(Received for publication, June 19, 1995; and in revised form, February 6, 1996)
From the
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 -antitrypsin, antithrombin, and C1
inhibitor readily polymerizes intracellularly and that polymerization
may lead to a reduced secretion efficiency.
Plasminogen activator inhibitor type 2 (PAI-2) ()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 -antitrypsin it has been
demonstrated that upon exposure to mild denaturing conditions at 4
°C the reactive center loop can be locked into the
-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
-sheet of another
molecule. The naturally occurring Z variant form of
-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
-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
-antitrypsin that predisposes to emphysema (Laurell
and Eriksson, 1963) (for review, see Carrell(1986)). The accumulation
of the Z form of
-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
-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.
To determine the expression level and the
secretion efficiency of PAI-2, about 80% confluent cultures (8
10
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-terminal sequence
analysis of purified secreted PAI-2 revealed that the external signal
peptide was correctly cleaved off (Mikus et al., 1993).
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.
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).
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.
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).
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.
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 -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 -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
-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
-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
-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.