(Received for publication, November 19, 1996, and in revised form, March 23, 1997)
From the Department of Biochemistry and Molecular Biology,
Joef Stefan Institute,
Jamova 39, 1000 Ljubljana, Slovenia
It is well known that the activities of the
lysosomal cysteine proteinases are tightly regulated by their
endogenous inhibitors, cystatins. Here we report a new inhibitor of
cysteine proteinases isolated from sea anemone Actinia
equina. The inhibitor, equistatin, is an acidic protein with pI
4.7 and molecular weight of 14,129. It binds tightly and rapidly to
cathepsin L (ka = 5.7 × 107
M1 s
1, Ki = 0.051 nM) and papain (ka = 1.2 × 107 M
1 s
1,
Ki = 0.57 nM). The lower affinity for
cathepsin B (Ki = 1.4 nM) was shown to
be due mainly to a lower second order association rate constant
(ka = 0.04 × 106
M
1 s
1). The inhibitor is
composed of 128 amino acids forming two repeated domains with 48%
identity. Neither of the domains shows any sequence homology to
cystatins, but they do show a significant homology to thyroglobulin
type-1 domains. A highly conserved consensus sequence motif of
Cys-Trp-Cys-Val together with conserved Cys, Pro, and Gly residues is
present in major histocompatibility complex class II-associated p41
invariant chain, nidogen, insulin-like growth factor proteins,
saxiphilin domain a, pancreatic carcinoma marker proteins
(GA733), and chum salmon egg cysteine proteinase inhibitor. In each of
the domains of the equistatin, the three residues are similarly
conserved, and the sequences Val-Trp-Cys-Val and Cys-Trp-Cys-Val are
present in domains a and b, respectively. We
suggest that equistatin belongs to a new superfamily of protein inhibitors of cysteine proteinases named thyroglobulin type-1 domain
inhibitors. This superfamily currently includes equistatin, major
histocompatibility complex class II- associated p41 invariant chain
fragment, and chum salmon egg cysteine proteinase inhibitor.
Sea anemones are known to be a rich source of variety of polypeptide neurotoxins (1, 2) and neuropeptides (3), but little is known about the presence of proteolytic enzymes and their inhibitors. A chymotrypsin-like protease was first isolated from Metridium senile and shown to possess the same zymogen activation and active site chemistry as the proteinase from mammalian pancreas (4). Early reports on the existence of proteinase inhibitors in different species of sea anemones (5-8) were followed by the isolation (9) and primary structure determination of an elastase inhibitor from Anemonia sulcata (10). The inhibitor was found to be a nonclassical Kazal-type inhibitor with respect to positioning of the half-cystines. More recently, the structure of a Kunitz-type proteinase inhibitor purified from the Caribbean sea anemone Stichodactyla heliantus has been determined by NMR spectroscopy (11).
Cysteine proteinases are members of one of the four mechanistic classes of proteinases and, together with their endogenous protein inhibitors, cystatins, play an important role in intracellular degradation (12). They have not yet been found in sea anemones.
In this article we describe the isolation of a new inhibitor of papain-like cysteine proteinases from sea anemone, Actinia equina, designated as equistatin, the kinetic properties of its interaction with papain-like cysteine proteinases, and its amino acid sequence.
Papain (2 × crystallized) and clostripain were purchased from Sigma (Germany), and Ep-475,1 a specific inhibitor of cysteine proteinases, was obtained from Peptide Research Foundation (Japan). The Staphylococcus aureus V8 proteinase was obtained from Miles (UK), and glycyl endopeptidase was a gift from Dr. Alan J. Barrett (The Babraham Institute, Cambridge, UK) and was prepared as described (13). Recombinant human cathepsin B and human cathepsin L were prepared as described previously (14, 15).
Inhibitor PurificationA. equina specimens were
collected on the northern coast of the Adriatic sea. The anemones (3 kg) were frozen, partially thawed, cut into small pieces, and
homogenized in 4.5 liters of deionized water. Nonsoluble material was
removed by centrifugation at 13,000 × g for 45 min.
The supernatant was adjusted to pH 10.5 and incubated at room
temperature for 1 h. Neutralization to pH 7.0 was followed by
additional centrifugation at 13,000 × g for 45 min.
