From the
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-A
Eosinophil major basic protein (MBP)
The complex of PAPP-A and
proMBP, denoted PAPP-A
Proteins used as standards or controls in
radioimmunoassays, PAPP-A
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
A mAb specific for
the propiece of proMBP, J163-15E10
For determinations of PAPP-A, an inhibition
radioimmunoassay was developed, utilizing
For establishing the standard curve, purified
PAPP-A
Rocket IEP was performed as described previously
(29) using
anti-PAPP-A
The analytical
ion exchange experiment (Fig. 2) shows that PAPP-A
From previous work it is known that the PAPP-A
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-A
To summarize, only pool v contained proMBP complexes that were present in substantial
amounts and different from PAPP-A
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.
The present investigation was prompted by the recent finding
that circulating PAPP-A is in fact a 2:2 complex with proMBP,
PAPP-A
Previously, several investigators have
used anti-PAPP-A
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-A
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
proMBP
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.
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) .
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).
We thank the staff at the Department of Gynecology and
Obstetrics, University Hospital for collecting and donating
pregnancy serum.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
proMBP 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.
(
)
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) .
pro-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-A
proMBP complex to determine the levels in
pregnancy serum of PAPP-A
antigen
(16, 17, 18, 19, 20) .
But anti-PAPP-A
proMBP 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.
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.
proMBP, 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-A
proMBP in
Freund's incomplete adjuvant. Eight weeks after the initial
immunization the animals were boosted intravenously with 100 µg of
PAPP-A
proMBP 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-A
proMBP
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).
I-PAPP-A
proMBP in
the radioimmunoassay described below (not shown).
, 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-A
proMBP, 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-A
proMBP standard curve.
All samples were reduced and alkylated
(24) prior to
measurement.
I-labeled
PAPP-A
proMBP
(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-A
proMBP (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.
proMBP 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.
proMBP (Dako, A230). Routinely, rocket IEP is used for
detection of PAPP-A
proMBP 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-A
proMBP (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-A
proMBP, 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.
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-A
proMBP complex. In this system, proMBP isolated from
reduced and carboxamidomethylated PAPP-A
proMBP (8, 10) elutes
later than PAPP-A
pro-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-A
proMBP.
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-A
proMBP 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.
proMBP
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).
proMBP complex
binds to immobilized nickel ions
(10) . Therefore, in order to
separate PAPP-A
proMBP 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-A
proMBP
(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.
proMBP 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-A
proMBP complexes escaped binding to the nickel affinity
column, probably due to shielding of the metal binding site of
PAPP-A
proMBP. 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-A
proMBP. 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.
proMBP.
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).
proMBP
(8, 10) . Consequently, polyclonal
anti-PAPP-A
proMBP 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-A
proMBP 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).
proMBP in different types of assays and reported
third trimester values of PAPP-A (now known to be
PAPP-A
proMBP
(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-A
proMBP. Probably, previous PAPP-A determinations have been
biased by the presence of surplus proMBP antigen.
proMBP, is present in other complexes. Interestingly,
because the highly acidic proMBP
(10, 13) was not
detected in fractions eluting later than the PAPP-A
proMBP
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.
angiotensinogen(
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 proMBP
angiotensinogen complexes are the most
abundant.
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) .
Table:
N-terminal sequence analysis of two high
molecular weight proMBP containing complexes
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.