From the
The Z (Glu
Comparison of the results with
similar findings of short chain polymers associated with the
antithrombin variant Rouen VI (Bruce, D., Perry, D., Borg, J.-Y.,
Carrell, R. W., and Wardell, M. R.(1994) J. Clin. Invest. 94,
2265-2274) suggests that polymerization is more complicated than
the mechanism proposed earlier. The Z, Siiyama, and Mmalton mutations
favor a conformational change in the antitrypsin molecule to an
intermediate between the native and latent forms. This would involve a
partial overinsertion of the reactive loop into the A sheet with
displacement of strand 1C and consequent loop-C sheet polymerization.
It is clear that the sequence of events
involved in proteinase inhibition requires the loop to be mobile and to
adopt a variety of conformations. A consequence of this is the
formation of the so-called ``latent'' conformation (by
analogy with the corresponding reactivable form of plasminogen
activator inhibitor-1(6) ), in which the intact loop is fully
incorporated into and stabilizes the A sheet (6, 9, 22). A second
consequence is the formation of loop-sheet polymers in which the
reactive center loop of one molecule is inserted into a
Here we examine a third antitrypsin deficiency variant,
Mmalton (Phe
Antitrypsin Mmalton was isolated from 130 ml of plasma and
eluted from the Q-Sepharose anion-exchange column as two distinct
peaks: one at a sodium chloride concentration similar to that of M and
Z antitrypsin (38) (denoted fraction 1) and the other at a
sodium chloride concentration similar to that of antitrypsin Siiyama (25) (denoted fraction 2). The protein in each of these
fractions was characterized separately. The antitrypsin in fraction 1
migrated as a 52-kDa band on SDS-PAGE (Fig. 1), but protein
estimation from the extinction coefficient resulted in underloading
when compared with M and Z antitrypsin controls. Amino acid analysis
confirmed the extinction coefficient (
We have previously shown that Z antitrypsin forms
intracellular inclusions in the liver of Z homozygotes by a unique
protein-protein interaction, loop-sheet polymerization(24) .
Similar loop-sheet polymers have been isolated from the plasma of an
individual who is a homozygote for the Siiyama mutation(25) .
This mutation, Ser
Antitrypsin Mmalton was isolated from the
plasma of an Mmalton/QO (null) bolton heterozygote as two distinct
peaks that represented monomer and polymer. Antitrypsin Mmalton
polymers were inactive as an inhibitor of bovine
The clue to the process limiting the
length of Mmalton polymers in vivo was provided by the finding
that a proportion of the molecules in the polymers isolated from plasma
had been cleaved at the reactive center (Fig. 1). This finding is
in keeping with a mechanism in which polymerization of antitrypsin
Mmalton occurs as a result of a preceding transition to the locking
conformation that is normally adopted to stabilize the
proteinase-inhibitor complex(47) . The combination of a
partially exposed reactive center loop together with a released strand
from the C sheet will allow the formation of sequential loop-sheet
linkages to give polymerization. In vitro, and presumably in
the protected environment of the endoplasmic pathway, this sequential
linkage will allow the formation of long chain polymers as shown in Fig. 5. However, within the plasma, the presence of extraneous
proteinases explains the truncation of polymerization by cleavage of
the exposed loop of the terminal molecule. The apparent absence of the
C-terminal peptide from the reactive loop-cleaved component of the
plasma polymers is both unexpected and intriguing (Fig. 2). This
peptide is usually tightly bound to the body of the molecule of serpins
cleaved in vitro and can only be dissociated by strong
denaturing conditions. The absence of the peptide from the cleaved
component of the Mmalton polymers implies that dissociation may follow
cleavage of the locking conformation in which strand 1C release has
occurred.
