|
|
|
|||||||
|
|||||||||
From the
The role of tumor suppressor proteins in the development of
malignancy has made the understanding of their molecular mechanisms of
action of great importance. Maspin is a tumor suppressor produced by a
number of cell types of epithelial origin. Exogenous recombinant maspin
has been shown to block the growth, motility, and invasiveness of
breast tumor cell lines in vitro and in vivo.
Although belonging to the the serine proteinase inhibitor (serpin)
superfamily of proteins, the molecular mechanism of maspin is currently
unknown. Here we show that the reactive site loop of maspin exists in
an exposed conformation that does not require activation by cofactors.
The reactive site loop of maspin, however, does not act as an inhibitor
of proteinases such as chymotrypsin, elastase, plasmin, thrombin, and
trypsin but rather as a substrate. Maspin is also unable to inhibit
tissue and urokinase type plasminogen activators. Stability studies
show that maspin cannot undergo the stressed-relaxed transition typical
of proteinase-inhibitory serpins, and the protein is capable of
spontaneous polymerization induced by changes in pH. It is likely,
therefore, that maspin is structurally more closely related to
ovalbumin and angiotensinogen, and its tumor suppressor activity is
independent of a latent or intrinsic trypsin-like serine
proteinase-inhibitory activity.
Maspin is a tumor suppressor protein expressed in normal human
mammary epithelial cells, but it is undetectable or is expressed at
very low levels in invasive primary or metastatic tumors and pleural
effusions(1) . The growth, motility, and metastatic potential of
mammary tumor cells are greatly reduced both in vitro and in vivo by transfection with the maspin cDNA. Exogenous
recombinant maspin has also been shown to block tumor cell invasion and
to significantly reduce cell motility in
vitro(1, 2) . Immunostaining studies demonstrate
that maspin is found in the extracellular matrix and at the plasma
membrane, but the the underlying biochemical basis of maspin function
is currently unknown(1) .
The cloned human maspin cDNA
encodes a 42-kDa protein that shares homology with the serpin
superfamily of protease inhibitors. Primary structure alignments have
indicated that maspin possesses an arginine residue at the P
We have demonstrated
previously that the tumor-suppressing ability of maspin is dependent on
the RSL, since proteolysis within this region abolishes the ability of
the protein to block invasion and motility (2). Here we show that the
RSL of recombinant maspin cannot inhibit trysin-like serine proteases.
We also provide structural evidence that the tumor suppressor function
of maspin is unlikely to involve protease inhibition.
Saccharomyces cerevisiae genomic
DNA was a gift from Dr. A. J. Brake (University of California, San
Francisco, CA). The yeast expression vector was obtained from the
laboratory of Dr. S. Hawkes (University of California, San Francisco,
CA), and yeast strain BJ2168 was purchased from the Berkeley type
culture collection.
AEBSF was from Calbiochem (La Jolla, CA),
Bradford assay reagents were from Bio-Rad, and Matrigel
Yeast cells were cultured in yeast
extract, peptone, 2% glucose (YEPD), and 2-ml samples were analyzed for
recombinant maspin expression after 24, 48, and 72 h of culture. Yeast
cells were pelleted by centrifugation and lysed, and the soluble and
insoluble fractions were prepared as described previously(15) .
Samples were analyzed by SDS-PAGE, and proteins were visualized by
staining with Coomassie Blue.
Maspin was purified
as follows. 5 ml of extraction buffer and 2 g of glass beads were
added/g wet weight of yeast, and samples were vortexed for 15 min at 4
°C (SP multi-tube vortexer, Baxter). The lysate was clarified by
centrifugation at 10,000 rpm for 20 min, and soluble maspin was
retained for further purification. This material was filtered (0.45
µm nylon filter, E and K Scientific Products, Campbell, CA) and
diluted 3
Throughout this
procedure, fractions containing maspin were identified by SDS-PAGE and
immunoblotting on nitrocellulose. The primary antibody was a rabbit
ABS4A polyclonal IgG antipeptide antibody described
previously(1) . The secondary antibody was a peroxidase-labeled
goat anti-rabbit IgG. Reactive bands were visualized with
4-chloro-1-napthol. Protein quantitation was performed using the
micro-Bradford assay.
1) Native gel electrophoresis at different pH values and ionic
strengths(17) . Continuous native gel electrophoresis (CNGE) was
performed at pH intervals between pH 6.5 and 9.0 employing a
Tris/glycine buffer system. At each pH, the effect of ionic strength
was determined by varying the concentration of Tris and glycine between
50 and 200 mM. Discontinuous native gel electrophoresis (DNGE)
utilized the same buffer system but different pH values for stacking
and resolving gels. The running buffer was 50 mM Tris, 50
mM glycine, pH 8.3.
