(Received for publication, March 3, 1994; and in revised form, November 17, 1994)
From the
The region COOH-terminal to the reactive center loop is highly
conserved in the serine protease inhibitor (serpin) family. We have
studied the structural consequences of three substitutions (Val
Met, Phe
Ser, and Pro
Ser) found in this region of C1 inhibitor in patients
suffering from hereditary angioedema. Equivalent substitutions have
been described in
1-antitrypsin and antithrombin III. The mutant
C1 inhibitor proteins were only partially secreted upon transient
transfection into COS-7 cells and were found to be dysfunctional.
Immunoprecipitation of conditioned media demonstrated that in the
intact, uncleaved form they all bind to a monoclonal antibody which
recognizes specifically the protease-complexed or reactive
center-cleaved normal C1 inhibitor. A second indication for an
intrinsic conformational change was the increased thermostability
compared to the normal protein. Furthermore, gel filtration studies
showed that the Val
Met and Pro
Ser mutant proteins, and to a lesser extent Phe
Ser, were prone to spontaneous multimerization. Finally, a
reduced susceptibility to reactive center cleavage by trypsin was
observed for all three mutants, and the cleaved Val
Met and Pro
Ser mutants failed to
adopt the conformation recognized by a cleavage-specific monoclonal
antibody. Investigation of plasmas of patients with the Val
Met or Pro
Ser substitutions showed
that these dysfunctional proteins circulate at low levels and are
recognized by the complex-specific antibody. These results strongly
indicate a conformational change as a result of these carboxyl-terminal
substitutions, such that anchoring of the reactive center loop at the
COOH-terminal side is not achieved properly. We propose that this
results in overinsertion of the loop into
-sheet A, which
subsequently leads to multimerization.
C1 inhibitor is a member of the serine protease inhibitor
(serpin) ()family. The inhibitory members of the serpins in
human plasma are important regulators of the coagulation, fibrinolytic
and complement cascades, and of proteases released from activated
neutrophils (see (1, 2, 3) for reviews).
They share a common structure, and the exact structural and kinetic
mechanism of inhibition is the subject of considerable investigation
and continuing debate. Attempted proteolytic attack on the so-called
P1-P1` peptidyl-bond (Schechter and Berger nomenclature, (4) )
in the reactive center results in the formation of very stable
complexes between inhibitor and target protease. The reactive center is
located in the COOH-terminal part on an exposed loop(5) , which
upon complete reactive center cleavage recedes into the central
-sheet A as the fourth of six strands(6) . This structural
transition induces an irreversible change to a more stable form, the
so-called S
R transition, which can be monitored by an increase
in thermostability (5) , conformation-specific monoclonal
antibodies(7, 8) , CD spectra, and resistance to
unfolding in denaturants(9) . The ability of the reactive
center loop to incorporate is an essential requirement for inhibitory
function. Experimental evidence for the proposal (10) that
complex formation of a serpin with target proteases involves partial
insertion of the loop into
-sheet A has been obtained recently for
antithrombin III(11) .
Studies with serpin mutants have
focused on the P14-P10 hinge region at the base of the loop, which is
highly conserved and contains predominantly alanines in inhibitory
serpins(12) . Substitutions are found in this region in the
non-inhibitory serpins ovalbumin and angiotensinogen (13) and
also in dysfunctional mutants of antithrombin III (ATIII) and C1
inhibitor in patients with thrombosis (14, 15) and
hereditary angioedema (HAE) type II(10, 16) ,
respectively. Mutations at P14 or P12, which were presumed to obstruct
correct fitting into underlying structures during incorporation of the
loop (10) , turn the serpin into a substrate for target
proteases. Recent evidence (17) indicates that such mutations
may not necessarily prevent loop incorporation, but cause a shift in
the branched reaction pathway (18) from inhibitory to
substrate-type reaction. The effect of mutations at P10 can be diverse;
an Ala Ser substitution in ATIII results in dysfunction toward
thrombin while factor Xa is still inhibited(19) . An engineered
P10 Ala
Pro mutation in
1-antitrypsin (
1-AT) does also
result in substrate-type behavior, but again it does not prevent the S
R transition, and consequently, insertion of the
loop(20) . Finally, a C1 inhibitor P10 Ala
Thr mutant
does not inhibit, nor is cleaved by, target proteases C1s, kallikrein
and factor XIIa(16) . This mutant was recently shown to be in a
conformation recognized by a complex-specific monoclonal antibody and
to be prone to multimer formation(21) .
