From the
Ecotin, a serine protease inhibitor found in the periplasm of
Escherichia coli, is unique in its ability and mechanism of
inhibiting serine proteases of a broad range of substrate specificity.
However, although the catalytic domain of human urokinase-type
plasminogen activator (uPA) has 40% identity to bovine trypsin and the
substrate specificities of these two proteases are virtually identical,
ecotin inhibits uPA almost 10,000-fold less efficiently than trypsin.
Ecotin was expressed on the surface of filamentous bacteriophage
(ecotin phage) to allow the isolation of more potent inhibitors of uPA
from a library of ecotin variants. The 142-amino acid inhibitor was
fused to the C-terminal domain of the M13 minor coat protein, pIII,
through a Gly-Gly-Gly linker and assembled into phage particles. The
ecotin phage were shown to react with anti-ecotin antibodies, revealing
a stoichiometry of approximately one ecotin per bacteriophage. The
ecotin displayed on the surface of phage inhibited trypsin with an
equilibrium dissociation constant of 6.7 nM, in close
approximation to that of free ecotin, indicating that phage-associated
ecotin is correctly folded and functionally active. Reactive-site amino
acids 84 and 85 of ecotin were then randomized and a library of 400
unique ecotin phage was created. Three hundred thousand members of the
library were screened with immobilized uPA and subjected to three
rounds of binding and in vitro selection. DNA sequence
analysis of the selected ecotin phage showed that ecotin M84R/M85R
predominated while ecotin M84R, M84K, and M84R/M85K were present at a
lower frequency. The four ecotin variants were overexpressed and
purified and their affinities toward uPA were determined. Each of the
selected ecotin variants exhibited increased affinity for uPA when
compared to wild-type ecotin with ecotin M84R/M85R showing a 2800-fold
increase in binding affinity.
Urokinase-type plasminogen activator (uPA)
High levels of
receptor-bound uPA are found on the surface of many cancer
cells
(12, 13, 14) . The role of uPA and its
receptor in tumor invasion and metastasis suggests two possible
approaches for chemotherapeutic intervention: one by blocking specific
interactions between the EGF-like domain of uPA and the uPA receptor,
and the other by specific inhibition of the proteolytic activity of
uPA. A truncated, soluble form of the uPA receptor was produced
genetically and shown to reduce the amount of uPA that bound to cells
expressing wild-type uPA receptor
(15) . By acting as a scavenger
for uPA, the soluble uPA receptor also inhibited the proliferation and
invasion of human cancer cells
(16) . Alternatively, a uPA mutant
which lacked proteolytic activity while retaining full receptor binding
affinity was shown to compete for cell surface receptors and, in turn,
inhibit metastasis (17). Finally, high-affinity urokinase receptor
antagonists were identified from a pentadecamer random peptide library
and were shown to compete with the EGF-like domain of uPA for binding
to the uPA receptor (18). Although a certain extent of success in
anti-invasion and anti-metastasis has been achieved in vitro using this approach, rapid clearance of uPA, uPA receptor
derivatives, or natural peptides may pose a problem if they are used as
therapeutic agents.
Less effort has been made toward developing
specific inhibitors for uPA, presumably because of the difficulty of
discriminating uPA from other serine proteases. Of the synthetic uPA
inhibitors that have been described to date, the 4-substituted
benzo[b]thiophene-2-carboxamidines were the most
potent and were shown to inhibit cell surface uPA as well as cell
surface uPA-mediated fibronectin degradation
(19) . The natural
macromolecular inhibitor of the plasminogen activators is the type 1
plasminogen activator inhibitor (PAI-1), a single-chain glycoprotein
with a molecular mass of approximately 50 kDa
(20) . However,
PAI-1 does not discriminate between the plasminogen activators,
inactivating tPA and uPA with nearly identical secondary rate
constants
(21) . Furthermore, high PAI-1 levels have recently
been found to associate with malignancy in a number of
cancers
(22, 23, 24, 25) . These findings
suggest that, in addition to functioning as a uPA inhibitor in normal
cells, PAI-1 or its complex may play a role in promoting growth or
spreading of cancers. These attributes disfavor the use of PAI-1 as a
therapeutic agent.
Ecotin is a dimeric serine protease inhibitor
found in the periplasm of Escherichia coli, where each unit of
the dimer contains 142 amino acids
(26, 27) . Ecotin has
been found to inhibit pancreatic serine proteases of a broad range of
specificity but not any known proteases from E.
