To ascertain the combination of mutations that are very close in sequential space, the GMK2 clone Table 1 was modified by site-directed mutagenesis to create the mutants YQ and YP, and th
Trang 1carboxypeptidase U (TAFIa) and demonstrates the
importance of CPU stability over proCPU concentration
in down-regulating fibrinolysis
Wolfgang Knecht1, Johan Willemse3, Hanna Stenhamre1, Mats Andersson2, Pia Berntsson1,
Christina Furebring2, Anna Harrysson1, Ann-Christin Malmborg Hager2, Britt-Marie Wissing1, Dirk Hendriks3and Philippe Cronet1
1 AstraZeneca R & D Mo¨lndal, Mo¨lndal, Sweden
2 Alligator Bioscience AB, Lund, Sweden
3 Laboratory of Medical Biochemistry, University of Antwerp, Wilrijk, Belgium
The fragile balance between the activities of the
coagu-lation cascade (thrombin generation) and the
fibrino-lytic system (plasmin generation) is essential to prevent
excessive blood loss upon damage of a blood vessel,
while maintaining the blood flow in parts of the
body distant from the injury Procarboxypeptidase U
[proCPU, thrombin-activatable fibrinolysis inhibitor
(TAFI), EC 3.4.17.20, MEROPS M14.009] belongs
to the metallocarboxypeptidase family and is a
human plasma zymogen, which is also known as thrombin-activatable fibrinolysis inhibitor (TAFI), plasma procarboxypeptidase B and procarboxypepti-dase R [1,2] ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis [3,4] The physiological role of proCPU and its activa-ted form, carboxypeptidase U (CPU) is outlined in Fig 1 ProCPU is synthesized in the liver and secreted into the plasma following the removal of its signal
Keywords
carboxypeptidase; coagulation; directed
evolution; fibrinolysis; protease
Correspondence
W Knecht, Molecular Pharmacology –
Target Production, AstraZeneca R & D
Mo¨lndal, 431 83 Mo¨lndal, Sweden
Fax: + 46 317763753
Tel: + 46 317065341
E-mail: wolfgang.knecht@astrazeneca.com
(Received 5 November 2005, accepted
19 December 2005)
doi:10.1111/j.1742-4658.2006.05110.x
Procarboxypeptidase U [proCPU, thrombin-activatable fibrinolysis inhib-itor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma ProCPU has been proposed to
be a molecular link between coagulation and fibrinolysis Upon activation
of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability The inherent instability of CPU is likely to be of major importance for the in vivo down-regulation of its activity, but the under-lying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underly-ing the instability of CPU’s activity We have shown that sunderly-ingle as well as
a few 2–4 mutations in human CPU can prolong the half-life of CPU’s activity at 37C from 0.2 h of wild-type CPU to 0.5–5.5 h for the mutants
We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteo-lytic digest by trypsin Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down-regu-lating fibrinolysis
Abbreviations
BEVS, Baculovirus expression vector system; CLT, clot lysis time; CPB, carboxypeptidase B; CPU, carboxypeptidase U; EPP, error prone PCR; Hip-Arg, hippuryl- L -arginine; ORF, open reading frame; PTCI, potato tuber carboxypeptidase inhibitor; TAFI, thrombin-activatable fibrinolysis inhibitor; WT, wild type.
