From the Department of Biochemistry and Molecular Biology, J. Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia
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ABSTRACT |
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Equistatin from sea anemone is a protein composed
of three thyroglobulin-type 1 domains known to inhibit papain-like
cysteine proteinases, papain, and cathepsins B and L. Limited
proteolysis was used to dissect equistatin into a first domain, eq d-1,
and a combined second and third domain, eq d-2,3. Only the N-terminal domain inhibits papain (Ki = 0.61 nM).
Remarkably, equistatin also strongly inhibits cathepsin D with
Ki = 0.3 nM but not other aspartic
proteinases such as pepsin, chymosin, and
HIV-PR. This activity resides on the eq
d-2,3 domains (Ki = 0.4 nM). Papain and
cathepsin D can be bound and inhibited simultaneously by equistatin at
pH 4.5, confirming the physical separation of the two binding sites.
Equistatin is the first inhibitor of animal origin known to inhibit
cathepsin D. The obtained results demonstrate that the widely
distributed thyroglobulin type-1 domains can support a variety of functions.
The recently discovered thyroglobulin type-1 domain inhibitors,
thyropins, are a group of proteins that have the ability to inhibit
both cysteine (1) and a group of as yet uncharacterized cation-dependent proteinases (2). Thyroglobulin type-1
domain is a structural element first found in thyroglobulin, a molecule that serves as the precursor of the thyroid hormone and in which three
different types of cysteine-rich domains are present (3). This domain
is exclusively present on the N-terminal section and is repeated 11 times (4). Similar type-1 domains, recognizable by the sequence motif
of Cys-Trp-Cys-Val, have been found in many other proteins including
saxiphilin (5, 6), nidogen (7), insulin-like growth factor-binding
proteins (8), pancreatic carcinoma marker proteins (GA-733) (9),
testican (10), major histocompatibility complex class II-associated p41
invariant chain (11), chum salmon egg cysteine proteinase inhibitor
(ECI)1 (12), and equistatin (13, 14). In these proteins
(except ECI and equistatin) the type-1 domain represents only part of the molecule. The function of these repetitive sequences is unknown, although there is evidence that the type-1 domain itself can act as an
inhibitor of cysteine proteinases, as shown in equistatin, ECI, and p41
fragment (11-15).
Equistatin is a protein isolated from sea anemone Actinia
equina. It is a reversible and tightly binding competitive
inhibitor of papain-like cysteine proteinases. Sequence data
(SWISS-PROT accession number P8149) have shown that the inhibitor has
an Mr of 21,755 and is composed of three
repeated thyroglobulin type-1 domains (13, 14). The first domain (eq
d-1) has 43% sequence identity with the second domain (eq d-2) and
49% with the third domain (eq d-3), whereas eq d-2 and eq d-3 show
32% identity. In the present study we have examined the inhibitory
activity of the thyroglobulin type-1 domains present in equistatin.
This has been done by dissecting the molecule by limited proteolysis into folded domain structures, isolating the domains and investigating their inhibitory activities against the cysteine proteinase: papain; a
number of aspartic proteinases: cathepsin D, pepsin, chymosin, and
HIV-PR; and serine proteinase: trypsin.
Materials--
Equistatin from A. equina and
Preparation of Equistatin Domain-1 (eq d-1) and Domain-2,3 (eq
d-2,3)--
Equistatin was subjected to limited proteolysis with
Vydac C18 Chromatography--
The N-terminal Sequence Determination--
An Applied Biosystems
liquid pulse sequencer 475A connected on line to a phenylthiohydantoin
analyzer 120A from the same manufacturer was used for automated amino
acid sequence analyses.
Polyacrylamide Gel Electrophoresis and
Microsequencing--
SDS-PAGE was performed using PhastSystem
apparatus (Pharmacia Biotech Inc.). Samples and molecular weight
markers ranging from 14,000-94,000 (Pharmacia) were run in the
presence of 5% SDS on an 8-25% gradient polyacrylamide gel.