As compared with the Z and Siiyama variants, antitrypsin
Mmalton is relatively stable. The majority of antitrypsin Mmalton
present in plasma is in the monomeric form, has essentially normal
inhibitory kinetics, and forms SDS-stable complexes with bovine
We thank Dr. L. Packman (Department of Biochemistry,
University of Cambridge) for peptide synthesis, amino acid analysis,
and N-terminal sequence analysis. We are indebted to the donor, T. P.,
who has consistently supported our research.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Lys) and Siiyama (Ser
Phe) deficiency variants of
-antitrypsin
result in the retention of protein in the endoplasmic reticulum of the
hepatocyte by loop-sheet polymerization in which the reactive center
loop of one molecule is inserted into a
-pleated sheet of a
second. We show here that antitrypsin Mmalton
(Phe
-deleted), which is associated with the same liver
inclusions, is also retained at an endoglycosidase H-sensitive stage of
processing in the Xenopus oocyte and spontaneously forms
polymers in vivo. These polymers, obtained from the plasma of
an Mmalton/QO (null) bolton heterozygote, were much shorter than other
antitrypsin polymers and contained a reactive center loop-cleaved
species. Monomeric mutant antitrypsin was also isolated from the
plasma. The monomeric component had a normal unfolding transition on
transverse urea gradient gel electrophoresis and formed polymers in
vitro more readily than M, but less readily than Z, antitrypsin.
The A
-sheet accommodated a reactive center loop peptide much less
readily than Z antitrypsin, which in turn was less receptive than
native M antitrypsin. The nonreceptive conformation of the A sheet in
antitrypsin Mmalton had little effect on kinetic parameters, the
formation of SDS-stable complexes, the S to R transition, and the
formation of the latent conformation.
-Antitrypsin (
-proteinase
inhibitor) is a 52-kDa 394-amino acid glycoprotein and the archetypal
member of the serpin
(
)superfamily(1) .
Members of this family each have a unique inhibitory specificity, but
share a similar molecular structure based on a dominant five-strand A
-sheet(2, 3, 4, 5, 6, 7, 8, 9, 10) .
The intact inhibitor has an exposed reactive center loop with the
P
-P
` residues
(
)acting as
``peptide bait'' for the cognate
proteinase(11, 12) . The precise conformation of the
reactive center loop in the active inhibitor when it docks with an
enzyme is unknown, but is likely to lie between the three-turn helical
conformation of ovalbumin (3, 10) and the canonical
structure of the small inhibitors(13, 14) . For serpins
to adopt a canonical conformation, the helical reactive center loop
must unfold with its N-terminal stalk inserted into the gap between
strands 3 and 5 of the A sheet(15, 16) . After docking
with the enzyme, the loop is presumed to insert further into the A
sheet to form an extended canonical structure that fits the
substrate-binding site of the cognate proteinase, the locking
conformation(17) . Dissociation of the enzyme-inhibitor complex
may result in the release of the intact serpin (18) or cleavage
of the loop at the P
-P
` residues(19) .
Reactive center loop cleavage results in a profound conformational
rearrangement in which the loop is fully inserted into the A sheet of
the molecule (2, 20, 21).
-pleated
sheet of a second. This interaction accounts for the biologically
important retention of Z (23, 24) and Siiyama (25) antitrypsin in the endoplasmic reticulum of the liver and
hence the accompanying plasma deficiency. Polymerization of Z
antitrypsin is blocked by annealing homologue reactive loop peptides to
the A sheet (24), and on this basis, it was reasonably deduced that
loop-sheet polymerization occurs due to the insertion of the loop of
one molecule into the A sheet of the next. The phenomenon of loop-sheet
polymerization is not restricted to antitrypsin and has now been
reported in plasminogen activator inhibitor-2 (26) and in
association with point mutations in
C1-inhibitor(27, 28) ,
-antichymotrypsin(29) , and
antithrombin(30) . Indeed, recent structural findings with
antithrombin (9, 30, 31) suggest an alternative
mechanism involving interaction between the loop of one molecule and
the C sheet of the next. The occurrence of C sheet-linked polymers of
antithrombin raised the likelihood that this is a general mechanism and
that antitrypsin also forms polymers by a C, rather than an A, sheet
interaction.
-deleted(32, 33, 34) ),
which gives the same liver inclusions (33, 35) and
plasma aggregates as Z (23) and Siiyama (25) antitrypsin.
The aggregates from plasma have been characterized and shown to have
the properties of loop-sheet polymers. The physical properties of these
polymers and the associated monomeric antitrypsin Mmalton, together
with data from the Z and Siiyama variants described
previously(24, 25) , fit convincingly with the C sheet,
rather than A sheet, mechanism of loop-sheet polymerization.
Study Patient
The donor was a 45-year-old male
who is a lifelong nonsmoker and has had no evidence of liver or lung
disease over a follow-up period of 18 years. He is the father of the
propositus, who had previously been identified as PI-type MmaltonZ
(36). DNA sequence analysis and isoelectric focusing confirmed him to
be an Mmalton/QO (null) bolton heterozygote(32, 37) ;
and therefore, all the circulating antitrypsin must be derived from the
Mmalton allele.