Trang 2peptide (prepeptide, see Fig 2) It can be activated
from its zymogen form to CPU by thrombin, plasmin
or most efficiently the thrombin–thrombomodulin
complex by cleavage after R114 [1,5,6](Fig 2) In
con-tact with a fibrin clot, CPU attenuates fibrinolysis
by removing carboxy-terminal lysines from partially
cleaved fibrin molecules, thereby diminishing its
cofac-tor activity for activation of plasminogen to plasmin
[7–9] Following its activation, CPU’s activity is
unsta-ble both in vivo and in in vitro experiments (as an
isolated protein), with reported half-lives at 37C from
8 to 15 min, hence the U in its name stands for
unsta-ble [10,11] The inherent and irreversiunsta-ble decay of
CPU’s activity is believed to be of major importance
for its in vivo down-regulation of activity and has been
linked to structural changes of the enzyme [3,12,13]
In vivo, CPU can also be inactivated by proteolytic
degradation, indicating more accessible and flexible
parts of the molecule exist It was therefore suggested
that the instability of CPU’s activity is due to intrinsic
structural lability of the enzyme, priming its
inactiva-tion [14]
Because of its prominent bridging function between
coagulation and fibrinolysis, the development of CPU
inhibitors as pro-fibrinolytic agents is an attractive
concept [15,16] But the instability of the enzyme has
prevented crystallization of CPU and the use of
struc-turally based drug design methods A
three-dimen-sional model of human proCPU based on the structure
of human pancreas procarboxypeptidase B, a closely
related protease exhibiting a higher stability, has been
published recently by Barbosa Pereira et al [17]
Recently, it was reported independently by two
separate groups that CPU prevents clot lysis from
proceeding into the propagation phase through a
threshold-dependent mechanism [18,19] The study of
this threshold phenomenon and, more generally, the
study of the effect of CPU on fibrinolysis, are also
severely complicated by its intrinsic instability of
activity
‘Directed evolution’ approaches allow the random generation of a large number of mutants followed by selection for the desired features Several proteins have been changed towards more desired properties using this approach Some examples include deoxyribonucleo-side kinases for changed substrate specificities [20,21], phosphotriesterase for improved catalytic rates [22], haem peroxidase for exotic environments (for example, inside a washing machine) [23], or amylase and sub-tilisin for improved thermostability [24,25]
In this study, we present the generation of CPU mutants with highly stable activity obtained by molecular evolution techniques and selection for decreased thermo-inactivation To achieve this we used
a directed evolution approach comprising the genera-tion of random libraries and recombinagenera-tion of advan-tageous mutations by Fragment-INduced Diversity (FINDTM) technology, as well as site-directed muta-genesis A high-throughput screen based on mamma-lian cells expressing proCPU mutants was developed
to select CPU variants with more thermostable activ-ity Seven proCPU mutants were selected and purified After activation by thrombin–thrombomodulin, three showed a remaining activity of more than 80% after 24-h incubation at 22C versus 20% for the wild type (WT), and two of these three showed a more than 25-fold increase in half-life of activity at 37 C Using one of the stable mutants, we have demonstrated the importance of CPU stability over proCPU concentra-tion in down-regulating fibrinolysis
Results
To investigate the role of exposed hydrophobic resi-dues on the stability of CPU’s activity, 13 point muta-tions were introduced in proCPU by site-directed mutagenesis and expressed in 3T3 cells (F135Q, I147S, F201T, I204Y, I205E, I204Y⁄ I205E, L214N, F244T, L281S, L335S, L376Q, T347I) Based on the alignment
of CPU sequence to the structure of carboxypeptidase
Fig 1 Physiological role of CPU CPU
attenuates fibrinolysis by removing
C-ter-minal exposed lysines from partially
degra-ded fibrin.
Trang 3B (CPB) [26] (Fig 2), these mutants were chosen to
replace hydrophobic amino acids of human CPU with
more hydrophilic residues located on the surface of
porcine CPB In addition, the T347I naturally
occur-ring variation in CPU was reported to double the
half-life (T1⁄ 2) of its activity at 37 C [11] and was
therefore included We found that the T347I mutant,
when tested in cell culture supernatant, was only 50%
more stable than our WT CPU with threonin at
posi-tion 347 (Table 1) Recently, Barbosa Pereira et al
[17] proposed, on the basis of their model of human
CPU, that the two consecutive I at positions 204 and
205 are exposed to the surface, and because they are
quite unique to CPU, might be of importance for the
process of CPU’s activity destabilization When we changed these two amino acids to their counterpart in porcine CPB (I204Y⁄ I205E), the T1 ⁄ 2 of the mutants’ activity was unchanged compared with WT CPU (data not shown)
In order to create a high number of mutants, ran-dom mutagenesis was done using either error prone PCR (EPP) or creating a library of mutants with the Genemorph PCR mutagenesis kit (GMK, Stratagene,
La Jolla, CA, USA) Sequencing of the full open read-ing frame (ORF) of randomly picked clones from these two approaches revealed a base mutation frequency of 0.41 ± 0.22% and 0.55 ± 0.23% per clone in 19 clones from the EPP library and in 17 clones from the
Fig 2 Multiple alignment of human preproCPU, human preproCPB and porcine proCPB The amino acid sequences of human preproCPU (accession number AAP35582.1), human preproCPB (accession number P15086) and porcine proCPB (accession number 1NSA) were aligned using CLUSTAL W [40] The pre- and the propeptide in preproCPU are shaded in black and grey, respectively Amino acid exchanges found in mutants with increased thermostability of CPU’s activity are marked in yellow ‘*’ means that the residues that column are identical
in all sequences in the alignment ‘:’ means that conserved substitutions have been observed ‘.’ means that semiconserved substitutions are observed.