coli(26) . Recently, ecotin has also been found to be a
highly potent anticoagulant and a reversible tight-binding inhibitor of
human factor Xa
(28) . Ecotin belongs to the
``substrate-like'' class of inhibitors
(29) with
Met-84 at the reactive-site (the P1 site)
(27) . A crystal
structure of ecotin complexed with trypsin showed that two trypsin
molecules bind to an ecotin dimer in a 2-fold symmetry
(30) . In
addition to the interactions through a primary site that includes the
reactive-site loop, ecotin makes a total of 9 hydrogen bonds to trypsin
through a secondary binding site located at the distal end of ecotin
relative to the reactive site. Modeling studies with ecotin and other
proteases including chymotrypsin and elastase indicates that similar
interactions could occur, along with other unique contacts. The
chelation of a target protease through the two binding sites is a
unique feature of ecotin since most serine protease inhibitors interact
with their target proteases predominately through their reactive-site
loop
(31) . The bidentate binding scheme utilized by ecotin may
allow fine tuning of protease inhibition toward specific targets
through protein engineering efforts. Although the catalytic domain of
uPA and trypsin are homologous
(7) and their substrate
specificities are virtually identical, ecotin is a poor inhibitor of
uPA proteolytic activity. We attempted to convert ecotin into a potent
uPA inhibitor using phage display to aid our understanding of
protease-inhibitor recognition and uPA function.
It was previously
shown that a bovine pancreatic trypsin inhibitor variant with altered
specificity and high affinity toward human neutrophil elastase could be
isolated from a library of bovine pancreatic trypsin inhibitor variants
by phage display technology
(32) . This demonstrated the
feasibility of studying protease-inhibitor interactions using this
technique. Phage display allows the expression of a diverse library of
peptides or protein variants on the surface of filamentous M13
bacteriophage
(33, 34) . This in turn allows the
isolation of individual phage particles that display desired binding
properties by an in vitro selection process. Since the
phenotype of each phage is directly linked to its genotype, specific
mutations in the displayed peptide or protein that confer a desired
function can be readily identified. Using this technology, a number of
antigen-antibody interactions have been studied
(34, 35) as well as hormone-receptor
interactions
(36, 37) , protein-nucleic acid
interactions
(38, 39) , and inhibitor-protease
interactions
(32, 40) .
We have shown that the phage
display approach can be broadly generalized to other protease
inhibitors by displaying ecotin on the surface of phagemid-derived
bacteriophage (ecotin phage). Ecotin was chosen because of its unusual
ability to inhibit trypsin, chymotrypsin, and elastase
(26) , and
its unique mechanism of inhibition
(30) . Its broad specificity
suggests a structural flexibility which would allow modifications via
protein engineering to confer novel properties. The crystal structure
of ecotin complexed with a variant of rat trypsin was solved
recently
(30) . Combined with information from the
three-dimensional structure, phage display can be used to search
designed libraries of ecotin mutants that affect interactions at the
inhibitor/protease interface. Variants with high affinity toward a
particular target protease can then be readily isolated and
characterized. Herein, we report the display of ecotin on the surface
of phagemid-derived bacteriophage, and the isolation of mutants with
high affinity toward uPA.
The ecotin gene for this construction was isolated from the
expression plasmid pTacTacEcotin
(41) and was inserted into the
multiple cloning site of pBluescript to generate pBSecotin. The
inserted DNA contained a Shine-Dalgarno sequence at the 5` end,
followed by the sequence encoding a 20-amino acid ecotin signal peptide
from the genomic clone (27), and the mature form of ecotin. The DNA
sequence coding for the COOH-terminal domain (residues 198-406)
of the gene III protein, which is embedded in the phage coat and is
essential for proper phage assembly
(49) , was generated by
polymerase chain reaction. This DNA fragment was fused to the end of
the ecotin gene of pBSecotin through a Gly-Gly-Gly linker
(Fig. 1). It should be noted that the ecotin gene and the
ecotin-gene III fusion were inserted in pBluescript in the same
orientation as the lacZ gene and disrupted the lacZ reading
frame. Ecotin and ecotin-pIII fusion were expressed from pBSecotin and
pBSeco-gIII, respectively, and the protein expression level was
increased approximately 5-fold by the addition of IPTG in the cultures
(data not shown). These results verified that the lac promoter
of pBluescript was utilized for transcription and the Shine-Dalgarno
sequence upstream of the ecotin gene was recognized for initiating
translation. However, phagemid propagation in the presence of IPTG
resulted in very low phage titer, perhaps due to the increased levels
of ecotin-pIII fusion which may be toxic to the cells. Since
ecotin-pIII fusion was expressed at a significant level in the absence
of IPTG without affecting phage titer, IPTG was not used in the phage
preparations. Under these conditions, the phage titer for pBSeco-gIII
was approximately 2 to 8
Our specific
target for binding selection is human uPA. It has been shown that
receptor binding of uPA through the EGF-like domain does not shield uPA
from the action of its endogenous inhibitors, PAI-1
(50, 51) and PAI-2
(52, 53) , nor does it modulate the
proteolytic activity of uPA
(54) . Since intact uPA is unstable
and readily degrades to its low molecular weight form (LMuPA) which
contains the protease domain
(7) , our binding selections were
carried out using LMuPA. Library phage displaying ecotin variants were
cycled through rounds of binding selection to isolate mutants with high
affinity. A strong consensus sequence was observed after only one cycle
of binding selection (). Of the 15 clones sequenced, 13
had Arg at the P1 position, while Met predominated at the P1`
position
(10) , along with a minor population of Arg
(3) ,
Lys
(1) , and Thr
(1) . It should be noted that only two
wild-type clones were observed from 15 sequenced clones, in spite of
the fact that wild-type ecotin phage accounted for approximately 40% of
the original library. This suggested that the panning process was not a
random selection. As the binding selection was carried through
subsequent cycles, a dramatic change in ecotin variant phage population
was observed. Wild-type ecotin phage were not detected after the second
cycle of selection. The observed population of the ecotin M84R phage
decreased from 53% in the first cycle to 20% in the second cycle and to
10% in the third cycle, while the population of ecotin M84R/M85R phage
that was initially less abundant increased from 20% in the first cycle
to 60% in the second cycle and to 70% in the third cycle.
