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Originally published In Press as doi:10.1074/jbc.M001384200 on July 25, 2000

J. Biol. Chem., Vol. 275, Issue 40, 31128-31133, October 6, 2000
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Expression of Recombinant Human Pregnancy-associated Plasma Protein-A and Identification of the Proform of Eosinophil Major Basic Protein as Its Physiological Inhibitor*

Michael T. OvergaardDagger , Jesper HaaningDagger , Henning B. BoldtDagger , Inger M. OlsenDagger , Lisbeth S. LaursenDagger , Michael Christiansen§, Gerald J. Gleich, Lars Sottrup-JensenDagger , Cheryl A. Conover||, and Claus OxvigDagger **

From the Dagger  Department of Molecular and Structural Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark, the § Department of Clinical Biochemistry, Statens Serum Institut, DK-2300 Copenhagen S, Denmark, and the  Department of Immunology and || Endocrine Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota 55905

Received for publication, February 18, 2000, and in revised form, June 6, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pregnancy-associated plasma protein-A (PAPP-A), originally known from human pregnancy serum, has recently been demonstrated to be a metzincin superfamily metalloproteinase involved in normal and pathological insulin-like growth factor (IGF) physiology. PAPP-A specifically cleaves IGF-binding protein (IGFBP)-4, one of six antagonists of IGF action, which results in release of IGF bound to IGFBP-4. IGFBP-4 is the only known PAPP-A substrate. Its cleavage by PAPP-A uniquely depends on the presence of IGF. We here report mammalian expression and purification of recombinant 1547-residue PAPP-A (rPAPP-A). The recombinant protein is secreted as a homodimer of about 400 kDa composed of two 200-kDa disulfide-bound subunits. Antigenically and functionally, rPAPP-A behaves like the native protein. In human pregnancy, PAPP-A is known to circulate as a 500-kDa disulfide-bound 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP. A comparison between rPAPP-A and pregnancy serum PAPP-A/proMBP complex surprisingly reveals a difference greater than 100-fold in proteolytic activity, showing that proMBP functions as a proteinase inhibitor in vivo. We find that polyclonal antibodies against PAPP-A abrogate all detectable IGFBP-4 proteolytic activity in pregnancy serum, pointing at PAPP-A as the dominating, if not the only, IGFBP-4 proteinase present in the circulation. We further show that pregnancy serum and plasma contain traces (<1%) of uncomplexed PAPP-A with a much higher specific activity than the PAPP-A/proMBP complex. The measurable activity of the PAPP-A/proMBP complex probably results from the presence of a minor subpopulation of partly inhibited PAPP-A that exists in a 2:1 complex with proMBP. Inhibition of PAPP-A by proMBP represents a novel inhibitory mechanism with the enzyme irreversibly bound to its inhibitor by disulfide bonds.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Human pregnancy-associated plasma protein-A (PAPP-A)1 was first isolated in 1974 from pregnancy serum along with other proteins believed to be of placental origin (1). The concentration in serum reaches about 50 mg/liter at the end of pregnancy (2, 3). PAPP-A was originally characterized as a high molecular weight homotetramer (1, 4, 5), but it has now been demonstrated that PAPP-A exists in pregnancy serum as a covalent, heterotetrameric 2:2 complex with the proform of eosinophil major basic protein (proMBP), PAPP-A/proMBP (6). The presence of proMBP in the circulation, as well as the existence of the PAPP-A/proMBP complex was revealed, in part, by the isolation of a PAPP-A and a proMBP peptide, linked together by a disulfide bond, from a digest of purified PAPP-A/proMBP (6).

The subunits of the PAPP-A/proMBP complex can be irreversibly separated by reduction of disulfide bonds and denaturation (6). In reducing SDS-PAGE, the PAPP-A subunit has an apparent molecular mass of 200 kDa (7), and its 1547-residue sequence is known from cloned cDNA (8). PAPP-A shows no global homology to any protein, but it contains sequence motifs, including an elongated zinc binding motif (HEXXHXXGXXH) at position 482-492 (8). This motif and a structurally important methionine residue, also thought to reside in PAPP-A at position 556, are strictly conserved within the metzincins, a superfamily of zinc peptidases: astacins, adamalysins (or reprolysins), serralysins, and matrixins (matrix metalloproteinases) (8-11). PAPP-A does not conform to other defining features of the individual established metzincin superfamily members, and interestingly, in PAPP-A the linear distance between the zinc binding motif and the conserved Met residue is 63 amino acids (8), whereas in other metzincins this distance is between 7 and 44 residues (11).