2) Transverse urea gradient gel
electrophoresis was carried out on maspin, trypsin-cleaved maspin,
human
3) Thermal stability studies were carried out
on maspin, trypsin-cleaved maspin, human
Maspin is structurally and
immunologically related to PAI-1(2) . We tested, therefore,
whether recombinant maspin had adopted the latent configuration that
PAI-1 can assume spontaneously at physiological pH and ionic strength.
It has been shown that partial unfolding of PAI-1 induced by
denaturants and detergents can restore PAI-1 inhibitory
activity(19) , but when we subjected maspin to similar
treatments, we were not able to induce any Arg-specific protease
inhibitory activity (results not shown).
SDS-PAGE analysis shows
maspin to be a substrate for chymotrypsin, elastase, plasmin, thrombin,
and trypsin (Fig. 2). We did not observe any SDS-heat
denaturation-resistant complexes formed with any of the proteases
tested. In contrast, PAI-1 and uPA formed an SDS and heat-stable
complex typical of inhibitory serpin-target protease interactions (Fig. 2). We observed complete degradation of maspin by 1:1 molar
ratios of chymotrypsin, elastase, plasmin, and trypsin, but at lower
protease concentrations, only plasmin, thrombin, and trypsin cleaved
maspin into discrete fragments. In each case, we observed a reduction
in the molecular mass of the protein to 38-kDa, suggesting that limited
proteolysis occurred. The protease most effective at inducing this
shift was trypsin, which did so even at the lowest protease to maspin
ratio tested (1:1,000). HPLC analysis of these samples gave similar
profiles and yielded two maspin peaks. One contained the 38 kDa band,
the other a 4.5 kDa band that was not resolved on 10% SDS-PAGE but
could be seen on 15% SDS-PAGE. In each case, analysis of the 4.5 kDa
band gave the sequence ILQHKDELNADHPFIYIIRH. This sequence is identical
to that starting at the putative P
Maspin is an extracellular tumor suppressor molecule. It has
been shown to block the growth, invasion, and metastatic properties of
mammary tumor cell lines in vitro and in
vivo(1, 2) . The expression of maspin is
down-regulated in breast cancer and is absent in most, or all, invasive
primary or metastatic tumors (1).
The classical inhibitory serpin
functions by presentation of a reactive site
P
Sequence alignments and immunological studies have shown that maspin
is structurally related to PAI-1 and PAI-2 (30 and 40% amino acid
identity, respectively). We therefore proposed that maspin may function
as a tumor suppressor by preventing uPA-mediated degradation of the
ECM. However, the data presented here do not support this hypothesis.
Maspin does not inhibit the proteases uPA, tissue-type plasminogen
activator, or plasmin via the simple 1:1 stoichiometric process of
classical serpin action. Instead, maspin is particularly sensitive to
limited proteolysis induced by trypsin and trypsin-like proteases. Thus
the RSL of maspin is already in an exposed active conformation
accessible to proteases that does not require co-factor activation for
interaction. We also demonstrate that such proteolysis does not induce
maspin to undergo the S to R transition. In this respect, it is similar
to the noninhibitory serpins, ovalbumin, and angiotensinogen. Therefore
we consider it unlikely that maspin possesses a classical serpin-like
protease inhibitory activity.
The polymerization phenomena observed
for maspin has been shown to occur with other serpins. However, for
most serpins, polymerization has so far only been shown to result from
denaturant, enzymatic, mutational, or thermally induced disruption of
hydrophobic bonds or salt bridges allowing mobility of the
RSL(26, 27, 28, 29, 30, 36) .