The clear supernatant was applied to a carboxymethyl papain-Sepharose
column (6 × 10 cm) previously equilibrated with 0.01 M Tris/HCl buffer, pH 8.0, containing 1 M NaCl
and 0.1% Brij. After thorough washing of the column, bound proteins
were eluted with 0.01 M NaOH. Fractions (20 ml) were
collected and assayed for inhibitory activity toward papain using
benzoyl-DL-Arg--naphthylamide as substrate (16). The
inhibitory fractions were pooled and concentrated by ultrafiltration (Amicon YM-5). The concentrate was applied to a Sephadex G-50 column
(4.5 × 140 cm) equilibrated with 0.01 M Tris/HCl buffer, pH 7.7, containing 0.1 M NaCl, and eluted at a flow rate of
18 ml/h. Inhibitory fractions with molecular weights of about 16,000 were pooled, concentrated (Amicon, YM-5), and dialyzed against 0.01 M Tris/HCl buffer, pH 7.2. The dialyzed sample was then
applied to a DEAE-Sephacel column (2 × 25 cm) equilibrated with
the same buffer. The column was washed extensively, and bound proteins were eluted with a linear salt gradient (0-0.1 M NaCl in
0.01 M Tris/HCl buffer, pH 7.2) at a flow rate 18 ml/h.
Equistatin eluted at 0.07 M NaCl.
SDS-PAGE and isoelectric focusing were performed on a PhastSystem apparatus (Pharmacia Biotech Inc.) following the manufacturer's instructions. The inhibitor and molecular weight markers ranging from Mr 14,400 to 94,000 were run in the presence of 0.5% SDS and 5% 2-mercaptoethanol on an 8-25% gradient polyacrylamide gel. The pI of the inhibitor was determined by calibrating the gel with isoelectric focusing marker proteins with pI values ranging from 3.5 to 8.15.
Protein Sequence DeterminationEquistatin was reduced
overnight with -mercaptoethanol at 37 °C and
S-pyridylethylated (17). Pyridylethylated equistatin was
hydrolyzed with glycyl endopeptidase as described (18). 4 nmol of
pyridylethylated equistatin were fragmented using 2% (w/w) S. aureus V8 proteinase in 0.5 M sodium lactate buffer, pH 4.0, at 37 °C for 20 h. Both enzyme hydrolyses were
performed in a final volume of 500 µl. Reactions were stopped by the
addition of trifluoroacetic acid. The resulting peptide mixtures were
separated by high performance liquid chromatography (Milton Roy Co.)
using a reverse phase ChromSpher C18 column equilibrated with 0.1%
(v/v) trifluoroacetic acid in water. Elutions was performed using
various linear gradients of 80% (v/v) acetonitrile containing 0.1%
(v/v) trifluoroacetic acid. The absorbance was monitored at 215 nm. Protein samples were hydrolyzed in 6.0 M HCl at 110 °C
for 24 h. Analyses of the peptide hydrolysates were performed on
an Applied Biosystems 421A amino acid analyzer with precolumn
phenylisothiocyanate derivatization. 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.
Protein
concentration of equistatin was determined by absorption measurements
at 280 nm using a molar absorption coefficient of 28,600 M1 cm
1 determined by the method
of Pace et al. (19) from the amino acid sequence or by the
method of Lowry et al. (20) using bovine serum albumin as
standard. The concentration of papain was determined spectrophotometrically using a molar absorption coefficient of 56,200 M
1 cm
1 (21).
The following buffers were used in
all kinetic and equilibrium studies: 0.1 M phosphate
buffer, pH 6.0, containing 5 mM dithiothreitol and 1 mM EDTA (for papain and cathepsin B) or 0.34 M
sodium acetate buffer, pH 5.5, containing 5 mM
dithiothreitol and 1 mM EDTA (for cathepsin L). Active site
titrations of cathepsins B and L were performed using cysteine
proteinase inhibitor Ep-475 as described previously (22). Papain,
further purified by affinity chromatography (23), 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 used to titrate equistatin as follows. Papain (0.1 µM final concentration) was incubated with increasing amounts of equistatin (0-0.2 µM final concentration) in 200 µl of 0.1 M phosphate buffer, pH 6.0, containing 5 mM dithiothreitol and 1 mM EDTA at 25 °C. After 15 min of incubation, 1800 µl of 100 µM Z-Phe-Arg p-nitroanilide was added, and the residual activity of papain was monitored as described previously at 410 nm with a Perkin-Elmer Lambda 18 spectrophotometer (22). The data were analyzed by computer fitting to the theoretical binding equation (24).
Kinetics of Inhibition of Papain and Cathepsins B and L by EquistatinThe kinetics of the reaction between equistatin and
papain, cathepsin B, and cathepsin L were analyzed by continuous
measurements of the loss of enzymatic activity in the presence of
substrate under pseudo first-order conditions with at least a 10-fold
molar excess of inhibitor. Equistatin in increasing concentrations and the fluorogenic substrate (10 µM Z-Phe-Arg
4-methyl-7-coumarylamide) were mixed in a cuvette with buffer (see
above) to a final volume of 1.97 ml. The enzyme (30 µl) was added,
and the release of product was monitored continuously at excitation and
emission wavelengths of 370 and 460 nm, respectively, by a Perkin-Elmer
LS50 spectrofluorimeter. The biphasic progress curves were recorded and
analyzed according to the model of slow tight binding kinetics using
the equation of Morrison (25): [P] = vst + (vz vs)(1
e
kt)/k, where [P] is 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 for the establishment of equilibrium between enzyme and inhibitor. ka (association rate constant) and
kd (dissociation rate constant) values were obtained
from the dependence of k on [I] according to the equations
k = ka·[I]/(1 + S/Km) + kd and
kd = k·vi/vz. The ka values were corrected for substrate competition
using the Km values of 65 µM for
papain (26), 2 µM for cathepsin L (27), and 150 µM for cathepsin B (16). Ki values were then determined from both individual rate constants
(=kd/ka). Less than 3% of the
substrate was hydrolyzed during the experiments throughout.
