From the Department of Biochemistry and Molecular
Biology, Medical University of South Carolina, Charleston, South
Carolina 29425 and the § Departamento de Biofisica, Escola
Paulista de Medicina, 100 Rua Tres de Maio, Sao Paulo 04044-020, Brazil
Received for publication, June 30, 2000, and in revised form, September 10, 2000
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ABSTRACT |
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Kallistatin is a heparin-binding serine
proteinase inhibitor (serpin), which specifically inhibits human tissue
kallikrein by forming a covalent complex. The inhibitory activity of
kallistatin is blocked upon its binding to heparin. In this study we
attempted to locate the heparin-binding site of kallistatin using
synthetic peptides derived from its surface regions and by
site-directed mutagenesis of basic residues in these surface regions.
Two synthetic peptides, containing clusters of positive-charged
residues, one derived from the F helix and the other from the region
encompassing the H helix and C2 sheet of kallistatin, were used to
assess their heparin binding activity. Competition assay analysis
showed that the peptide derived from the H helix and C2 sheet displayed
higher and specific heparin binding activity. The basic residues in
both regions were substituted to generate three kallistatin double mutants K187A/K188A (mutations in the F helix) and K307A/R308A and K312A/K313A (mutations in the region between the H helix and C2
sheet), using a kallistatin P1Arg variant as a scaffold. Analysis of
these mutants by heparin-affinity chromatography showed that the
heparin binding capacity of the variant K187A/K188A was not altered,
whereas the binding capacity of K307A/R308A and K312A/K313A mutants was
markedly reduced. Titration analysis with heparin showed that the
K312A/K313A mutant has the highest dissociation constant. Like
kallistatin, the binding activity of K187A/K188A to tissue kallikrein
was blocked by heparin, whereas K307A/R308A and K312A/K313A retained
significant binding and inhibitory activities in the presence of
heparin. These results indicate that the basic residues, particularly
Lys312-Lys313, in the region between the
H helix and C2 sheet of kallistatin, comprise a major heparin-binding
site responsible for its heparin-suppressed tissue kallikrein binding.
Kallistatin is a specific serine proteinase inhibitor (serpin) for
human tissue kallikrein (1). It inhibits tissue kallikrein by forming a
covalent enzyme-inhibitor complex (2). Our previous study showed that
kallistatin also has binding activity toward chymotrypsin, but it
behaves more like a substrate for chymotrypsin (1). In addition to
acting as a proteinase inhibitor, kallistatin also has hypotensive and
vasodilative effects independent of its inhibitory activity toward
tissue kallikrein (3, 4). Although the physiological functions of
kallistatin have not been well defined, previous studies indicate that
kallistatin acts as a multifunctional protein that exhibits unique
functions at different tissues (4).
The reactive-center loop, particularly the P1 residue, determines the
inhibitory specificity of a serpin. Substitutions of the P1 residue of
kallistatin dramatically change the inhibitory specificity of
kallistatin (1). High specificity and selectivity of kallistatin for
tissue kallikrein is determined by its unique Phe at P1 position and
probably also by other subsite-binding residues in the reactive-center
loop (1). Other than the reactive-center loop, heparin-binding sites of
a serpin can also regulate its inhibitory activity toward target serine
proteinase. Kallistatin is one of the heparin-binding serpins, which
include antithrombin, protein C inhibitor, plasminogen activator
inhibitor, heparin cofactor, and protease nexin. For most of the
heparin-binding serpins, heparin accelerates the inhibitory activity
toward their target serine proteinases (5-9). The mechanisms have been
explained by using a ternary complex and an allosteric model (5, 6, 10-13). Unlike most of these serpins, heparin suppresses the
inhibitory activity of kallistatin toward tissue kallikrein, whereas it
accelerates the interaction between kallistatin and chymotrypsin (1).
The mechanism by which heparin inhibits the interaction between
kallistatin and tissue kallikrein has not been explored.
The heparin-binding site of a serpin is pivotal not only in regulating
the inhibitory specificity of a serpin but also in directing a serpin
to its target tissues to perform its function (11, 14-16). It has been
demonstrated that heparan sulfate proteoglycans located on the
extracellular matrix and surface of vascular smooth muscle cells and
endothelial cells can trigger the inhibition of proteinases by bringing
serpins into close apposition with their target proteinases (14-16).
As in the case of other heparin-binding serpins, the heparin binding
activity of kallistatin may play an important role in its function on
cellular surfaces. The heparin-binding sites of proteins usually
contain clusters of basic amino acid residues, which provide positively
charged regions for binding to the acidic groups of heparin by
electrostatic interaction (17). Heparin-binding sites of antithrombin,
plasminogen activator inhibitor, protease nexin, and heparin cofactor
II have been located in the D helix (6, 11, 12, 18-20), whereas that
of protein C inhibitor is located in the H helix (21). The location of
the heparin-binding site in kallistatin, however, has not been identified.
In the present study, we attempted to identify the heparin-binding
sites in kallistatin by two approaches: 1) to design synthetic peptides
derived from surface regions of kallistatin that contain a high density
of basic residues and to assess their heparin binding capacity; 2) to
create kallistatin mutants by substituting basic residues with alanine
in the putative heparin-binding regions and to analyze the heparin
binding capacity of these mutants as well as the effects of heparin on
the inhibitory activity of kallistatin toward tissue kallikrein.
Identification of heparin-binding sites in kallistatin could provide
significant insights into the structural and functional relationship of
kallistatin, and pave the way for further studies of the regulation and
the physiological function of kallistatin.
Materials--
Escherichia coli strain TOP10, the
pTrc-His B expression vector was purchased from Invitrogen (San Diego,
CA); restriction enzymes, and isopropylthio- Molecular Modeling of Kallistatin--
The atomic coordinates of
the intact serpins, Design of Synthetic Peptides--
Surface regions of kallistatin
were determined by the kallistatin model created by the SYBYL
program and a program, PEPTIDESTRUCTURE, in Wisconsin Package
Version 10.0 (Genetics Computer Group (GCG), Madison, WI). The
synthetic peptides were designed according to surface regions that
encompass a high density of positively charged residues.
