Furin is a ubiquitous prototypical mammalian
kexin/subtilisin-like endoproteinase that is involved in the
proteolytic processing of a variety of proteins in the exocytic and
endocytic pathways, with cleavage occurring at the C terminus of the
minimal consensus furin recognition sequence Arg-Xaa-Xaa-Arg. In this
study, human proteinase inhibitor 8 (PI8), a widely expressed 45-kDa
ovalbumin-type serpin that contains two sequences homologous to the
minimal sequence for recognition by furin in its reactive site loop,
was tested for its ability to inhibit a recombinant soluble form of
human furin. PI8 formed an SDS-stable complex with furin and inhibited its amidolytic activity via a two-step mechanism with a
kassoc of 6.5 × 105
M
1 s
1 and an overall
Ki of 53.8 pM. Thus, PI8 inhibits furin in a rapid, tight binding manner that is characteristic of
physiological serpin-proteinase interactions. PI8 is not only the first
human ovalbumin-type serpin to demonstrate inhibitory activity toward furin, but it is also the first significant inhibitor of furin identified that is not a serpin reactive site loop mutant, either naturally occurring or engineered.
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INTRODUCTION |
The mammalian serine proteinase inhibitors, or serpins, are a
superfamily of proteins that regulate proteolytic events in a wide
variety of physiological processes including but not limited to blood
coagulation, viral and parasite pathogenicity, intracellular proteolysis, and tumor suppression (1). Serpins inhibit their target
proteinases by forming a 1:1 stoichiometric complex with the active
site of the proteinase, which is in most cases resistant to denaturants
(2). Serpins are composed of three
-sheets surrounded by eight
-helices and a reactive site domain that is highly divergent among
serpin family members and exists as a stressed loop with a canonical
conformation that confers the optimal conformation for high affinity
association with the substrate binding cleft of the cognate proteinase
(3-6). The
P1-P1
1
residues of the reactive site domain determine the inhibitory specificity of the serpin and act as a pseudosubstrate for the target
proteinase (6). Unlike a typical substrate, the serpin has the ability
to form a tight complex that may be essential for inactivation of the
proteinase (4, 7-9). Most serpins can interact with more than one
proteinase in vitro, but the affinity of such interactions
must be determined to suggest physiological relevance.
Ovalbumin represents the parent prototype of a unique family of serpins
whose members include plasminogen activator inhibitor-2 (PAI-2),2 an elastase
inhibitor isolated from monocyte-like cells, squamous cell carcinoma
antigen (SCCA), maspin, proteinase inhibitor (PI) 6, PI8, PI9, bomapin,
and SCCA2 (10, 11). PI6, PI8, and PI9 are unique among the mammalian
ovalbumin-type serpins in that they contain a cysteine residue in the
P1
position within the reactive site domain, which is also
present in the viral serpin CrmA (12). Ovalbumin-type serpins lack a
typical cleavable N-terminal signal sequence but have been found to
reside intracellularly (13, 14) or both intracellularly and
extracellularly (15-18). Therefore, it can be inferred that the
functions of members of the ovalbumin family of serpins may not be
strictly confined to the cytoplasm. It has been demonstrated that
individual mammalian ovalbumin-type serpins can inhibit a variety of
prototypic serine proteinases by distinctly different mechanisms using
a variety of kinetic parameters (19). Although most members of the
ovalbumin family of serpins exhibit defined proteinase inhibitory
activity, the true physiological targets of these serpins have not yet
been identified.
PI8 is a 45-kDa serpin that contains the sequence
Arg336-Asn337-Ser338-Arg339
at the P4-P1 positions in the reactive site
domain, as well as the sequence
Arg339-Cys340-Ser341-Arg342
at the P1-P3
positions, both of which conform
to the minimal sequence required for efficient processing by furin,
Arg-Xaa-Xaa-Arg (20). Additionally, PI8 was recently demonstrated to be
a potent inhibitor of the Bacillus subtilis dibasic
endoproteinase subtilisin A (21). A number of mammalian convertases
have been identified that demonstrate a high degree of functional and
structural similarity to yeast kexin and bacterial subtilisin, of which
furin is the prototype (reviewed in Ref. 22). Furin is a ubiquitously
expressed, membrane-associated, calcium-dependent serine
endoproteinase that cleaves a wide variety of precursor proteins in
both the exocytic and endocytic pathways. Furin cleaves at the C
terminus of Arg-Xaa-Xaa-Arg motifs and is involved in the proteolytic
processing of the von Willebrand factor precursor, pro-factor IX
precursor, the low density lipoprotein receptor-related protein,
pro-
-nerve growth factor, viral superantigens, and diphtheria toxin
(22). In addition, several viral coat proteins, including influenza
virus hemagglutinin, measles virus fusion protein, and HIV-1 gp160
(22), are cleaved and activated by furin, a process crucial in the
establishment of viral infectivity. In the present study, we
demonstrate that PI8 inhibits furin in a rapid, tight binding manner
that is characteristic of physiological serpin-proteinase
interactions.
