(Received for publication, August 17, 1995)
From the
Amyloid -protein (A
) is the major constituent of
senile plaques and cerebrovascular amyloid deposits in
Alzheimer's disease and is proteolytically derived from its
transmembrane parent protein the amyloid
-protein precursor
(A
PP). Although the physiological role(s) of secreted A
PPs
are not fully understood, several potential functions have been
described including the regulation of hemostatic enzymes factors XIa
and IXa and a role in cell adhesion. In the present study, we
investigated the proteolytic processing of A
PP by factor XIa
(FXIa). Incubation of the human glioblastoma cell line U138 stably
transfected to overexpress the 695 isoform of A
PP with FXIa
(2.5-5 nM) resulted in proteolytic cleavage of secreted
A
PP. Higher concentrations of FXIa (>25 nM) resulted
in loss in cell adherence. Coincubation of FXIa with purified,
recombinant Kunitz protease inhibitor domain of A
PP blocked both
the proteolytic processing of A
PP and the loss of cell adhesion.
The RHDS cell adhesion site of A
PP resides within residues
5-8 of the A
domain. Incubation of synthetic
A
peptide with increasing concentrations of
FXIa resulted in cleavage of A
between Arg
and
His
within the cell adhesion domain of the peptide.
FXIa-digested A
or A
PP
lost their abilities to serve as cell adhesion substrates
consistent with cleavage through this cell adhesion site. Together,
these results suggest a new potential biological function for FXIa in
the modulation of cell adhesion. In addition, we have shown that FXIa
can proteolytically alter A
and therefore possibly modify its
physiological and perhaps pathological properties.
Deposition of the amyloid -protein (A
) (
)in
senile plaques in the neuropil and in the walls of cerebral blood
vessels is a pathologic feature of Alzheimer's disease. A
is
a 39-42-amino acid protein that is proteolytically derived from
its transmembrane parent protein, amyloid
-protein precursor
(A
PP)(1, 2, 3, 4) . A
PP is
a multidomain protein that can be translated from alternatively spliced
transcripts from a single gene located on chromosome
21(5, 6, 7, 8, 9, 10, 11) .
The major mRNA species encode proteins of 695, 751, and 770 amino
acids. The latter two isoforms contain a 56-amino acid domain that is
homologous to Kunitz-type serine protease inhibitors
(KPI)(9, 10, 11) . These isoforms are
identical to the cell secreted inhibitor identified as protease nexin-2
(PN-2)(12, 13) . Secretory cleavage of A
PP occurs
within the A
domain, and therefore, processing through this
pathway precludes A
formation(14, 15) .
Although the physiological roles of secreted APP are not fully
understood, several potential functions have been ascribed to it. For
example, several laboratories have provided evidence that A
PP can
mediate both cell-cell and cell-surface adhesion in neural and
non-neural
cells(16, 17, 18, 19, 20) .
Recently, A
PP has been shown to be involved in regulating
intracellular calcium levels in neurons, thus providing protection to
these cells(21, 22) . Both the cell adhesion and
neuroprotective activities have been localized to the carboxyl-terminal
region of the secreted A
PP(17, 21, 22) .
In addition, growth-promoting activities of A
PP in non-neural and
neural cells have been reported(23, 24) . The region
responsible for this autocrine activity has been identified as the
sequence RERMS located in the middle portion of A
PP(24) .
Secreted forms of APP that contain the KPI domain have been
shown to inhibit several different serine proteases, which include
trypsin, chymotrypsin, and coagulation Factors XIa and
IXa(12, 25, 26, 27, 28, 29) .
Through inhibition of these latter two proteases, it has been suggested
that the KPI-containing isoforms of A
PP may play a role in
regulating hemostasis by acting as an anticoagulant(30) .
Factor XIa (FXIa) participates in the middle phase of the intrinsic
pathway of blood coagulation by converting the zymogen Factor IX to the
active serine protease Factor IXa. Factor IXa then converts Factor X to
Factor Xa, the next serine protease to participate in the coagulation
cascade ultimately leading to fibrin clot formation(31) .
In
the present study, we investigated the proteolytic processing of
APP by FXIa. We report here that FXIa can cleave the RHDS sequence
in the A
domain, altering the cell adhesive properties of both
A
and secreted A
PP. These data suggest a new potential
biological function for FXIa in the modulation of cell adhesion. These
findings also indicate that the KPI domain of A
PP can regulate the
activity of a protease that can process the A
domain, thus
altering its potential physiological and pathological properties.