Construction of Kallistatin Mutants Containing Mutations at
Putative Heparin-binding Sites--
The kallistatin variant P1Arg,
created as described previously (23), in the prokaryotic expression
vector pTrc His B was used as a backbone to construct kallistatin
mutants containing mutations at putative heparin-binding sites. The
expression vector adds a hexahistidine sequence at the N terminus of
the recombinant kallistatin for protein purification by metal-affinity
chromatography. The kallistatin variants K307A/R308A and K312A/K313A
were designed to contain alanine replacing the basic amino acid
residues in the putative heparin-binding sites of the region between
the H helix and C2 sheet and the other variant K187A/K188A in the F helix (Fig. 1). K307A/R308A and K312A/K313A were created by
site-directed mutagenesis of kallistatin P1Arg cDNA using a
sequence overlap-extension PCR method (24). The internal
primers
5'-CAACTTGTTGCGGGCGGCGAATTTTTACAAG-3'/5'-CTTGTAAAAATTCGCCGCCCGCAACAAGTTG-3' were employed to replace Lys307 and Arg308 with
Ala to create the kallistatin mutant, K307A/R308A, and
5'-GAATTTTTACGCGGCGCTAGAGTTGC-3'/5'-GCAACTCTAGCGCCGCGTAAAAATTC-3' were used to replace Lys312 and Lys313 with Ala
to create the mutant K312A/K313A. The mutated nucleotides are
underlined. The outside primers were 5'-CGTCTATGAGGCTAAC-3' and 5'-CTATGGTTTCGTGGGGT-3' for both mutants. The mutant fragments synthesized by PCR were cleaved with XhoI and
SalI and replaced the counterparts of the pTrc His-based
kallistatin P1Arg expression plasmids excised by the same
restriction enzymes. The mutant K187A/K188A was created by the PCR
method described previously. The primers 5'-TGATGGTGAGAGTTGCA-3' and
5'-TCCCTCGAGTTTCCGCCGCGACGTGGTCG-3', encompassing the
desired alanine codon substitutions (underlined) for
Lys187-Lys188 and a XhoI site, were
used to synthesize the mutant fragments of K187A/K188A by PCR. The PCR
fragment was cleaved with SmaI/XhoI and cloned
into the kallistatin P1Arg expression plasmid that had its
counterpart excised by the same restriction enzymes. The mutations
were confirmed by DNA sequencing.
Expression and Purification of the Kallistatin Mutants--
The
kallistatin mutants containing Ala substitutions at the putative
heparin-binding sites were expressed in a large scale of cell culture,
and then soluble cell lysates were isolated and purified by
nickel-affinity (nickel-nitrilotriacetic acid, Qiagen) and
heparin-affinity chromatography as described previously (23). Protein
purity was assessed by SDS-PAGE and staining by Coomassie Blue.
Concentrations of the mutants were determined by an enzyme-linked immunosorbent assay specific for human kallistatin (4).
Peptide Competition by Kallikrein Binding Assay--
Synthetic
peptides, dissolved in Me2SO at 5 µM, were
preincubated with 0.05, 0.1, and 0.2 unit/ml heparin in 17 µl of
reaction buffer, 20 mM sodium phosphate, pH 8.0, at
37 °C for 20 min. The same amount of Me2SO was added to
the control groups containing no synthetic peptides. Subsequently, 1 µl of the recombinant kallistatin (concentration, 0.4 mg/ml) was
added into the reaction (final concentration, 0.4 µM) for
a 10-min incubation at 37 °C. Finally, 2 µl of
125I-labeled tissue kallikrein at 1 × 104
cpm/µl was incubated with the mixture at 37 °C for 60 min. The reactions were stopped by SDS-loading buffer, resolved in 10% SDS-PAGE, and analyzed by autoradiography. A dose-dependent
peptide competition assay was also performed to demonstrate the
specific binding of the peptides to heparin. Different concentrations
of the synthetic peptides were used to compete with 0.4 µM kallistatin for 0.05 and 0.1 unit/ml of heparin in a
20-µl reaction mixture. The synthetic peptides, F peptide at 2.5, 5.0, 7.5, 10 and 15 µM, HC2 peptide at 1, 2, 3, 4 and 5 µM, and C4 peptide at 5, 10 and 20 µM, were
used in the dose-dependent peptide competition assay.
Fluorescence Titration of Kallistatin and the Mutants with
Heparin--
A solution of kallistatin wild type (0.116 µM) in 50 mM Tris-HCl, 100 mM
NaCl, 0.2% (w/v) PEG 6000, pH 7.4, at 20 °C was titrated with small
aliquots of a high concentration of heparin solution with minimal
dilution (<10%). The protein fluorescence ( Competitive Binding Assay--
Kallistatin wild type (0.116 µM) was titrated with small aliquots of low molecular
weight heparin in the same conditions as described above in the
presence of different concentrations of synthetic peptides. The
apparent Kd (Kd-app) for the interaction of each peptide was determined by plotting the
observed Kd (Kd-obs)
determined for the interaction of each peptide versus its
concentration in the assay.
Heparin Affinity Chromatography--
The relative affinity of
kallistatin P1Arg and the kallistatin mutants for immobilized heparin
was determined by heparin-affinity chromatography in a BioCAD SPRINT
system. Samples of approximately 40-100 µg in 2 ml of 20 mM sodium phosphate buffer, pH 7.0, 20 mM NaCl,
were loaded onto an HE1/M column, which was saturated with the same
buffer. The flow rate was controlled at 1 ml/min. The column was washed
with the same buffer until the absorbance reached the base line. The
mutant proteins were eluted with 50 ml of a NaCl gradient from 0 to 500 mM. The elution was monitored by absorbance at 280 nm. The
salt gradient and elution profiles were plotted, and the concentrations
of NaCl at elution peak were measured. Three different batches of each
recombinant protein were analyzed for their heparin binding capacity.