Purification of M, Z, and Mmalton Antitrypsin
M,
Z, and Mmalton antitrypsin were purified from 200, 250, and 130 ml of
plasma, respectively, by 50 and 75% ammonium sulfate fractionation
followed by thiol exchange and Q-Sepharose chromatography as detailed
previously (25, 38). The protein was stored in 50 mM Tris, 50
mM KCl, pH 7.4, with 0.1% (v/v) -mercaptoethanol, and
total protein was determined from the absorbance at 280 nm (39) and amino acid analysis. Purity was assessed by
7.5-15% (w/v) SDS-PAGE with the antitrypsin band being detected
by Western blot analysis (40) and amino-terminal sequencing
following transfer onto Problot membrane(41) . Mmalton polymers
were further purified by antitrypsin antibody affinity chromatography.
Bound antitrypsin was eluted with 0.2 M glycine, pH 2.5, and
collected onto 1 g of solid Tris to restore physiological pH. The Tris
was removed by concentration/dialysis into 50 mM Tris, 50
mM KCl, pH 7.4, in an Amicon concentrating cell.
Assessment of Antitrypsin Conformations
SDS,
nondenaturing, and transverse urea gradient polyacrylamide gel
electrophoresis; electron microscopy; heat stability assays; the
assessment of the rate of insertion of a synthetic reactive center loop
peptide (Ac-Ser-Glu-Ala-Ala-Ala-Ser-Thr-Ala-Val-Val-Ile) into the A
sheet of antitrypsin; and limited proteolysis of the reactive center
loop of M and Mmalton antitrypsin with Staphylococcus aureus V8 proteinase were performed as detailed previously(22) .
Limited proteolysis of the reactive center loop of antitrypsin with
papaya proteinase IV (Calbiochem-Novobiochem Ltd., Nottingham, United
Kingdom) was achieved by incubation in 30 mM potassium
phosphate, 30 mM NaCl, pH 7.0, for 2 h at 37 °C.
C-terminal peptides were separated by reverse-phase high performance
liquid chromatography (HPLC) on a PLRP-s column as described by
Pemberton et al.(42) .
Determination of the Association Kinetics of Antitrypsin
Mmalton with Bovine
The active-site values of M, Z, and Mmalton antitrypsin
were determined against bovine -Chymotrypsin and Human Neutrophil
Elastase
-chymotrypsin as described
previously(38) . Kinetic parameters for the interaction of
antitrypsin Mmalton with bovine
-chymotrypsin and human neutrophil
elastase were determined at 37 °C in the presence of substrate by
analyzing the progress curves for the formation of p-nitroanilide(38) .
Preparation of Latent M and Mmalton
Antitrypsin
Latent antitrypsin was prepared by incubating
isolated antitrypsin (final concentration of 0.2 mg/ml) at 67 °C
for 12 h in 20 mM Tris, pH 7.4, and 0.7 M sodium
citrate as described previously(22) . The citrate was removed by
dialysis against 20 mM Tris, pH 8.6, and the antitrypsin was
concentrated to 1.0 mg/ml. This material was then heated at 60 °C
for 3 h to convert any residual active antitrypsin to
polymer(38) . The presence of residual thermostable latent
antitrypsin was detected by nondenaturing PAGE and a concomitant loss
of inhibitory activity against bovine -chymotrypsin(22) .
Assessment of Antitrypsin Secretion from the Xenopus
Oocyte
The construction of Z and Mmalton mutants, in vitro transcription, and assessment of secretion from Xenopus oocytes were performed as described by Sidhar et
al.(43) .
)
of antitrypsin Mmalton to be 11.1 rather than 5.3 (39) for M
antitrypsin. Amino-terminal sequencing following transfer onto Problot
membrane showed that antitrypsin Mmalton in the early eluting peak of
fraction 1 was cleaved at the amino terminus (
DAAQK-),
whereas that of the main peak was intact (
EDPQG-) as
described previously for Z antitrypsin(38) . Fraction 2 migrated
as multiple bands on SDS-PAGE, and Western blot analysis revealed the
antitrypsin to be present as 52- and 48-kDa species (Fig. 1);
both were intact at the N terminus (
EDPQG-), and the 48-kDa
species had the same electrophoretic mobility as reactive center
loop-cleaved M antitrypsin. The cleaved protein was detected in all
four purifications of antitrypsin Mmalton, in Western blot analysis of
the starting material, and following antitrypsin antibody
immunopurification of fraction 2 (Fig. 2). The C-terminal peptide
liberated following reactive loop cleavage was not detected by SDS-PAGE
of fraction 2 (Fig. 2) or by reverse-phase HPLC. The specific
activities of fractions 1 and 2 were 66 and <1%, respectively, when
the total protein in fraction 1 was determined by amino acid analysis.