Figure 1:
Immunoblot analysis of secreted
APP in cultured medium from U138/695 cells incubated with FXIa. A, near confluent cultures of U183/695 cells were incubated
with increasing concentrations of FXIa for 18 h. The concentrations of
FXIa used were: none (lane 1), 2.5 nM (lane
2), 5 nM (lane 3), 25 nM (lane
4), 50 nM (lane 5), 250 nM (lane
6), and 100 nM thrombin (lane 7). B,
near confluent cultures of U138/695 cells were incubated alone (lane 1) or with 50 nM FXIa (lane 2), 50
nM FXIa and 100 nM KPI (lane 3), 100 nM KPI (lane 4), or 100 nM thrombin (lane
5) for 18 h. Aliquots of culture medium were analyzed for secreted
A
PP by SDS-PAGE and subsequent immunoblotting with mAb P2-1
as described under ``Experimental
Procedures.''
It
was noted that the 85-kDa truncated secreted A
PP was very
similar in size to that we and others have shown to be generated by
thrombin proteolysis(35, 37, 38) . Therefore,
we compared the effects of 50 nM FXIa and 100 nM
thrombin on A
PP proteolysis in U138/695 cells (Fig. 1A). The amino acid sequence at the thrombin
cleavage site in A
PP was determined to be EPR with cleavage
occurring on the carboxyl side of the arginine residue(37) .
This is the same sequence as the synthetic chromogenic substrate used
to assay FXIa activity in vitro(39) . Based on
sequence similarity and the size of the truncated secreted A
PP
protein, the initial cleavage site utilized by FXIa is most likely the
same as the thrombin cleavage site located at
Arg
-Ile
of the A
PP
sequence. FXIa cleavage of secreted A
PP
could
be inhibited when FXIa was preincubated with 100 nM KPI (Fig. 1B).
Figure 2:
Effect of FXIa on cell adhesion of
U138/695 cells. U138/695 cells were grown to 50% confluency, incubated
for 18 h alone (A) or with 50 nM FXIa (B),
100 nM KPI (C), 50 nM FXIa and 100 nM KPI (D), or 100 nM thrombin (E), and
then photographed using phase contrast microscopy. Magnification,
40.
Figure 3:
Immunoblot analysis of
A incubated with FXIa. A, purified
A
(25 µM) was incubated with
increasing concentrations of FXIa for 24 h. The concentrations of FXIa
used were: none (lane 1), 2.5 nM (lane 2), 5
nM (lane 3), 25 nM (lane 4), 50
nM (lane 5), and 250 nM (lane 6). B, purified A
(25 µM)
was incubated alone (lane 1) or with 50 nM FXIa (lane 2), 50 nM FXIa and 100 nM KPI (lane 3), 100 nM KPI (lane 4), or 100 nM thrombin (lane 5) for 24 h. Aliquots of the incubation
mix were analyzed for A
immunoreactivity by SDS-PAGE and
subsequent immunoblotting with mAb 6E10 as described under
``Experimental Procedures.''
To further identify the site of FXIa
cleavage within A, 10 µg of the peptide
was digested with 100 nM FXIa for 48 h. The digested peptide
was analyzed on a Tris/Tricine/SDS 10-20% polyacrylamide gel. The
Coomassie-stained gel revealed a truncated
3.4-kDa A
fragment (Fig. 4). In parallel experiments, the truncated A
peptide
was transferred to a polyvinylidene difluoride membrane and subjected
to amino-terminal sequence analysis. The resulting sequence derived
from five cycles of sequential Edman degradation is shown in Table 1. The amino-terminal sequence of the truncated A
peptide identified the FXIa cleavage between Arg
-His
within the RHDS cell adhesion domain.
Figure 4:
SDS-PAGE analysis of A digested with FXIa. Purified A
(10
µg) was incubated alone (lane 1) or with 100 nM FXIa (lane 2) at 37 °C for 24 h. The samples were
analyzed by SDS-PAGE followed by staining with Coomassie Brilliant
Blue.