PAI-2 also undergoes spontaneous polymerization at room temperature and
is currently the only other serpin able to do so under conditions close
to physiological(31) . Current structural data on a dimeric form
of antithrombin suggests that polymerization may be an an extension of
the dimerization process, whereby the RSL of one molecule inserts into
the C
We
have previously shown that trypsin cleavage of the RSL inactivates the
ability of maspin to block tumor cell invasion(2) . Such a
mechanism is probably used in vivo during the growth and
differentiation of mammary and other epithelial tissues, whereby
trypsin-like proteases that are capable of inactivating maspin's
suppressor functions are expressed transiently. Two likely protease
candidates implicated in cell growth and division, either one of which
could be a physiological inactivator of maspin, are the membrane-bound
trypsin-like protease hepsin and a recently discovered putative tumor
suppressor gene that also encodes a trypsin-like
protease(24, 25) . Hepsin is present at significant
levels in many different types of mammalian cells such as human
hepatoma cells, peripheral nerve cells, baby hamster kidney cells, and
mammary cancer cell lines. Furthermore, the levels of this protease are
greatly elevated in regions of active cell proliferation in the
developing mouse embryo. Given our recent observations that maspin is
also found in the prostate and gastrointestinal tract,
In summary, we have demonstrated that recombinant maspin is
unable to directly inhibit trypsin-like serine proteases involved in
the degradation of ECM, or undergo the S to R transition indicative of
an inhibitory function. However, the known involvement of maspin in
tumor growth, invasion, and metastasis suggests that this serpin has
evolved an RSL that functions as a proteolytic switch. Proteolysis
turns the switch off and allows epithelial cell growth and migration.
The RSL is therefore likely to bind components that are directly
involved in epithelial cell growth and invasion.
We thank V. Prochazka and T. Rigley for DNA
sequencing, I. C. Bathurst for helpful advice, and D. R. Pakianathan
for proofreading the manuscript.
Volume 270,
Number 26,
Issue of June 30, pp. 15832-15837, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
EVIDENCE THAT MASPIN IS NOT A PROTEASE INHIBITORY SERPIN (*)
ABSTRACT
INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
position, thereby suggesting that it might function as an
inhibitor of Arg-specific proteases involved in the invasion/metastatic
process(1) . Urokinase-type plasminogen activator (uPA)()appears to be the most important of these proteases.
For example, it has been shown that in certain types of cancer
individuals with elevated levels of uPA and its physiological
regulators, plasminogen activator inhibitor types 1 and 2 (PAI-1 and
PAI-2), have an increased risk of relapse and
death(3, 4) . However, data obtained from the
crystallographic structures of other serpin superfamily members
indicate that the identity of the P residue is insufficient
to accurately predict an inhibitory function for a serpin. Another
region of the molecule that is important in determining serpin function
is the ``hinge region'' of the reactive site loop
(RSL)(5, 6, 7) . This consists of a peptide
stretch that is located 9-15 residues amino-terminal to the
reactive site peptide bond. The hinge region helps present the reactive
site in an optimal configuration for docking, binding, and subsequent
inhibition of a target protease. In all inhibitory serpins identified
to date, the sequence through this region is highly conserved, while
serpins that do not function as protease inhibitors, such as ovalbumin
and angiotensinogen, are highly divergent in this region. The maspin
sequence within this region is divergent, and, thus, maspin might also
function in a capacity other than protease inhibition. Indeed, it has
been proposed previously that maspin might bind elements involved in
regulating cell shape change(8) . It has been shown that serpins
that are divergent within the hinge region, and have no known
inhibitory function, cannot undergo a transition from a stressed (S)
form to a relaxed (R) stable form following proteolysis within the
RSL(9, 34) . However, serpins that are protease
inhibitors, and some that have ligand binding functions, undergo this
transition(9, 10, 11, 12, 32) .
Thus, the S-R transition is considered to be a predictive marker for
serpins with inhibitory potential. The structural basis for the S-R
transition involves insertion of the RSL into the 
-pleated A sheet
following proteolysis(5, 33) .
Materials
pBluescript was purchased from
Stratagene (La Jolla, CA).
was from
Collaborative Biomedical Research (Bedford, MA). The Q-Sepharose and
S100HR resins were from Pharmacia Biotech Inc. Heparin-Superflow was
purchased from Sterogene (Arcadia, CA). The chromogenic substrates
S-2444 and S-2251 were from Kabi (Piscataway, NJ). PAI-1 was purchased
from Enzyme Research Laboratories (South Bend, IN). Unless specified,
all other reagents were purchased from Sigma.
Yeast Vector Construction, Growth, and Expression of
Recombinant Maspin
Maspin cDNA was isolated and modified by
polymerase chain reaction from vector pVL 1393/Mas (2) and
cloned into pHG10, a pBluescript vector containing the
glucose-regulated alcohol dehydrogenase II promoter from S.
cerevisiae(13) . The construction of pHG10 will be
described fully elsewhere. Briefly, a 1007-base pair glucose-regulated
alcohol dehydrogenase II promoter fragment was cloned by polymerase
chain reaction from S. cerevisiae genomic DNA with several
convenient restriction sites inserted to facilitate cloning of the
maspin cDNA and subsequent removal of the promoter/gene cassette. This
cassette was cloned as a NotI-XhoI fragment into a
yeast expression vector containing ura3, leu2d genes
and 2-µm sequences for selection and amplification in yeast. The
final yeast expression vector, designated pYMV4, was transformed into
yeast strain BJ2168 (14).