The value for fraction 1 compares with 77% for M antitrypsin and 50%
for Z antitrypsin purified using the same protocol; the reduced
activity of Z compared with M antitrypsin reflects contaminating
loop-sheet dimers.
Figure 1:
Left panel, 7.5-15% (w/v)
SDS-PAGE of Mmalton/QO (null) bolton fractions eluted from the
Q-Sepharose anion-exchange column. Lane1, molecular
mass markers (in kilodaltons); lane2, M antitrypsin
(5 µg); lane3, antitrypsin Mmalton fraction 1 (5
µg); lane4, antitrypsin Mmalton fraction 2 (20
µg); lane5, M antitrypsin cleaved at the
reactive center loop with S. aureus V8 proteinase (5 µg); lane6, M antitrypsin (5 µg). Middle
panel, Western blot for antitrypsin of 7.5-15% (w/v)
SDS-PAGE of Mmalton/QO (null) bolton fractions eluted from the
Q-Sepharose anion-exchange column. Lane1, M
antitrypsin (5 µg); lane2, antitrypsin Mmalton
fraction 1 (5 µg); lane3, antitrypsin Mmalton
fraction 2 (20 µg); lane4, M antitrypsin cleaved
at the reactive center loop with S. aureus V8 proteinase (5
µg); lane5, M antitrypsin (5 µg). Right
panel, Western blot for antitrypsin of 7.5-15% (w/v)
nondenaturing PAGE of Mmalton/QO (null) bolton fractions eluted from
the Q-Sepharose anion-exchange column. Lane1, M
antitrypsin (5 µg); lane2, antitrypsin Mmalton
fraction 1 (5 µg); lane3, antitrypsin Mmalton
fraction 2 (20 µg); lane4, M antitrypsin (5
µg).
Figure 2:
A,
7.5-15% (w/v) SDS-PAGE of Mmalton polymers and M antitrypsin
following immunopurification. Lane 1, molecular mass markers
(in kilodaltons); lane2, M antitrypsin (5 µg); lane3, M antitrypsin control eluted from the
antitrypsin affinity column; lane4, antitrypsin
Mmalton polymers (fraction 2) eluted from the antitrypsin affinity
column; lane5, M antitrypsin cleaved at the reactive
loop with S. aureus V8 proteinase (5 µg). Arrows represent native (N) and reactive center loop-cleaved (C) antitrypsin. Both bands were shown to be antitrypsin by
Western blot analysis (data not shown). B, overloaded
7.5-15% (w/v) SDS-PAGE to assess the C-terminal peptide from
antitrypsin Mmalton polymers (fraction 2) formed in vivo. Lane1, molecular mass markers (in kilodaltons); lane2, M antitrypsin (5 µg); lanes3 and 4, M antitrypsin cleaved at the reactive
loop with S. aureus V8 proteinase (5 and 10 µg,
respectively); lanes5-7, antitrypsin Mmalton
polymers (20, 40, and 60 µg, respectively). peptide shows
the electrophoretic mobility of the 4-kDa C-terminal peptide from
reactive loop-cleaved antitrypsin.
Western blot imaging of antitrypsin on
nondenaturing PAGE (Fig. 1) showed fraction 1 to be monomeric,
while fraction 2 contained high molecular mass polymers that were
present in Western blot analysis of starting plasma. As fraction 2
contains no monomeric antitrypsin, the reactive center loop-cleaved
antitrypsin must be an integral component of the polymers. The
conformation and stability of antitrypsin in these two fractions were
assessed by unfolding on transverse urea gradient gel electrophoresis.
