©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Angiotensinogen and Complement C3dg as Novel Proteins Binding the Proform of Eosinophil Major Basic Protein in Human Pregnancy Serum and Plasma (*)

Claus Oxvig (1), Jesper Haaning (1), Lene Kristensen (1), Jill M. Wagner (2), Inger Rubin (3), Torgny Stigbrand (4), Gerald J. Gleich (2), Lars Sottrup-Jensen (1)(§)

From the (1) Department of Molecular Biology, University of Aarhus, C. F. MAlle, Building 130, DK-8000 C, Denmark, the (2) Departments of Immunology and Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905, the (3) Department of Medical Biochemistry and Genetics, Panum Institute, University of Copenhagen, 2200 Copenhagen N, Denmark, and the (4) Department of Medical Biochemistry and Biophysics, University of Umeå, S-901 87 Umeå, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In sera from pregnant women, pregnancy-associated plasma protein-A (PAPP-A) circulates as a disulfide-bound complex (approximately 474 kDa) with the proform of eosinophil major basic protein (proMBP) (Oxvig, C., Sand, O., Kristensen, T., Gleich, G. J., and Sottrup-Jensen, L. (1993) J. Biol. Chem. 268, 12243-12246). We have produced monoclonal antibodies (mAbs) against the PAPP-AproMBP complex and established a radioimmunoassay utilizing a mAb recognizing the PAPP-A subunit. Surprisingly, serum levels of proMBP exceed those of PAPP-A four to 10-fold on a molar basis throughout pregnancy. This result prompted an investigation of the status of proMBP in pregnancy. Using a proMBP-specific mAb two novel proMBP complexes have been isolated by chromatographic techniques. Based on sequence analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reaction with specific antibodies, one is shown to be a 2:2 disulfide-bound complex (approximately 200 kDa) between proMBP and angiotensinogen. The other is a 2:2:2 complex (approximately 300 kDa) between proMBP, angiotensinogen, and complement C3dg. Circulating proMBP in pregnancy is thus present in three types of complexes. These results suggest that specific interactions between the complexed proteins occur in pregnancy, and the possibility is raised that their interactions are important in the pathophysiology of pregnancies associated with hypertension.


INTRODUCTION

Eosinophil major basic protein (MBP)() has been known for several years as a main constituent of the granules in eosinophilic leukocytes (1, 2, 3, 4, 5) . The 117-residue MBP is derived from a preproprotein of 222 amino acids, including a signal peptide of 16 or 17 residues (6, 7, 8) . The proform of the protein has not been isolated from eosinophils. However, proMBP, previously believed to be MBP (9) , was recently isolated from human pregnancy serum as a disulfide-bound complex with pregnancy-associated plasma protein-A (PAPP-A) (8, 10) . PAPP-A was first recognized in 1974 (11) , and recently the sequence of its 1547-residue subunit was determined (12) . ProMBP is strongly glycosylated containing 38.6% (w/w) of heterogeneous N- and O-linked glycan groups in addition to one glycosaminoglycan chain (10, 13) . The calculated average size is 38.3 kDa (10) , but in SDS-PAGE proMBP migrates as a weakly staining smear of 50-90 kDa (8) .

The complex of PAPP-A and proMBP, denoted PAPP-Apro-MBP, contains two PAPP-A subunits and two proMBP subunits, and has a calculated size of 474 kDa (8, 10) . By in situ hybridization both subunits have been shown to be of placental origin. Whereas proMBP mRNA is present only in X cells (14) , PAPP-A mRNA is localized both in the X cells and in the syncytiotrophoblast (15) . Previously, several investigators have used preparations of polyclonal antibodies against the PAPP-AproMBP complex to determine the levels in pregnancy serum of PAPP-A antigen (16, 17, 18, 19, 20) . But anti-PAPP-AproMBP is polyspecific, binding both subunits of the complex (8) , and values of PAPP-A might be biased in the presence of excess proMBP. Therefore, utilizing monoclonal antibodies of known specificity, we have compared the serum levels throughout pregnancy of the PAPP-A and proMBP subunits. We report here that the concentration of proMBP exceeds that of PAPP-A by several fold on a molar basis, and consequently, only a fraction of circulating proMBP is bound to PAPP-A. We also report that proMBP is covalently bound to angiotensinogen and complement C3dg. Previously, high molecular weight (HMW) angiotensinogen of undefined nature has been recognized in moderate quantities (15%) in plasma from pregnant women and in high quantities in hypertensive pregnant women (21) . We present data suggesting that what has previously been referred to as HMW angiotensinogen is actually a 2:2 complex between angiotensinogen and proMBP. A fraction of this complex further binds two molecules of complement C3dg in a 2:2:2 complex.