The potential of
serpins to form multimers by non-covalent intermolecular loop to sheet
bonding has been recognized recently. This phenomenon occurs upon
heating in intact, normal serpins (22) and is in fact
responsible for the observed thermal ``instability,'' as the
multimers escape immunological detection (see e.g.(23) ), or are removed upon centrifugation.
Multimerization can also occur after mild treatment with denaturants (24, 25) or after cleavage at P8 or P9 in
1-AT(22) . Furthermore, multimerization of intact
1-AT occurs in certain mutants at 37 °C, notably the common
1-AT-Z variant with a Glu
Lys mutation at the
turn leading into strand 4A(25) , and in the rare Siiyama
(Ser
Phe) and Malton (Phe
deleted)
variants(26) . The latter mutations are located in or near
helix B, which underlies the central
-sheet A. The affected
residues were predicted to play a role in opening of the A-sheet (27) by providing a ridge along which sheet 3A can slide.
The present study is concerned with three substitutions in the
region COOH-terminal to the reactive site in C1 inhibitor; Val
Met, Phe
Ser, and Pro
Ser. These residues are equivalent to Phe
,
Phe
, and Pro
in
1-AT, respectively (12) . The substitutions were identified in a screening for
mutations in exon 8 of the C1 inhibitor gene in HAE patients and are
located at generally highly conserved residues in serpins. Similar
mutations at equivalent positions have been found in
1-AT and
ATIII(28, 29, 30, 31) . In general,
they affect secretion of the mutant protein, and this was also found
for the three C1 inhibitor mutants, as illustrated by the reduced
levels of C1 inhibitor in the patients plasma. The observation that
they are, however, secreted to a certain extent in vitro(32) allowed investigation of the structural consequences
of each substitution. Evidence will be presented that, although they
are not in the direct vicinity of the reactive center loop or other
parts proposed to be involved in conformational changes(27) ,
these substitutions affect loop insertion and also favor
multimerization.
Gel filtration
was performed on TSK G3000 SW and G4000SW columns (TosoHaas,
Montgomeryville, PA) coupled to an Applied Biosystems HPLC system
(Warrington, U.K.). The buffer used was phosphate-buffered saline, pH
7.4 (PBS), 0.02% (v/v) Tween-20, 0.02% (w/v) NaN, and the
flow rate was 0.5 ml/min. Marker proteins were thyroglobulin (667 kDa),
apoferritin (443 kDa),
-amylase (200 kDa), albumin (68 kDa), and
ovalbumin (46 kDa); the void volume was monitored with dextran blue
(>2000 kDa). Unlabeled transfection media were concentrated
10-20-fold by repeated centrifugation (4500
g)
at 4 °C in Centricon-10 devices (Amicon, Beverly, MA) and stored at
-20 °C. Concentration was necessary to obtain a measurable
response in ELISA upon gel filtration. When analyzing recombinant C1
inhibitor, 50 µl of wild-type (rC1 inh-wt) concentrates was
injected onto the gel filtration column, and 100 µl of recombinant
C1 inhibitor mutants. With whole plasma, 10 µl of normal plasma and
25 µl of HAE plasma were diluted to 100 µl in running buffer
and injected. Collection of fractions was started at 9 min after
injection, just before elution of the void volume. Fractions were
collected with an E-6000c dropcounter/collector (Ismatec,
Zürich, Switzerland) and were stored in microtiter
plates at 4 °C until testing the following day.
ELISAs using mAb
RII or KOK12 as catching antibody were done as described(38) .