The proMBP subunit has a calculated peptide mass of 23 kDa (12, 13). In SDS-PAGE, however, proMBP migrates as a smear of 50-90 kDa that is not visible in Coomassie-stained gels (6), probably due to its strong and unusual glycosylation (7, 14). The mature form of the protein, MBP, is known from the eosinophil leukocyte as a cytotoxic protein, where the 206-residue proMBP is processed to generate mature, nonglycosylated MBP of 117 residues that is stored in granules (15). The propiece is highly acidic and is believed to mask the cytotoxicity of MBP during processing in the eosinophil (16). Cleavage of proMBP is believed not to occur in pregnancy serum, where the function of proMBP has remained unknown. Pregnancy serum contains a molar excess of proMBP compared with PAPP-A, which is disulfide-bound to angiotensinogen and complement C3dg (3).

PAPP-A and proMBP are both produced in the placenta during pregnancy but mainly in different cell types as shown by in situ hybridization. In this tissue, the vast majority of PAPP-A is synthesized in the syncytiotrophoblast, and all proMBP is synthesized in extravillous cytotrophoblasts (17). Clinically, depressed serum levels of PAPP-A are increasingly being used as a predictor of Down's syndrome pregnancies. Recent data show that measurements of proMBP also have a diagnostic value (18). Analyses by reverse transcriptase-polymerase chain reaction revealed that both PAPP-A and proMBP mRNA are present in several reproductive and nonreproductive tissues, although the levels are much lower than in the placenta (19).

Only recently, the putative metalloproteinase activity of PAPP-A has been experimentally confirmed (20). PAPP-A was partially purified from human fibroblast-conditioned medium and shown to be responsible for the known proteolytic activity of human fibroblast-conditioned medium against insulin-like growth factor-binding protein (IGFBP)-4. IGFBPs, of which six have been described, are important modulators of IGF-I and -II activity (21, 22). IGF bound to IGFBP cannot interact with its receptor, but bioactive IGF is released once the binding protein is cleaved. Interestingly, cleavage of IGFBP-4 by PAPP-A strictly requires the presence of IGF (20, 23). PAPP-A secretion has also been demonstrated from osteoblasts and marrow stromal cells (20), from granulosa cells (24), and from vascular smooth muscle cells,2 all of which have known IGF-dependent IGFBP-4 proteinase activity.

Here we report expression in mammalian cells of recombinant PAPP-A (rPAPP-A). This represents an important attainment for the further study of the role of PAPP-A in the IGF/IGFBP system and for the study of PAPP-A as a unique metalloproteinase. A comparison between rPAPP-A and PAPP-A/proMBP complex from pregnancy serum reveals a pronounced difference in proteolytic activity. We conclude that proMBP functions as a proteinase inhibitor in vivo. This finding establishes a biological role of proMBP outside the eosinophil leukocyte. It also represents a novel mode of proteinase inhibition with the enzyme covalently bound by disulfide bonds to its inhibitor.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmid Construction-- A PAPP-A expression plasmid was constructed from three overlapping partial PAPP-A cDNA clones, 29-2, pPA3, and pPA1 (8, 9). The BbeI-EcoRI fragment of 29-2, encoding PAPP-A residues 6-228,3 was excised by partial and full digestion, respectively, and ligated to a nucleotide fragment encoding an artificial signal peptide (MKDSCITVMAMALLSGFFFFAPASSYAA) plus residues 1-5 of PAPP-A. In brief, this latter fragment was generated in a polymerase chain reaction using a mixture of overlapping oligonucleotides (5'-CCTGCATCACTGTGATGGCCATGGCGCTGC-3', 5'-TGTCTGGGTTCTTTTTCTTCGCGCCGGCCTC-3', 5'-GAGCTATGCCGCGGAAGCTAGGGGCGCCAT-3', and 5'-GCGGCATAGCTCGAGGCCGGCGCGAAGAAA-3', 5'-AAGAACCCAGACAGCAGCGCCATGGCCATC-3', 5'-ACAGTGATGCAGGAATCCTTCATAAGCTTAG-3') as a template and primers containing a HindIII (5'-CTAAGCTTATGAAGGATT-3') and a BbeI (5'-ATGGCGCCCCTAGCTTCC-3') recognition site. The ligation product was cloned into the HindIII/EcoRI sites of pBluescript II (Stratagene) to generate pBN-228. The EcoRI-ClaI fragment of pPA3, encoding PAPP-A residues 229-784, was excised and ligated to the HindIII-EcoRI fragment of pBN-228 and cloned into the HindIII/ClaI sites of pBluescript II to generate pBN-784. Further, the ClaI-EcoRI fragment of pPA1, encoding PAPP-A residues 785-1547 and containing part of the 3'-untranslated region, was excised and ligated to the HindIII-ClaI fragment of pBN-784 and cloned into the HindIII/EcoRI sites of pBluescript II to generate pBN-1547. Because the ClaI site in the PAPP-A cDNA is sensitive to methylation, plasmids were propagated in an Escherichia coli dam- strain when required. Finally, the HindIII-XbaI fragment of pBN-1547, encoding the artificial signal peptide plus PAPP-A residues 1-1547, was cloned into the HindIII/XbaI sites of the mammalian expression vector pcDNA3.1+ (Invitrogen) to generate pcDNA3.1-PAPP-A. This construct was verified by sequence analysis. A deviation from the published PAPP-A cDNA sequence (8) was found at one site; nucleotides 78-803 (AGT) were not present, resulting in the absence of Val-27. In the original cDNA clones (29-2 and pPA3), the same deviation was observed upon resequencing. To allow for selection with hygromycin B (Invitrogen), the HindIII-XbaI fragment of pcDNA3.1-PAPP-A was excised and cloned into pcDNA3.1/Hygro(+) (Invitrogen) to obtain pcDNA3.1/Hygro-PAPP-A for generation of stably transfected cells. Plasmid DNA for transfection was prepared with QIAprep Spin Kit (Qiagen).