Electrophoretic separation under native conditions was carried out
using the same apparatus. The 12.5% homogeneous polyacrylamide gel and
buffer strips having a pH of approximately 4.1 were used. The complex
was made by mixing 10 µM porcine cathepsin D and 40 µM equistatin in 0.2 M sodium acetate buffer,
pH 4.0. Proteins were stained with Coomassie Blue R-250. To identify
proteins migrating on native PAGE, microsequencing of the separated
proteins was performed. After electrophoresis, the gel was soaked into
transfer buffer (25 mM Tris, 100 mM glycine,
and 20% methanol, pH 8.6) for 10 min. During this time a PVDF
difluoride membrane (Bio-Rad) was rinsed with 100% methanol and stored
in transfer buffer. The gel, sandwiched between a sheet of
polyvinylidene difluoride membrane and several sheets of blotting
paper, was assembled into a blotting apparatus (Biometra) and
electro-eluted for 1 h at 65 mV in transfer buffer. The PVDF
membrane was washed in deionized water for 5 min. Proteins
electroblotted onto PVDF membranes were stained with 0.05% Coomassie
Blue R-250 in 50% methanol and 10% acetic acid for 1 min and cut out.
Membranes were destained with 50% methanol, air-dried, and placed in
the cartridge block of the sequenator.
Determination of Protein Concentration--
Protein
concentrations were determined from absorbance at 280 nm using molar
absorption coefficients calculated from the amino acid sequences (19).
A molar absorption coefficient of 33,700 M Inhibition Kinetics of Cathepsin D--
Pepstatin, a tightly
binding inhibitor of cathepsin D, was used to titrate human cathepsin
D, pepsin, and chymosin. The active site titrated cathepsin D was then
used to determine the active concentrations of equistatin, eq d-1, and
eq d-2,3 as follows. Cathepsin D (final concentration, 0.1 µM) was incubated with increasing amounts of pepstatin
(final concentration, 0-1.5 µM) in 390 µl of 0.1 M sodium acetate buffer, pH 4.1. After 30 min of
incubation, 10 µl of 10 mM
H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH was added. The
initial cleavage rates were monitored as a function of decreasing absorbance at 300 nm with Perkin-Elmer Lambda 18 Spectrophotometer (21).
The equilibrium dissociation constants (Ki) for the
interaction between the equistatin, eq d-1, or eq d-2,3 and cathepsin D
were determined by the equilibrium method. The procedure (see previous
paragraph) was repeated with lower concentrations of cathepsin D (final
concentration, 6.4 nM) and inhibitors (final concentration,
0-10 nM). Ki was obtained from the
dependence of (vi/vo) on
I according to the equation
vi/vo =
(E/2){[E Inhibition Kinetics of Papain--
Papain purified by affinity
chromatography (18) had a thiol content of 0.92 ± 0.05 mol/mol of
enzyme as determined by reaction with 5,5'-dithiobis(2-nitrobenzoic
acid). Active site titrated papain was then used to determine the
active concentrations of eq d-1 as described previously (23) and that
of equistatin as follows. Papain (final concentration, 0.1 µM) mixed with cathepsin D (final concentration, 0.115 µM) was incubated with increasing amounts of equistatin
(final concentration, 0-0.2 µM) in 500 µl of 50 mM sodium acetate buffer, pH 4.5, containing 0.1 M NaCl, 5 mM dithiothreitol, and 1 mM EDTA. After 45 min of incubation at 25 °C, 500 µl
of 100 µM Z-Phe-Arg p-nitroanilide was added, and the residual activity of papain was monitored as described previously (23). The kinetics of inhibition of papain were analyzed according to Ref. 13. All experiments were done under pseudo first
order conditions with at least 10-fold molar excess of inhibitor. Papain activity was assayed in the presence of fluorogenic substrate Z-Phe-Arg 4-methyl-7-coumarylamide, and the release of product was
monitored continuously at excitation and emission wavelengths of 370 and 460 nm, respectively, using a Perkin-Elmer LS50B
spectrofluorimeter. All progress curves were fitted by non-linear
regression analysis to the integrated rate equation (24):
[P] = vst + (vz Equistatin was tested for inhibitory activity against papain, the
aspartic proteinases cathepsin D, pepsin, chymosin, and HIV-PR and
against serine proteinase, trypsin. It inhibits papain as previously
shown (13) and also cathepsin D but not other proteinases. In addition,
it has been reported that p41 fragment does not have any inhibitory
effect on cathepsin D (15). To check whether equistatin acts as a
substrate for cathepsin D, we incubated both in different molar ratios
for different periods of time and subjected the mixtures to
reverse-phase HPLC system. No degradation products were observed (Fig.