Recombinant Maspin Extraction and Purification
The
extraction of soluble recombinant protein from yeast was performed as
described previously (16) except that the extraction buffer used
was (0.1 M NaCl, 0.1 M Tris, 10 mM EDTA, 0.1
mM AEBSF, 0.1% Triton X-100, pH 7.5).
with TE (10 mM Tris, 1 mM EDTA, pH
8.0) containing 1 mM AEBSF prior to anion-exchange
chromatography on Q-Sepharose equilibrated in the same buffer (column
dimensions: -5
16 cm). Maspin was eluted by a NaCl
gradient (50-500 mM) in TE/AEBSF and concentrated.
Maspin was further purified by size fractionation on S100 HR (column
dimensions: -5
90 cm). The maspin peak from this column
was dialyzed into TE/AEBSF and further purified by affinity
chromatography on heparin-superflow (column dimensions: -5
5.5 cm). Maspin was eluted by a NaCl gradient (50-300
mM) in TE/AEBSF, concentrated, and dialyzed into
phosphate-buffered saline prior to further analysis.
Proteinase Inhibition/Catalysis
Studies
Recombinant maspin was incubated with varying
concentrations of chymotrypsin, elastase, trypsin, thrombin, urokinase,
or tissue plasminogen activator in phosphate-buffered saline, pH 7.4,
at 25 °C. For each assay, a fixed amount of maspin (1.1 µM final concentration) and logarithmic dilutions of enzyme (1-, 10-,
100-, or 1,000-fold) were incubated for periods of up to 12 h prior to
the addition of substrate (0.1 mM final concentration).
Residual enzyme activity was measured at 405 nm using the substrate
S-2444 for trypsin, thrombin, urokinase, and tissue plasminogen
activator; methoxy succinyl Ala-Ala-Pro-Val-paranitroanilide for
elastase and N-succinyl Ala-Ala-Pro-Phe-paranitroanilide for
chymotrypsin. The extracellular matrix (ECM) components chondroitin
sulfate, fibronectin, heparin, heparan sulfate, keratan sulfate,
hyaluronic acid, and reconstituted artificial basement membrane matrix
(Matrigel) were also preincubated with maspin for periods of up
to 12 h prior to the addition of the enzyme. The final assay
concentration of each of these was 1 mg/ml with the exception of
matrigel which was 0.1 mg/ml. Additional samples were analyzed for the
generation of cleavage products by SDS-PAGE after treatment with 0.1
mM AEBSF. Samples showing limited proteolysis were sequenced
as described below.
Protein Sequencing
Recombinant maspin and
proteolytically modified forms of recombinant maspin were analyzed by
reverse phase HPLC analysis on an aquapore RP-300 7-µm microbore
column (dimensions: 100 2.1-mm, inner diameter) using
5-80% CH
CN, 0.1% trifluoroacetic acid as the mobile
phase (11). Peaks corresponding to maspin cleavage products were
sequenced on an Applied Biosystems 476 pulsed liquid phase protein
sequencer (Foster City, CA).
Chemical and Heat Stability Studies
The stability
of trypsin-cleaved and uncleaved maspin was analyzed by the following.
-antitrypsin, and papain-inactivated
-antitrypsin according to Pace, Shirley, and
Thomson(18) .
-antitrypsin,
and papain-inactivated -antitrypsin. Aliquots (10
l) of 5 mg/ml maspin or 2.4 mg/ml
-antitrypsin in
phosphate-buffered saline were heated at different temperatures for 1 h
and then analyzed by CNGE employing a 50 mM Tris, 50 mM glycine, pH 8.3, buffer system.