Briefly, microtiter plates were coated with 2 µg/ml mAb in PBS
overnight at 4 °C. All subsequent incubations were done at room
temperature. Blocking was done with 3% (w/v) bovine serum albumin in
PBS for 30-45 min. Aliquots of HPLC fractions up to 100 µl
were incubated for 1.5 h followed by four washes with PBS, 0.1% (v/v)
Tween-20. Detection of adsorbed C1 inhibitor antigen was with
biotinylated rabbit polyclonal anti-C1 inhibitor antibodies (1 h),
followed by a 30-min incubation with streptavidin-coupled horseradish
peroxidase (Amersham), both diluted 1:1000 in PBS, 0.1% (v/v) Tween-20,
0.2% (w/v) gelatin. Development was with 3,3`,5,5`-tetramethylbenzidine
and reactions were stopped after 5 to 10 min with 2 M HSO
.
Figure 1:
Incubation of recombinant C1 inhibitor
mutants with C1s and immunoprecipitation with mAb KOK12.
[S]Methionine-labeled transfection media were
incubated with or without C1s (1.5-2 µg/ml final
concentration) for 2.5 h at 37 °C. PMSF was then added to a final
concentration of 5 mM and, after preclearing, specific
immunoprecipitation was done with mAb KOK12-Sepharose. After washing
and heating at 95 °C for 3 min, samples were subjected to SDS-PAGE
on 7.5% gels under non-reducing conditions. Recombinant C1 inhibitor
wild-type (wt) or mutant species are indicated at the top of
the figure. Lane M contains
C marker proteins,
and molecular mass is indicated in kDa on the left. Df denotes
the dye front. The lanes containing rF455S derive from a separate
experiment and are relatively overexposed to visualize the complex with
C1s.
Figure 2:
Thermostability of recombinant C1
inhibitor species. Aliquots of transfection media (100 µl of rC1
inh-F455S, 50 µl of other C1 inhibitor species) were incubated at
the indicated temperatures for 2 h. After centrifugation at 16,000
g for 20 min, total C1 inhibitor antigen in the
supernatant was determined by ELISA using mAb RII as catching antibody.
Results are expressed as percentage antigen remaining in solution
relative to a sample incubated at 37 °C. Symbols represent normal
C1 inh (wt, circles), rV451M (plus), rP476S (squares), and rF455S (triangles).
Figure 5:
Gel filtration and native PAGE of
heat-induced multimers of purified plasma C1 inhibitor. Purified plasma
C1 inhibitor in PBS was incubated at 50 or 55 °C for 1 h at a
concentration of 10 or 1 mg/ml. Aliquots were subsequently injected
onto the G4000SW column (A), or subjected to native PAGE on
4-15% gradient gels (B). Gel filtration profile of
samples containing 20 µg of C1 inhibitor protein incubated at 55
°C at original concentration of 10 mg/ml is indicated by a broken line, at 1 mg/ml by a stippled line, and of 1
mg/ml C1 inhibitor incubated at 37 °C by an unbroken line.
The scale on the left indicates minutes after injection. Elution times
(in min) of markers during a preceding run were as follows: dextran
blue, 10.0; thyroglobulin, 15.07; apoferritin, 16.9; -amylase,
18.5; ovalbumin, 20.26. Samples subjected to native PAGE contained 4
µg of C1 inhibitor protein and were visualized by Coomassie
staining. Lane 1, 1 mg/ml C1 inhibitor incubated at 37 °C. Lane 2, 1 mg/ml C1 inhibitor incubated at 50 °C. Lane
3, 1 mg/ml C1 inhibitor incubated at 55 °C. Lane 4,
10 mg/ml C1 inhibitor incubated at 55 °C. Lane M contains
marker proteins.
