From the
Recently inhibitors of the serpin family were shown to form
complexes with dichloroisocoumarine (DCI)-inactivated proteinases under
native conditions (Enghild, J. J., Valnickova, Z., ThI.,
and Pizzo, S. V.(1994) J. Biol. Chem. 269, 20159-20166).
This study demonstrates that serpin-DCI/proteinase complexes resist
dissociation when analyzed in reduced SDS-polyacrylamide gel
electrophoresis. Previously, SDS-stable serpin-proteinase complexes
have been observed only between serpins and catalytically active
proteinases. The stability of these complexes is believed to result
from an acyl-ester bond between the active site Ser
Serpins are the predominating serine proteinase inhibitors in
mammals and regulate the serine proteinases involved in blood
coagulation, fibrinolysis, complement activation, and
inflammation(1, 2) . Although the biological role of
many serpins is well understood, the serpin mechanism of proteinase
inhibition remains unclear. In contrast, small serine proteinase
inhibitors from the kunin and kazal families operate by a well
characterized ``standard mechanism''(3) . Several
hypotheses describing the interactions between serpins and proteinases
have been
presented(4, 5, 6, 7, 8, 9, 10, 11, 12) .
These hypotheses are based on the assumption that serpins contain an
exposed loop, similar to the RSL
Analysis of most serpin-proteinase complexes by SDS-PAGE
reveals the presence of an SDS-stable complex formed between the serpin
and the proteinase. These complexes are generally believed to be a
result of the SDS-induced distortion of the catalytic triad
(Ser
In this study we show that active site modified
proteinases are capable of forming SDS-stable complexes with serpins.
Moreover, SDS-stable complexes between serpins and active or
Ser
Similar to DCI/HNE, a complex is formed between
Much of our understanding of serine proteinase inhibitory
mechanisms comes from x-ray analyses of the small standard mechanism
inhibitors(19) . The standard mechanism of inhibition by
proteins such as kunins and kazals can be envisioned as a lock and key
interaction between the RSL and the substrate-binding sites of the
enzyme. Covalent bonds are not involved, and the complexes are not
stable to denaturing conditions because the interaction depends on the
conformation of the involved proteins. Some attempts to understand the
serpin mechanism of action have been made with the assumption that
serpins fulfill the requirements of the standard
mechanism(20, 21, 22) . As previously mentioned,
serpin-proteinase complexes deviate from the standard mechanism by
resisting dissociation in SDS-PAGE. The SDS-stable serpin-active
proteinase complexes are believed to be stabilized by a covalent
acyl-ester bond between the
Formation of
complexes between serpins and inactivated proteinases have previously
only been studied under native
conditions(12, 25, 26, 27) . Here we
show that serpin-inactive proteinase complexes including
By comparing
These results are at variance with previous
hypotheses on serpin-proteinase interactions. These hypotheses were
generated in part due to the appearance of COOH-terminal peptides in
SDS-PAGE of complexes. Association of a serpin and a proteinase
occasionally also results in the cleavage of a small amount of the
serpin instead of forming a complex. This reaction might account for
the occasional appearance of the free COOH-terminal peptide in
SDS-PAGE.
These studies show that SDS-stable serpin-DCI/proteinase
complexes are stabilized by covalent cross-links other than the
acyl-ester bond involving Ser
Complexes between
We thank Drs. Tim Oury, Hanne Gr, and Eva H.
Olsen for valuable suggestions.
ABSTRACT
INTRODUCTION
Experimental Procedures
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
of
the proteinase and the
-carbonyl group of the scissile bond in the
reactive site loop. We have further analyzed the structure of the
SDS-stable serpin-proteinase and serpin-DCI/proteinase complexes. The
results of these studies demonstrate the presence of the COOH-terminal
region of the serpin in both complexes. Since (i) modification of
Ser
does not prevent formation of SDS-stable complexes
and (ii) COOH-terminal peptides are present in both complexes, the
previously described mechanism does not sufficiently explain the
formation of SDS-stable complexes.
(
)of the
standard mechanism inhibitors, but vary in the way they predict how the
RSL is held in an inhibitory conformation to achieve tight binding
inhibition.
, His
, Asp
),
(
)which prevents the deacylation step necessary for
dissociation. An ester bond, not present in the native complex, is
thought to be formed between O
of Ser
and
the
-carbonyl of the scissile
bond(13, 14, 15) . Consequently, the RSL is
cleaved, releasing the COOH-terminal peptide of the serpin. However,
the structures of these SDS-stable complexes have never been
characterized and no direct evidence for the presence of this bond has
been presented. Since one of the deviations between serpins and
standard mechanism inhibitors is the ability to form SDS-stable
complexes, understanding the mechanism responsible for their formation
might provide valuable hints about the nature of the serpin mechanism
of inhibition.
