(Received for publication, June 9, 1995; and in revised form, July 25, 1995)
From the
The Alzheimer's disease related protein, amyloid
-protein precursor (A
PP), contains a domain homologous to
Kunitz-type serine protease inhibitors (KPI). The recombinant KPI
domain of A
PP is a potent inhibitor of coagulation factors XIa and
IXa and functions as an anticoagulant in vitro. Here we report
the expression, purification, and characterization of a reactive center
lysine mutant of the KPI domain of A
PP (KPI-Lys
). An
expression plasmid for the KPI-Lys
domain of A
PP
encoded amino acids 285-345 of the A
PP cDNA containing a
lysine substitution at arginine 17 in the KPI domain. The secreted
61-amino acid product was purified to homogeneity and functionally
characterized. The protease inhibitory properties of the KPI-Lys
domain were compared to those of the native KPI domain of
A
PP. Both KPI domains equally inhibited trypsin, chymotrypsin, and
coagulation factors IXa and Xa. However, the KPI-Lys
domain was an
25-fold less effective inhibitor of coagulation
factor XIa resulting in markedly less prolongation of the activated
partial thromboplastin time compared to the native KPI domain of
A
PP. On the other hand, the KPI-Lys
domain was an
10- and 5-fold better inhibitor of plasmin in a chromogenic
substrate assay and in a fibrinolytic assay, respectively, than the
native KPI domain of A
PP. Together, these studies suggest that the
KPI-Lys
domain has enhanced anti-fibrinolytic and
diminished factor XIa inhibitory properties compared to the native KPI
domain of A
PP.
The amyloid -protein precursor (A
PP) (
)is
the parent molecule to the Alzheimer's disease amyloid
-protein(1, 2, 3, 4) . A
PP
can be translated from predominantly three alternatively spliced mRNA
species to yield polypeptides of 695, 751, and 770 amino
acids(5, 6, 7) . The latter two species
contain an additional insert which codes for a domain that is
homologous to Kunitz-type serine protease inhibitors
(KPI)(5, 6, 7) . The KPI-containing isoforms
of A
PP are identical to the previously described cell-secreted
protease inhibitor, protease nexin-2 (PN-2)(8, 9) .
Messenger RNA encoding the KPI-lacking A
PP 695 isoform is found
primarily in brain. However, mRNA encoding the KPI-containing A
PP
751/770 isoforms are also abundant in brain and found in most
peripheral tissues, suggesting that these isoforms of A
PP may have
a common function throughout the body.
Several studies have
suggested a potential physiologic function for the KPI domain of
PN-2/APP in hemostasis. For example, measurements of protease
inhibition equilibrium constants revealed that PN-2/A
PP and its
recombinant KPI domain are very potent inhibitors of intrinsic blood
coagulation factors XIa and
IXa(10, 11, 12, 13, 14) .
In this regard it is noteworthy that PN-2/A
PP is an abundant
platelet
granule protein and is secreted in high concentrations
by platelets that are activated by physiologic
agonists(11, 15, 16, 17) . Together,
these findings suggest that secreted platelet PN-2/A
PP may play a
role in regulating the intrinsic blood coagulation cascade at sites of
vascular injury upon release by activated
platelets(10, 11, 15, 17) .
Fibrinolysis is another regulated proteolytic process that occurs at
sites of vascular injury. Previous studies have shown that
PN-2/A
PP and its recombinant KPI domain also inhibit plasmin but
to a lesser extent than coagulation factors XIa and
IXa(10, 12, 13, 14) .
Here we
report the high level expression, purification, and biochemical
characterization of a reactive center lysine mutant of the KPI domain
of APP (KPI-Lys
). The protease inhibitory,
anticoagulant and anti-fibrinolytic properties of the KPI-Lys
domain and the native KPI domain of A
PP were compared. The
results of these studies indicate that the KPI-Lys
domain
has enhanced anti-plasmin and diminished factor XIa inhibitory
properties compared to the native KPI domain of A
PP. These
properties suggest that KPI-Lys
may have utility as an
anti-fibrinolytic agent.
The principal
product of the second PCR, a 630-base pair fragment, was digested
with AgeI and HindIII, gel purified, ligated into the
large fragment of AgeI and HindIII digested pKPI200,
and transformed into bacteria. Plasmids from individual bacterial
transformants were tested initially for the presence of the diagnostic HaeIII restriction site and the DNA sequenced to confirm the
mutations and the integrity of the rest of the KPI sequence. The
resulting plasmid, designated pKPI-R17K (Fig. 1), contains the
promoter (5` AOX1) and terminator (3` AOX1) sequences
of the P. pastoris methanol-inducible alcohol oxidase gene and
the mutated KPI sequence in frame with the Saccharomyces cerevisiae
-mating factor prepro signal sequence. The transformation of
the KPI-R17K plasmid into P. pastoris was accomplished as
described previously(19) . The plasmid was linearized at the
unique StuI restriction site within the HIS4 gene
prior to transformation into the HIS4-negative strain of P. pastoris, GS115. Successful integration of the plasmid was
assessed by growth on minimal medium indicating the reconstitution of
the HIS4-positive phenotype. Expression and purification of
the KPI-Lys
domain were conducted as described previously
for the KPI domain of PN-2/A
PP(12) . Amino acid sequence
analysis of the purified protein confirmed the presence of the lysine
at position 17.