The
wild-type ecotin and ecotin mutants derived from the third cycle of
selection were cloned into the expression vector pTacTac. Proteins were
expressed in E. coli, and purified to >95% homogeneity
(data not shown). The equilibrium dissociation constants,
K
Ecotin was shown to be associated
with the filamentous phage by localizing ecotin antibodies to phage
particles. Ecotin phage also inhibited trypsin activity, suggesting
that ecotin is folded and active even when produced as a fusion
protein. In contrast, phagemids lacking ecotin-pIII on their surface
failed to inhibit trypsin activity, suggesting that inhibition occurred
through a specific interaction between trypsin and ecotin rather than a
nonspecific interaction between trypsin and phage particles. The
equilibrium dissociation constant for ecotin phage and trypsin was
estimated to be 6.7 nM. This value is within 3-fold of the
K
Ecotin phage were enriched from a pool of background phage when
biopanning against immobilized trypsin was carried out; an enrichment
of greater than 10
Having established conditions for binding selection, a
phage library was constructed with random mutations at the P1 (Met-84)
and P1` (Met-85) sites of ecotin in an attempt to search for high
affinity inhibitors for uPA. These two sites were chosen for
randomization because they flanked the scissile peptide bond.
Furthermore, the side chains of Met-84 and Met-85 have been shown to
interact with the S1 and S1` sites of trypsin in the three-dimensional
structure of the trypsin-ecotin complex. Like most substrate-like
inhibitors, these sites are major determinants for inhibition
specificity. A strong consensus sequence developed after one cycle of
selection of 3
Previous studies on the roles of the P1-P1`
residues of PAI-1 found that residues other than Arg or Lys at P1
displayed little or drastically reduced affinity for tPA or
uPA
(55) . Although ecotin and PAI-1 belong to different
inhibitor families and their mechanisms of action are quite
different
(31, 56) , our selection results are consistent
with that of a mutagenesis study of PAI-1 in that Arg and Lys were the
only residues found at the P1 site of ecotin. The P1 residue of ecotin,
Met-84, did not appear to interact strongly with the substrate binding
pocket of trypsin
(30) , suggesting that interactions between
ecotin and trypsin at sites other than the reactive center have a
significant contribution to the observed affinity. We hypothesize that
these interaction sites fail to interact with uPA properly so that a
basic residue at P1 that forms a strong electrostatic interaction with
Asp in the substrate binding pocket of uPA is required to confer
significant inhibition. A comparison of the crystal structures of
ecotin-trypsin
(30) and ecotin-collagenase
The
exact structural role of the P1` residue in binding with uPA is not
known, due to the unavailability of the uPA structure. The fact that
basic residues (Arg and Lys) were preferred at the P1` site suggests an
electrostatic interaction at the interface with uPA. Sequence alignment
of bovine trypsin and uPA
(7) revealed that an Asp is inserted
in a surface loop that, in trypsin, interacts with the prime sites of
the inhibitor
(30, 57) and, in the case of ecotin, may
form an electrostatic interaction with Arg or Lys at the P1` site.
Further evidence supporting this hypothesis came from a previous study
of the PAI-1-uPA interaction in which a Glu at the P1` site was found
to significantly reduce the second order rate constant of
inhibition
(55) , presumably by charge repulsion.
The specific
inhibition of proteases associated with various disease states holds
great promise as an alternative to current therapies. Highly specific
inhibitors of the proteases involved in cancer may prove to be a less
toxic alternative to the chemotherapies currently in use. Particular
stages of cancer are marked by an imbalance of proteolytic activity.
Elevated levels of proteases such as cathepsins B and D, collagenase
IV, and uPA are found in tumor tissues and correlate with tumor cell
invasion and metastasis
(3, 58, 59) . Specific
inhibition of a given protease by a macromolecular inhibitor can serve
as a starting point for subsequent development of small molecule
inhibitors. We have successfully displayed ecotin on the surface of
filamentous bacteriophage and demonstrated the use of this system to
engineer potent inhibitors for uPA. Owing to the unique structural
property of ecotin and the power of the in vitro selection,
the system described here could find a general application in
engineering specific ecotin-based inhibitors for target proteases,
particularly for those of clinical importance.