MATERIALS AND METHODS

Pregnancy Serum, Plasma, and Proteins

Serum samples from nine pregnant women were obtained in weeks 10, 17, 25, 33, and 40 (during labor). Two-ml samples were drawn and the serum stored at -20 °C (22) . Serum from five nonpregnant women was obtained and stored at -20 °C. For column chromatography, pooled pregnancy serum from various gestational ages stored at -20 °C was used. Blood from four normotensive pregnant women was drawn in EDTA-treated tubes. Plasma was obtained by centrifugation, stored on ice, and separately subjected to ion exchange chromatography within a few hours.

Proteins used as standards or controls in radioimmunoassays, PAPP-AproMBP, recombinant proMBP, and eosinophil MBP were purified as described previously (Refs. 10, 23, and 24, Footnote 2()).

Monoclonal Antibodies

Six to eight-week-old female BALB/c mice (Bomholtgaard, Denmark) were injected intraperitoneally with 100 µg of purified PAPP-AproMBP complex in Freund's complete adjuvant, and 4 weeks later the animals were given booster injections of 75 µg of PAPP-AproMBP in Freund's incomplete adjuvant. Eight weeks after the initial immunization the animals were boosted intravenously with 100 µg of PAPP-AproMBP on 3 consecutive days. Spleen cells were fused on day 4 as earlier described (25) with SP2/0 myeloma cells at a ratio of 1:0.7. Selection for positive hybridomas was done by enzyme-linked immunosorbent assay, using purified PAPP-AproMBP for coating (1 µg/ml) and rabbit anti-mouse IgG as second antibody. One fusion gave 11 hybrids. The antibodies were characterized as earlier described (26) . Clones T9, T10, and T46 were IgG, whereas clones T16, T17, T18, T23, T24, T31, T41, and T47 were IgG. All subclasses contained light chains of the type (data not shown).

The mAb T24, used in this study, recognizes an epitope in the PAPP-A polypeptide, because it does not recognize recombinant pro-MBP by dot blotting, and because proMBP is not able to inhibit binding of I-PAPP-AproMBP in the radioimmunoassay described below (not shown).

A mAb specific for the propiece of proMBP, J163-15E10, was used for Western and dot blotting experiments. mAbs used in a two-site MBP assay (J13-6B6 and J14-8A2) have been described earlier (22) , as well as mAb F8A2 directed against angiotensinogen (27) .

Radioimmunoassays and Rocket IEP

For determinations of pro-MBP antigen, a two-site radioimmunoassay was used (22) . The two-site assay utilizes mAbs recognizing epitopes on the mature (MBP) part of proMBP. Therefore, MBP purified from eosinophil granules as well as proMBP present in the PAPP-AproMBP complex are recognized in this assay and were both utilized as standards. Monomeric eosinophil MBP was stepwise diluted to concentrations between 19.20 and 0.14 nM and used for one standard curve. Likewise, purified PAPP-AproMBP, containing the subunits in a 2:2 molar ratio (8, 10), was diluted to subunit concentrations between 17.50 and 0.43 nM (cf. below in this section) and used for a second standard curve in the same experiment. The two standard curves gave virtually identical results. Data reported here are based on a PAPP-AproMBP standard curve. All samples were reduced and alkylated (24) prior to measurement.

For determinations of PAPP-A, an inhibition radioimmunoassay was developed, utilizing I-labeled PAPP-AproMBP (28) . Immulon-4 96-well plates (Dynatech Laboratories Inc., Chantilly, VA) were coated overnight at 4 °C with 100 µl/well Cappel rabbit anti-mouse-IgG (catalogue number 55480) diluted 100-fold in PBS (150 mM NaCl, 10 mM sodium phosphate, pH 7.4). Following washing three times with PT20 (0.1 M sodium phosphate, pH 7.5, containing 1% (v/v) Tween 20), remaining sites were blocked with 300 µl/well of STST (5% (w/v) skimmed milk powder, 0.3% (v/v) Tween 20, 150 mM NaCl, 2 mM CaCl, 20 mM Tris, pH 7.4) by incubating for 1 h at room temperature. Then, PAPP-A-specific mAb T24 was diluted in STST, and 50 µl was applied to each well. After a 3-h incubation at room temperature, the plates were washed as before. Unknown samples were diluted appropriately in STST, and 50 µl/well was added. With a delay of approximately 5 min, 50 µl of I-labeled PAPP-AproMBP (approximately 50,000 cpm/well) diluted in STST was added, and the plates were incubated 2 h at room temperature. Finally, after washing six times in PT20, the wells were counted in a scintillation counter (RIASTAR, Packard Instruments Co.). All incubations were done on a shaking platform, and samples were assayed at least in triplicate at different concentrations.