Heparin-suppressed Tissue Kallikrein Binding Assay--
Tissue
kallikrein binding activity of the kallistatin mutants containing Ala
substitutions in the putative heparin-binding sites was assessed by
kallikrein-binding assay in the presence of different concentrations of
heparin. Kallistatin P1Arg and mutants at concentration 0.6 µM were preincubated with different concentrations of
heparin, 0, 2.5, 5, 10, 20 and 30 units/ml, in 18.5 µl of 20 mM sodium phosphate buffer, pH 8.0, at 37 °C for 10 min.
Then 1.5 µl of 125I-labeled tissue kallikrein at 1 × 104 cpm/µl was added into the reactions, and the
mixtures were incubated at 37 °C for 90 min. The samples were
resolved on 10% SDS-PAGE under reducing condition and analyzed by autoradiography.
Effect of Heparin on Tissue Kallikrein Inhibition Assay--
The
effects of heparin on the inhibitory activity of the kallistatin
mutants were analyzed by preincubating 0.2 µM of the kallistatin mutants with 0, 2, 5, 10, 20, and 30 units/ml heparin in 45 µl of 20 mM sodium phosphate buffer, pH 8.0, at 37 °C
for 10 min. The reactions were then started by adding 5 µl of 2 ng/µl tissue kallikrein (final concentration, 6 nM) into
the reaction mixtures and incubating at 37 °C for 90 min. The
residual tissue kallikrein activity was determined by adding 20 µl of
the reaction mixture in 2 ml of buffer containing 50 mM
Tris-HCl, pH 8.0, and 30 µM of Val-Leu-Arg-MCA. The rate
of substrate hydrolysis was monitored at 380-nm excitation and 460-nm emission.
Circular Dichroism (CD) Spectroscopy--
The conformation of
the kallistatin mutants was estimated by CD spectroscopy. CD spectra
were obtained using a Jasco J710 spectropolarimeter at a
resolution of 0.2 nm, with a bandwidth of 2 nm. Ten spectra were
averaged for each derivative. The recombinant kallistatins were diluted
in 20 mM sodium phosphate, pH 7.0, 200 mM NaCl
at a concentration of 0.4 mg/ml using a cell of 0.02-cm path length.
Mean residue ellipticity was calculated according to the literature
(26). Protein concentrations were derived from UV spectroscopy and
confirmed by a specific enzyme-linked immunosorbent assay (4).
Molecular Modeling of Kallistatin--
The molecular structure of
kallistatin was created by homology modeling as shown in Fig.
1A. Because the
reactive-center loop is a highly variable region among serpins, there
is no good template for modeling the reactive-center loop of
kallistatin. Our previous study suggested that replacing the
residues in the hinge region of the reactive loop with bulky residues
may hinder a partial insertion of the reactive loop into A Design of Synthetic Peptide--
Surface regions of kallistatin
were determined by the program PEPTIDESTRUCTURE in Wisconsin Package
Version 10.0 (Genetics Computer Group (GCG)). A kallistatin model was
created by homology modeling using COMPOSER, in SYBYL, to assist in the
selection of the surface regions. Two surface regions, the F helix and
the loop between the H helix and C2 sheet, contain clusters of
positive-charged residues (Fig. 1B). To test whether these
two highly positive-charged regions are capable of binding with
heparin, two synthetic peptides corresponding to both regions were
synthesized: F peptide,
VGTIQLINDHVKKETRGKIV-NH2, spanning amino acids 176-195, and HC2 peptide,
RWNNLLRKRNFYKKLELHLP-NH2, spanning
amino acids 300-319. A scrambled peptide SCR, derived from HC2
peptide, was synthesized as RWNRLFRYLKNEPKLHNLKL-NH2. The
putative heparin-binding sites are underlined. A negative control C4 peptide derived from the surface region around the C4 sheet
was synthesized as PFISSRTTPKDFYVDENTTV-NH2, spanning amino
acids 222-242. All synthetic peptides were dissolved in Me2SO for use.
Peptide Competition by Kallikrein Binding Assay--
Heparin
binding activity of the synthetic peptides was compared by peptide
competition assay as shown in Fig. 2.
Peptides with heparin binding activity can prevent heparin from binding to kallistatin by competing for the heparin-binding sites.
Consequently, an effective competing peptide can free kallistatin from
heparin binding and thus restore its tissue kallikrein binding
activity. Kallistatin P1Arg forms a complex with tissue kallikrein in
the absence of heparin (lane 1). The complex formation was
inhibited in the presence of different concentrations of heparin
(lanes 2, 5, and 8). The F peptide,
representing the F helix region of kallistatin, did not reverse the
inhibition of the complex formation by 0.2 and 01 unit/ml heparin
(lanes 3 and 6). Only at a lower heparin
concentration, 0.05 unit/ml, did the F peptide start to restore the
tissue kallikrein binding activity of kallistatin (lane 9).
The HC2 peptide, spanning the H helix and C2 sheet, however, restored
the tissue kallikrein binding activity of kallistatin efficiently under
0.2, 0.1, and 0.05 unit/ml heparin (lanes 4, 7,
and 10). Fig. 3 shows
dose-dependent competition of the peptides with kallistatin
for heparin. The HC2 and F peptides compete
dose-dependently for heparin and reverse the inhibition of
complex formation (Fig. 3, A and B). The HC2
peptide at 1 µM starts to show competition for 0.2 unit/ml heparin, whereas the F peptide at 5 µM competes for 0.1 unit/ml heparin. The control peptide C4, spanning over the C4
sheet, did not reduce the heparin inhibition of the tissue kallikrein-kallistatin binding at concentrations as high as 20 µM (Fig. 3C). These results suggest that
heparin-binding sites of kallistatin are likely located within the F
helix and the region between the H helix and C2 sheet, and the latter
region is probably the major heparin-binding site of kallistatin.