(
)
and tissue-type plasminogen activator (tPA) are two serine
proteases that catalyze the conversion of the inactive precursor
plasminogen, to plasmin, a serine protease of broad substrate
specificity
(1, 2) . uPA has been found to be involved in
the activation of pericellular proteolysis during cell migration and
tissue remodeling, while the function of tPA is primarily connected to
intravascular clot dissolution
(3, 4) . Although no
three-dimensional structure currently exists for human uPA, it is
thought to be composed of three domains: an NH
-terminal
domain (residues 1-45), which has partial homology to
EGF
(5, 6) followed by a kringle domain (residues
50-131)
(5) , and a COOH-terminal protease domain (residues
159-411) with 40% identity to trypsin
(7) . The EGF-like
domain of uPA binds the membrane-bound urokinase receptor
(8) ,
localizing the proteolytic activity of uPA to the cell surface.
Receptor-bound uPA has been shown to cleave a 66-kDa extracellular
matrix protein
(9) as well as fibronectin
(10) . The
presence of uPA on the cell surface also allows the formation of
cell-surface plasmin, which is capable of degrading most components of
the extracellular matrix, either directly or through activation of
procollagenases
(3) . Cell surface uPA has been implicated in
mediating processes such as tumor growth, cell invasion, metastasis,
cell migration, and tissue remodeling
(3, 11) , all of
which require extracellular proteolytic activity.
Materials
Enzymes and reagents for molecular
cloning were purchased from New England Biolabs and were used following
the manufacturer's instructions. The E. coli strain
JM101 and the VCSM13 helper phage were from Stratagene. Low molecular
weight uPA (LMuPA) was obtained from American Diagnostica. Bovine
trypsin was from Sigma. The chromogenic substrate
Z-Gly-Pro-Arg-p-nitroanilide used for trypsin kinetics
analysis was from Bachem, and the chromogenic substrate
Z--Glu(
-t-butoxy)-Gly-Arg-p-nitroanilide
(Spectrozyme UK) used for LMuPA kinetics analysis was from American
Diagnostica. 4-Methylumbelliferyl p-guanidinobenzoate was from
Sigma.
-
S-dATP was from DuPont NEN. Sequenase Version
2.0 sequencing kit was from U. S. Biochemical Corp. Oligonucleotides
were synthesized with an Applied Biosystems 391 DNA synthesizer.
Plasmid and Library Constructions
The phagemid
pBSeco-gIII was constructed to produce ecotin on the surface of the
surface of filamentous phage. These phage are referred to as ecotin
phage. The ecotin expression plasmid pTacTacEcotin
(41) was
digested with the restriction endonucleases BamHI and
HindIII. The resulting DNA fragment encoding the ecotin gene
and its signal sequence was ligated to the large fragment of
BamHI/HindIII-digested pBluescript to produce
pBSecotin. The DNA sequence coding for amino acids 198-406 of
gene III of M13 was generated from M13 mp18 DNA using polymerase chain
reaction; the forward primer was 5`-GTC ACG AAG CTT CCA TTC GTT TGT GAA
TAT CAA GG-3`, and the reverse primer was 5`-GCA CGA AGC TTA AGA CTC
CTT ATT ACG CAG TAT G-3`. After HindIII digestion, the
polymerase chain reaction product was inserted into a HindIII
site at the 3` end of the ecotin gene of pBSecotin. The stop codon at
the COOH terminus of the ecotin gene was then removed, and a
Gly-Gly-Gly tether was introduced at the junction of the fusion gene.
This was achieved by site-directed mutagenesis
(42) using the
primer 5`-CG GTA GTT CGC GGC GGC GGA GCT GAA AGC GTC CAG-3`. The
resulting plasmid construct was named pBSeco-gIII. A pBSeco-gIII mutant
in which codons 84 and 85 and the third base pair of codon 83 of the
ecotin gene were deleted was constructed to provide control phage that
did not express the ecotin gene. This mutant, pBSeco-gIII, was
made by site-directed mutagenesis using the primer 5`-C AGT TCC CCG GTT
AGT AC GCC TGC CCG GAT GG-3`. A pBSeco-gIII library with random
mutations at codons 84 and 85 of the ``reactive-site'' loop
of ecotin was created by oligonucleotide-directed mutagenesis using the
oligonucleotide 5`-C AGT TCC CCG GTT AGT ACT NNS NNS GCC TGC CCG GAT
GG-3` (N = A/C/G/T; S = G/C) as the primer and
the uracilated, single-stranded pBSeco-gIII as the template. Also
introduced by this primer was a ScaI site, which enabled
facile differentiation between native templates and mutant templates.
The library of ecotin phage had 1024 possible DNA sequences that
resulted in 400 possible protein sequences.