Trang 4GMK library, respectively On the amino acid level
this corresponded to an average of 3.7 or 5.2
exchanges per enzyme for the error-prone PCR or the
Genemorph kit, respectively
In total, 24 600 clones, 14 600 from the EPP library
and 10 000 from the GMK library were screened for
improved thermostability of CPU activity in the
super-natant of mammalian cells in an HTS format The best
clones selected were more thoroughly analyzed using
an HPLC-based activity assay for CPU The most
sta-ble clone, GMK1, is five times more stasta-ble than WT
CPU From both libraries, about 1 in every 5000
clones exhibited a more than doubled T1⁄ 2 of activity
compared with WT The best clones selected from the
random mutagenesis approach, as well as the
site-direc-ted mutagenesis, are summarized in Table 1 and
consti-tute the basis for the first round of FINDTM
treatment It was also noted that all clones displayed
fewer mutations than the average number of mutations
present in randomly selected clones from both
libraries
To explore further combinations of activity
stabil-izing mutations identified in the first screening step
(Table 1) FINDTM was used For the first round of
FINDTMapproach, the following clones from Table 1
were used in two different combinations: in F1.1:
EPP1, EPP2, GMK1, GMK2 and, in F1.2: all clones
in Table 1 except WT FINDTM libraries were
expressed and 5000 clones of each library screened for
improved thermostability Table 2 summarizes clones
derived from this step As shown in Table 2, six clones
with improved T1⁄ 2 of their activity compared to the
parental clones could be found in the F1.1 treatment,
while only two clones were found in the F1.2 treat-ment with improved or equal properties, despite the higher number of clones put into this library It should also be mentioned here that the FINDTM treatment not only recombined existing mutations, but also intro-duces new mutations as observed in six out of the eight selected clones (Table 2)
To ascertain the combination of mutations that are very close in sequential space, the GMK2 clone (Table 1) was modified by site-directed mutagenesis to create the mutants YQ and YP, and the T1⁄ 2 of their activity was determined (Table 2) These combinations increased the stability of CPU’s activity, especially the
YQ mutant
Following the first round of FINDTM treatment, 50% of the mutants with improved thermal stability
of their activity appeared to bear mutations in the region encompassing residues 327–357 New mutants were made by site-directed mutagenesis, trying to combine the mutations leading to the strongest decrease in thermo-inactivation by site-directed muta-genesis The stability of their activity was evaluated either after expression in 3T3 cells or in insect cells using the Baculovirus expression vector system (BEVS) (Table 3) as an alternative expression system The S327P mutation was introduced because P is the corresponding amino acid to S327 in porcine CPB (Fig 2)
A second round of FINDTM treatment (F2) then included the clones: GMK2 + T347I, F1.1.C + R315H, F1.1.F + S327P, F1.1.A and YQ (see Tables 2
Table 1 Half-life (T1 ⁄ 2) of different CPU mutants’ activity at 37 C
created by site-directed or random mutagenesis WT and mutant
CPU were expressed in 3T3 cells and their stability was accessed
in the cell culture supernatant The remaining enzymatic activity
after incubation of CPU or its mutants at 37 C was determined
using a HPLC assay.
Clone
Amino acid
changes
in CPU
T1 ⁄ 2 at
37 C (min)
Method of generation
a
This mutation was not present in all PCR products derived from
this clone.
Table 2 Half-life (T1 ⁄ 2) of different CPU mutants’ activity at 37 C derived from the first round of FIND TM treatment and site-directed mutagenesis WT and mutant CPU were expressed in 3T3 cells and their stability was accessed in the cell culture supernatant The remaining enzymatic activity after incubation of CPU or its mutants
at 37 C was determined using a HPLC assay New mutations, not present in the parental clones are underlined.
Clone Amino acid changes in CPU
T1 ⁄ 2 at
37 C (h) F1.1.A I251T, H315R, S327C, N350S, H357Q 2.2 F1.1.B K166N, H315R, S327C, N350S, H357Q 1.5
a
These mutants were generated by site-directed mutagenesis from GMK2.