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EXPERIMENTAL PROCEDURES |
General Kinetic Methods--
Recombinant human furin was
prepared from a vaccinia virus construct (20). Recombinant human PI8
was prepared as described (21). The Km for furin and
the fluorogenic substrate Pyr-Arg-Thr-Lys-Arg-4-methylcoumaryl-7-amide (pERTKR-MCA, Bachem) as
well as the catalytically active concentration of furin were determined
as described previously (23). The Km for furin and
pERTKR-MCA was determined to be 3.2 µM. Active
site-titrated furin was used to determine the amount of PI8 needed for
a 1:1 molar binding stoichiometry for the determination of kinetic
constants. Furin (1.25 nM) was mixed with increasing
amounts of PI8 in a total volume of 180 µl in 100 mM
HEPES (pH 7.5) containing 0.5% Triton X-100 and 1 mM
CaCl2. Reactants were incubated for 30 min at 37 °C, and
pERTKR-MCA was added to a final concentration of 50 µM.
The enzymatically released 7-amido-4-methylcoumarin was then detected
at 25 °C using an SLM Instruments SLM-8000 spectrofluorimeter with
an excitation wavelength of 370 nm and an emission wavelength of 460 nm. The data were used to plot the enzymatic rate of substrate hydrolysis against the amount of PI8 used in the reaction. Linear regression to the x axis was used to calculate the amount of
PI8 required for a 1:1 molar binding stoichiometry with furin.
Slow Binding Inhibition Kinetics--
Inhibition progress curves
were obtained under pseudo-first order conditions by incubating the
reactants in 0.3 ml of the same buffer used for the titration of PI8.
Reactions were started by the addition of enzyme to a solution
containing the fluorogenic substrate and the appropriate inhibitor
concentration. Reactions for each experiment were started within
30 s, and the enzymatic production of 7-amido-4-methylcoumarin was
detected as described earlier. The final concentrations of the
reactants were 2 nM furin, 100 µM pERTKR-MCA,
and 4, 8, 12, and 16 nM PI8. Spontaneous substrate hydrolysis was measured in separate experiments and determined to be
negligible. The reactions were allowed to proceed until steady-state
velocity was attained, and the data were fitted to the integrated rate
equation for slow binding inhibition (24)
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(Eq. 1)
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by nonlinear regression using UltraFit 3.0 software (Biosoft) to
obtain values for the initial velocity (vo), the
steady-state velocity (vs), the initial
fluorescence (Ao), and the apparent first order
rate constant (k
) for the establishment of steady-state equilibrium of the proteinase-inhibitor complex. The data obtained from
nonlinear regression analysis were then used in various graphical transformations (25-29) to obtain the inhibition and rate constants for the interactions of PI8 with recombinant human furin.
Detection of SDS-Stable Furin-PI8 Complexes--
Antibodies
against recombinant human PI8 were generated in rabbits (30), and the
IgG fraction was purified by protein A-Sepharose column chromatography.
Furin and PI8 were incubated for 15 min at 37 °C, and the reaction
mixtures were subsequently subjected to 10% SDS-PAGE under reducing
conditions (31) and electrophoretically transferred to a nitrocellulose
membrane in 10 mM CAPS (pH 11) buffer containing 10%
methanol. The membrane was blocked with 1% nonfat dry milk in
Tris-buffered saline containing 0.02% azide, and complexes were
detected by incubating the membranes with rabbit anti-PI8 IgG, followed
by incubation with 125I-labeled protein A and
autoradiography.
