©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cleavage Specificity of Porcine Follipsin (*)

(Received for publication, May 15, 1995)

Junji Ohnishi (1) Takahiro Kihara (1) Takashi Hamabata (1) Kenji Takahashi (2) Takayuki Takahashi (1)(§)

From the  (1)Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060, Japan and the (2)Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(1) pocket for arginine. The specificity for pocket S(2) 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.


INTRODUCTION

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) (^1)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(r) = 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.


EXPERIMENTAL PROCEDURES

Materials

Fresh porcine ovaries were obtained from a local slaughterhouse. Synthetic peptide substrates containing MCA, angiotensin II, [Asn^1,Val^5]angiotensin II, serum thymic factor, mastoparan, and alpha-neo-endorphin were purchased from the Peptide Institute (Osaka, Japan). [Tyr,Phe]Osteocalcin, alpha-melanocyte-stimulating hormone (alphaMSH), neurotensin, oxidized insulin B-chain, trypsin, soybean trypsin inhibitor, benzamidine HCl, and aprotinin were obtained from Sigma. A mixture of molecular weight marker proteins for SDS-polyacrylamide gel electrophoresis (PAGE) was from Bio-Rad. Vectastain ABC kit for detection of antigens was obtained from Vector Laboratories, Inc. Human sctPA, two-chain tPA, and goat anti-human melanoma tPA antibody were purchased from Biopool AB (Umea, Sweden). Polyvinylidene difluoride transfer membrane was purchased from Millipore Corp. Other reagents used were of the highest grade available.

tPA Peptide Synthesis

A 10-residue peptide (Gln-Pro-Gln-Phe-Arg-Ile-Lys-Gly-Gly-Leu) containing a connecting part of the A/B chain sequence of human tPA was synthesized on an Applied Biosystems model 430A solid-phase synthesizer. The peptide was HPLC purified to >95% purity.

Enzyme Activity Assay

Enzyme activities toward peptide MCA substrates were assayed at pH 8.0 as described(13) . Human tPA activity was assayed at pH 8.0 using 0.1 mM Boc-Gln-Gly-Arg-MCA. The enzyme activity of the samples containing follipsin or trypsin was selectively determined with the same substrate in the presence of soybean trypsin inhibitor or aprotinin, strong inhibitors of both follipsin and trypsin.

Kinetic Parameters

Initial velocities, extrapolated from the plot of product versus time, were transformed into double-reciprocal plots of Lineweaver and Burk(14) . Maximum velocities (V(max)), K, and k values were obtained from intercepts of these plots.

Western Blotting

Samples of human tPA treated with follipsin were separated by SDS-PAGE (15) and transferred to polyvinylidene difluoride membrane(16) . The blotted membrane was incubated with mouse anti-human tPA monoclonal antibody at 1:2000 dilution and subsequently with biotinylated anti-mouse IgG antibody. The membrane was then incubated with avidin conjugated with horseradish peroxidase and stained with hydrogen peroxide and diaminobenzidine.

HPLC Separation and Analysis of Peptides

HPLC separation of peptides was performed with a reversed-phase SG-120 C-18 column (0.46 25 cm, Shiseido) on a Gilson HPLC using a solvent system of 0.1% trifluoroacetic acid and acetonitrile. The column was eluted at a flow rate of 1.0 ml/min and monitored at 220 or 230 nm. The peak fractions collected from HPLC of peptides were subjected to amino acid analysis using an Applied Biosystems automated Derivatizer-Analyzer (420A/130A).

NH(2)-terminal Amino Acid Sequence Analysis of tPA

Human tPA incubated with follipsin was electrophoresed by SDS-PAGE. The product polypeptides were electroblotted onto polyvinylidene difluoride membrane and analyzed directly for NH(2)-terminal sequences (17) in an Applied Biosystems model 477A sequenator with an on-line model 120A phenylthiohydantoin-derivative analyzer.