Figure 1:
Sequence of the DNA encoding
KPI-Lys domain and schematic representation of plasmid
pKPI-R17K. The nucleotide sequence of the 186 base pairs synthetic DNA
encoding the 61-amino acid KPI-Lys
domain is shown with
the mutations introduced at codons 17 and 18 indicated in italics. The four amino-terminal amino acids labeled with an asterisk (*) correspond to amino acid residues 285-288 (22) of A
PP that precede the KPI domain. The schematic
shows the configuration of the expression plasmid, pKPI-R17K,
illustrating the DNA encoding the KPI-Lys
sequence
attached to the S. cerevisiae
-mating factor prepro
secretion signal sequence. Expression is under control of the P.
pastoris alcohol oxidase promoter (5` AOX1) and
terminator (3` AOX1).
Inhibition equilibrium constants (K) for the purified KPI domains and factor XIa,
trypsin, chymotrypsin, and plasmin were determined by the method of
Bieth (21) as described previously (10, 12) .
The K
values for the purified KPI domains and
coagulation factor IXa were determined using a polylysine and
polyethylene glycol based factor X activation
assay(13, 14, 22, 23) . Factor Xa (1
nM) inhibitory activity was measured by incubation with the
purified KPI domains (5-10 nM) in 0.1 M
triethanolamine, pH 8.0, 0.1 M NaCl, containing 0.1%
polyethylene glycol (M
= 8000) and 100
µg/ml bovine serum albumin in triplicate in 96-well microtiter
plates. Incubations were conducted at 25 °C for at least 40 min to
allow for equilibrium between factor Xa and the KPI domains. At the end
of the incubation 0.3 M
tosyl-Gly-Pro-Arg-p-nitroanilide was added and hydrolysis
proceeded for 60 min at 25 °C. Graphical analysis yielded an
apparent K
(K
). Since the inhibition is
reversible a correction must be made for the K
of
the protease-substrate reaction. The K
for each
protease and its corresponding chromogenic substrate was determined
independently and the true K
was calculated using
the following equation:
A DNA encoding the mutant KPI-Lys domain was
prepared by site-directed mutagenesis and using PCR and the plasmid
KPI200 that encodes the native KPI domain of A
PP (Fig. 1).
DNA encoding the yeast-specific secretion signal of S. cerevisiae
-mating factor prepro signal sequence was attached to the DNA
encoding the KPI-Lys
domain and incorporated into a P.
pastoris expression vector to generate pKPI-R17K. The recombinant
plasmid pKPI-R17K (Fig. 1) was integrated into and expressed in
the methylotrophic yeast P. pastoris. Transcription from the P. pastoris AOX1 promoter included in pKPI-R17K occurs at very
high levels in P. pastoris cells grown in methanol providing
an inducible expression system particularly designed for foreign gene
expression(26) . Transformed P. pastoris cells were
first grown in a fermenter in the presence of glycerol and then
switched to methanol as the carbon source to induce expression of the
KPI-Lys
domain. The secreted KPI-Lys
domain
(>1.0 g/liter) comprised >80% of the protein in the fermentation
medium.
The expressed KPI-Lys domain was purified from
the fermentation medium by the same procedures previously described for
purification of the native KPI domain of A
PP(12) . As
shown in Fig. 2, the purified KPI-Lys
domain and
native KPI domain of A
PP had molecular masses of
6.5 kDa.
Amino acid sequencing of the purified KPI-Lys
domain
revealed the integrity of the expressed product and showed that the
amino terminus of the protein was properly processed upon secretion
(data not shown). Titration experiments with the purified
KPI-Lys
domain revealed a 1:1 stoichiometry for trypsin
inhibition, essentially the same for parallel titration experiments
with the purified native KPI domain of A
PP (Fig. 3).
Importantly, this result demonstrated that the KPI-Lys
domain, as the native KPI domain of A
PP, was fully active,
properly folded, and the disulfide bonds were in the correct
orientation when expressed and secreted by the Pichia cells.
This assessment was further supported by the finding that treatment of
either purified KPI domain with the reducing agent dithiothreitol
completely abolished inhibitory activity toward the target proteases
(data not shown).
Figure 2:
SDS-polyacrylamide gel electrophoresis
analysis of the purified native KPI domain and KPI-Lys domain. Five µg of the purified native KPI domain of A
PP
or the KPI-Lys
domain was subjected to electrophoresis on
a SDS/Tris/Tricine 10-20% gradient polyacrylamide gel. The
completed gel was stained with Coomassie Brilliant Blue R-250. Lane
1, native KPI domain of A
PP; lane 2, KPI-Lys
domain.