For establishing the standard curve, purified PAPP-AproMBP complex was used (10) . A preparation with an A of 0.572 corresponding to 1750 nM of PAPP-A subunit (10) was diluted (STST) in appropriate steps 100-fold to 2400-fold (17.50 nM to 0.73 nM PAPP-A subunit). For this range, a near-linear calibration curve was obtained in a semilogarithmic plot (not shown); correlation coefficient > 0.99.

Rocket IEP was performed as described previously (29) using anti-PAPP-AproMBP (Dako, A230). Routinely, rocket IEP is used for detection of PAPP-AproMBP during purification (10) . By this method, proMBP not complexed to PAPP-A is not detected.

Column Materials and Chemicals

Mono Q HR 5/5, DEAE-Sephacel, imino diacetic acid-Sepharose 4B, Sephacryl S-300 HR, and Q-Sepharose HP were from Pharmacia. All standard chemicals were from Sigma, Merck, or Amersham.

Column Chromatography

All chromatographic steps were performed at 4 °C, except for Mono Q and Q-Sepharose ion exchange chromatography, which was carried out at room temperature. Below are detailed conditions for purification of proMBP containing complexes corresponding to Fig. 3, A-C.


Figure 3: Isolation of proMBP complexes. A, preparative DEAE-Sephacel ion exchange. Nine-hundred ml of pooled pregnancy serum was loaded and gradient eluted as detailed under ``Material and Methods.'' The absorbance at 280 nm is shown by the solid line, and the gradient (at the column inlet) is indicated by the stippled line. (a, wash with PBS; b, start of gradient elution, PBS containing 0.15-1 M NaCl; c, wash with PBS containing 2 M NaCl). Fractions corresponding to the horizontal bar marked i (182-294) were positive for proMBP by dot blotting. B, nickel chelate chromatography. Pool i (A) was loaded onto a column charged with nickel ions and eluted with a decreasing pH gradient (stippled line) (d, column loading; e, wash with PBS containing 1 M NaCl; f, gradient elution from pH 7.4 to pH 4.5; g, wash with PBS, pH 4.5). Absorbance at 280 nm is shown as a solid line. The fall-through was pooled (ii, fractions 5-128), and fractions 256-298 (iii) were pooled. From previous work the latter pool is known to contain PAPP-AproMBP (10). C, Sephacryl S-300 gel chromatography of pool ii (B). ProMBP immunoreactivity was located in fractions 94-125, and two pools were made, iv (94-110) and v (111-125). Both pools were further separated on a Q-Sepharose ion exchange column (1 10 cm), as explained in the text.



A DEAE-Sephacel column (5 26 cm) (Fig. 3A) was loaded with 900 ml of pooled pregnancy serum, washed with PBS, and eluted at a flow rate of 200 ml/h. Elution was continued with a linear gradient formed from 750 ml of PBS and 750 ml of PBS containing 1 M NaCl. After the gradient was finished, the column was washed with 500 ml of PBS containing 2 M NaCl. The fraction size was 10 ml, except for fractions 1-50 (20 ml). The pool made (i) was loaded onto an imino diacetic acid-Sepharose 4B column (2.5 17 cm) (Fig. 3B) charged with nickel ions and equilibrated with PBS (100 ml/h, 10 ml/fraction). The column was then washed with PBS containing 1 M NaCl (150 ml) and eluted with a decreasing pH gradient formed from 600 ml of PBS, pH 7.4, and 600 ml of PBS adjusted to pH 4.5 with phosphoric acid. At the end of the gradient, PBS adjusted to pH 4.5 was pumped through the column. Two pools, ii and iii, were concentrated and subjected to gel chromatography on Sephacryl S-300. This column (5 100 cm) (Fig. 3C) was equilibrated and eluted with PBS (100 ml/h, 10 ml/fraction).

