INTRODUCTION

α₂-Macroglobulin is a glycoprotein that exists in plasma as a tetramer composed of identical subunits, with a total molecular weight of 725,000 (Barrett and Starkey, 1973; Sottrup-Jensen, 1989). It functions as an inhibitor of all four major classes of endoproteases. This inhibiting mechanism is unique in that limited proteolysis of α₂-macroglobulin at a “bait region” located near the middle of each subunit results in a conformational change in the inhibitor that physically entraps the protease. Because of this unique mechanism, proteases with molecular weights smaller than about 80,000 can be trapped by the inhibitor. Plasma kallikrein and plasmin are the largest proteases that the inhibitor is reported to bind (Barrett, 1981).

α₂-Macroglobulin is also known to be present in the follicular fluid of mammalian ovaries (Andersen et al, 1976; Curry et al., 1990; Gaddy-Kurten and Richards, 1991; Zhu and Woessner, 1991; Ohnishi et al., 1997). Recently, we have reported that the plasma kallikrein activity in human ovarian follicular fluid is largely associated with α₂-macroglobulin (Ohnishi et al., 1997). It has been well established that proteases bound to α₂-macroglobulin maintain their activity in regard to small substrates, but have diminished effect on macromolecular substrates (Barrett and Starkey, 1973; Barrett, 1981; Bieth et al., 1981; Gonias and Pizzo, 1983; Sottrup-Jensen, 1989; Sakurai et al., 1996; Uchino et al., 1993). Therefore, we presumed that little or no activity would be detected for α₂-macroglobulin fraction containing active plasma kallikrein regarding protein substrates. However, we observed the fraction inciting considerable activation of single-chain tissue-type plasminogen activator (sctPA), a protein known to be expressed in ovarian follicles upon ovulation (Beers, 1975; Beers et al., 1975; Ny et al., 1985; Reich et al., 1985; Reinthaller et al., 1990; Leonardsson et al., 1995). In this report, we show that a serine protease complexed with α₂-macroglobulin converts sctPA to two-chain tissue-type plasminogen activator (tPA). The present data also indicate that plasma kallikrein is not the enzyme responsible for this activation.

MATERIALS AND METHODS

RESULTS AND DISCUSSION

The protease inhibitor α₂-macroglobulin can be purified from the follicular fluid of human ovaries obtained during in vitro fertilization procedures by a series of column chromatographic steps on DE-52, Sephacryl S-300, and heparin-Cellulofine (Ohnishi et al., 1997). In this study, we obtained the α₂-macroglobulin fraction according to the above method. The chemical entity and purity of the fraction were confirmed by electrophoretic and immunological analyses, as described previously (Ohnishi et al., 1997). The sample thus prepared contained largely α₂-macroglobulin in free form. In addition, the sample was shown to contain in part α₂-macroglobulin complexed with plasma kallikrein. In view of the general understanding that proteases entrapped in α₂-macroglobulin no longer act on protein substrates because their free contact becomes sterically impossible (Barrett and Starkey, 1973; Barrett, 1981; Sottrup-Jensen, 1989), we assumed that the α₂-macroglobulin fraction would exhibit no significant activity toward protein substrates. However, in a preliminary experiment, sctPA (Mr = 60,000) upon incubation with the franction was found to be activated substantially. This unexpected result was initially thought to be due to a still-contaminating, unbound protease(s) in the α₂-macroglobulin sample. Therefore, the sample obtained from heparin-Cellulofine column chromatography was fractionated once more through a column of Sephacryl S-300 (Fig. 1). The fractions were monitored by assay with three MCA-substrates: Pro-Phe-Arg-MCA (substrate for kallikrein), Boc-Val-Leu-Lys-MCA (substrate for plasmin or endopeptidases with cleavage specificity for Lys-X peptide bonds), and Suc-Leu-Leu-Val-Tyr-MCA (substrate for proteasome). The enzyme activity toward Pro-Phe-Arg-MCA appeared in a single peak at the position corresponding to a molecular weight of 725,000. No detectable activity was observed in the fractions eluted at the position for lower molecular weight proteins. Little activity was detected with Boc-Val-Leu-Lys-MCA and Suc-Leu-Leu-Val-Tyr-MCA throughout the fractions. These results indicate that the sample obtained from the heparin-Cellulofine column did not contain proteolytic activities of plasmin, Lys-X-hydrolyzing endopeptidases, or proteasome. Therefore, we tentatively presumed that the α₂-macroglobulin fraction had not been seriously contaminated with unbound proteases. However, we cannot exclude completely the possibility that proteases having activities toward appropriate substrates other than the above three MCA substrates are present. The fractions 45–54 were pooled and used in the following experiments.

Fig. 1

Sephacryl S-300 column choromatography of α₂-macroglobulin fraction isolated from human ovarian follicular fluid. The α₂-macroglobulin fraction (1.2 mg) obtained from a heparin-Cellulofine column (Ohnishi et al., 1997) was concentrated, applied to a column (1.5 × 54 cm) of Sephacryl S-300, and eluted in 0.1 M Tris-HCl buffer (pH 8.0) containing 0.2 M NaCl. Flow rate was 20 ml/h, and fractions of 1 ml were collected. Absorbance at 280 nm and enzyme activities toward three MCA-substrates (Pro-Phe-Arg-MCA, Boc-Val-Leu-Lys-MCA, and Suc-Leu-Leu-Val-Tyr-MCA) of the fractions were determined. Little enzyme activity was detected with the latter two substrates, and only Pro-Phe-Arg-MCA-hydrolyzing activity is shown. Fractions 45–54 were pooled for further study. Elution positions of four molecular weight markers are indicated: a) porcine plasma α₂-macroglobulin (725 kDa); b) rabbit muscle aldolase (160 kDa); c) human plasma kallikrein (90 kDa); d) bovine serum albumin (65 kDa).

The α₂-macroglobulin fraction's effect on sctPA was examined. As shown in Fig. 2, sctPA was converted to its two-chain form when incubated with the fraction. The result illustrated in Fig. 3 is direct evidence that the protein possesses an enzyme activity toward sctPA. These observations suggest that sctPA is still to some extent accessible to a protease associated with α₂-macroglobulin. Since the fraction contained bound plasma kallikrein (Ohnishi et al., 1997), its part in sctPA activation was examined. Our previous data suggested that the enzyme activity detected with the substrate Pro-Phe-Arg-MCA was almost exclusively due to the action of bound plasma kallikrein (Ohnishi et al., 1997). Using the active site titrant p-nitrophenyl-p'-guanidinobenzoate HCl (Chase and Shaw, 1967), the active bound enzyme in the fraction was quantified. Fig. 4 shows the comparison of sctPA activation with plasma kallikrein and the α₂-macroglobulin fraction containing the same number of the active sites. sctPA was activated with the fraction faster than was free plasma kallikrein. It shoud be noted that activation by the fraction apparently accelerated with incubation time. It is not known at present why the activation proceeds non-linearly. We next conducted sctPA activation experiments using the α₂-macroglobulin fraction that had been previously treated with either anti-human α₂-macroglobulin IgG or anti-human plasma kallikrein IgG. As shown in Fig. 5A, the greater part of the fraction's Pro-Phe-Arg-MCA-hydrolyzing activity was immunoprecipitated with both IgGs. The result is consistent with our previous data (Ohnishi et al., 1997). Under these conditions, the sctPA-activating activity was largely immunoprecipitated by the α₂-macroglobulin IgG. However, the plasma kallikrein IgG-treated α₂-macroglobulin fraction was still capable of activating sctPA (Fig. 5B). These results clearly indicate that plasma kallikrein associated with α₂-macroglobulin is not involved in the activation of sctPA.

Fig. 2

Proteolytic conversion of sctPA to two-chain tPA by the α₂-macroglobulin fraction. Human sctPA (0.1 μg) was incubated at 37°C with the fraction (0.3 μg) in a total volume of 50 μl in 0.1 M Tris-HCl buffer (pH 8.0) containing bovine serum albumin (0.5 mg/ml). Aliquots (10 μl) of the mixture were taken for reducing SDS-PAGE/Western blot analysis at 0 (lane 1) and 4 hr (lane 2) of incubation. The polyvinylidene difluoride membrane with blotted proteins was first incubated with goat anti-human tPA IgG in a 1:5,000 dilution, and then with donkey anti-goat IgG in a 1:2,000 dilution. The 34 kDa chain of tPA is strongly detected by the antibody, but the 32 kDa chain is slightly visible in this detection system. Positions of molecular weight markers are shown on the left.

Fig. 3

Association of sctPA-activating activity with the α₂-macroglobulin fraction. The α₂-macroglobulin fraction (20 μg) was separately applied to two well positions of a 2–15% gradient polyacrylamide gel without SDS. After electrophoresis at 4°C, one lane was stained with Coomassie Brilliant Blue R-250, while the other one was sliced into pieces 3 mm wide for overnight extraction in 600 μl of 0.1 M Tris-HCl buffer (pH 8.0). Aliquots (487 ?l) of each extract were incubated with sctPA (0.3 μg) in a total volume of 500 μl at 37°C for 3 hr, then the mixtures were assayed for activated tPA activity using Boc-Gln-Gly-Arg-MCA in the presence of aprotinin. Activities relative to the highest value are shown.

Fig. 4

sctPA activation by the α₂-macroglobulin fraction and plasma kallikrein. Human sctPA (2 μg) was incubated at 37°C with the α₂-macroglobulin fraction (5.8 μg, enzyme activity of 0.18 nmol/min toward Pro-Phe-Arg-MCA) in a total volume of 1 ml in 0.1 M Tris-HCl buffer (pH 8.0) containing bovine serum ablumin (0.5 mg/ml). For comparison, a sctPA activation experiment was conducted under conditions using free human plasma kallikrein with the same number of the active sites. At the indicated times, aliquots of 200 μl of the incubation mixture were taken for determination of the activated tPA activity. Activities were assayed with Boc-Gln-Gly-Arg-MCA at 37°C in the presence of aprotinin, and are expressed as the amount of 7-amino-4-methylcoumarin released per 30 min. ○, sctPA activation by the α₂-macroglobulin fraction; •, sctPA activation by plasma kallikrein.

Fig. 5

Immunoprecipitation experiments of the bound sctPA-activating activity. The α₂-macroglobulin fraction (78 μg) was mixed with sheep anti-human α₂-macroglobulin IgG (120 μg) or rabbit anti-human plasma kallikrein IgG (100 μg) in 0.1 M Tris-HCl buffer (pH 8.0) containing 0.2 M NaCl in a volume of 100 μl and incubated at 4°C for 16 hr. One hundred microlitres of Staphylosorbe (10% v/v, binding capacity of 5 to 6 mg of human IgG/ml suspension) were added and the mixtures were incubated at 4°C for 3 hr with intermittent mixing every 30 min. After centrifuging at 10,000 × g for 10 min, the supernatants were assayed for enzyme activities. (A) The supernatants were directly assayed with Pro-Phe-Arg-MCA in order to detect plasma kallikrein (PK) activity. (B) The supernatants were incubated with human sctPA (0.2 μg) at 37°C for 3 hr, and then the activated tPA was assayed at 37°C with Boc-Gln-Gly-Arg-MCA in the presence of aprotinin. Enzyme acitivities are expressed as percentages of the controls that were similarly processed without antibodies.

The bound protease was stable. No significant loss of sctPA-activating activity of the fraction was observed in several months of incubation at pH 8.0 and 4°C. Fig. 6 shows the effect of pH on the sctPA-activating activity of the α₂-macroglobulin fraction. A bell-shaped curve revealed a large plateau at pH values in the ranges of 6–9. The effects of various protease inhibitors were examined and the results are shown in Fig. 7. The conversion of sctPA to tPA by the bound enzyme was strongly inhibited by aprotinin and DFP. Other inhibitors were without effect. These results indicate that a serine protease is involved in the conversion. We believe that auto-catalytic activation of sctPA is probably not a mechanism underlying the conversion of sctPA to tPA. If this would be the case, the conversion should have proceeded irrespective of whether or not aprotinin, which has no effect on tPA, was included in the reaction.

Fig. 6

Effect of pH on sctPA-activating activity. Human sctPA (0.4 μg) and the α₂-macroglobulin fraction were incubated at 37°C in a volume of 100 μl in 0.1 M sodium acetate buffer (pH 4, 5 or 6) or 0.1 M Tris-HCl buffer (pH 7, 8 or 9) or 0.1 M glycine-NaOH buffer (pH 10 or 11). After incubation for 1 hr, activated tPA activity was determined at 37°C in 0.1 M Tris-HCl buffer (pH 8.0) using Boc-Gln-Gly-Arg-MCA in the presence of aprotinin. Activities are expressed as the amount of 7-amino-4-methylcoumarin released per 30 min.

Fig. 7

Effects of various inhibitors on sctPA-activating activity. Human sctPA (0.2 μg) was incubated at 37°C with the α₂-macroglobulin fraction in 16 μl in 0.1 M Tris-HCl buffer (pH 8.0) in the presence of inhibitors. After 3 hr of incubation, the samples were mixed with 4 μl of 5 × SDS sample solvent, boiled, and electrophoresed in SDS under reducing conditions. Blotted sctPA and tPA were visualized as described in MATERIALS AND METHODS. Lane 1, sctPA only; lane 2, without inhibitors; lane 3, aprotinin (0.4 mg/ml); lane 4, antipain (40 μM); lane 5, benzamidine (2 mM); lane 6, PMSF (2 mM); lane 7, TLCK (0.4 mM); lane 8, TPCK (0.4 mM); lane 9, PCMB (0.4 mM); lane 10, E-64 (0.2 mM); and lane 11, DFP (4 mM). Note that aprotinin and DFP strongly inhibit tPA production.

We initiated the present study following the unexpected observation that the α₂-macroglobulin fraction obtained from the follicular fluid of human ovaries showed substantial proteolytic activity on the protein substrate sctPA. Because our attempt to dissociate and isolate the responsible enzyme from the protease/α₂-macroglobulin complex was unsuccessful, information regarding the protease is at present very limited. We previously reported (Ohnishi et al., 1997) that free plasma kallikrein and the α₂-macroglobulin fraction isolated from the human ovarian follicular fluid showed essentially the same substrate specificity when tested using various MCA substrates. These observations, together with the present finding that the bound plasma kallikrein of the α₂-macroglobulin fraction is not responsible for the activation of sctPA, suggest that the sctPA-activating enzyme perhaps exhibits diminished enzyme activity toward MCA substrates. However, further studies are necessary to know more about the enzyme itself. Furthermore, the mechanism of tPA production by the enzyme/α₂-macroglobulin complex as well as the biological meaning of its occurrence in human ovarian follicular fluid ramain to be elucidated. Nevertheless, this is the first demonstration that a protease complexed with α₂-macroglobulin exhibits detectable proteolytic activity toward sctPA.