Figure 5:
Effects of FXIa on the ability of
A and A
PP
to promote cell
adhesion of U138/695 cells. A suspension of U138/695 cells (3.4
10
) in serum-free culture medium were incubated at 37
°C for 1 h on 35-mm plastic dishes coated with (A) nothing (lane 1), 400 ng FXIa (lane 2), 65 ng KPI (lane
3), 400 ng FXIa and 65 ng KPI (lane 4), 2 µg
A
(lane 5), 2 µg
A
and 65 ng KPI (lane 6), 2 µg
A
digested with 400 ng FXIa (lane
7), or 2 µg A
digested with 400 ng
FXIa then incubated with 65 ng KPI (lane 8) or coated with (B) 10 µg of A
PP
(lane 1), 10
µg of A
PP
digested with 200 ng of FXIa (lane
2), or 10 µg of A
PP
digested with 200 ng of
thrombin (lane 3). Nonadherent cells were removed as described
under ``Experimental Procedures.'' Values are presented as
the means ± S.D. percentage of adherent cells counted in
3-5 fields of view compared with A
or
A
PP
.
The present studies show that FXIa, a target protease for
inhibition by the KPI domain of APP, proteolytically cleaves
secreted A
PP
from U138/695 cells. Initial cleavage
at low FXIa concentrations (
2.5 nM) occurred at the site
previously reported for thrombin cleavage at
Arg
-Ile
of A
PP
. It is
noteworthy that the amino acid sequence at the thrombin cleavage site
in A
PP is Glu
-Pro
-Arg
,
the same sequence as the synthetic chromogenic substrate used to assay
FXIa activity in vitro(39) . Additional cleavage sites
were utilized at higher concentrations of FXIa (Fig. 1A). Several potential functions have been
ascribed to secreted A
PP
, including cell adhesion,
growth supportive activity, and
neuroprotection(16, 17, 18, 19, 20, 21, 22, 23, 24) .
Proteolytic processing of A
PP
by FXIa may disrupt
one or more of these properties.
Reduced cell adhesion to the
substratum was observed in FXIa-treated U138/695 cells. This may have
resulted from disruption of the cell adhesion domain by FXIa. A cell
adhesion site in APP has been localized within the amino-terminal
region of the A
domain, which is present in secreted forms of the
protein(18) . To test this hypothesis, synthetic
A
was digested with increasing concentrations
of FXIa. The loss of A
immunoreactivity observed in Fig. 3was consistent with disruption of the mAb 6E10 epitope,
which has been mapped within the amino-terminal region of
A
(40) . Accompanying the loss of A
immunoreactivity,
we observed the appearance of a truncated A
peptide when treated
with FXIa (Fig. 4). Amino-terminal sequence analysis of the
first five amino acids of this truncated peptide yielded the A
sequence HDSGY (Table 1), consistent with cleavage through the
cell adhesion domain RHDS. It is noteworthy that the RHDS cleavage site
in the A
domain shows homology to the factor IX activation site
for FXIa(32) . Because cleavage through the RHDS sequence may
disrupt the cell adhesive properties of the A
domain, we compared
the abilities of A
and FXIa-cleaved
A
to serve as cell adhesion substrates.
A
promoted adhesion of U138/695 cells to the
substratum by nearly 20-fold over buffer-treated substratum (Fig. 5). However, FXIa-cleaved A
lost
its ability to serve as cell adhesive substrate. Similarly, FXIa
diminished the ability of secreted A
PP
to promote
cell adhesion. These results suggest that FXIa could disrupt adhesion
of cells that may use this region of the A
domain as an
extracellular matrix protein.
Together, these findings have
identified a new potential function for FXIa in the modulation of cell
adhesion. This suggests that in addition to a function in coagulation,
FXIa may participate in other roles of wound repair and tissue
remodeling at sites of vascular injury. It is noteworthy that platelets
activated by physiological agonists release large amounts of
PN-2/APP(27, 41) . Therefore, PN-2/A
PP
released by platelets at sites of vascular damage may participate in
both the regulation of coagulation and cell adhesion. Through its
intimate interactions with PN-2/A
PP, FXIa may also be involved in
both of these processes. The present data also show that the KPI domain
of PN-2/A
PP can regulate the activity of a target protease that
possesses the ability to modulate another potential biological function
on a distal region of the protein. Moreover, these findings demonstrate
that the KPI domain of PN-2/A
PP can regulate the activity of a
protease that can cleave the A
peptide. Proteolytic processing of
this nature could alter possible physiologic and perhaps pathologic
properties of A
.