation and
micro-injection of Xenopus laevis oocytes were as described by
Colman
(15) . Healthy oocytes were incubated for 7 h in
Barth's saline supplemented with 0.2 mCiml
L-[
S]methionine. At the end of
this period radiolabeling medium was replaced with Barth's saline
containing 10 m
M methionine and the incubation continued
overnight. Oocytes were homogenized and the oocyte extracts and
incubation media immunoprecipitated using anti-human
-antitrypsin (Dako) as described previously
(15, 16) . The products were analyzed on 12.5% (w/v)
SDS-polyacrylamide gels (SDS-PAGE)
(
)
followed by
fluorography using ``Amplify'' (Amersham International plc).
Quantitation of radio-labeled proteins was performed by scintillation
counting of excised gel fragments.
Endoglycosidase H Digestion
immunoadsorbed pellets
were suspended in 60 µl of 50 m
M Tris-HCl, pH 5.5, 1%
(w/v) SDS, 20% (v/v) glycerol, 1% (v/v) 2-mercaptoethanol and incubated
for 5 min at 95 °C. After cooling, 10 µl of 1 milliunit/µl
endoglycosidase H containing 1 m
M phenylmethylsulfonyl
fluoride was added, and samples were digested for 16 h at 37 °C
(17) and then analyzed by SDS-PAGE as described above.
Xenopus Oocyte Processing of the Siiyama
Variant
Expression of PiM-, PiZ-, and
S-encoding RNAs in Xenopus oocytes produced a
54-kDa partially glycosylated intracellular form of the inhibitor
(Fig. 1) and a 56-kDa secreted protein. However, it was evident
from the fluorograph that secretion of normal, M
-antitrypsin was far greater than that of both Z and
S
variants. The estimated molecular mass of secreted
-antitrypsin as reported by various authors, ranges
over 52-58 kDa, depending on the electrophoretic conditions and
molecular mass standards used. Immunoprecipitated protein was treated
with endoglycosidase H
(18) prior to analysis by SDS-PAGE to
determine the intracellular location of the 54-kDa
-antitrypsin seen in oocytes. As shown in
Fig. 1
, the intracellular 54-kDa species was converted to a
single band of molecular mass 46 kDa on endoglycosidase H digestion.
Intracellular PiZ and S
proteins were sensitive to
digestion, indicating that both mutant proteins are retained in a
pre-Golgi compartment, probably the ER. Digestion with the enzyme had
no effect on secreted M, Z, and S
proteins which were
terminally glycosylated.
Figure 1:
Synthesis of
M, Z, and S antitrypsins in Xenopus oocytes
and the endoglycosidase H sensitivity of secreted and retained forms.
Twenty oocytes were injected with messenger RNA for a given antitrypsin
variant and radiolabeled with
L-[
S]methionine. Radiolabeled proteins
were immunoprecipitated from cell extracts and incubation media then
either separated by SDS-PAGE or digested with endoglycosidase H prior
to electrophoresis. O represents oocyte extract and S represents material secreted into the medium. Control oocytes were
injected with an equivalent volume of distilled water. Molecular mass
was determined by the co-migration of standard protein
markers.
To quantify differences in the extent of
secretion of -antitrypsin PiM, PiZ, and S
bands were excised and counted and the experiment repeated with
different batches of oocytes to eliminate, as far as possible, oocyte
variation. Fig. 2( open bars) shows the amount of
inhibitor secreted expressed as a percentage of the total
immunoprecipitable material. S
(13.7% secreted
± 1.2) and Z (10.0% secreted ± 1.0) variants are
similarly retained in oocytes whereas M
-antitrypsin
(63.3% secreted ± 3.9) is more readily secreted from these cells
during the incubation.
Figure 2:
Quantitation of the relative amounts of
Phe
Leu M, Z, and S
antitrypsins
secreted from microinjected oocytes. Bands were excised from gels and
radioactivity determined by liquid scintillation counting. Amounts of
secreted antitrypsin are expressed as a percentage of the total
immunoprecipitable protein. Each column represents the mean of at least
seven different experiments using five different animals. Each
experiment involved the labeling of at least 20 oocytes. Values shown
are expressed as the arithmetic mean ± the standard error of the
mean. Shaded columns denote constructs bearing the Phe
Leu change.
Effect of the Phe
The Phe Leu Mutation on
-Antitrypsin Secretion
Leu mutant was constructed using polymerase chain reaction
mutagenesis of M and Z cDNAs. The double mutant Phe
Leu, Ser
Phe was constructing using M cDNA as
template. The effect of the Phe
Leu mutation on M,
Z, and S
antitrypsin secretion is shown in
Fig. 2
( hatched bars). A 3-fold enhancement of Z
antitrypsin secretion was recorded in the chimer Phe
Leu, Glu
Lys (28.9% secreted ±
4.2). Moreover, the S
Leu
chimer was
secreted (68.9% ±3.1) as efficiently as normal M antitrypsin.