Gel Electrophoresis, Dot Blotting, and Western Blotting

For SDS-PAGE, the Tris-glycine system (30) was used (10-20% gradient gels of size 0.5 80 100 mm). Gels were stained with Coomassie Brilliant Blue or blotted (31) onto ProBlott PVDF membranes (Applied Biosystems) for immunological detection. Since it was found that high molecular weight complexes, including PAPP-AproMBP, were poorly electroblotted, nonreduced gels were submerged for 1 h in PBS, pH 8.0, containing 10 mM dithiothreitol prior to electroblotting in order to break interchain disulfide bridges of the complexes. All blots were blocked with 2% (w/v) Tween 20, and equilibrated in TST (50 mM Tris, 500 mM NaCl, 0.1% (w/v) Tween 20, pH 9.0). Primary antibody was diluted in TST containing 0.5% (v/v) fetal calf serum, and the blot was incubated for 1 h at 37 °C. Following washing in TST, the blot was incubated at room temperature with peroxidase-conjugated secondary antibodies (Dako, P260 for mouse mAbs, and P217 for rabbit polyclonal antibodies) diluted in TST. The blot was washed as before and developed in 35 mM citric acid, 67 mM sodium phosphate, pH 5.0, containing 1 mM 3,3`-diaminobenzidine and 0.01% (v/v) hydrogen peroxide. For detection of proMBP and angiotensinogen, the mAbs J163-15E10 and F8A2 (27) , respectively, were used. Anti-complement C3d (Dako, A063) was used for detection of complement C3dg. Material spotted directly onto a moistened PVDF membrane was air-dried and processed after reduction for 10 min with 10 mM dithiothreitol in PBS, pH 8.0. For detection of proMBP using mAb J163-15E10, the effect of reduction is conspicuous. Probably, the relevant proMBP epitope is less exposed in a nonreduced complex compared to the reduced complex.

Protein Sequence Analysis

Material blotted onto ProBlott PVDF membranes was stained with Coomassie Brilliant Blue. Bands of interest were excised and subjected to N-terminal sequence analysis on an Applied Biosystems 477A instrument equipped with an on-line 120A high performance liquid chromatography system.

Visualization of Three-dimensional Structures

RasMol v2.4 (Roger Sayle, Glaxo Research and Development, Greenford, Middlesex, United Kingdom) was run under Microsoft Windows and used to display the three-dimensional structure of bovine antithrombin III (32) . The program was obtained by anonymous FTP (ftp.dcs.ed.ac.uk), and the coordinate file was from the Brookhaven Protein Data Bank.


RESULTS

PAPP-A and ProMBP Levels throughout Gestation

Mice were immunized with the PAPP-AproMBP complex, and a series of monoclonal antibodies were produced and tested. mAb T24, recognizing the PAPP-A subunit of the complex, was used to establish an inhibition radioimmunoassay as detailed under ``Materials and Methods.'' Sera from a series of nine women were obtained at weeks 10, 17, 25, 33, and 40, and the levels of PAPP-A subunit were measured. The samples were also assayed for the levels of proMBP using a two-site MBP radioimmunoassay. Results presented in Fig. 1 show that, on a molar basis, the concentration of the proMBP subunit exceeds that of the PAPP-A subunit several fold. In the first and second trimester, the molar amount of proMBP is approximately 10 times higher than PAPP-A, but in the third trimester, due to stabilization of the proMBP level, it exceeds PAPP-A approximately 4-fold. In serum from five nonpregnant women, PAPP-A was not detectable, and proMBP was estimated at approximately 7 nM.


Figure 1: Radioimmunoassay of PAPP-A and proMBP subunits. Serial serum samples from nine pregnant women (weeks 10, 17, 25, 33, and 40) were analyzed using specific mAbs as detailed under ``Materials and Methods.'' Average values are plotted, and the vertical bars indicate standard deviations. Expressed in mass per volume, 100 nM PAPP-A monomer (198.5 kDa (10)) corresponds to 19.9 mg/liter, and 100 nM proMBP monomer (38.3 kDa (10)) corresponds to 3.8 mg/liter.