1A). In contrast, equistatin
was found to be a good substrate for trypsin, and this fact was used
for the separation of the thyroglobulin type-1 domains by a limited
proteolysis with
INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References
-trypsin were prepared as described (Refs. 13 and 16, respectively).
Cathepsin D was purified from human and porcine liver using a procedure
slightly modified from that previously described (17). Papain
(crystallized twice) was obtained from Sigma and further purified by
affinity chromatography (18). Pepstatin (a specific inhibitor of
aspartic proteinases), porcine pepsin, and calf chymosin were also
purchased from Sigma, and chromogenic substrate
H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH was from Novabiochem.
The HIV-PR and its substrate
Ala-Thr-His-Gln-Val-Tyr-Phe(NO2)-Val-Arg-Lys-Ala were
generously provided by Dr. Bruce Korant.
-trypsin, 500 µg being incubated with 5 µg of
-trypsin in 0.5 ml of 0.1 M Tris/HCl buffer, pH 8.0, for 40 min at
37 °C. The reaction was stopped by the addition of trifluoroacetic acid.
-trypsin digest of
equistatin was separated by high performance liquid chromatography
(Milton Roy Co.) using a reverse-phase Vydac C18 column equilibrated
with 5% acetonitrile containing 0.1% (v/v) trifluoroacetic acid.
Elution was performed using a linear gradient of 80% (v/v)
acetonitrile containing 0.1% (v/v) trifluoroacetic acid. Absorbance
was monitored at 215 nm. Peptide fractions were collected and assayed
for inhibitory activities toward papain and cathepsin D. The same HPLC
conditions were used when the mixture of equistatin and cathepsin D was
applied. Prior to application the inhibitor and the enzyme were
incubated in 0.1 M sodium acetate buffer, pH 3.7, at
37 °C. The incubation time was 2 h, and the concentration of
the proteinase was 30% (w/w).
1
cm
1 was calculated for equistatin, 13,355 M
1 cm
1 for eq d-1, 20,345 M
1 cm
1 for eq d-2,3, 49,320 M
1 cm
1 for cathepsin D, 52,185 M
1 cm
1 for pepsin, and 54,090 M
1 cm
1 for chymosin. The
concentration of papain was determined spectrophotometrically using a
molar absorption coefficient of 56,200 M
1
cm
1 (20).
I
Ki] + [(Ki + I
E)2
4KiE]1/2}, where
vi and vo are velocities
with and without inhibitor, respectively, I is the inhibitor
concentration, and E is the enzyme concentration (22). The
potential inhibitory properties of equistatin against pepsin (12.5 nM), chymosin (12 nM), and HIV-PR (30 nM) were tested in a same way as against cathepsin D.
vs)(1
e-kt)/k, where [P]
presents the product concentration, vz and
vs are the initial and the steady-state
velocities, respectively, t is time, and k is the
observed pseudo-first order rate constant. The second order rate
constant ka was calculated from the slope of the
plot k versus [I] (slope =
ka [I]/(1 + S/Km) + kd. The
dissociation rate constants, ka, were obtained from
individual measurements kd = kvi/vz, and the
equilibrium inhibition constant, Ki, was calculated
from Ki=kd/ka.