Tissue Culture, Transfection, and Protein Expression-- Human embryonic kidney 293T cells (293tsA1609neo) (25) were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, nonessential amino acids, and gentamicin (Life Technologies, Inc.). Cells were plated onto 6-cm tissue culture dishes and were transfected 18 h later by calcium phosphate coprecipitation (26) using 10 µg of DNA (pcDNA3.1-PAPP-A). After a further 48 h, the supernatants were harvested and cleared by centrifugation. For generating 293T cell lines that stably express PAPP-A, cells were transfected with FspI-linearized pcDNA3.1/Hygro-PAPP-A by the same method, selected for resistance to 400 µg/ml hygromycin B (Invitrogen). Single colonies were picked and expanded, and stable cell lines were maintained in medium with 100 µg/ml hygromycin B. COS-7 cells (27) were maintained in the same medium but transfected with SuperFect (Qiagen) according to the protocol of the manufacturer.

ELISA-- The levels of recombinant PAPP-A in the supernatants were measured by a standard sandwich ELISA. PAPP-A polyclonal antibodies, anti-(PAPP-A/proMBP),4 were used for capture, and a PAPP-A monoclonal antibody (mAb) (234-2, 234-5, 234-4, 234-6, 234-3, or 234-7) (28) followed by peroxidase-conjugated anti-(mouse IgG) (P260, DAKO) was used for detection. PAPP-A/proMBP purified from pregnancy serum (7) or purified rPAPP-A was used to establish standard curves. The amount of protein in the standards was determined by amino acid analysis (29).

Protein Fractionation-- Purification of rPAPP-A from cell culture supernatants was accomplished by a procedure of precipitation, heparin chromatography (30, 31), and gel filtration. Recombinant PAPP-A was precipitated from cell culture supernatants (50 ml) by 10% (w/v) PEG 6000. The precipitate was dissolved in 50 mM Tris, 50 mM NaCl, pH 8.0, containing standard protease inhibitors and loaded onto a HiTrap heparin-Sepharose (5 ml) (Amersham Pharmacia Biotech) equilibrated with the same buffer. Bound proteins were eluted by a linear increase in the salt concentration to 1000 mM over 30 min at 1 ml/min. Recombinant PAPP-A eluted mainly as a single peak around 600 mM NaCl. Finally, pooled fractions were concentrated by ultrafiltration and chromatographed at 0.5 ml/min on a Superose 6 HR 10/30 (Amersham Pharmacia Biotech) equilibrated with phosphate-buffered saline. PAPP-A/proMBP was purified from term pregnancy serum as described previously (7).

For analytical purposes, pooled pregnancy serum, pregnancy plasma, or culture supernatant was run on a Mono Q HR 10/10 column (Amersham Pharmacia Biotech) equilibrated with 50 mM Tris, 50 mM NaCl, pH 8.0. Prior to column loading, samples were diluted by the addition of 2 volumes of water. Elution was performed with a linear salt gradient from 50 to 1000 mM over 40 min at 1 ml/min, and fractions of 1 ml were collected. For all runs, the salt concentration was 50 mM in fraction 10 and 1000 mM in fraction 50. In Fig. 5A, the absorbance at 280 nm shown was recorded in a second run of one-tenth of the same sample and multiplied by a factor of 10. The two chromatograms obtained were superimposable, and the shape and position of the larger PAPP-A peak (around fraction 43) was the same. The smaller PAPP-A peak (around fraction 24) also had the same position in the two runs, but with the ELISA used, the levels of PAPP-A antigen could not be determined accurately in those fractions from the second run. Thus, ELISA values of the first run are shown.

Miscellaneous Procedures-- SDS-PAGE was performed in Tris-glycine gels (10-20% or 15%) (32) or in precast 3-8% Tris acetate gels (Novex). Separated proteins were visualized by Coomassie staining of gels or first blotted onto a polyvinylidine difluoride membrane for sequence analysis on an Applied Biosystems 477A sequencer equipped with an on-line HPLC (33). For immunovisualization, blots were blocked with 2% Tween 20 and equilibrated in 50 mM Tris, 500 mM NaCl, 0.1% Tween 20, pH 9.0 (TST). Primary antibody (mAb 234-2 for PAPP-A, and mAb 234-10 for proMBP) (28) was diluted in TST containing 0.5% fetal bovine serum, and blots were incubated for 1 h at 37 °C. For detection of reduced PAPP-A, polyclonal antibodies4 were used. Incubation with peroxidase-conjugated secondary antibodies (P260 or P217; DAKO) diluted in TST was done for 1 h at room temperature. Blots were developed using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). Hydrolysis and quantification of amino acids and amino sugars was carried out as described previously (7).