Competitive Binding Assay--
The heparin binding activity of
peptide HC2, which is relatively higher than that of peptide F2, was
further assessed by competitive binding assay. Titration of kallistatin
with heparin was performed in the presence of HC2 peptide at
concentrations of 0.984 and 5.920 µM or its scrambled
peptide SCR at 0.584 and 4.543 µM. Fig. 4 shows the equilibrium binding of
kallistatin with heparin in the presence of HC2 and SCR peptides. Table
I shows that the shift in the
Kd-obs value of kallistatin with heparin is more efficient in the presence of peptide HC2 as compared with SCR
under a low peptide concentration.
Kd-app for peptides HC2 and SCR were
calculated as the x intercept for the plot of Kd-obs versus peptide
concentration (Fig. 5). The obtained values were 0.395 µM for HC2 peptide and 2.574 µM for SCR peptide. These results indicate that peptide
HC2 derived from the sequence between the H helix and C2 sheet can
specifically bind to heparin not only by the positively charged
residues but also by the sequence and structure of the peptide.
Construction, Expression, and Purification of Kallistatin Mutants
Containing Mutations at Putative Heparin-binding Sites--
The role
of individual basic residues in the F helix and the region between the
H helix and C2 sheet of kallistatin was further defined by
site-directed mutagenesis. The kallistatin variant P1Arg, which has the
highest inhibitory activity toward human tissue kallikrein (1), was
used as a model for mutagenesis. The basic residues
Lys187-Lys188 in the F helix, and
Lys307-Arg308 and
Lys312-Lys313 in the region between the H helix
and C2 sheet were substituted with Ala, a neutral amino acid. Three
kallistatin double mutants, K187A/K188A, K307A/R308A, and
K312A/K313A, with double Ala substitutions for the basic residues, were
engineered. The recombinant kallistatins were expressed and purified to
apparent homogeneity (data not shown).
Heparin Binding Activity of Kallistatin Mutants--
Heparin
binding activity of kallistatin mutants was assessed by
heparin-affinity chromatography. The heparin binding activity of the
kallistatin variants with mutations in the putative heparin-binding sites was compared to kallistatin P1Arg. The results of the NaCl gradient elution pattern from a heparin-affinity column are shown in
Fig. 6 and summarized in Table
II. Kallistatin P1Arg, without mutation
in the putative heparin-binding sites, was eluted at 340 mM
NaCl. K187A/K188A, containing Ala substitutions for basic residues in
the F helix, was eluted at 330 mM NaCl, indicating that the
substitutions did not significantly interfere with its heparin binding
activity. Both K307A/R308A and K312A/K313A, containing mutations in the
region between the H helix and C2 sheet, showed reduced heparin binding
activity as indicated by their elution was at 270 and 220 mM NaCl, respectively. These results suggest that the basic
residues in the region between the H helix and C2 sheet are the
heparin-binding sites of kallistatin.
Dissociation Constants of Kallistatin and the Mutants--
The
relative heparin binding activity of K312A/K313A and K307A/R308A
mutants is lower than that of kallistatin P1Arg as measured by
heparin-affinity chromatography. Definitive heparin binding activity
was further determined by Kd. An accurate
Kd determination can be obtained when the titration
is performed at a protein concentration close to or lower than the
Kd value (25). Fig. 7
shows the relative fluorescence change
( Tissue Kallikrein Binding Activity of the Kallistatin Mutants in
the Presence of Heparin--
A tissue kallikrein binding assay was
performed in the presence of heparin to illustrate the effects of
heparin on the tissue kallikrein binding activity of the kallistatin
mutants as shown in Fig. 8. The complex
formation between tissue kallikrein and kallistatin P1Arg was inhibited
by heparin in a dose-dependent manner. The tissue
kallikrein binding activity of K187A/K188A was also inhibited by
heparin in the same manner. However, both K307A/R308A and K312A/K313A
variants retained their relatively strong binding to tissue kallikrein
as compared with P1Arg and K187A/K188A in the presence of heparin.
K312A/K313A appears to be completely resistant to heparin inhibition at
concentration as high as 30 units/ml. These results further confirm the
location of the heparin-binding site and indicate that the basic
residues in the heparin binding region of kallistatin are responsible
for its ability to suppress tissue kallikrein's activity.
The Inhibitory Activity of the Kallistatin Mutants toward Tissue
Kallikrein in the Presence of Heparin--
The inhibition of tissue
kallikrein activity by kallistatin mutants can be quantified by
measuring the residual amidolytic activities of tissue kallikrein
following incubation with kallistatin. The results are shown in Fig.
9. After 90-min incubation, the enzymatic
activity of tissue kallikrein was almost completely inhibited by all of
the kallistatin mutants in the absence of heparin. Preincubation with
heparin at 5 units/ml suppressed the inhibitory activity of kallistatin
P1Arg and K187A/K188A and completely restored the tissue kallikrein
activity. K307A/R308A and K312A/K313A mutants require a higher
concentration of heparin to suppress their inhibitory activity toward
tissue kallikrein. For K307A/R308A, preincubation with 5 units/ml
heparin caused approximately 50% restoration of the tissue kallikrein
activity. The tissue kallikrein activity was restored gradually to
about 85% when 30 units/ml heparin was used. K312A/K313A, however,
appeared to be more resistant to the inhibition by heparin. Even at 30 units/ml heparin, K312A/K313A still inhibited about 70% of tissue
kallikrein after the 90-min reaction. These results indicate that
Lys312-Lys313 acts as a major heparin-binding
site responsible for the inhibitory effect of heparin on tissue
kallikrein activity.
Estimation of the Conformation of the Kallistatin Mutants--
The
conformation of kallistatin mutants, P1Arg, K307A/R308A, and
K312A/K313A, were analyzed by CD spectroscopy. The spectra showed that
the wavelength-scanning profiles of K307A/R308A and K312A/K313A were
identical to that of the P1Arg mutant (Fig.
10). The results indicate that the
double Ala substitutions for the basic residues do not perturb the
secondary structure of kallistatin and that these mutants retain the
same structural integrity. The data demonstrate that the reduced
heparin affinity is not due to a conformational change.
In this study we identified the location of a major
heparin-binding site in the kallistatin molecule by peptide competition assay and by site-directed mutagenesis and protein engineering. Two
putative heparin-binding regions, the F helix and a region between the
H helix and C2 sheet, were identified by a sequence analysis program
and a molecular model of kallistatin. The HC2 peptide derived from the
region between the H helix and C2 sheet displays a strong and specific
heparin binding activity in competition assay. Mutagenesis at the basic
residues of the kallistatin molecule by double Ala substitutions showed
that K307A/R308A and K312A/K313A mutants, containing mutations in the
region between the H helix and C2 sheet, had reduced heparin binding
activity and were relatively resistant to the inhibition by heparin for
tissue kallikrein binding. The K187A/K188A mutant, containing Ala
substitutions in the F helix, retained the same heparin binding
activity as kallistatin, and both of their inhibitory activities were
suppressed by heparin. These results indicate that the major
heparin-binding site responsible for the inhibitory effect of heparin
is located in the region between the H helix and C2 sheet of kallistatin.
Heparin is a glycosaminoglycan that is a highly negative-charged
molecule. The heparin-binding sites of proteins usually consist of
consecutive basic amino acids that can bind to the acidic groups of
heparin by electrostatic interaction (17). Two regions in kallistatin
encompass clusters of basic amino acids: residues 174-186,
DHVKKETRGKIVD in the F helix, and residues 303-316, NLLRKRNFYKKLEL corresponding to the region between the H helix and C2 sheet. In a
peptide competition assay, the concentration of HC2 peptide required
was less than 1/10 of that of the F peptide to show competition with
kallistatin for heparin binding, suggesting the region between the H
helix and C2 sheet has a strong heparin binding activity. The
Kd-app values for peptide HC2 and its
scrambled peptide SCR are separated by almost a factor of 10 (0.395 and
2.574 µM for HC2 and SCR, respectively). This indicates
that the specific heparin binding of HC2 peptide is contributed not
only to the positively charged residues, but also to a specific
structure and residue composition.
Although the peptide competition assay showed the heparin-binding
capability of both regions, the results did not provide direct
relevance of those basic clusters to the heparin binding activity of
kallistatin. There are three consecutive basic residues, Lys187-Lys188,
Arg306-Lys307-Arg308, and
Lys312-Lys313, distributed in both regions in
kallistatin. To further address the importance of these basic clusters
in heparin binding, double Ala substitution mutants, K187A/K188A,
K307A/R308A, and K312A/K313A, were created to evaluate their heparin
binding activity.
As assessed by heparin-affinity chromatography, the K187A/K188A mutant
retained the same heparin-affinity capacity as P1Arg kallistatin.
Moreover, the K187A/K188A mutant exhibited the same extent of
heparin-suppressed effect on tissue kallikrein inhibition as
kallistatin P1Arg. These results indicate that
Lys187-Lys188 of kallistatin does not play a
significant role in heparin binding. The lack of the heparin binding
activity in the F helix can be explained by the scattering of three
acidic residues, Asp184, Glu189, and
Asp196, in this region that neutralizes the net positive
charge of the F helix.
Consistent with the results of the competition assay, the
relative heparin affinity of K307A/R308A and K312A/K313A mutants decreased as estimated by heparin-affinity chromatography, indicating that the basic residues,
Arg306-Lys307-Arg308 and
Lys312-Lys313, are responsible for heparin
binding. Although
Arg306-Lys307-Arg308 has higher
positive-charged density than Lys312-Lys313,
the significantly lower dissociation constant of the K312A/K313A mutant
indicates that Lys312-Lys313 are crucial
heparin-binding residues of kallistatin.
Studies of the heparin-binding sites of serpins have shown that several
basic residues in different regions coordinate for heparin binding but
not all the heparin-binding sites are related to heparin-accelerated
inhibition of proteinases (11-12, 21). Therefore, it is possible that
regions other than the loop between the H helix and C2 sheet in
kallistatin could also be involved in heparin binding. Nevertheless,
the heparin-suppressed tissue kallikrein-binding assays showed
resistance of both K307A/R308A and K312A/K313A to heparin suppression,
indicating that the basic residues in the loop between the H helix and
C2 sheet comprise not only a heparin-binding region but also are
related to heparin-suppressed tissue kallikrein inhibition.
Additionally, the CD spectra of the mutants showed that the double
mutagenesis did not perturb the secondary structure of kallistatin,
demonstrating that the reduced heparin affinity and resistance to
heparin suppression are not due to a conformational change. Compared
with Lys307-Arg308,
Lys312-Lys313 appeared to contribute more in
heparin binding, because mutant K312A/K313A displayed less relative
heparin affinity than mutant K307A/R308A. In agreement with the heparin
affinity data, the inhibitory activity of K312A/K313A toward tissue
kallikrein displayed a lower sensitivity to heparin suppression as
compared with that of K307A/R308A toward tissue kallikrein in the
heparin-suppressed inhibition assay. Taken together, our study
indicates that Lys312-Lys313 in kallistatin is
a major heparin-binding site that is responsible for heparin-suppressed
tissue kallikrein inhibition.
The heparin-binding sites of several serpins, such as antithrombin,
heparin cofactor II, protease nexin I, and plasminogen activator
inhibitor I, have been localized primarily in the D helix (6, 11, 12,
18-20). Protein C inhibitor, however, has a distinctive
heparin-binding site located in the H helix (21). Kallistatin does not
contain a high density of basic residues nor a conserved distribution
of basic residues in the D helix as analyzed by amino acid sequence
alignment with these serpins (Fig.