Chromatographic Analysis of Pregnancy Serum

Since pregnancy serum on a molar basis contains excess proMBP compared with PAPP-A antigen, we investigated the distribution of proMBP in serum. A sample of pooled pregnancy serum was subjected to ion exchange chromatography, as shown in Fig. 2. PAPP-AproMBP was detected in late eluting fractions, in agreement with previous results (10) . Dot blotting revealed the presence of proMBP antigen in several fractions preceding those containing the PAPP-AproMBP complex. In this system, proMBP isolated from reduced and carboxamidomethylated PAPP-AproMBP (8, 10) elutes later than PAPP-Apro-MBP (not shown). Thus, it was concluded that the highly acidic proMBP (10, 13) does not exist in serum as a monomer or polymers hereof. Rather, it exists in complex with plasma proteins other than PAPP-A. At least some of these complexes are less strongly retained on Mono Q than PAPP-AproMBP.


Figure 2: Ion exchange chromatography of pregnancy serum. Six-hundred µl of pooled pregnancy serum was diluted with 2400 µl of water and loaded onto a Mono Q HR 5/5 ion exchange column: fraction size, 500 µl; flow rate, 0.5 ml/min. The column was equilibrated with PBS, and elution was performed by increasing the concentration of sodium chloride in the PBS buffer as shown by the stippled line. The solid line shows absorbance recorded at 275 nm. The upper horizontal bar indicates fractions positive for pro-MBP in dot blotting (34-60), and the lower bar shows PAPP-A-containing fractions located by rocket IEP (45-60).



In a separate Sephacryl S-300 gel chromatography experiment (conditions as for Fig. 3C below), pregnancy serum was analyzed (not shown). ProMBP was detected in fractions corresponding to 0.41-0.62 v (V = 0.40 v). From this column, PAPP-AproMBP elutes at approximately 0.47-0.55 v. The results indicate that complex(es) of proMBP with as yet unknown plasma proteins are of high molecular weight.

Purification of ProMBP Complexes from Pregnancy Serum

Attempts to purify the putative proMBP complex(es) by affinity chromatography utilizing the mAb J163-15E10 were unsuccessful, probably because the proMBP epitope recognized by this mAb is hidden in nonreduced complexes of proMBP with other proteins.

The analytical ion exchange experiment (Fig. 2) shows that PAPP-AproMBP and the putative proMBP-containing complexes elute at high salt concentration, well separated from most plasma proteins. Evidently, these species are strongly anionic, most likely largely resulting from the negatively charged carbohydrate and glycosaminoglycan groups of proMBP (10, 13) . To isolate these putative complexes, a large scale experiment using 900 ml of pooled pregnancy serum was performed. The serum was loaded directly onto a DEAE-Sephacel ion exchanger (Fig. 3A) as detailed under ``Materials and Methods.'' The dimensions of this column were chosen so that only the strongly negatively charged proteins were retained. Only the late eluting proMBP containing fractions were pooled (pool i).

From previous work it is known that the PAPP-AproMBP complex binds to immobilized nickel ions (10) . Therefore, in order to separate PAPP-AproMBP from putative complexes of proMBP and other proteins, pool i was loaded onto a nickel affinity column (Fig. 3B). The fall-through and proteins eluted with a decreasing pH gradient were collected and analyzed by dot blotting. ProMBP was detected in fractions corresponding to pools ii and iii (Fig. 3B). Of those, pool iii is known to contain PAPP-AproMBP (10) . To exclude several irrelevant proteins of lower molecular weight evident in SDS-PAGE (not shown), these pools were concentrated and separately subjected to Sephacryl S-300 gel chromatography.

From pool ii, two proMBP containing pools were obtained, iv and v (Fig. 3C). To further purify the proMBP complexes, each pool was loaded onto a Q-Sepharose ion exchanger (not shown) and eluted with a linear salt gradient (0.15-1 M NaCl). We found this step useful, although the material seemed very heterogeneous in ion exchange chromatography. Pool iv contained PAPP-AproMBP in small amounts, as seen from Western blotting (Fig. 4, lane 1). In dot blotting, the material reacted with each of the mAbs J163-15E10 and T24, recognizing the proMBP and PAPP-A subunit, respectively (not shown). Thus, a minor fraction of the PAPP-AproMBP complexes escaped binding to the nickel affinity column, probably due to shielding of the metal binding site of PAPP-AproMBP. In contrast, pool v contained substantial amounts of proMBP in two bands of lower molecular weight (Fig. 4, lane 2).