Figure 3:
Titration of the protease inhibitory
activities of the purified native KPI domain and KPI-Lys domain with trypsin. Ten nanomolar trypsin was incubated with
increasing concentrations of purified native KPI domain (
) or
KPI-Lys
domain (
) and the residual trypsin activity
was measured using the chromogenic substrate
carbobenzoxy-Val-Gly-Arg-4-nitroanilide as described under
``Experimental Procedures.'' The data represent the means
± S.D. of three separate
determinations.
The protease inhibitory properties of the purified
KPI-Lys domain were compared to those of purified native
KPI domain of A
PP (Table 1). The protease inhibition
equilibrium constants obtained for the inhibition of trypsin,
chymotrypsin, and coagulation factors IXa and Xa by the purified
KPI-Lys
domain were essentially the same as those obtained
for the inhibition of these proteases by the purified native KPI domain
of A
PP (Table 1). However, the KPI-Lys
domain
was an
25-fold less effective inhibitor of coagulation factor XIa
than the native KPI domain of A
PP. This finding indicates that the
arginine residue at the reactive center of the KPI domain of A
PP
is important for optimal inhibition of factor XIa. On the other hand,
the KPI-Lys
domain was found to be an
10-fold better
inhibitor of plasmin compared to the native KPI domain of A
PP (Table 1). Neither KPI domain inhibited urokinase.
Since the
KPI-Lys domain inhibited factors XIa, IXa, and Xa, we
performed studies to determine if it inhibited the coagulant activity
of normal human plasma (Fig. 4). The native KPI domain of
A
PP at 1 µM prolonged the APTT >2-fold over pooled
normal plasma as previously reported(13) . In contrast, only at
concentrations
50 µM or more was the APTT prolongation
>2-fold by the KPI-Lys
domain. At 50 µM,
the native KPI domain of A
PP prolonged the APTT
15-fold. These
data suggest that the KPI-Lys
domain has diminished
anticoagulant properties in in vitro clotting assays compared
to the native KPI domain of A
PP. Since clotting in the APTT assay
involves the activity of numerous coagulation factors in the intrinsic
pathway, the weaker anticoagulant activity of the KPI-Lys
domain observed is consistent with the above finding that it is a
less effective inhibitor of coagulation factor XIa than the native KPI
domain of A
PP (Table 1).
Figure 4:
Inhibition of plasma coagulation by
purified native KPI domain and KPI-Lys domain. Pooled
normal human plasma was incubated with increasing concentrations of
purified native KPI domain (
) or KPI-Lys
domain
(
) in microtiter plates for 10 min at room temperature. At the
conclusion of the incubation time, the APTT was measured as described
under ``Experimental Procedures.'' The data represent the
mean ± S.D. of three separate determinations and are presented
as fold prolongation of the clotting time over a simultaneously
performed uninhibited sample.
The KPI-Lys domain
was found to have enhanced inhibition of plasmin in a chromogenic
substrate assay compared to the native KPI domain of A
PP (Table 1). Therefore, we conducted studies to compare the
abilities of the KPI-Lys
domain and the native KPI domain
of A
PP to inhibit plasmin degradation of its natural substrate,
fibrin. The KPI-Lys
domain inhibited plasmin in the in
vitro fibrinolytic assay starting at concentrations >0.1
µM and with an IC
of
1 µM (Fig. 5). In contrast, the native KPI domain of A
PP
inhibited plasmin in the same assay starting at concentrations >1
µM and with an IC
of
5 µM,
consistent with its less effective inhibition of plasmin in the
chromogenic substrate assay (Table 1).
Figure 5:
Inhibition of fibrinolysis by purified
native KPI domain and KPI-Lys domain. Fibrin microtiter
plates were prepared by incubating 5.4 µM fibrinogen and
1.5 nM thrombin in a final volume of 100 µl of 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM
CaCl
, in the absence or presence of increasing
concentrations of the native KPI domain of A
PP (
) or the
KPI-Lys
domain (
). Then, 5 µl of 0.6 µM plasmin was added to the fibrin wells and incubated shaking for 16
h at 37 °C in a humidified environment. The extent of fibrinolysis
was determined as a loss in absorbance at 405 nm in a V
microtiter plate
reader.
Together, these studies
indicate that the KPI-Lys domain possesses enhanced
antiplasmin and diminished factor XIa inhibitory and anticoagulant
properties compared to the native KPI domain of A
PP. Although the
present studies show that the native KPI domain of A
PP is an
inhibitor of plasmin and fibrinolysis in in vitro assays, its
additional anticoagulant properties may interfere in the normal genesis
of fibrin clots. On the other hand, KPI-Lys
domain
exhibits increased anti-fibrinolytic activity yet it is a much less
effective anticoagulant in in vitro assays. Since the
inhibition of factors IXa and Xa by the KPI-Lys
domain is
similar to that of the native KPI domain, the Arg
Lys mutation
of KPI-Lys
may result in an inhibitor which overall is a
better anti-fibrinolytic agent. This hypothesis needs to be examined in in vivo models. An anti-fibrinolytic inhibitor may be useful
in cardiopulmonary bypass as an alternative to aprotinin
administration.