Thus the Phe
Leu mutation fully restores secretion
of the S
variant to that of the normal phenotype and
reduces retention of Z antitrypsin.
Effect of the Loop Hinge Mutants on
The 20-residue reactive
center loop of -Antitrypsin Secretion
-antitrypsin extends 15 residues
(P
P
) amino-terminal and 5 residues
(P
`
P
`) carboxyl-terminal to the
reactive center. The influence of residues P
and
P
(near the base of the loop) on the secretory
properties of M and Z antitrypsins was examined by replacing the normal
residues with those present in the noninhibitory serpin ovalbumin;
namely P
Thr
Arg and P
Ala
Val. Fig. 3displays the extent of secretion of antitrypsin as a
percentage of the total immunoprecipitable protein. M P
Arg
shows a significant reduction in secretion (41.7% ± 2.3)
compared with normal M antitrypsin (62.4% ± 3.1). No significant
difference was observed between Z antitrypsin (12.4% ± 1.3) and
the Z P
Arg double mutant (13.7% ± 1.3). The
secretion defect observed with Z antitrypsin is partially corrected if
the P
alanines are substituted for the larger valine
residues as indicated by the increased secretion of Z
P
Val (25.2% ± 2.7) compared with Z antitrypsin.
Figure 3:
Quantitation of the relative amounts of
PVal and P
Arg loop hinge M, Z, and
S
antitrypsins secreted from microinjected oocytes.
Bands were excised from gels and radioactivity determined by liquid
scintillation counting. Amounts of secreted antitrypsin are expressed
as described in the legend to Fig. 2. Each column represents the mean
of at least four different experiments using oocytes from five
animals.
Secretion-defective
It is now realized that the abnormalities of serpins
associated with deficiency have a common molecular pathology in that
they can spontaneously undergo a conformational transition which
results in partial retention within the endoplasmic pathway and,
particularly with -Antitrypsin
Variants
-antitrypsin, is accompanied by
polymer formation. The liver disease in Z homozygotes is coincident
with the intracellular retention of polymerized inhibitor in inclusion
bodies within the endoplasmic reticulum of the hepatocytes
(2, 19) . Recently another variant,
-antitrypsin S
, was shown to have the
same association with plasma deficiency and the identical histological
finding of hepatic inclusions of mutant inhibitor
(3, 7) . Both the Z and S
mutants have
amino acid substitutions, that although well separated from each other,
will theoretically have the same effect, that is to open the A sheet
between the third and fifth strands and thus promote the process of
loop sheet polymerization
(4) .
-antitrypsin will form long chain polymers with
equal facility
(7) , and Fig. 1demonstrates that they are
secreted from oocytes with comparable efficiency, Z 10.0% and
S
13.7% of immunoprecipitable material. In mammalian
cells only a small proportion of nonsecreted Z protein accumulates in
the ER; the majority is rapidly degraded
(20) . We attempted to
measure the degradation of nonsecreted material in oocytes by
quantifying
-antitrypsin before and after the chase
incubation. We were unable to detect significant proteolysis, mainly
because the slow rate of equilibration of the large intracellular amino
acid pool means that labeling continues well into the chase incubation.
Other experiments following the fate of retained Z protein in oocytes
suggest that little degradation occurs over the time scale of our
experiments
(21) . The synthesis of Z antitrypsin does stimulate
the activity of a number of lysosomal enzymes in injected oocytes
(22) , but this response may not facilitate the removal of
retained protein since, in mammalian cells, degradation has been shown
to occur in a post ER nonlysosomal compartment
(23) . Both Z and
S
variants are retained in a similar, if not
identical, pre-Golgi compartment within the oocyte as indicated by the
pattern of endoglycosidase H digestion. These findings emphasize the
link between loop sheet insertion and the secretory block and suggest
that, as with Z, the plasma deficiency of the S
variant can be explained solely in terms of a failure in protein
export. We have investigated the phenomenon of loop sheet
polymerization by two approaches involving the prevention of entry of
the reactive center loop into the A sheet. First, mutations which close
the gap between strands s3A and s5A and then mutations in the
loop-hinge region which restrict entry into the sheet due to steric
hindrance.