Figure 4: SDS-PAGE and immunoblot analyses of fractions containing proMBP complexes. A, Western blots utilizing proMBP-specific mAb. Lane 1, nonreduced material originating from pool iv containing traces of PAPP-AproMBP that escaped binding to the nickel column; lane 2, nonreduced material from pool v showing two novel proMBP-containing bands; lane 3, nonreduced material further purified from pool iii, containing predominantly PAPP-AproMBP. B, Coomassie Brilliant Blue-stained SDS-PAGE. Lane 4, nonreduced material from pool v as in lane 2; lane 5, nonreduced material from pool v containing only proMBP-positive species; lane 6, same material as in lane 5 but reduced. C, samples as loaded in lane 5 and immunologically detected using angiotensinogen-specific mAb (lane 7) and anti-complement C3d (lane 8). Positions of size markers (kDa) are indicated.



Pool iii, was subjected to the same procedure of gel filtration and ion exchange chromatography as pool ii (not shown). The prominent band shown by Western blotting after Q-Sepharose ion exchange (Fig. 4, lane 3) is PAPP-AproMBP. A faint band of lower molecular weight is present, but since the N-terminal sequence could not be determined, this material was not further investigated.

To summarize, only pool v contained proMBP complexes that were present in substantial amounts and different from PAPP-AproMBP.

Sequence Analysis of ProMBP-containing Complexes

The material obtained from pool v contained four major components of approximate sizes 300, 200, 95, and 85 kDa, respectively, in nonreducing SDS-PAGE (Fig. 4, lane 4). All bands were electroblotted and subjected to sequence analysis. For the two proMBP-positive bands (approximately 300 and 200 kDa), the results are shown in . As expected, both bands contained the proMBP sequence in two different forms (8) , sequences 1 and 2. Sequence 3 was compatible with the presence of intact angiotensinogen (33) in both bands. In addition, the N-terminal sequence of complement C3dg (34) was revealed in the approximately 300-kDa band (sequence 4).

The material in the two bands of lower molecular weight not containing proMBP was also identified. The approximately 95-kDa band turned out to represent the strongly acidic HC2/bikunin (35) , having the N-terminal of both bikunin (36) and HC2 (37) . The material in the approximately 85-kDa band also contained an intact bikunin chain. But in contrast to the 95-kDa band, it contained two truncated variants of the HC2 chain, shortened by 96 and 102 residues, respectively.

Late eluting fractions from the Q-Sepharose column contained only traces of HC2/bikunin, but still considerable amounts of the two proMBP-positive species (Fig. 4, lane 5). Reducing SDS-PAGE of the same material (Fig. 4, lane 6) revealed the expected components of the two proMBP complexes. Clearly, angiotensinogen was present at approximately 60 kDa and complement C3dg at about 40 kDa. ProMBP is barely seen in Coomassie Brilliant Blue-stained gels, but clearly visible at 50-90 kDa in Western blots (8, 10) . Sequence analysis of all bands blotted onto PVDF membrane confirmed their identities. Immunoblotting of the nonreduced material using a mAb recognizing angiotensinogen and polyclonal anti-complement C3d further verified the identities of the components (Fig. 4, lanes 7 and 8).

To investigate whether the complex containing C3dg was an artifact resulting from complement activation during preparation of serum, plasma samples (600 µl) from four normotensive pregnant women were chromatographed separately on Mono Q using conditions given in Fig. 2 . ProMBP-containing fractions were pooled and concentrated, and by immunoblotting as in Fig. 4(lanes 7 and 8) angiotensinogen and complement C3dg were detected in the same complexes (not shown).

Concerning the stoichiometry of the complexes, we believe the approximately 200-kDa band is a 2:2 complex of proMBP and angiotensinogen. First, the apparent molecular weight is compatible with twice the sum of 38.3 kDa (calculated size of proMBP (10) ) and approximately 60 kDa (angiotensinogen). Second, the yields of the components were similar in sequence analysis of the nonreduced complex. For example, the recovery of Val in cycle 3 () was 45 pmol, reflecting the amount of angiotensinogen. The amount of Leu in cycles 1 and 2 was 10 and 34 pmol, respectively, reflecting an equimolar amount of proMBP.

The approximately 300-kDa band most likely represents a 2:2:2 complex of proMBP, angiotensinogen, and complement C3dg. Compared with the lower band, the increment in size is in fair agreement with the addition of two molecules of C3dg. Likewise, the yields observed in sequence analysis support the presence of equimolar amounts of the three constituents.