Phage Preparations
For the preparation of
pBluescript, pBSeco-gIII, and pBSeco-gIII bacteriophage, plasmid
DNAs were transformed into a male strain (F`) of JM101. A single colony
selected on ampicillin plates was grown in 3 ml of 2YT medium (16 g of
tryptone, 10 g of yeast extract, 5 g of NaCl/liter) containing 60
µg/ml ampicillin at 37 °C for 7 h. The culture was diluted into
30-100 ml of 2YT/ampicillin, grown to A
= 0.25, and infected with the helper phage VCSM13 at a
multiplicity of infection of approximately 100 helper phage per cell.
The infected culture was allowed to grow at 37 °C with shaking for
approximately 12 h. Phage particles were harvested by precipitation
with 5% polyethylene glycol and resuspended in 1 ml of TBS buffer (150
mM NaCl, 50 mM Tris-HCl, pH 7.4). Phage titers
typically ranged from 5
10
to 2
10
cfu/ml culture. For library phage preparation, the mutagenesis
reaction mixture was ethanol-precipitated, redissolved in water,
electroporated into F` JM101, and plated on 150-mm LB/ampicillin
plates. Cells from the plates were recovered in 5 ml of LB/ampicillin
and diluted in 50 ml of 2YT/ampicillin to an A
= 0.25, and then infected with VCSM13 helper phage. The
infected culture was grown for 12 h at 37 °C with shaking, and the
phage were harvested as described above.
Immunoblotting Analysis of Ecotin Fusion
Phage
Approximately 5 10
cfu were loaded in
duplicate onto a single 1% agarose gel with 25 mM Tris, 250
mM glycine (pH 8.6) as the running buffer
(43) . The gel
was electrophoresed at 6 mA constant current for 16 h. One set of
samples was transblotted onto a nitrocellulose filter. The filter was
immunostained for ecotin by allowing it to react with rabbit
anti-ecotin antibodies followed by reaction with horseradish
peroxidase-conjugated goat anti-rabbit IgG antibodies. The other set of
samples were denatured by soaking the gel in 0.5 N NaOH for 4
h, washed by soaking in water for 4-8 h, and stained with
ethidium bromide.
Inhibition of Trypsin Activity by Ecotin-pIII Fusion
Phage
pBSeco-gIII and pBSeco-gIII phage were suspended in
trypsin assay buffer (50 mM Tris-HCl, 100 mM NaCl, 20
mM CaCl
, pH 8.0) and adjusted to 1.5
10
cfu/ml. Various volumes of phage solution were
incubated with 0.5 nM trypsin in a total volume of 125 µl
of trypsin assay buffer, 0.01% Tween 80 in a 96-well microtiter plate
at room temperature for 20 min. After adding 125 µl of 0.1
mM substrate Z-Gly-Pro-Arg-p-nitroanilide, the
residual trypsin activity was measured by monitoring the increase of
optical density at 405 nm.
Binding Enrichments
Polystyrene Petri dishes (35
mm, Falcon) were coated with 1 ml of 10 µg/ml bovine trypsin or
LMuPA in phosphate-buffered saline (137 mM NaCl, 2.7
mM KCl, 10 mM NaHPO
, 1.8
mM K
HPO
, pH 7.5) overnight, and excess
binding sites were blocked with 5% non-fat dry milk solution for 2 h.
For control experiments, Petri dishes were coated with 5% non-fat dry
milk solution. Phage were added to the dishes in buffer containing 1 ml
of phosphate-buffered saline, 0.5% Tween 20 and incubated overnight
with gentle agitation at room temperature. Solutions containing the
phagemid were then removed and the dishes were washed 9 times with 5 ml
of phosphate-buffered saline/Tween 20. Each wash was approximately 1
min. Bound phage were serially eluted by incubation with 1 ml of 0.1
N HCl/glycine solution (pH 2.2) with gentle shaking for 15 min
at room temperature. Three elutions were performed. The eluates were
neutralized with 185 µl of 1 M Tris-HCl (pH 8.8). For
biopanning against immobilized trypsin, a mixture of pBSeco-gIII phage
(1.1
10
cfu) and pBluescript phage (2.8
10
cfu) was used. An aliquot of the appropriately diluted
solution of each wash and elution was used to infect 100 µl of
saturated JM101 cells. After incubation for 15 min at 37 °C, the
infected cells were plated on LB/ampicillin plates containing IPTG and
5-bromo-4-chloro-3-indolyl
-D-galactoside. The cfu ratio
of pBSeco-gIII phage to pBluescript phage was calculated by the number
of white and blue colonies, respectively. For biopanning against
immobilized LMuPA, a mixture of 2.5
10
cfu library
phage were used. Phage from the third elution were amplified for the
next cycle of panning.