Measurement of PAPP-A Proteolytic Activity-- IGFBP-4 proteolysis was assayed as described previously (23, 34). Approximately 2 ng of rPAPP-A contained in 293T culture medium was incubated for 16 h at 37 °C in 2 mM CaCl2, 50 mM Tris, pH 7.5, with 10,000 cpm of purified 125I-labeled IGFBP-4 (35) in the absence and presence of 5 nM IGF-II (Bachem), standard protease inhibitors, and 5 µg/ml polyclonal anti-(PAPP-A/proMBP).4 The total reaction volume was 25 µl. Intact IGFBP-4 and its proteolytic fragments were separated by reducing 15% SDS-PAGE and were visualized by autoradiography. Likewise, the activity of the PAPP-A/proMBP complex was compared with the activity of rPAPP-A in two parallel time course experiments scaled up to larger reaction volumes. Equal amounts of sample A (pool of term pregnancy serum diluted, approximately 6×, to 7 µg/ml of PAPP-A/proMBP as determined by ELISA calibrated with purified PAPP-A/proMBP) and sample B (as sample A, but rPAPP-A supernatant added to 7 µg/ml as determined by ELISA calibrated with purified rPAPP-A) were incubated with 125I-labeled IGFBP-4 in the presence of 5 nM IGF-II. Aliquots of the reactions were stopped at selected time points by freezing. The experiment with sample A was also carried out in the presence of 5 µg/ml purified rabbit polyclonal IgG, either anti-(PAPP-A/proMBP)4 or irrelevant polyclonal antibodies (anti-alpha 2-macroglobulin, A033; DAKO). A similar time course experiment was performed for comparison of PAPP-A activity in selected column fractions.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of Recombinant PAPP-A in Mammalian Cells-- Human embryonic kidney 293T cells were transiently transfected with pcDNA3.1-PAPP-A encoding a signal peptide followed by the entire PAPP-A polypeptide (residues 1-1547).3 Two days post-transfection, the supernatant contained about 5 µg/ml of recombinant PAPP-A (rPAPP-A) as measured by a PAPP-A-specific ELISA. PAPP-A was not detectable in supernatants from mock-transfected cells. The rPAPP-A antigen was recognized in ELISA by all monoclonal antibodies available, and the integrity of the protein was verified by Western blotting (not shown). Similar results were obtained with transfected COS-7 cells. To establish a stable cell line for production of rPAPP-A, 293T cells were transfected with pcDNA3.1/Hygro-PAPP-A and selected for resistance to hygromycin B. A population of stably transfected cells was maintained, and further, individual cells were cloned.

Purification and Analysis of Recombinant PAPP-A-- Recombinant PAPP-A contained in culture medium was precipitated essentially quantitatively with 10% polyethylene glycol 6000, redissolved, and further purified by heparin chromatography (30, 31) and gel filtration as detailed under "Materials and Methods." Our previously published procedure for purification of the PAPP-A/proMBP complex from pregnancy serum (7) proved useless, because unlike PAPP-A/proMBP, which is fully soluble in 16% polyethylene glycol 6000, rPAPP-A precipitated in the range of 4-12% polyethylene glycol. Purified rPAPP-A migrated faster in SDS-PAGE than the disulfide-bound 2:2 PAPP-A/proMBP complex purified from pregnancy serum (Fig. 1, lanes 3 and 4). Reduced rPAPP-A and PAPP-A from reduced PAPP-A/proMBP both migrated as a band around 200 kDa (Fig. 1, lanes 1 and 2). Thus, rPAPP-A is secreted as a dimer of about 400 kDa. A limited degree of degradation that could not be prevented is apparent for both species after reduction of disulfide bonds (Fig. 1, lanes 1 and 2).


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  		Fig. 1.   SDS-PAGE (10-20%) of purified rPAPP-A expressed in mammalian cells and PAPP-A/proMBP purified from pregnancy serum. Lane 1, reduced rPAPP-A; lane 2, reduced PAPP-A/proMBP; lane 3, unreduced rPAPP-A; lane 4, unreduced PAPP-A/proMBP. About 2 µg of protein was loaded in lanes 1 and 3, and 5 µg was loaded in lanes 2 and 4. Protein bands were visualized by Coomassie staining. ProMBP released from PAPP-A/proMBP upon reduction (lane 2) is not visible due to its unusual glycosylation (6, 7, 14).