Trang 5and 3) Libraries created from these clones by FINDTM
technology were expressed, screened and characterized
as described in material and methods A total of about
14 200 clones were screened Table 4 summarizes clones
derived from this second round of FINDTM The
same mutation combination as in the best clone made
by site-directed mutagenesis (YQ + S327C) was also generated by this second round of FINDTM treatment and identified by the screening The subsequently increased activity stabilization during the different steps
of directed evolution and screening is illustrated in Fig 3, displaying the most stable clones found in each step
From the mutants created, seven clones (F1.2.A; F1.1.F, YQ, YQ + S327C, F1.2.A + R315H, F1.1.F + N348S, F1.1.F + H355Y) were chosen for expression using the BEVS and purification of the mutants for analysis as purified protein WT proCPU and mutants were expressed in Sf9 insect cells as C-terminal His-tagged proteins and purified from the supernatant of a 1-L culture using IMAC Figure 4 shows as examples the homogenity of the WT proCPU-CHis and YQ proCPU-CHis preparations (0-min samples)
The parameters determined for these mutants are summarized in Table 5 In contrast to the screening and previous characterization in crude cell superna-tants, assays were now carried out in a defined buffer
of 50 mm Hepes, pH 7.4 The T1⁄ 2 of CPU activity at
37C increased from 0.2 h for WT CPU to more than
5 h for the two most stable clones (Table 5) It appears that the T1⁄ 2 of activity measured directly in the supernatant of the cell cultures deviates from the T1⁄ 2
of the purified proteins in a defined buffer system It is likely that cell culture medium components influence the thermo-inactivation of the mutants This was con-firmed by putting purified YQ + S327C back into insect cell culture medium, which prolonged the T1⁄ 2
of activity at 37C (data not shown)
A second estimation of the thermal stability of activity of each mutant was measuring activity after
Table 3 Half-lives (T1 ⁄ 2) of different CPU mutants’ activities at
37 C made from clones in Tables 1 and 2 by site-directed
muta-genesis WT and mutant CPU were expressed in 3T3 cells or in
insect cells (as indicated) and their stability was accessed in the cell
culture supernatant The remaining enzymatic activity after
incuba-tion of CPU or its mutants at 37 C was determined using a HPLC
assay The T1 ⁄ 2 of the parental clone is shown in brackets for easy
comparison.
Clone Amino acid changes in CPU T1 ⁄ 2 at 37 C (h)
F1.1.C + R315H K166N, S327C, H357P 1.6 (4.4)
F1.1.A + R315H I251T, S327C, N350S,
H357Q
0.7 (2.2)
F1.1.F + H355Y b S327C, S348N, H355Y,
H357Q
4 (2.9) F1.1.F + S327P S327P, S348N, H357Q 0.3 c (2.9)
YQ + S327Cb,d S327C, H355Y, H357Q 26 (3)
a Very low expression level did not allow T1 ⁄ 2 determinations for
GMK2 + T347I b These mutants were expressed in insect cells and
have an 8xHis tag as described in Experimental procedures c
Activ-ity was determined using the Hippuricase assay.dThe same
combi-nation was independently found within the second FIND TM
treatment (see Table 4).
Fig 3 Subsequent increase in stability of activity during the directed evolution process of CPU T1 ⁄ 2 data at 37 C for the most stable clones as determined in the supernatant of 3T3 cells are presented More results for the different steps are presented in the correspond-ing tables: Random mutagenesis (Table 1), first FIND TM (Table 2), second FIND TM ⁄ site-directed mutagenesis (Tables 3 and 4).
Table 4 Half-life (T1 ⁄ 2) of different CPU mutants’ activity at 37 C
derived from the 2nd round of FIND TM treatment WT and mutant
CPU were expressed in 3T3 cells and their stability was accessed
in the cell culture supernatant The remaining enzymatic activity
after incubation of CPU or its mutants at 37 C was determined
using a HPLC assay Mutations not found in the parental clones are
underlined.
Clone
Amino acid changes
in CPU
T1 ⁄ 2 at
37 C (h)
H357Q
2.9
a Identical to YQ + S327C (see Table 3).