-modified proteinases are shown to contain the
COOH-terminal region of the serpin, indicating that the
P
-P
peptide bond is intact in both complexes.
Thus, other cross-links than the acyl-ester bond between the
O
of Ser
and the
-carbon of the
scissile bond account for the stability of serpin-proteinase complexes.
Materials
DCI and porcine trypsin were from
Sigma. Recombinant PI was from Cooper Laboratories,
Mountain View, CA. HNE was a kind gift of Drs. Weislaw Watorek and Jan
Potempa, University of Georgia, Athens, GA.
ACT and
cathepsin G (cat G) were from ART Biochemicals, Athens, GA.
SDS-PAGE
Proteins were analyzed in 5-15%
linear gradient gels using the buffer system of Bury(16) .
Automated Edman Degradation
Automated Edman
degradation was carried out in an Applied Biosystems model 477A
sequencer with on-line analysis of the phenylthiohydantoins using an
Applied Biosystems model 120A HPLC apparatus. Some samples were
transferred to polyvinylidene difluoride (PVDF) membranes by
electroblotting before Edman degradation (17).
Preparation of
PI- and
ACT-Proteinase
Complexes
PI or
ACT (3
µg) were incubated in 100 mM HEPES, pH 8.0, with
increasing amounts of proteinase for 5 min at 25 °C, followed by
addition of DCI to a final concentrations of 0.25 mM.
Complexes between
PI or
ACT and
DCI-inactivated proteinases were prepared by preincubating HNE,
trypsin, or cat G with 0.25 mM DCI in 100 mM HEPES,
pH 8.0, for 15 min at 25 °C. Following inactivation
PI or
ACT (3 µg) were incubated
with increasing amount of the appropriate DCI-inactivated proteinase
for 30 min at 37 °C. The concentrations of both active and
DCI-inactivated proteinases were varied between 0- and 2-fold (HNE), 0-
and 3-fold (trypsin), or 0- and 3.5-fold (cat G) molar excess. All
samples were reduced with 10 mM dithiothreitol (DTT) and
boiled 2 min in SDS-PAGE sample buffer prior to electrophoresis.
Matrix-assisted Laser Desorption Ionization
Time-of-flight (MALDI-TOF) Mass Spectrometry of Purified
Serpin-Proteinase Complexes
Native and inactive complexes were
formed as described above followed by reduction with DTT and alkylation
with a 2-fold molar excess of iodoacetic acid in 8 M guanidinium HCl. The complexes were separated from unbound
proteinase and autolytic fragments by reverse phase HPLC on a RP C2/C18
column in a SMART system (Pharmacia Biotech Inc.) and analyzed by
SDS-PAGE, Edman degradation, and mass spectrometry (MS). Mass spectra
were acquired in a prototype linear time-of-flight mass spectrometer
(Applied Biosystems, Uppsala, Sweden) equipped with a 0.7-m flight tube
and a 337 nm nitrogen laser. Spectra were sampled at 300 MHz using
-cyano-4-hydroxycinnamic acid as matrix.
Edman Degradation of COOH-terminal CNBr Peptides Derived
from
Complexes between
PI-DCI/Trypsin
PI and trypsin and DCI/trypsin were subjected to
SDS-PAGE and transferred to a PVDF membrane. The 65-kDa
PI-trypsin or
PI-DCI/trypsin
complexes were excised and incubated in HCl:methylsulfide for 5 min at
23 °C to convert potential methionine sulfoxide residues to
methionine. The PVDF membrane-bound sample was treated with CNBr
overnight to cleave Met-Xaa peptide bonds. The resulting CNBr fragments
were subsequently subjected to two or three cycles of Edman
degradation. In cycle 3 or 4, orthophthaldialdehyde (OPA) was applied
to block all non-Pro NH
termini. Edman degradation was
subsequently continued on fragments with available
NH
-terminal Pro residues.
SDS-stable Complexes between
Inactivation of HNE,
trypsin, and cat G was performed as described in Ref. 18. Titrations of
PI
and Active or DCI-inactivated HNE
PI with active HNE (Fig. 1A) and
DCI/HNE (Fig. 1B) were analyzed by reduced SDS-PAGE. All
the reaction products were identified by Edman degradation following
electrotransfer to PVDF membranes. The formation of the usual 65-kDa
PI/HNE complex was almost complete at a 1:1 molar
ratio, and only small amounts of modified
PI were
observed in the presence of excess HNE (Fig. 1A, lanes 4-9). Two distinct
P
-P
-modified forms of
PI (40
and 41 kDa) can be distinguished in lanes5 and 6. In the lower band additional cleavage was identified at the
Thr
-Asp
peptide bond in
PI.