Sequence analysis of intact rPAPP-A monomer revealed the expected N-terminal sequence, except that is was preceded by an alanine residue (Ala-Glu-Ala-Arg-Gly-Ala-Thr-Glu). The number of N-acetylglucosamine monosaccharides per rPAPP-A monomer was found through acid hydrolysis to be 22. However, PAPP-A isolated from PAPP-A/proMBP contains 44 N-acetylglucosamine monomers per PAPP-A polypeptide chain (7). This difference in glycosylation appears to be reflected by the slight difference in size of the faint degradation products of rPAPP-A and PAPP-A from PAPP-A/proMBP (Fig. 1, lanes 1 and 2).

Functional Characterization of Recombinant PAPP-A-- The activity of rPAPP-A against IGFBP-4, the only known PAPP-A substrate, was analyzed. As expected, IGFBP-4 was cleaved in the presence but not in the absence of IGF (Fig. 2, lanes 3 and 4). The activity of rPAPP-A was inhibited by polyclonal anti-(PAPP-A/proMBP), EDTA, and 1,10-phenanthroline (Fig. 2, lanes 5-7), but not by TIMP-1, a broad spectrum inhibitor of matrix metalloproteinases (Fig. 2, lane 8). Supernatants from mock-transfected cells, with or without added IGF, did not contain IGFBP-4 proteolytic activity (Fig. 2, lanes 1 and 2).


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  		Fig. 2.   IGF dependent cleavage of IGFBP-4 by rPAPP-A. Recombinant PAPP-A was incubated with radiolabeled IGFBP-4 in the absence (lane 3) and in the presence (lane 4) of IGF-II. The activity of rPAPP-A with IGF-II was completely inhibited by the addition of 5 µg/ml polyclonal anti-(PAPP-A/proMBP) (lane 5), by 5 mM EDTA (lane 6), and by 5 mM 1,10-phenanthroline (lane 7) but not by 5 ng/ml TIMP-1 (lane 8). Supernatant from mock-transfected 293T cells did not show IGFBP-4 degradation without (lane 1) or with (lane 2) added IGF-II. Intact IGFBP-4 (32 kDa) and its two degradation products were separated by SDS-PAGE after reduction and visualized by autoradiography.

Activity Comparison of Recombinant PAPP-A and PAPP-A/proMBP-- An initial experiment revealed that approximately 50 ng of PAPP-A/proMBP complex purified from pregnancy serum was required to obtain the same degree of IGFBP-4 cleavage as 0.1 ng of rPAPP-A (data not shown). Hypothetically, this difference could be caused by a reduction in the activity of PAPP-A/proMBP during chromatographic processing. We therefore compared in a time course experiment the IGFBP-4 proteolytic activity of unfractionated pregnancy serum and the activity of pregnancy serum with rPAPP-A added to the same concentration as pregnancy serum PAPP-A (Fig. 3, A and B). Estimated from this experiment, the difference in specific activity between PAPP-A in pregnancy serum and rPAPP-A is about 100-fold. We conclude that PAPP-A as present in pregnancy serum is strongly inhibited by its binding to proMBP. Hence, proMBP is a proteinase inhibitor.


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  		Fig. 3.   Inhibition of the proteolytic activity of PAPP-A by covalently bound proMBP. Panel A, the activity of PAPP-A, as found in unfractionated pregnancy serum, was measured by incubating radiolabeled IGFBP-4 with diluted pregnancy serum in the presence of added IGF-II. Aliquots of the reactions were stopped at selected time points (2-360 min), and they were run side by side on reducing SDS-PAGE. Degradation was visualized by autoradiography. B, same as A, but the pregnancy serum was adjusted with cell culture medium to contain an equal amount of pregnancy serum PAPP-A and rPAPP-A. C, same as A, but with polyclonal anti-(PAPP-A/proMBP) added (5 µg/ml). Time points above each lane are durations of reactions in minutes.

Further Analysis of the PAPP-A Activity in Pregnancy Serum-- Incubation of pregnancy serum with polyclonal anti-(PAPP-A/proMBP) (Fig. 3C) abolished cleavage of IGFBP-4. Longer incubation with more pregnancy serum in the presence of inhibitory anti-(PAPP-A/proMBP) did not show any IGFBP-4 degradation (Fig. 4). As judged from this, PAPP-A is the dominating, perhaps the only, proteinase in pregnancy serum capable of IGFBP-4 cleavage. Based on these experiments, we sought to find out whether pregnancy serum contains traces of uninhibited PAPP-A, not complexed with proMBP. The distribution of PAPP-A antigen in fractions from a Mono Q column loaded with pregnancy serum showed the expected, broad, and late eluting PAPP-A/proMBP peak around fraction 43 (Fig. 5A). However, this was preceded by a much smaller, less broad peak with a maximum in fraction 24. Interestingly, the elution position and shape of this peak corresponded to that of rPAPP-A when analyzed in the same chromatographic system (Fig. 5B). Note that the PAPP-A axes in Fig. 5 have logarithmic scales.


