(Received for publication, May 15, 1995)
From the
Follipsin purified from the follicular fluid of porcine ovaries
was studied for its specificity against various synthetic and peptide
substrates. The enzyme cleaved only by an endopeptidase activity at the
amide and peptide bonds of Arg-X, indicating strict specificity of the
S pocket for arginine. The specificity for pocket S
appears to favor either hydrophobic or basic side chains. A
10-residue peptide containing a portion of the activation site of human
tissue plasminogen activator was synthesized and tested with the
enzyme. The peptide was cleaved by follipsin at the Arg-Ile bond, as
expected from the specificity deduced above. Furthermore, the enzyme
successfully converted single-chain precursor tissue plasminogen
activator (sctPA) to its active, two-chain form by cleaving the
corresponding peptide bond. Comparison of the rates of single-chain
precursor tissue plasminogen activator activation and tissue
plasminogen activator peptide hydrolysis revealed that the former is a
more efficient substrate than the latter.
Germ cells in mammalian ovaries grow to maturity in individual follicles(1) . A single primordial follicle, which contains a germ cell enveloped by only one layer of flattened follicular cells, is transformed into a ripe, preovulatory follicle with thousands of highly differentiated cells. Each fully matured folicle contains large amounts of fluid in the follicular space. It is generally thought that the constituents of the fluid are derived from either plasma or follicular tissue itself. Among the proteins present in the fluid are proteolytic enzymes which have attracted special attention in connection with follicular maturation and/or ovulation(2, 3, 4, 5) . A number of studies (6, 7, 8, 9, 10, 11) have established that the ovulatory process is mediated by plasmin generated from plasminogen by plasminogen activator.
In the previous
study(12) , we detected an enzyme activity in the follicular
fluid of porcine ovaries capable of hydrolyzing synthetic,
arginine-containing peptide 4-methylcoumaryl-7-amide (MCA) ()substrates. The activity was found to increase several
times during follicle maturation. Subsequent studies including
purification and characterization (13) revealed that the enzyme
follipsin is a novel peptidase belonging to the serine proteinase
family. The enzyme consists of two different polypeptide chains having M
= 45,000 and 32,000 and is shown to be
structurally homologous with human plasma kallikrein and factor XIa.
The physiological role of follipsin is not known at present. In order to determine the physiological substrate(s), it is necessary to understand its substrate specificity in detail. The aim of the present study is to investigate the action of follipsin toward various synthetic and peptide substrates. Based on the results, the specificities of some of the side chain-binding pockets of the enzyme were deduced. We also describe the proteolytic conversion of human single-chain precursor tissue-type plasminogen activator (sctPA) to its mature form (tPA) by follipsin.
Figure 1: Hydrolysis of tPA peptide by follipsin. tPA peptide (0.4 µmol) was incubated at 37 °C with 11.6 pmol of follipsin in 100 µl of 40 mM Tris-HCl, pH 8.0. After the indicated incubation periods, 5-µl aliquots were mixed with 35 µl of 0.1% trifluoroacetic acid, passed through a 0.4-µm filter, and applied to a reversed-phase HPLC column equilibrated with 0.1% trifluoroacetic acid. The column was then eluted with a linear gradient (0-50%) of acetonitrile containing 0.1% trifluoroacetic acid, and the absorbance at 230 nm was monitored. Preparative HPLC was performed using 50 µl of 4-h sample. Eluates corresponding to peaks a-c were collected for amino acid composition analyses. Based on these data, the deduced sequences of the peptides are: a, Ile-Lys-Gly-Gly-Leu; b, Gln-Pro-Gln-Phe-Arg; and c, Gln-Pro-Gln-Phe-Arg-Ile-Lys-Gly-Gly-Leu.
Figure 2:
Activation of human sctPA by follipsin.
Human sctPA (110 pmol) was incubated alone () or with 1.8 pmol of
follipsin (
) or trypsin (
) in 60 mM Tris-HCl (pH
8.0) in a volume of 100 µl. Aliquots (10 µl) of the incubation
mixture were taken at the indicated times, and the activity of
activated tPA toward Boc-Gln-Gly-Arg-MCA was selectively determined by
assaying in the presence of aprotinin (40 mg/ml in assay). Mean values
for two determinations are shown.
As shown in Fig. 3, incubation
of sctPA with follipsin brought about the production of two polypeptide
chains having M = 34,000 and 32,000 with a
concomitant decrease in the staining of the 61-kDa precursor protein.
These results indicate that proteolytic cleavage at a single internal
peptide bond underlies the activation of tPA by follipsin. In order to
determine the cleavage site, the NH
-terminal amino acid
sequences of the products were analyzed. A single amino acid sequence
(Ile-Lys-Gly-Gly-Leu-) was obtained for the 32-kDa polypeptide. This
sequence corresponds to part of sctPA
(Ile
-Leu
) (20) indicating that the
product is derived from the COOH-terminal portion of the precursor
protein. The sequence analysis of 34-kDa polypeptide, which is derived
from the NH
-terminal portion of sctPA, did not give clear
results because of lower yields of phenylthiohydantoin amino acids.
This is presumably due to heterogeneity in the NH
-terminal
region of the polypeptide. In fact, Wallen et al. (21) documented that the NH
-terminal amino acid
sequences of sctPA purified from the culture medium of human melanoma
cells always contain several sets of amino acid sequneces.
Nevertheless, the results clearly indicate that the
Arg
-Ile
peptide bond in sctPA was
selectively cleaved by follipsin.
Figure 3:
Conversion of sctPA to tPA by follipsin.