Figure 3:
Gel filtration of recombinant C1 inhibitor
species on G3000SW column. Transfection media were concentrated 15-fold
(rC1 inh-wt, rV451M, and rP476S) or 10-fold (rF455S) by
ultrafiltration, and separated over a TSK G3000SW column at a flow rate
of 0.5 ml/min. Fractions of 4 drops (82-85 µl; one fraction
equivalent to 10 s) were collected, starting at 9 min after
injection. After addition of 20 µl of PBS, 0.1% (v/v) Tween-20, 100
µl was tested in ELISA for total C1 inhibitor antigen using mAb RII (A), and subsequently in ELISA with the complex-specific mAb
KOK12 (B). Position of the void volume (>667 kDa) and
elution of marker proteins is indicated at the top. rC1 inh-wt eluted
as a single peak at the same position as the purified plasma protein
(not shown for clarity, see also Fig. 4). Response of rP476S is
indicated by closed squares, rV451M by plus symbols,
and rF455S by closed triangles. Color reaction was
overdeveloped, which caused high responses to reach a maximum and
display significant tailing.
Figure 4: Gel filtration of recombinant C1 inhibitor species on G4000SW column. Transfection media were concentrated 15-fold (rC1 inh-wt and rP476S), or 20-fold (rF455S), and separated over a G4000SW column. Fractions of 12 drops (250-260 µl; one fractions equalled approximately 31 s) were collected, and tested in RII-ELISA (A), or KOK12-ELISA (B). Fractions of rC1 inh-wt were tested at a 1:4 dilution in a 100 µl volume, and rC1 inh mutants were tested at 100 µl undiluted. The position of the void volume (>2000 kDa) and elution of marker proteins is indicated at the top. Response of rC1 inh-wt is indicated by open circles, rP476S by closed squares, and rF455S by closed triangles.
Attempts to
purify and visualize S-labeled C1 inh mutants by native
gel electrophoresis in separate monomeric and multimeric forms were
unsuccessful, due to the low amounts of these secretion deficient
proteins available, and, more importantly, their tendency to
multimerize during manipulation. Therefore, heat-induced multimers of
purified plasma C1 inhibitor were used as a reference to estimate the
degree of multimerization. Upon incubation for 1 h at 55 °C at a
concentration of 10 mg/ml, purified plasma protein eluted from the
G4000SW column mainly at the void volume position (
2000 kDa), and
at 1 mg/ml a broad multimer peak at 1000-2000 kDa position was
obtained (Fig. 5A). The majority of multimers obtained
at 10 mg/ml C1 inhibitor were too large to enter the separating gel
when subjected to native PAGE (Fig. 5B, lane
4). Based on the migration of marker proteins, the smallest C1
inhibitor multimer that was clearly visible appeared to be a trimer.
The size of the multimers obtained at 1 mg/ml appeared to range from 4
to 10 C1 inhibitor molecules (Fig. 5B, lane
3), and these eluted from the gel filtration column with a broad
peak at 12.07 min. It could be deduced, therefore, that the average
size of the mutant rC1 inhibitor multimers is smaller than this because
they eluted at approximately 13.5 min (see Fig. 4, fraction
9).
Figure 6:
Quantitative comparison of rC1 inhibitor
species in RII and KOK12 ELISA. Fresh transfection media were tested in
2-fold serial dilutions in ELISA with mAb RII () or mAb KOK12
(
). To ensure identical conditions, the top half of the
microtiter plates was coated with RII, and the bottom half with KOK12.
The outcome of a similar experiment where Tween-20 was omitted from the
media or the sample incubation step of the ELISA was identical (not
shown).
Figure 7:
Incubation of recombinant C1 inhibitor
mutants with trypsin and immunoprecipitation with mAbs RII and KII.
Metabolically labeled transfection media were incubated with or without
trypsin at a final concentration of 0.05 or 5 µg/ml for 2 h at 37
°C. SBTI and APMSF were then added to final concentrations of 10
µg/ml and 100 µM, respectively. After preclearing,
samples were split in two and immunoprecipitated with mAb RII-Sepharose (top panel) or mAb KII-Sepharose (bottom panel). C1
inhibitor species and trypsin concentrations are indicated at the top.
Additional bands in lane 10, top panel, apart from
the C1 inhibitor band at 104 kDa, are C marker proteins
accidentally carried over from the marker lane M at the
left.