Monomeric antitrypsin Mmalton (fraction 1) unfolded with a profile
similar to that of native M and Z antitrypsin (Fig. 3). M and
Mmalton antitrypsin cleaved at the reactive center loop with S.
aureus V8 proteinase failed to unfold in 8 M urea,
indicating that the A -sheet had been stabilized by insertion of
the reactive loop peptide. Similarly, M and Mmalton antitrypsin
polymers formed by heating at 60 °C and antitrypsin Mmalton
polymers isolated from the plasma were resistant to unfolding in 8 M urea. The antitrypsin Mmalton polymers isolated from the
plasma migrated farther into the gel than M and Mmalton antitrypsin
polymers formed by heating at 60 °C. This reflects the
nondenaturing PAGE and electron micrograph observations (Fig. 4)
that circulating antitrypsin Mmalton polymers are shorter and hence of
lower molecular mass than M and Mmalton antitrypsin polymers formed in vitro.
Figure 3:
7.5% (w/v) transverse urea gradient gel
electrophoresis of M, Z, and Mmalton antitrypsin monomers and polymers.
All lanes contained 50 µg of protein; the left of each gel
represents 0 M urea, and the right represents 8 M urea. Toprowleft, native M
antitrypsin; toprowright, native Z
antitrypsin; secondrowleft, native
antitrypsin Mmalton monomer; secondrowright, M antitrypsin cleaved at the reactive center loop
with S. aureus V8 proteinase; thirdrowleft, antitrypsin Mmalton cleaved at the reactive center
loop with S. aureus V8 proteinase; thirdrowright, M antitrypsin polymers formed by heating M
antitrypsin (0.2 mg/ml) at 60 °C for 3 h; bottomrowleft, antitrypsin Mmalton polymers formed by heating
antitrypsin Mmalton (0.2 mg/ml) at 60 °C for 3 h; bottomrowright, Western blot for antitrypsin of
Mmalton polymers isolated from plasma.
Figure 4:
Electron micrographs of antitrypsin
Mmalton monomers (upper panel) and polymers (middle
panel) isolated from plasma. Shown in the lower panel,
for comparative purposes, are M antitrypsin polymers formed by heating
M antitrypsin (0.25 mg/ml) at 65 °C for 12 h. All preparations were
stained with 1% (w/v) uranyl acetate. As antitrypsin Mmalton polymers
isolated from plasma were only partially pure, the polymers were shown
to be antitrypsin by the attachment of polyclonal antitrypsin
antibodies, which were then visualized by negative staining and
electron microscopy (data not shown).
Isolated monomeric antitrypsin Mmalton
polymerized less readily than Z antitrypsin, but more readily than M
antitrypsin, after heating at 0.6 mg/ml and 40 °C (Fig. 5).
The resulting polymers showed similar electrophoretic mobility to Z
antitrypsin polymers formed under identical conditions. Monomeric
antitrypsin Mmalton accepted an 11-mer antithrombin reactive center
loop peptide at a significantly slower rate than M or Z antitrypsin (t for M antitrypsin = 30-60 min, for Z
antitrypsin = 2 h, and for Mmalton = 24 h) (Fig. 6). This implies that the Phe deletion closes
or distorts the gap between strands 3 and 5 of the A sheet or that the
reactive center loop adopts a conformation that hinders exogenous
peptide insertion.
Figure 5:
7.5-15% (w/v) nondenaturing PAGE
showing the polymerization of M (upper panel), Mmalton (middle panel), and Z (lower panel) antitrypsin (0.6
mg/ml) in 50 mM Tris, 50 mM KCl, pH 7.4, at 40
°C. All lanes contained 10 µg of protein. Lane1, 0 h; lane2, 12 h; lane3, 24 h; lane4, 48 h; lane5, 72 h; lane6, 6 days; lane7, 12 days.
Figure 6:
7.5-15% (w/v) nondenaturing PAGE
showing the rate of insertion of an 11-mer antithrombin reactive center
loop peptide into M, Z, and Mmalton antitrypsin. Antitrypsin (0.6 mg/ml
in storage buffer) was incubated with an equal volume of a 100-fold
molar excess of peptide at 37 °C. Shown are M (upperpanel), Z (middle panel), and Mmalton (lower
panel) antitrypsin at the following time points: lane1, 0 h; lane2, 0.5 h; lane3, 1 h; lane4, 2 h; lane5, 4 h; lane6, 12 h; lane7, 24 h; lane8, 48 h; lane9, 72 h. Each lane contained 5 µg of protein. N, native M, Z, or Mmalton antitrypsin; BC, protein
containing the synthetic reactive center loop
peptide.