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  		Fig. 4.   Inhibition of the IGFBP-4 proteolytic activity of pregnancy serum by polyclonal antibodies against PAPP-A. Extended incubation (24 h) and twice the amount of pregnancy serum (corresponding to 4 ng of PAPP-A/25-µl reaction volume) showed IGFBP-4 degradation (lane 1), also in the presence of irrelevant polyclonal antibodies (rabbit anti-alpha -2-macroglobulin, 5 µg/ml) (lane 2). With polyclonal anti-(PAPP-A/proMBP) (5 µg/ml), no degradation was seen (lane 3).


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  		Fig. 5.   Anion exchange chromatography on a Mono Q HR 10/10 column. A, pregnancy serum (3.2 ml) was diluted with water, loaded onto the column, and eluted with a gradient of increasing salt concentration. The flow rate was 1 ml/min, and fractions of 1 ml were collected. The concentration of PAPP-A antigen was measured in all fractions by ELISA and plotted onto the chromatogram. B, culture medium (3 ml) containing rPAPP-A was diluted and loaded onto the column. Elution was performed as for A, and PAPP-A ELISA values were plotted on the resulting chromatogram. Note that the PAPP-A axes have logarithmic scales.

Comparison of activity against IGFBP-4 demonstrated that the specific activity of PAPP-A antigen was higher, greater than 25-fold, in the early than in the late eluting peak (Fig. 6, A and B). Thus, most likely the PAPP-A antigen eluting around fraction 24 is uncomplexed PAPP-A. However, because the two PAPP-A peaks are not well separated, the fractions of the early peak also contain a relatively high amount of PAPP-A/proMBP complex. This interpretation was confirmed by Western blotting of the material in the two peaks using a PAPP-A-specific monoclonal antibody (Fig. 7, lanes 1 and 2). The slightly faster migration of dimeric rPAPP-A (see above) loaded on the same gel (Fig. 7, lane 3) is apparent. A parallel Western blot with a proMBP-specific monoclonal antibody verified that proMBP was present in the upper but not in the lower PAPP-A band of the early peak and in the late eluting PAPP-A band (Fig. 7, lanes 4 and 5). As expected, after reduction of both the early and the late eluting PAPP-A only one band, corresponding to the intact PAPP-A monomer, was visible (Fig. 8, lanes 1 and 2).


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  		Fig. 6.   IGFBP-4 proteolytic activity in chromatographic fractions. The activity of pregnancy serum PAPP-A eluting early and late from an anion exchanger (Fig. 5A) was evaluated. Fraction 43 (late eluting PAPP-A) was diluted 100-fold to contain the same concentration of PAPP-A as fraction 24 (early eluting PAPP-A), and equal amounts were incubated with radiolabeled IGFBP-4 in the presence of IGF-II. Aliquots of the reactions were stopped at selected time points (0-48 h) and analyzed. Time points above each lane are durations of reactions in hours. A, PAPP-A antigen from fraction 24; B, PAPP-A antigen from fraction 43.


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  		Fig. 7.   Western blots of chromatographic fractions separated in 3-8% nonreducing SDS-PAGE. Pregnancy serum PAPP-A eluting early and late from an anion exchanger (Fig. 5A) was analyzed with PAPP-A and proMBP-specific mAbs. Fractions around fraction 24 (fractions 22-25, Fig. 5A) were pooled, and PAPP-A antigen was purified by heparin chromatography and analyzed by Western blotting using a PAPP-A mAb (lane 1) and a proMBP mAb (lane 4). Likewise, heparin-purified PAPP-A antigen from fractions around fraction 43 (fractions 41-45, Fig. 5A) was analyzed using the PAPP-A mAb (lane 2) and the proMBP mAb (lane 5). For comparison, rPAPP-A was loaded on the gel developed with the PAPP-A mAb (lane 3). All samples were nonreduced.


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  		Fig. 8.   Western blot of chromatographic fractions separated in 10-20% reducing SDS-PAGE. Early (lane 1) and late (lane 2) eluting PAPP-A antigen further purified by heparin chromatography (see legend to Fig. 7) was analyzed by Western blotting using polyclonal antibodies against PAPP-A. Purified PAPP-A/proMBP (7) was loaded for comparison (lane 3). All samples were reduced, and the expected band of approximately 200 kDa was observed in all lanes.