RESULTS

Activities toward MCA-containing Substrates

Previously, we have shown that follipsin preferentially hydrolyzes Arg-X bonds using synthetic peptide substrates containing MCA(13) . A detailed specificity study was carried out for the enzyme toward substrates with Arg at the P(1) position. Among the substrates tested, Boc-Gln-Arg-Arg-MCA is the best substrate for follipsin based on its k/K value (Table 1). Z-Arg-Arg-MCA is a poor substrate for the enzyme, having the lowest k among the tested substrates. The k/K value for Z-Phe-Arg-MCA is about 6-fold greater than that for Z-Arg-Arg-MCA.



Hydrolysis of Peptide Substrates by Follipsin

Peptide substrates containing Arg and/or Lys residues were tested for hydrolysis by follipsin. Relative hydrolysis rates were calculated for the peptides, and the results are summarized in Table 2. Cleavage occurred only on the COOH-terminal side of Arg residues. The specificity is thus consistent with that found with MCA substrates. Interestingly, the enzyme rapidly hydrolyzed peptides having hydrophobic amino acids (Phe, Leu, and Pro) at P(2) position. The Arg-Phe peptide bond of [Tyr,Phe]osteocalcin was not hydrolyzed by the enzyme at all.



Hydrolysis of Synthetic tPA Peptide

A decapeptide (Gln-Pro-Gln-Phe-Arg-Ile-Lys-Gly-Gly-Leu) containing a proteolytic activation site of human sctPA was synthesized. It contains 1 internal residue each of Arg and Lys. Incubation of the peptide with follipsin resulted in two new HPLC peaks (Fig. 1). Amino acid analysis of the peptides revealed that hydrolysis takes place at the Arg-Ile bond.


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.



Activation of Human sctPA by Follipsin

Human sctPA, which has little or no activity as assayed with the substrate Boc-Gln-Gly-Arg-MCA, was activated by follipsin (Fig. 2). The tPA activity increased rapidly within 60 min and then slowly leveled off. In contrast, tPA activity caused by trypsin activation first increased up to a level comparable to that observed with follipsin and declined thereafter. This is probably due to extensive hydrolysis of the activated tPA by trypsin. Consistent with the previous observations from several laboratories(18, 19) , sctPA was not activated autocatalytically.


Figure 2: Activation of human sctPA by follipsin. Human sctPA (110 pmol) was incubated alone () or with 1.8 pmol of follipsin (bullet) 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(r) = 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(2)-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(2)-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(2)-terminal region of the polypeptide. In fact, Wallen et al. (21) documented that the NH(2)-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 NH(4)HCO(3) (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(2)-terminal amino acid sequence analysis.



Kinetic Parameters of Follipsin on sctPA and the tPA Peptide

In order to compare the nature of follipsin action on sctPA and the tPA peptide, the kinetic parameters for these substrates were determined. As shown in Table 3, similar k values were obtained for the two substrates. However, the k/K value of the enzyme for the protein substrate was approximately 7 times greater than for the peptide substrate. This is mainly due to the difference in the affinities for the two substrates.




DISCUSSION

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(1) 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(2). 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(2) 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(2) site for hydrolysis.

The specificity of subsite S(2) 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^1,Val^5]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(1) and S(2) 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(2) 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.^2 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) .


FOOTNOTES

*
This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education and Culture, Japan and Research Grants from the Akiyama Foundation and the Naito Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Div. of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060, Japan. Tel. and Fax: 011-706-2748.

(^1)
The abbreviations used are: MCA, 4-methylcoumaryl-7-amide; PAGE, polyacrylamide gel electrophoresis; Boc, t-butyloxycarbonyl; Z, benzyloxycarbonyl; tPA, active two-chain tissue-type plasminogen activator; sctPA, single-chain precursor tissue-type plasminogen activator; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.

(^2)
J. Ohnishi and T. Takahashi, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Ronald T. MacFarland for critical reading of this manuscript and Yasuko Sakurai for her technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.