Human sctPA (758 pmol) was mixed with follipsin (26 pmol) in 50 mM NHHCO
(pH 8.0) in a volume of 2.0 ml, and
incubated at 37 °C. Aliquots (10 µl) of the reaction mixture
were taken at 0 (lane 1), 0.25 (lane 2), 0.5 (lane 3), 1 (lane 4), and 1.5 h (lane 5) of
incubation for SDS-PAGE/Western blot analysis under reducing
conditions, as described under ``Experimental Procedures.''
The remainder (1.8 ml, 2-h incubation) of the sample was lyophilized
and dissolved in a small volume of SDS-PAGE sample solvent for
NH
-terminal amino acid sequence
analysis.
The present study clearly establishes that porcine follipsin
is an endopeptidase capable of hydrolyzing Arg-X bonds of both
MCA-containing substrates and peptides. No hydrolyzing activity at
Lys-X bonds could be found for the enzyme with either synthetic or
peptide substrates. These results support the suggestion that substrate
recognition subsite S of the enzyme can be occupied only by
Arg.
The K values of follipsin for the
MCA substrates tested in this study were found to be fairly close to
each other. Similarly the enzyme showed similar k
values for the substrates with the exception of Z-Arg-Arg-MCA. The difference in the k
/K
values of Z-Phe-Arg-MCA and Z-Arg-Arg-MCA suggests some
preference for amino acid residues with a hydrophobic side chain in
S
. Such a subsite specificity was more clearly shown in the
experiments using peptide substrates. Hydrophobic amino acids (Phe,
Leu, and Pro) are commonly present at the P
position in the
peptide that are hydrolyzed by follipsin. In this regard, the cleavage
profile of [Tyr
,Phe
]osteocalcin
is noteworthy. This peptide was presumed to be cleaved at two sites
because it contains paired Arg residues. However, the enzyme cleaved
only at the Arg-Arg bond, indicating that Phe
, but not
Arg
, of the peptide binds the S
site for
hydrolysis.
The specificity of subsite S for hydrophobic
amino acid residues is not absolute. This subsite could be occupied by
basic amino acids (Arg and Lys) since the synthetic substrates
Boc-Gln-Arg-Arg-MCA and Boc-Leu-Lys-Arg-MCA (13) are well
hydrolyzed by follipsin. However, acidic side chains are apparently not
favored. This notion is supported by the following observations.
[Asn
,Val
]Angiotensin II was
hydrolyzed by the enzyme to some extent, whereas angiotensin II was
completely resistant to hydrolysis. In addition, no cleavage occurred
on the COOH-terminal side of the Arg residue of oxidized insulin
B-chain. Aspartyl (angiotensin II) and glutamyl (oxidized insulin
B-chain) residues are at a position 1 residue to the left of the Arg in
these peptides.
Although the specificity of other side chain-binding
pockets cannot be deduced clearly, our results appear to indicate that
they have broad specificities. We presume that as far as the results
obtained with synthetic and peptide substrates are concerned, the
specificity of follipsin is dependent primarily on S and
S
side chain recognition, and the number and nature of
other residues in the substrates are only of secondary importance.
Since follipsin is present in the fluids of mature follicles in
mammalian ovaries, it is reasonable to postulate that the enzyme is
involved in a proteolytic event associated with follicular maturation
and/or ovulation. Recent studies indicate that follicular rupture in
ovulation is mediated by plasmin generated from plasminogen by tPA (6, 7, 8, 9, 10, 11) and that
tPA is secreted as the inactive single-chain precursor into the fluid
from granulosa cells of matured follicles(22) . Therefore, the
activation of tPA must be a prerequisite for degradation of the
follicle wall although a proteinase(s) responsible for the activation
has not yet been identified. The amino acid sequence
(-Pro-Gln-Phe-Arg-Ile-Lys-Gly-Gly-Leu
-, in
human tPA residue number) around the activation site is conserved
completely in tPAs from human(20) , rat(23) , and
mouse(24) . Considering that when Phe is located at the P
position follipsin efficiently cleaves peptide bonds on the
COOH-terminal side of Arg residues, the Arg
-Ile
bond in sctPA could be cleaved by the enzyme. In the present
study, we demonstrated that follipsin is indeed capable of specifically
cleaving this bond and converting sctPA to active tPA in
vitro.
Comparison of the rates of sctPA activation and of the
tPA peptide hydrolysis by follipsin revealed that the former is a more
efficient substrate than the latter based on the k/K
values. It is
interesting that the K
value for sctPA
was one-fifth that for the peptide substrate, and is 1-2 orders
of magnitude smaller than those for the synthetic MCA substrates. Such
a high affinity for sctPA may reflect the presence of additional
substrate interaction sites other than the catalytic domain on the
enzyme molecule. In a manner analogous to the important role of the
noncatalytic heavy chain of plasma kallikrein in the expression of
coagulant activity, neutrophil aggregation, and elastase release (25, 26, 27, 28) , the 45-kDa
polypeptide of follipsin (13) perhaps facilitates its specific
interaction with sctPA.
The present finding that porcine follipsin
specifically activates human sctPA in vitro prompts us to
speculate its role in the process of follicle wall degradation upon
ovulation. To establish the validity of this assumption, tPA activation
experiments using proteins from the same species are required. Since
neither porcine sctPA nor human follipsin is available, such
experiments were not conducted in the present study. However, we have
recently detected a follipsin-like enzyme activity in follicular fluid
from human ovaries obtained during in vitro fertilization
procedures. The purification of the enzyme from the fluid
is thus under way in our laboratory. From a physiological point of
view, it would be particularly interesting to compare its efficiency in
tPA activation with those of plasmin, grandular kallikrein, and factor
XIa, the enzymes known to activate tPA in vitro(19) .