Expression and Purification of Recombinant Ecotin and
Ecotin Mutants
Ecotin and ecotin mutants were produced in
bacteria from the expression vector pTacTacEcotin
(41) . The
expression and purification procedures were as follows. JM101 cells
were freshly transformed with expression plasmid DNA. A single colony
selected from ampicillin plates was used to inoculate 3 ml of LB
containing 60 µg/ml ampicillin. The cultures were grown at 37
°C for 9 h and diluted to 1 liter of LB/ampicillin. Following
growth at 37 °C for 1 h, IPTG was added to the cultures to a final
concentration of 0.2 mM, and continued to grow for 12 h at for
37 °C. Cells were harvested and treated with lysozyme in a solution
containing 25% sucrose, 10 mM Tris-HCl (pH 8.0). The
periplasmic fraction was dialyzed against 10 mM sodium citrate
(pH 2.8). Following the dialysis, the supernatant was adjusted to pH
7.4 with 1 M Tris-HCl (pH 8.0), and to 0.3 M NaCl.
The solution was heated in boiling water for 10 min, and then cooled to
room temperature. The precipitate was removed by centrifugation, and
the supernatant was dialyzed against water overnight at 4 °C. The
solution containing the ecotin was loaded onto a Vydac C4 reverse-phase
high performance liquid chromatography column (2.2 25 cm) which
was equilibrated with 0.1% trifluoroacetic acid. The column was washed
and then eluted with a linear gradient of 34-37% acetonitrile,
0.1% trifluoroacetic acid at a flow rate of 10 ml/min over 30 min.
Fractions were analyzed with SDS-polyacrylamide gel
electrophoresis
(44) , and the ones containing pure ecotin were
pooled and lyophilized. Purified ecotin was redissolved in buffer
containing 10 mM Tris-HCl (pH 7.4) and stored at 4 °C. The
concentrations of ecotin and ecotin mutants were determined using a
calculated molar extinction coefficient (45) of 2.2
10
cm
M
and were in
good agreement with that from the Bradford assay (data not
shown)
(46) .
Determination of Equilibrium Dissociation
Constants
LMuPA was titrated with 4-methylumbelliferyl
p-guanidinobenzoate to obtain an accurate concentration of
enzyme active sites. Various concentrations of ecotin or ecotin mutants
were incubated with human LMuPA in a total volume of 990 µl of
buffer containing 50 mM NaCl, 50 mM Tris-HCl (pH
8.7), 0.01% Tween 80. The final concentrations of LMuPA used for the
determination of equilibrium dissociation constants,
K, of ecotin or ecotin mutants were as
follows: 0.5 nM (M84R/M85R); 1.0 nM (M84R, M84K, and
M84R/M85K); 7.2 nM (wild-type). The final concentrations of
ecotin mutants ranged from 0.6 to 40 nM, and the
concentrations of wild-type ecotin ranged from 1.25 to 50
µM. Following a 30-min incubation at room temperature to
reach equilibrium, 10 µl of 10 mM substrate Z-
-Glu
(
-t-butoxy)-Gly-Arg-p-nitroanilide was added and the rate
of p-nitroaniline formation was measured by monitoring the
change of absorption at 410 nm in a 10-min period. The data were fit to
the equation derived for kinetics of reversible tight-binding
inhibitors
(47, 48) by nonlinear regression analysis,
and the values for apparent K
were
determined.
RESULTS
Design of the Phage Display System
Ecotin was
expressed on the surface of phagemid-derived bacteriophage to establish
an in vitro selection for ecotin variants. The pBluescript
vector was chosen for ecotin phagemid construction due to its high copy
number, small size, and stability. The presence of the M13 origin of
replication permits efficient packaging of phage particles when
bacteria carrying the pBluescript phagemid are infected with helper
phage. The presence of the -complementation factor permits
monitoring of the phagemid by
-galactosidase activity, and the
-lactamase gene permits quantitation of phagemid titer by cfu.
10
cfu/ml of culture.
Figure 1:
Schematic
representation of the DNA fragment encoding the ecotin-pIII fusion and
the lac regulatory sequence of the phagemid pBluescript. Ecotin was
fused to the carboxyl-terminal domain (residues 198-406) of M13
pIII through a Gly-Gly-Gly linker. The amino acid and nucleotide
sequences between BamHI restriction site and the start of the
ecotin signal peptide (S.P.), at the junction of the gene
fusion, and at the end of gene III are shown. Transcription of the
fusion protein is under control of the lac promoter/operator sequence
of the phagemid pBluescript, and secretion is directed by a 20-amino
acid signal peptide from the ecotin genomic clone. The fusion construct
was inserted between BamHI and HindIII restriction
sites of pBluescript in the same direction as lacZ gene and
disrupted the lacZ reading frame. pBluescript contains the
-lactamase gene which provides ampicillin resistance and allows
quantification of the ecotin phage by colony forming
units.
Characterization of the Ecotin-pIII
Fusion
pBSeco-gIII phage were fractionated by electrophoresis in
a 1% agarose gel and analyzed for their interaction with anti-ecotin
antibodies. Fig. 2shows that ecotin co-migrated with intact
phage particles (lane 1), indicating that the ecotin-pIII
fusion was expressed and incorporated into the phage particles. In
contrast, phagemids lacking the ecotin-pIII fusion (pBluescript,
lane 2) or having a deletion/frameshift mutation within
ecotin-pIII fusion (pBSeco-gIII, lane 3) did not interact
with anti-ecotin antibodies. These results established that ecotin was
displayed on phage only in the context of a correct ecotin-pIII fusion.