Equistatin was purified from
A. equina by a procedure similar to that used for the
isolation of cysteine proteinase inhibitors of human origin (28).
Initially, the supernatant was exposed to alkaline pH to dissociate the
complexes between the inhibitor and other proteins. The most selective
purification step, affinity chromatography on carboxymethyl
papain-Sepharose, then allowed separation of papain-inhibiting proteins
from the majority of noninhibitory proteins. This was followed by gel
filtration on Sephadex G-50 (Fig. 1A), where
the low molecular weight inhibitor (equistatin) was separated from high
Mr inhibitor(s) of cysteine proteinases, which
were not further characterized. Final purification was achieved by
DEAE-Sephacel chromatography, from which the inhibitor eluted as a
single peak at 0.07 M NaCl (Fig. 1B). About 5 mg
of pure equistatin was obtained from 3 kg (fresh weight) of sea
anemones.
SDS-PAGE and Analytical Isoelectric Focusing
On SDS-PAGE
under reducing conditions, equistain migrates as a single band with
Mr of about 16,000 (Fig.
2A). The molecular weight is higher than the
molecular weights of either stefins or cystatins
(Mr ~ 11,000 and 13,000, respectively) but
lower than those of kininogens (Mr ~ 50,000-100,000) (12). The stefins, the cystatins, and the kininogens
are proteins with similar sequences and, until recently, were the
only known endogenous inhibitors of papain-like cysteine proteinases.
On analytical isoelectric focusing, the inhibitor is shown to be an
acidic protein with a pI value of 4.7. Very faint bands with pI values
of 4.9 and 4.5, probably corresponding to the isoforms of the inhibitor
(see below for explanation), could also be seen (Fig.
2B).
Amino Acid Sequence of Equistatin
The major and minor
N-terminal amino acid sequences, labeled NI-1 and
NI-2, respectively, are shown in Fig.
3A. Sequence analyses of the peptides derived
from glycyl endopeptidase digestion provided the amino acid sequence of
the whole molecule (Fig. 3A). The largest peptide,
G-3, spanned the middle part of the inhibitor and overlapped with both NI sequences. The C-terminal sequence was
confirmed by peptides G-5 and G-(5+6), which
ended with a pyridylethylated Cys residue that is not a glycyl
endopeptidase cleavage site. Additional overlapping peptides,
designated as E peptides (Fig. 3A) were obtained
by S. aureus V8 proteinase digestion. During protein
sequence analysis we have observed sequence polymorphism mainly in the
middle part of the molecule (Fig. 3A). However, the yield of
these residues was lower than 20% when compared with the main
sequence. The observed sequence heterogeneity together with the results
of isoelectric focusing reveals the presence of at least two closely
related isoforms. As the isolation procedure involves the use of many
anemone specimens, the difference in amino acid composition could arise
from allelic polymorphism.
The inhibitor comprises 128 amino acid residues including these 11 cysteines and has a molecular weight of 14,129. The inhibitor has no potential glycosylation sites of the Asn-X-(Thr/Ser) type.
Amino Acid Sequence ComparisonAlignment of equistatin
residues 1-64 with 65-128 shows that the inhibitor consists of a
tandem repeat with 48% identity and 60% similarity (Fig.
3B). This indicates that equistatin derives from a single
ancestral gene that was duplicated and modified during evolution.
However, neither domain shows any sequence homology with the members of
the cystatin superfamily (12, 29). A Blast (30) search of the
Swiss-Prot data bases (31) revealed high sequence similarity with
thyroglobulin type-1 domain, a domain of about 65 amino acid residues
that repeats 10 times in the N-terminal part of thyroglobulin (32). A
number of other proteins containing the thyroglobulin type-1 domain
motif were found. These proteins display a variety of physiological
functions in different organisms. Major histocompatibility complex
class II-associated p41 invariant chain fragment and chum salmon egg
cysteine proteinase inhibitor are potent inhibitors of papain-like
cysteine proteinases (33, 34), the former being involved in antigen
presentation (35). Nidogen is a glycoprotein that probably plays a
central role in the supramolecular organization of basement membranes
and is tightly associated with laminin (36). Insulin-like growth
factor-binding proteins act as inhibitors of insulin-like growth factor
(37). Saxiphilin, characterized by a high affinity for a neurotoxin, saxitoxin (38, 39), and a tumor-associated cell surface antigen known
also as GA733 are proteins whose functions are not yet elucidated (40).