11). The heparin-binding site in
kallistatin is in the region between the H helix and C2 sheet, which is
adjacent to the H helix. In this region, kallistatin has a greater
basic charge density than other serpins. Molecular modeling of
kallistatin illustrates that
Arg306-Lys307-Arg308 is located at
the C-terminal end of the H helix and
Lys312-Lys313 at the N-terminal end of the C2
sheet. The heparin-binding residues, Arg306-Lys307-Arg308, in
kallistatin are conserved with the basic residues, 276-278, in the
C-terminal boundary of the H helix in protein C inhibitor (Fig. 7).
These conserved basic residues in protein C inhibitor are not related
to heparin-accelerated proteinase inhibition, although they can bind
heparin strongly (21). The heparin-binding residues
Arg269-Lys270, responsible for
heparin-accelerated proteinase inhibition, in the H helix in protein C
inhibitor, however, is absent in kallistatin. Instead, kallistatin has
the major heparin-binding site Lys312-Lys313 at
the N-terminal boundary of the C2 sheet, which is unique among serpins
(Fig. 11). The turn between the H helix and C2 sheet in kallistatin has
a 3-4 residue insertion as compared with those in other serpins (Fig.
11). This insertion extends C2 sheet and bulges the loop outward
according to the kallistatin model.
Lys312-Lys313 thus protrudes from the turn and
is positioned toward the reactive-center loop of kallistatin. At this
position, binding of heparin may easily affect the conformation of the
reactive-center loop or docking of the loop into a reactive cleft of a
serine proteinase.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactoside were from
Life Technologies, Inc. (Gaithersburg, MD); Taq polymerase
was from PerkinElmer Life Sciences (Norwalk, CT);
nickel-nitrilotriacetic acid-agarose was from Qiagen (Santa Clarita,
CA); the POROS HE/1 column was from PerSeptive Biosystems (Cambridge,
MA); Low molecular mass heparin (4500 Da) was purchased from
Rhône-Poulenc (Aventis) and Upjohn (Kalamazoo, MI);
D-Val-Leu-Arg-MCA (methylcoumarinamide) was from Enzyme
System Products (Livermore, CA); human tissue kallikrein was purified
as described previously (22); synthetic peptides were synthesized and
provided by Dr. L. Juliano (from the Department de Biofisica, Escola
Paulista de Medicima, Sao Paulo, Brazil).
1-antitrypsin (1qlp), ovalbumin (1ova),
antithrombin (1ant), and cleaved form of protein C inhibitor (1pai)
were obtained from the Protein Data Bank at Rutgers University.
A molecular model was created using the homology modeling module,
Composer, in the SYBYL program (version 6.5, Tripos, Inc.). The
topologically equivalent residues across these serpins were determined
first based on sequence homology. A structural alignment of the serpins
was then performed with the equivalent residues as a starting point.
This alignment determined the structurally conserved regions
(SCRs)1 as well as the
average framework of the SCRs. SCRs of kallistatin were generated by
using fragments of the homologs to construct the backbone of the SCRs
and a rule-based procedure to generate the side chains. The
structurally variable regions were constructed by using fragments from
known structures, which are compatible with the rest of the model, and
then using sequence information to postulate which single fragment is
best for use in the final model. The kallistatin model was then
refined by side-chain torsion relieving and energy minimization. The
backbone of the whole model was constrained and then energy minimized
by steepest descent until the maximum derivative was less than 50 kcal/(mol·Å). The constraint was then removed, and additional
minimization was performed until the maximum derivative was less than 5 kcal/(mol·Å) using steepest-descent algorithm. Finally, conjugate
gradient minimization continued until the maximum derivative was less
than 0.1 kcal/(mol·Å).
ex = 280 nm,
em = 340 nm, 10-nm slits) was measured in a 1-ml
quartz cuvette, thermostabilized, and held in a Shimadzu RF-1501
spectrofluorometer under constant agitation. The dependence of the
relative fluorescence change, i.e.
F/F0 = (F0
Fobs)/F0, where
F0 and Fobs represent starting and observed fluorescence values, respectively, can be analyzed by nonlinear least-squares data fitting by the binding equation (Eq. 1) (25) using the Grafit program (version 3.0, Erithacus
Software Ltd.).
In this equation, P0 is the total protein
concentration, H0 represents the heparin
concentration, Kd is the dissociation constant, and
(Eq. 1)
Fmax/F0 is
the maximum relative fluorescence change. For kallistatin P1Arg,
K312A/K313A, and K307A/R308A mutants, the concentrations in the assay
were 0.170, 0.141, and 0.160 µM, respectively.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet
and thus converting kallistatin from an inhibitor to a substrate for
tissue kallikrein (data not shown). We assumed that the partial
insertion of the hinge region is critical for the inhibitory
conformation of kallistatin. Therefore, the atomic coordinates of
antithrombin were chosen to model the reactive-center loop of
kallistatin, P16-P5', because the reactive loop of antithrombin has the
same number of residues as kallistatin and the hinge region of the
reactive loop was partially inserted into the A
-sheet as predicted
for serpins (27). The conformational energy of the kallistatin model is
approximately
1300 kcal·mol
1, indicating an
acceptable conformation without serious steric clash. The F helix, H
helix, and C2 sheet are indicated in Fig. 1. The length of the
kallistatin molecule is about 72 Å as measured from the protruding
reactive loop to the distal end of the molecule. The F helix is distant
from the reactive loop, and the distance measured between the C
of
the basic residues Lys187-Lys188 in the F helix
and the C
of the reactive center is about 46-48 Å. The loop
between the H helix and C2 sheet crosses through the reactive loop and
locates the basic residues
Arg306-Lys307-Arg308 and
Lys312-Lys313 in a proximate position to the
reactive center with distances of 20-25 and 11-13 Å,
respectively.
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Fig. 1.