Figure 8: Gel filtration of normal and HAE plasma. Plasma samples were injected onto the G4000SW column immediately after thawing (A and B) or after incubation at 37 °C for 1 h (C). 48 fractions of 125-130 µl (one fraction equivalent to 15-15.5 s) were collected and tested in 1:5 dilution in the RII-ELISA (A and C), or KOK12-ELISA (B). Results with plasma from a healthy member of family 30 are indicated by open circles, a HAE patient from family 30 (P476S substitution) by closed squares, and a HAE patient from family 10 (V451M) by plus symbols. Note that color development in the different assays was not arrested at identical times, and responses in the different panels cannot quantitatively be compared.
Of the approximately 30 residues forming the COOH-terminal
region in serpins, consisting of s1C, s4B, and s5B (P362-P391 in
1-AT), 17 are strictly or highly conserved in the entire protein
family(12) . F455 and P476 in C1 inhibitor correspond to
residues F370 and P391 in
1-AT and are strictly conserved, V451 is
less well conserved and corresponds to F366 in
1AT and an alanine
in ovalbumin. These residues are located in close proximity in the
three-dimensional structure, forming a hydrophobic stack in the core of
the protein (see Fig. 9, and below). The importance of this
region has already been inferred from studies on stop- and frameshift
mutations in
1-AT(50, 51) , and substitutions in
ATIII(29, 31) , which prevent or significantly reduce
secretion. More recently, clues that substitutions in this region
produce conformational changes emerged from studies with ATIII
mutants(30, 52) . However, a direct role for the
conserved COOH terminus of serpins in determining reactive center loop
conformation or in inducing multimerization has not been proposed
before. The somewhat unexpected findings reported here with three
different C1 inhibitor mutants may provide further insight into the
nature of the structural changes involved.
Figure 9:
Local environment of the substituted
residues. Depicted is a C-trace of the COOH-terminal region of
cleaved
1-AT(6) , showing predominantly
-sheet B in
the plane of the picture and
-sheet C in perpendicular view to the
left. The side chains and van der Waals surfaces of the residues which
correspond with the substitutions in C1 inhibitor studied here are
shown (F366, F370, and P391), as are the side chains of F208 and V388
which contribute to the conserved hydrophobic pocket. The position of
the P1` residue S359 is indicated. For clarity,
-sheet A and
helices A, B, and D are removed. Coordinates of cleaved
1-AT were
obtained from the Protein Data Bank (entry 7API) and were visualized
with InsightII software (Biosym, San Diego,
CA).
The results with
conformation-specific mAbs and the thermostability assay are strong
indications for an intrinsic conformational change in the C1 inhibitor
mutants. When taken separately, neither binding to mAb KOK12 nor the
increase in thermostability might be considered unequivocal proof for
loop overinsertion, since unknown changes affecting other regions of
the protein could in principle result in the same properties. The
combination of these two observations, however, is highly suggestive of
insertion of the reactive center loop as the underlying event, since it
has been postulated and experimentally demonstrated that loop insertion
is linked to both complex formation and increased
stability(10, 11, 22, 40, 53) .
Similarly, the presence of high molecular weight material in the C1
inhibitor mutant preparations could be explained by nonspecific
aggregation resulting from misfolding and subsequent exposure of
hydrophobic regions, rather than by the more ordered process of loop to
sheet multimerization. Such misfolded proteins are, however, unlikely
to be secreted(54) , and aggregated material would not be
expected to be limited in size, as was observed with these C1 inhibitor
mutants. Based on these considerations, and current concepts of serpin
structure and function (2, 3, 11) , we favor
the explanation that the substitutions in C1 inhibitor cause
overinsertion of the reactive center loop, which subsequently renders
the protein prone to intermolecular multimerization. Conceivably, the
equivalent substitutions which have been found in ATIII and 1-AT
mutants (28, 29, 30, 31) may have
the same effect. In support of this is the decreased heparin affinity
of several ATIII mutants(31) , which is also observed when the
reactive center loop is inserted(53) .