Fig. 4A shows a schematic diagram of the
regions of similarity between equistatin and proteins containing the
thyroglobulin type-1 domain, and Fig. 4B shows a sequence
alignment relative to the thyroglobulin type-1 domains of equistatin.
By introducing only a few short gaps in the alignment, the amino acid
identities between the thyroglobulin type-1 domains in equistatin and
those in all the other proteins listed in Fig. 4B are
approximately 40% for the 49 C-terminal amino acids of both domains.
The cysteine-rich sequence motif Cys-Trp-Cys-Val and the positions of
some other amino acids (Cys-24, Cys-60, Pro-22, Gln-34, Gly-28, and
Gly-49; equista in Fig. 4B) were found to be
highly conserved among all related repeats, indicating that the
proteins are probably evolutionarily related.
Active Site Titration and Kinetics of Inhibition
The sequence
data suggest that the two sequentially homologous parts of equistatin
(Fig. 3B) may form two potential proteinase binding sites.
The binding stoichiometry of papain (active concentration 95%) and
equistatin was therefore determined by titration monitored by the loss
of enzymatic activity. 0.95 ± 0.04 mol of equistatin was needed
to saturate 1 mol of papain, indicating that the two proteins formed an
equimolar complex (Fig. 5). It could be suggested that
binding of one proteinase molecule to equistatin prevents binding of
the second proteinase molecule, probably by steric hindrance. However,
there are a number of other possibilities. (i) One of the domains is
not inhibitory at all, as observed in the kininogens (41). (ii) One of
the domains has substantially lower affinity for proteinases, as found
for the mucus proteinase inhibitor interaction with various serine
proteinases (42). (iii) Both domains bind to the same proteinase
molecule but only one of them binds to the active site; the other binds
to another site distant from the active site, as reported for rhodiin
binding to thrombin (43). Additional spectroscopic and structural
studies involving mutant proteins will therefore be needed to clarify which of the above hypotheses is correct.
The kinetics of binding of equistatin to papain and cathepsins B and L were studied under pseudo first-order conditions assuming 1:1 binding stoichiometry (see above). The pseudo first-order rate constants were found to increase linearly with increasing concentrations of inhibitor [I], in agreement with the proposed binding mechanism (25). Values of the second-order rate constants (ka), the dissociation rate constants (kd), and the equilibrium constants (Ki) are presented in Table I. Rapid binding of equistatin to cathepsin L and papain was observed, but the complexes with papain were ~10-fold less stable, with a 5-fold lower association rate constant and a 2-fold higher dissociation rate constant. The rate of complex formation between equistatin and cathepsin B was substantially slower. Its ka value is >30-fold lower than those for cathepsin L and papain, also reflected in the increased Ki value although the overall effect is partially compensated by a lower kd value. Cathepsin B (44) differs from papain (45) and its homologue cathepsin L (46) by having an additional loop of about 20 amino acids, which partially occludes the active site, thus interfering with inhibitor binding (47).
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The kinetic and equilibrium constants for the interaction of equistatin with cathepsins L and B and papain are similar to those reported for the interactions of these enzymes with cystatins (12, 22, 47). The Ki values are also in reasonable agreement with those obtained for various forms of chum salmon egg cysteine proteinase inhibitor (34, 48) although they differ significantly from the values for the p41 form of invariant chain fragment. The latter was found to be a stronger inhibitor of cathepsin L (~10-fold) and a weaker inhibitor of papain (~3-fold) but did not inhibit cathepsin B at all (33).
In conclusion, a new protein inhibitor of papain-like cysteine proteinases was isolated from sea anemone A. equina. The inhibitor, equistatin, is distinct from cystatins but shares significant sequence homology with two other chum salmon egg cysteine proteinase inhibitors, p41 invariant chain fragment and cysteine proteinase inhibitor. The three inhibitors were therefore suggested to form a new superfamily of cysteine proteinase inhibitors. The thyroglobulin type-1 domain motif, common to all three inhibitors, has been identified in a variety of other proteins. Whether this highly conserved thyroglobulin type-1 element indeed acts as an inhibitor of cysteine proteinases in these proteins remains to be established as well as the mechanism of binding to cysteine proteinases.
We thank Dr. Aleksander Lucu for providing us with sea anemones and Dr. Iztok Dolenc and Robert Kuhelj for their gifts of cathepsins L and B, respectively. We also thank Dr. Roger H. Pain for critical reading of the manuscript.