At higher HNE concentrations (Fig. 1A, lanes
7-9), a complex of 54 kDa is observed. This smaller
PI/HNE complex was generated as a result of a cleavage
of the Ala
-Gln
peptide bond in the HNE
moiety releasing a 11.6-kDa NH
-terminal fragment.
Figure 1:
SDS-PAGE of complexes between serpins
and active or inactive proteinases. PI (3 µg) was
titrated with HNE (panelA) or DCI/HNE (panelB) and analyzed by SDS-PAGE. Lane1,
PI; lanes 2-9, molar ratios of
HNE:
PI or DCI/HNE:
PI (0.2, 0.6, 0.8,
1.0, 1.2, 1.6, 1.8, and 2.0, respectively); lane10,
size marker. In panels C and D, titrations are shown
of
PI with trypsin and DCI/trypsin, respectively. Lane1,
PI; lanes2-9, molar ratios of trypsin:
PI
or DCI/trypsin:
PI (0.25, 0.50, 1.0, 1.2, 1.5, 2.0,
2.5, and 3.0, respectively); lane 10, 4 µg of DCI/trypsin.
In panelE and F, titrations are shown of
ACT with cat G and DCI/cat G, respectively. Lane1,
ACT; lanes 2-9, molar
ratios of cat G:
ACT or DCI/cat G:
ACT
(0.29, 0.58, 1.17, 1.47, 1.76, 2.35, 2.93, and 3.5, respectively); lane10, 4.75 µg of active cat
G.
A
concentration-dependent formation of SDS-stable complexes between
PI and DCI/HNE was observed (Fig. 1B).
In contrast to the reaction between
PI and active HNE,
some virgin
PI was observed even in the presence of
high concentrations of DCI/HNE (Fig. 1B, lanes
7-9). However, we detected no COOH-terminal
PI peptide and only insignificant amounts of the
54-kDa complex. The latter complex is due to a autoproteolytic cleavage
of HNE before the DCI inactivation. These results demonstrate that,
like active HNE, DCI/HNE forms an SDS-stable complex with
PI.
SDS-stable Complexes between
To study if the ability
of DCI/HNE to form SDS-stable complexes with PI and
Active or DCI-inactivated Trypsin
PI was
restricted to HNE, we analyzed the interaction between
PI and trypsin. A titration of
PI
with active trypsin demonstrated the 65-kDa
PI-trypsin
complex (Fig. 1C). As seen with HNE, a 54-kDa
intermediate complex is formed with excess proteinase. Several closely
spaced bands can be discerned due to various cleavage events both in
the trypsin and
PI moiety. Edman degradation of the
smallest intermediate band revealed two NH
-terminal
sequences starting at Thr
in the
PI and
at the Ser
in the trypsin moiety. The 14-kDa double band
was apparent in all lanes, including the trypsin control (Fig. 1C, lane 10), and represents
autoproteolytic fragments of trypsin. Below the 14-kDa double band,
small amounts of the 4.1-kDa COOH-terminal
PI-peptide
are apparent (Fig. 1C, lanes8 and 9).
PI and DCI/trypsin (Fig. 1D). Complexes
of 54 kDa are observed as well and are probably due to fragmentation of
the trypsin molecule before DCI inactivation, as described above for
the HNE. As expected, we did not detect modified
PI or
COOH-terminal peptide in SDS-PAGE. These results show that formation of
SDS-stable complexes between
PI and DCI-inactivated
proteinases is not a property of DCI/HNE alone.
SDS-stable Complexes of
To further investigate the
ability of serpins to form SDS-stable complexes with active and
inactive proteinases, we examined the interaction between
ACT and
Active or DCI-inactivated Cat G
ACT and cat G. The reaction products are similar to
those observed between
PI and trypsin (Fig. 1E). The Asn
-Gln
bond of
ACT was cleaved in the 95-kDa complex between
ACT-cat G. Two intermediate complexes are readily
formed when an excess of cat G is used (Fig. 1E, lanes 4-9). The upper dominant 86-kDa complex between
ACT and active cat G was cleaved between residues
Leu
-Gln
in the cat G moiety and between the
Asn
-Gln
peptide bond in the
ACT moiety. Several polypeptides below 30 kDa were
observed. By comparing active cat G run alone (Fig. 1F, lane10) and DCI/cat G (Fig. 1E, lane10), these were judged to be degradation
products of cat G. In Fig. 1F a titration of
ACT with DCI/cat G is shown. Again, a
concentration-dependent formation of SDS-stable complexes between a
serpin and an inactive proteinase was demonstrated. However, complex
formation was less pronounced using this pair of serpin-DCI/proteinase
but still significant.