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RESULTS |
Inhibition of Human Furin by PI8--
Preliminary studies
indicated that the interaction between furin and PI8 obeyed slow
binding inhibition kinetics, as the amidolytic activity of furin
inhibited by PI8 attained steady-state equilibrium and the data were
successfully fitted to Equation 1. On average, ten PI8 molecules were
required to form a stable inhibitory complex with one molecule of
furin, as determined by titration. The kinetic characterization of the
inhibition of furin by PI8 was performed using PI8 concentrations
ranging from two to eight times the molar concentration of furin. A
family of inhibition progress curves representative of the interaction
between furin and PI8 at the chosen PI8 concentrations is shown in Fig.
1. As expected, steady-state equilibrium
was achieved more readily as the concentration of PI8 in the reaction
mixture increased. Data obtained from the inhibition progress curves
were fitted to Equation 1 by nonlinear regression analysis, and the
results indicated that the initial velocity, vo,
was inversely proportional to the concentration of PI8 for each set of
progress curves. This suggests that the slow onset of the inhibition of
furin by PI8 follows the two-step mechanism
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where a loose proteinase-inhibitor (PI) complex is rapidly formed,
followed by a slow isomerization to the tight PI
complex (25). This
observation was confirmed by plotting
vmax/vo against the PI8
concentration, which indicated a linear relationship with a positive
slope (data not shown). The Ki for the formation of
the initial loose complex was calculated from the slope of the line
using the relationship
vmax/vo = Km[I]/[S]Ki + (1 + Km/[S]), and was estimated to be 7.2 ± 1.2 nM (n = 4). In addition, the apparent first
order rate constant k
was found to increase as PI8
concentration increases, which is consistent with the proposed
mechanism.

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Fig. 1.
Progress curves from slow binding kinetics
for the inhibition of human furin by PI8. Furin (2 nM)
was reacted with 0, 4, 8, 12, and 16 nM PI8 in 100 mM HEPES (pH 7.5)/0.5% Triton X-100/1 mM
CaCl2 at 25 °C in the presence of 0.1 mM
pERTKR-MCA. The reactions were monitored continuously for 5 h, and
the data were fitted to Equation 1 to generate values for the variables vo, vs,
Ao, and k . To negate any effects of
substrate depletion, the upper limit of product formation used to
determine the steady-state rates was within the linear range of the
uninhibited furin control.
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The overall second order association rate constant
kassoc was determined by plotting
log([P]
[P]t) as a function of time,
where [P]
= vo/k
and [P]t is the fluorescence measured at various times
between 0 and 20 min for individual progress curves (data not shown)
(28). The slope of the lines obtained is equal to
0.43[I]{kassoc/(1 + [S]/Km)}, from which
kassoc was calculated to be 6.5 ± 0.1 × 105 M
1 s
1
(n = 4). The overall inhibition constant
Ki
was determined from plots of both
vmax/vs and (vo
vs)/vs
versus PI8 concentration (data not shown). The slopes
of these plots are equal to
Km/[S]Ki
, from which
Ki
was calculated to be 53.8 ± 10.4 pM (n = 4).
To determine the rate constant for the reverse isomerization step
k
2 of the furin-PI8 tight complex, a plot of
k
against vo/vs was generated.
This plot was linear (data not shown), and k
2
was calculated directly from the slope of the line to be 2.5 ± 0.3 × 10
5 s
1 (n = 4).
Using the relationship t1/2 = 0.693/k
2, a half-life of 7.7 h was
estimated for the reverse isomerization of the tight complex to the
loose complex. The value of the rate constant for the formation of the
tight complex k2 was determined by fitting a
plot of k
versus PI8 concentration (data not
shown) to the hyperbolic equation (24)
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(Eq. 2)
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by nonlinear regression analysis. Using this method,
k2 was estimated to be 3.3 ± 0.2 × 10
3 s
1 (n = 4). To verify
that the interaction of furin and PI8 occurs by the suggested mechanism
and to justify the use of a hyperbola to describe the relationship
between k
and [PI8], a double-reciprocal plot of
1/(k
k
2) versus
1/[PI8] was generated (Fig. 2) using
the values obtained from Equations 1 and 2 that is linear and crosses
the positive y axis at a point approximately equal to
1/k2 for the mechanism suggested earlier (29).
The y intercept of the plot in Fig. 2 was used to determine
a value for k2 of 2.1 ± 0.8 × 10
3 s
1 (n = 4), which is
reasonably close to the value of k2 determined by Equation 2. More importantly, the plot in Fig. 2 justifies the
manipulation of data and determination of kinetic constants according
to the suggested mechanism.