Uncomplexed, dimeric PAPP-A has not previously been demonstrated in pregnancy serum. To verify that complex formation with proMBP is not a consequence of blood coagulation, separate runs of freshly drawn EDTA-treated pregnancy plasma were analyzed on the same system. Again, two PAPP-A peaks were found, and the difference in heights of the early and late eluting peak was the same as for serum (not shown). Thus, the vast majority of PAPP-A is present in both pregnancy serum and plasma as PAPP-A/proMBP complex, but a minor fraction (<1%) of PAPP-A is present as an uncomplexed PAPP-A dimer. This uncomplexed fraction has much higher specific activity.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have expressed in mammalian cells the entire 1547-residue human PAPP-A. Biochemical analyses show that 1) the expressed protein is immunoreactive against all available monoclonal antibodies in ELISA and in Western blotting, 2) rPAPP-A is secreted as a homodimer of about 400 kDa, 3) reduction of the recombinant molecule yields monomers slightly smaller than the 200-kDa subunit from pregnancy serum PAPP-A because of a lower degree of glycosylation, 4) rPAPP-A cleaves IGFBP-4 in an IGF-dependent manner, and 5) the activity of rPAPP-A can be abrogated by standard metalloproteinase inhibitors and by polyclonal anti-(PAPP-A/proMBP), but not by TIMP-1, which is specific for matrix metalloproteinases (36). We further find that 6) the activity of rPAPP-A and pregnancy serum PAPP-A differs 100-fold, demonstrating a biological role of proMBP as a proteinase inhibitor of PAPP-A, and 7) the PAPP-A activity of pregnancy serum, inhibitable with PAPP-A antibodies, is in part due to a minor fraction (<1%) of uncomplexed PAPP-A present in both pregnancy serum and plasma. An expression system for recombinant PAPP-A provides a biochemical basis for future studies of PAPP-A and the biological systems in which it functions. PAPP-A is particularly interesting because its cleavage of IGFBP-4 uniquely depends on the presence of IGF (20, 23). In addition, PAPP-A is interesting structurally because it is distinct in several regards from the previously classified four members of the metzincin superfamily of metalloproteinases, to which it belongs (9-11). In this report, we have mainly focused on the physiological inhibition of PAPP-A.

Many proteinases require a propeptide to assist folding and/or secretion (37, 38). However, because the expressed rPAPP-A is antigenic, functional (Fig. 2), and secreted abundantly into the culture medium as a dimer of the expected size (Fig. 1), the PAPP-A propeptide (8, 9) is required neither for folding nor secretion. Often a propeptide functions to retain the proteolytic activity of a zymogen, which becomes active in the extracellular compartment only after propeptide cleavage (37). But the four-residue stretch immediately preceding residue 1 of PAPP-A, Arg-Gln-Gln-Arg (8), resembles the consensus furin cleavage site (39). Thus, also in vivo PAPP-A is likely to be secreted as an active proteinase following intracellular cleavage. Other regions of the PAPP-A polypeptide, possibly on the C-terminal side of the proteolytic domain, could be important for proper folding and secretion as found recently for meprin A, another metzincin metalloproteinase (40).

Comparison of rPAPP-A with PAPP-A as present in pregnancy serum reveals a strikingly lower IGFBP-4 proteolytic activity of the latter (Fig. 4, A and B). This difference, about 100-fold, cannot be caused by a diffusible inhibitor because rPAPP-A was also assayed in the context of pregnancy serum. Rather, it can be ascribed to proMBP as it is bound covalently to PAPP-A in pregnancy serum. The relatively low activity of pregnancy serum could be caused by unidentified IGFBP-4 proteinases rather than PAPP-A (41, 42), but since the addition of polyclonal PAPP-A antibodies abolished all detectable activity (Fig. 3C and Fig. 4), PAPP-A seems to be the dominating, if not the only, IGFBP-4 proteinase in pregnancy serum.

It was tempting to speculate that the low activity of pregnancy serum PAPP-A is based on low amounts of PAPP-A circulating uncomplexed to proMBP. PAPP-A without proMBP would be expected to elute earlier than PAPP-A/proMBP from an anion exchange column, because proMBP is heavily substituted with acidic carbohydrate groups, including glycosaminoglycan (7, 14). To test the hypothesis, we chromatographed pregnancy serum on a Mono Q column and measured the concentration of PAPP-A in fractions of the eluate (Fig. 5A). Indeed, a small peak preceding the broad, late eluting PAPP-A/proMBP peak was observed whose position correlated with the position of rPAPP-A in a separate column run (Fig. 5B). The peak heights differed by a factor of about 100; the same distribution was observed with pregnancy plasma and is therefore not influenced by blood coagulation (not shown). Measurement of activity in PAPP-A peak fractions revealed that the early peak contained material of much higher specific activity than the late peak (Fig. 6), and Western blotting demonstrated the presence of a PAPP-A species of 400 kDa in the early but not in the late peak (Fig. 7, lanes 1 and 2). This species, also not reactive with a proMBP-specific monoclonal antibody (Fig. 7, lane 4), represents dimeric pregnancy serum PAPP-A. Because the two peaks are not well separated, the early eluting peak also contained 500-kDa PAPP-A/proMBP complex, which lowers the specific PAPP-A activity of this peak (Fig. 7). In conclusion, proMBP dramatically inhibits the activity of PAPP-A in pregnancy serum by having formed a covalent complex with PAPP-A. The measurable PAPP-A activity of pregnancy serum stems from a fraction of PAPP-A, less than 1%, which is present as an uninhibited PAPP-A dimer. However, since late eluting PAPP-A is not completely inactive (Fig. 6), low amounts of a hypothetical, incompletely inhibited 2:1 PAPP-A/proMBP complex may also be contributing. Such a complex would largely coelute with 2:2 PAPP-A/proMBP complex due to the extreme heterogeneity of the proMBP carbohydrates (7, 14).