The inhibitory conformation requires the partial
insertion of the loop into the A
sheet(14, 15, 16) , and it is plausible that
distortion of this domain may perturb the loop and so affect the
inhibitory kinetics with serine proteinases. Antitrypsin Mmalton had an
essentially normal association rate constant and K value with human neutrophil elastase (2.9 ± 0.03
10
M
s
and <5 pM (Fig. 7A), respectively,
compared with 5.3 ± 0.06
10
M
s
and <5
pM for M antitrypsin(38) ) and with bovine
-chymotrypsin (3.0 ± 0.02
10
M
s
and <5
pM, respectively, compared with 2.6 ± 0.2
10
M
s
and
9.2 ± 2.7 pM for M antitrypsin(38) ) and was
able to form SDS-stable complexes (Fig. 7B). Limited
proteolysis of the reactive loop with S. aureus V8 proteinase
(which cleaves at P
-P
) and papaya proteinase IV
(which cleaves at P
-P
) showed no differences
between Mmalton and native M antitrypsin (data not shown).
Figure 7:
A,
progress curve showing antitrypsin Mmalton inhibition of human
neutrophil elastase. The reaction was started with 155 pM elastase, and the curves represent 0 (), 66 (
), 132
(
), 197 (▾), 263 (
), and 329 (
) pM antitrypsin Mmalton. B, M and Mmalton antitrypsin complex
formation with bovine
-chymotrypsin. The enzyme and inhibitor were
incubated together in 50% (v/v) reaction buffer at room temperature for
15 min before heating at 95 °C for 2 min and separating by
7.5-15% SDS-PAGE. All lanes contained 5 µg of protein, and
ratios are based on activity. Molecular mass markers (in kilodaltons)
are shown on the left. Lane1, M antitrypsin; lanes2-4, M antitrypsin:chymotrypsin ratios of
1:0.5, 1:1 and 1:2, respectively; lane5, Mmalton
antitrypsin monomer; lanes 6-8, antitrypsin Mmalton
monomer:chymotrypsin ratios of 1:0.5, 1:1, and 1:2,
respectively.
Distortion of the A sheet did not prevent the insertion of the
reactive center loop peptide after cleavage as antitrypsin Mmalton
monomer was able to undergo the S to R (active to cleaved) transition (Fig. 8). More specifically, antitrypsin Mmalton was induced to
polymerize upon heating at temperatures in excess of 60 °C, and
this was prevented by the insertion of the reactive loop peptide into
the A sheet. Similarly, heating both M and Mmalton antitrypsin at 67
°C for 12 h in 0.7 M sodium citrate produced a
thermostable monomeric species (data not shown) that we have recently
characterized as the latent conformation(22) .
Figure 8:
Heat stability of native () and
reactive center loop-cleaved (
) antitrypsin Mmalton (0.2 mg/ml).
The xaxis represents increasing temperature, and the yaxis represents the percentage of antigen remaining
in the solution after filtration measured against the value at 30
°C. The assay was performed in 20 mM Tris, pH 7.4, and all
points are the average of duplicate values.
In vitro studies demonstrated that purified Z antitrypsin was able to
polymerize more readily than antitrypsin Mmalton, which in turn
polymerized more readily than M antitrypsin. To confirm this
observation in vivo and to show that antitrypsin Mmalton is
retained at the same point of processing as Z antitrypsin, these
mutants, along with an M antitrypsin control, were expressed in Xenopus oocytes. M antitrypsin secreted from the oocytes was
65 ± 7.6% (S.E.) of the total synthesized antitrypsin. Both Z
and Mmalton antitrypsin had reduced secretion of 11.1 ± 3.3 and
19.8 ± 3.1%, respectively. The experiments were performed on at
least four occasions for each variant, and on each occasion, the
secretion was the sum of 20 injected oocytes. The retained Z and
Mmalton antitrypsin were sensitive to digestion with endoglycosidase H (Fig. 9), localizing them to a pre-Golgi compartment, most likely
the endoplasmic reticulum.
Figure 9:
12.5% (w/v) SDS-PAGE showing the digestion
of intracellular and secreted forms of antitrypsin with endoglycosidase
H. Samples 1-3 are oocyte extracts from M, Z, and
Mmalton antitrypsin, respectively, with (+) and without (-)
endoglycosidase H (Endo H) digestion. Samples 4-6 are the corresponding secreted forms of the inhibitor with and
without enzyme.