Since the mutant C1
inhibitor proteins tended to multimerize, it could be put forward that
intrinsic binding to mAb KOK12 and the increased thermostability are
properties that merely reflect the presence of pre-existing multimers.
The available data argue against this and instead point to a direct
relationship between loop overinsertion and multimer formation. First,
intrinsic binding to mAb KOK12 is an important indicator of a
conformation resembling the complexed state. The notion of a
conformational change during complex formation which involves a
partially inserted loop is at the center of current models of serpin
structure and function(10, 11) . Second, recognition
by mAb KOK12 of the C1 inhibitor mutants preceded multimerization, as
demonstrated by the monomer fractions on gel filtration of both
recombinant and natural C1 inhibitor mutants (Fig. 3, Fig. 4, and Fig. 8). Furthermore, in ELISA and
immunoprecipitation experiments of fresh media, binding to mAb KOK12 is
virtually quantitative, and even a fraction of native normal rC1
inhibitor, which does not spontaneously multimerize, can be detected.
Lastly, no mechanism other than loop or exogenous peptide insertion
into -sheet A is known at present which would explain the increase
in thermostability (Fig. 2). The concept of thermal instability
as solely representing a structural disintegration at elevated
temperatures has been refined recently. In the case of
1-AT, it is
now known that as temperature increases, the process of multimerization
itself occurs simultaneously with a loss of detectable
antigen(25) . Significantly,
1-AT-Z (E342K), which
spontaneously multimerizes at 37 °C, has a similar thermal
transition point as the normal
1-AT-M(25) , indicating
that pre-existing multimers do not obligatory alter the
thermostability. Therefore, a shift in stability profile most probably
results from an intrinsic conformational change which is in principle
independent of multimerization. How this change might lead to
multimerization, and the difference between these multimers and
heat-induced multimers of the normal protein, is addressed below in
more detail.
The C1 inhibitor mutant proteins were predominantly
retained intracellularly. Pulse-chase experiments demonstrated that
rV451M and rP476S accumulated in the cell during an 8-h
chase(32) , but that the intracellular fraction of rF455S was
degraded. Clearly, correct folding of C1 inhibitor is
dependent on the affected residues, and this is achieved only when this
cluster of conserved residues is able to anchor the reactive center
loop at the COOH-terminal side, and to counteract the tendency of the
loop to insert into
-sheet A. The secreted mutant proteins
apparently are liable to loop insertion, resulting in exposure of the
KOK12 epitope. The overinsertion of the loop would subsequently cause
weakening of the bonds between sheet 3A and 5A, which in turn renders
the molecule prone to intermolecular loop to sheet
polymerization(21, 25) . This suggested mechanism of
multimer formation is slightly different than that proposed for the
1-AT-Z and Siiyama (S53F) mutants(25, 26) , for
which locking of
-sheet A in an open state has been postulated to
be the primary event.
The recent elucidation of the crystallographic
structure of intact ATIII by two separate groups (39, 55) has provided yet another mechanism by which
serpins may multimerize. Both ATIII structures consist of dimers of an
intact inhibitory molecule of which part of the reactive center loop
(P3-P7) replaces sheet 1C of either a cleaved (55) or a latent (39) second ATIII molecule. As discussed in more detail in (39) , this novel mode of loop-sheet 1C bonding may contribute
to the process of multimerization, instead of exclusive loop-sheet A
bonding. Moreover, we postulate that loop-sheet 1C bonding may play a
role in the C1 inhibitor mutants described here, since these probably
already have weakened tethering of sheet 1C to the body of the molecule
as a result of the substitutions. This novel mechanism also explains
the puzzling observation that heating of normal C1 inhibitor yields
very large multimers (see Fig. 5), while certain mutants
spontaneously form multimers of a distinctly smaller size. In addition,
with two C1 inhibitor mutants tested, rP476S (this study) as well as
the A436T mutant reported earlier(21) , multimers are formed
that do not grow larger upon heating. Indeed, the C1
inhibitor mutants display the predicted small size (dimer-tetramer) of
multimers formed by loop-sheet 1C bonding.
