doi:10.1016/S0022-2836(02)01102-6 available online at http://www.idealibrary.com on
w
B
J. Mol. Biol. (2002) 324, 237–246
Activation Mechanism of Pro-astacin: Role of the Propeptide, Tryptic and Autoproteolytic Cleavage and
Importance of Precise Amino-terminal Processing
Irene Yiallouros1†, Reinhild Kappelhoff1†, Oliver Schilling1
Frank Wegmann1, Mike W. Helms1, Astrid Auge1
Gertrud Brachtendorf1, Eva Große Berkhoff1, Bernd Beermann2
Hans-Jürgen Hinz2, Simone König3, Jasna Peter-Katalinic3 and
Walter Stöcker1*
1
Institute of Zoophysiology
University of Münster
Hindenburgplatz 55, D-48143
Munster, Germany
2
Institute of Physical Chemistry
University of Münster
Schlossplatz 4/7, D-48149
Munster, Germany
3
Institute of Medical Physics
and Biophysics, University of
Münster, Robert-Koch-Str 31
D-48149 Munster, Germany
Astacin (EC 3.4.24.21) is a prototype for the astacin family and for the metzincin superfamily of zinc peptidases, which comprise membrane-bound
and secreted enzymes involved in extracellular proteolysis during tissue
development and remodelling. Generally, metzincins are translated as
pro-enzymes (zymogens), which are activated by removal of an N-terminal pro-peptide. In astacin, however, the mode of zymogen activation
has been obscured, since the pro-form does not accumulate in vivo. Here
we report the detection of pro-astacin in midgut glands of brefeldin Atreated crayfish (Astacus astacus ) by immunoprecipitation and mass spectrometry. We demonstrate that the pro-peptide is able to shield the active
site of mature astacin as a transient inhibitor, which is degraded slowly.
In vitro studies with recombinant pro-astacin in the absence of another
protease reveal a potential of auto-proteolytic activation. The initial cleavage in this autoactivation appears to be an intramolecular event. This is
supported by the fact that the mutant E93A-pro-astacin is incapable of
autoactivation, and completely resistant to cleavage by mature astacin.
However, this mutant is cleaved by Astacus trypsin within the pro-peptide. This probably reflects the in vivo situation, where Astacus trypsin
and astacin work together during pro-astacin activation. In a first step,
trypsin produces amino-terminally truncated pro-astacin derivatives.
These are trimmed subsequently by each other and by astacin to yield
the mature amino terminus, which forms a salt-bridge with Glu103 in the
active site. The disruption of this salt-bridge in the mutants E103A and
E103Q results in extremely heat labile proteins, whose catalytic activities
are not altered drastically, however. This supports a concept according to
which the linkage of Glu103 to the precisely trimmed amino terminus is
a crucial structural prerequisite throughout the astacin family.
q 2002 Elsevier Science Ltd. All rights reserved
*Corresponding author
Keywords: astacin; metzincins; zinc peptidase; pro-enzyme; activation
mechanism
† These two authors contributed equally to this work.
Present addresses: G. Brachtendorf, F. Wegmann, Institute of Cell Biology, ZMBE, Von-Esmarch-Straße 56, D-48149
Münster, Germany; M. W. Helms, Institute of Clinical Chemistry and Laboratory Medicine, Albert-Schweitzer-Str. 33,
D-48149 Münster, Germany; A. Auge, Institute of Physiological Chemistry and Pathobiochemistry, Waldeyerstr. 15,
D-48149 Münster, Germany; O. Schilling, EMBL, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany.
Abbreviations used: BMP1, bone morphogenetic protein 1; Dns, dansyl, 5-(dimethylamino)-naphthalene-1-sulfonyl;
E-64, L -trans-epoxysuccinyl-leucylamide-(4-guanido)-butane; IPTG, isopropyl-b-D -thiogalactopyranoside; MMP,
matrix metalloproteinase; PBS, phosphate-buffered saline; PVDF, polyvinylidene fluoride; STANA, succinyl-Ala-AlaAla-4-nitroanilid; TFA, trifluoroacetic acid; brefeldin A (BFA), g,4-dihydroxy-2-[6-hydroxy-1-heptenyl]-4cyclopentanecrotonic acid lambda-lactone.
E-mail address of the corresponding author: [email protected]
0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved
238
Introduction
The zinc endo-peptidase astacin (EC 3.4.24.21), a
prototype of the astacin family1 – 3 and the metzincin superfamily4 is synthesized in the hepatopancreas of the crayfish Astacus astacus L and stored
as an active protease in the stomach.5 There it acts
as a collagenolytic digestive enzyme, while other
members of the astacin family like the BMP1/tolloid-like enzymes,6 – 9 the meprins10 – 12 or the hatching proteases13,14 catalyse the limited proteolysis of
extracellular matrix components during embryonic
development and tissue assembly.