Phe, is located in the B helix
and similarly results in the formation of hepatic
inclusions(44) . The only other identified mutation that
predisposes to the formation of inclusions is Phe
deletion, which has been variously named Mmalton (33, 34, 37),
Mnichinan(45) , and Mcagliari(46) . We demonstrate here
that as predicted(24) , this point mutation also favors the
formation of loop-sheet polymers. Furthermore, several associated
findings provide strong support for the proposal (9, 28, 30, 31) that the polymerization
occurs through the linking of the reactive loop of one molecule to the
C, rather than the A, sheet of the next. Specifically, the findings are
compatible with the occurrence of a spontaneous and inappropriate
change in conformation of the variant antitrypsin toward that normally
adopted by the inhibitor in the stable complex with the
proteinase(47) . The structure of this ``locking''
conformation has not been determined, but there is evidence to support
the conclusion (47) that it involves a partial reincorporation of the
loop (48) together with release of strand 1C of the
molecule(28) .
-chymotrypsin and
were resistant to unfolding on transverse 0-8 M urea
gradient gels. These data support our hypothesis that polymers result
from the linkage of the reactive loop of one molecule with the
-pleated sheet of a second. Such an interaction obscures the
scissile P
-P
` bond of the reactive center
loop, rendering the protein inactive, but also stabilizes the A sheet
of the receptor molecule. Antitrypsin Mmalton formed long chain
polymers in vitro (Fig. 5), but the polymers isolated
from plasma were predominantly only 2-5 molecules in length (Fig. 1). This differed from the plasma findings in the Siiyama
homozygote, in which the polymers were formed by 15-20 molecules
of antitrypsin Siiyama (25). The findings do, however, resemble those
recently described for a variant of antithrombin, Rouen VI Asn
Asp(30) , which forms short chain polymers. These
polymers were truncated at 2-3 units by the complete conversion
of the terminal molecule to the latent form. We initially thought that
antitrypsin Mmalton might similarly undergo a spontaneous transition to
the latent form and hence result in short polymers. Although it was
shown that the variant antitrypsin could be induced into the latent
form, this conformation was not detected in either the monomeric or
polymeric fraction from plasma.
-chymotrypsin. Moreover, Mmalton has the same limited proteolysis
profile as antitrypsin following digestion with bovine
-chymotrypsin (P
-P
`), S. aureus V8 proteinase (P
-P
), and papaya proteinase
IV (P
-P
). This compares with Z antitrypsin,
which has modified inhibitory kinetics (38, 49, 50) and is not susceptible to cleavage
at P
-P
(38) . Monomeric antitrypsin
Mmalton forms long chain polymers on incubation in vitro at a
faster rate than M antitrypsin, but at a slower rate than Z antitrypsin (Fig. 5). All of these findings fit with a common molecular
pathology that explains the accumulation of these three variants of
antitrypsin within the endoplasmic reticulum of the liver. We believe
the shared abnormality is a tendency for overinsertion of the reactive
loop in the inhibitor molecule to give a spontaneous conformational
change with release of strand 1C. The consequent formation of loop-C
sheet-linked polymers will vary in rate depending on the nature of the
underlying mutation. Polymerization is more marked in the Siiyama
variant, in which all the plasma antitrypsin is
polymerized(25) , and less so in the Z variant, which is
predominantly present in plasma as the monomer, although the
inaccessibility of its P
-P
bond to
proteolysis infers partial insertion of its loop(38) . Mmalton
circulates in a virtually normal functional form, but its decreased
rate of formation of binary complexes (Fig. 6) indicates that its
own loop is likely to be overinserted and to inhibit homologue peptide
insertion. The difference in severity of the molecular lesions of the
two antitrypsin variants is confirmed by the reduced secretion of Z as
compared with Mmalton in the Xenopus oocyte expression system.
Z and Mmalton, but not M, antitrypsin were retained within the oocyte
at a pre-Golgi stage most likely to be in the endoplasmic reticulum.
Interestingly, the secretion of antitrypsin Mmalton was almost twice
that of Z antitrypsin, reflecting the reduced rate of forming
loop-sheet polymers. These and other
results(9, 30, 31) , taken together with the
observations of Eldering et al.(28) , favor the
conclusion that mutations that produce hepatic inclusions lead to
overinsertion of the reactive loop and a likely C sheet mechanism of
polymerization, although they do not exclude the alternative
possibility of a loop-A sheet interaction.
-P
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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.