Synthetic peptides derived from the surface
regions of kallistatin. The synthetic peptides encompassing
the putative heparin-binding sites were designed according to the
surface regions of kallistatin determined by PEPTIDESTRUCTURE in the
Wisconsin Package and by a kallistatin model created by COMPOSER in
SYBYL. Two surface regions around the F helix and the region between
the H helix and C2 sheet containing clusters of basic residues were
selected to synthesize the peptides, designated as F peptide and HC2
peptide. Another surface region around the C4 sheet was selected as a
negative control, designated as C4 peptide. A, the
kallistatin structure was created by homology modeling. The F helix, H
helix, and C2 sheet are labeled. The cyanic areas represent
the regions of the synthetic peptides as indicated. The basic residues
in both regions are shown as labeled. B, the secondary
structural features of kallistatin are overlined in the
amino acid sequence. The regions of the synthetic peptides are
shaded. The basic residues in the putative heparin-binding
regions are in boxes. The boldface characters
indicate the names of the synthetic peptides.
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Fig. 2.
Effects of the synthetic peptides on the
heparin-inhibited complex formation between kallistatin and tissue
kallikrein. F and HC2 peptides at 5 µM were used to
compete with 0.04 µM kallistatin for 0.2, 0.1, and 0.05 unit/ml heparin in 18 µl of reaction buffer, 20 mM sodium
phosphate, pH 8.0, at 37 °C for 20 min. A tissue kallikrein binding
assay was conducted by adding 2 µl of 125I-labeled tissue
kallikrein at 1 × 104 cpm/µl in each reaction at
37 °C for 60 min. The tissue kallikrein-kallistatin complexes were
analyzed by SDS-PAGE and autoradiography. The synthetic peptides and
the concentrations of heparin added in each tissue kallikrein-binding
assay are as indicated on each lane. -, without addition of
peptide.
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Fig. 3.
Dose-dependent effects of the
synthetic peptides on the heparin-inhibited complex formation between
kallistatin and tissue kallikrein. Different concentrations of the
synthetic peptides were used to compete with kallistatin for heparin in
the tissue kallikrein-binding assay. CNTL, tissue kallikrein
binding assay without addition of heparin and peptide. A,
HC2 peptide at different concentrations, 1, 2, 3, 4, and 5 µM, was added to compete with kallistatin for 0.1 unit/ml
heparin. B, F peptide at concentrations of 2.5, 5, 7.5, 10 and 15 µM was added to compete with kallistatin for 0.05 unit/ml heparin. C, C4 peptide at concentrations 5, 10, and
20 µM, was added to compete with kallistatin for 0.05 unit/ml heparin.
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Fig. 4.
Fluorescence titration of kallistatin for
heparin binding in the presence of the synthetic peptides. Heparin
was titrated in a solution of kallistatin at a concentration of 0.116 µM ( ) in the presence of 0.984 µM HC2
peptide (
), 5.920 µM HC2 peptide (
), 0.584 µM SCR (
), and 4.454 µM SCR (
).
Dissociation constant (Kd-obs) determination for kallistatin in
the presence of the synthetic peptides
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Fig. 5.
Apparent dissociation constants
(Kd-app) determination for HC2 and
SCR peptides. The x intercept for the plot of
Kd-obs versus peptide
concentration indicates Kd-app for
peptides HC2 ( ) and SCR (
).
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Fig. 6.
Heparin binding capacity of the kallistatin
mutants. The heparin binding activity of kallistatin P1Arg,
K187A/K188A, K307A/R308A, and K312A/K313A was assessed by
heparin-affinity chromatography. Approximately 40-100 µg of
kallistatins were loaded onto a heparin-affinity column (1.7-ml bed
volume) equilibrated with 20 mM sodium phosphate buffer, pH
7.0, 20 mM NaCl. The samples were eluted with 50 ml of NaCl
gradient from 0 to 500 mM in phosphate buffer at a flow
rate of 1.0 ml/min with detection at OD280. The
concentration of NaCl was monitored by a conductivity meter in the
BioCAD SPRINT system. The elution peaks for the kallistatin mutants are
as indicated.
Relative heparin affinity of the kallistatin mutants analyzed by
chromatography
F/F0) with increasing heparin
concentrations for kallistatin wild type (Fig. 7A), P1Arg
(Fig. 7B), K307A/R308A (Fig. 7C), and K312A/K313A (Fig. 7D). Table III
summarizes the Kd values for kallistatin and the
mutants, as well as their respective protein concentrations used for
the measurement. Kallistatin wild type and kallistatin P1Arg have
comparable Kd values. The Kd of
K307A/R308A and K312A/K313A mutants are 2.5- and 16-fold higher than
that of kallistatin P1Arg, respectively, indicating decreased heparin affinity of K307A/R308A and K312A/K313A mutants. The results
demonstrate that Lys307, Arg308,
Lys312, and Lys313 are responsible for heparin
binding activity of kallistatin, particularly Lys312 and
Lys313 residues.
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Fig. 7.
Equilibrium binding of heparin to
kallistatins monitored by fluorescence titration. A,
Heparin was titrated into solutions of kallistatin wild type at
concentration 0.116 µM; B, P1Arg at 0.170 µM; C, K307A/R308A at 0.160 µM;
and D, K312A/K313A at 0.141 µM in 50 mM Tris-HCl, 100 mM NaCl, 0.2% (w/v) PEG 6000, pH 7.4, at 20 °C. The relative change in kallistatin fluorescence
was monitored by ex = 280 nm,
em = 340 nm, in 10-nm slits.
Dissociation constant (Kd) determination for kallistatin and
the mutants with heparin
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Fig. 8.