As predicted from the cDNA and genomic DNA
sequences15,16 astacin is synthesized as a pre-proenzyme. The cDNA sequence of the pre-pro-segment of astacin encodes 49 amino acid residues
with a signal peptide of 15 residues and a prosequence of 34 residues. For activation of pro-astacin, the amino-terminal pro-peptide has to be
removed by cleavage between Gly2 1 and Ala1,
since in the X-ray crystal structure17 the aminoterminal alanine residue of mature astacin is saltbridged to the side-chain carboxylate of Glu103
(Figure 1). This highly conserved residue is contained in the active-site consensus sequence 92HExxHxxGxxHE-103 as a direct neighbour of
His102, one of the three histidine residues ligating
the catalytic zinc ion together with Tyr149 and a
water molecule.18
It has been suggested that processing of proastacin occurs on its way from the hepatopancreas
to the stomach5 but the mechanism of activation
and the responsible protease(s) have remained
unknown so far. A comparison of the pro-peptides
of the astacin family members indicates several
possible modes of activation. Some astacins exhibit
an arginine or lysine residue in position 2 1 which
permits direct processing by trypsin as is the case
Figure 1. The (magenta) water-mediated salt-bridge
between Ala1 and Glu103 in mature astacin. In the proenzyme, the N-terminal pro-peptide prevents this interaction. MOLSCRIPT34 was used for modelling.
Activation Mechanism of Pro-astacin
for meprin.3 Others like the BMP1/tolloid-like
enzymes contain a furin cleavage site (R-X-K/R-R)
in positions 2 4 to 2 1.2 Astacin itself does not contain a tryptic or a furin-type cleavage site preceding its N-terminal residue.15,16
In the present study, we immunoprecipitate proastacin from crayfish midgut glands and demonstrate that mature astacin degrades its own propeptide very slowly and is transiently inhibited by
it. We observe that recombinant pro-astacin is able
to activate itself autoproteolytically in vitro, and
we provide evidence that, under physiological conditions, Astacus trypsin and astacin work together
successively during pro-astacin activation. Furthermore, we show that the substitutions of Glu103 by
Gln or Ala have only minor influence on catalysis,
but render the enzyme extremely unstable.
Results
Immunoprecipitation of pro-astacin from
crayfish hepatopancreas
By 2D PAGE of immunoprecipitated protein
from hepatopancreas extracts of brefeldin A-treated animals with anti-astacin antibodies, we
resolved a cluster of spots around 29 kDa with an
average pI of 3.6 and a spot of about 22 kDa and a
pI of 3.4, respectively (Figure 2). The 22 kDa spot
was identified as astacin by matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF)
mass spectrometry according to three assignable
astacin fragments. The spot of higher molecular
mass (29 kDa) was identified as pro-astacin, since
it contained the pro-peptide specific fragment
ALYYNDGMFEGDIK. Non-reducing conditions
were used for preparative 2D PAGE to reduce
interference by antibody light chains migrating at
the same position as pro-astacin on reducing 2D
PAGE gels. This resulted in the same characteristic
spatial arrangement of pro-astacin and astacin
spots as seen under reducing conditions (not
shown) by staining with Coomassie brilliant blue
or by immunodetection. The pI values of reduced
Figure 2. Non-reducing 2D-PAGE and staining with
Coomassie brilliant blue of the immunoprecipitation
from crayfish hepatopancreas extract using anti-astacin
antibodies. The acrylamide concentration in the second
dimension was 12%.
239
Activation Mechanism of Pro-astacin
most pro-peptide fragments, their identity and
their position within the protein sequence could
be derived from the correlation of their respective
m/z values for molecular [M þ n H]nþ ions,
detected in the electrospray ionization quadrupole
time-of-flight (ESI-QTOF) mass map (Table 1). The
resulting fragmentation pattern is in accordance
with the cleavage specificity of astacin (Figure
3(a)). The fragmentation of the pro-peptide by astacin was also monitored at a ten times lower concentration of enzyme (0.1 mM) in the presence of
the chromogenic substrate succinyl-Ala-Ala-Ala-4nitroanilid (STANA). Under these conditions, aryl
amidase activity could be observed only after
about 90% of the pro-peptide was degraded
(Figure 3(b)), indicating that the pro-peptide and/
or derived fragments inhibit STANA cleavage by
astacin. The degradation can be described as a
pseudo first-order decay, with an apparent rate
constant of:
kapp ¼ 2:1 £ 1024 s21 ð^9:3 £ 1026 s21 Þ
Figure 3. Fragmentation of the pro-peptide by mature
astacin. (a) First line: amino-terminal sequence of prepro-astacin, indicating the putative cleavage site of the
signal peptidase and the known amino terminus of
mature astacin. Residues of the mature form of astacin
are numbered according to the published amino acid
sequence35 and the X-ray crystal structure17 starting with
Ala1. In the pro-peptide and signal peptide, the residues
are numbered in reverse order with Gly 2 1 being the
neighbour of Ala1. Other lines: alignment of pro-peptide
fragment sequences. The arrows indicate the major cleavage sites by astacin in sequential order from top to bottom. NDGMFE does not correspond to a major m/z
value and is therefore shown in parentheses. (b) Pro-peptide degradation by astacin in the presence of the lowaffinity substrate STANA. 0.1 mM astacin was incubated
with 10 mM pro-peptide in the presence of 1.0 mM
STANA in 50 mM Hepes– NaOH at pH 8.0 and 25 8C.