Trang 6incubation at 22C for 24 h (Table 5) Mutants
YQ + S327C, F1.2.A and F1.1.F + H355Y were
again the most stable and only one mutant, F1.1.F +
N348S, lost more than 50% of its activity
In order to exclude profound effects of the
muta-tions on enzymatic activity and inhibitor binding
affin-ity, the Km for hippuryl-l-arginine (Hip-Arg), the
specific activity at 24 mm Hip-Arg and the IC50for the
specific inhibitor PCI were determined As can be seen
in Table 5, the Kmvalues of the mutants shift to lower values, while all mutants except F1.1.F show an increased specific activity To detect any changes in the positioning of the propeptide, or, in other words, to see if the contact region between the catalytic domain and the prodomain was changed by the mutations, we also measured the residual activity without activation
by thrombin–thrombomodulin A correct positioning
of the propeptide should keep the residual activity on
Fig 4 Tryptic digest of WT and YQ proCPU-CHis (A) SDS PAGE of a bovine trypsin digest of WT proCPU-CHis (1.3 lgÆlane)1) and YQ pro-CPU-CHis (2 lgÆlane)1) Two proCPU-CHis to bovine trypsin ratios (w ⁄ w) were used: (i) 1 : 20 and (ii) 1 : 100 Digests were run at 26 C for the times indicated and then separated by SDS ⁄ PAGE and the gel was Coomassie stained Two major degradation products of WT- and YQ-proCPU-CHis became visible and are indicated by arrows in the figure (B) WT and YQ proCPU-CHis were digested by bovine trypsin as described under (A) (i) for the times indicated Fifteen micrograms per lane were separated by SDS ⁄ PAGE and transferred to a polyvinylid-ene difluoride membrane for N-terminal sequencing (Amidoblack staining) The bands indicated by numbers were identified as starting at the N-terminus with (i) a mixture of A115 and F23, (ii) a mixture of A115 and F23, (iii) Y353 and (iv) A115.
Table 5 Kinetic and stability parameters for purified WT and mutant CPUs The T1⁄ 2 of activity at 37 C in cell culture medium is shown in brackets for easy comparison Specific activity was determined at 24 m M Hip-Arg and the IC50of PCI at 4 m M Hip-Arg The specific activity for 24 m M Hip-Arg without activation by thrombin–thrombomodulin is given in brackets.
T1 ⁄ 2
at 37
C (h)
Activity left after
24 h at 22 C in
% (mean ± SD)
K m
(m M )
Specific activity (UÆmg)1)
IC50 PTCI (l M )
Trang 7the same level as for WT-proCPU until it is cleaved
away and proCPU activated to CPU The activities
ranged from 1.6 to 4.4 UÆmg)1, with 1.9 UÆmg)1 for
WT-proCPU-CHis, or, as a percentage of the specific
activity after activation, from 1.7 to 5.8% with 3.6%
for WT-proCPU-CHis At 4 mm Hip-Arg as substrate
in the assay, inhibition of all mutants is achieved at
somewhat lower concentrations of potato tuber
carb-oxypeptidase inhibitor (PTCI) The YQ mutant,
cho-sen because it had the most stable activity of the
purified mutants having only two mutations, was used
for further extensive characterization
To determine if the increased thermal stability of
CPU activity is connected to an increased
thermosta-bility of the protein itself, we monitored the thermal
unfolding of WT and YQ proCPU-CHis Compared
with the WT, the midpoint temperature (Tm) of the
protein-unfolding transition has increased for YQ
proCPU-CHis by 10.4C (Fig 5a) Because in YQ
proCPU-CHis, H355 and H357 are replaced by
nonio-nizable amino acids, we monitored thermal unfolding
also at different pH values (Fig 5b) Approaching low
pH values, when histidines become fully protonated, a
pronounced drop of Tm was seen with WT
proCPU-CHis, while only a marginal one was recorded with
YQ proCPU-CHis The drop in Tm from pH 7.4 to
pH 4.5 was 12.8C for WT proCPU-CHis but only
2.3C for YQ proCPU-CHis This indicates a role of
H355 and⁄ or H357 in the thermal stability of proCPU
Furthermore, we digested WT proCPU-CHis and YQ
proCPU-CHis with bovine trypsin (Fig 4), which
resulted in the case of WT proCPU-CHis in one
prominent degradation product of approximately
25 kDa and a weak, probably intermediate band at
about 38 kDa (arrows in Fig 4A), while for YQ
proCPU-His, a strong band at 38 kDa became visible
but none was visible at about 25 kDa Subsequently,
N-terminal sequencing of these bands identified a
clea-vage site between R352 and Y353 in WT
proCPU-CHis, but not in the YQ mutant Consequently, the
two mutations of YQ make the mutant less sensitive
to tryptic digestion close to the positioning of its two
mutations
Next, we compared the affinity of the enzyme for
synthetic and physiological substrates, and determined
the Kmconstants of native CPU from plasma,