Expression and Purification of Biologically Active
Maspin from Yeast
The intracellular expression of recombinant
maspin in yeast was maximal at 48 h. At this time, a major band of 42
kDa was observed in the soluble fraction obtained from yeast
transformed with the expression vector pYMV4 but not in control yeast (Fig. 1). Only a small amount of this material was observed in
the insoluble fraction, indicating that the extraction procedure was
efficient at solubilizing the bulk of maspin expressed. No recombinant
protein was observed in the yeast growth media. Recombinant maspin
comprised at least 40% of the total soluble yeast protein and was
readily purified by the chromatographic methods used. The protein bound
to Q-Sepharose and was eluted with 0.15 M NaCl. Gel-filtration
on S100HR resolved a peak containing 42-kDa maspin from a high
molecular mass contaminant. Affinity chromatography on
heparin-superflow yielded a peak eluting at 0.15 M NaCl that
was >98% pure maspin as judged by SDS-PAGE (Fig. 1) and
reverse phase HPLC (data not shown). The average yield of purified
recombinant maspin/g wet weight of yeast from several different
preparations was 13.5 mg. This material has been shown to be fully
biologically active in its ability to block tumor cell invasion through
an artificial membrane in an in vitro assay(2) .
Figure 1:
Expression and
purification of recombinant maspin from yeast. Lane 1,
markers; lane2, 48-h time point from control yeast; lane3, 48-h time point from pYMV4-transfected yeast; lane4, maspin from Q-Sepharose; lane5, maspin from S100-HR; lane6,
purified maspin from heparin-superflow.
Maspin Does Not Complex with or Inhibit Arg-specific
Serine Proteases Involved in Degradation of the ECM
Sequence
alignment studies reported by Zou et al.(1) suggest
that maspin may have an inhibitory specificity for proteases that
cleave at arginine residues. Since maspin is capable of blocking tumor
invasion and metastasis, we proposed that it might inhibit proteases
involved in ECM breakdown. shows the residual activities
of the proteases tested after incubation with a 10-fold excess of
maspin for 1 h. Similar results were obtained after 5 min, 6 h, and 12
h of incubation. Maspin was unable to block the cleaving activity of
any of the proteases tested at any of the protease/inhibitor ratios
used. On the basis that many serpins require an extracellular matrix
co-factor for expression of their inhibitory activity (e.g. PAI-1, vitronectin) and that recombinant maspin binds, albeit
weakly, to heparin, we tested the possibility that maspin also requires
co-factor activation of its inhibitory activity. However, none of the
purified extracellular matrix components activated an inhibitory
function (). We have shown previously that maspin blocks
invasion through matrigel over a 24-h period(2) . We tested,
therefore, the possibility that matrigel contains an unidentified
maspin-activating co-factor. We did not, however, observe any
activation of a latent chymotrypsin, trypsin, or uPA inhibitory
function in the presence of matrigel.
residue,
indicating proteolysis to have occurred within the RSL. We could not
obtain sequence from the amino terminus of recombinant maspin or the
38-kDa degradation products, suggesting that each is blocked. This
indicates that no proteolytic modification of the N terminus occurred
with these three proteases. We did not observe significant cleavage of
maspin by uPA (Fig. 2B) or tissue-type plasminogen
activator (data not shown) at any of the protease to maspin ratios
tested.
Figure 2:
Proteolysis of maspin. Maspin and enzyme
were incubated as described in the text, and aliquots were
electrophoresed on 10% SDS-PAGE under reducing conditions. A, C, D, E, and F represent 1-h
incubations at 37 °C of enzyme and maspin at the molar ratio
indicated. They are the maspin degradation profiles after treatment
with plasmin, thrombin, trypsin, chymotrypsin and elastase,
respectively. B represents the incubation of uPA and maspin at
an enzyme unit to mol of maspin ratio. G is the interaction of
uPA and PAI-1. Lane1, uPA; lane2,
PAI-1; lane3, uPA + PAI-1 after 5-min
incubation (C* is the uPA:PAI-1
complex).
Recombinant Maspin Polymerizes in Response to Changes in
pH
Intact and trypsin-cleaved maspin showed one major band at
all pH values and ionic strengths upon CNGE studies (Fig. 3).
However, in the case of intact maspin, some protein did not migrate
into the gel. This phenomenon was not observed with trypsin-cleaved
maspin. Trypsin-cleaved maspin migrated slightly faster on CNGE than
uncleaved maspin, suggesting that it possessed a slightly higher net
negative charge under these conditions. In contrast, both uncleaved and
cleaved maspin gave laddering patterns upon DNGE under all conditions
tested (Fig. 3). Human -antitrypsin migrated as
a single band on either CNGE or DNGE but possessed considerably higher
negative charge than either form of maspin.
Figure 3:
Continuous and discontinuous native gel
electrophoresis of maspin. A and B are maspin and
trypsin-cleaved maspin electrophoresed on 7.5% CNGE. Buffer conditions
were 50 mM Tris, 50 mM glycine, pH 8.3. C, D, and E are maspin, trypsin-cleaved maspin, and
human -antitrypsin electrophoresed on 7.5% DNGE. The
pH values of the stacking and resolving gels were 6.9 and 8.9. Buffer
conditions: 50 mM Tris, 40 mM glycine.
