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INTRODUCTION |
Recent investigations indicate that when the proteins of the
plasma kallikrein/kinin system (high molecular weight kininogen (HK),1 prekallikrein (PK),
and factor XII (XII)) assemble on their binding sites, putative
receptor(s) on endothelial cells, PK is activated to kallikrein, and
factor XII is activated secondarily (1-3). Activation of this
proteolytic system on endothelial cells is independent of a negative
charged, artificial surface (1). PK is activated to kallikrein by a
membrane-associated cysteine protease whose activity is recognized only
when PK binds to HK on endothelial cells (1). These data along with the
finding that metabolic inhibitors, antimycin A and
2-deoxy-D-glucose, and pronase abolish HK binding to
endothelial cells (4), suggest that the kininogen-binding site,
putative receptor, is a physiochemical structure.
Recent evidence also suggests that the kininogen receptor on
endothelial cells may be comprised of one or more candidate proteins. A
33-kDa protein identified as gC1qR has been recognized as a kininogen-binding protein (5, 6). However, this protein does not bind
low molecular weight kininogen, a requirement of kininogen binding to
endothelial cells (7), and very few copies are found on the endothelial
cell membrane (8). Furthermore, investigations have shown that
endothelial cell urokinase plasminogen activator receptor (uPAR) can
also serve as a kininogen-binding site on endothelial cells (9).
However, this protein is not found on the platelet surface and alone
cannot account for the total number of kininogen-binding sites that
number 10 million versus 0.25 million for uPAR (10).
Additional studies from our own laboratory have identified that
cytokeratin 1 (CK1) is a kininogen-binding protein, and CK1 is found on
the surface of endothelial cells (11). The present studies map the HK
binding region on CK1 and show that peptides of the kininogen-binding
site on CK1 block PK activation on HUVEC. These data indicate that CK1
is a component of the kininogen multiprotein receptor which
participates in PK activation.
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EXPERIMENTAL PROCEDURES |
Materials--
A biotinylation kit and ImmunoPure streptavidin
horseradish peroxidase dihydrochloride (3,3',5,5'-tetramethylbenzidine
dihydrochloride) were supplied by Pierce. Prestained and low molecular
weight standards, nitrocellulose, and polyacrylamide were purchased
from Bio-Rad. Purified human cytokeratin was obtained from DAKO Corp.,
Carpintera, CA. Human umbilical vein endothelial cells (HUVEC),
endothelial cell growth medium, and trypsin-neutralizing buffer were
purchased from Clonetics, San Diego, CA. Trypsin-EDTA was obtained from Life Technologies, Inc. GST gene fusion system was purchased from Amersham Pharmacia Biotech.
Proteins, Peptides, and Antibodies--
Single chain high
molecular weight kininogen (HK) with a specific activity of 13 units/mg
in 4 mM sodium acetate-HCl and 0.15 M NaCl, pH
5.3, was purchased from Enzyme Research Laboratories, Inc., South Bend,
IN. HK was biotinylated according to the procedure of Pierce (see Ref.
12). Briefly, 5 mg of HK was dialyzed against 0.01 M sodium
phosphate, 0.15 M NaCl, pH 7.4. Sulfo-NHS-LC-biotin was
added to HK to give 12-fold molar excess of sulfo-NHS-LC-biotin to HK.
After incubation for 2 h in ice, the sample was then loaded onto
10 ml of Econo-PacI0 DG column (Bio-Rad). Biotinylated HK (biotin-HK) was monitored by absorbance at 280 nm using an extinction coefficient of 7.0 for HK and a protein assay (Bio-Rad). Biotin-HK had
a specific activity of 17 units/mg. Low molecular weight kininogen (LK)
was prepared as previously reported (7). Prekallikrein (PK) was
purchased from Enzyme Research Laboratories, South Bend, IN.
Peptides corresponding to specific amino acid sequences of CK1 were
prepared (13). The numbers assigned to the location of the amino acids
of the following synthetic peptides are from Johnson et al.
(13). Peptides G137YGGGYGPVCSPGGIQEVTI156
(GYG20), N157QS LLQPLNVEIDPEIQKVK176
(NQS20), S177REREQIKSLNNQFASFIDK196 (SRE20),
V197RFLEQQNKVLQTKWELLQQ216 (VRF20),
V217 DTSTRTHNLEPYFESFINN236 (VDT20),
S237RRRVDQLKSDQS RLDSELK256 (SRR20),
N257MQDMVEDYRNKYEDEINKR276 (NMQ20),
VEGPQLTGLISNQIQ (Scrambled PGG15 (see below)),
biotin-KG143PVCSPGGIQEVTINQSLLQ162-amide, and
acetyl-CAEVKAQYEDIAQKSKAE-amide sequences were synthesized by Quality
Controlled Biochemicals, Hopkinton, MA. Peptides G143
PVCSPGGIQEVTIN157 (GPV15),
P148GGIQEVTINQSLLQ162 (PGG15), and
E153VTINQSLLQPLNVE166 (EVT14) were synthesized
at the Protein and Carbohydrate Structure Facility of the University of
Michigan (Ann Arbor, MI). Additional peptides made at the University of
Michigan were HVLDHGHKHKHGHGHGKHKNKGKK (HVL) and HKHGHGHGKHKNKGKKNGKH
(HKH) both from domain 5 of HK, LDCNAEVYVVPWEKKIYPTVNCQPLGM (LDC) from
domain 3 of kininogens, and FNQTQPERGDNNLTR (FNQ) from human
coagulation factor X (12). The peptides were made by solid-phase
synthesis using FMOC (N-(9-fluorenyl)methoxycarbonyl) moiety
chemistry. All peptides were purified by preparative reverse-phase high
performance liquid chromatography and characterized by amino acid
analysis and mass spectroscopy. In this article, each peptide from
cytokeratin 1 is named using the one-letter code of the first three
amino acid residues followed by the number of amino acids in the
peptide unless otherwise stated.