recom-binant WT CPU and YQ CPU for Hip-Arg and
bra-dykinin using an arginine kinase-based kinetic assay
[27] Data are presented in Table 6 No differences
were seen in the Kmvalues of the three CPUs for
bra-dykinin and Hip-Arg when the kinetic assay was used,
proving that the mutations in the YQ proCPU did not
alter the affinity of the carboxypeptidase for synthetic
and physiological substrates However, when the Km for Hip-Arg was measured using HPLC (Table 5), YQ shows Kmvalue similar to the kinetic assay, while WT CPU does not
Fig 5 Thermal unfolding of WT CHis and YQ proCPU-CHis The thermal unfolding of WT and YQ proCPU-CHis was mon-itored using the fluorescent dye Sypro orange The unfolding pro-cess results in increase in fluorescence, which was monitored (A) shows the means of three independent unfolding curves in 50 m M
Hepes pH 7.4 and the solid line present the best fit of equation 1
to all data (B) shows the Tmof thermal unfolding curves at differ-ent pH values (best fit of equation 1 to all data ± SEM of the fit).
d , YQ proCPU-CHis; s, WT proCPU-CHis Buffers used were
50 m M sodium acetate, pH 4.5, 50 m M Mes pH 5.6–6.5, 50 m M
Hepes, pH 7.4.
Table 6 Comparison of Km constants of native, WT and YQ CPU for Hip-Arg and bradykinin using an continuous enzyme assay.
K m values are expressed in lMÆL)1and are the mean ± SEM of a duplicate measurement.
Trang 8The hypothesis that CPU down-regulates fibrinolyis
by a threshold dependent mechanism was recently
pub-lished [18,19] As long as the CPU activity remains
above this threshold (reported to be 8 UÆL)1),
fibrinoly-sis does not accelerate but stays in its initial phase [19]
The study of this threshold phenomenon is severely
complicated by the intrinsic instability of CPU’s
activ-ity YQ proCPU-CHis was consequently tested for its
antifibrinolytic potential in an in vitro clot lysis assay
and used for confirmation of the threshold hypothesis
We reconstituted proCPU-depleted plasma with
increasing amounts of the activated stable YQ mutant
or with WT CPU (CPU activities ranging from 0 to
237 UÆL)1) and used these in clot lysis experiments, as
described previously [19,28] Recovery of the added
CPU was in the range of 96–103%, as measured with a
kinetic plasma assay [27] The final t-PA concentration
used was 40 ngÆmL)1 The stable YQ mutant was able
to prolong the in vitro clot lysis time (CLT) in a way that
can be theoretically expected based on its stability
The decay of CPU can be expressed using the
fol-lowing simplified equation
N¼ N0· e–k⁄ t where k¼ ln(2) ⁄ T, T ¼ half life of CPU
Rearrangement of this formula gives the equation:
t¼ [T log(2))1]· [log(No ⁄ N)], where t is the time
above the threshold, N0the initial CPU activity and N
the threshold activity value
This equation indicates that the time above the threshold is linearly related with the CPU half life and only logarithmically with the initial CPU activity (gen-erated from proCPU by first order kinetics) The hypo-thesis that this time above the threshold determines the CLT is strongly confirmed and illustrated in Figs 6 and 7
Figure 6 shows representative clot lysis profiles at different YQ CPU concentrations Increasing the enzyme activity below the ‘threshold value’ did not show a significant increase in CLT However, each doubling of the CPU activity in excess of the ‘thresh-old value’ increased CLT with one CPU mutant half life Plotting log (CPU activity added) versus CLT clearly confirms the CPU threshold hypothesis The estimated threshold value in our experiments was
12 UÆL)1 which corresponds very well with the
8 UÆL)1described by Leurs et al [19]
Figure 7 illustrates the linear relationship between CPU stability and CLT Adding 40 UÆL)1WT CPU to proCPU-depleted plasma increases CLT by 22 min However, the addition of 40 UÆL)1 YQ CPU (with a 7.5- fold increased stability) increases CLT by
153 min, which corresponds very well with the increase one theoretically can expect (i.e 7.5· 22 min) When the selective CPU inhibitor PTCI (20 lgÆmL)1) was added from the start, no significant prolongation of CLT was seen by adding YQ or WT CPU
1 YQ t1/2
Fig 6 Threshold hypothesis confirmation The graph shows representative clot lysis profiles of proCPU depleted plasma reconstituted with increasing concentrations of activated YQ mutant (concentrations ranging from 0 UÆL)1to 237 UÆL)1) The threshold value is estimated by plotting log of the CPU activity added versus the clot lysis time (inset) Each doubling of the enzyme activity above the threshold value increases clot lysis time with one CPU mutant half-life.