These data indicate that
mild pH changes are sufficient to induce polymerization of both forms
of maspin but not of the prototypic serpin
-antitrypsin.
Recombinant Maspin Does Not Undergo the S to R Transition
Fig. 4shows the urea-induced unfolding transitions of
maspin and -antitrypsin. It can be seen that native
-antitrypsin undergoes a marked decrease in mobility
at approximately 1.5 M urea, while an RSL-cleaved form of the
molecule does not. These findings concur with those previously reported
by Mast et al.(26) and support the hypothesis that
inhibitory serpins exist in a stressed state that can be converted to a
relaxed state by cleavage within the RSL of the molecule. Recombinant
native maspin showed two bands at low urea concentrations, but as the
urea concentration increased beyond 0.5 M, these two bands
fused to migrate as one. RSL cleaved maspin only showed one band at all
urea concentrations, but it did show a small decrease in mobility at
low urea concentrations. Heat denaturation studies showed both forms of
maspin to have similar stability characteristics. They were equally
unstable, readily denaturing, and losing the ability to migate as
monomers on CNGE between 40 and 50 °C (Fig. 5). In contrast,
native -antitrypsin was more stable, denaturing
between 50 and 60 °C, while RSL-cleaved
-antitrypsin was stable up to 80 °C (Fig. 5). The differences in stability of the uncleaved molecules
may be due in part to differences in glycosylation as
-antitrypsin has a much higher negative charge than
maspin, and, as we would predict, intracellularly produced recombinant
maspin is not glycosylated.
Figure 4:
Transverse urea gradient gel
electrophoresis of maspin. A and B are the
urea-induced unfolding transitions of native human
-antitrypsin and papain-cleaved
-antitrypsin on a 7.5% polyacrylamide gel. C and D are the urea-induced unfolding transitions of
uncleaved and trypsin-cleaved maspin. Resolving gel and running buffer
conditions were 50 mM Tris, 50 mM borate, pH
8.7.
Figure 5:
Thermal
denaturation of maspin. Samples were analyzed as described in the text
and are as follows: A, native human
-antitrypsin; B, papain-cleaved
-antitrypsin; C, maspin; D,
trypsin-cleaved maspin.
These results demonstrate that purified
recombinant maspin does not undergo a transition from a stressed form
to a more heat-stable relaxed form.
()The related
serpins PAI-1 and PAI-2 are also implicated in tumor invasion and
metastasis, but their role is less evident since they, as well as their
target protease urokinase (uPA), are overexpressed in many
cancers(3, 4) .
-P peptide bond with the
appropriate P
specificity for the target enzyme. PAI-1 and
PAI-2 present arginine as the P residue and accordingly
their Arg-specific target proteases (uPA or tissue-type plasminogen
activator) attempt to cleave this bond but become trapped in a 1:1
stoichiometric complex that is resistant to heat- or detergent-induced
denaturation(20) . In addition, PAI-1 and PAI-2 will complex
with, and inhibit, other cognate proteases such as plasmin, thrombin,
and trypsin, albeit with somewhat slower association rate
constants(21, 22) . The peptide sequence in the RSL of
inhibitory serpins is also sensitive to limited proteolysis by
proteases that are not inhibited by serpins. Such proteolysis has been
shown to inactivate all of the inhibitory serpins tested so far by
inducing their transition from a stressed form to a relaxed form in
which the once contiguous amino acids can be found at opposite ends of
the resulting molecule. The structural basis for this transition
involves insertion of the RSL peptide sequence amino-terminal to the
site of proteolysis into the major -pleated sheet A structure
present in serpins(5) . However, the extent of insertion
occurring prior to proteolysis will most likely differ from serpin to
serpin given the recent crystal structures of a mutant inhibitory form
of -antichymotrypsin and a dimerized form of
antithrombin III(23, 35) . The structure of
-antichymotrypsin revealed no insertion, while the
structure of dimerized antithrombin showed two residues to be inserted.
-pleated sheet of another molecule(35) . The data
presented here indicate that maspin may polymerize by a similar
mechanism. The demonstration that maspin cleaved at the RSL also
polymerizes indicates that the RSL is still available for insertion
into another maspin molecule and supports the finding that maspin does
not undergo the S-R transition. This data indicate that maspin is more
closely structurally related to PAI-2 and ovalbumin, than PAI-1.
its
role as a tumor suppressor is most likely not confined to mammary
tissue.