Km values of 65 µM were used for
papain (25), 2 µM for cathepsin L (26), and 150 µM for cathepsin B (27).
RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References
-trypsin. Two major peaks were obtained on
reverse-phase HPLC (Fig. 1B). The molecular weights, estimated by the SDS-PAGE under non-reducing conditions, were about
7,000 and 14,000 (Fig. 2A).
N-terminal sequences of the fragments locate them in the sequence of
the equistatin molecule as shown in Fig.
3. Their sizes, determined by SDS-PAGE
under nonreducing conditions (Fig. 2A), are consistent with
their being domains. The smaller fragment starts with the N terminus of
the equistatin and therefore corresponds to the first domain (eq d-1). The larger fragment revealed two sequences, starting with
Ala68 and Val152. The
Lys67-Ala68 bond is positioned at the
beginning of the second domain (Fig. 3). Equistatin as isolated is
substantially nicked between Arg151 and Val152,
but the fragments are linked by a disulfide
bond.2 A narrow double band,
visible on SDS-PAGE (Fig. 2A), suggests the presence of
cleavage by trypsin very near to the C terminus. The fragment with
N-terminal Ala68 thus represents the combined second and
third domains, eq d-2,3 (Fig. 3).
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Fig. 1.
HPLC analyses of equistatin.
Chromatograms shows the HPLC elution profile of equistatin after
incubation with different enzymes. In panel A the equistatin
was incubated with cathepsin D in a final molar ratio of 2:1, and in
panel B the equistatin was fragmented using 1% (w/w)
-trypsin. Identities of peaks were based on the N-terminal
sequences.
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Fig. 2.
Electrophoretic analyses of equistatin.
A, SDS-PAGE under nonreducing conditions of dissected
equistatin. Lane 1, intact equistatin; lane 2,
the combined second and third domains of equistatin (eq d-2,3);
lane 3, first domain of equistatin (eq d-1); lane
4, molecular weight standards. B, native PAGE of the
formation of the equistatin-cathepsin D complex. Lane 1,
porcine cathepsin D; lane 2, equistatin and porcine
cathepsin D mixed 30 min before electrophoresis; lane 3,
equistatin. The gels were stained with Coomassie Blue, and bands were
identified by microsequence analysis.
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Fig. 3.
Schematic diagram of the function of
equistatin fragments used in this study. Sites of proteolytic
cleavage are indicated by gaps and located by residue numbers. Cleavage
sites obtained by the action of -trypsin are indicated by
arrows. The pairing of cysteine residues in the disulfide
bond is indicated by a horizontal line connecting cysteine
residues.
It was shown previously that the molar binding stoichiometry of equistatin and papain is 1:1 (13). When cathepsin D was titrated with equistatin, 1 mol of the latter was also needed to saturate 1 mol of cathepsin D (Fig. 4A). Even more, by titrating papain with intact equistatin in the presence of cathepsin D, we demonstrated that the two inhibitory activities of equistatin are structurally separate. The residual activities of both papain and cathepsin D are listed in Table I.
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To assign the inhibitory activities to individual domains of equistatin, a kinetic analysis of the inactivation of papain and cathepsin D was performed. The results are given in Table II. The N-terminal domain, eq d-1, exhibited practically the same inhibitory characteristics against papain as intact equistatin, p41 fragment, and ECI (12, 13, 15). The two-domain C-terminal fragment, eq d-2,3, showed little or no inhibition of papain.