DISCUSSION

The present investigation was prompted by the recent finding that circulating PAPP-A is in fact a 2:2 complex with proMBP, PAPP-AproMBP (8, 10) . Consequently, polyclonal anti-PAPP-AproMBP is polyspecific (8) and cannot be used in assays aimed at measuring serum PAPP-A. PAPP-A devoid of proMBP has not been demonstrated. Only as part of the PAPP-AproMBP complex can it be isolated from serum (10). We have determined, for the first time, serum levels of the PAPP-A subunit, and in the same samples levels of the proMBP subunit, throughout gestation. We demonstrate that on a molar basis, levels of proMBP are several fold higher than those of PAPP-A (Fig. 1).

Previously, several investigators have used anti-PAPP-AproMBP in different types of assays and reported third trimester values of PAPP-A (now known to be PAPP-AproMBP (8) ) at approximately 50 mg/liter or more (16, 17, 18, 19) . Our results suggest that the late pregnancy level of PAPP-A antigen is approximately 25 mg/liter, corresponding to 30 mg/liter of PAPP-AproMBP. Probably, previous PAPP-A determinations have been biased by the presence of surplus proMBP antigen.

Our results show that serum concentrations of PAPP-A continue to rise, almost linearly, throughout pregnancy. This is in contrast to earlier reports describing an exponential increase in early pregnancy and leveling off in the third trimester (17, 18, 19) . Those results are likely to be in error as a consequence of the early, steep rise in serum proMBP. The proMBP profile determined here is in accordance with previous results (9, 22, 38) . Noteworthy, the molar ratio of PAPP-A and proMBP is not constant during pregnancy.

Analytical ion exchange and gel chromatography of pooled pregnancy serum confirmed the presence of proMBP in excess of PAPP-A (Fig. 2) and showed that proMBP, besides its existence in PAPP-AproMBP, is present in other complexes. Interestingly, because the highly acidic proMBP (10, 13) was not detected in fractions eluting later than the PAPP-AproMBP complex, noncomplexed proMBP or polymers, hereof, most likely do not exist in serum. By gel filtration chromatography we showed that all proMBP-containing complexes are of high molecular weight.

As detailed under ``Results,'' we isolated two related disulfide bound complexes of proMBP. One is a 2:2 complex of proMBP with angiotensinogen, the other further contains two molecules of complement C3dg in a 2:2:2 complex. The results were obtained by sequence analysis of intact complexes () and of their components following reduction, by estimates of molecular weights in SDS-PAGE (Fig. 4, lane 5), and by reaction with specific mAbs and polyclonal antibodies in Western blotting (Fig. 4, lanes 7 and 8).

Regarding the 2:2:2 complex of proMBP and angiotensinogen with complement C3dg, to our knowledge there have been no reports of high molecular weight C3dg in pregnancy. However, increased second and third trimester plasma levels of both C3 and C3d have been described (39) .

Previously, high molecular weight (HMW) variants of angiotensinogen have been observed in both normal and pregnancy serum (40) . In normal serum, these variants elute mainly in the void volume upon gel filtration (40) and constitute about 5% of total angiotensinogen, but in pregnancy serum they amount to 15% in the third trimester of normal pregnancies (21, 41) . In hypertensive pregnancies, the proportion of HMW angiotensinogen is further increased, and it can become the predominant form (21) .

In nonpregnancy serum, the 5% HMW angiotensinogen present is not likely to be a complex between angiotensinogen and proMBP, because of the very low levels of proMBP measured here. But the additional 10% HMW angiotensinogen in pregnancy serum, on the other hand, is likely constituted by the complexes we have identified. First, in gel chromatography, the proMBPangiotensinogen(complement C3dg) complexes would be expected to elute as poorly resolved peaks between the void volume and the elution position of low molecular weight (LMW) angiotensinogen, as observed for HMW angiotensinogen (40, 41) . Second, the total amount of angiotensinogen in late pregnancy is approximately 5000 nM (calculated from a titer of 6.7 µg of angiotensin I/ml) (42) , and 10%, hereof, equals the surplus of proMBP relative to PAPP-A. We cannot exclude that, besides PAPP-A, proMBP binds proteins other than angiotensinogen. But our results strongly suggest that the proMBPangiotensinogen complexes are the most abundant.