Trang 9Due to its physiological role and the need for a very
tight regulation in the blood coagulation cascade, it is
likely that CPU has been selected for intrinsic
instabil-ity, which ensures rapid inactivation of its activity at
the site of action The irreversible decay in activity has
been shown to be accompanied by structural changes
of CPU [12,13], it is therefore very likely that the loss
of activity is caused by structural changes of the
enzyme triggered upon activation This instability is a
serious challenge when dealing with overexpression
and purification of the protein The mechanism behind
CPU’s activity inactivation is still not fully understood,
but several aspects contributing to CPU’s instability
are illuminated by our work
CPB is a close homologue to CPU, but has a
signifi-cantly higher stability Aligning the CPU sequence
onto the CPB structure [26] reveals the presence of
numerous potentially exposed hydrophobic amino
acids in CPU Exposed hydrophobic residues lead to
aggregation, and replacing exposed hydrophobic
resi-dues with more polar resiresi-dues has been reported to
stabilize proteins [29,30] Of the 12 hydrophobic to
hydrophilic point mutations carried out in CPU, only
one, L376Q (clone A), had a stabilizing effect, in this
case, of about 33% All the other mutants either did
not change the T1⁄ 2 of CPU’s activity more than
± 20%, or, in the case of I147S, did not express at all
(data not shown), suggesting that the instability does
not result from hydrophobically driven aggregation of
the protein This is further confirmed by the existence
of a natural variant of CPU, where T347 is subsituted
by an I Although accentuating the hydrophobic
character of the protein surface, the mutation induces
a stabilization of the protein (Table 1 and [11]) Random evolution of the enzyme has allowed us to identify mutants of 2.5 to five-fold increased T1⁄ 2 in activity (Table 1), with one or two mutations per clone The following first round of FINDTM treatment pro-longed T1⁄ 2 from 12 min for the WT to 4.4 h for clone F1.1.C Further combination by rational site-directed deletion or addition of mutations (Table 3) resulted in more than half of the cases in a decrease of T1⁄ 2 A fur-ther round of FINDTMtreatment did not improve T1⁄ 2 further compared with a combination of mutations pre-viously found by site-directed mutagenesis, but inde-pendently produced the same combination of mutations that were also determined to display the most stable activity (YQ + S327C¼ F2.C) An overall view of the evolution process is presented in Fig 3
A number of mutants with modifications in this region of the polypeptide chain were expressed in insect cells, purified and characterized (Table 5) The mutant displaying the most stable activity at 37C had mutations at the positions S327, H355 and H357, and this is also reflected by the selection of proteins to
be purified, that all have at least two mutations at these positions The T1⁄ 2 of activity of the purified mutants determined in a defined buffer system, as used during purification procedures, differed significantly from T1⁄ 2 determined in mammalian or insect cell cul-ture supernatant From a practical point of view, to allow for high-throughput mutant screening, thermo-stability had to be measured in cell culture superna-tants The corresponding values obtained from purified proteins show that cell medium itself and⁄ or unknown substances secreted by the cells sometimes strongly
Fig 7 CPU stability versus proCPU concentration in influencing clot lysis time The graph shows the effect of adding increasing activities of
WT CPU and YQ CPU on the clot lysis time, clearly showing the importance of the CPU stability over proCPU concentration.