Secretion and Sheet Accessibility
The Smutation (Ser
Phe) is located in the B helix
which underlies the A sheet and provides a surface on which strand s3A
slides in order for the sheet to open
(8) . Substitution with a
large, aromatic side chain at this position is thought to lock the
sheet in the open conformation and thereby promote loop sheet
polymerization
(7, 8) . Recently, Yu and colleagues
(11) have reported that substitution of the Phe residue at
position 51 by a small, nonpolar residue such as leucine enhances the
thermal stability and decreases heat-induced polymerization of wild
type M
-antitrypsin. Here we show that the stabilizing
properties of this change at position 51 have an ameliorating influence
on the Z and S
secretion-defective mutations
(Fig. 2). This effect, however, is not equivalent for both
dysfunctional proteins; secretion of the Z/Phe
Leu
double mutant is increased nearly 3-fold, whereas secretion of
S
/Phe
Leu is equal to M
-antitrypsin (a greater than 5-fold increase). The
increased effect on S
may simply be a matter of
proximity in that removal of Phe
may correct an aberrant
conformation of the B helix, induced by the introduction of a third
contiguous phenylalanine at position 53, and thus allow closure of the
A sheet. The location of the Z mutation, at the hinge of the reactive
center loop, will influence both A sheet opening and loop mobility.
Changes at position 51 are liable to reverse the former but not the
latter and would be predicted to allow only a partial correction of the
Z secretory defect.
Secretion and Loop Mobility
A profound structural
transformation from a stressed S conformation to a more ordered,
heat-stable, and relaxed R state is observed upon reactive center
cleavage of inhibitory serpins. This S to R transition is dependent
upon the insertion of the mobile reactive center loop into sheet
A after cleavage of the P
-P
` peptide bond.
Inappropriate insertion of the intact loop into
sheet A
is a feature of the polymerization of aberrant serpins, but partial insertion of the uncleaved loop into the
sheet is thought to
facilitate formation of the canonical form of the active inhibitor
(24, 25, 26) . The reactive center loops of all
inhibitory serpins are characterized by the conservation of small
hydrophobic amino acids, particularly at positions P
,
P
, and P
at the base of the loop
(27) . These residues are orientated with their side chains
facing the hydrophobic interior of the molecule
(6) , and as a
consequence, there is a constraint on their size and polarity if loop
insertion is to occur. The absence of inhibitory activity and the S to
R transition in ovalbumin and angiotensinogen can be explained by the
appearance of larger and/or more polar residues in these critical
positions
(27) . Similarly, several natural mutants of
antithrombin III, C1-inhibitor, and other serpins have been identified
with point mutations at positions P
and P
,
and in most cases these are proteinase substrates, not inhibitors
(26, 28) . Schulze et al. (29) have
shown that substitution of P
Thr by Arg converts a reactive
center mutant of
-antitrypsin from an inhibitor to a
substrate that fails to undergo a detectable conformational change.
However, a contradictory case has been made by Hood et al. (30) , who constructed a P
Thr
Arg
-antitrypsin which retained the ability to complex
with several cognate proteinases and underwent the S to R transition.
-antitrypsin from oocytes was not
improved by the replacement of P
Thr by arginine. This may
mean that aggregation is unhampered by partial exclusion of strand 4A
from the sheet, alternatively the gap between strands 3A and 5A may be
wider as a result of the lysine residue at position 342 and thus able
to accommodate the larger and more polar arginine side chain. The
assertion that an arginine residue at P
does not prevent
polymerization of the Z variant, and presumably does not materially
affect loop insertion, is in general agreement with the observation
that such a change in the normal inhibitor is compatible with the S
R transition and maintenance of inhibitory function
(30) . Nevertheless, the P
Arg mutation may have
other structural consequences in addition to its effect on loop
mobility, since the secretion of M type
-antitrypsin
P
Thr
Arg was significantly reduced, but not to the
level observed for Z
-antitrypsin.
and P
were more
effective in overcoming the block in secretion imposed by the Z
mutation, causing a 2-fold increase in export of this mutant
-antitrypsin (Fig. 3). Recent results
(28) indicate that insertion up to P
is required
for the release of strand 1 from the C sheet. The presence of valine at
P
, and perhaps P
, will inhibit this insertion
and hence the availability of the S1C position for C sheet
polymerization. Similarly, a naturally occurring mutation of the human
C1-inhibitor with P
Ala
Glu was not an effective
inhibitor and did not undergo the S to R conformational change and also
showed no tendency to polymerize
(26) . Alanine to valine
substitutions at both P
and P
will
consequently serve to hinder this more extensive incorporation of the
loop, although single replacement with threonine at P
does
not prevent M type antitrypsin from undergoing the S to R transition
(31) . Thus an increase in secretion of Z antitrypsin with
valine residues in both the P
and P
positions
is most readily explained by a C sheet mechanism of polymerization.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.