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Fig. 2.
1/(k -k 2)
versus 1/[PI8] for the interaction of PI8 with human
furin. Values for k were generated as described in the
legend to Fig. 1. The value of k 2 was
determined from a plot of k against
vo/vs. The line crosses the
positive y axis at a point approximately equal to
1/k2, as described for a two-step binding
mechanism.
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Formation of a PI8-Furin SDS-Stable Complex--
Complex formation
between furin and PI8 was visualized by Western blotting using rabbit
anti-human PI8 IgG. As shown in Fig. 3,
incubation of furin with PI8 resulted in the formation of an SDS-stable
complex that migrated with an apparent molecular mass of ~225 kDa
following reduction with 2-mercaptoethanol. In the absence of reducing
agent, the complex migrated with an apparent molecular mass of ~200
kDa (data not shown). Additionally, no complex was observed following
incubation of furin with a PI8 preparation that had been previously
heat denatured (data not shown). The formation of a tight, SDS-stable
complex between furin and PI8 is also consistent with the inhibition
mechanism described earlier.

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Fig. 3.
SDS-stable complex formation between human
furin and PI8. Complexes were allowed to form, subsequently
boiled, reduced, and subjected to 10% SDS-PAGE and immunoblotting with
rabbit anti-human PI8 IgG. Lane 1, furin; lane 2,
furin + PI8; lane 3, PI8.
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DISCUSSION |
In the present study, we have performed a detailed kinetic
analysis of the inactivation of human furin by PI8. PI8 inhibited furin
via a two-step mechanism characterized by the rapid formation of an
initial loose complex followed by a slow isomerization to a tight,
stable complex that was visualized by SDS-PAGE followed by Western
blotting. The apparent molecular mass of the complex was approximately
225 kDa, which is significantly higher that the predicted mass of
~140 kDa. Although the reason for the anomalously high molecular mass
of the complex is unknown and will require further investigation, the
aberrant migration may arise either from aggregation of furin-PI8
complexes or incomplete denaturation of the complex by SDS. It is
unlikely, however, that this apparently higher molecular mass occurs as
a result of an alternative binding stoichiometry, because furin has
only one catalytic center and PI8 has only one reactive site loop to
facilitate the interaction. A second immunoreactive species migrating
at approximately 42 kDa was seen in lane 2 of Fig. 3, which
most likely represents PI8 cleaved as a result of the interaction with
furin. The overall inhibition constant for the inactivation of furin by
PI8 was 53.8 pM, indicating that PI8 is a potent inhibitor
of this proteinase. The initial loose complex of furin and PI8 had a
Ki of 7.2 nM, which is similar to the
Ki values of 8 and 6.6 nM for the
inhibition of plasmin and chymotrypsin by
2-antiplasmin, respectively (26). The furin-PI8 loose complex is converted to the
tight complex at a rate of 3.3 × 10
3
s
1, which is comparable with the rates reported for
chymotrypsin-
2-antiplasmin and
plasmin-
2-antiplasmin complexes (26). In addition, the kassoc for furin and PI8 was determined to be
6.5 × 105 M
1
s
1, which was lower than the rate of inhibition of
subtilisin A by PI8 (1.2 × 106
M
1 s
1) (21) but exceeded the
rates of inhibition of plasma kallikrein by C1-inhibitor (6.9 × 104 M
1 s
1) (32) and
human thrombin and coagulation factor Xa by PI8 (1.0 × 105 M
1 s
1 and
7.5 × 104 M
1
s
1, respectively) (21), as well as the rate of inhibition
of granzyme B by CrmA (2.9 × 105
M
1 s
1) (33). These comparisons
indicate that the kinetic constants for the inhibition of furin by PI8
are of physiological significance. Furthermore, PI8 is the only
ovalbumin-type serpin, as well as the only naturally occurring
intracellular human serpin not associated with a disease state,
demonstrated to be a significant inhibitor of furin. Previously, only
peptide chloromethylketones and an
1-antitrypsin
(
1-AT) variant have been described as significant inhibitors of furin.
1-AT Portland is an engineered
variant of
1-AT Pittsburgh (34) that carries an
additional Ala355
Arg mutation in its reactive site
domain to provide the minimal consensus sequence for efficient
recognition and processing by furin (35). PI8 contains the sequence
Arg336-Asn337-Ser338-Arg339
at the P4-P1 positions in the reactive site
domain that, based upon sequence alignment, is presumably recognized by
the substrate binding cleft of furin in this interaction.