Effects of heparin on the tissue kallikrein
binding activity of the kallistatin mutants. Tissue kallikrein
binding assays for the kallistatin mutants were performed in the
presence of heparin. Different concentrations of heparin, 0, 2.5, 5, 10, 20, and 30 units/ml, were preincubated with 0.6 µM
kallistatin P1Arg, K187A/K188A, K307A/R308A, and K312A/K313A,
respectively, in 20 mM sodium phosphate buffer, pH 8.0, at
37 °C for 10 min. The reaction mixtures were then incubated with
1.5 × 104 cpm of 125I-labeled tissue
kallikrein for an additional 90 min. The results of the tissue
kallikrein-binding assay for the individual mutants are shown as
indicated.
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Fig. 9.
Effects of heparin on the inhibitory activity
of the kallistatin mutants toward tissue kallikrein. The
kallistatin mutants, P1Arg ( ), K187A/K188A (
), K307A/R308A (
),
and K312A/K313A (
), at 0.2 µM were preincubated with
0, 2, 5, 10, 20, and 30 units/ml heparin in 20 mM sodium
phosphate buffer, pH 8.0, at 37 °C for 10 min. The mixtures were
then incubated with 6 nM tissue kallikrein at 37 °C for
90 min. The remaining tissue kallikrein activity was measured by
D-Val-Leu-Arg-MCA hydrolysis.
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Fig. 10.
CD spectra of kallistatin P1Arg,
K307A/R308A, and K312A/K313A. The wavelength scanning applied on
the CD analysis was from 190 to 250 nm.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 11.
Amino acid sequence alignment of the
heparin-binding sites in serpins. The sequences of the D helix and
the region between the H helix and C2 sheet of kallistatin
(HKS), protein C inhibitor (PCI),
antithrombin (ATIII), heparin cofactor II (HCII),
protease nexin I (PN-I), and plasminogen activator
I (PAI-I) are aligned with the sequences of antitrypsin
(AT) and antichymotrypsin (ACT), which are
non-heparin-binding serpins. Basic residues are
double-underlined.
Our results showed that the heparin affinity of kallistatin is not as high as other heparin-binding serpins. Nevertheless, the Kd value (0.185 µM) for kallistatin with heparin are only two times higher than the Kd (0.084 µM) described for heparin-antithrombin interaction in a solution with similar ionic strength (28), suggesting that kallistatin affinity for heparin is physiologically significant. Our previous study has shown that heparin affects the inhibitory activity of kallistatin differently, depending on the target proteinase (1). Heparin suppresses the inhibition toward plasma kallikrein and human and rat tissue kallikrein, whereas it accelerates the inhibition toward thrombin, activated protein C, and chymotrypsin by 5- to 50-fold (1). Similarly, heparin promotes the inhibitory activity of protein C inhibitor by about 30-fold toward thrombin and activated protein C, but inhibits the inhibitory activity toward human tissue kallikrein (21, 29).
The mechanisms of the heparin-accelerating proteinase inhibition have been explained by: (a) allosteric effects in which heparin induces conformational changes favorable for the binding of serpins with proteinases (10-13), and (b) a ternary complex model in which heparin acts as a template to bind serpin and proteinase and thus enhancing the association of both molecules (5, 6). Compared with kallistatin and protein C inhibitor, the serpins with heparin-binding sites in the D helix have greater inhibition enhancement (>1000-fold) upon heparin binding (6). It has been speculated (6) that the difference in rate enhancement can be accounted for by heparin binding through the H helix, where it positions the serpins in a less favorable orientation for reacting with the heparin-bound proteinases than when heparin binds through the D helix. The location of kallistatin's heparin binding region may also explain the modest acceleration response to heparin by the same speculation, because the heparin binding region is adjacent to the H helix according to the molecular model of kallistatin.
The mechanism of the heparin-suppressed proteinase inhibition of kallistatin and protein C inhibitor is still not well defined. A study has speculated that the heparin-suppressed effect of protein C inhibitor is caused by an allosteric effect or steric hindrance upon heparin binding, although direct evidence is absent (30). While analyzing the reaction of kallistatin and tissue kallikrein in the presence of heparin by SDS-PAGE, we noticed that the cleaved form of kallistatin did not increase and the formation of kallistatin-kallikrein complex was still suppressed (data not shown). This suggests that heparin does not convert kallistatin to a substrate for tissue kallikrein. Therefore, the heparin suppression effect could be caused by slowing down or blockage of the association of kallistatin with kallikrein. The region between the H helix and C2 sheet in kallistatin stretches through the reactive-center loop that places the heparin-binding residues in proximity to the reactive-center loop as shown in the molecular model of kallistatin (Fig. 1). The proximity between the heparin-binding sites and the reactive-center loop suggests possible mechanisms for heparin-suppressed proteinase inhibition. First, binding of heparin to these sites may generate steric hindrance that obstructs the insertion of the reactive-center loop into the reactive cleft of tissue kallikrein. Second, heparin binding may induce a conformational change of the reactive-center loop that reduces the inhibitory activity of kallistatin. Third, because the electrostatic interactions between the basic residues in the heparin-binding sites and the acidic residues around the reactive cleft of tissue kallikrein may efficiently position the reactive-center loop of kallistatin into the reactive cleft, heparin could mask the basic residues and thus interfere with the enzyme-inhibitor interaction.
In summary, we have shown that the region between the H helix and C2
sheet in kallistatin is a major heparin-binding domain related to
heparin-suppressed tissue kallikrein inhibition. The basic amino acids
Lys312-Lys313 in this region are the
heparin-binding residues that are significantly resistant to the
heparin-suppressed effect. The identification of the heparin-binding
sites in kallistatin is critical for further investigation of the
physiological function and regulation of kallistatin.
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ACKNOWLEDGEMENT |
---|
We thank Dr. Erika Büllesback for technical support on circular dichroism spectroscopy work.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL 44083.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 843-792-9927; Fax: 843-792-4850; E-mail: chaoj@musc.edu.
Published, JBC Papers in Press, October 2, 2000, DOI 10.1074/jbc.M005791200
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ABBREVIATIONS |
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The abbreviations used are: SCR, structurally conserved regions; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; CD, circular dichroism; PEG, polyethylene glycol.
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