A partial view of the
C-backbone of cleaved
1-AT, with the region surrounding the
residues which are substituted in C1 inhibitor highlighted, is shown in Fig. 9. The local structure in intact ovalbumin is essentially
identical. It is evident that the affected residues are part of a
hydrophobic pocket, which is highly conserved among the serpins. The
three substitutions studied here most probably disrupt formation of
this pocket. Other conserved residues which are likely to contribute to
the local structural integrity are F208 in s4C, and V388 in
s5B(12) . Interestingly, several of the residues involved
(F208, F370, and P391) have also been implicated in the transition to
the latent form in plasminogen activator inhibitor-1
(PAI-1)(56) . In addition, we note that although the residue
corresponding to F366 in
1-AT is generally phenylalanine or an
aliphatic residue, PAI-1 is the only serpin with a methionine at this
position, as in the case of the C1 inh-V451M mutant studied here. It
may therefore be possible that the typical features of latent PAI-1, i.e. almost complete loop insertion, pivoting of s1C around
F370, and distortion of s3C and s4C(56) , also play a role in
the conformational changes observed in the C1 inhibitor mutants. Such
structural features would also agree with the occurrence of loop-sheet
1C bonding mentioned above.
The mutant C1 inhibitor proteins were
all three quantitatively immunoprecipitated by mAb KOK12 and had
moderately increased thermostability in the intact form. With respect
to complex formation with C1s and binding to mAb KII, however, rF455S
behaved differently from rV451M and rP476S and instead resembled the
normal protein to a certain extent. Binding to mAb KII upon trypsin
cleavage is linked to the structural transition of complete loop
incorporation into sheet A. Since trypsin-cleaved mutants rV451M and
rP476S were unable to bind to mAb KII (Fig. 7), this indicates
that complete loop insertion is somehow perturbed. This observation
implies that subtle structural differences exist between C1 inhibitor
F455S and the other two mutants. One possibility for such differences
might be the configuration of -sheet A, where strands 3 and 5
normally should be able to separate completely in order to accommodate
the reactive center loop as strand 4 upon cleavage.
Investigation of
plasmas of two HAE families demonstrated that in vivo the C1
inhibitor V451M and P476S mutant proteins are also partially secreted.
Again, this is similar to findings with ATIII and 1-AT, where low
levels of circulating dysfunctional mutants are sometimes
found(31, 50) . Thus, the phenotype of HAE in these C1
inhibitor-deficient patients falls between the classically defined type
I and II(47) . Accurate quantification of both functional and
antigenic C1 inhibitor levels, in combination with the telltale binding
to mAb KOK12, may lead to early recognition of this phenotype in future
screening. The C1 inhibitor mutant proteins appeared to circulate in
monomeric form in plasma, but multimerized upon incubation at 37 °C in vitro. This was also found for
1-AT-Z, where slow
multimerization of the purified protein from a homozygous patient took
place in vitro at 37 °C(24, 25) . In the
case of C1 inhibitor, the absence of detectable multimers in
circulation might be due to the presence of the normal protein, other
factors that may be limiting, or increased clearance of multimers. In
addition, the tendency of the recombinant C1 inhibitor mutants to
multimerize could possibly be increased compared to the plasma protein,
due to the addition of Tween-20 to the transfection media. Non-ionic
detergents like Nonidet P-40 and Tween-20 increase multimerization in
normal plasma
1-AT (22) and C1 inhibitor.
Multimers can however sometimes be observed directly in plasma,
as in the case of
1-AT-Siiyama (26) and C1 inhibitor
A436T(21) .
In conclusion, we have shown that substitutions at conserved COOH-terminal positions in C1 inhibitor result in a protein configuration which is increasingly recognized among members of the serpin family. Since mutations leading to overinsertion of the loop and/or multimerization can apparently be located in diverse regions in serpins (helix B, the turn between s5A and sS4A, the reactive center loop itself, and the extreme COOH terminus), it may be expected that novel sites which produce this configuration will be identified.