Black dots, decay of the pro-peptide according to RPHPLC of 100 ml samples. Broken line, fit to a pseudo
first-order decay with an apparent rate constant of
kapp ¼ 2.1 £ 1024 s21 ^ (9.3 £ 1026 s21). Black lines, astacin
activity toward STANA in the presence or in the absence
of pro-peptide. STANA activity reaches the slope of the
curve measured in the absence of the pro-peptide only
after more than 90% of the pro-peptide is degraded.
and non-reduced astacin are pH 3.519 and pH 3.4,
respectively.
Kinetics of pro-peptide fragmentation and
fragment identification
Several proteases, e.g. papain-like cysteine proteases are inhibited efficiently by their own propeptides.20 In the case of astacin, the synthetic propeptide (50 mM) was degraded completely by
1 mM astacin within 120 minutes. Reversed phase
(RP) HPLC analysis revealed that astacin cleaves
the pro-peptide at several sites (Figure 3(a)). For
ð1Þ
Expression, folding, and activation of proastacin and E93A-pro-astacin
Pro-astacin was expressed in Escherichia coli
BL21(DE3) cells, purified by Ni-NTA-affinity chromatography, and folded by dilution and removal
of reducing agents and guanidinium chloride. In
contrast to mature astacin, the pro-enzyme did not
bind to the Pro-Leu-Gly-NHOH inhibitor affinity
column (data not shown). This indicates that the
pro-peptide shields the active site in the zymogen.
Folded pro-astacin (10 mM) kept at 8 8C in
50 mM Tris – HCl buffer (pH 8.5) was shown to be
capable of autoproteolytic activation, since it was
partially converted to mature astacin within two
days; the conversion was complete after two
weeks (Figure 4(a); 8 8C corresponds to physiological conditions; at higher temperatures the autoactivation is accomplished faster). According to
amino-terminal sequencing, the pro-enzyme
started with the sequence MSPI, indicating that it
still carried the initial methionine residue while
the final autoproteolytic cleavage product yielded
AAIL, the exact amino terminus of mature wildtype astacin (Figures 3(a) and 4(a)). As seen in
Figure 4(a), the autoproteolytic activation results
in a series of differently processed intermediates,
which run between the pro-enzyme and mature
astacin on SDS-PAGE. It has not been possible so
far to resolve these intermediates by N-terminal
sequencing, indicating a series of cleavages within
the pro-peptide resulting in a heterogeneous mixture of products. On zymography gels, only the
mature form, but not the pro-astacin precursor
exhibited gelatinolytic activity (Figure 4(b)). Interestingly, the rate of pro-astacin to astacin conversion could be enhanced strongly upon addition of
Astacus trypsin at a concentration of 10 nM, but
240
Activation Mechanism of Pro-astacin
Table 1. Correlation of deconvoluted m/z values of peptide components detected in the HPLC-separated fractions by
ESI-QTOF MS with their theoretical counterparts
Rt (minutes)
14.0
15.2
16.4
19.1
19.8
20.8
22.3
22.9
23.9
m/z (deconvoluted)
754.43
470.3
910.51
2838.44
952.58
725.41
1393.70
714.36
1904.94
1576.82
952.47
714.36
1462.72
2839.32
Assignment
RAGRQPA
IIPE/A or RQPA/R or QPAR/V
RAGRQPAR
SPIIPEAARALYYNDGMFEGDIKLR
IIPEAARAL or SPIIPEAAR
RQPARV or SPIIPEA
NDGMFEGDIKLR
DIKLRA
RALYYNDGMFEGDIKL
SPIIPEAARALYYN or GMFEGDIKLRAGRQ
IIPEAARAL or SPIIPEAAR
DIKLRA
SPIIPEAARALYY
PEAARALYYNDGMFEGDIKLRAGRQ or EAARALYYNDGMFEGDIKLRAGRQP
not by an equimolar concentration of mature astacin (Figure 5).
With the aim to avoid autoproteolytic processing
of pro-astacin and in order to check whether the
autoproteolytic activation of pro-astacin was intramolecular or intermolecular, we produced another
mutant, E93A-pro-astacin. The corresponding
mutation in mature astacin had been shown to render the enzyme inactive.21 E93A-pro-astacin could
be expressed, folded and purified to homogeneity
and was shown to be resistant against autocatalytic
cleavage (Figure 4(a)). The E93A mutant was resistant to cleavage by mature astacin, but it was
cleaved by crayfish trypsin within the pro-peptide
at the peptide bond between Arg2 3 and Val2 2,
as verified by protein sequencing.