Comparison of HNE-
The complex
between PI and
DCI/HNE-
PI Complexes
PI and DCI/HNE is slightly larger (Fig. 2, lane3) than the complex between
PI and active HNE (Fig. 2, lane2). If the postulated cleavage of the
P
-P
peptide bond in the
PI/HNE complex occurs, the 4.1-kDa COOH-terminal
peptide is released from the complex following SDS-PAGE. However, the
observed mass difference between the two complexes is minute and not
explained by the absence of a 4.1-kDa polypeptide from the
PI/HNE complex. Edman degradation of both complexes,
following transfer to PVDF membranes, demonstrated that the
Thr
-Asp
peptide bond in the
PI/HNE complex had been cleaved. The NH
termini of the
PI-DCI/HNE complex were intact.
Proteolysis of Thr
-Asp
releases a 1.1-kDa
peptide and explains the size difference between the two complexes (Fig. 2). This indicates that SDS-stable
PI/HNE
complexes contain the COOH-terminal peptide. Consequently, the
stability of this complex cannot be due to an acyl-ester bond between
the active site Ser
and the
-carboxyl of the P
residue, since this would require a nucleophilic attack at the
P
-P
bond followed by deacylation and release
of the COOH-terminal peptide.
Figure 2:
Comparison
of the PI/HNE and
PI-DCI/HNE
complexes. Complexes of
PI and HNE or DCI/HNE were
analyzed by SDS-PAGE. Lane1,
PI
(1.5 µg); lane2, complex between
PI (2.5 µg) and HNE (1.9 µg); lane3, complex between
PI (2.5 µg) and
DCI/HNE (1.9 µg); lane4, HNE (2.0 µg); lane5, size marker.
MALDI-TOF MS of
Additional
evidence for the presence of the COOH-terminal peptide in the denatured
complexes was obtained by MS. Since the heterogeneous glycosylation of
plasma PI-DCI/Trypsin and
PI-Trypsin Complexes
PI, HNE, and cat G potentially could complicate
data interpretation, we decided to concentrate on the non-glycosylated
complexes between recombinant
PI and trypsin. The
masses of trypsin,
PI,
PI-DCI/trypsin, and
PI-trypsin
determined by MALDI-TOF are compared to the theoretical masses
calculated from the primary structures (). The masses of
trypsin and
PI alone are in accordance with the
expected intact molecules. The
PI-DCI/trypsin and
PI-trypsin complexes were stable during MS analysis,
confirming the covalent nature of the complexes. SDS-PAGE of the
HPLC-purified complexes (data not shown) revealed that free
PI and
PI-trypsin or
PI-DCI/trypsin complexes coelute and are clearly
separated from free trypsin. However, peaks corresponding to both
PI and free trypsin or DCI/trypsin were observed
during MS analysis, suggesting that some dissociation had occurred.
This dissociation may be due to the much higher irradiation needed for
desorption of the complexes. Dissociation due to the high irradiance
also reduces mass accuracy as indicated in . However, the
determined mass of 67.9 kDa for the
PI-DCI/trypsin
complex is consistent with a complex between intact
PI
and DCI/trypsin containing the COOH-terminal peptide. Prior to MS
analysis the complex was analyzed by Edman degradation following
SDS-PAGE and electrotransfer to PVDF membranes, confirming that the
NH
termini of both proteins were intact. The
PI-trypsin complex subjected to MS was similarly
analyzed by Edman degradation, revealing NH
-terminal
truncations of
PI and trypsin at
Lys
-Thr
in
PI and
Lys
-Ser
in the trypsin molecule. The
determined mass of 53.0 kDa is in agreement with the theoretical
molecular weight of this truncated
PI-trypsin complex (). These observations support the SDS-PAGE data (Fig. 2) and suggest that denatured complexes between
PI and active trypsin, like the complexes between
PI and DCI/trypsin, contain the COOH terminus of
PI.
Identification of the COOH-terminal Peptide Sequence in
PVDF
membrane-bound samples were treated with CNBr to generate peptides from
the COOH-terminal region in PI-Trypsin Complexes
PI-trypsin and
PI-DCI/trypsin complexes. Edman degradation of the
generated fragments resulted in the release of several
phenylthiohydantoin derivatives in the first two cycles. Before the
third or fourth cycle, OPA was automatically applied to the sample (Fig. 3). This treatment blocks all primary NH
groups
but leaves secondary NH
groups of Pro residues unaffected.