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The kinetics of binding of equistatin to cathepsin D was performed using a synthetic substrate that contains a chromophore, the nitrophenylalanine residue, in the P1' position. Using H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH as substrate means that 6.4 nM enzyme is the minimum concentration usable, so that the determination of subnanomolar Ki values cannot be precisely determined. However, the equilibrium dissociation constant for the interaction between cathepsin D and equistatin (Ki = 0.3 nM) indicates that equistatin is a strong inhibitor of cathepsin D (Fig. 4B). For the papain active fragment, eq d-1, Ki is >1 µM. This is at least several orders of magnitude higher than the Ki value for intact equistatin, indicating that the inhibitory active site of the equistatin must be located on other domains. The eq d-2,3 domains exhibited the same inhibition characteristics as the whole equistatin molecule (Ki = 0.4 nM). Additionally, the formation of a tight complex between cathepsin D and equistatin was demonstrated by native PAGE (Fig. 2B) and confirmed by microsequence analysis. On SDS-PAGE without reduction, equistatin migrates as a single band with an Mr of about 22,000 (Fig. 2A), whereas under reducing conditions it showed single band with an Mr of about 16,000 (13). On the other hand, equistatin on native PAGE gives one major and one minor band, both having the same N-terminal sequence and molecular weight. The minor band probably reflects a molecule of equistatin differing slightly in covalent structure, e.g. glycosylation, amide content, etc. Equistatin showed no inhibitory activity against other aspartic proteinases, pepsin, chymosin, and HIV-PR, even at 2 µM concentrations of inhibitor, thus showing a high degree of specificity for cathepsin D.
Multiplication of inhibitory reactive sites has also occurred in several families of serine proteinase inhibitors (28-30). In the case of kininogen, two (D2 and D3) of three cystatin-like domains can simultaneously inhibit papain-like cysteine proteinases (31, 32). The subtle differences between both domains have significant implications for protein interactions. The D2 domain is able to inhibit calpain, and this feature is unique among cystatins. The region responsible for the calpain inhibition is distinct from the inhibitory region for papain-like cysteine proteinases (33). Another example is the IAP (inhibitor of apoptosis proteins) family of proteins where two or three copies of the BIR (baculovirus IAP repeat) domain are present. Not all BIRs are equivalent in their ability to inhibit caspases (34).
Our results show clearly that the different thyroglobulin type-1
domains present in equistatin, despite their amino acid sequence similarity, can inhibit proteinases of different classes. The first
domain inhibits the cysteine proteinases very strongly, and the second
and/or third domains inhibit cathepsin D equally strongly. This is
particularly clear in the case of equistatin, because this protein is
composed only of thyroglobulin type-1 domains. One molecule of
cathepsin D binds somewhere on eq d-2,3. The cleavage characterized
above may result in a change in conformation and hence function. It is
therefore not possible to say, from the properties of equistatin as
isolated, whether the uncleaved C-terminal domain has inhibitory
activity or not. Alternatively, if the cleavage has no effect on the
structure of domain 3, it could be that domain 3 binds cathepsin D, in
which case the results show that domain 2 would not. The resolution of
the problem will require the production of individual domains by
recombinant technology. Efforts to identify the targets of each of the
individual thyroglobulin type-1 domains within thyropins and to
understand the structural basis of their differential inhibition of
proteinases will provide additional insights into the mechanisms by
which thyropins regulate the proteolytic activity.
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ACKNOWLEDGEMENTS |
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We thank Dr. Selma Berbi, Dr.
Lejla Begi
-Odoba
i
, and Tina Lenassi for cathepsin
D, Dr. Igor Kri
aj and Adrijana Leonardi for performing
N-terminal sequences, and Dr. Roger Pain for helpful discussion and
critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Ministry of Science and Technology of the Republic of Slovenia Grant J1-7422-0106 (to V.T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should addressed. Tel.: 386-61-1773623;
Fax: 386-61-273594; E-mail: brigita.lenarcic{at}ijs.si.
The abbreviations used are: ECI, salmon egg cysteine proteinase inhibitor; HIV-PR, human immunodeficiency virus protease; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride.
2
B. Lenari
, unpublished observations.
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REFERENCES |
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