Peptide acetyl-CAEVKAQYEDIAQKSKAE-amide (CAE18)
(A351-E367) was used to immunize goats for the
production of antisera (anti-CAE18) at Quality Controlled Biochemicals,
Inc., Hopkinton, MA (13). Peptide CAE18 is seen in a few cytokeratins
and is coded by exon 5 of cytokeratin 1 (13). Antisera produced were
affinity purified on a column that immobilized its respective peptide.
Monoclonal antibodies C1801 and C2931, which are mixtures of
anti-cytokeratin clones, and mouse IgG were supplied by Sigma.
Monoclonal antibody C1801 had antibodies against cytokeratins 1, 5, 6, and 8. Monoclonal antibody C2931 had antibodies against cytokeratins 4, 5, 6, 8, 10, 13, and 18.
Gel Electrophoresis and Immunoblot Analysis--
Proteins were
separated on a 12% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and then transferred to nitrocellulose
membranes at 8 mA overnight. The electroblots then were incubated in
blocking buffer (5% (wt/v) dry milk with 0.1% (w/v) BSA, 0.05% Tween
20, 0.15 M NaCl, and 20 mM Tris-HCl, pH 7.4)
for 1 h (14). Thereafter, the membranes were incubated with
monoclonal antibodies C1801 or anti-CAE18 antibody at 1 µg/ml for
2 h. After washing, the nitrocellulose membrane was incubated with
horseradish peroxidase-conjugated anti-mouse or anti-goat antibody,
respectively (1:2,000), for 2 h. The specific reactivity of
antibodies with the electroblotted proteins were detected with the ECL
system (Amersham Pharmacia Biotech). All steps were carried out at room temperature.
Isolation of CK1 cDNAs from Keratinocyte and Endothelial
gt11 Libraries--
Five oligonucleotide primers were prepared to
include the coding sequences of exons 1-5, 1-7, or 3-5 of human CK1
(13). Each sense primer contained a 5'-encoding sequence, an
EcoRI restriction enzyme site, and a translation initiation
sequence (15). Each antisense primer had a SalI restriction
site at the 3' end. The two sense primers were
5'-G10TCACTATCAACCAGAGCCTT30-3' from exon 1 and
5'-T355ATGAGGATGAAATCAACAAGCGG378-3' from exon
3. The antisense primers were 5'-TCACTCAGCTTTGCTCTTCTGGGC-3' from base
pairs 631-651 of exon 5 and 5'-TCACTCCAGATCCAGGGCCAGCTT-3' from base
pairs 961-981 of exon 7. The sequence numbers of the previous
nucleotides are from Steinert et al. (16).
Nested polymerase chain reaction was employed to isolate CK1 cDNA
from either a keratinocyte or human umbilical vein endothelial
gt11
expression library (17). The keratinocyte and endothelial cell cDNA
libraries were generously provided by Drs. J. T. Elder and David
Ginsburg, respectively, of the University of Michigan. By using 20 pmol
of commercially available sense and antisense primers for
gt11
(Promega, Madison, WI), polymerase chain reaction on the cDNA
expression libraries were performed in a 100-µl reaction mixture
according to the standard methods (18). The reaction products (10 µl)
were analyzed by electrophoresis on 1% agarose gels and stained with
ethidium bromide. Ten-µl aliquots of the polymerase chain reaction
products then were used as templates for the second reaction. Fifty
picomoles of primer pairs for human CK1, as shown above, were selected.
The reactions were incubated at 94 °C for 2 min and then were
amplified for 30 cycles at 94 °C for 1 min, 54 °C for 1 min, and
72 °C for 2 min with a final extension time for 15 min at 72 °C
on Perkin-Elmer DNA thermal cycler 480. The cDNAs of the exons of
interest from either the keratinocyte or endothelial cell cDNA
library were gel-purified from 1% agarose gels. The fidelity of the
cDNAs for the desired fragments of human CK 1 were confirmed by DNA
sequence analysis.
Expression of Recombinant Cytokeratin 1 (rCK1)--
The purified
cDNA were cloned into the EcoRI-SalI
restriction sites of vector pGEX-5X-1 (Amersham Pharmacia Biotech) and
transformed into INV
F'-competent cells (Invitrogen, Carlsbad, CA).
All positive colonies were screened and analyzed by restriction digest
using EcoRI and SalI. Additional digestion by
BglI was performed to confirm the proper orientation of the
insert. Recombinant CK1 proteins were produced in Escherichia
coli from pGEX-5X-1 plasmid by the GST gene fusion system
(Amersham Pharmacia Biotech). Briefly, CK1 fusion proteins expression
were induced by adding 0.6 mM
isopropyl-D-thiogalactoside to the E. coli
transformed with the pGEX-5X-1 recombinants. Induced cultures were
incubated for an additional 4 h at 25-30 °C to express GST-CK1
fusion proteins. The bacteria were harvested and lysed by sonicating
for 4 cycles (30 s each) at 4 °C. Solubilization of the fusion
proteins was performed by 1% Triton X-100, and it was centrifuged at
10,000 × g for 10 min to remove the insoluble material
and applied directly to glutathione-Sepharose 4B. The GST-CK1 fusion
proteins were washed and eluted from the column by the manufacturer's
elution buffer (Amersham Pharmacia Biotech). The isolated GST-CK1
fusion proteins were digested with 1% (w/w) bovine factor Xa for
16 h at room temperature. The cleaved GST fusion protein was then
re-applied to the glutathione-Sepharose 4B affinity column, and the
recombinant CK1 proteins were recovered in the fractions not binding to
the column, whereas the GST mostly remained bound. The recombinant
proteins were confirmed to be CK1 by immunoblotting with antibodies
C1801 and anti-CAE18. The isolated recombinant CK1s (rCK1) were named
according to the size of the protein as determined by reduced
SDS-PAGE.
Endothelial Cell Culture--
HUVEC were obtained and cultured
according to the recommendations of Clonetics Corp. Cells between the
1st and 5th passage were subcultured onto fibronectin-treated, 96-well
plates 24 h prior to the start of the experiment as previously
reported (19). Cell viability was determined using trypan blue
exclusion. Cell number were determined by counting on a hemocytometer.
Direct Biotin-HK Binding to Recombinant Cytokeratin
1--
Recombinant CK128, rCK131, and
Deleted1-6rCK131 at 1 µg/well were incubated
in microtiter plate cuvette wells in 0.1 M
Na2CO3, pH 9.6, overnight at 37 °C. After
blocking the wells with 0.2% BSA for 1 h at 37 °C, the wells
were washed and then incubated with biotin-HK (7 nM) in
0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4, containing 1% BSA and 0.05% Tween 20 in the absence (
Zn2+) or presence (+Zn2+) of 50 µM Zn2+ or a 50-fold molar excess of HK (HK)
in the presence of 50 µM Zn2+ for up to
2 h at 37 °C. The relative binding of biotin-HK binding to the
cytokeratins was determined using ImmunoPure streptavidin horseradish peroxidase conjugate (Pierce) and peroxidase-specific fast
reacting substrate, 3,3',5,5'-tetramethylbenzidine dihydrochloride (Pierce), as described previously (4). Bound biotin-HK was quantified
by measuring the absorbance of the reaction mixture at 450 nm using a
microplate auto reader EL 311 (Bio-Tek Instrument, Winooski, VT). Total
biotin-HK binding to each recombinant protein in the presence or
absence of 50 µM Zn2+ was determined and
compared with the level of binding seen in the presence of zinc ion and
a 50-fold molar excess of HK.
Inhibition of Biotin-HK Binding to
rCK128--
Recombinant CK128 was coupled to
microtiter plates in 0.1 M Na2CO3,
pH 9.6, overnight at 37 °C. After blocking the wells with 0.2% BSA
for 1 h at 37 °C, the wells were washed and then incubated with
biotin-HK (7 nM) in 0.01 M sodium phosphate,
0.15 M NaCl, pH 7.4, containing 1% BSA and 0.05% Tween 20 and 50 µM Zn2+ in the absence or presence of
a 50-fold molar excess of HK or various concentrations of peptides
(HVL, HKH, LDC, and FNQ) or low molecular weight kininogen (LK) for
1 h at 37 °C. After incubation the level of specific biotin-HK
binding to the rCK128 was determined as described above by
subtracting nonspecific binding, i.e. the level of binding
seen in the presence of a 50-fold molar excess of HK, from that
determined in the presence of each of the peptides.
Inhibition of Biotin-HK Binding to Endothelial Cells by
Recombinant Cytokeratin 1 and Peptides of CK1--
Confluent
monolayers of HUVEC were washed three times with HEPES-Tyrode's buffer
(137 mM NaCl, 3 mM KCl, 12 mM
NaHCO3, 0.4 mM NaH2PO4,
1 mM MgCl2, 14.7 mM HEPES, 5.5 mM dextrose, and 3.5 mg/ml bovine serum albumin (BSA), pH
7.35) (4) containing 2 mM CaCl2, 1 mM MgCl2, and 50 µM
Zn2+. Biotin-HK (7 nM) in the same buffer were
added to the monolayers for 60 min at 37 °C in the absence or the
presence of increasing concentrations of HK, various rCK1s, or
synthetic peptides of the protein portion of CK1. The relative amount
of biotin-HK bound to the cells was determined as described above.
Specific biotin-HK binding was determined after nonspecific binding
(i.e. binding in the presence of a 50-fold molar excess HK)
was subtracted from that seen with biotin-HK alone or in the presence
of the various protein and peptide inhibitors.
Inhibition of Biotin-HK Binding to Native CK1--
Additional
investigations were performed to determine if peptides to a HK binding
region on CK1 blocked biotin-HK binding to native CK1. Human purified
cytokeratin from Dako (1 µg/well) was incubated in microtiter plate
wells in 0.1 M Na2CO3, pH 9.6, overnight at 37 °C. After blocking the wells with 0.2% BSA,
biotin-HK (7 nM) in HEPES-Tyrode's buffer, pH 7.35, containing containing 2 mM CaCl2, 1 mM MgCl2, and 50 µM
Zn2+ was incubated up to 2 h at 37 °C in the
absence or presence of a 50-fold molar excess of HK or various
overlapping synthetic peptides to CK1. The amount of biotin-HK bound
was demonstrated as described above for biotin-HK bound to rCK1 or
endothelial cells.
Direct Biotin-GPV20 Binding to HK--
Further experiments were
performed to determine if biotin-GPV20 directly binds to HK linked to
microtiter plates. HK (1 µg/ml) was incubated in microtiter plate
wells in 0.1 M Na2CO3, pH 9.6, overnight at 37 °C. After blocking the wells with 0.2% BSA,
increasing concentrations of biotin-GPV20 in HEPES-Tyrode's buffer, pH
7.35, containing containing 2 mM CaCl2, 1 mM MgCl2, and 50 µM
Zn2+ was incubated up to 2 h at 37 °C in the
absence or presence of 50-fold molar excess of HK or peptide GPV15. The
amount of biotin-HK bound was demonstrated as described above for
biotin-HK bound to endothelial cells.
Influence of Cytokeratin 1 Peptides on Prekallikrein Activation
on Endothelial Cells--
Investigations were performed to determine
if competing HK binding to endothelial cells by peptides to its binding
domain on cytokeratin 1 could influence PK activation. Confluent
monolayers of endothelial cells were incubated with 20 nM
HK in HEPES-Tyrode's buffer containing 50 µM
Zn2+ (1) in the absence or presence of 100 or 500 µM GYG20, GPV15, or PGG15 for 1 h at 37 °C. After
incubation, the cells were washed, and PK (20 nM) was added
for an additional hour of incubation. At the conclusion of the
incubation, the cells were washed and 0.4 mM
H-D-Pro-Phe-Arg-p-nitroanilide (S2302)
(Dia-Pharm, Franklin, OH) was added in the same buffer and hydrolysis
proceeded for 1 h at 37 °C. Additional experiments were
performed to determine if increasing concentrations of peptide PGG15
(0.003-500 µM) inhibited biotin-HK binding (7 nM) and PK activation in a
concentration-dependent fashion.
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RESULTS |
Characterization of Recombinant Cytokeratins--
In order to map
the domains on CK1 which HK binds, recombinant CK1s were expressed in
E. coli by a GST gene fusion system (Figs.
1 and 2).
Since both the amino- and carboxyl-terminal globular end domains of CK1
are highly enriched with glycine, and glycine is toxic to cells, we
designed recombinant proteins that avoided these regions (13, 16).
Three recombinant proteins were produced. Recombinant CK114
was protein-coded by exons 3-5; rCK128 was protein partially coded by exon 1 and all of exons 2-5; and rCK131
was protein partially coded by exons 1 and 7 and all of exons 2-6 (Fig. 1). A fourth recombinant protein
(Deleted1-6rCK131) was identical in size and
immunoreactivity to rCK131 but was missing the first 6 amino acids of its amino terminus (data not shown). The name of each
recombinant protein is based upon its expected size or the migration of
the isolated fragment on 12% SDS-PAGE (data not shown). The GST-CK1
fusion proteins migrated on 12% SDS-PAGE with an apparent molecular
mass of 40- (rCK114), 55- (rCK128), and 58-kDa
(rCK131) protein bands when stained with Coomassie Blue
(Fig. 2, top panel). Since GST migrated with the approximate
molecular mass of 26-31 kDa, small amounts of GST alone comigrated
with isolated rCK128 and rCK131. Therefore we examined the isolated rCK1s by immunoblot for molecular mass. Using
monoclonal antibody C1801 on immunoblot, detectable major bands of the
rCK1s were seen at 14, 28, and 31 kDa, respectively, corresponding to
the predicted size of the recombinant CK1 proteins (Fig. 2,
middle panel). Using a monoclonal antibody that is not directed to CK1 (C2931), no protein bands were seen (data not shown).
Furthermore, using an affinity purified polyclonal antibody reared to a
peptide from exon 5 of CK1 (anti-CAE18), the isolated rCK114, rCK128, and rCK131 were
also detected by immunoblot at the predicted size (Fig. 2, bottom
panel). The higher bands seen on the immunoblots with
rCK128 and rCK131 with both antibodies probably
represented residual intact fusion protein detected by immunoblot.
These results indicated that the recombinant proteins were recognized
by antibodies that identify cytokeratin 1.

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Fig. 1.
Diagram of exon structure of human
cytokeratin 1 and overlapping recombinant proteins and peptides used to
characterize the high molecular weight kininogen binding domain.
The middle structure shows a schematic diagram of
the first 7 exons of human cytokeratin 1 according to Johnson et
al. (13). The numbers within each exon diagram
represent the number of amino acids (aa) in each of the
exons. The three dark lines above the exon diagram represent
the span of each of three recombinant CK1s (rCK131,
rCK128, and rCK114) that were used to locate
the HK binding region. The three letters and 20 between the heavy black lines below the exon diagram
(GYG20, NQS20, SRE20, VRF20, VDT20, SRR20, and
NMQ20) represent 7 sequential 20 amino acid peptides that
partially span the protein coded by exons 1-3. The three peptides
(GPV15, PGG15, and EVT14) below peptides GYG20 and NQS20 represent
peptides used for the fine mapping of the HK-binding site on
cytokeratin 1. The full sequence of each of these peptides along with
their location on cytokeratin 1 is given under "Experimental
Procedures."
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Fig. 2.
Characterization of recombinant CK1.
Top panel, SDS-PAGE. Two to 3 µg of three
glutathione transferase (GST) recombinant cytokeratin 1 (rCK1) fusion
proteins (GST-rCK114, GST-rCK128,
and GST-rCK131) were subjected to 12% SDS-PAGE
after reduction with 2% -mercaptoethanol and boiling. Isolated
rCK1s (rCK114, rCK128, and
rCK131) separated from the GST fusion protein
also were subjected to 12% SDS-PAGE and electroblotted onto
nitrocellulose paper. An immunoblot was performed with monoclonal
antibody C1801 (middle panel, C 1801 Ab) and
anti-CAE18 (bottom panel, Anti-CAE18) as
described under the "Experimental Procedures." The top
figure is a photograph of a SDS-PAGE stained with Coomassie Blue R250,
and the middle and bottom figures are photographs
of autoradiograms of chemiluminescence. The numbers to the
right of the gels represent molecular mass markers in
kilodaltons.
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Biotin-HK Binds to Recombinant CK1--
Initial investigations
determined if the rCK1s could bind HK in a Zn2+-requiring
mechanism as native CK1 (11) (Fig. 3).
Recombinant CK1 (rCK131, rCK128, and
Deleted1-6rCK131) were coupled to microtiter
plates followed by treatment with biotin-HK (Fig. 3). Incubation of
biotin-HK with the microtiter plates coated with the rCK131
and rCK128, but not
Deleted1-6rCK131, resulted in increased
specific biotin-HK binding over time only in the presence of 50 µM Zn2+ (Fig. 3). These data indicated that
the HK specifically bound directly to rCK131 and
rCK128 but not
Deleted1-6rCK131, and the binding was Zn2+-dependent. These data
suggested that the six amino acids at the amino terminus of
rCK131 and rCK128, but not
Deleted1-6rCK131, participated in HK binding
to CK1 (Table I).

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Fig. 3.
Biotin-HK binds to rCK1s. Recombinant
CK131 (A), rCK128 (B),
and Deleted1-6rCK131 (C) were
linked to microtiter plates. After blocking, the cuvette wells were
washed and then incubated with biotin-HK (7 nM) in the
absence ( ) or presence ( ) of 50 µM Zn2+
or a 50-fold molar excess of HK ( ) in the presence of 50 µM Zn2+ (see "Experimental Procedures").
Total biotin-HK binding to each recombinant protein in the presence or
absence of 50 µM Zn2+ was determined and
compared with the level of binding seen in the presence of zinc ion and
a 50-fold molar excess of HK. The data presented are the mean ± S.E. of triplicate points of three independent experiments.
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Table I
The variable influence of overlapping amino acid sequences of
recombinant proteins and peptides coded by exon 1 of CK1 on biotin-HK
binding to endothelial cells
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Domains of HK That Participate in Biotin-HK Binding to
rCK128--
Since biotin-HK bound to certain rCK1, studies
were first performed to determine which cell binding domain(s) on
kininogen bound to CK1 (5, 12). Investigations determined which
synthetic peptides from each cell binding region on domains 3 and 5 of
kininogen blocked biotin-HK binding to rCK128 (12, 20)
(Fig. 4). By using peptides from domain 5 of HK (HVL, HKH) and domain 3 of both kininogens (LDC), there was
progressive inhibition of biotin-HK binding to rCK128 over
a range of concentrations from 1 to 300 µM (Fig. 4). At
300 µM, HVL, HKH, and LDC blocked biotin-HK binding to
rCK128 by 90 ± 3.8, 70 ± 0.5, and 74 ± 1.7%, respectively. Under the same conditions of this experiment, 0.5 µM LK blocked the biotin-HK binding to rCK128
by 77 ± 1.4%. Alternatively, a peptide from human coagulation
factor X (FNQ) had no significant inhibitory effect. These data
indicated that HK interacted with rCK128 by regions on both
its heavy chain domain 3 and light chain domain 5.

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Fig. 4.
Determination of the domains of HK that
inhibit biotin-HK binding to rCK128. Recombinant
CK128 was coupled to microtiter plates. After blocking, the
cuvette wells were washed and then incubated with biotin-HK (7 nM) and 50 µM Zn2+ in the absence
or presence of a 50-fold molar excess of HK, various peptides (HVL,
HKH, LDC, and FNQ), or low molecular weight kininogen (LK)
at the indicated concentrations. After incubation, the level of
specific biotin-HK binding to the HUVEC was determined by subtracting
nonspecific binding, i.e. the level of binding seen in the
presence of a 50-fold molar excess of HK, from that determined in the
presence of each of the peptides or LK. The figure represents the
mean ± S.E. of triplicate points from 3 to 4 separate
experiments.
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Recombinant CK1 That Inhibit Biotin-HK Binding to
HUVEC--
Investigations next turned to map the HK binding region on
CK1. Since HK bound to CK1 (11) and rCK128 and
rCK131 (Fig. 3), we postulated that rCK1s may inhibit
biotin-HK binding to HUVEC if they contained a cell binding domain for
kininogens. Recombinant CK128 inhibited biotin-HK binding
to HUVEC with an IC50 of 0.4 µM (Fig.
5A). Alternatively,
rCK114 did not inhibit binding (Fig. 5A).
Recombinant CK131 also inhibited biotin-HK binding to HUVEC with an IC50 of 0.5 µM (Fig. 5B).
At 1 µM, rCK128 and rCK131
inhibited biotin-HK binding 65 and 75%, respectively (Fig. 5,
A and B). Alternatively, a six-amino acid,
amino-terminal deletion mutant of rCK131
(Deleted1-6rCK131) did not inhibit binding of biotin-HK to HUVEC to the same extent (Fig. 5B and Table I). Rather, 1 µM Deleted1-6rCK131
blocked HK binding by 25%, a level of inhibition similar to that seen
with GST alone. These data suggested that protein coded by exon 1 of
CK1 including the amino-terminal portion of rCK131
contained a region that participated in HK binding to CK1.

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Fig. 5.
Inhibition of biotin-HK binding to HUVEC by
recombinant cytokeratin 1. Monolayers of HUVEC were incubated with
biotin-HK (7 nM) in the absence or presence of increasing
concentrations of isolated recombinant cytokeratins or GST protein.
A shows the concentration-dependent inhibition of
biotin-HK binding by rCK114 ( ) and rCK128
( ). B shows the concentration-dependent
inhibition of biotin-HK binding by GST ( ), rCK131 ( ),
or Deleted1-6rCK131 ( ). The figure
represents the mean ± S.E. of the percent biotin-HK bound when
compared with uninhibited binding of each data point in triplicate from
3 to 5 separate experiments.
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Inhibition of Biotin-HK Binding to HUVEC by Peptides of
CK1--
Investigations next were performed to map the biotin-HK
binding region on CK1 by using 7 sequential 20-amino acid peptides that
spanned portions of the protein partially coded by exons 1 and 3 and
all of exon 2 (amino acids 137-276, Fig. 1 and Table I) (13). The
peptide strategy was developed to confirm the recombinant protein
strategy. On close inspection, the rCK1s began 17 amino acids
carboxyl-terminal to the end of the glycine-rich region of the amino
terminus of the protein coded by exon 1 of CK1 (Table I). Only peptide
GYG20, which is 17 amino acids amino-terminal to the amino terminus of
rCK128 and rCK131, inhibited biotin-HK binding
to HUVEC with IC50 of 35 µM (Figs.
6A, Table I). None of the
remaining 6 sequential peptides, containing amino acids 157-276,
significantly inhibited biotin-HK binding to HUVEC (Fig. 6,
A and B). These data also suggested that a
HK-binding site on CK1 was localized to protein coded by exon 1.

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Fig. 6.
Inhibition of biotin-HK binding to HUVEC by
CK1 peptides. Biotin-HK (7 nM) was incubated with
endothelial cells in the absence or presence of 50-fold molar excess HK
or various concentrations of 20 amino acid, sequential peptides of
human CK1 (A, GYG20 ( ), SRE20 ( ), and VRF20 ( );
B, NQS20 ( ), VDT20 ( ), SRR20 ( ), and NMQ20 ( );
see "Experimental Procedures"). After incubation, the level of
bound biotin-HK was determined as described under "Experimental
Procedures." The data are mean ± S.E. of triplicate
determinations from three different experiments.
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Fine Mapping of the HK-binding Site on CK1--
In order to map
further the sequence on CK1 that bound HK, we synthesized three
overlapping 15 amino acid peptides localized to the inhibitory region
of the protein produced by exon 1 (Fig. 1 and Table I) and determined
which peptide(s) produced the greatest inhibition of biotin-HK binding
(Fig. 7). Peptides GPV15 and PGG15 inhibited biotin-HK binding to HUVEC with IC50 at 18 and 9 µM, respectively (Fig. 7A and Table I). These
data indicated that a biotin-HK binding domain on HUVEC is localized to
a 20-amino acid sequence on the protein coded by exon 1 of CK1.
Peptides GPV15 and PGG15 also blocked biotin-HK binding to cytokeratin with IC50 of 20 and 3 µM, respectively, as
shown in Fig. 7B. The next overlapping peptide, EVT14, did
not achieve an IC50 at 300 µM on HUVEC or
cytokeratin, respectively (Fig. 7). The specificity of these
interactions was shown by the fact that a scrambled peptide of PGG15
did not inhibit biotin-HK binding to HUVEC or purified cytokeratin
(Fig. 7, A and B). These data indicated that a
biotin-HK binding domain for CK1 is localized to a 20-amino acid
sequence between Gly143 and Gln162 on the
protein coded by exon 1 of CK1.

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Fig. 7.
Fine mapping of the binding region of CK1 for
biotin-HK. A, studies on endothelial cells. Biotin-HK
(7 nM) was incubated with endothelial cells in the absence
or presence of 50-fold molar excess HK or various concentrations of 15 amino acid, overlapping peptides coded by exon 1 of human CK1 (GPV15
( ), PGG15 ( ), EVT14 ( ), or a scrambled PGG15 ( ), see
"Experimental Procedures"). B, studies on cytokeratin.
Cytokeratin purchased from Dako was linked to microtiter plates. After
blocking, the cuvette wells were washed and then incubated with
biotin-HK (7 nM) and various concentrations of 15 amino
acid, overlapping peptides coded by exon 1 of human CK1 (GPV15 ( ),
PGG15 ( ), EVT14 ( ), or a scrambled PGG15 ( ); see
"Experimental Procedures"). After incubation, the level of bound
biotin-HK in both panels was determined as described under
"Experimental Procedures." The data are mean ± S.E. of
triplicate determinations from three different experiments.
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Ability of Biotin-GPV20 to Bind to HK--
In order to show
further the specificity of the above 20-amino acid region of the
non-glycine-rich region of the protein coded by exon 1 of CK1 to bind
to HK, a biotinylated form of the peptide GPV20 was prepared.
Increasing concentrations of biotin-GPV20 bound to HK linked to
microtiter plates, and this binding was blocked by a 50-fold molar
excess peptide GPV15 or HK (Fig. 8).

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Fig. 8.
The direct binding of biotin-GPV20 to
HK. HK was linked to microtiter plates overnight. After blocking
the wells, the cuvettes were washed and then incubated with increasing
concentrations biotin-GPV20 (*GPV20) in the absence ( ,
*GPV20 alone) or presence ( , *GPV20 + HK) of 50-fold molar excess HK
or peptide GPV15 ( , *GPV20 + GPV15). The level of biotin-HK bound
was determined as described under "Experimental Procedures." The
data are mean ± S.E. of triplicate determinations from three
different experiments.
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Influence of CK1 Peptides on Prekallikrein Activation on
HUVEC--
Previous investigations have shown that the binding of HK
to endothelial cells is essential for PK activation by a
cell-associated cysteine protease (1). Investigations next were
performed to determine if peptides of the HK binding domain on CK1
blocked PK activation on HUVEC (Fig.
9A). Increasing concentrations
of peptides GYG20, GPV15, or PGG15 were inhibitory to PK activation on
endothelial cells. Peptides GYG20 at 500 µM blocked PK
activation by 45 ± 3%. Peptides GPV15 or PGG15 were more potent
inhibitors of PK activation producing 80% inhibition at 100 µM and 95% inhibition at 500 µM. These
data indicated that blocking HK binding to endothelial cells with
peptides of its binding domain on CK1 blocked PK activation on
endothelial cells.

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Fig. 9.
Influence of CK1 peptides on PK activation on
HUVEC. A, confluent monolayers of endothelial cells
were incubated with 20 nM HK in the absence
(HK+PK) or presence of 100 or 500 µM GYG20,
GPV15, or PGG15. After incubation, the cells were washed, and PK (20 nM) was added. The cells then were washed again and 0.4 mM H-D-Pro-Phe-Arg-p-nitroanilide
was added and hydrolysis of the substrate was measured for 1 h.
B, confluent monolayers of endothelial cells were incubated
with 20 nM HK or biotin-HK in the absence or presence of
increasing concentrations of PGG15 for 1 h at 37 °C. After
incubation, the cells were washed, and those wells that were incubated
with unlabeled HK received PK (20 nM) for an additional
hour of incubation. These cells then were washed again, and 0.4 mM H-D-Pro-Phe-Arg-p-nitroanilide
was added in the same buffer, and hydrolysis of the substrate ( , PK
activation) was measured for an hour. After the first incubation, those
wells that received biotin-HK ( , % biotin-HK bound) were examined
for the level of bound biotin-HK as described under "Experimental
Procedures." In both panels, the data represent the mean ± S.E.
of three triplicate experiments.
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Further studies were performed to determine if there was a correlation
between inhibition of biotin-HK binding to HUVEC and inhibition of
prekallikrein activation by peptide PGG15 (Fig. 9B). As the
concentration of peptide PGG15 increased from 0.003 to 100 µM, there was a progressive decrease in prekallikrein
activation and HK binding (Fig. 9B). The IC50 of
peptide PGG15 on prekallikrein activation and HK binding was 1 and 10 µM, respectively. These data indicated that at 3 µM peptide PGG15, there was over 80% inhibition of PK
activation when there was about 20% inhibition of HK binding.
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DISCUSSION |
This study has four major findings. First, synthetic peptides of
domains 3 and 5 of HK inhibited the biotin-HK binding to rCK128. These data are similar to those found previously
that HK interacts with purified cytokeratin by the same regions of the
protein (i.e. its heavy and light chain) that interact with endothelial cells (11, 12, 20). Second, rCK128 and
rCK131 are inhibitors of biotin-HK binding to HUVEC with
IC50 of 0.4 to 0.5 µM. Previously, we
reported that commercially prepared cytokeratin partially blocked
biotin-HK binding to HUVEC (11). The present data confirm those
findings and suggest that the HK binding competitor may be localized to
a portion of cytokeratin 1 coded by exon 1. Further investigations
revealed that a biotin-HK binding domain on cytokeratin 1 is localized
to 20 amino acids (Gly143 to Gln162) beyond the
glycine-rich region in the globular region of human CK1 of the protein
coded by exon 1. Last, peptides of the HK binding region on cytokeratin
1 interfere with prekallikrein activation on endothelial cells (1, 3).
These data confirm that HK binding to HUVEC is an essential step for
endothelial cell prekallikrein activation (1, 3).
Both the amino- and carboxyl-terminal globular end domains of CK1 are
known to be enriched with glycine. CK1 is known to be insoluble because
of its glycine-rich regions (21). Preparation of the full-length
recombinant CK1 and peptides extending through the glycine-rich region
for this investigation was not feasible. We succeeded in preparing
recombinant protein fragments of CK1 carboxyl-terminal to its
glycine-rich region. It was helpful to find that both
rCK131 and rCK128 directly bind to HK and
inhibit biotin-HK binding to HUVEC. These data suggest that the six
amino acids ( ... VTINQS ... ) seen in rCK131 but
not Deleted1-6rCK131 participate in HK binding
to isolated cytokeratin and cytokeratin on HUVEC (11). The observation
that inhibition of biotin-HK binding by rCK131 and
rCK128 is not stronger is probably due to the fact that
only a small portion of the domain on CK1 that HK binds is actually
contained in these recombinant proteins. Combining synthetic peptide
studies with the recombinant protein data serves to define better the
region on the protein coded by exon 1 of CK1 that interacts with HK. By
using native sequences, scrambled peptides, and biotinylated peptides
of CK1, we are able to localize a 20-amino acid region from
Gly143 to Gln162 which binds HK. Since we were
unable to prepare either peptides or recombinant proteins more
amino-terminal to this region, we cannot exclude an additional role of
the first 136 amino acids of CK1 in HK binding. Our data map one site
on CK1 where HK binds even though HK itself interacts with CK1 by
regions on both its heavy and light chains.
The possible role of CK1 in the plasma kallikrein/kinin system was
recognized by our observation that biotin-HK binds to commercially prepared cytokeratin in a concentration-dependent fashion
in the presence of Zn2+ (11). In addition to showing that
various forms of recombinant CK1 interfere with HK binding to HUVEC,
the present report shows that PK activation is inhibited by peptides of
CK1 that block HK from binding to endothelial cells. Inhibition of
prekallikrein activation by CK1 peptides of the cell binding region of
HK occurred at a 10-fold lower concentration than inhibition of HK
binding itself. It is not known at this time whether the effect of the CK1 peptide to block PK activation is merely the inhibition of HK
binding to HUVEC, an essential requirement for PK activation on these
cells, or an actual interference with PK activation that is signaled
through CK1 on the membrane of HUVEC (1, 3). The finding, however, that
a 3 µM concentration of peptide PGG15 blocks 80% of
prekallikrein activation while it only inhibits 20% of HK binding
suggests that HUVEC CK1 may participate in the events that lead to PK
activation (1). These data also suggest that modulation of expression
of CK1 could result in regulation of prekallikrein activation and thus
any proteolytic reactions dependent upon it, i.e. bradykinin
liberation and factor XII and single chain urokinase activation (1, 3).
This interpretation indicates a new regulatory mechanism of the plasma
kallikrein/kinin system.
The present report solidifies the recognition of CK1 as a
kininogen-binding protein, putative receptor, on the membranes of HUVEC. Recent preliminary studies from another laboratory (22) also
confirm that HK binds to CK1. CK1 along with gC1qR and urokinase plasminogen activator receptor may constitute a multiprotein receptor complex for kininogens on cells in the intravascular compartment (23).
Regulation of kininogen binding on cells in the intravascular compartment may have a number of consequences on vascular biology. The
presence of HK on endothelial cells is a necessary component for PK
assembly and activation (1-3). PK activation on endothelial cells
results in bradykinin liberation, a potent mediator of vascular cell
stimulation, and cellular fibrinolysis independent of tissue plasminogen activation and fibrin (1-3). Further investigations are
needed to determine how the binding of the HK·PK complex to CK1
initiates prekallikrein activation.