The finding that proMBP inhibits PAPP-A in pregnancy serum establishes a biological role of this protein outside the eosinophil leukocyte from where it originally is known. It also represents a novel mode of proteinase inhibition because the enzyme is covalently bound by disulfide bonds to its inhibitor. To our knowledge, no analogous example of this exists in the literature. In the placenta, PAPP-A and proMBP are synthesized in different cell types (17), and therefore the covalent PAPP-A/proMBP complex must form in the extracellular compartment after secretion. Complex formation does require a specific interaction between the two proteins. However, there is not a need for proMBP to interact specifically with the active site of PAPP-A, as long as IGFBP-4 cannot reach this due to steric hindrance or due to a proMBP-induced allosteric change in the conformation of PAPP-A. A model based on steric hindrance is in favor because disulfide linkage between a cysteine residue in proMBP and Cys-381 of PAPP-A, close to the active site in the primary structure, has been demonstrated (6). However, based on the known presence of free sulfhydryl groups in mature eosinophil MBP (43), a specific interaction between one cysteine residue of proMBP and the active site zinc atom of PAPP-A must be considered as a possible inhibitory mechanism. This would be in analogy with the propeptide cysteine switch mechanism known from other metzincins (38, 44).

What is the significance of the PAPP-A/proMBP complex in vivo? An unusually high IGFBP-4 proteinase activity resulting from uncomplexed PAPP-A may be required locally for placental development. However, because of the high PAPP-A concentration in pregnancy serum, proMBP's inhibitory function is likely to be important for circulating PAPP-A that uninhibited would cause a dramatic increase in IGFBP-4 proteinase activity in the circulation. An inhibitory role may also be relevant outside of pregnancy, although the synthesis of both PAPP-A and proMBP is much lower (19). In tissues, the proliferation of certain cell types may be stimulated, in an autocrine or paracrine manner, via their secretion of PAPP-A that in turn would cause an increased level of bioactive IGF. Neighboring cells of a different kind that synthesize and secrete proMBP have the potential to control this proliferation by inhibiting the enzymatic activity of PAPP-A. Not many individual cell types have been analyzed yet, but it is known that cultured fibroblasts synthesize PAPP-A, and not proMBP (20), and in the placenta PAPP-A and proMBP are synthesized in different cell types as shown by in situ hybridization (17).

However, although proMBP is likely to play an inhibitory role for PAPP-A in the circulation, its role may be different elsewhere. A hypothesis where PAPP-A activity is not inhibited but rather controlled or regulated by proMBP is attractive; it is tempting to speculate that PAPP-A/proMBP represents a latent form of PAPP-A that can become active under given circumstances, possibly at the cell surface.

    ACKNOWLEDGEMENTS

We thank L. Bale, L. Kristensen, and P. Lind for excellent technical assistance and S. Mohan for donating recombinant IGFBP-4 produced in E. coli. We thank the staff at the Department of Obstetrics and Gynecology, Aarhus University Hospital, for donating pregnancy serum.

    FOOTNOTES

* This work was supported by grants from the Danish Natural Science Research Council, the Danish Medical Research Council, the Novo Nordic Foundation, and the Alfred Benzon Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed. Fax: 45 8612 3178; E-mail: co@mbio.aau.dk.

Published, JBC Papers in Press, July 25, 2000, DOI 10.1074/jbc.M001384200

2 Bayes-Genis, A., Schwartz, R. S., Ashai, K., Lewis, D. A., Overgaard, M. T., Christiansen, M., Oxvig, C., Holmes, D. R., Jr., and Conover, C. A. Arterioscler. Thromb. Vasc. Biol., in press.

3 In this paper, PAPP-A is numbered with the N-terminal Glu as residue 1, and the cDNA sequence is numbered with the codon encoding Glu-1 (GAG) as nucleotides 1-3 as in Ref. 8. In the deposited sequence record (GenBankTM accession number X68280), this Glu is residue 5, and the corresponding nucleotides are 13-15.

4 Polyclonal anti-(PAPP-A/proMBP) was raised in rabbits using highly purified PAPP-A/proMBP (7) (M. T. Overgaard, M. Christiansen, and C. Oxvig, unpublished).

    ABBREVIATIONS

The abbreviations used are: PAPP-A, pregnancy-associated plasma protein-A; rPAPP-A, recombinant PAPP-A; MBP, eosinophil major basic protein; proMBP, the proform of eosinophil major basic protein; PAGE, polyacrylamide gel electrophoresis; IGFBP, insulin-like growth factor-binding protein; IGF, insulin-like growth factor; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; TIMP-1, tissue inhibitors of metalloproteinases-1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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