To determine whether the ecotin molecules displayed on the surface of
phage were active, the ability of ecotin phage to inhibit the
proteolytic activity of trypsin was determined. Ecotin phage were
incubated with rat trypsin for 20 min and the residual proteolytic
activity was measured. Fig. 3shows that trypsin activity
decreased as the concentration of ecotin phage increased, whereas
trypsin activity remained essentially unchanged when incubated with
pBSeco-gIII
phage. Based on the observation that approximately 10%
of the phagemid particles were infectious and that, on average, each
virion contained one copy of the ecotin-pIII fusion protein as judged
by its immunochemical activity compared to a known amount of free
ecotin protein (data not shown), a measure of the affinity of ecotin
phage for trypsin could be calculated. An apparent equilibrium
dissociation constant, K
, of 6.7
nM for ecotin phage and trypsin was calculated using
reversible tight-binding kinetics. These results suggested that ecotin
was correctly folded and active as a fusion with pIII.
Figure 2:
Characterization of ecotin-pIII fusion
phage. Phage samples were loaded in duplicate and fractionated on a
single 1% agarose gel. One set of samples was stained with ethidium
bromide (panel A). The other set of samples was transblotted
onto a nitrocellulose filter, which was immunostained for ecotin
(panel B) (see ``Experimental Procedures'' for
details). Lane 1, pBSeco-gIII; lane 2, pBluescript;
lane 3, pBSeco-gIII; lane 4, pBSeco-gIII P1/P1`
library. See ``Experimental Procedures'' for
details.
Figure 3:
Inhibition of rat trypsin activity by
pBSeco-gIII phage. Various concentrations of phage were mixed with rat
trypsin in a 96-well microtiter plate. Following a 20-min incubation at
room temperature, the substrate, Z-Gly-Pro-Arg-p-nitroanilide,
was added to the mixture. Residual trypsin activity in percentage is
expressed as the ratio of the rate of
Z-Gly-Pro-Arg-p-nitroanilide hydrolysis in the presence of
phage to that in the absence of phage at various phage concentrations.
Data were average of two independent experiments and agreed within 12%.
Circle, pBSeco-gIII; triangle,
pBSeco-gIII.
Binding Enrichments on Trypsin-coated
Dishes
Biopanning against immobilized trypsin was carried out to
determine whether ecotin phage could be selectively enriched from a
background phage population. Bovine trypsin was used to coat the bottom
of polystyrene Petri dishes by incubating a 10 µg/ml trypsin
solution in the polystyrene dishes. After extensive washing, the
presence of immobilized trypsin on the dish was verified by its ability
to hydrolyze the chromogenic substrate
Z-Gly-Pro-Arg-p-nitroanilide (data not shown). A mixture of
ecotin phage and pBluescript phage at an approximately 1:2800 ratio was
incubated with the trypsin-coated dishes overnight at room temperature.
After extensive washing, the bound phage were eluted with low pH buffer
(0.1 N HCl/glycine, pH 2.2). These conditions are known to
dissociate the trypsin-ecotin complex
(27) . Because the
insertion of the ecotin-gIII fusion gene in pBluescript disrupted the
gene coding for lacZ, F` JM101 cells infected with pBSeco-gIII phage
yielded white colonies on ampicillin plates containing IPTG and
5-bromo-4-chloro-3-indolyl -D-galactoside. This permitted
discrimination between pBluescript phage (blue colonies) and ecotin
phage (white colonies). The results in show that, starting
with a ratio of ecotin phage to pBluescript phage of 1:2800, the ratio
of ecotin phage to pBluescript phage exceeded 4:1 after one cycle of
binding and elution on trypsin-coated dishes. Thus, a single step of
selection yielded greater than 10
-fold enrichment. In
contrast, only a slight enrichment (10-fold) was found when the
experiment was performed with dishes coated with 5% milk. Subsequent
elutions also showed greater than 10
-fold enrichments on
trypsin-coated dishes.
Library Construction and the Search for a High Affinity
Urokinase Inhibitor
Oligonucleotide-directed mutagenesis was
used to introduce random mutations at the P1
(84) and P1`
(85) sites of ecotin. These amino acids are in the reactive-site
loop of the inhibitor and flank the scissile peptide bond. The sequence
NNS, where n = A/C/G/T, and S = G/C, was
incorporated into each codon to create a mutant library with all
possible amino acid substitutions, 1024 possible DNA sequences and 400
possible protein sequences. After the mutagenesis reaction, the DNA
mixture was ethanol-precipitated and electroporated into JM101 cells.