Interestingly, PI8 contains a second sequence
Arg339-Cys340-Ser341-Arg342
at the P1-P3
positions in the reactive site
domain, which also may be involved in the interaction of PI8 with furin
or another mammalian convertase. The precise sequence in the PI8
reactive site domain involved in the interaction between furin and PI8 is presently unknown.
In order for PI8 to inhibit furin in vivo, PI8 must
presumably enter the secretory pathway. The ovalbumin-type serpins
PAI-2, SCCA, and maspin each lack a cleavable N-terminal signal
sequence, but all can be found extracellularly. PAI-2 contains two
hydrophobic regions proximal to the N terminus, designated as
H1 and H2, that are involved in its secretion
through facultative polypeptide translocation. H2 is also
homologous to the uncleaved secretion signal in ovalbumin (17). PAI-2
constructs carrying a deleted H1 or H2 region
transfected into Chinese hamster ovary cells demonstrated decreased
glycosylation and secretion, whereas mutants displaying increased
hydrophobicity in the H1 or H2 regions
exhibited increased glycosylation and secretion. PI8 shares 62 and 65%
amino acid sequence identity with the H1 and H2
regions in PAI-2, respectively (Fig. 4).
However, if amino acid mismatches are allowed to be resolved by the
presence of a hydrophobic amino acid in the PI8 sequence, as shown by
an asterisk in Fig. 4, PI8 shares 69 and 100% identity with
the H1 and H2 regions of PAI-2, respectively. These hydrophobic regions near the N terminus in PI8 may permit secretion of PI8 in a manner analogous to PAI-2 and, ultimately, the
interaction of furin and PI8. Potential PI8 secretion may also be
regulated at the level of transcription. In the case of PAI-2, both the
cytosolic and secreted forms are encoded by a single mRNA as seen
by Northern blot analysis (17). However, a Northern blot of
poly(A)+ mRNA from a wide variety of human tissues
revealed two PI8 transcripts of 3.8 and 1.4 kilobases (12). In this
connection, yeast invertase and human gelsolin are two proteins that
each have cytoplasmic and secreted forms. The cytoplasmic and secreted
forms of invertase are encoded by two mRNA forms transcribed from a
single gene through the use of different transcriptional initiation
sites, where the transcript for the cytoplasmic form initiates within
the invertase signal sequence (36). The cytoplasmic and secreted forms
of gelsolin are also encoded by two mRNA forms transcribed from a single gene through the alternative use of two promoters. The 5
region
of the mRNA coding for cytosolic gelsolin is derived from two exons
that encode an untranslated sequence and translation starts at an
internal exon common to both mRNAs, whereas the 5
end of the
mRNA coding for secreted gelsolin is derived from a single unique
exon that encodes an N-terminal signal sequence (37). Thus, the two
distinct PI8 mRNA species may serve a purpose analogous to those of
invertase and gelsolin.

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Fig. 4.
Amino acid sequence identity between the
H1 and H2 regions in PAI-2 and the
corresponding regions in PI8. Mismatches in which a hydrophobic
amino acid is present in PI8 are designated by an
asterisk.
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The mammalian processing endoproteinase furin is actively involved in
normal cellular processes and has also been linked to pathological
situations. In this regard, furin is involved in the cleavage and
activation of diphtheria toxin, a process that is important for
cytotoxicity, and is also a processor and activator of the coat
proteins of such viruses as influenza virus, measles virus, and HIV-1,
a process essential for virus infectivity. The interaction of furin
with PI8 results in the rapid formation of a tight complex with a
relatively long half-life. Because PI8 contains two hydrophobic regions
proximal to the N terminus nearly identical to the internal secretion
signals in PAI-2, as well as two forms of PI8 mRNA, also seen with
yeast invertase and human gelsolin, it is not unreasonable to suggest
that a fraction of the PI8 synthesized may be secreted and interact
with furin under normal conditions or in response to specific stimuli.
Therefore, PI8 may regulate the activity of furin and, in turn, such
events as pro-protein processing and virus infectivity by its
secretion, either through facultative polypeptide translocation
facilitated by hydrophobic interactions or by alternative
transcriptional initiation to produce mRNA encoding PI8 that
contains a cleavable N-terminal signal sequence.