Expression, folding and functional analysis of
E103A-astacin and E103Q-astacin
The water-mediated salt-bridge between the
side-chain of Glu103, which resides in the active
site and the ammonium group of Ala1, the amino
terminus of mature astacin, has been considered
to be crucial for the structure and function of the
enzyme, since the free amino terminus becomes
available only upon removal of the pro-peptide
(Figure 1).17 To study the role of this salt-bridge,
the mutants E103A-astacin and E103Q-astacin
were produced by site-directed mutagenesis and
expressed in E. coli BL21(DE3) cells. Subsequent to
the isolation of the mutant astacins from cell
lysates by Ni-NTA-affinity chromatography and
folding, the mutants were purified by Pro-LeuGly-NHOH affinity chromatography.21 Each of the
resulting preparations shows a single distinct
band on SDS-PAGE (Figure 6(a)).
Circular dichroism spectra of the folded mutants
were very similar to those of recombinant wildtype astacin and crayfish astacin, indicating the
typical secondary structure of astacin (Figure 6(b)).
Both the E103A and the E103Q mutant retain catalytic activity (Table 2). E103Q-astacin shows kinetic constants similar to those of the recombinant
wild-type astacin, and the kcat/Km-value of E103Aastacin is even about threefold higher.
In order to probe the effect of the Glu103 substitution on the structural stability, the enzyme variants were heated to 54 8C and the catalytic
activity monitored. This treatment led to a rapid
and complete loss of activity in the mutants
E103A-astacin and E103Q-astacin, with half-lives
of t1/2 ¼ 22 seconds and t1/2 ¼ 27 seconds, respectively. Crayfish astacin and recombinant wild-type
astacin proved to be much more stable, their
activity being decreased to 43% and 30% within
the incubation window of ten minutes, respectively
(Figure 7). The rapid loss of catalytic activity of the
mutant enzymes illustrates the important contribution of the salt-bridge to the overall structural
stability of astacin.
Discussion
Immunoprecipitation of pro-astacin
Amounts of pro-astacin sufficient for immunoprecipitation, staining with Coomassie brilliant
blue by following PAGE, and detection by MALDITOF analysis could be concentrated in hepatopancreas extracts upon pre-treatment of animals with
brefeldin A, which blocks vesicular transport
along the secretory pathway. Without this treatment, the concentration of pro-astacin remains so
low that it can be detected only by using antibody
techniques, even after stimulating enzyme
synthesis.5,22 The data provide evidence that proastacin is present only transiently during secretion.
Expression of pro-astacin
In some proteases, e.g. the pro-hormone convertase furin, the pro-peptide has the function of an
intramolecular chaperone.23 This seems to be not
the case in astacin, because the recombinant
mature enzyme, lacking the pro-part, folds readily
into the stable active conformation. This was
observed upon expression of astacin in insect cells
241
Activation Mechanism of Pro-astacin
Figure 5. Time-course of the activation of pro-astacin.
Pro-astacin (10 mM) was incubated alone (continuous
curve), in the presence of 10 nM crayfish trypsin (shortdash curve) or 10 nM astacin (long-dash curve). Astacin
activity released from pro-astacin was monitored by the
STANA assay (10 mM substrate). The latter curve was
corrected for the activity caused by the added mature
astacin (10 nM). Inset: SDS/14% polyacrylamide gel
stained with Coomassie brilliant blue. E93A-pro-astacin
(5 mg) was incubated alone or with 0.1 mM crayfish trypsin or 0.1 mM astacin. Cleavage by trypsin produced a
new band with about the same size as mature astacin
(23 kDa). Amino-terminal sequencing revealed that the
cleavage occurred between Arg3 and Val2 in the propeptide (see Figure 3(a)).
Figure 4. Autoproteolytic activation of pro-astacin.
(a) An SDS/14% polyacrylamide gel stained with Coomassie brilliant blue showing 5 mg of recombinant proastacin 60 hours after folding and subsequent dialysis,
and 5 mg astacin from autoproteolytic activation of the
shown recombinant pro-astacin after further storage for
two weeks at 8 8C. The sequences of both proteins were
determined by N-terminal sequencing. Crayfish astacin
(5 mg) and the inactive E93A-pro-astacin mutant (5 mg)
are used as standards. (b) Gelatine zymography (0.1%
(w/v) gelatine immobilized in an SDS/14% polyacrylamide gel) of 250 ng of the proteins shown in (a). Proastacin and E93A-pro-astacin are inactive; crayfish
enzyme migrates below the recombinant proteins, since
it lacks a His-tag.
(unpublished results) and in bacteria.15,21 Also, the
procollagen C-proteinase (BMP1) can be expressed
functionally without the pro-peptide,24 indicating
that the pro-peptide is not a general folding prerequisite in astacins.
Pro-peptide fragmentation
Mature astacin cleaves its synthetic pro-peptide
at several sites. Envisioning the cleavage specificity
of astacin,25 the pro-peptide should be a reasonably
good substrate. Surprisingly, it is turned over only
very slowly and it temporarily inhibits astacin.
Full activity was regained only after about 90% of
the added pro-peptide was digested. Hence, the
pro-peptide seems to shield the active site while it
is degraded slowly as a competing substrate.
Autoproteolytic activation
In the absence of another protease, recombinant
pro-astacin is capable of autoproteolytic activation.