Approximately 5 10
individual clones were obtained
in a single electroporation. A ScaI restriction digest of DNA
from 20 individual clones revealed that approximately 60% of the
transformants were derived from mutagenesis and 40% were background
resulting from the single-stranded DNA template. Based on the
nucleotide sequences of 64 mutated codons from the library, the
frequencies of nucleotide occurrence were: n = 23% A,
19% C, 31% G, and 27% T; S = 30% C, 70% G. Although the
occurrence of nucleotides in this library is not evenly distributed,
especially at the third position of the codon, the large number of
individual transformants (3
10
) greatly exceeds the
4.8
10
transformants necessary for a 99% confidence
level that each member of the library is represented.
, of the mutants toward LMuPA were
determined using tight-binding kinetics
(47, 48) and are
shown in I. All mutants exhibited significantly higher
affinity than that of wild-type ecotin. The double mutant, ecotin
M84R/M85R, was the most potent, with a 2800-fold increase in affinity
toward uPA.
DISCUSSION
There is an extensive array of naturally evolved
macromolecular inhibitors that regulate the proteolytic activities of
serine proteases. Although the three-dimensional structure of the
various scaffolds may differ among these inhibitors, usually a
reactive-site loop complementary to the substrate specificity of the
proteases is presented to the target protease in such a way to cripple
the active-site machinery of the enzyme. A classic example of this type
of interaction can be found in the bovine pancreatic trypsin
inhibitor-trypsin complex. It was previously shown that phage display
can be used to alter the inhibitory profile of bovine pancreatic
trypsin inhibitor against the serine protease, human neutrophil
elastase
(32) . It had yet to be shown that this approach could
be applied to other protease inhibitors. Ecotin is unique among the
serine protease inhibitors in having a dyad-related secondary binding
site that permits recognition of a wide range of proteases through a
network-linked array of interactions. By showing that ecotin can be
displayed on the surface of filamentous phage and can be remodeled to
inhibit uPA, the application has been broadened to show that protein
engineering methods can be used to design an even greater array of
potent macromolecular inhibitors.
of free ecotin (2.3 nM). The
lowered affinity of the ecotin-pIII fusion may be due to blocked access
for trypsin by the phage particle, or may simply be experimental error
due to the inherent difficulty of direct measurement of the number of
phage particles and ecotin molecules incorporated. Nevertheless, this
result suggested that the phage-associated ecotin was highly active and
virtually unhindered by the presence of the associated bacteriophage.
-fold was achieved after the first
elution of the bound phage in the first cycle of binding selection.
This enrichment was comparable to those reported for antigen-antibody
(33, 34) and hormone-hormone receptor
(36) interactions.
Subsequent elutions (elution 2 and elution 3) yielded lower numbers of
phage, but the enrichment ratio essentially remained unchanged. In
contrast, almost no enrichment was observed when biopanning was carried
out in the absence of a target protease. These results showed that the
presence of trypsin on the plate was required for enrichment,
suggesting that binding occurred by specific interaction between ecotin
and trypsin.
10
phage; all except two wild-type
ecotin phage had Arg at the P1 site, with ecotin M84R/M85
predominating. Arg and Lys were the only residues found at the P1 site
in the subsequent binding selection cycles, and Arg was also the
favored residue at the P1` site. Examination of the equilibrium
dissociation constant, K
, using
tight-binding kinetics showed that all ecotin mutants from the third
cycle of selection exhibited high affinity toward uPA and represented
an 800-2,800-fold increase in potency relative to wild-type
ecotin. The engineered protease inhibitors selected here are the most
potent uPA inhibitors described to date; using comparable assay
conditions the affinity of ecotin M84R/M85R for uPA
(K
= 1 nM) was 160-fold
higher than that of a synthetic inhibitor, 4-benzodioxolanylethenly
benzo[b]thiophene-2-carboxamidine
(K
= 160 nM), which was
the most potent synthetic uPA inhibitor previously
described
(19) .
(
)
complexes revealed a significant displacement at the
binding loops of ecotin, suggesting that these loops adopted different
conformations when encountering different proteases in order to attain
an optimal complementary fit. This property is unique among the known
protease inhibitors, in which the reactive loops are less affected by
complex formation and their canonical conformations remain essentially
unchanged when complexed with different proteases
(31) . Based on
these observations, mutations can be introduced at the
inhibitor-protease interface to remodel ecotin to attain improved
binding affinity and selectivity. Phage display can then provide an
efficient means of screening a conformationally diverse library from
which an inhibitor with the desired specificity can be selected.
Table:
Binding enrichments of ecotin phage on trypsin-
or milk-coated plates
Table:
Identity of ecotin mutants from the P1/P1`
library after various cycles of binding selection to immobilized LMuPA
Table:
Apparent equilibrium dissociation constants
for ecotin and ecotin mutants with LMuPA
-D-galactoside; LMuPA, low molecular
weight urokinase-type plasminogen activator; pIII, gene III product of
M13 bacteriophage; PAI-1, type 1 plasminogen activator inhibitor; tPA,
tissue-type plasminogen activator; Z, carbobenzoxy.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.