Consequently, the number of phenylthiohydantoin derivatives acids was
reduced, and from cycle 3 or 4 to cycle 10 we obtained the sequence
(Pro)-Pro-Glu-Val-Lys-Phe-Asn-Lys. This sequence corresponds to
P
or P
to P
of the
COOH-terminal region of
PI and confirms that the
region COOH-terminal to the reactive site is present in the SDS-stable
PI-trypsin and
PI-DCI/trypsin
complexes.
Figure 3:
Identification of the COOH-terminal
sequence in PI-DCI/trypsin and
PI-trypsin. Electroblotted complexes were cleaved with
CNBr and subjected to 2 or 3 cycles of Edman degradation.
NH
-terminal residues other than Pro were blocked with OPA.
Continued Edman degradation revealed the sequence of P
or
P
to P
of the COOH-terminal peptide. P indicates CNBr cleavage sites.
-carboxyl of the P
residue
and O
of the proteinase Ser
. This
mechanism involves a denaturation-induced distortion of the catalytic
triad whereby the COOH-terminal peptide is released and an acyl-ester
bond is formed and
trapped(1, 13, 14, 15, 23, 24) .
This mechanism seems to be based primarily on a comparison to standard
mechanism inhibitors and two observations; (i) the peptide
COOH-terminal to the P
residue is released following
denaturation of the complex, and (ii) the formed complex is dissociated
by nucleophiles such as hydrazine(25) .
PI-DCI/HNE,
PI-DCI/trypsin, and
ACT-DCI/cat G resist dissociation when analyzed in
reduced SDS-PAGE. This observation is incompatible with the scenario
described above, since DCI inactivates serine proteinases by modifying
the active site Ser
(18, 28) .
Consequently, the formation of an acyl ester bond involving O
of Ser
is not possible and other cross-links must
account for the stability seen. Moreover, cleavage of the RSL
P
-P
peptide bond cannot occur since the
involved proteinases are catalytically inactive (Fig. 1). This
was confirmed by the absence of free COOH-terminal peptides following
analysis of the complexes by SDS-PAGE (Fig. 1, panels B, D, and F), mass spectrometry (), and
Edman degradation (Fig. 3).
PI/HNE and
PI-DCI/HNE in reduced
SDS-PAGE, we noticed a small size difference (Fig. 3). This small
size difference could not readily be explained by the expected loss of
the 4.1-kDa COOH-terminal peptide from the
PI/HNE
complex. Edman degradation of the
PI/HNE complex
revealed that the NH
terminus of
PI had
been truncated, releasing the peptide
Glu
-Thr
. The loss of this 1.1-kDa
peptide is consistent with the size difference noticed on SDS-PAGE (Fig. 2). This result suggested that the SDS-stable
PI/HNE complexes contained the COOH-terminal peptide,
as a size difference of 5.2 kDa would have been seen if this peptide
was removed in addition to the NH
-terminal peptide. To
further investigate whether the COOH-terminal peptides were present in
PI-DCI/trypsin and
PI-trypsin, the
complexes were purified and analyzed by MALDI-TOF MS () and
Edman degradation. These analyses confirmed that the COOH-terminal
peptide is present in complexes between serpins and active as well as
inactive proteinases.
of the proteinase and the
-carbonyl of the P
residues. In addition, data from
SDS-PAGE (Fig. 2), mass spectrometry (), and Edman
degradation of electroblotted complexes indicate that SDS-stable
complexes between serpins and active proteinases are stabilized by the
same mechanism as serpin-DCI/proteinase complexes.
Table: Mass spectrometry of denatured
PI-trypsin complexes
PI and porcine trypsin were formed as described under
``Experimental Procedures'' and purified by reverse-phase
HPLC. Complexes and controls were analyzed by MALDI-TOF mass
spectrometry. The observed masses are compared to the calculated masses
in the presence and absence of the COOH-terminal peptide of
PI.
PI,
-proteinase inhibitor;
ACT,
-antichymotrypsin; cat G,
cathepsin G; DCI, 3,4-dichloroisocoumarin; DCI/HNE,
3,4-dichloroisocoumarin-inactivated HNE; DCI/trypsin,
3,4-dichloroisocoumarin-inactivated porcine trypsin; DCI/cat G,
3,4-dichloroisocoumarin-inactivated cathepsin G; DTT,
dithiothreitol; HNE, human neutrophil elastase (EC 3.4.21.37); PAGE,
polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride;
MALDI-TOF, matrix-assisted laser desorption ionization-time-of-flight;
MS, mass spectrometry; OPA, orthophthaldialdehyde; HPLC, high
performance liquid chromatography.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.