Several lines of evidence suggest that the initial
cleavage in the autoactivation process might be
intramolecular. One indication is that pro-astacin
does not bind to a column with immobilized ProLeu-Gly-NHOH and is inactive toward peptide
and protein substrates like gelatine. Hence, the
active site of pro-astacin seems to be blocked by
the pro-domain. Furthermore, the mutant E93Apro-astacin was shown to be resistant to autoproteolytic processing, which would rule out the
possibility of activation cleavage by an accidentally
co-purified bacterial protease. Another argument
supporting an intramolecular cleavage is that the
E93A-pro-astacin is not processed upon addition
of mature astacin. Moreover, the pro-astacin to
astacin conversion is not accelerated upon addition
of active astacin. Therefore, it is very likely that the
pro-enzyme cuts the pro-peptide slowly in an
intramolecular fashion.
242
Activation Mechanism of Pro-astacin
Figure 7. Inactivation of the astacin-variants by heat
denaturation. The proteins (80 nM) were incubated at
54 8C. Samples were withdrawn at several time-points
between zero and ten minutes, and the residual enzyme
activity was determined using 0.23 mM Dns-Pro-LysArg-Ala-Pro-Trp-Val substrate and 2.0 £ 1029 M enzyme
in 0.1 M Tris – HCl buffer (pH 8.0). The activities are displayed in relation to the initial activities before incubation. E103A-astacin, filled triangles; E103Q-astacin,
open circles; recombinant wild-type astacin, filled circles;
crayfish astacin, open squares.
Figure 6. Site-directed mutagenesis of Glu103. (a) SDS/
14% polyacrylamide gel of the renatured and purified
E103A-astacin (5 mg), E103Q-astacin (5 mg) and crayfish
astacin (5 mg) stained with Coomassie brilliant blue.
(b) Representative CD spectra of the E103Q-astacin
(0.44 mg/ml; open squares) in comparison with recombinant wild-type astacin (1.1 mg/ml; filled triangles) and
crayfish astacin (0.7 mg/ml; open circles) at 20 8C. The
average value of 20 spectra each is shown.
Activation mechanism
In a previous study, active astacin cleaved an 18meric synthetic peptide mimicking the activation
site of pro-astacin and it was deduced that astacin
might activate its zymogen.22 However, the slow
autoactivation of pro-astacin is not in agreement
with the rapid pro-astacin to astacin conversion
seen in vivo.5 If this and the in vivo concentrations
of active astacin (1 mg/ml) and trypsin (3 mg/
ml)26 are taken into account, it seems that under
physiological conditions the activation mechanism
of pro-astacin follows a different route. We have
demonstrated in the present work that Astacus
trypsin, but not astacin cleaves pro-astacin and
accelerates its activation. Tryptic cleavage eventually results in a derivative, whose peptide chain
starts at Val2 (Figures 3(a) and 5). These data
suggest that the activation of pro-astacin minimally
requires two steps. The initial event is catalysed by
Astacus trypsin and produces a series of premature astacin species, the shortest of which starts
at Val2. Such premature derivatives and mature
astacin are responsible for subsequent cleavages
resulting in the mature amino terminus. It has
been shown that amino-terminally elongated
forms of mouse meprin A were enzymatically
active, albeit thermally unstable.27 The activation
mechanism of pro-astacin exhibits similarities
with that of other metzincins. In several proMMPs, for example, an initial cut by another proteinase in the so-called bait region is followed by
autoproteolytic cleavages, which eventually release
the mature N terminus.28
Table 2. Kinetic parameters for the hydrolysis of Dns-PKRAPWV by astacin mutants compared with recombinant
wild-type astacin (25 8C, 0.1 M Tris – HCl buffer (pH 8.0))
Astacin (1 £ 1029M)
Wild-type
E103Q
E103A
Km £ 1024 (M)
kcat (s21)
kcat/Km £ 105 (M21s21)
Activity (%)
Dns-PKRAPWV (M)
3.2 (^0.7)
3.0 (^0.7)
6.0 (^0.7)
57.9 (^4.9)
65.3 (^6.0)
305.6 (^20.4)
1.8 (^0.3)
2.2 (^0.8)
5.1 (^0.9)
100
122
283
5 £ 1026 2 1.0 £ 1023
1 £ 1025 2 1.5 £ 1023
1 £ 1025 2 1.5 £ 1023
243
Activation Mechanism of Pro-astacin
Importance of precise amino-terminal trimming
and salt-bridge formation
The precise trimming of the amino terminus
seems to be a key element in the pro-astacin to
astacin conversion, since it enables the correct formation of the buried salt-bridge between the
amino-terminal ammonium group and the carboxyl group of Glu103. This carboxylate group is
anchored in a network of hydrogen bonds involving several water molecules, the backbone carbonyl group of Asp140, the guanidinium group of
Arg106, the carboxyl group of Asp186 and the
hydroxyl group of Ser145. The latter is part of the
conserved Met-turn (SXMHY) consensus sequence
of the astacins.29 Hence, the basement of the zincbinding site and the amino-terminal segment are
connected tightly. However, mutations of Glu103
do not influence catalysis significantly (Table 2). In
the E103Q mutant, the glutamine side-chain is still
able to form hydrogen bonds, whereas in the
E103A-mutant the Ala side-chain is neither saltbridged to Ala1 nor integrated into the hydrogen
bonding network beneath the active site. This
should increase the flexibility and might explain
the elevated activity of the E103A-mutant.
Obviously, the gain in flexibility and catalytic efficiency is accompanied by a dramatic loss of overall
stability, as seen in the heat-denaturation assays.
Our data support the concept that Glu103 is not
critical for catalysis, but important for structural
stability mediated by the salt-bridge to the amino
terminus after release of the pro-peptide. The conservation of this position throughout the astacin
family suggests that the ability to form this saltbridge is a general structural requirement for
other
astacins
including
BMP1/tolloid-like
enzymes and meprin.
Materials and Methods
Animals
Adult A. astacus L were obtained from a commercial
crayfish farm (Dr M. Keller, Augsburg, Germany). The
animals were kept in darkened tanks containing aerated
fresh water at 13 8C, and fed on a mixed diet once a
week.
Chemicals
If not stated otherwise, all chemicals were obtained
from Amersham Bioscience, Freiburg; Applichem,
Darmstadt; Serva, Heidelberg; Biorad, Munich; Bachem,
Heidelberg; GIBCO Life Technologies, Eggenstein;
Sigma/Aldrich, Deisenhofen; Merck, Darmstadt;
GERBU, Gaiberg; New England Biolabs, Bad Schwalbach, Germany.
Enzymes
Astacin (accession numbers: EC 3.4.24.21; SWISSPROT P07584; PIR A25829; cDNA AJ242595; genomic
X95684; PDB 1AST, 1IAA, 1IAB, 1IAC, 1IAD, 1IAE,
1QJI, 1QJJ) was prepared as described,5,26 lyophilised
and stored at 220 8C. Concentrations were determined
spectrophotometrically using the molar extinction coefficient e280 nm ¼ 42,800 M21 cm21.30 Astacus trypsin from
A. astacus L. was purified and assayed as described.26
Pro-peptide
The pro-peptide of astacin, SPIIPEAARALYYNDGMFEGDIKLRAGRQPARVG, was synthesized at the Institute
of Biochemistry I, University of Erlangen, Germany and
stored at 220 8C as a lyophilisate. Concentration was
determined by absorption at 274 nm, based on the calculated molar extinction coefficient of (2x ) tyrosine
e274 nm ¼ 2,800 M21 cm21.31
Antibodies
Polyclonal antibodies were raised in rabbits against
the synthetic pro-peptide of astacin (anti pro-astacin
antiserum; SeqLab, Göttingen, Germany) and against
purified astacin from crayfish stomach (anti-astacin antiserum; Charles River WIGA GmbH, Kisslegg, Germany).
Astacin antibodies were affinity-purified before immunoprecipitation using the respective antigen as ligand.
Immunoprecipitation of pro-astacin from
hepatopancreas extracts
Crayfish were starved for five to seven days before the
gastric fluid was removed. Then 750 mg (1 mg/ml in 40%
(v/v) methanol) brefeldin A (g,4-dihydroxy-2-[6-hydroxy-1-heptenyl]-4-cyclopentanecrotonic acid lambdalactone; BFA; Sigma, Deisenhofen, Germany) were
injected into the hemolymph of the animals. After two
hours, the crayfish were sacrificed and the hepatopancreas glands removed. The intact glands were washed
thoroughly with a modified Tris-buffered Van Harreveld’s freshwater crustacean saline (200 mM NaCl,
5 mM KCl, 2.5 mM MgCl2·6H2O, 15 mM CaCl2·2H2O,
5 mM maleic acid, 5 mM Tris – HCl (pH 7.5)) to remove
contaminations of gastric fluid. Subsequently, the tissue
was dissected and stirred on ice for two to three hours
in lysis buffer in the presence of proteinase inhibitors
(50 mM Tris – HCl (pH 8.2), 1% (w/v) sodium deoxycholate, 1% (w/v) Triton X-100, 10 mM EDTA, 10 mM 1,10phenanthroline, 5 mM Pefabloc, 10 mg/ml E-64, 100 mg/
ml pepstatin A) before the extract was ground further in
a potter. After centrifugation (13,000 g, 20 minutes, 4 8C),
the supernatant (about 17 ml) was used for
immunoprecipitation.
Antibodies were coupled to protein A-Sepharose CL4B beads (Amersham Bioscience, Freiburg, Germany):
the beads were swollen in PBS (pH 7.4) at 20 ml/g for
ten minutes, incubated for two to three hours with antibodies (13.6 mg/ml of swollen beads) and washed three
times with 0.1 M potassium phosphate (pH 8.2). Before
immunoprecipitation the hepatopancreas extract was
incubated for two to three hours with protein A-Sepharose and cleared by centrifugation at 13,000 g to eliminate
unspecific binding. The supernatant was treated for ten
minutes at 7 8C with IgG-protein A-Sepharose for immunoprecipitation and centrifuged. The pellet was washed
three times with 0.1 M potassium phosphate, (pH 8.0),
1 M sodium chloride and finally with water. The precipitated proteins were separated from protein A-Sepharose by incubation for ten minutes at room temperature
244
in re-hydration buffer (9 M urea, 2% (w/v) Chaps, 1.5%
Servalyt 3-7, bromophenole blue) and centrifugation.
Electrophoresis
SDS-PAGE and Western blot analysis were performed
according to standard procedures.21
Two-dimensional PAGE was performed using
immobilized pH-gradients in the first dimension.32 IPGstrips (Immobiline DryStrip gels; Amersham Bioscience,
Freiburg, Germany) with a linear pH range of 3 – 6.5
were re-hydrated in the sample solution and electrofocussing occurred sequentially for 4.5 hours at 300 V,
4.5 hours at 1900 V and 15 hours at 3500 V. In the second
dimension, the samples were run under non-reducing
conditions on SDS-PAGE.
N-terminal sequencing
For N-terminal sequencing of proteins from SDS/
polyacrylamide gels the corresponding bands were
blotted onto polyvinylidene fluoride (PVDF) membrane,
excised from the blot membrane and submitted to
Edman degradation (SeqLab, Göttingen, Germany or
the core facility at the Institute of Physiological Chemistry and Pathobiochemistry, University of Münster).
Construction of the cDNA encoding pro-astacin
Based on pET3a-pre-pro-astacin15 the pro-astacin
cDNA was amplified with the sense primer 50 GTGGTGCATATGTCGCCCATCATCCCAGAGCG-3 0 ,
containing an Nde I restriction site and the start ATG
(TIBMolBiol, Berlin, Germany), and the antisense primer
50 -GAGAGTCGACGGATCCTAGTGATGGTGATGGTG
. . . . . . . . . . . . . . . . . . . . . . . . ::
0
ATGCCTTAGGCTACACTC-3
containing the restric. . . ::
tion sites SalI, BamHI, a 6. .£. .His-tag
. . . . . . . . and a stop codon
(in bold face).15 The amplification was carried out in a
thermocycler (Hybaid, Middlesex, UK) using the programme: (i) one cycle of 120 seconds at 94 8C; (ii) ten
cycles of 15 seconds at 94 8C, 30 seconds at 63 8C, and
15 seconds at 72 8C; (iii) 15 cycles of 15 seconds at
94 8C, 30 seconds at 63 8C, and 60 seconds plus a five
seconds increment per cycle at 72 8C; (iv) one cycle of
seven minutes at 72 8C. The PCR product was cloned
into the vector pCRe2.1 (Invitrogen, Groningen, NL)
and finally into pET3a using the restriction sites Nde I
and Bam HI.
Site-directed mutagenesis
Mutations were introduced into the recombinant wildtype astacin cDNA15 using the PCR-based QuickChangee site-directed mutagenesis kit (Stratagene, Heidelberg, Germany). The changed triplet (underlined) is
positioned in the middle of each sequence of the complementary primer pairs (BIG Biotech GmbH, Freiburg,
Germany):
E103A sense 50 -GGCTTCTACCATGCGCACACCC
GTATGG-30 ; E103A antisense 50 -CCATACGGGTGT
GCGCATGGTAGAAGCC-30 ; E103Q sense 50 -CATTGG
CTTCTACCATCAGCACACCCGTATGG-30 ; E103Q antisense 50 -CCATACGGGTGTGCTGATGGTAGAAGCCA
ATG-30 .
For PCR amplification of the whole plasmid, the following protocol was used: (i) one cycle of 30 seconds at
95 8C; (ii) 12 cycles of 30 seconds at 95 8C, 60 seconds at
Activation Mechanism of Pro-astacin
55 8C, and 10.5 minutes at 68 8C. After Dpn I-restriction
of the parental template DNA, Epicurian Colie XL1Blue super competent cells were transformed with the
nicked circular plasmids pET3a-E103A-astacin and
pET3a-E103Q-astacin. Closures of the nicks are effected
in the cells by cellular repair mechanisms. The resulting
mutants were confirmed by DNA sequencing (SeqLab,
Göttingen, Germany).
E93A-pro-astacin was synthesized by Nco I/Bam HIrestriction of pET3a-E93A-astacin21 and ligation of the
corresponding fragment into Nco I/Bam HI-treated
pET3a-pro-astacin.
Protein expression, purification, and folding
Expression of pro-astacin and the astacin variants in
E. coli BL21(DE3) cells was induced with 1 mM or
0.4 mM isopropyl-b-D -thiogalactopyranoside (IPTG;
Sigma, Deisenhofen, Germany) as described.15
Purification and folding of recombinant astacin and
pro-astacin was performed essentially as described21 but
with the following modifications: in the case of astacin
20 mM imidazole was added to each buffer run over the
nickel nitrilotriacetic acid affinity chromatography column (Ni-NTA; Qiagen, Hilden, Germany). In the case of
pro-astacin, Ni-NTA affinity chromatography was run
in the absence of inhibitors and imidazole. Folding was
performed in presence of 0.3 mM oxidized and 3 mM
reduced glutathione without Pro-Leu-Gly-NHOH. The
pH value of the subsequent dialysis buffer was 8.5. The
centrifuged solution was concentrated in centrifugal filter units (cutoff value 10 kDa; Eppendorf, Cologne,
Germany) to a volume of about 500 ml. The Pro-LeuGly-NHOH affinity-chromatography column used for
purification of mature astacin and astacin mutants was
equilibrated with 0.05 M Tris – HCl buffer (pH 7.0). After
sample application, the column was washed with
0.25 M NaCl in 0.1 M Tris – HCl (pH 7.0). Elution was
achieved with non-buffered 0.1 M Tris, 0.5 M NaCl.
Enzymatic assays
Proteolytic activity of non-reduced proteins was
assayed by zymography using SDS-PAGE with gels containing 0.1% (w/v) gelatine (Sigma, Deisenhofen,
Germany) as described.21
Continuous assays were run in the presence of the
chromogenic substrate STANA19 at a concentration of
3 mM in 50 mM Hepes –NaOH buffer (pH 8.0) at 25 8C.
Absorbance at 405 nm was recorded with a Lambda 40
spectrophotometer (Perkin – Elmer, Norwalk, USA).
Quenched fluorescence activity assays were performed using the substrate Dns-Pro-Lys-Arg-Ala-ProTrp-Val in a Perkin – Elmer LS50B Fluorescence
Spectrometer.19,25
Reversed phase HPLC of pro-astacin fragments
Pro-peptide (50 mM) was incubated with 1.0 mM astacin in 50 mM Hepes – NaOH (pH 8.0) for 120 minutes at
room temperature. Under these conditions, the propeptide is degraded completely. A portion of this sample
(100 ml) was separated by reversed phase (RP)-HPLC on
a ET280/8/4 Nucleosil 5 C18 column (Macherey&Nagel,
Düren, Germany) using a solvent gradient of 0 – 70%
acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA) at a
flow rate of 1 ml/minute. Peptides were detected at
214 nm with a 1000S Diode Array Detector (Applied
245
Activation Mechanism of Pro-astacin
Biosystems, Ramsey, USA). Elution peaks were integrated by weighing the appropriate peak areas cut out
of the chromatography chart. Pro-peptide fragments
were assigned according to mass spectrometric analysis.
Degradation of the pro-peptide by astacin can be
described by a pseudo first-order reaction neglecting
competition with the cleavage of subsequent fragments:
½Pro-peptidet ¼ ½Pro-peptidet0 £ e2kapp t
where kapp is the pseudo first-order rate constant. The
software Grafit 4.0 (Erithacus Software, Staines, UK)
was used for data analysis.
For mass spectrometry, RP-HPLC elution peaks were
vacuum-dried, stored at 2 20 8C and redissolved in 0.1%
(v/v) acetic acid immediately before MS analysis.
Circular dichroism (CD) spectroscopy
For CD spectroscopy, samples were analysed in
10 mM potassium phosphate buffer (pH 8.0) at 20 8C
using a thermostatically controlled quartz cuvette of
0.5 mm path-length in a type “CD6” spectropolarimeter
(Jobin Yvon, Paris, France). Spectra were taken every
0.5 nm from 185 nm to 260 nm with an accuracy of
^1 nm. They have been accumulated 20 times and averaged. The results were expressed as mean residue ellipticity: ½QMRE ¼ ðMRW £ uobs =c £ dÞ; where uobs is the
observed ellipticity (in mdeg.) at the respective wavelength, MRW is the mean residue weight of the enzyme
(MRW ¼ M/n, M ¼ 23581 g/mole, n ¼ 207 amino acid
residues). The cuvette path-length is given in cm and c
is the protein concentration in mg/ml.
Mass spectrometry
MALDI-TOF analysis of tryptic cleavage products was
performed on a Voyager System 4189 (Applied Biosystems, Weiterstadt, Germany) by ChromaTec GmbH
(Greifswald, Germany) with an acquisition mass range
of 1000– 4000 Da. The theoretical peptide fragments
obtained by trypsinolysis of proastacin were calculated
for non-reducing conditions using the sequence analysis
tool PEPTIDE MASS of the SWISS-PROT database.33
For the mass spectrometric analysis of pro-peptide
fragments, measurements were performed in the positive ion mode on an ESI-QTOF mass spectrometer
(Micromass, Manchester, UK) using the first quadrupole
as a broadband filter and a TOF detector. The sample solution was admitted using nanospray needles, which produced a stable spray for at least ten minutes. Most
samples provided abundant ions after one minute spraying. The m/z values of molecular [M þ n H]þn ions produced from peptide components in the fractions
analysed were correlated to theoretical masses of the
pro-peptide fragments to identify their origin and the
cleavage site. Only the most abundant ions were
assigned to their theoretical clips in the sequence.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (DFG).
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Edited by R. Huber
(Received 13 May 2002; received in revised form 27 September 2002; accepted 1 October 2002)