Trang 10prolonged or decreased the stability of the activity of
the CPU mutants (Table 5) This is most striking for
YQ + S327C, with T1⁄ 2 of 5.5 h in Hepes buffer,
6.8 h in mammalian cell culture medium and 26 h in
insect cell culture medium For mutants containing the
S327C mutation, conditions, such as pH, determining
how fast oxidation of the cystein might occur, may
play a role
Because most of the purification and in vitro assays
are carried out at room temperature, we also determined
the activity after 24-h incubation at 22C Of all
puri-fied mutants, three showed a remaining activity of more
than 80% after 24-h incubation at 22C versus 20% for
the WT, and two of these three showed a more than
25-fold increase in T1⁄ 2 of activity at 37 C The decreased
Kmand mostly increased specific activity may partially
reflect the improved stability of activity, especially at
low substrate concentrations during Kmdeterminations,
resulting in a higher velocity than for WT CPU and
thereby decreasing the observed Km in comparison to
WT CPU This hypothesis is supported by the use of
a newly developed continuous coupled enzyme assay
instead of the discontinuous HPLC assay that
demon-strated similar Kmvalues of the native and WT, and the
YQ mutant CPU with a synthetic and physiological
substrate of CPU There seem to be no major changes
in the positioning of the propeptide, as indicated by
residual activities of the mutants close to WT-proCPU
IC50 values for the inhibition by a specific inhibitor
PTCI [16] are maximally five-fold lower than for the
WT With the exception of stability of activity, the CPU
mutants appear surprisingly similar to the WT in their
enzymatic properties
Marx et al [31] also described the generation of
forms of CPU with a highly stable activity, but in
con-trast to the work presented here, this refers to a hybrid
of CPU ending at position 314 (Fig 2) fused to the
following C-terminal part of human CPB This
chi-mera had a half-life of 1.5 h at 37C We therefore
show here that a stabilization of CPU’s activity that is
more than that which naturally occurs can already be
achieved with only one or a few mutations in the
region following position 314 in CPU
Fifty per cent of the residues mutated in the clones
selected from the first round of FINDTM are located
in a distinct region encompassing residues 327–357
(Table 2 and Fig 2), as well as the naturally occurring
and activity stabilizing mutation T347I The mutants
with the most stable activity are achieved by
combina-tions of few conservative mutacombina-tions, S327C, H355Y
and H357Q Can the effects of the mutations reported
here and the reasons for the increased stability of
activity if connected to structural changes be rationally
explained? The three residues correspond to P300, Y327 and P329 in porcine CPB (numbering according
to Fig 2) Keeping a strict orientation of the side chains, replacing P300 with a serine would leave the H-bond to the OH group of the side-chain nonsatis-fied, thereby destabilizing the protein Based on the CPB structure, H355 lies in close proximity to a cluster
of charged residues: R324, K326, H330 and E360 Introducing a Q at position 355 is likely to favour the formation of H-bonds with one or several of these resi-dues, attenuating the charge repulsions between some
of the basic amino acids The stabilization induced by the replacement of H357 by a Y is more difficult to explain, but the aromatic nature of the side chain is likely to interact favourably with the hydrophobic clus-ter made up of I316, F318, A337 and V341 Another contribution to the low stability of the WT proCPU is the close spatial proximity of the three His residues at
330, 355 and 357 In the YQ mutant, two histidines are replaced by nonionizable amino acids Although not very pronounced at physiological pH, partial charges on the His could induce a destabilizing charge–charge repulsion effect This hypothesis is sup-ported by the findings that WT proCPU-CHis is less stable in thermal unfolding at low pH, when H330, H355 and H357 would be protonated, while the drop
of stability of YQ proCPU-CHis is a lot less pro-nounced (Fig 5b)
These observations suggest that our mutations improve residue interactions in this region, leading to
an improved structural stability of the protein Limited trypsinolysis of WT and YQ proCPU-CHis further corroborate this scenario, as trypsin cleavage occurs at R352 in WT CPU, but not in the mutant harbouring the H355Y⁄ H357Q mutations (Fig 4)
Recently, the hypothesis was put forward that CPU can down-regulate fibrinolysis through a threshold-dependent mechanism [19] We used the stable
YQ CPU mutant to test this hypothesis The antifi-brinolytic potential of the stable mutant was tested in
an in vitro clot lysis assay The YQ mutant was able to prolong in vitro clot lysis time in a way that can be expected based on the stability of its activity Thus YQ
is the first described stable CPU form with conserved antifibrinolytic potential This threshold hypothesis [19] could be confirmed by adding activated YQ pro-CPU-CHis to proCPU depleted plasma and plotting CLT versus the log of the CPU activity added The threshold value in our experiments was 12 UÆL)1, which is in good agreement to the value reported by Leurs et al [19] of 8 UÆL)1 As long as CPU remains above this activity value, fibrinolysis does not proceed into the acceleration phase The threshold hypothesis