It has been proposed (43) that angiotensinogen is secreted as a 60-kDa monomer, and that HMW angiotensinogen is composed entirely of LMW angiotensinogen subunits. Evidently, only a fraction of HMW angiotensinogen is present as homopolymers in pregnancy serum. As illustrated in Fig. 5, most likely angiotensinogen contains no intrachain disulfide bridges. But angiotensinogen polymers may result from air oxidation of its cysteine residues or from sulfhydryl-disulfide interchange in case one or more of the cysteine residues are bound to e.g. glutathione.


Figure 5: Schematic representation of the backbone structure of bovine antithrombin III, a member of the serpin family. C atoms (32) corresponding to positions of cysteine residues in angiotensinogen upon alignment of the sequences of antithrombin III and angiotensinogen (44, 45) are shown as spheres (Val-5, right; Lys-115, upper; Asp-201, lower; Thr-281, left). None of the cysteine residues of angiotensinogen are likely to form intrachain disulfide bridges, since the minimal distance between any two -carbons of these residues is 27.9 Å.



The mechanism of assembly of the complexes between pro-MBP, angiotensinogen and complement C3dg is not known. However, since no monomeric proMBP has been found in pregnancy serum, and since proMBP present in the PAPP-AproMBP complex is a disulfide-bridged dimer (8, 10) , it is likely that the novel complexes identified here also contain a bridged dimer of proMBP. ProMBP may be secreted as a monomer which readily dimerizes, or it may be secreted as a dimer. In any case, proMBP, like eosinophil MBP (5) , probably contains free SH-groups in addition to disulfide bridges. We therefore suggest that one proMBP dimer binds two molecules of angiotensinogen as a result of thiol-disulfide exchange or air oxidation. A fraction of these 2:2 complexes further binds two molecules of complement C3dg, which contains both a disulfide bridge (46) and a free sulfhydryl group resulting from hydrolysis of the thiol ester in C3 (47) .

The assembly of the complexes identified here is likely to take place in the placenta, since not only proMBP (14) and PAPP-A (15) , but also angiotensinogen, are expressed in placental tissue (48) . Furthermore, it has been shown recently that in all regions of placenta, HMW angiotensinogen constitutes the major proportion (60-70%) of the total angiotensinogen (49) . Since the two novel complexes were detected in pregnancy serum and plasma, the presence of C3dg in the 2:2:2 complex is not an artifact resulting from complement activation during preparation of serum. Whether this complex is formed locally in the placenta or in the plasma is not known. In any case, the finding here that the predominant part of pro-MBP in pregnancy serum is associated with only three proteins, PAPP-A, angiotensinogen, and C3dg, points toward specific interactions. This observation brings together proteins of diverse functional roles. It is of immediate interest to investigate the relationship of proMBP to hypertensive pregnancies, previously associated with HMW angiotensinogen (21) .

  
Table: N-terminal sequence analysis of two high molecular weight proMBP containing complexes

Phenylthiohydantoin-derivatives identified are given in standard three-letter code. Regarding yields, see main text. Materials subjected to sequence analysis are the approximately 200-kDa and approximately 300-kDa bands (Fig. 4, lane 4), also visualized as the proMBP-positive bands of lane 2. For both bands, sequences 1 and 2 originate from proMBP which has two N termini, one residue out of phase (8), and sequence 3 is the N-terminal of angiotensinogen (33). Sequence 4 (approximately 300 kDa band) originates from complement C3dg (34).



FOOTNOTES

*
The work was supported by the Danish Biomembrane Research Center, the Swedish Medical Research Council, and by Grants AI 09728, AI 15231, and HD 22924 from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Fax: 45-8942-2679; oxvig@biobase.dk.

The abbreviations used are: MBP, human eosinophil major basic protein; HMW, high molecular weight; LMW, low molecular weight; IEP, immunoelectrophoresis; mAb, monoclonal antibody; PAPP-A, human pregnancy-associated plasma protein-A; PBS, phosphate-buffered saline; proMBP, proform of human eosinophil major basic protein; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride.

P. Popken-Harris, M. McGrogan, D. A. Loegering, J. L. Checkel, H. Kubo, L. L. Thomas, J. N. Moy, L. Sottrup-Jensen, J. L. Snable, M. T. Kikuchi, and G. J. Gleich, submitted for publication.


ACKNOWLEDGEMENTS

We thank the staff at the Department of Gynecology and Obstetrics, University Hospital for collecting and donating pregnancy serum.


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