Table: Effect of maspin +/- ECM components
on proteinase activity
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Bots and J. P. Medema Serpins in T cell immunity J. Leukoc. Biol., November 1, 2008; 84(5): 1238 - 1247. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L.-j. Shao, H. Y. Shi, G. Ayala, D. Rowley, and M. Zhang Haploinsufficiency of the Maspin Tumor Suppressor Gene Leads to Hyperplastic Lesions in Prostate Cancer Res., July 1, 2008; 68(13): 5143 - 5151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. J. Askew, S. Cataltepe, V. Kumar, C. Edwards, S. M. Pace, R. N. Howarth, S. C. Pak, Y. S. Askew, D. Bromme, C. J. Luke, et al. SERPINB11 Is a New Noninhibitory Intracellular Serpin: COMMON SINGLE NUCLEOTIDE POLYMORPHISMS IN THE SCAFFOLD IMPAIR CONFORMATIONAL CHANGE J. Biol. Chem., August 24, 2007; 282(34): 24948 - 24960. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Al-Ayyoubi, B. S. Schwartz, and P. G. W. Gettins Maspin Binds to Urokinase-type and Tissue-type Plasminogen Activator through Exosite-Exosite Interactions J. Biol. Chem., July 6, 2007; 282(27): 19502 - 19509. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Tsoli, P. K Tsantoulis, A. Papalambros, B. Perunovic, D. England, D. A Rawlands, G. M Reynolds, D. Vlachodimitropoulos, S. L Morgan, C. A Spiliopoulou, et al. Simultaneous evaluation of maspin and CXCR4 in patients with breast cancer J. Clin. Pathol., March 1, 2007; 60(3): 261 - 266. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Khalkhali-Ellis Maspin: The New Frontier Clin. Cancer Res., December 15, 2006; 12(24): 7279 - 7283. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Cella, A. Contreras, K. Latha, J. M. Rosen, and M. Zhang Maspin is physically associated with {beta}1 integrin regulating cell adhesion in mammary epithelial cells FASEB J, July 1, 2006; 20(9): 1510 - 1512. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Yin, X. Li, Y. Meng, R. L. Finley Jr., W. Sakr, H. Yang, N. Reddy, and S. Sheng Tumor-suppressive Maspin Regulates Cell Response to Oxidative Stress by Direct Interaction with Glutathione S-Transferase J. Biol. Chem., October 14, 2005; 280(41): 34985 - 34996. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Benarafa and E. Remold-O'Donnell The ovalbumin serpins revisited: Perspective from the chicken genome of clade B serpin evolution in vertebrates PNAS, August 9, 2005; 102(32): 11367 - 11372. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. H. P. Law, J. A. Irving, A. M. Buckle, K. Ruzyla, M. Buzza, T. A. Bashtannyk-Puhalovich, T. C. Beddoe, K. Nguyen, D. M. Worrall, S. P. Bottomley, et al. The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix J. Biol. Chem., June 10, 2005; 280(23): 22356 - 22364. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Al-Ayyoubi, P. G. W. Gettins, and K. Volz Crystal Structure of Human Maspin, a Serpin with Antitumor Properties: REACTIVE CENTER LOOP OF MASPIN IS EXPOSED BUT CONSTRAINED J. Biol. Chem., December 31, 2004; 279(53): 55540 - 55544. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ngamkitidechakul, D. J. Warejcka, J. M. Burke, W. J. O'Brien, and S. S. Twining Sufficiency of the Reactive Site Loop of Maspin for Induction of Cell-Matrix Adhesion and Inhibition of Cell Invasion: CONVERSION OF OVALBUMIN TO A MASPIN-LIKE MOLECULE J. Biol. Chem., August 22, 2003; 278(34): 31796 - 31806. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Bass, A.-M. M. Fernandez, and V. Ellis Maspin Inhibits Cell Migration in the Absence of Protease Inhibitory Activity J. Biol. Chem., November 27, 2002; 277(49): 46845 - 46848. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Sood, M. S. Fletcher, L. M. Gruman, J. E. Coffin, S. Jabbari, Z. Khalkhali-Ellis, N. Arbour, E. A. Seftor, and M. J. C. Hendrix The Paradoxical Expression of Maspin in Ovarian Carcinoma Clin. Cancer Res., September 1, 2002; 8(9): 2924 - 2932. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Chipuk, L. V. Stewart, A. Ranieri, K. Song, and D. Danielpour Identification and Characterization of A Novel Rat Ov-Serpin Family Member, Trespin J. Biol. Chem., July 12, 2002; 277(29): 26412 - 26421. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. E. Blacque and D. M. Worrall Evidence for a Direct Interaction between the Tumor Suppressor Serpin, Maspin, and Types I and III Collagen J. Biol. Chem., March 22, 2002; 277(13): 10783 - 10788. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Biliran Jr. and S. Sheng Pleiotrophic Inhibition of Pericellular Urokinase-type Plasminogen Activator System by Endogenous Tumor Suppressive Maspin Cancer Res., December 1, 2001; 61(24): 8676 - 8682. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Y Shi, W. Zhang, R. Liang, S. Abraham, F. S. Kittrell, D. Medina, and M. Zhang Blocking Tumor Growth, Invasion, and Metastasis by Maspin in a Syngeneic Breast Cancer Model Cancer Res., September 1, 2001; 61(18): 6945 - 6951. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Maass, T. Hojo, M. Ueding, J. Lüttges, G. Klöppel, W. Jonat, and K. Nagasaki Expression of the Tumor Suppressor Gene Maspin in Human Pancreatic Cancers Clin. Cancer Res., April 1, 2001; 7(4): 812 - 817. [Abstract] [Full Text]
|
|
|
|
|
|
C. N. Rao, S. S. Lakka, Y. Kin, S. D. Konduri, G. N. Fuller, S. Mohanam, and J. S. Rao Expression of Tissue Factor Pathway Inhibitor 2 Inversely Correlates during the Progression of Human Gliomas Clin. Cancer Res., March 1, 2001; 7(3): 570 - 576. [Abstract] [Full Text]
|
|
|
|
|
|
R. McGowen, H. Biliran Jr., R. Sager, and S. Sheng The Surface of Prostate Carcinoma DU145 Cells Mediates the Inhibition of Urokinase-type Plasminogen Activator by Maspin Cancer Res., September 1, 2000; 60(17): 4771 - 4778. [Abstract] [Full Text]
|
|
|
|
|
|
T. Liu, P. A. Pemberton, and A. D. Robertson Three-state Unfolding and Self-association of Maspin, a Tumor-suppressing Serpin J. Biol. Chem., October 15, 1999; 274(42): 29628 - 29632. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Sheng, B. Truong, D. Fredrickson, R. Wu, A. B. Pardee, and R. Sager Tissue-type plasminogen activator is a target of the tumor suppressor gene maspin PNAS, January 20, 1998; 95(2): 499 - 504. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. A. Pemberton, A. R. Tipton, N. Pavloff, J. Smith, J. R. Erickson, Z. M. Mouchabeck, and M. C. Kiefer Maspin Is an Intracellular Serpin That Partitions into Secretory Vesicles and Is Present at the Cell Surface J. Histochem. Cytochem., December 1, 1997; 45(12): 1697 - 1706. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Tsujimoto, N. Tsuruoka, N. Ishida, T. Kurihara, F. Iwasa, K. Yamashiro, T. Rogi, S. Kodama, N. Katsuragi, M. Adachi, et al. Purification, cDNA Cloning, and Characterization of a New Serpin with Megakaryocyte Maturation Activity J. Biol. Chem., June 13, 1997; 272(24): 15373 - 15380. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Yavelow, A. Tuccillo, S. S. Kadner, J. Katz, and T. H. Finlay {alpha}1-Antitrypsin Blocks the Release of Transforming Growth Factor-{alpha} from MCF-7 Human Breast Cancer Cells J. Clin. Endocrinol. Metab., March 1, 1997; 82(3): 745 - 752. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Riewald and R. R. Schleef Human Cytoplasmic Antiproteinase Neutralizes Rapidly and Efficiently Chymotrypsin and Trypsin-like Proteases Utilizing Distinct Reactive Site Residues J. Biol. Chem., June 14, 1996; 271(24): 14526 - 14532. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Riewald and R. R. Schleef Molecular Cloning of Bomapin (Protease Inhibitor 10), a Novel Human Serpin That Is Expressed Specifically in the Bone Marrow J. Biol. Chem., November 10, 1995; 270(45): 26754 - 26757. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Becerra, A. Sagasti, P. Spinella, and V. Notario Pigment Epithelium-derived Factor Behaves Like a Noninhibitory Serpin J. Biol. Chem., October 27, 1995; 270(43): 25992 - 25999. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |