©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Intracellular Polymerization of the Serpin Plasminogen Activator Inhibitor Type 2 (*)

(Received for publication, June 19, 1995; and in revised form, February 6, 1996)

Peter Mikus (1) (2) Tor Ny (1)(§)

From the  (1)Department of Medical Biochemistry and Biophysics, Umeå University, S-901 87 Umeå, Sweden and the (2)Institute of Molecular and Physiological Genetics, SAS Bratislava, Slovak Republic

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Plasminogen activator inhibitor type 2 (PAI-2) is synthesized in two molecular forms: an intracellular, nonglycosylated form and an extracellular, glycosylated form. The bitopological distribution of PAI-2 is caused by an inefficient internal secretion signal. In addition, the secretion efficiency of PAI-2 seems to differ, depending on the cell type, differentiation state, and culture conditions. In recombinant cell clones designed for the synthesis of the secreted form of PAI-2, the fraction of secreted PAI-2 decreased with increasing expression levels. Subcellular fractionation of cell clones with higher expression levels revealed that PAI-2 accumulating in the cell was mainly associated with the organelles of the secretory pathway. Electrophoresis under nondenaturating conditions revealed that the PAI-2 retained at higher expression levels was mainly polymerized.

Polymers of PAI-2 were also detected in cytosolic extracts prepared from human placenta and phorbol ester-stimulated U 937 cells, indicating that intracellular polymerization of PAI-2 may occur in the cytosols of cells that normally express PAI-2 under physiological conditions.

When purified PAI-2 or cellular extracts were incubated at 37 °C for 24 h most of the PAI-2 protein was found to polymerize. Polymer formation was prevented by the addition of synthetic peptides with sequences corresponding to residues P2 to P14 in the reactive center loop of PAI-2 and antithrombin. These synthetic peptides also caused dissociation of prepolymerized purified PAI-2 and PAI-2 polymers in cellular extracts. Incubation with unrelated peptides of the same size had no effect on polymer formation or dissociation of preformed polymers, indicating that polymerization of PAI-2 occurs by the loop-sheet mechanism.

Taken together, our data suggest that the wild-type form of PAI-2, like some natural pathological genetic variants of alpha(1)-antitrypsin, antithrombin, and C1 inhibitor readily polymerizes intracellularly and that polymerization may lead to a reduced secretion efficiency.


INTRODUCTION

Plasminogen activator inhibitor type 2 (PAI-2) (^1)is a member of the serine protease inhibitor family of proteins (serpins) that inhibits urokinase-type plasminogen activator and the two-chain form of tissue-type plasminogen activator (Lecander et al., 1984; Carell and Travis, 1985; Kruithof et al., 1986). PAI-2 is produced by placenta, macrophages, and cell lines of different origin (Kawano et al., 1968; Lecander et al., 1984; Vassalli et al., 1984; Åstedt et al., 1985; Kruithof et el., 1986), but its physiological function is not well understood. Although PAI-2 is synthesized from a single mRNA (Belin et al., 1989), it is found in two distinct forms that vary in molecular mass and cellular localization: an intracellular, nonglycosylated form with a molecular mass of about 47 kDa and an extracellular, glycosylated form with a more heterogeneous molecular mass of about 60 kDa (Genton et al., 1987; Wohlwend et al., 1987; Belin et al., 1989). PAI-2 contains an internal, noncleaved secretion signal that seems to be inefficient by design, thereby allowing the bitopological distribution of the protein (von Heijne et al., 1991). The relative distribution of intracellular and secreted forms of PAI-2 also seems to depend on the cell type, culture conditions, and differentiation state of the cell, from almost all of PAI-2 being secreted (Ye et al., 1988) to the majority remaining intracellular (Genton et al., 1987; Wohlwend et al., 1987; Ny et al., 1989).

An unusual feature of the inhibitors that belong to the serpin family is the mobility of their reactive center loops. The reactive loops of serpins can adopt varying conformations, and a mobile reactive center loop seems to be required for the inhibitory activity (reviewed by Carrell and Evans(1992); Gettins et al.(1993)). For the inhibitory serpin alpha(1)-antitrypsin it has been demonstrated that upon exposure to mild denaturing conditions at 4 °C the reactive center loop can be locked into the beta-pleated A sheet, forming a more thermostable, inactive, latent-like conformation similar to the latent form of plasminogen activator inhibitor type 1 (PAI-1) (Carrell et al., 1991). However, at elevated temperatures and particularly at higher concentrations the same denaturing conditions favor formation of noncovalent polymers (Schulze et al., 1990; Evans 1991; Mast et al., 1992; Lomas et al., 1992). These polymers are formed by a mechanism denoted loop-sheet polymerization, whereby the reactive center loop of one molecule is inserted into the gap in the beta-sheet of another molecule. The naturally occurring Z variant form of alpha(1)-antitrypsin for which 4% of northern Europeans are heterozygotes contains a mutation at the base of the reactive center loop of the molecule (Carrell, 1986). This polymorphic variant is even more prone to polymerization than the wild type and forms polymers even under physiological conditions (Lomas et al., 1992). In patients that are homozygous for the Z allele only about 15% of the mutated alpha(1)-antitrypsin is secreted into plasma, while the remaining 85% accumulates in the endoplasmic reticulum of hepatocytes, which causes two major clinical sequels: the formation of intracellular inclusions associated with hepatocellular damage (Sharp et al., 1969) and the deficiency of circulating alpha(1)-antitrypsin that predisposes to emphysema (Laurell and Eriksson, 1963) (for review, see Carrell(1986)). The accumulation of the Z form of alpha(1)-antitrypsin in the secretory pathway is caused by spontaneous loop-sheet polymerization of mutated antitrypsin (Lomas et al., 1992). Siiyama antitrypsin is another abnormal variant of alpha(1)-antitrypsin with impaired secretion (Lomas et al., 1993b). Also for this natural mutant accumulation in the endoplasmic reticulum of hepatocytes due to loop-sheet polymerization has been reported (Lomas et al., 1993b).

During purification and biochemical characterization of recombinant PAI-2 we noted that PAI-2 readily polymerized even in the absence of denaturing agents (Mikus et al., 1993). In this report we have studied the secretion efficiency of PAI-2 from cells expressing different levels of the secreted form of PAI-2. We find that in cells with high expression levels PAI-2 spontaneously polymerizes in the secretory pathway by the loop-sheet polymerization mechanism, which leads to a reduced secretion efficiency. Polymerization of the intracellular form of PAI-2 was also observed in the cytosol of cells and tissues that are normally producing PAI-2, e.g. in human placenta and phorbol ester-stimulated U 937 cells. This indicates that intracellular polymerization of PAI-2 may occur also in cells that express PAI-2 at physiological levels.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes, T4 DNA ligase, and polynucleotide kinase were from Boehringer Mannheim (Germany). Dulbecco's modified Eagle's medium and BHK21 medium were from Life Technologies, Inc. (Paisley, United Kingdom). Phorbol 12-myristate 13-acetate (PMA) was from Sigma. Synthetic peptides P2-P14, PH, and OZ were from the Biomedical Center, University of Uppsala (Sweden). Chromogenic substrate S-2444 was from Kabi Vitrum (Stockholm, Sweden). TintElize and PAI-2 monoclonal antibody MAI 21 were from Biopool AB (Umeå, Sweden). Galactosyl transferase monoclonal antibody GalTf was kindly provided by Prof. Eric Berger (Institute of Physiology, Zürich, Switzerland). The ECL Western blotting system was purchased from Amersham (Amersham Place, Buckinghamshire, United Kingdom).

Construction of Expression Plasmids

Plasmids pPM 4 and pSFV-SP-PAI-2 containing the coding sequence of PAI-2 in-frame fused with a cleavable signal peptide derived from PAI-1 were subcloned into the SV40-driven expression vector pSVAstop 1 (Zettlmeissl et al., 1987) and the Semliki forest virus (SFV) expression vector pSFV 1 (Liljeström and Garoff, 1991), respectively, as described previously (von Heijne et al., 1991; Mikus et al., 1993).

Cell Culture

Chinese hamster ovary (CHO) cells and human fibrosarcoma cells HT 1080 were grown in the Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Baby hamster kidney (BHK) cells were cultured in the BHK 21 medium supplemented with 5% fetal calf serum, and human histiocytic lymphoma cells U 937 were grown in the RPMI 1640 medium supplemented with 10% fetal calf serum. To induce the expression of PAI-2 in U 937 cells, cells were incubated for 24 h in the presence of 30 ng/ml PMA.

Expression of PAI-2 in Transfected and Infected Cells

Cells were transfected with plasmid pPM 4 mixed with plasmid pSVneo (Southern and Berg, 1982) to a ratio of 19:1 using the calcium phosphate precipitation method (Chen and Okayama, 1987), and stable clones were isolated as described previously (Ny et al., 1989, Mikus et al., 1993). Infectious virus particles were prepared as described (Liljeström and Garoff, 1991; Liljeström et al., 1991). Preparation, storage, and infections with recombinant PAI-2-expressing virus were performed as described previously (Mikus et al., 1993).

To determine the expression level and the secretion efficiency of PAI-2, about 80% confluent cultures (8 times 10^5 cells) of PAI-2 expressing stable cell clones or cells infected with SFV expression construct were grown for 24 h at 37 °C. After this period conditioned medium was collected, and corresponding cellular extracts (1 ml) were prepared (Ny et al., 1989) and assayed for PAI-2 antigen by specific enzyme-linked immunosorbent assay (Biopool) and ECL Western blotting (Amersham) as described by the suppliers.

NH(2)-terminal sequence analysis of purified secreted PAI-2 revealed that the external signal peptide was correctly cleaved off (Mikus et al., 1993).

Preparation of Human Placental Extracts

Fresh term placenta was immediately put on ice, cut into small pieces, and homogenized in a Downs homogenizer in ice-cold phosphate-buffered saline buffer (150 mM NaCl, 10 mM sodium phosphate, pH 7.3). Cellular debris was removed by centrifugation at 600 times g for 10 min at 4 °C, after which the supernatant was directly analyzed for the presence of PAI-2 polymers or stored at -80 °C for further use. The protein concentration of the placental extracts was about 25 µg/ml, and the extracts contained approximately 600 ng of PAI-2 antigen/ml.

To test whether the formation of PAI-2 polymers could occur during the preparation of extracts, we incubated purified PAI-2 (500 ng/ml) at 4 °C for various periods of time. Under these conditions no polymer formation was observed after 24 h.

Binary Complex Formation

The binary complexes between PAI-2 and synthetic 13-mer peptides corresponding to residues P2 to P14 of the reactive center loop of PAI-2 (RCL-PAI-2, TEAAAGTGGVMTG) and the reactive center loop of a structurally related serpin antithrombin (RCL-AT, SEAAASTAVVIAG) were formed as described (Schulze et al., 1990; Carrell et al., 1991; Evans, 1991; Lomas et al., 1992). RCL-PAI-2 and RCL-AT are predicted to insert into the groove in the beta-sheet A, thereby preventing loop-sheet polymerization. As controls for the reactive center loop peptides, two negative control peptides predicted not to insert were designed. Synthetic peptide TG (GFMQQIQKGSYPD) is derived from PAI-2, where it constitutes the transglutaminase acceptor site (Jensen et al., 1994). Synthetic peptide PH (APATKGPGRVIYA) is unrelated to serpins and only chosen based on its identical size.

Purified recombinant PAI-2 (Mikus et al., 1993) (0.1 mg/ml) was incubated in 10 mM Tris-HCl buffer, pH 7.5, with a 100-fold molar excess of the synthetic peptide at 37 °C for 24 h. PAI-2 inhibitory activity and antigen in the samples were determined by direct chromogenic activity assay and immunoblotting as described (Mikus et al., 1993). The relative amounts of monomeric and polymerized PAI-2 were assayed by electrophoresis under nondenaturating conditions followed by densitometric scanning of ECL Western blots.

To study the dissociation of polymers, purified recombinant PAI-2 (0.1 mg/ml) was prepolymerized in 10 mM Tris-HCl buffer, pH 7.5 by incubation at 37 °C for 24 h. After this period a 100-fold molar excess of peptide was added, and the samples were incubated at 37 °C for different periods of time. Polymerization of PAI-2 was visualized by electrophoresis under nondenaturating conditions followed by immunoblotting and confirmed by loss of inhibitory activity determined as described (Mikus et al., 1993).

Subcellular Fractionation

A fraction enriched in Golgi vesicles was isolated by flotation in a sucrose gradient as described (Rios et al., 1992). Briefly, a crude cellular homogenate from approximately 10^7 PAI-2 expressing CHO cells transfected with plasmid pPM4 was centrifuged (9,000 times g, 10 min at 4 °C) to produce a nuclear pellet and postnuclear supernatant. The supernatant was adjusted to 37% (w/v) sucrose and overlaid with 2 volumes of 35% sucrose and 1 volume of 27% sucrose in 10 mM Tris-HCl, pH 7.4. Gradients were centrifuged for 2.5 h at 85,000 times g, and the bands at the 35-27% interface were collected and stored at -80 °C. The presence of Golgi membranes in the fractions was examined by ECL Western blotting using antibodies directed against galactosyl transferase (Rios et al., 1992, 1994). Galactosyl transferase was only detected in a fraction containing organelles of the secretory pathway and not in the homogenate and nuclear pellet.


RESULTS

PAI-2 Forms Polymers in the Secretory Pathway at Higher Expression Levels

To study the secretion efficiency of PAI-2 at different expression levels we used the PAI-2 expression plasmid pPM 4 containing an in-frame fused signal peptide derived from PAI-1. As shown previously, the secreted form of PAI-2 from this expression construct is correctly processed, and the external signal peptide is cleaved off (Mikus et al., 1993). CHO and BHK cells were transfected with plasmid pPM 4, and individual stable transfected clones with different expression levels were isolated. As shown in Table 1the level of PAI-2 expression varied almost 10-fold among these clones. By measuring the PAI-2 content in cellular extracts and conditioned medium we found that the secretion efficiency of PAI-2 varied with the level of expression, such that the secretion efficiency was lower when the expression level was higher. Only about 50% of PAI-2 was secreted from CHO cells with the highest PAI-2 expression (clone 2.4), while 98% of PAI-2 was secreted from cells with low levels of expression (clone 4.5). Similar results were obtained also for stable transfected BHK cells, where the secretion of PAI-2 varied from 30 to 95%, depending on the level of expression (Table 1).



The amounts of the different molecular forms of PAI-2 in cellular extracts and medium of CHO clones expressing different levels of PAI-2 were also analyzed by SDS-PAGE followed by immunoblotting (Fig. 1). As shown previously, the molecular mass of intracellular and extracellular PAI-2 is about 47 and 60 kDa, respectively. In the clone 2.4, which has the highest expression level, about 50% of PAI-2 was found inside the cell, whereas almost all of PAI-2 was secreted into the medium in the clone 4.5, which has the lowest expression level.


Figure 1: Distribution of intracellular and extracellular PAI-2 at different expression levels. CHO cells transfected with pPM4 expressing high (2.4), medium (5.1), and low (4.5) levels of PAI-2 were cultured, and cellular extracts (E) and conditioned medium (M) were prepared and fractionated by SDS-PAGE. PAI-2 antigen was assayed by ECL Western blotting as described under ``Experimental Procedures.'' The minor 42-kDa molecular mass form of PAI-2 found in cellular extracts of clones expressing high and medium levels of PAI-2 represents a proteolytically cleaved form of PAI-2 (Mikus et al., 1993). The positions of molecular mass standards are indicated in kDa.



In order to study the secretion of PAI-2 at higher expression levels, we used an efficient Semliki forest virus-based expression system (Liljeström and Garoff, 1991). After infection with PAI-2-expressing virus, the level of expression was about 14-fold higher as compared with the cell clone transfected with plasmid pPM 4 with the highest PAI-2 expression level. As shown in Table 2only about 35-40% of PAI-2 protein was secreted in the three different cell lines tested.



To exclude the possibility that the reduced secretion efficiency of PAI-2 was due to the expression system or a consequence of the high expression per se, we performed control experiments where the secretion efficiency of PAI-1 was determined in cells infected with PAI-1-expressing virus. Although the level of PAI-1 expression was approximately the same as in the cells infected with PAI-2-expressing virus, PAI-1 was secreted very efficiently in all cell lines tested, revealing 97-98% secretion (Table 2). These results suggest that the correlation between low secretion and high expression is a feature specific to the PAI-2 protein and is not caused by an inability of cells to secrete higher amounts of protein.

In order to determine more precisely the localization of PAI-2 that resides inside the cells, we performed subcellular fractionation of PAI-2 expressing CHO cells transfected with plasmid pPM4. Cells were homogenized, nuclei were pelleted, and the postnuclear supernatant was loaded on sucrose gradient. The fractions were examined for their content of Golgi membranes and PAI-2 by SDS-PAGE followed by immunoblot analysis. In agreement with previous reports (Rios et al., 1992, 1994), the Golgi-specific marker galactosyl transferase was only detected in a fraction containing organelles of the secretory pathway (Fig. 2, lane 4). In cells with high PAI-2 expression levels about 80% of the intracellular PAI-2 was retained in this fraction. The Golgi-enriched fraction revealed heterogeneous molecular forms of PAI-2 with molecular masses corresponding to the intracellular nonglycosylated form, up to the fully glycosylated secreted molecules (Fig. 2, lane 2). These molecular forms most likely represent intermediates of PAI-2 glycosylation.


Figure 2: Molecular forms of PAI-2 in cellular extract, Golgi-enriched fraction, and conditioned medium from CHO cells transfected with pPM4 expressing high levels of PAI-2. Cellular extract (lane 1), Golgi-enriched fraction (lane 2), and conditioned medium (lane 3) were fractionated by SDS-PAGE, and PAI-2 antigen was assayed by ECL Western blotting. The Golgi-enriched fraction was also assayed for galactosyl transferase antigen (4). The positions of molecular mass standards are indicated in kDa.



To determine whether the accumulation of PAI-2 in the secretory pathway at higher expression levels was caused by polymerization, a Golgi-enriched fraction from CHO cells with high and low expression levels of PAI-2 was prepared and analyzed by electrophoresis under nondenaturating conditions. As shown in Fig. 3, PAI-2 in the Golgi-enriched fraction from cells with a low level of PAI-2 expression was mostly in the monomeric form, whereas it was mostly polymerized in cells with a high expression level. Polymerization of PAI-2 in the secretory pathway is therefore a likely reason for the low secretion efficiency of cells expressing higher amounts of PAI-2.


Figure 3: Polymerized forms of PAI-2 in the Golgi-enriched fraction from cells expressing high levels of PAI-2. Golgi-enriched fraction was prepared from CHO cells with a low level of PAI-2 expression (clone 4.5, lane A) and from cells with a high level of PAI-2 expression (clone 2.4, lane B) and analyzed by electrophoresis under nondenaturating conditions followed by ECL Western blotting. Lane A contains 1 µg of total protein; lane B contains 100 ng of total protein. The mobilities of the monomeric and polymeric forms of PAI-2 are indicated by arrows.



PAI-2 Polymerizes Spontaneously by the Loop-sheet Polymerization Mechanism

A decreased secretion efficiency due to intracellular polymerization has previously been described for the Z mutation of alpha(1)-antitrypsin (Lomas et al., 1992). In that case it was shown that the polymers were formed by the loop-sheet polymerization mechanism. Because loop-sheet polymerization can be prevented by the formation of binary complexes where a synthetic peptide homologous to the reactive center loop is inserted into the A sheet of the inhibitor (Schulze at al., 1990; Lomas et al., 1992), we incubated purified PAI-2 with an excess of a 13-mer synthetic peptide RCL-PAI-2 with the sequence corresponding to residues P2 to P14 of the reactive center loop of PAI-2.

Purified monomeric PAI-2 (90% active) (Fig. 4, lane 1) was incubated in 10 mM Tris-HCl, pH 7.5, at 37 °C for 24 h. After this treatment most PAI-2 spontaneously polymerized (lane 2) with a corresponding loss of inhibitory activity, suggesting that insertion of the reactive center loop of one PAI-2 molecule into the A sheet of another molecule has taken place (Schulze et al., 1990; Evans, 1991). This polymerization was prevented by the addition of a 100-fold molar excess of RCL-PAI-2 (lane 3). Polymerization was also prevented by incubation with excess of the synthetic peptide RCL-AT corresponding to residues P2 to P14 of the reactive center loop of antithrombin (lane 4). Consistent with binary complex formation only 3 and 5% of the inhibitory activity remained after incubation with RCL-PAI-2 and RCL-AT, respectively. As shown in lanes 5 and 6, incubation in the presence of 100-fold molar excess of two negative control peptides predicted not to insert into the A sheet did not prevent formation of PAI-2 polymers, indicating the sequence specificity required for binary complex formation.


Figure 4: Effect of reactive center loop peptide on the spontaneous polymerization of PAI-2. Purified recombinant PAI-2 was incubated in the absence or presence of a 100-fold molar excess of synthetic peptides. After 24 h at 37 °C the samples were analyzed by nondenaturating gel electrophoresis. Lane 1, starting material, 90% active purified monomeric PAI-2; lane 2, PAI-2 incubated in the absence of peptides (remaining activity 5%); lane 3, PAI-2 incubated in the presence of the synthetic peptide RCL-PAI-2 corresponding to the reactive center loop of PAI-2 (remaining activity 3%); lane 4, PAI-2 incubated in the presence of synthetic peptide RCL-AT corresponding to the reactive center loop of antithrombin (remaining activity 5%); lane 5, PAI-2 incubated with the negative control peptide TG corresponding to the transglutaminase acceptor site of PAI-2 (remaining activity 4%); lane 6, PAI-2 incubated with the negative control peptide PH (remaining activity 5%).



It was also possible to dissociate polymers of purified PAI-2 formed under physiological conditions by incubation in the presence of synthetic peptides corresponding to residues P2 to P14 of PAI-2 and antithrombin. As shown in Fig. 5dissociation of prepolymerized recombinant PAI-2 (lane 2) was obtained following incubation in the presence of a 100-fold molar excess of the P2-P14 peptides RCL-PAI-2 (lane 3) and RCL-AT (lane 4). Incubation of prepolymerized PAI-2 alone or with a 100-fold molar excess of negative control peptides did not result in any dissociation of PAI-2 polymers (Fig. 5, lanes 5-7).


Figure 5: Reversal of polymerization by incubation of prepolymerized PAI-2 with reactive center loop peptide. Purified PAI-2 was prepolymerized and incubated with synthetic peptides as described under ``Experimental Procedures.'' Lane 1, purified recombinant monomeric PAI-2 (90% active); lane 2, prepolymerized PAI-2 (remaining activity 5%); lanes 3 and 4, prepolymerized PAI-2 incubated at 37 °C in the presence of peptides RCL-PAI-2 and RCL-AT for 30 h (3 and 5% active, respectively); lanes 5 and 6, prepolymerized PAI-2 incubated with negative control peptides TG and PH for 30 h (5% active); lane 7, prepolymerized PAI-2 incubated at 37 °C for 30 h (5% active).



Naturally Expressed PAI-2 Can Polymerize in the Cytosol under Normal Physiological Conditions

To investigate if polymerization of PAI-2 occurs in cells that are naturally producing PAI-2, we analyzed cellular extracts from the human histiocytic lymphoma cell line U 937 and from placenta for the presence of PAI-2 polymers. As shown in Fig. 6, lane 2, extracts of untreated U 937 cells contain mostly the monomeric form of PAI-2. However, following treatment with PMA, which causes a 10-fold induction of PAI-2 expression, a larger fraction of PAI-2 was found to be polymerized (Fig. 6, lane 3). Also, extracts prepared from human placenta (Fig. 6, lane 4), as well as from CHO cells that are overexpressing the wild-type form of PAI-2 (Fig. 6, lane 5) contained polymers of PAI-2. This indicates that polymerization may occur both in cells that express PAI-2 under physiological conditions and in recombinant cells that overexpress the wild-type form of PAI-2.


Figure 6: Polymerized forms of PAI-2 in cells and tissue that naturally produce PAI-2. Extracts were prepared as described under ``Experimental Procedures'' and analyzed by nondenaturating gel electrophoresis. Lane 1, purified recombinant PAI-2 (10 ng); lane 2, PAI-2 from untreated U 937 cells, expression level 0.8 pg/cell/24 h (1 µg of total protein was loaded); lane 3, PAI-2 from PMA-treated U 937 cells, expression level 9 pg/cell/24 h (100 ng of total protein was loaded); lane 4, PAI-2 from human placenta (200 ng of total protein); lane 5, PAI-2 from CHO cells infected with wild-type PAI-2 expressing SFV recombinant virus (25 ng of total protein).



When cellular extracts prepared from placenta (Fig. 7A, lane 2) or PMA-treated U 937 cells (Fig. 7B, lane 2) were incubated at 37 °C alone for an extended period of time, the fraction of PAI-2 polymers increased slightly (Fig. 7, A and B, lane 7). The fraction of polymerized PAI-2 also increased following incubation in the presence of negative control peptides TG and PH predicted not to insert into the A sheet of PAI-2 (lanes 5 and 6). However, incubation under the same conditions in the presence of the specific peptides RCL-PAI-2 and RCL-AT fully prevented further polymerization and caused a partial dissociation of PAI-2 polymers (Fig. 7, A and B, lanes 3 and 4). Taken together, these results suggest that PAI-2 polymers found in cells that are normally producing PAI-2 have been formed by the loop-sheet polymerization mechanism.


Figure 7: Partial dissociation of PAI-2 polymers in placental and cellular extracts following incubation with reactive center loop peptides. Extracts from placenta (A) and PMA-treated U 937 cells (B) incubated in the absence or presence of synthetic peptides for 30 h at 37 °C were analyzed by nondenaturating gel electrophoresis. Lane 1, purified recombinant PAI-2 migrating mostly as a monomer; lane 2, freshly prepared extracts before incubation; lanes 3 and 4, extracts incubated in the presence of peptides RCL-PAI-2 and RCL-AT, respectively; lanes 5 and 6, extracts incubated in the presence of negative control peptides TG and PH, respectively; lane 7, extracts incubated alone for 30 h at 37 °C.



To determine the intracellular localization of polymerized PAI-2 in cells that normally express PAI-2, subcellular fractionation of placental cells was performed. The intracellular PAI-2 was found to be nonglycosylated and retained in the cytosolic fraction. No PAI-2 antigen was detectable in the Golgi-enriched fraction (data not shown), suggesting that polymerization in cells that naturally produce PAI-2 takes place in the cytoplasm, before the protein enters the secretory pathway.


DISCUSSION

PAI-2 is a member of the serpin family of proteins that inhibits urokinase-type plasminogen activator and the two-chain form of tissue-type plasminogen activator (Lecander et al., 1984; Carell and Travis, 1985; Kruithof et al., 1986). The inhibitory members of this family are able to adopt a variety of conformations as a result of a mobile reactive center loop. In active serpins the reactive center loop constitutes an exposed loop acting as a bait for the target protease (Carrell and Evans, 1992; Schreuder et al., 1994). In cleaved and latent inhibitors this reactive center loop is fully inserted into the A sheet of the molecule (Loebermann et al., 1984; Mottonen et al., 1992). The conformation of the reactive center loop in the native complex with cognate proteases has been a matter of dispute (Potempa et al., 1994; Patston et al., 1991; Carrell et al., 1991; Schulze et al., 1992). However, recent findings suggest that cleavage of the reactive center loop by protease and subsequent loop insertion induce the conformational changes required to lock the inhibitor-protease complex (Fa et al., 1995; Lawrence et al., 1995; Wilczynska et al., 1995). In addition, some serpins can form polymers by the so called loop-sheet polymerization mechanism, whereby the reactive center loop of one molecule inserts into the A sheet of another molecule (Schulze et al., 1990; Evans, 1991; Mast et al., 1992; Lomas et al., 1992). In this study we report that PAI-2 is unusually prone to form polymers by the loop-sheet polymerization mechanism and that intracellular polymers of PAI-2 form at physiological expression levels and in cells that naturally synthesize PAI-2.

By using an expression system where PAI-2 has been engineered to enter the secretory pathway, we studied the secretion efficiency of PAI-2 at different expression levels. As shown in Table 1and Table 2the secretion efficiency decreases when the level of PAI-2 expression increases. This is specific for the PAI-2 protein and not caused by a generally impaired secretion at high expression levels, since the secretion efficiency of PAI-1 is high and unaltered at expression levels where the secretion of PAI-2 is greatly impaired (Table 2).

At higher expression levels most of the extracellular form of PAI-2 that remains inside the cell is retained in the organelles of the secretory pathway. As shown by SDS-PAGE this PAI-2 consists of heterogeneous molecular forms that may represent intermediates of PAI-2 glycosylation (Fig. 2, lane 2). Some lower molecular forms of PAI-2 that are also detected in the secretory organelles probably constitute PAI-2 molecules recognized as abberant that are being degraded.

As shown by electrophoresis under nondenaturating conditions, the minor fraction of PAI-2 detected in organelles of the secretory pathway at low expression levels is mainly monomeric, whereas at high expression levels a much larger portion of the PAI-2 that is retained is polymerized (Fig. 3). These data suggest that the secretory form of PAI-2, like the Z form of alpha(1)-antitrypsin, can polymerize in the secretory pathway and that this may be the reason for the reduced secretion efficiency at higher expression levels.

Polymerization of PAI-2 also occurs in cells that are naturally producing PAI-2. Unstimulated U 937 cells produce low amounts of PAI-2 (Wohlwend et al., 1987). As shown in Fig. 6, lane 2, this PAI-2 is mainly monomeric. However, following PMA stimulation, which leads to a 10-fold induction of PAI-2 synthesis (Genton et al., 1987; Wohlwend et al., 1987), a large fraction of PAI-2 is polymerized (Fig. 6, lane 3).

Placenta is a rich source of PAI-2 synthesis in the organism. During pregnancy the concentration of PAI-2 in plasma increases almost linearly, reaching levels of 100-300 ng/ml at term, followed by a decrease to nondetectable levels within about a week postpartum (Lecander and Åstedt, 1986; Åstedt et al., 1987). As shown in Fig. 6, lane 4 extracts prepared from fresh term placenta contain a large fraction of PAI-2 polymers.

Because of the poor efficiency of the secretion signal of PAI-2 only about 15% of PAI-2 enters the secretory pathway and becomes secreted, while about 85% remains in the cytosol (Genton et al., 1987). In keeping with this, intracellular polymerization of PAI-2 in cells that normally express PAI-2 occurs in the cytosol. However, when PAI-2s secretion signal is improved, polymerization mainly occurs in the organelles of the secretory pathway. In both cases polymerization is observed at higher expression levels, indicating that this process is concentration-dependent and occurs at a point where a critical concentration is approached. As shown here both nonglycosylated and glycosylated PAI-2 can form polymers, and polymerization can take place in different cellular compartments. In addition, PAI-2 purified to homogeneity spontaneously forms polymers in vitro, indicating that the ability to polymerize is characteristic for PAI-2 and that no additional factors are needed.

Attempts were made to determine whether PAI-2 polymerizes by the so called loop-sheet polymerization mechanism, where the reactive center loop of one molecule interacts with the gap in the beta-sheet of another molecule (Schulze et al., 1990; Evans, 1991).

Incubation of PAI-2 at 37 °C for 24 h results in almost complete polymerization (Fig. 4, lane 2), whereas in the presence of synthetic peptides corresponding to residues P2 to P14 of the reactive center loop of PAI-2 or antithrombin this polymerization is prevented (Fig. 4, lanes 3 and 4). The sequence of the reactive center loop of antithrombin is very similar to that of PAI-2, and a peptide with this sequence can also form a binary complex with related serpin alpha(1)-antitrypsin (Schulze et al., 1990; Lomas et al., 1992, 1993a). In contrast, incubation in the presence of a negative control peptide corresponding to a unique interhelical loop of PAI-2 exposed to the solvent (Jensen et al., 1994) and with an unrelated peptide of the same size does not prevent polymer formation (Fig. 4, lanes 5 and 6). Dissociation of PAI-2 polymers and reappearance of the monomeric form also occurs during prolonged incubation of prepolymerized PAI-2 in the presence of the reactive center loop peptides (Fig. 5, lanes 3 and 4), but not in the absence of peptide or in the presence of negative control peptides predicted not to insert into the A sheet (Fig. 5, lanes 5-7). In a similar fashion incubation of cellular extracts in the presence of the reactive center loop peptides leads to partial dissociation of polymers (Fig. 7, A and B, lanes 3 and 4), whereas incubation alone or in the presence of the negative control peptides leads to further polymerization. Altogether these findings indicate that PAI-2 polymers formed in vitro and found inside cells are formed by loop-sheet polymerization.

Loop-sheet polymerization was first suggested as an explanation for the noncovalent polymerization of alpha(1)-antitrypsin at elevated temperatures (Schulze et al., 1990). Later it was demonstrated that the formation of loop-sheet polymers was a more general characteristic of serpins that could be induced upon exposure to mild denaturating conditions, especially at higher temperatures and concentrations (Evans, 1991). In the majority of inhibitory serpins formation of loop-sheet polymers has been observed only in vitro following induction by mild denaturation (Evans, 1991). The exception to this are some naturally occurring pathological variants of alpha(1)-antitrypsin (Lomas et al., 1992, 1993a, 1993b), C1 inhibitor (Aulak et al., 1993), and antithrombin (Bruce et al., 1994).

To our knowledge, PAI-2 is the only ``normal'' serpin so far described that spontaneously forms polymers in vitro as well as intracellularly under physiological conditions. It is interesting to note that the wild-type form of PAI-2 seems to be much more prone to form polymers than the pathologic Z form of antitrypsin, and all attempts to induce the locked latent like conformation resulted in polymerization (Mikus et al., 1993).

Loop-sheet polymerization may partly explain previous rather puzzling observations of variations in PAI-2 secretion among different cell lines. The reduced growth rate and instable expression in eucaryotic cell lines expressing higher levels of PAI-2 (Mikus et al., 1993) are likely to be due to intracellular polymerization.

The reasons why PAI-2 exists in both intra- and extracellular forms are not known, and no physiological role for any of these forms has so far been firmly established. PAI-2 may play a role to secure hemostasis during pregnancy (Åstedt et al., 1987), and PAI-2 stored in the cytosol of monocytes/macrophages may represent a reservoir of inhibitory activity that can be released by cell suffering (Belin et al., 1989). Since PAI-2 readily forms polymers it is possible that cells that store PAI-2 intracellularly have mechanisms that can dissociate the polymers to regain active inhibitor molecules.

In this study we have shown that PAI-2 forms intracellular polymers under physiological conditions by the loop-sheet polymerization mechanism. Some naturally occurring mutants of other serpins have also been shown to polymerize by the same mechanism, leading to pathological conditions (Lomas et al., 1992, 1993a, 1993b; Bruce et al., 1994). However, in contrast to these serpins it is the normal form of PAI-2 that polymerizes. To our knowledge there is so far no pathological condition that involves a PAI-2 deficiency or that involves polymerization of PAI-2. Further investigation will be necessary to determine whether the tendency to polymerize has significance for the physiological function of PAI-2 in hemostasis and inflammation or is just a fortuitous consequence of its structural organization.


FOOTNOTES

*
This work was supported by the Swedish Natural Science Research Council grant B-AA/BU 08473-308 and the Swedish Society for Medical Research. 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 all correspondence should be addressed: Dept. of Medical Biochemistry and Biophysics, Umeå University, S-901 87 Umeå, Sweden. Tel.: 46 90 16 65 65; Fax: 46 90 13 64 65; tor.ny{at}medchem.umu.se.

(^1)
The abbreviations used are: PAI-1, PAI-2, plasminogen activator inhibitor type 1 and type 2, respectively; BHK, baby hamster kidney; CHO, Chinese hamster ovary; PMA, phorbol 12-myristate 13-acetate; serpin, serine protease inhibitor, SFV, Semliki forest virus; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Peter Liljeström and Sergei Aleshkov for the SFV expression plasmids and Sven Carlsson for helpful discussions. The galactosyl transferase antibody was a generous gift from Prof. E. G. Berger (Geneva, Switzerland).


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